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Referee #3

Evidence, reproducibility and clarity

Summary:

Parkin, a E3 ubiquitin ligase, is involved in the clearance of damaged mitochondrial via mitophagy. Upon mitochondrial damage, the activated Parkin ubiquitinates many mitochondrial substrates, leading to the recruitment of mitophagy effectors. However, the mechanism of substrate recognition by Parkin is still not known.

In this manuscript, Koszela et al. utilized diverse biochemical assays and biophysical approaches, combined with AlphaFold prediction, to identify a conserved region in the flexible linker between the Ubl and RING0 domains of Parkin that recognizes mitochondrial GTPase Miro1 via a stretch of hydrophobic residues and is critical for its ubiquitination activity on Miro1. This manuscript reveals the mechanisms by which Parkin recognizes and ubiquitinates substrate Miro1, providing a biochemical explanation for the presence of Parkin at the mitochondrial membrane prior to activation by mitochondrial damage. This study also provides insights into mitochondrial homeostasis and may facilitate new therapeutic approaches for Parkinson's disease.

Major Comments:

• The authors should expand the background introduction to include the biological function of Miro1, the domain architecture of Miro1 and more context of Miro1 K572 ubiquitination in mitophagy.
• Figure 1B is confusing. Due to the presence of various bands, it is hard to assign specific bands in each lane. In addition, there are various unlabeled bands that makes things unclear. The authors should include loading controls to clearly discern pParkin, Ube1, Ube2L3, and all substrates.
• In Figure 1B, it was not possible to identify the ubiquitination bands of E2 enzyme UBE2L3 and the E1 enzyme UBE1. Please indicate these bands on the gel.
• Since ubiquitinated Miro1 and Mfn1 are similar in molecular weight (Fig. 1b), the authors should show a western blot against the Miro1 and Mfn1 tag as done in the supplementary information, At least for the competition assays involving both Miro1 and Mfn1.
• The conclusion that Miro1 is pParkin's preferred substrate is not convincing. In the competition assay used to show substrate preference, Miro1 is at a five-fold higher concentration than the other substrates and 25-fold higher than FANCI/D2. This would ultimately drive pParkin's interaction with Miro1. This is further highlighted by the fact that it adding Mfn1 in excess has a similar effect. The competition assay should be done at equimolar concentrations of Miro1 and substrate. More convincing would be a competition assay where substrate ubiquitination is quantified at several different concentrations of Miro1.
• In Figure 1F, it is unclear what is defined as "high" or "low" ubiquitination levels statistically. Some of the changes in ubiquitination levels are extremely subtle (ex. mitoNEET and FancI/D2 in the presence and absence of Miro1 and Mfn1). In some cases, I find it extremely difficult to tell if there is any change in the ubiquitination levels when comparing lanes containing excess of different substrates. I would like to see band quantifications of this experiment in triplicate to support the conclusions drawn from the competition assay.
• The authors used both unmodified and phosphorylated Parkin for the crosslinking experiments and observe no difference in the intensity of the bands. However, this is not sufficient to draw any conclusion about the affinity between phosphorylated Parkin and Miro1 (which was done in lines 341-343). The authors should comment on why they did not test pParkin binding with Miro1, especially given the statement:

"In our assays in the absence of pUb, pParkin must interact with its substrates without the action of pUb, likely through 158 transient, low affinity interactions" - The reference to Parkin115-124 as a "Substrate Targeting Region (STR)" is misleading. This would imply that this motif in Parkin is responsible for general substrate recognition when there is no direct evidence of this. In Figure 5F, the authors create a synthetic peptide based off the STR sequence. Although this sequence was effective in inhibiting the ubiquitination of Miro1, it was ineffective against Mfn1. This would indicate that Mfn1 relies on a completely different set of interactions for ubiquitination by Parkin. I suggest that the authors tone down the language in describing this region and rename this region (perhaps "Miro1 Targeting Region (MTR)"?). - The authors appear to confuse plDDT and PAE scores in Figure 5B. The PAE describes the expected positional error of each residue in the model. The plot should be colored in terms of Expected Position Error (Ångstrom), not plDDT scores.

Minor Comments:

• Figure 1A would benefit from a schematic showing the domain architecture. If the goal is to appreciate the length of the linker, then showing the actual amino acid length would be beneficial.
• In Supplementary Figure 2D, the authors performed the MST experiment with His6-Smt3-tagged Parkin. The group had previously shown that the presence of the tag artificially interferes with autoubiquitination, potentially by forming intramolecular interactions. The SEC, Native Page, and ITC data of untagged Parkin with Miro1 provide sufficient evidence that the interaction between the two are weak. The authors should consider removing the MST data, since they are not congruent with the other experiments.
• The ITC data in Supplementary Figure 2C look promising. It would be nice if the authors could try to quantify the Kd of their STR peptides to Miro1
• Are STR peptides 1 and/or 2 unable to inhibit ubiquitination of other Parkin substrates besides Mfn1? Do these other substrates utilize the STR for recognition? AlphaFold modeling may provide some insight on Parkin recognition of other substrates.
• The authors shold consider using AlphaFold3 to model the interaction of pParkin with Miro1 compares to unmodified Parkin.
• Please label the protein names in Figure 4A for a better presentation.
• Page 2, line 37. "...by a 65-residue flexible region (linker) to a unique to Parkin RING0 domain..." should be "...by a 65-residue flexible region (linker) to a unique Parkin RING0 domain...". The second "to" should be omitted.
• Page 3, Line 48: "fulfill", not "fulfil"
• Page 5, line 110. In sentence, "...phosphorylation at Ser65 of Parkin...", it is better to explicitly state that this phosphorylation happens on the Parkin Ubl domain.
• Page 7, line151. Figure 1F should be Figure 1G.
• Page 11, line 241. In sentence "...Miro1 residues R263, R265 and D228...", do the authors mean R261 and not R265?

Significance

Parkin is an E3 ubiquitin ligase that is activated to ubiquitinate diverse substrates on the mitochondrial membrane in response to mitochondrial damage, thereby recruiting mitophagy effectors. This study reveals the mechanisms by which Parkin recognizes and ubiquitinates Miro1, providing insights into mitochondrial homeostasis and facilitating new therapeutic approaches for Parkinson's disease.

Readers with a background in protein ubiquitination and mitochondrial homeostasis might be interested in this study. My expertise includes protein ubiquitination and structural biology. However, I do not have sufficient expertise to evaluate the NMR experiments in this manuscript.

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Author response:

The following is the response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Nitta et al, in their manuscript titled, "Drosophila model to clarify the pathological significance of OPA1 in autosomal dominant optic atrophy." The novelty of this paper lies in its use of human (hOPA1) to try to rescue the phenotype of an OPA1 +/- Drosophilia DOA model (dOPA). The authors then use this model to investigate the differences between dominant-negative and haploinsufficient OPA1 variants. The value of this paper lies in the study of DN/HI variants rather than the establishment of the drosophila model per se as this has existed for some time and does have some significant disadvantages compared to existing models, particularly in the extra-ocular phenotype which is common with some OPA1 variants but not in humans. I judge the findings of this paper to be valuable with regards to significance and solid with regards to the strength of the evidence.

Suggestions for improvements:

(1) Stylistically the results section appears to have significant discussion/conclusion/inferences in section with reference to existing literature. I feel that this information would be better placed in the separate discussion section. E.g. lines 149-154.

We appreciate the reviewer’s suggestion to relocate the discussion, conclusions, and inferences, particularly those that reference existing literature, to a separate discussion section. For lines 149–154, we placed them in the discussion section (lines 343–347) as follows. “Our established fly model is the first simple organism to allow observation of degeneration of the retinal axons. The mitochondria in the axons showed fragmentation of mitochondria. Former studies have observed mitochondrial fragmentation in S2 cells (McQuibban et al., 2006), muscle tissue (Deng et al., 2008), segmental nerves (Trevisan et al., 2018), and ommatidia (Yarosh et al., 2008) due to the LOF of dOPA1.”

For lines 178–181, we also placed them in the discussion section (lines 347–351) as follows. “Our study presents compelling evidence that dOPA1 knockdown instigates neuronal degeneration, characterized by a sequential deterioration at the axonal terminals and extending to the cell bodies. This degenerative pattern, commencing from the distal axons and progressing proximally towards the cell soma, aligns with the paradigm of 'dying-back' neuropathy, a phenomenon extensively documented in various neurodegenerative disorders (Wang et al., 2012). ”

For lines 213–217, 218–220, and 222–223, we also placed them in the discussion section (lines 363– 391) as follows. “To elucidate the pathophysiological implications of mutations in the OPA1 gene, we engineered and expressed several human OPA1 variants, including the 2708-2711del mutation, associated with DOA, and the I382M mutation, located in the GTPase domain and linked to DOA. We also investigated the D438V and R445H mutations in the GTPase domain and correlated with the more severe DOA plus phenotype. The 2708-2711del mutation exhibited limited detectability via HA-tag probing. Still, it was undetectable with a myc tag, likely due to a frameshift event leading to the mutation's characteristic truncated protein product, as delineated in prior studies (Zanna et al., 2008). Contrastingly, the I382M, D438V, and R445H mutations demonstrated expression levels comparable to the WT hOPA1. However, the expression of these mutants in retinal axons did not restore the dOPA1 deficiency to the same extent as the WT hOPA1, as evidenced in Figure 5E. This finding indicates a functional impairment imparted by these mutations, aligning with established understanding (Zanna et al., 2008). Notably, while the 2708-2711del and I382M mutations exhibited limited functional rescue, the D438V and R445H mutations did not show significant rescue activity. This differential rescue efficiency suggests that the former mutations, particularly the I382M, categorized as a hypomorph (Del Dotto et al., 2018), may retain partial functional capacity, indicative of a LOF effect but with residual activity. The I382M missense mutation within the GTPase domain of OPA1 has been described as a mild hypomorph or a disease modifier. Intriguingly, this mutation alone does not induce significant clinical outcomes, as evidenced by multiple studies (Schaaf et al., 2011; Bonneau et al., 2014; Bonifert et al., 2014; Carelli et al., 2015). A significant reduction in protein levels has been observed in fibroblasts originating from patients harboring the I382M mutation. However, mitochondrial volume remains unaffected, and the fusion activity of mitochondria is only minimally influenced (Kane et al., 2017; Del Dotto et al., 2018). This observation is consistent with findings reported by de la Barca et al. in Human Molecular Genetics 2020, where a targeted metabolomics approach classified I382M as a mild hypomorph. In our current study, the I382M mutation preserves more OPA1 function compared to DN mutations, as depicted in Figures 5E and F. Considering the results from our Drosophila model and previous research, we hypothesize that the I382M mutation may constitute a mild hypomorphic variant. This might explain its failure to manifest a phenotype on its own, yet its contribution to increased severity when it occurs in compound heterozygosity.

(2) I do think further investigation as to why a reduction of mitochondria was noticed in the knockdown. There are conflicting reports on this in the literature. My own experience of this is fairly uniform mitochondrial number in WT vs OPA1 variant lines but with an increased level of mitophagy presumably reflecting a greater turnover. There are a number of ways to quantify mitochondrial load e.g. mtDNA quantification, protein quantification for tom20/hsp60 or equivalent. I feel the reliance on ICC here is not enough to draw conclusions. Furthermore, mitophagy markers could be checked at the same time either at the transcript or protein level. I feel this is important as it helps validate the drosophila model as we already have a lot of experimental data about the number and function of mitochondria in OPA+/- human/mammalian cells.

We thank the reviewer for the insightful comments and suggestions regarding our study on the impact of mitochondrial reduction in a knockdown model. We concur with the reviewer’s observation that our initial results did not definitively demonstrate a decrease in the number of mitochondria in retinal axons. Furthermore, we measured mitochondrial quantity by conducting western blotting using antiCOXII and found no reduction in mitochondrial content with the knockdown of dOPA1 (Figure S4A and B). Consequently, we have revised our manuscript to remove the statement “suggesting a decreased number of mitochondria in retinal axons. However, whether this decrease is due to degradation resulting from a decline in mitochondrial quality or axonal transport failure remains unclear.” Instead, we have refocused our conclusion to reflect our electron microscopy findings, which indicate reduced mitochondrial size and structural abnormalities. The reviewer’s observation of consistent mitochondrial numbers in WT versus mutant variant lines and elevated mitophagy levels prompted us to evaluate mitochondrial turnover as a significant factor in our study. Regarding verifying mitophagy markers, we incorporated the mito-QC marker in our experimental design. In our experiments, mito-QC was expressed in the retinal axons of Drosophila to assess mitophagy activity upon dOPA1 knockdown. We observed a notable increase in mCherry positive but GFP negative puncta signals one week after eclosion, indicating the activation of mitophagy (Figure 2D–H). This outcome strongly suggests that dOPA1 knockdown enhances mitophagy in our Drosophila model. The application of mito-QC as a quantitative marker for mitophagy, validated in previous studies, offers a robust approach to analyzing this process. Our findings elucidate the role of dOPA1 in mitochondrial dynamics and its implications for neuronal health. These results have been incorporated into Figure 2, with the corresponding text updated as follows (lines 159–167): “Given that an increase in mitophagy activity has been reported in mouse RGCs and nematode ADOA models (Zaninello et al., 2022; Zaninello et al., 2020), the mitoQC marker, an established indicator of mitophagy activity, was expressed in the photoreceptors of Drosophila. The mito-QC reporter consists of a tandem mCherry-GFP tag that localizes to the outer membrane of mitochondria (Lee et al., 2018). This construct allows the measurement of mitophagy by detecting an increase in the red-only mCherry signal when the GFP is degraded after mitochondria are transported to lysosomes. Post dOPA1 knockdown, we observed a significant elevation in mCherry positive and GFP negative puncta signals at one week, demonstrating an activation of mitophagy as a consequence of dOPA1 knockdown (Figure 2D–H).”  

(3) Could the authors comment on the failure of the dOPA1 rescue to return their biomarker, axonal number to control levels. In Figure 4D is there significance between the control and rescue. Presumably so as there is between the mutant and rescue and the difference looks less.

As the reviewer correctly pointed out, there is a significant difference between the control and rescue groups, which we have now included in the figure. Additionally, we have incorporated the following comments in the discussion section (lines 329–342) regarding this significant difference: “In our study, expressing dOPA1 in the retinal axons of dOPA1 mutants resulted in significant rescue, but it did not return to control levels. There are three possible explanations for this result. The first concerns gene expression levels. The Gal4-line used for the rescue experiments may not replicate the expression levels or timing of endogenous dOPA1. Considering that the optimal functionality of dOPA1 may be contingent upon specific gene expression levels, attaining a wild-type-like state necessitates the precise regulation of these expression levels. The second is a nonautonomous issue. Although dOPA1 gene expression was induced in the retinal axons for the rescue experiments, many retinal axons were homozygous mutants, while other cell types were heterozygous for the dOPA1 mutation. If there is a non-autonomous effect of dOPA1 in cells other than retinal axons, it might not be possible to restore the wild-type-like state fully. The third potential issue is that only one isoform of dOPA1 was expressed. In mouse OPA1, to completely restore mitochondrial network shape, an appropriate balance of at least two different isoforms, lOPA1 and s-OPA1, is required (Del Dotto et al., 2017). This requirement implies that multiple isoforms of dOPA1 are essential for the dynamic activities of mitochondria.”

(4) The authors have chosen an interesting if complicated missense variant to study, namely the I382M with several studies showing this is insufficient to cause disease in isolation and appears in high frequency on gnomAD but appears to worsen the phenotype when it appears as a compound het. I think this is worth discussing in the context of the results, particularly with regard to the ability for this variant to partially rescue the dOPA1 model as shown in Figure 5.

As the reviewer pointed out, the I382M mutation is known to act as a disease modifier. However, in our system, as suggested by Figure 5, I382M appears to retain more activity than DN mutations. Considering previous studies, we propose that I382M represents a mild hypomorph. Consequently, while I382M alone may not exhibit a phenotype, it could exacerbate severity in a compound heterozygous state. We have incorporated this perspective in our revised discussion (lines 375-391).

“Notably, while the 2708-2711del and I382M mutations exhibited limited functional rescue, the D438V and R445H mutations did not show significant rescue activity. This differential rescue efficiency suggests that the former mutations, particularly the I382M, categorized as a hypomorph (Del Dotto et al., 2018), may retain partial functional capacity, indicative of a LOF effect but with residual activity. The I382M missense mutation within the GTPase domain of OPA1 has been described as a mild hypomorph or a disease modifier. Intriguingly, this mutation alone does no induce significant clinical outcomes, as evidenced by multiple studies (Schaaf et al., 2011; Bonneau et al., 2014; Bonifert et al., 2014; Carelli et al., 2015). A significant reduction in protein levels has been observed in fibroblasts originating from patients harboring the I382M mutation. However, mitochondrial volume remains unaffected, and the fusion activity of mitochondria is only minimally influenced (Kane et al., 2017; Del Dotto et al., 2018). This observation is consistent with findings reported by de la Barca et al. in Human Molecular Genetics 2020, where a targeted metabolomics approach classified I382M as a mild hypomorph. In our current study, the I382M mutation preserves more OPA1 function compared to DN mutations, as depicted in Figures 5E and F. Considering the results from our Drosophila model and previous research, we hypothesize that the I382M mutation may constitute a mild hypomorphic variant. This might explain its failure to manifest a phenotype on its own, yet its contribution to increased severity when it occurs in compound heterozygosity.”

(5) I feel the main limitation of this paper is the reliance on axonal number as a biomarker for OPA1 function and ultimately rescue. I have concerns because a) this is not a well validated biomarker within the context of OPA1 variants b) we have little understanding of how this is affected by over/under expression and c) if it is a threshold effect e.g. once OPA1 levels reach <x% pathology develops but develops normally when opa1 expression is >x%. I think this is particularly relevant when the authors are using this model to make conclusions on dominant negativity/HI with the authors proposing that if expression of a hOPA1 transcript does not increase opa1 expression in a dOPA1 KO then this means that the variant is DN. The authors have used other biomarkers in parts of this manuscript e.g. ROS measurement and mito trafficking but I feel this would benefit from something else particularly in the latter experiments demonstrated in figure 5 and 6.

The reviewer raised concerns regarding the adequacy of axonal count as a validated biomarker in the context of OPA1 mutants. In response, we corroborated its validity using markers such as MitoSOX, Atg8, and COXII. Experiments employing MitoSOX revealed that the augmented ROS signals resulting from dOPA1 knockdown were mitigated by expressing human OPA1. Conversely, the mutant variants 2708-2711del, D438V, and R445H did not ameliorate these effects, paralleling the phenotype of axonal degeneration observed. These findings are documented in Figure 5F, and we have incorporated the following text into section lines 248–254 of the results:

“Furthermore, we assessed the potential for rescuing ROS signals. Similar to its effect on axonal degeneration, wild-type hOPA1 effectively mitigated the phenotype, whereas the 2708-2711del, D438V, and R445H mutants did not (Figure 5F). Importantly, the I382M variant also reduced ROS levels comparably to the wild type. These findings demonstrate that both axonal degeneration and the increase in ROS caused by dOPA1 downregulation can be effectively counteracted by hOPA1. Although I382M retains partial functionality, it acts as a relatively weak hypomorph in this experimental setup.”

Moreover, utilizing mito-QC, we observed elevated mitophagy in our Drosophila model, with these results now included in Figure 2D–H. Given the complexity of the genetics involved and the challenges in establishing lines, autophagy activity was quantified by comparing the ratio of Atg8-1 to Atg8-2 via Western blot analysis. However, no significant alterations were detected across any of the genotypes. Additionally, mitochondrial protein levels derived from COXII confirmed consistent mitochondrial quantities, showing no considerable variance following knockdown. These insights affirm that retinal axon degeneration and mitophagy activation are present in the Drosophila DOA model, although the Western blot analysis revealed no significant changes in autophagy activation. Such findings necessitate caution as this model may not fully replicate the molecular pathology of the corresponding human disease. These Western blot findings are presented in Figure S4, with the following addition made to section lines 255–263 of the results:

“We also conducted Western blot analyses using anti-COXII and anti-Atg8a antibodies to assess changes in mitochondrial quantity and autophagy activity following the knockdown of dOPA1. Mitochondrial protein levels, indicated by COXII quantification, were evaluated to verify mitochondrial content, and the ratio of Atg8a-1 to Atg8a-2 was used to measure autophagy activation. For these experiments, Tub-Gal4 was employed to systemically knockdown dOPA1. Considering the lethality of a whole-body dOPA1 knockdown, Tub-Gal80TS was utilized to repress the knockdown until eclosion by maintaining the flies at 20°C. After eclosion, we increased the temperature to 29°C for two weeks to induce the knockdown or expression of hOPA1 variants. The results revealed no significant differences across the genotypes tested (Figure S4A–D).”

In assessing the effects of dominant negative mutations, measurements including ROS levels, the ratio of Atg8-1 to Atg8-2, and the quantity of COXII protein were conducted, yet no significant differences were observed (Figure S6). This limitation of the fly model is mentioned in the results, noting the observation of the axonal degeneration phenotype but not alterations in ROS signaling, autophagy activity, or mitochondrial quantity as follows (line 287–290):

“We investigated the impacts of dominant negative mutations on mitochondrial oxidation levels, mitochondrial quantity, and autophagy activation levels; however, none of these parameters showed statistical significance (Figure S6).”

The reviewer also inquired about the effects of overexpressing and underexpressing OPA1 on axonal count and whether these effects are subject to a threshold. In response, we expressed both wild-type and variant forms of human OPA1 in Drosophila in vivo and assessed their protein levels using Western blot analysis. The results showed no significant differences in expression levels between the wild-type and variant forms in the OPA1 overexpression experiments, suggesting the absence of a variation threshold effect. These findings have been newly documented as quantitative data in Figure 5C. Furthermore, we have included a statement in the results section for Figure 6A, clarifying that overexpression of hOPA1 exhibited no discernible impact, as detailed on lines 274–276.

“The results presented in Figure 5C indicate that there are no significant differences in the expression levels among the variants, suggesting that variations in expression levels do not influence the outcomes.”

(6) Could the authors clarify what exons in Figure 5 are included in their transcript. My understanding is transcript NM_015560.3 contains exon 4,4b but not 5b. According to Song 2007 this transcript produces invariably s-OPA1 as it contains the exon 4b cleavage site. If this is true, this is a critical limitation in this study and in my opinion significantly undermines the likelihood of the proposed explanation of the findings presented in Figure 6. The primarily functional location of OPA1 is at the IMM and l-OPA1 is the primary opa1 isoform probably only that localizes here as the additional AA act as a IMM anchor. Given this is where GTPase likely oligomerizes the expression of s-OPA1 only is unlikely to interact anyway with native protein. I am not aware of any evidence s-OPA1 is involved in oligomerization. Therefore I don't think this method and specifically expression of a hOPA1 transcript which only makes s-OPA1 to be a reliable indicator of dominant negativity/interference with WT protein function. This could be checked by blotting UAS-hOPA1 protein with a OPA1 antibody specific to human OPA1 only and not to dOPA1. There are several available on the market and if the authors see only s-OPA1 then it confirms they are not expressing l-OPA1 with their hOPA1 construct.

As suggested by the reviewer, we performed a Western blot using a human OPA1 antibody to determine if the expressed hOPA1 was producing the l-OPA1 isoform, as shown in band 2 of Figure 5D. The results confirmed the presence of both l-OPA1 and what appears to be s-OPA1 in bands 2 and 4, respectively. These findings are documented in the updated Figure 5D, with a detailed description provided in the manuscript at lines 224-226. Additionally, the NM_015560.3 refers to isoform 1, which includes only exons 4 and 5, excluding exons 4b and 5b. This isoform can express both l-OPA1 and s-OPA1 (refer to Figure 1 in Song et al., J Cell Biol. 2007). We have updated the schematic diagram in the figure to include these exons. The formation of s-OPA1 through cleavage occurs at the OMA1 target site located in exon 5 and the Yme1L target site in exon 5b of OPA1. Isoform 1 of OPA1 is prone to cleavage by OMA1, but a homologous gene for OMA1 does not exist in Drosophila. Although a homologous gene for Yme1L is present in Drosophila, exon 5b is missing in isoform 1 of OPA1, leaving the origin of the smaller band resembling s-OPA1 unclear at this point.

Reviewer #2 (Public Review):

The data presented support and extend some previously published data using Drosophila as a model to unravel the cellular and genetic basis of human Autosomal dominant optic atrophy (DOA). In human, mutations in OPA1, a mitochondrial dynamin like GTPase (amongst others), are the most common cause for DOA. By using a Drosophila loss-of-function mutations, RNAi- mediated knockdown and overexpression, the authors could recapitulate some aspects of the disease phenotype, which could be rescued by the wild-type version of the human gene. Their assays allowed them to distinguish between mutations causing human DOA, affecting the optic system and supposed to be loss-of-function mutations, and those mutations supposed to act as dominant negative, resulting in DOA plus, in which other tissues/organs are affected as well. Based on the lack of information in the Materials and Methods section and in several figure legends, it was not in all cases possible to follow the conclusions of the authors.

We appreciate the reviewer's constructive feedback and the emphasis on enhancing clarity in our manuscript. We recognize the concerns raised about the lack of detailed information in the Materials and Methods section and several figure legends, which may have obscured our conclusions. In response, we have appended the detailed genotypes of the Drosophila strains used in each experiment to a supplementary table. Additionally, we realized that the description of 'immunohistochemistry and imaging' was too brief, previously referenced simply as “immunohistochemistry was performed as described previously (Sugie et al., 2017).” We have now expanded this section to include comprehensive methodological details. Furthermore, we have revised the figure legends to provide clearer and more thorough descriptions.

Similarly, how the knowledge gained could help to "inform early treatment decisions in patients with mutations in hOPA1" (line 38) cannot be followed.

To address the reviewer's comments, we have refined our explanation of the clinical relevance of our findings as follows. We believe this revision succinctly articulates the practical application of our research, directly responding to the reviewer’s concerns about linking the study's outcomes to treatment decisions for patients with hOPA1 mutations. By underscoring the model’s value in differential diagnosis and its influence on initiating treatment strategies, we have clarified this connection explicitly, within the constraints of the abstract’s word limit. The revised sentence now reads: "This fly model aids in distinguishing DOA from DOA plus and guides initial hOPA1 mutation treatment strategies."

Reviewer #3 (Public Review):

Nitta et al. establish a fly model of autosomal dominant optic atrophy, of which hundreds of different OPA1 mutations are the cause with wide phenotypic variance. It has long been hypothesized that missense OPA1 mutations affecting the GTPase domain, which are associated with more severe optic atrophy and extra-ophthalmic neurologic conditions such as sensorineural hearing loss (DOA plus), impart their effects through a dominant negative mechanism, but no clear direct evidence for this exists particularly in an animal model. The authors execute a well-designed study to establish their model, demonstrating a clear mitochondrial phenotype with multiple clinical analogs including optic atrophy measured as axonal degeneration. They then show that hOPA1 mitigates optic atrophy with the same efficacy as dOPA1, setting up the utility of their model to test disease-causing hOPA1 variants. Finally, they leverage this model to provide the first direct evidence for a dominant negative mechanism for 2 mutations causing DOA plus by expressing these variants in the background of a full hOPA1 complement.

Strengths of the paper include well-motivated objectives and hypotheses, overall solid design and execution, and a generally clear and thorough interpretation of their results. The results technically support their primary conclusions with caveats. The first is that both dOPA1 and hOPA1 fail to fully restore optic axonal integrity, yet the authors fail to acknowledge that this only constitutes a partial rescue, nor do they discuss how this fact might influence our interpretation of their subsequent results.

As the reviewer rightly points out, neither dOPA1 nor hOPA1 achieve a complete recovery. Therefore, we acknowledge that this represents only a partial rescue and have added the following explanations regarding this partial rescue in the results and discussion sections.

Result:

Significantly —> partially (lines 207 and 228) Discussion (lines 329–342):

In our study, expressing dOPA1 in the retinal axons of dOPA1 mutants resulted in significant rescue, but it did not return to control levels. There are three possible explanations for this result. The first concerns gene expression levels. The Gal4-line used for the rescue experiments may not replicate the expression levels or timing of endogenous dOPA1. Considering that the optimal functionality of dOPA1 may be contingent upon specific gene expression levels, attaining a wild-type-like state necessitates the precise regulation of these expression levels. The second is a non-autonomous issue. Although dOPA1 gene expression was induced in the retinal axons for the rescue experiments, many retinal axons were homozygous mutants, while other cell types were heterozygous for the dOPA1 mutation. If there is a non-autonomous effect of dOPA1 in cells other than retinal axons, it might not be possible to restore the wild-type-like state fully. The third potential issue is that only one isoform of dOPA1 was expressed. In mouse OPA1, to completely restore mitochondrial network shape, an appropriate balance of at least two different isoforms, l-OPA1 and s-OPA1, is required (Del Dotto et al., 2017). This requirement implies that multiple isoforms of dOPA1 are essential for the dynamic activities of mitochondria.

The second caveat is that their effect sizes are small. Statistically, the results indeed support a dominant negative effect of DOA plus-associated variants, yet the data show a marginal impact on axonal degeneration for these variants. The authors might have considered exploring the impact of these variants on other mitochondrial outcome measures they established earlier on. They might also consider providing some functional context for this marginal difference in axonal optic nerve degeneration.

In response to the reviewer’s comment regarding the modest effect sizes observed, we acknowledge that the magnitude of the reported changes is indeed small. To explore the impact of these variants on additional mitochondrial outcomes as suggested, we employed markers such as MitoSOX, Atg8, and COXII for validation. However, we could not detect any significant effects of the DOA plus-associated variants using these methods. We apologize for the redundancy, but to address Reviewer #1's fifth question, we present experimental results showing that while the increased ROS signals observed upon dOPA1 knockdown were rescued by expressing human OPA1, the mutant variants 2708-2711del, D438V, and R445H did not ameliorate this effect. This outcome mirrors the axonal degeneration phenotype and is documented in Figure 5F. The following text has been added to the results section

lines 248–254:

“Furthermore, we assessed the potential for rescuing ROS signals. Similar to its effect on axonal degeneration, wild-type hOPA1 effectively mitigated the phenotype, whereas the 2708-2711del, D438V, and R445H mutants did not (Figure 5F). Importantly, the I382M variant also reduced ROS levels comparably to the wild type. These findings demonstrate that both axonal degeneration and the increase in ROS caused by dOPA1 downregulation can be effectively counteracted by hOPA1. Although I382M retains partial functionality, it acts as a relatively weak hypomorph in this experimental setup.”

Moreover, utilizing mito-QC, we observed elevated mitophagy in our Drosophila model, with these results now included in Figure 2D–H. Given the complexity of the genetics involved and the challenges in establishing lines, autophagy activity was quantified by comparing the ratio of Atg8-1 to Atg8-2 via Western blot analysis. However, no significant alterations were detected across any of the genotypes. Additionally, mitochondrial protein levels derived from COXII confirmed consistent mitochondrial quantities, showing no considerable variance following knockdown. These insights affirm that retinal axon degeneration and mitophagy activation are present in the Drosophila DOA model, although the Western blot analysis revealed no significant changes in autophagy activation. Such findings necessitate caution as this model may not fully replicate the molecular pathology of the corresponding human disease. These Western blot findings are presented in Figure S4, with the following addition made to section lines 255–263 of the results:

“We also conducted Western blot analyses using anti-COXII and anti-Atg8a antibodies to assess changes in mitochondrial quantity and autophagy activity following the knockdown of dOPA1. Mitochondrial protein levels, indicated by COXII quantification, were evaluated to verify mitochondrial content, and the ratio of Atg8a-1 to Atg8a-2 was used to measure autophagy activation. For these experiments, Tub-Gal4 was employed to systemically knockdown dOPA1. Considering the lethality of a whole-body dOPA1 knockdown, Tub-Gal80TS was utilized to repress the knockdown until eclosion by maintaining the flies at 20°C. After eclosion, we increased the temperature to 29°C for two weeks to induce the knockdown or expression of hOPA1 variants. The results revealed no significant differences across the genotypes tested (Figure S4A–D).”

In assessing the effects of dominant negative mutations, measurements including ROS levels, the ratio of Atg8-1 to Atg8-2, and the quantity of COXII protein were conducted, yet no significant differences were observed (Figure S6). This limitation of the fly model is mentioned in the results, noting the observation of the axonal degeneration phenotype but not alterations in ROS signaling, autophagy activity, or mitochondrial quantity as follows (line 287–290):

“We investigated the impacts of dominant negative mutations on mitochondrial oxidation levels, mitochondrial quantity, and autophagy activation levels; however, none of these parameters showed statistical significance (Figure S6).”

Despite these caveats, the authors provide the first animal model of DOA that also allows for rapid assessment and mechanistic testing of suspected OPA1 variants. The impact of this work in providing the first direct evidence of a dominant negative mechanism is under-stated considering how important this question is in development of genetic treatments for DOA. The authors discuss important points regarding the potential utility of this model in clinical science. Comments on the potential use of this model to investigate variants of unknown significance in clinical diagnosis requires further discussion of whether there is indeed precedent for this in other genetic conditions (since the model is nevertheless so evolutionarily removed from humans).

As suggested by the reviewer, we have expanded the discussion in our study to emphasize in greater detail the significance of the fruit fly model and the MeDUsA software we have developed, elaborating on the model's potential applications in clinical science and its precedents in other genetic disorders. Our text is as follows (lines 299–318):

“We have previously utilized MeDUsA to quantify axonal degeneration, applying this methodology extensively to various neurological disorders. The robust adaptability of this experimental system is demonstrated by its application in exploring a wide spectrum of genetic mutations associated with neurological conditions, highlighting its broad utility in neurogenetic research. We identified a novel de novo variant in Spliceosome Associated Factor 1, Recruiter of U4/U6.U5 Tri-SnRNP (SART1). The patient, born at 37 weeks with a birth weight of 2934g, exhibited significant developmental delays, including an inability to support head movement at 7 months, reliance on tube feeding, unresponsiveness to visual stimuli, and development of infantile spasms with hypsarrhythmia, as evidenced by EEG findings. Profound hearing loss and brain atrophy were confirmed through MRI imaging. To assess the functional impact of this novel human gene variant, we engineered transgenic Drosophila lines expressing both wild type and mutant SART1 under the control of a UAS promoter.

Our MeDUsA analysis suggested that the variant may confer a gain-of-toxic-function (Nitta et al.,  2023). Moreover, we identified heterozygous loss-of-function mutations in DHX9 as potentially causative for a newly characterized neurodevelopmental disorder. We further investigated the pathogenic potential of a novel heterozygous de novo missense mutation in DHX9 in a patient presenting with short stature, intellectual disability, and myocardial compaction. Our findings indicated a loss of function in the G414R and R1052Q variants of DHX9 (Yamada et al., 2023). This experimental framework has been instrumental in elucidating the impact of gene mutations, enhancing our ability to diagnose how novel variants influence gene function.”

Recommendations for the Authors:

Reviewer #1 (Recommendations For The Authors):

Overall I enjoyed reading this paper. It is well presented and represents a significant amount of well executed study. I feel it further characterizes a poorly understood model of OPA1 variants and one which displays significant differences with the human phenotype. However I feel the use of this model with the author's experiments are not enough to validate this model/experiment as a screening tool for dominant negativity. I have therefore suggested the above experiments as a way to both further validate the mitochondrial dysfunction in this model and to ensure that the expressed transcript is able affect oligomerization as this is a pre-requisite to the authors conclusions.

We assessed the extent to which our model reflects mitochondrial dysfunction using COXII, Atg8, and MitoSOX markers. Unfortunately, neither COXII levels nor the ratio of Atg8a-1 to Atg8a-2 showed significant variations across genotypes that would clarify the impact of dominant negative mutations. Nonetheless, MitoSOX and mito-QC results revealed that mitochondrial ROS levels and mitophagy are increased in Drosophila following intrinsic knockdown of dOPA1. These findings are documented in Figures 2, 5, and S6.

Regarding oligomer formation, the specifics remain elusive in this study. However, the expression of dOPA1K273A, identified as a dominant negative variant in Drosophila, significantly

disrupted retinal axon organization, as detailed in Figure S7. From these observations, we hypothesize that oligomerization of wild-type and dominant negative forms in Drosophila results in axonal degeneration. Conversely, co-expression of Drosophila wild-type with human dominant negative forms does not induce degeneration, suggesting that they likely do not interact.

Reviewer #2 (Recommendations For The Authors):

Materials and Methods:

The authors used GMR-Gal4 to express OPA1-RNAi. I) GMR is expressed in most cells in the developing eye behind the morphogenetic furrow. So the defects observed can be due to knock- down in support cells rather than in photoreceptor cells.

We have added the following sentences in the result (lines 194–196)."The GMR-Gal4 driver does not exclusively target Gal4 expression to photoreceptor cells. Consequently, the observed retinal axonal degeneration could potentially be secondary to abnormalities in support cells external to the photoreceptors.”

OPA1-RNAi: how complete is the knock-down? Have the authors tested more than one RNAi line?

We conducted experiments with an additional RNAi line, and similarly observed degeneration in the retinal axons (Figure S2 A and B; lines 178–179).

The loss-of-function allele, induced by a P-element insertion, gives several eye phenotypes when heterozygous (Yarosh et al., 2008). Does RNAi expression lead to the same phenotypes?

A previous report indicated that the compound eyes of homozygous mutations of dOPA1 displayed a glossy eye phenotype (Yarosh et al., 2008). Upon knocking down dOPA1 using the GMR-Gal4 driver, we also observed a glossy eye-like rough eye phenotype in the compound eyes. These findings have been added to Figure S3 and lines 192–194.

There is no description on the way the somatic clones were generated. How were mutant cells in clones distinguished from wild-type cells (e. g. in Fig. 4).

In the Methods section, we described the procedure for generating clones and their genotypes as follows (lines 502–505): "The dOPA1 clone analysis was performed by inducing flippase expression in the eyes using either ey-Gal4 with UAS-flp or ey3.5-flp, followed by recombination at the chromosomal location FRT42D to generate a mosaic of cells homozygous for dOPA1s3475." Furthermore, we have created a table detailing these genotypes. In these experiments, it was not possible to differentiate between the clone and WT cells. Accordingly, we have noted in the Results section (lines 201–203): "Note that the mutant clone analysis was conducted in a context where mutant and heterozygous cells coexist as a mosaic, and it was not possible to distinguish between them.”

Why were flies kept at 29{degree sign}C? this is rather unusual.

Increased temperature was demonstrated to induce elevated expression of GAL4 (Kramer and Staveley, Genet. Mol. Res., 2003), which in turn led to an enhanced expression of the target genes. Therefore, experiments involving knockdown assays or Western blotting to detect human OPA1 protein were exclusively conducted at 29°C. However, all other experiments were performed at 25°C, as described in the methods sections: “Flies were maintained at 25°C on standard fly food. For knockdown experiments (Figures 1C–E, 1F–H, 2A–H, 3B–K, 5F, S1, S2 A and B, and S6A), flies were kept at 29°C in darkness.” Furthermore, “We regulated protein expression temporally across the whole body using the Tub-Gal4 and Tub-GAL80TS system. Flies harboring each hOPA1 variant were maintained at a permissive temperature of 20°C, and upon emergence, females were transferred to a restrictive temperature of 29°C for subsequent experiments.”

Legends:

It would be helpful to have a description of the genotypes of the flies used in the different experiments. This could also be included as a table.

We have created a table detailing the genotypes. Additionally, in the legend, we have included a note to consult the supplementary table for genotypes.

Results:

Line 141: It is not clear what they mean by "degradation", is it axonal degeneration? And if so, what is the argument for this here?

In the manuscript, we addressed the potential for mitochondrial degradation; however, recognizing that the expression was ambiguous, the following sentence has been omitted: "Nevertheless, the degradation resulting from mitochondrial fragmentation may have decreased the mitochondrial signal.”

Fig. 2: Axons of which photoreceptors are shown?

We have added "a set of the R7/8 retinal axons" to the legend of Figure 2.

Line 167: The authors write that axonal degeneration is more severe after seven days than after eclosion. Is this effect light-dependent? The same question concerns the disappearance of the rhabdomere (Fig. 3G–J).

We conducted the experiments in darkness, ensuring that the observed degeneration is not light- dependent. This condition has been added to the methods section to clarify the experimental conditions.

Line 178/179: Based on what results do they conclude that there is degeneration of the "terminals" of the axons?

Quantification via MeDUsA has enabled us to count the number of axonal terminals, and a noted decrease has led us to conclude axonal terminal degeneration. We have published two papers on these findings. We have added the following description to the results section to clarify how we defined degeneration (lines 174–176): "We have assessed the extent of their reduction from the total axonal terminal count, thereby determining the degree of axonal terminal degeneration (Richard JNS 2022; Nitta HMG 2023).

Line 189: They write: ".. we observed dOPA1 mutant axons...". How did they distinguish es mutant from the controls?

Fig. 5 and Fig. 6: How did they distinguish genetically mutant cells from genetically control cells in the somatic clones?

Mutant clone analysis was conducted in a context where mutant and heterozygous cells coexist as a mosaic, and it was not possible to distinguish between them. Accordingly, this point has been added to lines 201–203, “Note that the mutant clone analysis was conducted in a context where mutant and heterozygous cells coexist as a mosaic, and it was not possible to distinguish between them.” and the text in the results section has been modified as follows: （Before）“To determine if dOPA1 is responsible for axon neurodegeneration, we observed the dOPA1 mutant axons by expressing full- length versions of dOPA1 in the photoreceptors at one day after eclosion and found that dOPA1 expression significantly rescued the axonal degeneration” —> （After）“To determine if dOPA1 is responsible for axon neurodegeneration, we quantify the number of the axons in the dOPA1 eye clone fly with the expression of dOPA1 at one day after eclosion and found that dOPA1 expression partially rescued the axonal degeneration”

Line 225/226: It is not clear to me how their approach "can quantitatively measure the degree of LOF".

To address the reviewer's question and clarify how our approach quantitatively measures the degree of loss of function (LOF), we revised the statement (lines 238–247):

"Our methodology distinctively facilitates the quantitative evaluation of LOF severity by comparing the rescue capabilities of various mutations. Notably, the 2708-2711del and I382M mutations demonstrated only partial rescue, indicative of a hypomorphic effect with residual activity. In contrast, the D438V and R445H mutations failed to show significant rescue, suggesting a more profound LOF. The correlation between the partial rescue by the 2708-2711del and I382M mutations and their classification as hypomorphic is significant. Moreover, the observed differences in rescue efficacy correspond to the clinical severities associated with these mutations, namely in DOA and DOA plus disorders. Thus, our results substantiate the model’s ability to quantitatively discriminate among mutations based on their impact on protein functionality, providing an insightful measure of LOF magnitude.”

Discussion:

Line 251, 252 and line 358: What is "the optic nerve" in the adult Drosophila?

In humans, the axons of retinal ganglion cells (RGCs) are referred to as the optic nerve, and we posit that the retinal axons in flies are similar to this structure. In the introduction section, where it is described that the visual systems of flies and humans bear resemblance, we have appended the following definition (lines 107–108): “In this study, we defined the retinal axons of Drosophila as analogous to the human optic nerve.”

Line 344: These bands appear only upon overexpression of the hOPA1 constructs, so this part of the is very speculative.

Confirmation was achieved using anti-hOPA1, demonstrating that myc is not nonspecific. These results have been added to Figure 5D. Furthermore, the phrase “The upper band was expected as” has been revised to “From a size perspective, the upper band was inferred to represent the full-length hOPA1 including the mitochondria import sequence (MIS).” (lines 464–465)

I was missing a discussion about the increase of ROS upon loss/reduction of dOPA1 observed by others and described here. Is there an increase of ROS upon expression of any of the constructs used?

We demonstrated that not only axonal degeneration but also ROS can be suppressed by expressing human OPA1 in the genetic background of dOPA1 knockdown. Additionally, rescue was not possible with any variants except for I382M. Furthermore, we assessed whether there were changes in ROS in the evaluation of dominant negatives, but no significant differences were observed in this experimental system. These findings have been added to the discussion section as follows (lines 318–328). “Our research established that dOPA1 knockdown precipitates axonal degeneration and elevates ROS signals in retinal axons. Expression of human OPA1 within this context effectively mitigated both phenomena; it partially reversed axonal degeneration and nearly completely normalized ROS levels. These results imply that factors other than increased ROS may drive the axonal degeneration observed post-knockdown. Furthermore, while differences between the impacts of DN mutations and loss-of- function mutations were evident in axonal degeneration, they were less apparent when using ROS as a biomarker. The extensive use of transgenes in our experiments might have mitigated the knockdown effects. In a systemic dOPA1 knockdown, assessments of mitochondrial quantity and autophagy activity revealed no significant changes, suggesting that the cellular consequences of reduced OPA1 expression might vary across different cell types.”

Reviewer #3 (Recommendations For The Authors):

Consider being more explicit regarding literature that has or has failed to test a direct dominant negative effect by expressing a variant in question in the background of a full OPA1 complement. My understanding is that this is the first direct evidence of this widely held hypothesis. This lends to the main claim promoting the utility of fly as a model in general. The authors might also outline this in the introduction as a knowledge gap they fill through this study.

In the introduction, we have incorporated a passage that highlights precedents capable of distinguishing between LOF and DN effects, and we note the absence of models capable of dissecting these distinctions within an in vivo organism. This study aims to address this gap, proposing a model that elucidates the differential impacts of LOF and DN within the context of a living model organism, thereby contributing to a deeper understanding of their roles in disease pathology. We added the following sentences in the introduction (lines 71–80).

“In the quest to differentiate between LOF and DN effects within the context of genetic mutations, precedents exist in simpler systems such as yeast and human fibroblasts. These models have provided valuable insights into the conserved functions of OPA1 across species, as evidenced by studies in yeast models (Del Dotto et al., 2018) and fibroblasts derived from patients harboring OPA1 mutations (Kane et al., 2017). However, the ability to distinguish between LOF and DN effects in an in vivo model organism, particularly at the structural level of retinal axon degeneration, has remained elusive. This gap underscores the necessity for a more complex model that not only facilitates molecular analysis but also enables the examination of structural changes in axons and mitochondria, akin to those observed in the actual disease state.”

The authors should clarify the language used in the abstract and introduction on the effect of hOPA1 DOA and DOA plus on the dOPA1- phenotype. Currently written as "none of the previously reports mutations known to cause DOA or DOA plus were rescued, their functions seems to be impaired." but presumably the authors mean that these variants failed to rescue to the dOPA1 deficient phenotype.

We thank the reviewer for the constructive feedback. We acknowledge the need for clarity in our description of the effects of hOPA1 DOA and DOA plus mutations on the dOPA1- phenotype in both the abstract and the introduction. The current phrasing, "none of the previously reported mutations known to cause DOA or DOA plus were rescued, their functions seem to be impaired," may indeed be confusing. To address your concern, we have revised this statement to more accurately reflect our findings: "Previously reported mutations failed to rescue the dOPA1 deficiency phenotype." For Abstract site, we have changed as following. "we could not rescue any previously reported mutations known to cause either DOA or DOA plus.”→ “mutations previously identified did not ameliorate the dOPA1 deficiency phenotype.”

DOA plus is associated with a multiple sclerosis-like illness; as written it suggests that the pathogenesis of sporadic multiple sclerosis and that associated with DOA plus share and underlying pathogenic mechanism. Please use the qualifier "-like illness." 

We have added the term “multiple sclerosis-like illness” wherever “multiple sclerosis” is mentioned.

Reviewer #1 (Public Review):

This article by Navratna et al. reports the first structure of human HGSNAT in an acetyl-CoA-bound state. Through careful structural analysis, the authors propose potential reasons why certain human mutations lead to lysosomal storage disorders and outline a catalytic mechanism. The structural data are of good quality, and the manuscript is clearly written. This study represents an important step toward understanding the mechanism of HGSNAT and is valuable to the field. I have the following suggestions:

(1) The authors should characterize whether the purified protein is active. Otherwise, how does one know if the detergent used maintains the protein in a biologically relevant state? The authors should at least attempt to do so. If these prove to be challenging, at the very least, the authors should try a cell-based assay to demonstrate that the GFP tag does not interfere with the function.

(2) In Figure 5, the authors present a detailed schematic of the catalytic cycle, which I find to be too speculative. There is no evidence to suggest that this enzyme undergoes isomerization, similar to a transporter, between open-to-lumen and open-to-cytosol states. Could it not simply involve some movements of side chains to complete the acetyl transfer?

Author response:

The following is the authors’ response to the original reviews.

(1) The authors should characterize whether the purified protein is active. Otherwise, how does one know if the detergent used maintains the protein in a biologically relevant state? The authors should at least attempt to do so. If these prove to be challenging, at the very least, the authors should try a cell-based assay to demonstrate that the GFP tag does not interfere with the function. The authors would need to establish an in vitro assay using purified protein and assess the level of Acetyl-CoA in the reaction (there are commercial kits and a long list of literature showing how to measure this). They could also follow the HS acetylation reaction by e.g. HPLC-MS or NMR (among other methods).

The cryo-EM sample was prepared without the exogenous addition of ligand, as noted in the manuscript. However, we see that acetyl-CoA was intrinsically bound to the protein, indicating the ability of GFP-tagged HGSNAT protein to bind the ligand. Upon dialysis, we see release of acetyl-CoA from the protein, which we have confirmed by LC-MS analysis (Fig S9). We purified the protein at a pH optimal for acetyl-CoA binding, as suggested by Bame, K. J. and Rome, L. H. (1985) and Meikle, P. J. et al., (1995). Because we see acetyl-CoA in a structure obtained using a GFP fusion, we argue that GFP does not interfere with protein stability and ability to bind to the co-substrate. As demonstrated by existing literature HGSNAT catalyzed reaction is compartmentalized spatially and conditionally. The binding of acetyl-CoA happens towards the cytosol and is optimal at pH 7-0.8.0, while the transfer of the acetyl group to heparan sulfate occurs towards the luminal side and is optimal at pH 5.0-6.0. We attempted measuring HGSNAT catalyzed reaction by monitoring decrease in acetyl-CoA in presence of D-glucosamine (acetyl group acceptor) using a coupled enzyme acetyl-CoA assay kit from SIGMA (MAK039) that converts acetyl-CoA to a fluorescent product measurable at Ex/Em of 535/587 nm. We noticed a decrease in the level of acetyl-CoA in the presence of HGSNAT-ACO complex (blue) and apo HGSNAT (red); the difference compared to the ACO standard (gray) was not significant. While optimizing the assay, Xu et al. (2024, Nat Struct Mol Biol) published structural and biochemical characterization of HGSNAT, showing that detergent-purified HGSNAT is active.

Author response image 1.

Acetyl-CoA levels in absence and presence of HGSNAT purified in digitonin. Decrease in the levels of 10 mM acetyl-CoA was measured in presence of 10 mM D-glucosamine and 30 nM HGSNAT at pH 7.5.

(2) In Figure 5, the authors present a detailed schematic of the catalytic cycle, which I find to be too speculative. There is no evidence to suggest that this enzyme undergoes isomerization, similar to a transporter, between open-to-lumen and open-to-cytosol states. Could it not simply involve some movements of side chains to complete the acetyl transfer? The speculative nature of this assumption needs to be clearly acknowledged throughout the manuscript and discussed in more detail. The authors could use HDX-MS or introduce cysteine residues in the hypothetical inward- and outward-facing cavities and test accessibility by incubating the purified protein with maleimides or other agents reacting with free cysteine.

We thank the reviewers for this insightful critique. Yes, the enzyme could likely achieve catalysis by simple side chain movements without undergoing extensive isomerization steps, as depicted in Figure 5. We also agree with the reviewer that HDX-MS could be the best way to monitor the substrate-induced conformational dynamics within HGSNAT experimentally. In the absence of data supporting large movements during the acetyl transfer reaction, figure 5 is speculative. We have now edited Figure 5 in the revised version of the manuscript based on the observations we made in this study.

(3) The acetyl-CoA-bound state is described as the open-to-lumen state. Indeed, from Figure 1C, the lumen opening appears much larger than the cytosol opening. Is there any small tunnel that connects the substrate site to the cytosol? In other words, is this state accessible to both the lumen and the cytosol, albeit with a larger opening toward the lumen? This question arises because, in Figure S5, the tunnel calculated by MOLE seems to also connect to the cytosol.

Yes, it is likely that the ACOS is accessible via lumen and cytosol to varying degrees, as evidenced by MOLE prediction. However, binding of the bulky nucleoside head group of acetyl-CoA at ACOS blocks the cytosolic entrance in the confirmation discussed in this manuscript. MOLE prediction was performed on a structure devoid of acetyl-CoA, and it is possible that the protein doesn’t essentially undergo isomerization between open-to-lumen and open-to-cytosol confirmations during acetyl transfer. Likely, ACOS is always accessible from both the lumen and cytosol, but depending on the substrates or products bound, the accessibility could be limited to either the lysosomal lumen or cytosol. We have rewritten all the statements mentioning an open-to-lumen confirmation to reflect this argument.

(4) The authors state, "Interestingly, in most of the detergent conditions we tested, HGSNAT was predominantly dimeric (Fig S1C-H)," and also mention, "In all the detergents we tested, HGSNAT eluted as a dimer, a testament to the extensive side-chain interaction network." The dimerization is said to be mediated by a disulfide bond. I would be surprised if the detergents the authors tested could break a disulfide bond. Therefore, can this observation truly serve as a testament to an "extensive" side-chain interaction network?

We agree with the reviewer that detergents are unlikely to break a disulfide bond. To address this comment, we generated a C334A mutant of HGSNAT and extracted it from cells in 1% digitonin. It is still expressed as a dimer (Fig S8E). However, upon heating the detergent solubilized protein, we noticed that the FSEC peak for C334A shows a monomeric HGSNAT (Fig S8I and S8K). We hypothesize that in the absence of C334 disulfide, the extensive hydrophobic side-chain interaction network displayed in Figure 2C is responsible for maintaining the integrity of the dimer. Heating disturbs these non-disulfide interactions, thereby rendering the protein monomer.

(5) Apart from the cryo-EM structure, the article does not provide any other experimental evidence to support or explain a molecular mechanism. Due to the complete absence of functional assays, mutagenesis analysis, or other structures such as a ternary complex or an acetylated enzyme intermediate, the mechanistic model depicted in Figure 5 should be taken with caution. This uncertainty needs to be clearly described in the manuscript text. Performing additional mutagenesis experiments to test key hypotheses, or further discussing relevant data from the literature, would strengthen the manuscript.

We agree with the reviewer on the lack of supporting evidence for the mechanistic models proposed in Fig 5. They were made based on previously reported biochemical characterization of HGSNAT by Rome & Crain (1981), Rome et al. (1983), Miekle et al. (1995), and Fan et al. (2011). However, we agree with the reviewer that this schematic is not experimentally proven and is speculative at best. We have edited Figure 5 in the revised version of the manuscript. In addition, we have also performed mutagenesis analysis to study the stability of mutants (Fig S8) and performed LC-MS analysis to identify endogenously bound acetyl-CoA (Fig S9) to strengthen parts of the manuscript. We have discussed our findings in the results and modified the discussion according to these suggestions.

(6) It is discussed that H269 is an essential residue that participates in the acetylation reaction, possibly becoming acetylated during the process. However, there is no solid experimental evidence, e.g. mutagenesis analysis or structural analysis, in this or previous articles, that demonstrates this to be the case. Providing more information, ideally involving additional experimental work, would strengthen this aspect of the mechanism that is proposed. This would require establishing an in vitro assay, as described in 1).

H269, as a crucial catalytic residue, was suggested by monitoring the effect of chemical modifications of amino acids on acetylation of HGSNAT membranes by Bame, K. J. and Rome, L. H. (1986). We generated N258I and H269A mutants of HGSNAT and analyzed their stability. We noticed a greater destabilization in N258I compared to H269A (Fig S8). We believe this is because of the loss of ability to bind acetyl-CoA, as the TMs around a catalytic core of the protein in our cryo-EM structure were stabilized by interactions with acetyl-CoA. Recently, Xu et al. (2024, Nat Struct Mol Biol) suggested that they do not observe acetylated histidine in their structure. However, our structure and that reported by Xu et al. (2024) are obtained at cytosolic pH. Perhaps, acetylation of H269 occurs at acidic lysosomal pH. Extensive structural and catalytic investigation of HGSNAT at low pH is required to rule out H269 acetylation as a step in the HGSNAT catalyzed reaction.

(7) In the discussion part, the authors mention previous studies in which it was postulated that the catalytic reaction can be described by a random order mechanistic model or a Ping Pong Bi Bi model. However, the authors leave open the question of which of these mechanisms best describes the acetylation reaction. The structure presented here does not provide evidence that could support one mechanism or the other. The authors could explore if an in vitro experimental measurement of protein activity would provide any information in this regard.

We agree with the reviewer that a more detailed kinetic analysis is necessary to define the bisubstrate reaction mechanism of HGSNAT. All the existing structural data on two isoforms of HGSNAT is obtained at basic pH. As a result, the existing structures do not unambiguously demonstrate the bisusbtrate mechanism of HGSNAT. We believe low pH structural characterization and a detailed kinetic and structural characterization of HGSNAT in membrane mimetics like nanodiscs could provide more insights into the mechanism. However, these studies are a future undertaking and are not a part of this manuscript.

(8) Although the authors map the mutations leading to MPS IIIC on the structure and use FoldX software to predict the impact of these mutations on folding and fold stability, there is no experimental evidence to support FoldX's predictions. It would be ideal if an additional test for these predictions were included in the manuscript. The authors could follow the unfolding of purified mutants by SEC, FSEC, or changes in intrinsic fluorescence to assess protein stability.

As suggested here, we prepared HGSNAT MPSIIIC variants and tested their expression and stability (please see Fig S8). These results have been included in the revised version of the manuscript.

(9) Some sidechains that have quite strong sidechain density are missing atoms. I would be particularly careful with omitting sidechains that pack in the hydrophobic core, as this can tend to artificially reduce the clash score. Check F81, L62, P91 and V87, for example.

We have revisited the modeling of these regions and deposited new coordinates.

(10) W316 seems to have the wrong rotamer.

This has been corrected in the new coordinate file that has been released.

(11) N134 and N433 seem to have extra density. Are these known glycosylation sites?

As per Hrebicek M. et al., 2006 and Feldhammer M. et al., 2009, there are five predicted glycosylation sites: N66, N114, N134, N433, and N602. However, we see evidence for NAG density at N114, N134, and N433. These have now been modeled in the structure.

(12) At the C-terminal residue (Ile-635), the very C-terminal carboxylate is modeled pointing to a hydrophobic environment. It seems more likely to me that the Ile sidechain is packing here, with the C-terminal carboxylate facing the solvent.

Thank you for pointing this out. We have edited the orientation of the Ile sidechain accordingly.

Presentation and wording of results/methods:

- Figure S3 legend "At places with missing density, the side chains were trimmed to C- alpha" - this is incorrect, I think the authors mean C-beta.

We have corrected this error in the revised version of the manuscript.

- Figure S3 legend - the authors refer to a gray mesh, where a transparent surface is displayed.

Thanks for pointing this error out. We have corrected this in the revised version.

- Some colloquial/vague wording in the main text (a lot of sentences starting with "Interestingly, ...". Making the wording more specific would help the reader I think.

We have edited out ‘interestingly’ from the document and have re-written parts of the manuscript, per reviewers’ suggestion, for brevity.

- Figure S2 legend, "throughout the processing workflow the resolution of luminal domain was used as a guidepost" - it is not entirely clear to me what this means in this context, perhaps revise the wording?

We have rephrased this line in the revised draft of the manuscript.

- Figure S2 and methods, Local refinements of LD and TMD are mentioned, but not indicated on the processing workflow.

We have included a new Fig S2 & edited the legend, including these changes, per the reviewers’ suggestions.

Reviewer #2 (Public Review):

The authors addressed the question of how mitochondrial proteins that are dually localized or only to a minor fraction localized to mitochondria can be visualized on the whole genome scale. For this, they used an established and previously published method called BiG split-GFP, in which GFP strands 1-10 are encoded in the mitochondrial DNA and fused the GFP11 strand C-terminally to the yeast ORFs using the C-SWAT library. The generated library was imaged under different growth and stress conditions and yielded positive mitochondrial localization for approximately 400 proteins. The strength of this method is the detection of proteins that are dually localized with only a minor fraction within mitochondria, which so far has hampered their visualization due to strong fluorescent signals from other cellular localizations. The weakness of this method is that due to the localization of the GFP1-10 in the mitochondrial matrix, only matrix proteins and IM proteins with their C-termini facing the matrix can be detected. Also, proteins that are assembled into multimeric complexes (which will be the case for probably a high number of matrix and inner membrane-localized proteins) resulting in the C-terminal GFP11 being buried are likely not detected as positive hits in this approach. Taking these limitations into consideration, the authors provide a new library that can help in the identification of eclipsed protein distribution within mitochondria, thus further increasing our knowledge of the complete mitochondrial proteome. The approach of global tagging of the yeast genome is the logical consequence after the successful establishment of the BiG split-GFP for mitochondria. The authors also propose that their approach can be applied to investigate the topology of inner membrane proteins, however, for this, the inherent issue remains that it cannot be excluded that even the small GFP11 tag can impact on protein biogenesis and topology. Thus, the approach will not overcome the need to assess protein topology analysis via biochemical approaches on endogenous untagged proteins.

Reviewer #3 (Public Review):

Summary:

Here, Bykov et al move the bi-genomic split-GFP system they previously established to the genome-wide level in order to obtain a more comprehensive list of mitochondrial matrix and inner membrane proteins. In this very elegant split-GFP system, the longer GFP fragment, GFP1-10, is encoded in the mitochondrial genome and the shorter one, GFP11, is C-terminally attached to every protein encoded in the genome of yeast Saccharomyces cerevisiae. GFP fluorescence can therefore only be reconstituted if the C-terminus of the protein is present in the mitochondrial matrix, either as part of a soluble protein, a peripheral membrane protein, or an integral inner membrane protein. The system, combined with high-throughput fluorescence microscopy of yeast cells grown under six different conditions, enabled the authors to visualize ca. 400 mitochondrial proteins, 50 of which were not visualised before and 8 of which were not shown to be mitochondrial before. The system appears to be particularly well suited for analysis of dually localized proteins and could potentially be used to study sorting pathways of mitochondrial inner membrane proteins.

Strengths:

Many fluorescence-based genome-wide screens were previously performed in yeast and were central to revealing the subcellular location of a large fraction of yeast proteome. Nonetheless, these screens also showed that tagging with full-length fluorescent proteins (FP) can affect both the function and targeting of proteins. The strength of the system used in the current manuscript is that the shorter tag is beneficial for the detection of a number of proteins whose targeting and/or function is affected by tagging with full-length FPs.

Furthermore, the system used here can nicely detect mitochondrial pools of dually localized proteins. It is especially useful when these pools are minor and their signals are therefore easily masked by the strong signals coming from the major, nonmitochondrial pools of the proteins.

Weaknesses:

My only concern is that the biological significance of the screen performed appears limited. The dataset obtained is largely in agreement with several previous proteomic screens but it is, unfortunately, not more comprehensive than them, rather the opposite. For proteins that were identified inside mitochondria for the first time here or were identified in an unexpected location within the organelle, it remains unclear whether these localizations represent some minor, missorted pools of proteins or are indeed functionally important fractions and/or productive translocation intermediates. The authors also allude to several potential applications of the system but do little to explore any of these directions.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

The study reports on a previously unrecognized function of ATG6 in plant immunity. The work is valuable because it proposes a direct interaction between ATG6 and a well-studied salicylic acid receptor protein, NPR1, which may interest researchers investigating plant immunity regulation. While the data presented are compelling, more information regarding the specificity of ATG6's role would improve the overall impact of the study, especially with an eye towards consistency with prior work.

We also genuinely thank the editor and reviewers for the constructive and helpful suggestions and comments. These comments have greatly improved the quality and thoroughness of our manuscript. We have carefully studied these comments and have made the appropriate changes as far as possible. Additionally, some minor errors were also corrected during the revision process. New text is shown in blue in the revised manuscript. Our responses to the reviewer's comments are provided below each respective comment.

Public Reviews:

Reviewer #1 (Public Review):<br /> Summary:<br /> The authors showed that autophagy-related genes are involved in plant immunity by regulating the protein level of the salicylic acid receptor, NPR1.<br /> Strengths:<br /> The experiments are carefully designed and the data is convincing. The authors did a good job of understanding the relationship between ATG6 and NRP1.

Thank you very much for recognizing our research.

Weaknesses:<br /> - The authors can do a few additional experiments to test the role of ATG6 in plant immunity.<br /> I recommend the authors to test the interaction between ATGs and other NPR1 homologs (such as NPR2).

Thanks to your valuable feedback, it was discovered that the Arabidopsis NPRs family comprises six members: NPR1, NPR2, NPR3, NPR4, NPR5/PETIOLE 1 (BOP1), and NPR6/BOP2. NPR3/4 function in tandem as negative regulators to modulate SA signaling and plant immune responses (Ding et al., 2018). Similar to NPR1, NPR2 acts as a positive regulator of SA signaling (Castello et al., 2018). NPR5/BOP1 and NPR6/BOP2 primarily participate in the regulation of plant growth and development (McKim et al., 2008). This study specifically investigates the correlation between ATG6 and NPRs in plant resistance to pathogenic bacteria. Consequently, we experimentally confirmed the interaction between ATG6 and NPR1, NPR3, and NPR4 (Fig. 1 and Fig. S1 in the revised manuscript). It would be intriguing to further explore the interactions between ATG6 and other NPRs in the context of regulating plant growth and development in future research endeavors.

-The concentration of SA used in the experiment (0.5-1 mM) seems pretty high. Does a lower concentration of SA induce ATG6 accumulation in the nucleus?

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). Consequently, to investigate the function of NPR1, many scientists and research groups typically employ higher concentrations of SA (e.g., 0.5 mM, 1 mM, or even 5 mM) to elucidate its role (Spoel et al., 2009; Fu et al., 2012; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a). In our study, we observed an interaction between ATG6 and NPR1. To enhance the detection of the NPR1 protein, we standardized the SA concentration (Arabidopsis was treated with 0.5 mM SA; Tobacco was treated with 1 mM SA) used in our experiments. Subsequently, we analyzed the nuclear accumulation ATG6 or NPR1 using a relatively high SA concentration (Arabidopsis was treated with 0.5 mM SA; Tobacco was treated with 1 mM SA), consistent with concentrations used in previous studies (Spoel et al., 2009; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a).

-Does the silencing of ATG6 affect the cell death (or HR) triggered by AvrRPS4?

Thank you for pointing this out. In this study, we examined changes in Pst DC3000/avrRps4-induced cell death in Col, amiRNAATG6 # 1, amiRNAATG6 # 2, npr1, NPR1-GFP, ATG6-mCherry and ATG6-mCherry × NPR1-GFP plants. The results of Taipan blue staining showed that Pst DC3000/avrRps4-induced cell death in npr1, amiRNAATG6 # 1 and amiRNAATG6 # 2 was significantly higher compared to Col (Fig. S15 in the revised manuscript). Conversely, Pst DC3000/avrRps4-induced cell death in ATG6-mCherry, NPR1-GFP and ATG6-mCherry × NPR1-GFP was significantly lower compared to Col. Notably, Pst DC3000/avrRps4-induced cell death in ATG6-mCherry × NPR1-GFP was significantly lower compared ATG6-mCherry and NPR1-GFP (Fig. S15 in the revised manuscript). These results suggest that ATG6 and NPR1 cooperatively inhibit Pst DC3000/avrRps4-induced cell dead. The relevant description can be found in lines 394-404 of the revised manuscript.

-SA and NPR1 are also required for immunity and are activated by other NLRs (such as RPS2 and RPM1). Is ATG6 also involved in immunity activated by these NLRs?

Thank you for your valuable comments. The most notable event in the NLR-mediated ETI immune response is the induction of hypersensitive response-programmed cell death (HR-PCD) (Jones and Dangl, 2006; Yuan et al., 2021). SA plays a dual role in the ETI response. On one hand, the accumulation of SA during the R gene-mediated ETI defense response is directly linked to the onset of HR-PCD (Nawrath and Metraux, 1999). SA and NPR1 can enhance the ETI response by regulating the expression of downstream target genes (Falk et al., 1999; Feys et al., 2001; Ding et al., 2018; Liu et al., 2020). On the other hand, the activation of SA signaling can have a negative regulatory effect on HR-PCD during the ETI response. High levels of SA have been shown to significantly inhibit HR-PCD triggered by the avrRpt2 effector (Rate and Greenberg, 2001; Devadas and Raina, 2002; Jurkowski et al., 2004). Rate et al. discovered that the inhibition of HR-PCD by SA relies on NPR1 (Rate and Greenberg, 2001).

Arabidopsis AtATG6 or its homologs in other species (such as NbBECLIN1, TaATG6s, etc.) have been identified as positive regulators in plant immunity, playing a crucial role in inhibiting cell death and preventing invasion by pathogenic microorganisms (Liu et al., 2005; Patel and Dinesh-Kumar, 2008; Yue et al., 2015). Patel et al. demonstrated that, akin to autophagy-deficient mutants previously documented, AtATG6 antisense (AtATG6-AS) plants treated with Pst DC3000/avrRpm1 exhibited diffuse cell death, indicating the necessity of ATG6 in restricting cell death (Patel and Dinesh-Kumar, 2008). In tobacco, deficiencies in BECLIN 1 result in the onset of diffuse HR-PCD, underscoring the essential role of BECLIN 1 in limiting HR-PCD (Liu et al., 2005). Despite the genetic evidence supporting the critical function of ATG6 in plant immunity, the precise molecular mechanisms through which ATG6 impedes the invasion of pathogenic microorganisms remain elusive.

In our study, we uncovered that ATG6 interacts with NPR1 to hinder pathogen invasion and inhibit the initiation of cell death. In animals, members of the NLR family have been observed to interact with the autophagy-related protein LC3 to inhibit the survival of pathogen (Zhang et al., 2019). Similar mechanisms may exist in plants. However, it remains to be explored whether NLR directly induces the activation of ATG6 through interaction or the relationship between NPR1-ATG6 interactions and NLR-mediated plant immunity, necessitating further investigation.

Reviewer #2 (Public Review):

Summary:

The manuscript by Zhang et al. explores the effect of autophagy regulator ATG6 on NPR1-mediated immunity. The authors propose that ATG6 directly interacts with NPR1 in the nucleus to increase its stability and promote NPR1-dependent immune gene expression and pathogen resistance. This novel role of ATG6 is proposed to be independent of its role in autophagy in the cytoplasm. The authors demonstrate through biochemical analysis that ATG6 interacts with NPR1 in yeast and very weakly in vitro. They further demonstrate using overexpression transgenic plants that in the presence of ATG6-mcherry the stability of NPR1-GFP and its nuclear pool is increased.

However, the overall conclusions of the study are not well supported experimentally. The significance of the findings is low because of their mostly correlational nature, and lack of consistency with earlier reports on the same protein.

Thank you for your valuable and constructive suggestions. In this article, we unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity (Fig. 8 in the revised manuscript). ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and formation of SINCs-like condensates. This may be of interest to researchers studying the regulation of plant immunity. While there may be minor flaws in our current study, the significance of these findings cannot be overstated, as they have the potential to redirect scientific attention towards uncovering novel functions for autophagy genes.

Based on the integrity and quality of the data as well as the depth of analysis, it is not yet clear if ATG6 is a specific regulator of NPR1 or if it is affecting NPR1's stability indirectly, through inducing an elevation of SA levels in plants. As such, the current study demonstrates a correlation between overexpression of ATG6, SA accumulation, and NPR1 stability, however, whether and how these components work together is not yet demonstrated.

Thanks to your valuable feedback. Although as the reviewer said there may be some flaws in our data from the current results, scientific research is an ongoing process and I am confident that future studies will be even better. From the results given to us at the moment at least this study reports a previously undiscovered function of ATG6 in plant immunity. We propose a direct interaction between ATG6 and a well-studied salicylic acid receptor protein, NPR1. We unveil a novel relationship in which ATG6 positively regulates NPR1 in plant immunity (Fig. 8 in the revised manuscript). ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and formation of SINCs-like condensates. This may be of interest to researchers studying the regulation of plant immunity.

Based on the provided biochemical data, it is not yet clear if the ATG6 functions specifically through NPR1 or through its paralogs NPR3 and NPR4, which are negative regulators of immunity. It is quite possible that interaction with NPR1 (or any NPR) is not the major regulatory step in the activity of ATG6 in plant immunity. The effect of ATG6 on NPR1 could well be indirect, through a change in the SA level and redox environment of the cell during the immune response. Both SA level and redox state of the cell were reported to induce accumulation of NPR1 in the nucleus and increase in stability.

Thanks to your valuable feedback. In this study, we validated the interaction between ATG6 and NPR1 through various approaches and identified the key regions mediating their interaction. Our findings indicate that ATG6 interacts with NPR1 to synergistically enhance plant resistance by regulating NPR1 protein levels, stability, nuclear accumulation, and the formation of SINC-like condensates. These results clearly demonstrate the involvement of ATG6 in the regulation of NPR1.Furthermore, we also found that ATG6 interacts with NPR3/4 (Fig. S1 in the revised manuscript). This is particularly relevant given that NPR3 and NPR4 have been shown to act as adaptors for the ubiquitin E3 ligase Cullin 3 (CUL3) to regulate the degradation of NPR1. Therefore, whether ATG6 regulates NPR1 through its interactions with NPR3/4 is an intriguing question worth exploring in future studies. We appreciate the reviewer's concerns and are committed to addressing them in our future research to further elucidate the complex regulatory mechanisms involving ATG6, NPR1, and other key players in plant immunity.

Another major issue is the poor quality of the subcellular analyses. In contradiction to previous studies, ATG6 in this study is not localized to autophagosome puncta, which suggests that the soluble localization pattern presented here does not reflect the true localization of ATG6. Even if the authors propose a novel, non-canonical nuclear localization for ATG6, they still should have detected the canonical autophagy-like localization of this protein.

Thanks to your valuable feedback. We conducted predictions at NLS Mapper (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi) and identified two bipartite NLSs in ATG6, with the sequences "MRKEEIPDKSRTIPIDPNLPKWVCQNCHHS" and "DPNLPKWVCQNCHHS LTIVGVDSYAGKFFNDP". To further elucidate the nuclear localization of ATG6, we introduced Agrobacterium tumefaciens carrying ATG6-GFP into nls-mCherry tobacco leaves through transient transformation. Subsequently, we observed the localization of ATG6-GFP, along with the canonical autophagy-like patterns. Our findings revealed fluorescence signals of ATG6-GFP in both the cytoplasm and nuclei (Figure 2b). The nuclear-localized ATG6-GFP overlapping with the nuclear-localized marker, nls-mCherry (indicated by white arrows). Additionally, we observed punctate patterns indicative of canonical autophagy-like localization of ATG6-GFP fluorescence signals (indicated by red circles). Based on these results, we are more confident about the authenticity of ATG6's nuclear localization. The revised manuscript includes clearer images to support our observations.

Recommendations for the Authors:

Reviewer #2 (Recommendations For The Authors):

The duration and concentration of SA treatments are quite variable between experiments which makes comparisons difficult.

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). Consequently, to investigate the function of NPR1, many scientists and research groups typically employ higher concentrations of SA (e.g., 0.5 mM, 1 mM, or even 5 mM) to elucidate its role (Spoel et al., 2009; Fu et al., 2012; Lee et al., 2015; Saleh et al., 2015; Skelly et al., 2019; Zavaliev et al., 2020; Chen et al., 2021a). In our study, we observed an interaction between ATG6 and NPR1. To enhance the detection of the NPR1 protein, we standardized the SA concentration used in our experiments. In this study, for the treatment of Arabidopsis, we followed the protocols outlined in Saleh et al. and Spoel et al., utilizing 0.5 mM SA (Spoel et al., 2009; Saleh et al., 2015). For tobacco treatment, we adopted the methodology described in the study by Zavaliev et al., administering 1 mM SA (Zavaliev et al., 2020).

The methods section does not explain some of the essential experimental conditions and reagents used in the study.

Thank you for pointing this out. Due to word limitations we have placed the detailed experimental methods and reagents in Supplemental Data 1. In Supplemental Data 1, we provide a comprehensive overview of the experimental flow and conditions employed in our study.

Lines 62-63: the C-terminal domain of all NPRs has a name (already defined as SA-binding domain (SBD)). Also, it would be worth referring to the structure of NPR1 (Kumar et al 2022, Nat) as the source of information about its domains.

Thank you for pointing this out, we have changed this description in the revised manuscript (lines 62-63).

Lines 66-69: NPR1 doesn't form monomers. A recent study showed that the basic functional unit of NPR1 is a dimer (Kumar et al 2022, Nat).

Thank you for pointing this out. In the revised manuscript (line 67) " monomers " has been changed to “dimer”.

Lines 89-95 and elsewhere: the term "invasion" has a very specific meaning and it doesn't necessarily refer to disease. A pathogen can invade the plant but cause no disease (e.g. ETI). Most plant genetic immune mechanisms act after pathogen invasion, not before it. Those cited works reported the disease resistance, not the invasion resistance.

Thank you for pointing this out. We've changed the incorrect description in the revised manuscript (line 91).

Lines 113-119: the truncation at the aa328 includes half of the ANK domain (repeats 1 and 2), not just BTB. The C-terminal truncation variant contains the other half (repeats 3 and 4) of the ANK domain, not the entire ANK domain. It also contains the SBD, not just the NLS. So, this kind of analysis cannot determine the role of ANK domain in the interaction, nor it can conclusively determine if the interaction is through SBD. The interaction should be tested with the SBD domain only in order to make this conclusion.

Thank you for pointing this out, we have removed the inappropriate description and made the appropriate changes in the revised manuscript (lines 114 and 115).

In Figure S1, the equally strong interaction of atg6 is found for NPR3/NPR4. Does that mean that atg6 functions also through these other NPRs? What's the significance of these data compared to NPR1-ATG6 interaction? This is especially important, because both NPR3 and NPR4 are predominantly nuclear proteins, and they are unlikely to significantly overlap with autophagy components in the cytoplasm.

NPR1 and its paralogues NPR3/NPR4, which frequently interact with other proteins to regulate plant immune responses (Backer et al., 2019; Chen et al., 2019). To identify ATGs that interact with NPRs, we performed yeast two-hybrid (Y2H) screens using NPRs as bait. Interestingly, ATG6 interacted with NPR1, NPR3 and NPR4, respectively, and different concentrations of SA treatment did not significantly affect their interaction (Fig. S1a). NPR1 is an important positive regulator of the plant immune response (Chen et al., 2021b). In Arabidopsis and N. benthamian, ATG6 or its homologues was reported to act as a positive regulator to enhance plant disease resistance to P. syringae pv. tomato (Pst) DC3000 and Pst DC3000/avrRpm1 bacteria (Patel and Dinesh-Kumar, 2008), N. benthamiana mosaic virus (TMV) (Liu et al., 2005). Therefore, in this study we focused on investigating the biological significance of the interaction between ATG6 and NPR1. Whether the interaction between ATG6 and NPR3/4 also has an effect on plant immunity is a question that remains to be explored in future studies.

In Figure 1c and elsewhere: why not use the anti-mCherry antibody to detect atg6-mcherry? Are we seeing the correct protein band of atg6-mcherry? Also, it is not clear what antibodies they used throughout the study: the sources and specificities of antibodies are not provided.

Thank you for pointing this out. We initially synthesized the ATG6 antibody (anti-ATG6, 1:200, peptide, C-KEKKKIEEEERK, Abmart) in order to detect the endogenous ATG6 protein, and we also tested the specificity and potency of the ATG6 antibody (results are shown in Fig. S17). Additionally, in order to determine the location of the ATG6-mCherry bands, we also detected ATG6-mCherry in ATG6-mCherry Arabidopsis using the ATG6 antibody, and we also used Col as a control (results are shown in Fig. S4). These results show that our synthesized ATG6 antibody can effectively and clearly immunize to both ATG6 and ATG6-mCherry. Therefore, in this study, we used the ATG6 antibody to analyze both ATG6-mCherry and endogenous ATG6. Detailed antibody information is presented in Supplementary Data 1, table S4

In Figures 1d, 2a, and 2b, the subcellular localization pattern of atg6 contradicts what was published before (Fujiki et al 2007, Plant Phys; Liu et al 2018, FPlS; Xu et al 2017, Autophagy; Li et al 2018, Nat. Comm.). As an autophagy protein, atg6 was shown to localize to cytoplasmic puncta (autophagosomes), like atg8. No nuclear localization was found in those studies. The lack of puncta and the strong nuclear accumulation are signs that the localization of atg6 reported here has to be interpreted with caution. With the data provided, I am not convinced yet that we are looking at the correct ATG6 subcellular localization. Even if the authors propose a novel, non-canonical localization for atg6, they still should have detected the canonical autophagy-like localization of this protein.

Thanks to your valuable feedback. To further elucidate the nuclear localization of ATG6, we introduced Agrobacterium tumefaciens carrying ATG6-GFP into nls-mCherry tobacco leaves through transient transformation. Subsequently, we observed the localization of ATG6-GFP, along with the canonical autophagy-like patterns. Our findings revealed fluorescence signals of ATG6-GFP in both the cytoplasm and nuclei (Figure 2b). The nuclear-localized ATG6-GFP overlapping with the nuclear-localized marker, nls-mCherry (indicated by white arrows). Additionally, we observed punctate patterns indicative of canonical autophagy-like localization of ATG6-GFP fluorescence signals (indicated by red circles). Based on these results, we are more confident about the authenticity of ATG6's nuclear localization. The revised manuscript includes clearer images to support our observations.

It would make more sense to include the BiFC data (fig. S2) in the main figure, instead of the co-localization (fig. 1d) which cannot serve as evidence for interaction.

Thank you for the feedback. We accept your suggestion. In Fig.1, we have replaced the co-localization image with a BiFC (Bimolecular Fluorescence Complementation) image to better illustrate the interaction.

In Figure S2, the bifc signals have to be quantified to qualify as evidence for interaction. also, a subcellular marker has to be used (e.g. nuclear mcherry). From the current poor-quality images, one cannot determine where in the cell the presumed interaction takes place, nucleus or cytoplasm, or both. Also, no puncta are seen in these images.

Thank you for pointing this out. Despite the lack of clarity in the images we provided, our BiFC results unequivocally demonstrate the interaction between ATG6 and NPR1 in both the cytoplasm and nucleus. Notably, as the reviewer pointed out, punctate signals were not observed in our images. This lack of punctate signals is consistent with previous studies (Figure 2) that have also shown BiFC results between autophagy-associated proteins ATG8s and their interacting partners. For instance, Fig 1G (Marshall et al. 2019, Cell), Fig 2F (Marshall et al. 2019, Cell), Fig 4B (Macharia et al. 2019, BMC Plant Biology), and Fig 3 (Zhou et al. 2018, Autophagy) all did not exhibit punctate signals, aligning closely with our findings.

In Figure S3a, the nuclear localization is shown for stomata. It is known that stomata are especially strong expressors of the transgenes, and localization there could be an artefact of overaccumulation of the fusion protein. Also, why do they present the localization of atg6-gfp, if the analysis and the cross were made with atg6-mcherry?

Thank you for pointing this out. In our previous experiments, we observed the localization of ATG6 in the nucleus of Arabidopsis thaliana plants overexpressing ATG6-GFP (Fig. S3a). To clearly visualize the location of the nucleus, we used the cytosolic DAPI dye, which readily stained the nuclei of the stomatal guard cells. This allowed us to easily identify the nuclear regions for our observations. Additionally, in Fig. 2a and Fig.S3b, we detected the fluorescence signal of ATG6-mCherry within the nucleus, further confirming the nuclear localization of ATG6. Moreover, the nuclear and cytoplasmic fractions were separated. Under SA treatment, ATG6-mCherry and ATG6-GFP were detected in the cytoplasmic and nuclear fractions in N. benthamiana (Fig. 2c and d). Similarly, ATG6 was also detected in the nuclear fraction of UBQ10::ATG6-GFP and UBQ10::ATG6-mCherry overexpressing plants (Fig. 2e and f).

In Figure S3b, the images are low resolution and of poor quality. Why atg6-mcherry is expressed in a single cell if these are transgenic plants? The nuclear co-localization with npr1-gfp has to be shown more clearly with high res. images and also be quantified, because the expression of atg6-mcherry is not as uniform as npr1-gfp.

Thank you for pointing this out. Contrary to the reviewer's assertion, the ATG6-mCherry fluorescence signal depicted in Figure S3b was not exclusive to a single cell. In fact, this fluorescence was also evident in other cells, albeit with relatively weaker intensity. This disparity in fluorescence intensity may be attributed to the irregularities in leaf structure at the time of image capture using the microscope. To bolster our conclusion, we further examined the fluorescence signals in the cells of the root elongation zone in ATG6-mCherry x NPR1-GFP, as depicted in the figure below. Our observations revealed that the fluorescence signals of ATG6-mCherry exhibited uniform distribution, with detection in both the cytoplasm and nucleus. We have replaced the original unclear image with a high-quality image.

Lines 138-143: In fig. S3d, it would make more sense to show the WB on the hybrid npr1-gfp/atg6-mcherry plants with both anti-gfp and anti-mcherry antibodies to detect the free mcherry/gfp. Since the analysis of the level of free FP is done, then why didn't they test the free mcherry levels in Figure S4a? This would be more important than testing the free GFP in ATG6-GFP plants, because the imaging of atg6-mcherry was done in the hybrid plants (fig. S3b).

Thank you for pointing this out. We initially synthesized the ATG6 antibody (anti-ATG6, 1:200, peptide, C-KEKKKIEEEERK, Abmart) in order to detect the endogenous ATG6 protein, and we also tested the specificity and potency of the ATG6 antibody (results are shown in Fig. S17). Additionally, in order to determine the location of the ATG6-mCherry bands, we also detected ATG6-mCherry in ATG6-mCherry Arabidopsis using the ATG6 antibody, and we also used Col as a control (results are shown in Fig. S4). These results show that our synthesized ATG6 antibody can effectively and clearly immunize to both ATG6 and ATG6-mCherry. Therefore, in this study, we used the ATG6 antibody to analyze both ATG6-mCherry and endogenous ATG6. Detailed antibody information is presented in Supplementary Data 1, table S4. In the previous experiments, we procured the mCherry antibody (mCherry-Tag Monoclonal Antibody(6B3), BD-PM2113, China) to immunolabel ATG6-mCherry. However, we encountered challenges with the potency of this mCherry antibody, and considering our budget constraints, as well as the availability of our self-synthesized ATG6 antibody, we chose not to pursue the purchase of another antibody from a different company for the continuation of the Western Blot experiment.

In Figure 2c, there's no atg6-mcherry detected at time 0, in either cytoplasm or nucleus, yet the microscope images in panel a show strong accumulation in both compartments.

Thank you for pointing this out. Previous studies ATG6 can also be degraded via the 26s proteasome pathway (Qi et al., 2017). We speculate that this phenomenon might be attributed to the rapid turnover of ATG6 at time 0.

Lines 156-160: this statement is unsupported by the data. In fig. S5, the bands for native atg6 in the nuclear fraction are extremely weak, and they do not show the reverse pattern of change along the time points compared to the cytoplasmic fraction, which would indicate that the nuclear fraction is complementary to the cytoplasmic pool of the protein. The result more likely suggests that the majority of the ATG6 is in the cytoplasm, and that the weak bands detected in the nucleus are either background signal, or a contamination from the cytoplasmic pool. At this low protein level or poor immuno-detection the background signal is inevitable due to overexposure. Even though the actin marker is not detected in the nuclear fraction, it doesn't necessarily mean that there's no contamination from the cytoplasm in the nuclear fraction. The actin is just too abundant and can be detected at lower exposure.

Thank you for pointing this out. In Fig. S5, we detected the subcellular localization of endogenous ATG6, although the image quality was somewhat low. Nevertheless, the cytosolic and nuclear localization of ATG6 could be clearly observed. In addition to this, we also verified the cytosolic and nuclear localization of ATG6 in Arabidopsis using confocal fluorescence microscopy and nucleoplasmic separation experiments. Actin and H3 were used as cytoplasmic and nucleus internal reference, respectively. (Fig. 2e and f). Furthermore, we observed the cytosolic and nuclear localization of ATG6 when we expressed ATG6-GFP or ATG6-mCherry in tobacco leaves through cis-transfection experiments (Fig. 2a-d). These results are consistent with the prediction of the subcellular location of ATG6 in the Arabidopsis subcellular database (https://suba.live/) (Fig. S3c). The reviewer's feedback has been valuable in helping us present these findings more clearly. We acknowledge the limitations in the image quality for the endogenous ATG6 localization, but we believe the combination of multiple experimental approaches, including the use of fluorescent protein fusions, provides robust evidence for the cytosolic localization of ATG6 in plant cells. Moving forward, we will continue to investigate the significance of ATG6's subcellular distribution and its potential dual roles in both the nucleus and the cytosol, particularly in the context of its interaction with the key immune regulator NPR1. We appreciate the reviewer's constructive comments, as they will help us strengthen the presentation and interpretation of our findings.

In Figure 3a the images are of too low resolution to see the co-localization. The focal planes of the top and bottom panels are quite different: the top is focused on stomata, the bottom - on pavement cells. So, the number of the NPR1-GFP nuclei between these two focal planes is dramatically different. Also, it looks like the atg6-mcherry in these plants are predominantly in the cytoplasm, not the nucleus as the authors claim. A higher resolution and higher quality of images are required to determine this.

Thank you for pointing this out. To ensure the clarity and accuracy of our confocal images, we have supplied a clearer image as supplementary evidence. The Bright images distinctly show that both sets of images are in the same plane of focus. Furthermore, in the figure (third one in the fourth column), the nucleus localization of ATG6-mCherry is clearly visible, and that ATG6-mCherry is co-localized with NPR1-GFP in the nucleus, as indicated by the white arrow.

In Figure 3b, it is not indicated what exactly was measured and in what condition, mock or SA. If these are numbers of nuclei, then it should be indicated what size of the area was sampled, not just "section", and both mock and SA should be included in the measurements. Also, how many independent images have been sampled? what does the error bar represent? What does "normal" mean? Shouldn't this be a mock treatment?

Thank you for pointing out this. The term "Normal" in this context refers to mock treatment, and we have revised the description for clarity. In Figure 3b, the graph illustrates the count of nuclear localizations of NPR1-GFP in ATG6-mCherry × NPR1-GFP and NPR1-GFP Arabidopsis plants following SA treatment. Statistical data were obtained from three independent experiments, each comprising five individual images, resulting in a total of 15 images analyzed for this comparison. Detailed descriptions were also added to the revised manuscript (Lines 568-570, 800-804).

Lines 167-168: the proposed increase of NPR1-GFP in the nucleus could be simply due to a higher accumulation of SA in the hybrid plants, not because of the direct interaction of atg6.

Thank you for pointing out this. Our results confirmed that ATG6 overexpression significantly increased nuclear accumulation of NPR1 (Fig. 3). Notably, the ratio (nucleus NPR1/total NPR1) in ATG6-mCherry × NPR1-GFP was not significantly different from that in NPR1-GFP, and there is a similar phenomenon in N. benthamiana (Fig. 3c-f). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 might result from higher levels and more stable NPR1, rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. Furthermore, we found that under SA treatment, the protein levels of NPR1 were significantly higher in the ATG6-mCherry × NPR1-GFP line compared to the NPR1-GFP line (Fig. 5a). Notably, even in the absence of differences in SA levels between the two lines, we observed that ATG6 could delay the degradation of NPR1 under normal conditions (Fig. 6). These findings suggest that ATG6 employs both SA-dependent and SA-independent mechanisms to maintain the stability of the key immune regulator NPR1. In summary, we therefore suggest that the increased nuclear accumulation in NPR1 cells is a dual effect of SA and ATG6.

Lines 202-204: "Increased nuclear accumulation" implies increased translocation. However, they found that the ratio of NPR1-GFP does not change (Figure 3), so the reason for higher nuclear accumulation is not translocation, but abundance.

Thank you for pointing out this. Our results confirmed that ATG6 overexpression significantly increased nuclear accumulation of NPR1 (Fig. 3). ATG6 also increases NPR1 protein levels and improves NPR1 stability (Fig. 5 and 6). Therefore, we consider that the increased nuclear accumulation of NPR1 in ATG6-mCherry x NPR1-GFP plants might result from higher levels and more stable NPR1 rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. To verify this possibility, we determined the ratio of NPR1-GFP in the nuclear localization versus total NPR1-GFP. Notably, the ratio (nucleus NPR1/total NPR1) in ATG6-mCherry × NPR1-GFP was not significantly different from that in NPR1-GFP, and there is a similar phenomenon in N. benthamiana (Fig. 3c-f). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 might result from higher levels and more stable NPR1, rather than the enhanced nuclear translocation of NPR1 facilitated by ATG6. Further we analyzed whether ATG6 affects NPR1 protein levels and protein stability. Our results show that ATG6 increases NPR1 protein levels under SA treatment and ATG6 maintains the protein stability of NPR1 (Fig. 5 and 6). These results suggested that the increased nuclear accumulation of NPR1 by ATG6 result from higher levels and more stable NPR1. The corresponding description is shown in revised manuscript (lines 338~352).

Lines 204-205: the co-localization in Figure 1d cannot be interpreted as interaction.

Thank you for the feedback. We have replaced the co-localization image with a BiFC (Bimolecular Fluorescence Complementation) image to better illustrate the interaction in Fig 1d.

What age of plants were used for the analysis in Figures 4 and S7? The age of the plant might significantly affect the free SA levels under control conditions.

Thank you for the feedback. In Figures 4 and S7, 3-week-old plants were used to determine salicylic acid (SA) levels and the expression of target genes. Figures 4 and S7 figure notes provide detailed descriptions (lines 818-819).

In Figure 5a they treat with SA, but the analysis in Figure S10 is done with the pathogen, so how can these data be correlated?

Thank you for pointing out this. Previous studies have demonstrated that pathogen infestation rapidly increases the salicylic acid (SA) content in plants, and the elevated SA then activates plant immune responses. Therefore, both pathogen treatment and direct SA treatment can activate SA-dependent plant immune responses. The NPR1 protein is known for its instability. In Figure 5a, we utilized a 0.5 mM SA treatment to assess the changes in NPR1 protein levels, as the impact of SA treatment is more immediate and pronounced.

Lines 241-242: In Figure 5b, it is not clear why there's no detection of NPR1-GFP and atg6-mcherry at time 0?? The levels of proteins in the transient assay are sufficiently high for detection by WB.

Thank you for pointing this out. The NPR1 protein is known to be unstable and prone to degradation through the 26S proteasome pathway (Spoel et al., 2009; Saleh et al., 2015). In addition, previous studies ATG6 can also be degraded via the 26s proteasome pathway (Qi et al., 2017). We speculate that this phenomenon might be attributed to the rapid turnover of NPR1 and ATG6 at time 0.

In Figures 5c-d, the quality of these images is very poor, and they do not clearly show the signs. What structure was exactly measured in these images? There are so many fluorescent bodies there, that it is not clear what are we looking at. Also, it is not clear why they did not show the mcherry channel? It would be important to see if the bodies in SA-treated plants show co-localization with atg6-mcherry autophagosomes (if these exist at all).

Thank you for pointing this out. Interestingly, similar to previous reports (Zavaliev et al., 2020), SA promoted the translocation of NPR1 into the nucleus, but still a significant amount of NPR1 was present in the cytoplasm (Fig. 3c and e). Previous studies have shown that SA increased NPR1 protein levels and facilitated the formation of SINCs in the cytoplasm, which are known to promote cell survival (Zavaliev et al., 2020). We therefore observed the fluorescence signal of SINCs-like condensates in the cytoplasm of tobacco leaves. After 1mM SA treatment, more SINCs-like condensates fluorescence were observed in N. benthamiana co-transformed with ATG6-mCherry + NPR1-GFP compared to mCherry + NPR1-GFP (Fig. 5c-d and Supplemental movie 1-2). We have a clearer demonstration in the supplemental video movie 1-2. Additionally, we observed that SINCs-like condensates signaling partial co-localized with certain ATG6-mCherry autophagosomes fluorescence signals.

Lines 245-247: so, is it atg6 or SA that increases the NPR1 levels? If this is due to SA, then the whole study doesn't have novelty, because we already know from previous works that SA increases the stability of npr1.

Thank you for pointing this out. Indeed, previous studies have shown that salicylic acid (SA) increases NPR1 levels and protein stability (Spoel et al., 2009; Saleh et al., 2015). In our experiments, we found that under SA treatment, the protein levels of NPR1 were significantly higher in the ATG6-mCherry × NPR1-GFP line compared to the NPR1-GFP line (Fig. 5a). Additionally, free SA levels were also significantly elevated in the ATG6-mCherry × NPR1-GFP line under pathogen challenge (Pst DC3000/avrRps4), but not under normal conditions (Fig. 4a). Furthermore, even in the absence of differences in SA levels between the two lines, we observed that ATG6 could delay the degradation of NPR1 under normal conditions (Fig. 6). These findings represent one of our new discoveries. These findings suggest that ATG6 employs both SA-dependent and SA-independent mechanisms to maintain the stability of the key immune regulator NPR1.

Lines 313-316: npr1 and atg6 can function independently from each other, so the term "jointly" is misleading. Based on the overall data provided in this manuscript it cannot be concluded that the two proteins work in one complex to control plant immunity.

Thank you for pointing this out. In the revised manuscript "jointly" has been changed to “cooperatively”.

Lines 369-374: this speculation is beyond the main hypothesis claiming that atg6 functions through npr1. If atg6 can activate the transcription alone, then what is the significance of its activation of npr1? How can one distinguish between the two?

Thank you for pointing this out. Transcription activation by transcription factors typically requires at least two conserved structural domains: a transcription activation domain and a DNA-binding domain. However, ATG6 does not possess these two typical conserved structural domains found in canonical transcription factors. Given this structural context, it is unlikely that ATG6 would be able to directly activate transcription on its own. The lack of the canonical transcription factor domains in ATG6 suggests that it may not be able to function as a direct transcriptional activator. Previous studies have shown that acidic activation domains (AADs) in transcriptional activators (such as Gal4, Gcn4 and VP16) play important roles in activating downstream target genes. Acidic amino acids and hydrophobic residues are the key structural elements of AAD (Pennica et al., 1984; Cress and Triezenberg, 1991; Van Hoy et al., 1993). Chen et al. found that EDS1 contains two ADD domains and confirmed that EDS1 is a transcriptional activator with AAD (Chen et al., 2021a). Here, we also have similar results that ATG6 overexpression significantly enhanced the expression of PR1 and PR5 (Fig. 4b-c and S9), and that the ADD domain containing acidic and hydrophobic amino acids is also found in ATG6 (148-295 AA) (Fig. S14). We speculate that ATG6 might act as a transcriptional coactivator to activate PRs expression synergistically with NPR1.

Lines 389-400: the cell death due to AvrRPS4 in Col-0 ecotype is extremely weak as there's no complete receptor complex for this effector. So, one has to use a very high dose to induce cell death in Col-0, certainly higher than the one used for bacterial growth. The authors used the same dose in both assays, so it is likely that what we see as "cell death" is not an effector-triggered response, but rather symptom-associated for the virulent pathogen.

Thank you for pointing this out. Indeed, as the reviewer pointed out, most cell death assays use higher concentrations of Pst DC3000/avrRps4 or Pst DC3000/avrRpt2, but they typically treat Arabidopsis for a relatively short period, usually less than 1 day(Hofius et al., 2009; Zavaliev et al., 2020). In this study, although we used relatively low Pst DC3000/avrRps4 (0.001) injections, we detected cell death under a relatively long period of Pst DC3000/avrRps4 infestation (3 days). Pst DC3000/avrRps4-infested plants multiply significantly in host cells, and therefore we assumed that the propagated pathogens after 3 days of incubation would be sufficient to induce intense cell death. Consequently, we chose this concentration of Pst DC3000/avrRps4 for the experiment.

Lines 407-416: why do you expect "delay of degradation" with autophagy inhibitor? Shouldn't it be the opposite? In Figure S14, if we compare the bands between 120min and 120min+ConA+WM, the effect of autophagy inhibitors is actually quite strong (0.47 vs 0.22), with about 50% more degradation of NPR1 in their presence. So, the conclusion that the degradation of NPR1 is autophagy-independent is wrong according to this result.

Thank you for pointing this out. We have revised the inaccurate description, as outlined in the revised manuscript (lines 413-425).

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Author response:

Reviewer #1 (Public Review):  

Weaknesses:  

The weakness of this study lies in the fact that many of the genomic datasets originated from novel methods that were not validated with orthogonal approaches, such as DNA-FISH. Therefore, the detailed correlations described in this work are based on methodologies whose efficacy is not clearly established. Specifically, the authors utilized two modified protocols of TSA-seq for the detection of NADs (MKI67IP TSA-seq) and LADs (LMNB1-TSA-seq). Although these methods have been described in a bioRxiv manuscript by Kumar et al., they have not yet been published. Moreover, and surprisingly, Kumar et al., work is not cited in the current manuscript, despite its use of all TSA-seq data for NADs and LADs across the four cell lines. Moreover, Kumar et al. did not provide any DNA-FISH validation for their methods. Therefore, the interesting correlations described in this work are not based on robust technologies.    

An attempt to validate the data was made for SON-TSA-seq of human foreskin fibroblasts (HFF) using multiplexed FISH data from IMR90 fibroblasts (from the lung) by the Zhuang lab (Su et al., 2020). However, the comparability of these datasets is questionable. It might have been more reasonable for the authors to conduct their analyses in IMR90 cells, thereby allowing them to utilize MERFISH data for validating the TSA-seq method and also for mapping NADs and LADs. 

We disagree with the statement that the TSA-seq approach and data has not been validated by orthogonal approaches and with the conclusion that the TSA-seq approach is not robust as summarized here and detailed below in “Specific Comments”.  TSA-seq is robust because it is based only on the original immunostaining specificity provided by the primary and secondary antibodies plus the diffusion properties of the tyramide-free radical. TSA-seq has been extensively validated by microscopy and by the orthogonal genomic measurements provided by LMNB1 DamID and NAD-seq.  This includes: a) the initial validation by FISH of both nuclear speckle (to an accuracy of ~50 nm) and nuclear lamina TSA-seq  and the cross-validation of nuclear lamina TSA-seq with lamin B1 DamID in a first publication (Chen et al, JCB 2018, doi: 10.1083/jcb.201807108); b) the further validation of SON TSA-seq by FISH in a second publication ((Zhang et al, Genome Research 2021, doi:10.1101/gr.266239.120); c) the cross-validation of nucleolar TSA-seq using NAD-seq and the validation by light microscopy of the predictions of differences in the relative distributions of centromeres, nuclear speckles, and nucleoli made from nuclear speckle, nucleolar, and pericentric heterochromatin TSA-seq in the Kumar et al, bioRxiv preprint (which is in a last revision stage involving additional formatting for the journal requirements) doi:https://doi.org/10.1101/2023.10.29.564613; d) the extensive validation of nuclear speckle, LMNB1, and nucleolar TSA-seq generated in HFF human fibroblasts using published light microscopy distance measurements of hundreds of probes generated by multiplexed immuno-FISH MERFISH data (Su et al, Cell 2020, https://doi.org/10.1016/j.cell.2020.07.032), as we described for nucleolar TSA-seq in the Kumar et al, bioRxiv preprint and to some extent for LMNB1 and SON TSA-seq in the current manuscript version (see Specific Comments with attached Author response image 2).

Reviewer 1 raised concerns regarding this FISH validation given that the HFF TSA-seq and DamID data was compared to IMR90 MERFISH measurements.  The Su et al, Cell 2020 MERFISH paper came out well after the 4D Nucleome Consortium settled on HFF as one of the two main “Tier 1” cell lines.  We reasoned that the nuclear genome organization in a second fibroblast cell line would be sufficiently similar to justify using IMR90 FISH data as a proxy for our analysis of our HFF data. Indeed, there is a high correlation between the HFF TSA-seq and distances measured by MERFISH to nuclear lamina, nucleoli, and nuclear speckles (Author response image 1).  Comparing HFF SON-TSA-seq data with published IMR90 SON TSA-seq data (Alexander et al, Mol Cell 2021, doi.org/10.1016/j.molcel.2021.03.006), the HFF SON TSA-seq versus MERFISH scatterplot is very similar to the IMR90 SON TSA-seq versus MERFISH scatterplot.  We acknowledge the validation provided by the IMR90 MERFISH is limited by the degree to which genome organization relative to nuclear locales is similar in IMR90 and HFF fibroblasts. However, the correlation between measured microscopic distances from nuclear lamina, nucleoli, and nuclear speckles and TSA-seq scores is already quite high. We anticipate the conclusions drawn from such comparisons are solid and will only become that much stronger with future comparisons within the same cell line.

Author response image 1.

Scatterplots showing the correlation between TSA-seq and MERFISH microscopic distances. Top: IMR90 SON TSA-seq (from Alexander et al, Mol Cell 2021) (left) and HFF SON TSA-seq (right) (x-axis) versus distance to nuclear speckles (y-axis). Bottom: HFF Lamin B1 TSA-seq (x-axis) versus distance to nuclear lamina (y-axis) (left) and HFF MKI67IP (nucleolar) TSA-seq (x-axis) versus distance to nucleolus (y-axis) (right).

In our revision, we will add justification of the use of IMR90 fibroblasts as a proxy for HFF fibroblasts through comparison of available data sets. 

Reviewer #2 (Public Review):  

Weaknesses:  

The experiments are largely descriptive, and it is difficult to draw many cause-and-effect relationships. Similarly, the paper would be very much strengthened if the authors provided additional summary statements and interpretation of their results (especially for those not as familiar with 3D genome organization). The study would benefit from a clear and specific hypothesis.

We acknowledge that this study was hypothesis-generating rather than hypothesis-testing in its goal. This research was funded through the NIH 4D-Nucleome Consortium, which had as its initial goal the development, benchmarking, and validation of new genomic technologies.  Our Center focused on the mapping of the genome relative to different nuclear locales and the correlation of this intranuclear positioning of the genome with functions- specifically gene expression and DNA replication timing. By its very nature, this project has taken a discovery-driven versus hypothesis-driven scientific approach.  Our question fundamentally was whether we could gain new insights into nuclear genome organization through the integration of genomic and microscopic measurements of chromosome positioning relative to multiple different nuclear compartments/bodies and their correlation with functional assays such as RNA-seq and Repli-seq.

Indeed, as described in this manuscript, this study resulted in multiple new insights into nuclear genome organization as summarized in our last main figure.  We believe our work and conclusions will be of general interest to scientists working in the fields of 3D genome organization and nuclear cell biology.  We anticipate that each of these new insights will prompt future hypothesis-driven science focused on specific questions and the testing of cause-and-effect relationships. 

Given the extensive scope of this manuscript, we were limited in the extent that we could describe and summarize the background, data, analysis, and significance for every new insight. In our editing to reach the eLife recommended word count, we removed some of the explanations and summaries that we had originally included. 

As suggested by Reviewer 2, in our revision we will add back additional summary and interpretation statements to help readers unfamiliar with 3D genome organization.

Specific Comments in response to Reviewer 1:

(1)  We disagree with the comment that TSA-seq has not been cross-validated by other orthogonal genomic methods.  In the first TSA-seq paper (Chen et al, JCB 2018, doi: 10.1083/jcb.201807108), we showed a good correlation between the identification of iLADs and LADs by nuclear lamin and nuclear speckle TSA-seq and the orthogonal genomic method of lamin B1 DamID, which is reproduced using our new TSA-seq 2.0 protocol in this manuscript.  Similarly, in the Kumar et al, bioRxiv preprint (doi:https://doi.org/10.1101/2023.10.29.564613), we showed a general agreement between the identification of NADs by nucleolar TSA-seq and the orthogonal genomic method of NAD-seq.  (We expect this preprint to be in press soon; it is now undergoing a last revision involving only reformatting for journal requirements.) Additionally, we also showed a high correlation between Hi-C compartments and subcompartments and TSA-seq in the Chen et al, JCB 2018 paper. Specifically, there is an excellent correlation between the A1 Hi-C subcompartment and Speckle Associated Domains as detected by nuclear speckle TSA-seq.  Additionally, the A2 Hi-C subcompartment correlated well with iLAD regions with intermediate nuclear speckle TSA-seq scores, and the B2 and B3 Hi-C subcompartments with LADs detected by both LMNB TSA-seq and LMNB1 DamID.  More generally, Hi-C A and B compartment identity correlated well with predictions of iLADs versus LADs from nuclear speckle and nuclear lamina TSA-seq.

(2)  In the Chen et al, JCB 2018 paper we also qualitatively and quantitatively validated TSA-seq using FISH.  Qualitatively, we showed that both nuclear speckle and nuclear lamin TSA-seq correlated well with distances to nuclear speckles versus the nuclear lamina, respectively, measured by immuno-FISH.

Quantitatively, we showed that SON TSA-seq could be used to estimate the microscopic mean distance to nuclear speckles with mean and median residuals of ~50 nm.  First, we used light microscopy to show that the spreading of tyramide-biotin signal from a point-source of TSA staining fits well with the exponential decay predicted theoretically by reaction-diffusion equations assuming a steady rate of tyramide-biotin free radical generation by the HRP enzyme and a constant probability throughout the nucleus of free-radical quenching (through reaction with protein tyrosine residues and nucleic acids).  Second, we used the exponential decay constant measured by light microscopy together with FISH measurements of mean speckle distance for several genomic regions to fit an exponential function and to predict distance to nuclear speckles genome-wide directly from SON TSA-seq sequencing reads.  Third, we used this approach to test the predictions against a new set of FISH measurements, demonstrating an accuracy of these predictions of ~50 nm.

(3)  The importance of the quantitative validation by immuno-FISH of using TSA-seq to estimate mean distance to nuclear speckles is that it demonstrates the robustness of the TSA-seq approach.  Specifically, it shows how the TSA-seq signal is predicted to depend only on the specificity of the primary and secondary antibody staining and the diffusion properties of the tyramide-biotin free radicals produced by the HRP peroxidase.  This is fundamentally different from the significant dependence on antibodies and choice of marker proteins for molecular proximity assays such as DamID, ChIP-seq, and Cut and Run/Tag which depend on molecular proximity for labeling and/or pulldown of DNA.

This robustness leads to specific predictions.  First, it predicts similar TSA-seq signals will be produced using antibodies against different marker proteins against the same nuclear compartment.  This is because the exponential decay constant (distance at which the signal drops by one half) for the spreading of the TSA is in the range of several hundred nm, as measured by light microscopy for several TSA staining conditions.  Indeed, we showed in the Chen et al, JCB 2018 paper that antibodies against two different nuclear speckle proteins produced very similar TSA-seq signals while antibodies against LMNB versus LMNA also produced very similar TSA-seq signals.  Similarly, we showed in the Kumar et al preprint that antibodies against four different nucleolar proteins showed similar TSA-seq signals, with the highest correlation coefficients for the TSA-seq signals produced by the antibodies against two GC nucleolar marker proteins and the TSA-seq signals produced by the antibodies against two FC/DFC nucleolar marker proteins.

Author response image 2.

Comparison of TSA-seq data from different cell lines versus IMR90 MERFISH.  The observed correlation between SON (nuclear speckle) TSA-seq versus MERFISH is nearly as high for TSA-seq data from HFF as it is for TSA-seq data from the IMR90 cell line (Alexander et al, Mol Cell 2021) in which the MERFISH was performed. The correlations for SON, LMNB1 (nuclear lamina) and MKI67IP (nucleolus) versus MERFISH are highest for HFF TSA-seq data as compared to TSA-seq data from other cell lines (H1, K562, HCT116).  Comparison of measured distances to nuclear locale (y-axis) versus TSA-seq scores (x-axis) from different cell lines labeled in red. Left to right: SON, LMNB1, and MKI67IP.  Top to bottom: SON TSA-seq versus MERFISH for two TSA-seq replicates; TSA-seq from HFF, H1, K562, and HCT116 versus MERFISH.

Second, it predicts that the quantitative relationship between TSA-seq signal and mean distance from a nuclear compartment will depend on the convolution of the predicted exponential decay of spreading of the TSA signal produced by a point source with the more complicated staining distribution of nuclear compartments such as the nuclear lamina or nucleoli.  We successfully used this concept to explain the differences emerging between LMNB1 DamID and TSA-seq signals for flat nuclei and to recognize the polarized distribution of different LADs over the nuclear periphery.

(4)  After our genomic data production and during our data analysis, a valuable resource from the Zhuang lab was published, using MERFISH to visualize hundreds of genomic loci in IMR90 cells. We acknowledge that the much more extensive validation of TSA-seq by the multiplexed immuno-FISH MERFISH data is dependent on the degree to which the nuclear genome organization is similar between IMR90 and HFF fibroblasts.  However, the correlation between distances to nuclear speckles, nucleoli, and the nuclear lamina measured in IMR90 fibroblasts and the nuclear speckle, nucleolar, and nuclear lamina TSA-seq measured in HFF fibroblasts is already striking (See Author response image 1).  With regard to SON TSA-seq, the MERFISH versus HFF TSA-seq correlation is close to what we observe using published IMR90 SON TSA-seq data (correlation coefficients of 0.89 (IMR90 TSA-seq) versus 0.86 (HFF TSA-seq).  Moreover, this correlation is highest using TSA-seq data from HFF cells as compared to the three other cell lines. (see Author response image 2).  We believe these correlations can be considered a lower bound on the actual correlations between the FISH distances and TSA-seq that we would have observed if we had performed both assays on the same cell line. 

(5)  Currently, we still require tens of millions of cells to perform each TSA-seq assay.  This requires significant expansion of cells and a resulting increase in passage numbers of the IMR90 cells before we can perform the TSA-seq. During this expansion we observe a noticeable slowing of the IMR90 cell growth as expected for secondary cell lines as we approach the Hayflick limit.  We still do not know to what degree nuclear organization relative to nuclear locales may change as a function of cell cycle composition (ie percentage of cycling versus quiescent cells) and cell age.  Thus, even if we performed TSA-seq on IMR90 cells we would be comparing MERFISH from lower passages with a higher percentage of actively proliferating cells with TSA-seq from higher passages with a higher percentage of quiescent cells. 

We are currently working on a new TSA-seq protocol that will work with thousands of cells.  We believe it is better investment of time and resources to wait until this new protocol is optimized before we repeat TSA-seq in IMR90 cells for a better comparison with multiplexed FISH data. 

Specific Comments in response to Reviewer 2:

(1)  As we acknowledge in our Response summary, we were limited in the degree to which we could actually follow-up our findings with experiments designed to test specific hypotheses generated by our data.  However, we do want to point out that our comparison of wild-type K562 cells with the LMNA/LBR double knockout was designed to test the long-standing model that nuclear lamina association of genomic loci contributes to gene silencing.  This experiment was motivated by our surprising result that gene expression differences between cell lines correlated strongly with differences in positioning relative to nuclear speckles rather than the nuclear lamina.  Despite documenting in these double knockout cells a decreased nuclear lamina association of most LADs, and an increased nuclear lamina association of the “p-w-v” fiLADs identified in this manuscript, we saw no significant change in gene expression in any of these regions as compared to wild-type K562 cells.  Meanwhile, distances to nuclear speckles as measured by TSA-seq remained nearly constant.

We would argue that this represents a specific example in which new insights generated by our genomics comparison of cell lines led to a clear and specific hypothesis and the experimental testing of this hypothesis.

In response to Reviewer 2, we are modifying the text to make this clearer and to explicitly describe how we were testing the hypothesis that distance to nuclear lamina is correlated with but not causally linked to gene expression and how to test this hypothesis we used a DKO of LMNA and LBR to change distances relative to the nuclear lamina and to test the effect on gene expression.

Reviewer #2 (Public Review):

Summary:

In this study, Liu et al. explore the interplay between G-quadruplexes (G4s) and R-loops. The authors developed novel techniques, HepG4-seq and HBD-seq, to capture and map these nucleic acid structures genome-wide in human HEK293 cells and mouse embryonic stem cells (mESCs). They identified dynamic, cell-type-specific distributions of co-localized G4s and R-loops, which predominantly localize at active promoters and enhancers of transcriptionally active genes. Furthermore, they assessed the role of helicase Dhx9 in regulating these structures and their impact on gene expression and cellular functions.

The manuscript provides a detailed catalogue of the genome-wide distribution of G4s and R-loops. However, the conceptual advance and the physiological relevance of the findings are not obvious. Overall, the impact of the work on the field is limited to the utility of the presented methods and datasets.

Strengths:

(1) The development and optimization of HepG4-seq and HBD-seq offer novel methods to map native G4s and R-loops.

(2) The study provides extensive data on the distribution of G4s and R-loops, highlighting their co-localization in human and mouse cells.

(3) The study consolidates the role of Dhx9 in modulating these structures and explores its impact on mESC self-renewal and differentiation.

Weaknesses:

(1) The specificity of the biotinylation process and potential off-target effects are not addressed. The authors should provide more data to validate the specificity of the G4-hemin.

(2) Other methods exploring a catalytic dead RNAseH or the HBD to pull down R-loops have been described before. The superior quality of the presented methods in comparison to existing ones is not established. A clear comparison with other methods (BG4 CUT&Tag-seq, DRIP-seq, R-CHIP, etc) should be provided.

(3) Although the study demonstrates Dhx9's role in regulating co-localized G4s and R-loops, additional functional experiments (e.g., rescue experiments) are needed to confirm these findings.

(4) The manuscript would benefit from a more detailed discussion of the broader implications of co-localized G4s and R-loops.

(5) The manuscript lacks appropriate statistical analyses to support the major conclusions.

(6) The discussion could be expanded to address potential limitations and alternative explanations for the results.

Reviewer #3 (Public Review):

Summary:

The authors developed and optimized the methods for detecting G4s and R-loops independent of BG4 and S9.6 antibody, and mapped genomic native G4s and R-loops by HepG4-seq and HBD-seq, revealing that co-localized G4s and R-loops participate in regulating transcription and affecting the self-renewal and differentiation capabilities of mESCs.

Strengths:

By utilizing the peroxidase activity of G4-hemin complex and combining proximity labeling technology, the authors developed HepG4-seq (high throughput sequencing of hemin-induced proximal labelled G4s) , which can detect the dynamics of G4s in vivo. Meanwhile, the "GST-His6-2xHBD"-mediated CUT&Tag protocol (Wang et al., 2021) was optimized by replacing fusion protein and tag, the optimized HBD-seq avoids the generation of GST fusion protein aggregates and can reflect the genome-wide distribution of R-loops in vivo.

The authors employed HepG4-seq and HBD-seq to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). The data indicate that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptionally active genes.

Combined with Dhx9 ChIP-seq and co-localized G4s and R-loops data in wild-type and dhx9KO mESCs, the authors confirm that the helicase Dhx9 is a direct and major regulator that regulates the formation and resolution of co-localized G4s and R-loops.

Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops-associated genes.

In conclusion, the authors provide an approach to studying the interplay between G4s and R-loops, shedding light on the important roles of co-localized G4s and R-loops in development and disease by regulating the transcription of related genes.

Weaknesses:

As we know, there are at least two structure data of S9.6 antibody very recently, and the questions about the specificity of the S9.6 antibody on RNA:DNA hybrids should be finished. The authors referred to (Hartono et al., 2018; Konig et al., 2017; Phillips et al., 2013) need to be updated, and the authors' bias against S9.6 antibodies needs also to be changed. However, as the authors had questioned the specificity of the S9.6 antibody, they should compare it in parallel with the data they have and the data generated by the widely used S9.6 antibody.

Although HepG4-seq is an effective G4s detection technique, and the authors have also verified its reliability to some extent, given the strong link between ROS homeostasis and G4s formation, and hemin's affinity for different types of G4s, whether HepG4-seq reflects the dynamics of G4s in vivo more accurately than existing detection techniques still needs to be more carefully corroborated.

Author response:

eLife assessment

This useful study describes an antibody-free method to map G-quadruplexes (G4s) in vertebrate cells. While the method might have potential, the current analysis is primarily descriptive and does not add substantial new insights beyond existing data (e.g., PMID:34792172). While the datasets provided might constitute a good starting point for future functional studies, additional data and analyses would be needed to fully support the major conclusions and, at the same time, clarify the advantage of this method over other methods. Specifically, the strength of the evidence for DHX9 interfering with the ability of mESCs to differentiate by regulating directly the stability of either G4s or R-loops is still incomplete.

We thank the editors for their helpful comments.

Given that antibody-based methods have been reported to leave open the possibility of recognizing partially folded G4s and promoting their folding, we have employed the peroxidase activity of the G4-hemin complex to develop a new method for capturing endogenous G4s that significantly reduces the risk of capturing partially folded G4s. We will be happy to clarify the advantage of our method.

In the Fig. 7, we applied the Dhx9 CUT&Tag assay to identify the G4s and R-loops directly bound by Dhx9 and further characterized the differential Dhx9-bound G4s and R-loops in the absence of Dhx9. Dhx9 is a versatile helicase capable of directly resolving R-loops and G4s or promoting R-loop formation (PMID: 21561811, 30341290, 29742442, 32541651, 35905379, 34316718). Furthermore, we showed that depletion of Dhx9 significantly altered the levels of G4s or R-loops around the TSS or gene bodies of several key regulators of mESC and embryonic development, such as Nanog, Lin28a, Bmp4, Wnt8a, Gata2, and Lef1, and also their RNA levels (Fig.7 I). The above evidence is sufficient to support the transcriptional regulation of mESCs cell fate by directly modulating the G4s or R-loops within the key regulators of mESCs.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Non-B DNA structures such as G4s and R-loops have the potential to impact genome stability, gene transcription, and cell differentiation. This study investigates the distribution of G4s and R-loops in human and mouse cells using some interesting technical modifications of existing Tn5-based approaches. This work confirms that the helicase DHX9 could regulate the formation and/or stability of both structures in mouse embryonic stem cells (mESCs). It also provides evidence that the lack of DHX9 in mESCs interferes with their ability to differentiate.

Strengths:

HepG4-seq, the new antibody-free strategy to map G4s based on the ability of Hemin to act as a peroxidase when complexed to G4s, is interesting. This study also provides more evidence that the distribution pattern of G4s and R-loops might vary substantially from one cell type to another.

We appreciate your valuable points.

Weaknesses:

This study is essentially descriptive and does not provide conclusive evidence that lack of DHX9 does interfere with the ability of mESCs to differentiate by regulating directly the stability of either G4 or R-loops. In the end, it does not substantially improve our understanding of DHX9's mode of action.

In this study, we aimed to report new methods for capturing endogenous G4s and R-loops in living cells. Dhx9 has been reported to directly unwind R-loops and G4s or promote R-loop formation (PMID: 21561811, 30341290, 29742442, 32541651, 35905379, 34316718). To understand the direct Dhx9-bound G4s and R-loops, we performed the Dhx9 CUT&Tag assay and analyzed the co-localization of Dhx9-binding sites and G4s or R-loops. We found that 47,857 co-localized G4s and R-loops are directly bound by Dhx9 in the wild-type mESCs and 4,060 of them display significantly differential signals in absence of Dhx9, suggesting that redundant regulators exist as well. We showed that depletion of Dhx9 significantly altered the RNA levels of several key regulators of mESC and embryonic development, such as Nanog, Lin28a, Bmp4, Wnt8a, Gata2, and Lef1, which coincides with the significantly differential levels of G4s or R-loops around the TSS or gene bodies of these genes (Fig.7). The comprehensive molecular mechanism of Dhx9 action is indeed not the focus of this study. We will work on it in the future studies. Thank you for the comments.

There is no in-depth comparison of the newly generated data with existing datasets and no rigorous control was presented to test the specificity of the hemin-G4 interaction (a lot of the hemin-dependent signal seems to occur in the cytoplasm, which is unexpected).

The specificity of hemin-G4-induced peroxidase activity and self-biotinylation has been well demonstrated in previous studies (PMID: 19618960, 22106035, 28973477, 32329781). In the Fig.1A, we compared the hemin-G4-induced biotinylation levels in different conditions. Cells treated with hemin and Bio-An exhibited a robust fluorescence signal, while the absence of either hemin or Bio-An almost completely abolished the biotinylation signals, suggesting a specific and active biotinylation activity. To identify the specific signals, we have included the non-label control and used this control to call confident HepG4 peaks in all HepG4-seq assays.

The hemin-RNA G4 complex has also been reported to have mimic peroxidase activity and trigger similar self-biotinylation signals as DNA G4s (PMID: 32329781, 31257395, 27422869). Therefore, it is not surprising to observe hemin-dependent signals in the cytoplasm generated by cytoplasmic RNA G4s.

In the revised version, we will include careful comparison between our data and previous datasets.

The authors talk about co-occurrence between G4 and R-loops but their data does not actually demonstrate co-occurrence in time. If the same loci could form alternatively either R-loops or G4 and if DHX9 was somehow involved in determining the balance between G4s and R-loops, the authors would probably obtain the same distribution pattern. To manipulate R-loop levels in vivo and test how this affects HEPG4-seq signals would have been helpful.

Single-molecule fluorescence studies have shown the existence of a positive feedback mechanism of G4 and R-loop formation during transcription (PMID: 32810236, 32636376), suggesting that G4s and Rloops could co-localize at the same molecule. Dhx9 is a versatile helicase capable of directly resolving R-loops and G4s or promoting R-loop formation (PMID: 21561811, 30341290, 29742442, 32541651, 35905379, 34316718). Although depletion of Dhx9 resulted in 6,171 Dhx9-bound co-localized G4s and R-loops with significantly altered levels of G4s or R-loops, only 276 of them (~4.5%) harbored altered G4s and R-loops, suggesting that the interacting G4s and R-loops are rare in living cells. Nowadays, the genome-wide co-occurrence of two factors are mainly obtained by bioinformatically intersection analysis. We agreed that the heterogenous distribution between cells will give false positive co-occurrence patterns. We will carefully discuss this point in the revised version. At the same time, we will make efforts to develop a new method to map the co-localized G4 and R-loop in the same molecule in the future study.

This study relies exclusively on Tn5-based mapping strategies. This is a problem as global changes in DNA accessibility might strongly skew the results. It is unclear at this stage whether the lack of DHX9, BLM, or WRN has an impact on DNA accessibility, which might underlie the differences that were observed. Moreover, Tn5 cleaves DNA at a nearby accessible site, which might be at an unknown distance away from the site of interest. The spatial accuracy of Tn5-based methods is therefore debatable, which is a problem when trying to demonstrate spatial co-occurrence. Alternative mapping methods would have been helpful.

In this study, we used the recombinant streptavidin monomer and anti-GP41 nanobody fusion protein (mSA-scFv) to specifically recognize hemin-G4-induced biotinylated G4 and then recruit the recombinant GP41-tagged Tn5 protein to these G4s sites. Similarly, the recombinant V5-tagged N-terminal hybrid-binding domain (HBD) of RNase H1 specifically recognizes R-loops and recruit the recombinant protein G-Tn5 (pG-Tn5) with the help of anti-V5 antibody. Therefore, the spatial distance of Tn5 to the target sites is well controlled and very short, and also the recruitment of Tn5 is specifically determined by the existence of G4s in HepG4-seq and R-loops in HBD-seq.

Reviewer #2 (Public Review):

Summary:

In this study, Liu et al. explore the interplay between G-quadruplexes (G4s) and R-loops. The authors developed novel techniques, HepG4-seq and HBD-seq, to capture and map these nucleic acid structures genome-wide in human HEK293 cells and mouse embryonic stem cells (mESCs). They identified dynamic, cell-type-specific distributions of co-localized G4s and R-loops, which predominantly localize at active promoters and enhancers of transcriptionally active genes. Furthermore, they assessed the role of helicase Dhx9 in regulating these structures and their impact on gene expression and cellular functions.

The manuscript provides a detailed catalogue of the genome-wide distribution of G4s and R-loops. However, the conceptual advance and the physiological relevance of the findings are not obvious. Overall, the impact of the work on the field is limited to the utility of the presented methods and datasets.

Strengths:

(1) The development and optimization of HepG4-seq and HBD-seq offer novel methods to map native G4s and R-loops.

(2) The study provides extensive data on the distribution of G4s and R-loops, highlighting their co-localization in human and mouse cells.

(3) The study consolidates the role of Dhx9 in modulating these structures and explores its impact on mESC self-renewal and differentiation.

We appreciate your valuable points.

Weaknesses:

(1) The specificity of the biotinylation process and potential off-target effects are not addressed. The authors should provide more data to validate the specificity of the G4-hemin.

The specificity of hemin-G4-induced peroxidase activity and self-biotinylation has been well demonstrated in previous studies (PMID: 19618960, 22106035, 28973477, 32329781). In the Fig.1A, we compared the hemin-G4-induced biotinylation levels in different conditions. Cells treated with hemin and Bio-An exhibited a robust fluorescence signal, while the absence of either hemin or Bio-An almost completely abolished the biotinylation signals, suggesting a specific and active biotinylation activity.

(2) Other methods exploring a catalytic dead RNAseH or the HBD to pull down R-loops have been described before. The superior quality of the presented methods in comparison to existing ones is not established. A clear comparison with other methods (BG4 CUT&Tag-seq, DRIP-seq, R-CHIP, etc) should be provided.

Thank you for the suggestions. We will include the comparisons in the revised version.

(3) Although the study demonstrates Dhx9's role in regulating co-localized G4s and R-loops, additional functional experiments (e.g., rescue experiments) are needed to confirm these findings.

Dhx9 has been demonstrate as a versatile helicase capable of directly resolving R-loops and G4s or promoting R-loop formation in previous studies (PMID: 21561811, 30341290, 29742442, 32541651, 35905379, 34316718). We believe that the current new dataset and previous studies are enough to support the capability of Dhx9 in regulating co-localized G4s and R-loops.

(4) The manuscript would benefit from a more detailed discussion of the broader implications of co-localized G4s and R-loops.

Thank you for the suggestions. We will include a more detailed discussion in the revised version.

(5) The manuscript lacks appropriate statistical analyses to support the major conclusions.

We apologized for this point. Whereas we have applied careful statistical analyses in this study, lacking of some statistical details make people hard to understand some conclusions. We will carefully add details of all statistical analysis.

(6) The discussion could be expanded to address potential limitations and alternative explanations for the results.

Thank you for the suggestions. We will include a more detailed discussion about this point in the revised version.

Reviewer #3 (Public Review):

Summary:

The authors developed and optimized the methods for detecting G4s and R-loops independent of BG4 and S9.6 antibody, and mapped genomic native G4s and R-loops by HepG4-seq and HBD-seq, revealing that co-localized G4s and R-loops participate in regulating transcription and affecting the self-renewal and differentiation capabilities of mESCs.

Strengths:

By utilizing the peroxidase activity of G4-hemin complex and combining proximity labeling technology, the authors developed HepG4-seq (high throughput sequencing of hemin-induced proximal labelled G4s), which can detect the dynamics of G4s in vivo. Meanwhile, the "GST-His6-2xHBD"-mediated CUT&Tag protocol (Wang et al., 2021) was optimized by replacing fusion protein and tag, the optimized HBD-seq avoids the generation of GST fusion protein aggregates and can reflect the genome-wide distribution of R-loops in vivo.

The authors employed HepG4-seq and HBD-seq to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). The data indicate that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptionally active genes.

Combined with Dhx9 ChIP-seq and co-localized G4s and R-loops data in wild-type and dhx9KO mESCs, the authors confirm that the helicase Dhx9 is a direct and major regulator that regulates the formation and resolution of co-localized G4s and R-loops.

Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops-associated genes.

In conclusion, the authors provide an approach to studying the interplay between G4s and R-loops, shedding light on the important roles of co-localized G4s and R-loops in development and disease by regulating the transcription of related genes.

We appreciate your valuable points.

Weaknesses:

As we know, there are at least two structure data of S9.6 antibody very recently, and the questions about the specificity of the S9.6 antibody on RNA:DNA hybrids should be finished. The authors referred to (Hartono et al., 2018; Konig et al., 2017; Phillips et al., 2013) need to be updated, and the authors' bias against S9.6 antibodies needs also to be changed. However, as the authors had questioned the specificity of the S9.6 antibody, they should compare it in parallel with the data they have and the data generated by the widely used S9.6 antibody.

Thank you for the updating information about the structure data of S9.6 antibody. We politely disagree the specificity of the S9.6 antibody on RNA:DNA hybrids. The structural studies of S9.6 (PMID: 35347133, 35550870) used only one RNA:DNA hybrid to show the superior specificity of S9.6 on RNA:DNA hybrid than dsRNA and dsDNA. However, Fabian K. et al has reported that the binding affinities of S9.6 on RNA:DNA hybrid exhibits obvious sequence-dependent bias from null to nanomolar range (PMID: 28594954). We will include the comparison between S9.6-derived data and our HBD-seq data in the revised version.

Although HepG4-seq is an effective G4s detection technique, and the authors have also verified its reliability to some extent, given the strong link between ROS homeostasis and G4s formation, and hemin's affinity for different types of G4s, whether HepG4-seq reflects the dynamics of G4s in vivo more accurately than existing detection techniques still needs to be more carefully corroborated.

Thank you for pointing out this issue. In the in vitro hemin-G4 induced self-biotinylation assay, parallel G4s exhibit higher peroxidase activities than anti-parallel G4s. Thus, the dynamics of G4 conformation could affect the HepG4-seq signals (PMID: 32329781). In the future, people may need to combine HepG4-seq and BG4s-eq to carefully explain the endogenous G4s. We will carefully discuss this point in the revised version.

Auf gut Norddeutsch: Morgen ist auch noch ein Tag.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

This paper by Beath et. al. identifies a potential regulatory role for proteins involved in cytoplasmic streaming and maintaining the grouping of paternal organelles: holding sperm contents in the fertilized embryos away from the oocyte meiotic spindle so that they don't get ejected into the polar body during meiotic chromosome segregation. The authors show that by time-lapse video, paternal mitochondria (used as a readout for sperm and its genome) is excluded from yolk granules and maternal mitochondria, even when moving long distances by cytoplasmic streaming. To understand how this exclusion is accomplished, they first show that it is independent of both internal packing and the engulfment of the paternal chromosomes by maternal endoplasmic reticulum creating an impermeable barrier. They then test whether the control of cytoplasmic steaming affects this exclusion by knocking down two microtubule motors, Katanin and kinesis I. They find that the ER ring, which is used as a proxy for paternal chromosomes, undergoes extensive displacement with these treatments during anaphase I and interacts with the meiotic spindle, supporting their hypothesis that the exclusion of paternal chromosomes is regulated by cytoplasmic streaming. Next, they test whether a regulator of maternal ER organization, ATX-2, disrupts sperm organization so that they can combine the double depletion of ATX-2 and KLP-7, presumably because klp-7 RNAi (unlike mei-1 RNAi) does not affect polar body extrusion and they can report on what happens to paternal chromosomes. They find that the knockdown of both ATX-2 and KLP-7 produces a higher incidence of what appears to be the capture of paternal chromosomes by the meiotic spindle (5/24 vs 1/25). However, this capture event appears to halt the cell cycle, preventing the authors from directly observing whether this would result in the paternal chromosomes being ejected into the polar body. 

Strengths: 

This is a useful, descriptive paper that highlights a potential challenge for embryos during fertilization: when fertilization results in the resumption of meiotic divisions, how are the paternal and maternal genomes kept apart so that the maternal genome can undergo chromosome segregation and polar body extrusion without endangering the paternal genome? In general, the experiments are well-executed and analyzed. In particular, the authors' use of multiple ways to knock down ATX-2 shows rigor. 

Weaknesses: 

The paper makes a case that this regulation may be important but the authors should do some additional work to make this case more convincing and accessible for those outside the field. In particular, some of the figures could include greater detail to support their conclusions, they could explain the rationale for some experiments better and they could perform some additional control experiments with their double depletion experiments to better support their interpretations. Also, the authors' inability to assess the functional biological consequences of the capture of the sperm genome by the oocyte spindle should be discussed, particularly in light of the cell cycle arrest that they observe. 

These general comments are addressed in the more specific critiques below.

Reviewer #2 (Public Review): 

Summary 

In this manuscript, Beath et al. use primarily C. elegans zygotes to test the overarching hypothesis that cytoplasmic mechanisms exit to prevent interaction between paternal chromosomes and the meiotic spindle, which are present in a shared zygotic cytoplasm after fertilization. Previous work, much of which by this group, had characterized cytoplasmic streaming in the zygote and the behavior of paternal components shortly after fertilization, primarily the clustering of paternal mitochondria and membranous organelles around the paternal chromosomes. This work set out to identify the molecular mechanisms responsible for that clustering and test the specific hypothesis that the "paternal cloud" helps prevent the association of paternal chromosomes with the meiotic spindle. 

Strengths 

This work is a collection of technical achievements. The data are primarily 3- and 4-channel time-lapse images of zygotes shortly after fertilization, which were performed inside intact animals. There are many instances in which the experiments show extreme technical skill, such as tracking the paternal chromosomes over large displacements throughout the volume of the embryo. The authors employ a wide variety of fluorescent reporters to provide a remarkably clear picture of what is going on in the zygote. These reagents and the novel characterization of these stages that they provide will be widely beneficial to the community. 

The data provide direct visualization of what had previously been a mostly hypothetical structure, the "paternal cloud," using simultaneous labeling of paternal DNA and mitochondria in combination with a variety of maternal proteins including maternal mitochondria, yolk granules, tubulin, and plasma membrane. Together, these images provided convincing evidence of the existence of this specified cytoplasmic domain. They go on to show that the knockdown of the ataxin-2 homolog ALX-2, a protein previously shown to affect ER dynamics, disrupted the paternal cloud, identifying a role for ER organization in this structure. 

The authors then used the system to test the functional consequences of perturbing the cytoplasmic organization. Consistent with the paternal cloud being a stable structure, it stayed intact during large movements the authors generated using previously published knockdowns (of mei-1/katanin and kinesin-13/kpl-7) that increased cytoplasmic streaming. They used this data to document instances in which the paternal chromosomes were likely to have been attached to the spindle. They concluded with direct evidence of spindle fibers connecting to the paternal chromatin upon knockdown of ATX-2 in combination with increased cytoplasmic streaming, providing strong, direct support for their overarching hypothesis. 

Weaknesses 

While the data is convincing, the narrative of the paper could be streamlined to highlight the novelty of the experiments and better articulate the aims. For example, the cloud of paternal mitochondria and membranous organelles was previously shown, but Figures 1-2 largely reiterate that observation. The innovation seems to be that the combination of ER, yolk, and maternal mitochondrial markers makes the existence of a specified domain more concrete. There are also some instances where more description is needed to make the conclusions from the images clear. 

These general comments are addressed in the more specific critiques below.

The manuscript intersperses what read like basic characterizations of fluorescent markers that, as written, can distract from the main story. The authors characterized the dynamics of ER organization throughout the substages of meiosis and the permeability of the envelope of ER that surrounds the paternal chromatin, but it could be more clearly established how the ability to visualize these structures allowed them to address their aims.

We have added the following after the initial description of ER morphology changes: (ER morphology was used to determine cell-cycle stages during live imaging reported below in Fig. 6.)

More background on what was previously known about ER organization in M-phase and the role of ataxin proteins specifically may help provide more continuity. 

We have added references to transitions to ER sheets during mitotic M-phase in HeLa cells and Xenopus extracts.

Reviewer #3 (Public Review): 

Summary: 

This study by Beath et al. investigated the mechanisms by which sperm DNA is excluded from the meiotic spindle after fertilization. Time-lapse imaging revealed that sperm DNA is surrounded by paternal mitochondria and maternal ER that is permeable to proteins. By increasing cytoplasmic streaming using kinesin-13 or katanin RNAi, the authors demonstrated that limiting cytoplasmic streaming in the embryo is an important step that prevents the capture of sperm DNA by the oocyte meiotic spindle. Further experiments showed that the Ataxin-2 protein is required to hold paternal mitochondria together and close to the sperm DNA. Finally, double depletion of kinesin-13 and Ataxin-2 suggested an increased risk of meiotic spindle capture of sperm DNA. 

Overall, this is an interesting finding that could provide a new understanding of how meiotic spindle capture of sperm DNA and its accidental expulsion into the polar body is prevented. However, some conceptual gaps need to be addressed and further experiments and improved data analyses would strengthen the paper. 

- It would be helpful if the authors could discuss in good detail how they think maternal ER surrounds the sperm DNA

We have added 2 references to papers about nuclear envelope re-assembly from Shirin Bahmanyar’s lab and suggest the ER envelope is a halted intermediate in nuclear envelope reassembly.

and why is it not disrupted following Ataxin disruption. 

We have been attempting to disrupt ER structures in the meiotic embryo for the last 5 years by depleting profilin, BiP, atlastin, ATX-2 and by optogenetically packing ER into a ball in the middle of the oocyte.  None of these treatments prevent envelopment of the sperm DNA by maternal ER.  None of these treatments remove ER from the spindle envelope and none remove ER from the plasma membrane.  These treatments mostly result in “large aggregates” of ER that we have not examined by EM.  Wild speculation: any disruption of the ER strong enough to prevent ER envelopment around chromatin would be sterile because the M to S transition in the mitotic zone of the germline would be blocked.  Rapid depletion of ATX-2 to the extent shown by rigorous data in this manuscript does not prevent ER envelopment around chromatin.  We chose not to speculate about the reasons for this because we do not know why.

- Since important phenotypes revealed in RNAi experiments (e.g. kinesin-13 and ataxin-2 double depletion) are not very robust, the authors should consider toning down their conclusions and revising some of their section headings. I appreciate that they are upfront about some limitations, but they do nonetheless make strong concluding sentences. 

We have changed the discussion of the klp-7 atx-2 double depletion to: “The capture of the sperm DNA by the meiotic spindle in ATX-2 KLP-7 double depleted embryos suggests that the integrity of the exclusion zone around the sperm DNA might insulate the sperm DNA from spindle microtubules.  However, a much larger number of klp-7(RNAi) singly depleted and atx-2(degron) singly depleted time-lapse sequences are needed to rigorously support this idea. “

- The discussion section could be improved further to present the authors' findings in the larger context of current knowledge in the field. 

We have expanded the discussion as suggested.

- The authors previously demonstrated that F-actin prevents meiotic spindle capture of sperm DNA in this system. However, the current manuscript does not discuss how the katanin, kinesin-13 and Ataxin-2 mechanisms could work together with previously established functions of F-actin in this process. 

We have added pfn-1(RNAi) to the discussion section.

- How can the authors exclude off-target effects in their RNAi depletion experiments? Can kinesin-13, katanin, and Ataxin phenotypes be rescued for instance? 

For ataxin-2 phenotypes, two completely independent controls for off target effects are shown.  GFP(RNAi) on a strain with and endogenous ATX-2::GFP tag vs GFP(RNAi) on a strain with no tag on the ATX-2.  ATX-2::AID with or without auxin.  For kinesin-13 and katanin, we did not do a rigorous control for off-target effects of RNAi.  However, the effects of these depletions on cytoplasmic microtubules have been previously reported by others

- How are the authors able to determine if the paternal genome was actually captured by the spindle? Does lack of movement definitively suggest capture without using a spindle marker? 

mKate::tubulin labels the spindle in each capture event.  This can be seen in Video S3. for mei-1(RNAi) and Figure 9 for atx-2 klp-7 double depletions.

(1) Major issues: 

The images provided are not convincing that mitochondria are entirely excluded from the regions with yolk granules from the images provided. Please provide insets of magnified images of the paternal mitochondria in Figure 1E to more clearly show the exclusion even when paternal mitochondria are streaming. Providing grayscale images, individual z-sections and/or some quantification of this data might also be more convincing to this reviewer. 

We have modified Fig. 1 by adding single wavelength magnified insets to more clearly show that paternal mitochondria are in a “black hole” in the maternal yolk granules during  cytoplasmic streaming.

Figure 2 -This figure can be retitled to highlight that the paternal organelle cloud is impermeable to mitochondria and conserved. 

The legend has been re-titled as suggested.

Figure 3B, An image of the DNA within the ring of maternal ER especially since the maternal ER ring is used as a proxy for the paternal chromosomes in later figures would strengthen the authors' claims.

We have added a panel showing DAPI-stained DNA in the center of the ER ring and paternal mitochondria cloud. 

Why is the faster time scale imaging significant? I think this could be more clearly set up in the paper. Perhaps rapid imaging of maternal mito-labeled kca-1(RNAi) embryos would better show the difference in time scale, with the expectation that the paternal cloud forms and persists while the ER invades. 

We are not sure what the reviewer means.  5 sec time intervals were used throughout the paper.  We are also not sure how kca-1(RNAi) would help.  Movement of the entire oocyte into and out of the spermatheca is what limits the ability to keep a fusing sperm in focus.  kca-1(RNAi) would prevent cytoplasmic streaming but not ovulation movements.

Figure 4 - The question about the permeability of the ER envelope seems to come out of nowhere as written. It isn't clear how it contributes to the larger story about preventing sperm incorporation in the spindle.

This section of the results is introduced with: “If the maternal ER envelope around sperm DNA was sealed and impermeable during meiosis, this could both prevent the sperm DNA from inducing ectopic spindle assembly and prevent the sperm DNA from interacting with meiotic spindle microtubules.” 

The data in Figure 4 would probably not be expected to be in this paper based on the paper title. Maybe the title needs something about ER dynamics? "eg. ATX-2 but not an ER envelope" isolates the paternal chromatin? 

In Figure 5, it seems that RNAi of klp-7 and Mei-1 had slightly different effects on short-axis displacement of the ER envelope (klp-7 affecting it more dramatically than mei-1) and slightly different effects on interaction with the meiotic spindle (capture vs streaming past the spindle). The authors mention in their discussion that the difference in the interaction with the meiotic spindle might reflect the effects that loss of Mei-1 may have on the spindle but could it also be a consequence of the differences in cytoplasmic streaming observed?

With our current data, the only statistically significant difference between cytoplasmic streaming of the sperm contents in mei-1(RNAi) vs klp-7(RNAi) is that excessive streaming persists longer into metaphase II in klp-7(RNAi).  We have added a sentence describing this difference to the results.  If differences in streaming were the cause of different capture frequencies, then klp-7(RNAi) would cause more capture events than mei-1(RNAi) but the opposite was observed.  We have avoided too much discussion here because the frequency of capture events is too low to demonstrate statistically significant differences between mei-1(RNAi), klp-7(RNAi), and atx-2(degron) + klp-7(RNAi) without a very large increase in the number of time-lapse sequences.  

Also, the authors should find a way to represent this interaction with the meiotic spindle in a quantitative or table form to allow the reader to observe some of the patterns they report more easily.

We have added a table to Fig. 9 that summarizes capture data.

Finally, can the authors report when they observe the closest association with the meiotic spindle: Does it correlate with the period of greatest displacement (AI) or are they unlinked? 

The low frequency of capture events makes it difficult to test this rigorously.

Figure 6- 'Endogenously tagged ATX-2 was observed throughout oocytes and meiotic embryos without partial co-localization with ER.' How can the authors exclude co-localization with ER? 

We have changed the wording to: “Endogenously tagged ATX-2 was observed throughout oocytes and meiotic embryos (Fig. 6A; Fig. S2).  ATX-2 did not uniquely  co-localize with ER (Fig. S2).“

The rationale for why the authors think that the integrity of sperm organelles is important to keep the genomes apart is not clear to this reviewer and needs to be explained better. Moving the discussion of the displacement experiments in Figure S3 from the end of the results section to the ATX-2 knockdown section would help accomplish this. 

We have added the sentence: “The frequency of sperm capture by the meiotic spindle (Fig. 9D) was significantly higher than wild-type controls in klp-7(RNAi) atx-2(AID) double depleted embryos (p=0.011 Fisher’s exact test).   Although the number of single mutant embryos analyzed was too low to demonstrate a significant difference between single and double mutant embryos,  these results qualitatively support the hypothesis that limiting cytoplasmic streaming and maintaining the integrity of the ball of paternal mitochondria are both important for preventing capture events between the meiotic spindle and sperm DNA.”

It looks like, in the double knockdown of ATX-2 and KLP-7, the spread of paternal mitochondria is less affected than when only ATX-2 is depleted. What effect does this result have on the observation that the incidence of sperm capture appears to increase in the double depletion? What does displacement of the ER ring look like in the double depletion? Is it additive, consistent with their interpretation that both limiting cytoplasmic streaming and maintaining the integrity of the ball of paternal mitochondria is required to keep the genomes separate? 

We cannot show a significant difference between single a double knockdowns without increasing n by alot.  We did not analyze ER ring displacement in the double mutant.

Is the increased incidence of capture in the double-depleted embryos significant? 

We have added the sentence: “The frequency of sperm capture by the meiotic spindle (Fig. 9D) was significantly higher than wild-type controls in klp-7(RNAi) atx-2(AID) double depleted embryos (p=0.011 Fisher’s exact test).   Although the number of single mutant embryos analyzed was too low to demonstrate a significant difference between single and double mutant embryos,  these results qualitatively support the hypothesis that limiting cytoplasmic streaming and maintaining the integrity of the ball of paternal mitochondria are both important for preventing capture events between the meiotic spindle and sperm DNA.”

What do the authors make of the cell cycle arrest observed when paternal chromosomes are captured? Is there an argument to be made that this arrest supports the idea that preventing this capture is actively regulated and therefore functionally important? 

We chose not to discuss the mechanism of this arrest because considerably more work would be required to prove that it is not caused by a combination of imaging conditions and genotype.  The low frequency of these capture + arrest events would make it very difficult to show that the arrest does not occur after depleting a checkpoint protein.

(2) Minor concerns: 

Top of page 4: "streaming because depletion tubulin stops cytoplasmic streaming (7)" should be "streaming because depletion of tubulin stops cytoplasmic streaming (7)" 

The ”of” has been inserted.

Page 6: "This result indicated that the volume of paternal mitochondria excludes maternal mitochondria and yolk granules but not maternal ER." The authors have only shown this for maternal mitochondria, not yolk granules. 

We have deleted the mention of yolk granules here.

Page 7: "These results suggest that all maternal membranes are initially excluded from the sperm at fusion." Should be "These results show that maternal ER are initially excluded from the sperm at fusion. Since maternal mitochondria and yolk granules are excluded later, this suggests that all maternal membranes are initially excluded from the sperm at fusion." 

We have changed this sentence as suggested.

It's not clear why the authors show other types of movement that might be quantified when cytoplasmic streaming is affected in Figure 5A and only quantify long-axis and short-axis displacement. 

We have deleted the other types of movement from the schematic.  Although these parameters were quantified, we did not include this data in the results so it would be confusing for the reader to have them in the schematic.

Bottom of page 7: Mention that the GFP::BAF-1 was maternally provided. 

We have added “Maternally provided..”

Missing an Arrow on Figure 1A 9:20. 

We removed the text citation to an arrow in Fig. 1A because we moved most of the description of the ER ring to Fig. 3 to address other reviewer suggestions.

Supplemental videos should be labeled appropriately to indicate what structures are labeled. It is currently difficult to understand what is being shown. 

(3) Issues with the Discussion section: 

"The simplest explanation is that cytoplasm does not mix during the 45 min from GVBD to pronucleus formation due to the high viscosity of cytoplasm." - Citation page 12. 

We have changed the sentence to: “The simplest hypothesis is that maternal and paternal cytoplasm might not mix during the 45 min from GVBD to pronucleus formation due to the high viscosity of cytoplasm.” 

"The higher frequency of capture of the sperm DNA by the meiotic spindle in ATX-2 KLP-7 double depleted embryos compared with either single depletion suggests that the integrity of the exclusion zone around the sperm DNA may insulate the sperm DNA from spindle microtubule" - Pages 12-13 reference the figures. 

This sentence has been rewritten in response to other comments but the new sentence now references revised Fig. 9.

"ATX-2 is required to maintain the integrity of the ball of paternal mitochondria around the sperm DNA, but the mechanism is unknown." - Page 13 reference figure. 

A reference to Figs 7 and 8 has been inserted.

" In control embryos, the sperm contents rarely came near the meiotic spindle in agreement with a previous study that found that male and female pronuclei rarely form next to each other (6). Streaming of the sperm contents was most commonly restricted to a jostling motion with little net displacement, circular streaming in the short axis of the embryo, or long axis streaming in which the sperm turned away from the spindle before the halfway point of the embryo. Depletion of MEI-1 or KLP-7 resulted in longer excursions of the sperm contents in the long axis of the embryo toward the spindle but frequent capture of the sperm by the spindle was only observed in mei-1(RNAi)." - Page 13, the corresponding figures need to be referenced for these sentences. 

We have inserted figure references.

"In capture events observed after double depletion of ATX-2 and KLP-7, a bundle of microtubules was discernible extending from the spindle into the ER envelope surrounding the sperm DNA. Such bundles were not observed in mei-1(RNAi) capture events, likely because of the previously reported low density of microtubules in mei-1(RNAi) spindles (36, 37)." - Pages 13-14 references figures here. 

We have inserted figure references.

"The higher frequency of capture of the sperm DNA by the meiotic spindle in ATX-2 KLP-7 double depleted embryos compared with either single depletion suggests that the integrity of the exclusion zone around the sperm DNA may insulate the sperm DNA from spindle microtubules." - This should be toned down since this phenotype is not robust. 

We have changed this to: “The capture of the sperm DNA by the meiotic spindle in ATX-2 KLP-7 double depleted embryos suggests that the integrity of the exclusion zone around the sperm DNA might insulate the sperm DNA from spindle microtubules.  However, a much larger number of klp-7(RNAi) singly depleted and atx-2(degron) singly depleted time-lapse sequences are needed to rigorously support this idea. “

ATX-2 depletion alters ER morphology but does not impact the maternal ER envelope - could the authors provide a potential explanation for this? 

In the discussion, we cite papers showing that ATX-2 depletion affects many different cellular processes so the effect we see on paternal mitochondria might have nothing to do with the ER ring.   We have been attempting to disrupt ER structures in the meiotic embryo for the last 5 years by depleting profilin, BiP, atlastin, ATX-2 and by optogenetically packing ER into a ball in the middle of the oocyte.  None of these treatments prevent envelopment of the sperm DNA by maternal ER.  None of these treatments remove ER from the spindle envelope and none remove ER from the plasma membrane.  These treatments mostly result in “large aggregates” of ER that we have not examined by EM.  Wild speculation: any disruption of the ER strong enough to prevent ER envelopment around chromatin would be sterile because the M to S transition in the mitotic zone of the germline would be blocked.  Rapid depletion of ATX-2 to the extent shown by rigorous data in this manuscript does not prevent ER envelopment around chromatin.  We chose not to speculate about the reasons for this because we do not know why.

It would be good to have representative images of what the altered spindle looks like in MEI-1-depleted oocytes. 

The structure of MEI-1-depleted spindles has been described in the cited references.

"Depletion of MEI-1 or KLP-7 resulted in longer excursions of the sperm contents in the long axis of the embryo toward the spindle but frequent capture of the sperm by the spindle was only observed in mei-1(RNAi)" - It is intriguing that this does not happen in the double depletion experiments of kinesin-13 and ATX-2. The authors should perhaps discuss this. 

This does happen in KLP-7 ATX-2 double depleted embryos as shown in Fig. 9.

(4) Missing citations: 

"This analysis was restricted to embryos from anaphase I through anaphase II because our streaming data and that of Kimura 2020 indicate that the sperm contents have not moved significantly before anaphase I." - This needs an appropriate citation. Page 10. 

We have inserted citations here.

" The simplest explanation is that cytoplasm does not mix during the 45 min from GVBD to pronucleus formation due to the high viscosity of cytoplasm." - Citation page 12. Not referencing figures in the discussion. 

We have changed the sentence to: “The simplest hypothesis is that maternal and paternal cytoplasm might not mix during the 45 min from GVBD to pronucleus formation due to the high viscosity of cytoplasm.” 

"The higher frequency of capture of the sperm DNA by the meiotic spindle in ATX-2 KLP-7 double depleted embryos compared with either single depletion suggests that the integrity of the exclusion zone around the sperm DNA may insulate the sperm DNA from spindle microtubule" - Pages 12-13 reference the figures. 

A reference to the revised Fig. 9 has been inserted in the revised version of this sentence.

"ATX-2 is required to maintain the integrity of the ball of paternal mitochondria around the sperm DNA, but the mechanism is unknown." 

References to Figs. 7 and 8 have been inserted.

Page 13 reference figure 

" In control embryos, the sperm contents rarely came near the meiotic spindle in agreement with a previous study that found that male and female pronuclei rarely form next to each other (6). Streaming of the sperm contents was most commonly restricted to a jostling motion with little net displacement, circular streaming in the short axis of the embryo, or long axis streaming in which the sperm turned away from the spindle before the halfway point of the embryo. Depletion of MEI-1 or KLP-7 resulted in longer excursions of the sperm contents in the long axis of the embryo toward the spindle but frequent capture of the sperm by the spindle was only observed in mei-1(RNAi)." Page 13, the corresponding figures need to be referenced for these sentences. 

We have inserted citations here.

"In capture events observed after double depletion of ATX-2 and KLP-7, a bundle of microtubules was discernible extending from the spindle into the ER envelope surrounding the sperm DNA. Such bundles were not observed in mei-1(RNAi) capture events, likely because of the previously reported low density of microtubules in mei-1(RNAi) spindles (36, 37)." Pages 13-14 references figures here. 

We have inserted citations here.

(5) Referencing wrong figures in the text: 

Figure 5 - In the figure legend there is a 5C but there is no 5C panel in the figure. 

A C has been inserted in Fig. 5.

Figure 6A - "Dark holes were observed suggesting exclusion from the lumens of larger membranous organelles (Fig. 6A; Fig. S2)." Page 10. 

6A has been changed to 6C.

Figure 6A is showing background autofluorescence in WT oocytes so I am not certain why it is cited here. 

The Figure citation has been corrected to 6B, C.

Figure 8 - I could not find the supplemental data file with the individual mitochondria distance measurements. 

We are including the Excel file with the revised submission.

The last sentence of the first paragraph should be re-worded to be more concise ". In C. elegans, the nucleus is positioned away from the site of future fertilization so that the meiosis I spindle assembles at the opposite end of the ellipsoid zygote from the site of fertilization (2-4). " 

Every word of this sentence is important.

Last sentence second paragraph typo "These microtubules are thought to drive meiotic cytoplasmic streaming because depletion tubulin stops cytoplasmic streaming (7) and depletion of the microtubule-severing protein katanin by RNAi results in an increased mass of cortical microtubules and an increase in cytoplasmic streaming (8)." Pages 3-4. 

“of” has been inserted.

(6) Typos in the introduction should be corrected: 

Ataxin or kinesin-13 are not mentioned in the introduction but these are a big focus of the paper. 

Gong et al 2024 written instead of number citation (page 5), no citation in References.

This has been corrected. 

Supplemental videos should be labeled appropriately to indicate what structures are labeled. It is currently difficult to understand what is being shown.

pas fou

Reviewer #2 (Public Review):

Summary:

Hiramatsu et al. investigated how cognate neurotransmitter receptors with antagonizing downstream effects localize within neurons when co-expressed. They focus on mapping the localization of the dopaminergic Dop1R1 and Dop2R receptors, which correspond to the mammalian D1- and D2-like dopamine receptors, which have opposing effects on intracellular cAMP levels, in neurons of the Drosophila mushroom body (MB). To visualize specific receptors in single neuron types within the crowded MB neuropil, the authors use existing dopamine receptor alleles tagged with 7 copies of split GFP to target reconstitution of GFP tags only in the neurons of interest as a read-out of receptor localization. The authors show that both Dop1R1 and Dop2R, with differing degrees, are enriched in axonal compartments of both the Kenyon Cells cholinergic presynaptic inputs and in different dopamine neurons (DANs), which project axons to the MB. Co-localization studies of dopamine receptors with the presynaptic marker Brp suggest that Dop1R1 and, to a larger extent Dop2R, localize in the proximity of release sites. This localization pattern in DANs suggests that Dop1R1 and Dop2R work in dual-feedback regulation as autoreceptors. Finally, they provide evidence that the balance of Dop1R1 and Dop2R in the axons of two different DAN populations is differentially modulated by starvation and that this regulation plays a role in regulating appetitive behaviors.

Strengths:

The authors use reconstitution of GFP fluorescence of split GFP tags knocked into the endogenous locus at the C-terminus of the dopamine receptors as a readout of dopamine receptor localization. This elegant approach preserves the endogenous transcriptional and post-transcriptional regulation of the receptor, which is essential for studies of protein localization.

The study focuses on mapping the localization of dopamine receptors in neurons of the mushroom body. This is an excellent choice of system to address the question posed in this study, as the neurons are well-studied, and their connections are carefully reconstructed in the mushroom body connectome. Furthermore, the role of this circuit in different behaviors and associative memory permits the linking of patterns of receptor localization to circuit function and resulting behavior. Because of these features, the authors can provide evidence that two antagonizing dopamine receptors can act as autoreceptors within the axonal compartment of MB innervating DANs. The differential regulation of the balance of the two receptors under starvation in two distinct DAN innervations provides evidence of the role that regulation of this balance can play in circuit function and behavioral output.

Weaknesses:

The approach of using endogenously tagged alleles to study localization is a strength of this study, but the authors do not provide sufficient evidence that the insertion of 7 copies of split GFP to the C terminus of the dopamine receptors does not interfere with the endogenous localization pattern or function. Both sets of tagged alleles (1X Venus and 7X split GFP tagged) were previously reported (Kondo et al., 2020), but only the 1X Venus tagged alleles were further functionally validated in assays of olfactory appetitive memory. Despite the smaller size of the 7X split-GFP array tag knocked into the same location as the 1X venus tag, the reconstitution of 7 copies of GFP at the C terminus of the dopamine receptor, might substantially increase the molecular bulk at this site, potentially impeding the function of the receptor more significantly than the smaller, single Venus tag. The data presented by Kondo et al. 2020, is insufficient to conclude that the two alleles are equivalent.

The authors' conclusion that the receptors localize to presynaptic sites is weak. The analysis of the colocalization of the active zone marker Brp whole-brain staining with dopamine receptors labeled in specific neurons is insufficient to conclude that the receptors are localized at presynaptic sites. Given the highly crowded neuropil environment, the data cannot differentiate between the receptor localization postsynaptic to a dopamine release site or at a presynaptic site within the same neuron. The known distribution of presynaptic sites within the neurons analyzed in the study provides evidence that the receptors are enriched in axonal compartments, but co-labeling of presynaptic sites and receptors in the same neuron or super-resolution methods are needed to provide evidence of receptor localization at active zones. The data presented in Figures 5K-5L provides compelling evidence that the receptors localize to neuronal varicosities in DANs where the receptors could play a role as autoreceptors.

Given the highly crowded environment of the mushroom body neuropil, the analysis of dopamine receptor localization in Kenyon cells is not conclusive. The data is sufficient to conclude that the receptors are preferentially localizing to the axonal compartment of Kenyon cells, but co-localization with brain-wide Brp active zone immunostaining is not sufficient to determine if the receptor localizes juxtaposed to dopaminergic release sites, in proximity of release sites in Kenyon cells, or both.

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This study defines a fundamental aspect of protein kinase signalling in the protist parasite Toxoplasma gondii that is required for acute and chronic infections. The authors provide compelling evidence for the role of SPARK/SPARKEL kinases in regulating cAMP/cGMP signalling, although evidence linking the loss of these kinases to changes in the phosphoproteome is incomplete. Overall, this study will be of great interest to those who study Toxoplasma and related apicomplexan parasites.

We thank the reviewers for their thoughtful and positive evaluation of our work. Below, we have addressed all of the public reviews and recommendations for the authors in point-by-point responses. Additionally, we include with this resubmission RT-qPCR data where we observe no significant change in transcript levels for the relevant AGC kinases, supporting the hypothesis that SPARK/SPARKEL–regulation is post-translational.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Herneisen et al characterise the Toxoplasma PDK1 orthologue SPARK and an associated protein SPARKEL in controlling important fate decisions in Toxoplasma. Over recent years this group and others have characterised the role of cAMP and cGMP signalling in negatively and positively regulating egress, motility, and invasion, respectively. This manuscript furthers this work by showing that SPARK and SPARKEL likely act upstream, or at least control the levels of the cAMP and cGMP-dependent kinases PKA and PKG, respectively, thus controlling the transition of intracellular replicating parasites into extracellular motile forms (and back again).

The authors use quantitative (phospho)proteomic techniques to elegantly demonstrate the upstream role of SPARK in controlling cAMP and cGMP pathways. They use sophisticated analysis techniques (at least for parasitology) to show the functional association between cGMP and cAMP signalling pathways. They therefore begin to unify our understanding of the complicated signalling pathways used by Toxoplasma to control key regulatory processes that control the activation and suppression of motility. The authors then use molecular and cellular assays on a range of generated transgenic lines to back up their observations made by quantitative proteomics that are clear in their design and approach.

The authors then extend their work by showing that SPARK/SPARKEL also control PKAc3 function. PKAc3 has previously been shown to negatively regulate differentiation into bradyzoite forms and this work backs up and extends this finding to show that SPARK also controls this. The authors conclude that SPARK could act as a central node of regulation of the asexual stage, keeping parasites in their lytic cell growth and preventing differentiation. Whether this is true is beyond the scope of this paper and will have to be determined at a later date.

Strengths:

This is an exceptional body of work. It is elegantly performed, with state-of-the-art proteomic methodologies carefully being applied to Toxoplasma. Observations from the proteomic datasets are masterfully backed up with validation using quantitative molecular and cellular biology assays.

The paper is carefully and concisely written and is not overreaching in its conclusions. This work and its analysis set a new benchmark for the use of proteomics and molecular genetics in apicomplexan parasites.

Weaknesses:

This reviewer did not identify any weaknesses.

Reviewer #2 (Public Review):

Summary:

The manuscript by Herneisen et al. examines the Toxoplasma SPARK kinase orthologous to mammalian PDK1 kinase. The extracellular signals trigger cascades of the second messengers and play a central role in the apicomplexan parasites' survival. In Toxoplasma, these cascades regulate active replication of the tachyzoites, which manifests as acute toxoplasmosis, or the development into drug-resilient bradyzoites characteristic of the chronic stage of the disease. This study focuses on the poorly understood signaling mechanisms acting upstream of such second messenger kinases as PKA and PKG. The authors showed that similar to PDK1, Toxoplasma SPARK appears to regulate several AGC kinases.

Strengths:

The study demonstrated a strong association of the SPARK kinase with an elongin-like SPARKEL factor and an uncharacterized AGC kinase. Using a set of standard assays, the authors determined the SPARK/SPARKEL role in parasite egress and invasion. Finally, the study presented evidence of the SPARK/SPARKEL involvement in the bradyzoite differentiation.

Weaknesses:

Although the study can potentially uncover essential sensing mechanisms operating in Toxoplasma, the evidence of the SPARK/SPARKEL mechanisms is weak. Specifically, due to incomplete data analysis, the SPARK/SPARKEL-dependent phosphoregulation of AGC kinases cannot be evaluated. The manuscript requires better organization and lacks guidance on the described experiments. Although the study is built on advanced genetics, at times, it is unnecessarily complicated, raising doubts rather than benefiting the study.

The evidence for the SPARK/SPARKEL interaction is demonstrated through diverse experimental approaches that are internally consistent. Five separate mass spectrometry experiments, with replicates and appropriate controls, with tags on either SPARK or SPARKEL, showed that SPARK and SPARKEL form a strong interaction (Figure 1A, 1D, 1E; Figure 1—figure supplement 1). Global mass spectrometry experiments assessing the impact of  SPARK or SPARKEL depletion showed similar features (a reduction in PKG and PKA abundance and up-regulation of bradyzoite-associated proteins; Figure 3C–D). The phenotypes associated with SPARK and SPARKEL depletion phenocopy one another in all cell biological assays we tested (Figure 2A, 2D and PMID: 35484233; Figure 2E–J; Figure 4E–F; Figure 6A–B). Measuring the abundance of SPARK and SPARKEL in unenriched samples was challenging, but immunoblotting and proteomics suggest that depletion of one factor leads to down-regulation of the other (Figure 2B, 2C; Figure 3—figure supplement 1), which explains the genetic and cell biological phenocopying described above. We note that “further biochemical studies are required to discern the regulatory interactions between SPARK and SPARKEL” (first submission lines 590-591) and are beyond the scope of this work.

The evidence for SPARK/SPARKEL regulation of AGC kinase activity is demonstrated through diverse experimental approaches that are also internally consistent. PKA C1 and PKG abundance levels decrease in parasites depleted of SPARK/SPARKEL, as measured by mass spectrometry (Figure 3A and 3C) and cell-based assays for PKA C1/R (Figure 4D–F). Comparisons of the global SPARK-, PKA R-, PKG-, and PKA C3-depleted phosphoproteomes suggest that PKA and PKG activity is reduced upon SPARK depletion whereas the activity of an unrelated factor (PP1) is unaffected (Figure 4G–H, Figure 4—figure supplement 1, Figure 5D–E, Figure 7I–J). Parasites depleted of SPARK are hypersensitized to a PKG inhibitor (Figure 5B–C). SPARK, PKA, and PKG are proximal in cellulo (Figure 3I) and SPARK co-purifies with PKA C3 (Figure 7A). The kinetic-phase phenotypes associated with SPARK and SPARKEL depletion (PMID: 32379047, Figure 2A, 2D–2J) are consistent with reduced PKG activity (PMID: 28465425) and only develop after PKG has been depleted as shown by proteomics experiments (Figure 2E-J and Figure 3C). Other studies have shown that the effects of reduced PKG activity are dominant to reduced PKA C1 activity (PMID: 29030485). The replicative-phase phenotypes associated with SPARK and SPARKEL depletion are consistent with reduced PKA C3 activity (PMID: 27247232 and herein). Mechanistically, PKG and PKA C1 activity must be lower in SPARK-depleted parasites because the abundances of these kinases are lower (Figure 3A, 3C). The mechanism of regulation may be more complex in the case of PKA C3, as SPARK depletion did not cause a reduction in PKA C3 abundance as measured by cellular assays (Figure 7B–F), but PKA C3 activity decreased (Figure 7I–K). We concede that multiple mechanisms may lead to the reduction in PKA C1 and PKG abundances, such as decreased activation loop phosphorylation and autophosphorylation at other stabilizing sites or enhanced ubiquitin ligase activity leading to active degradation of the kinases; we have moved speculation regarding such mechanisms to the Discussion.

Although the reviewer commented that the manuscript “requires better organization” in the public review, no specific recommendations were provided to the authors. Therefore, we did not change the organization of the manuscript. We added an additional paragraph to the Discussion to reiterate key findings: “A prior study identified SPARK as a regulator of parasite invasion and egress following 24 hours of kinase depletion (Smith et al., 2022). Unexpectedly, we observed that three hours of SPARK or SPARKEL depletion were insufficient to impact T. gondii motility or calcium-dependent signaling, indicating that the phenotypes associated with SPARK and SPARKEL depletion develop over time. Quantitative proteomics revealed that PKA and PKG abundances began to decrease after more than three hours of SPARK depletion. Proximity labeling experiments also suggested that SPARK, PKA, and PKG are spatially associated within the parasite cell. We propose a model in which SPARK down-regulation coincides with reduced PKG and PKA activity due to diminished protein levels.” This work built upon genetic and proteomic approaches recently described by our group, which we cited in the text and extensive methods section. We added additional experimental detail where noted in the reviewer’s recommendations to the authors.

The study utilizes advanced genetics because biochemical tools for eukaryotic parasites are limited. For example, no antibodies for T. gondii SPARK, PKA subunits, or PKG exist; to say nothing of phosphosite-specific antibodies, which are common in the mammalian cell signaling field. Therefore, to measure the relationship between SPARK, SPARKEL, and PKA subunits, we had to generate strains in which multiple proteins were tagged with epitopes for downstream analysis. The genetic experiments included appropriate controls and were internally consistent with results obtained using orthogonal approaches, such as mass spectrometry.

Reviewer #3 (Public Review):

Summary:

This paper focuses on the roles of a toxoplasma protein (SPARKEL) with homology to an elongin C and the kinase SPARK that it interacts with. They demonstrate that the two proteins regulate the abundance of PKA and PKG, and that depletion of SPARKEL reduces invasion and egress (previously shown with SPARK), and that their loss also triggers spontaneous bradyzoite differentiation. The data are overall very convincing and will be of high interest to those who study Toxoplasma and related apicomplexan parasites.

Strengths:

The study is very well executed with appropriate controls. The manuscript is also very well and clearly written. Overall, the work clearly demonstrates that SPARK/SPARKEL regulate invasion and egress and that their loss triggers differentiation.

Weaknesses:

(1) The authors fail to discriminate between SPARK/SPARKEL acting as negative regulators of differentiation as a result of an active role in regulating stage-specific transcription/translation or as a consequence of a stress response activated when either is depleted

We demonstrate a novel function for SPARK and SPARKEL as negative regulators of differentiation. The pathways leading to differentiation are being actively studied. Up-regulation of a positive transcriptional regulator of chronic differentiation, BFD1, is sufficient to trigger differentiation in vitro in the absence of other stressful growth conditions (PMID: 31955846). SPARK or SPARKEL depletion results in up-regulation of proteins that are up-regulated upon BFD1 overexpression. Whether BFD1 overexpression or SPARK and SPARKEL depletion triggers cellular stress pathways is beyond the scope of the current work, which focused instead on the immediate effect of these pathways on AGC kinases. Study of the effect of the various kinases on the parasite phosphoproteome shows that the putative targets of PKA C3 are specifically downregulated upon SPARK knockdown, indicating PKA C3 activity is indeed decreased in the latter condition.

(2) The function of SPARKEL has not been addressed. In mammalian cells, Elongin C is part of an E3 ubiquitin ligase complex that regulates transcription and other processes. From what I can tell from the proteomic data, homologs of the Elongin B/C complex were not identified. This is an important issue as the authors find that PKG and PKA protein levels are reduced in the knockdown strains

Our experiments suggest that SPARK and SPARKEL form a complex, and down-regulation of one complex member leads to down-regulation of the other. Thus in all tested assays, knockdown of SPARK and SPARKEL phenocopy one another. Further biochemical and structural work will be required to determine the mechanism by which SPARKEL regulates SPARK.

Nearly all studies of the function of elongin C have been conducted in mammalian cells. Proteins with elongin C domains may serve alternative and unexplored functions in unicellular eukaryotes. We searched for the presence of Elongin A/B and known Elongin C complex members in the T. gondii genome and were unable to identify orthologs, explaining why these proteins were not identified in mass spectrometry experiments. Please see our response in Recommendations for the Authors, Reviewer 3 point 2.

Beyond the concerns raised by the review team, we have identified and corrected the following errors or omissions in the first submission of the manuscript:

- Line 176 of the first submission referred to a “peptide sequence match (PSM)”, which we have changed to “peptide-spectrum match”.

- We recolored and relabeled the lines in Figure 5A so that it is easier to match a specific peptide with a specific line; and also corrected a mislabeling.

- Figure 7B SPARK panel was incorrectly centered. The raw files can be viewed in Figure 7—source data 2.

- Figure 7—figure supplement 1D was missing an x-axis label.

- Line 1172 referred to “Supplementary File X”, which we corrected to “Supplementary File 3”.

- We have updated references to preprints that have since been published, including PMID: 38093015, 37933960, 37966241, and 37610220.

Editors comments:

The proteomics data reported in this study underpin the major findings and are very comprehensive. As noted in the reviews, it is strongly recommended that the authors normalize the levels of detected phosphopeptides against the levels of the parent protein in the different mutant lines in order to identify changes in protein phosphorylation that are linked to protein kinase activity rather than protein degradation. A focus on changes that occur at early time points following protein knock-down may also help to identify the main targets of each kinase.

Please see our response to Reviewer 2 Recommendations for the Authors, points 1 and 2.

Reviewer #1 (Recommendations For The Authors):

During my reading, I only found one small mistake. In Figure 7F, the x-axis is missing the word 'PKA'.

We have updated the x-axis to read “SPARK-AID/PKA C3-mNG (h. + IAA)”.

All information, code, and reagents are clearly explained.

Reviewer #2 (Recommendations For The Authors):

How the phosphoproteome was analyzed needs to be clarified. The normalization step, computing the ratio of the phosphopeptide to the protein (peptide) intensity, appears omitted. It is the most critical step of the analysis. The minor shifts between protein and phosphosite intensity seem negligible, as seen in Figure 4 AB. The significant changes can only be deduced by calculating this ratio. In the current state, the presented results are inconclusive. The manuscript contains overreaching and often unsupported statements because the data has not been appropriately filtered. Related to this topic, it is advisable to use well-accepted terminology and complete words when describing proteome and phosphoproteome. The interexchange of a "peptide" and a "phosphopeptide" in the text confuses and misleads.

To clarify the phosphoproteome analysis:

We cite a previous description of the phosphoproteomics sample preparation workflow (lines 1124-1125 of the first submission for example). Our quantitative phosphoproteomics experiments comprise two datasets generated from the same multiplexed samples. The samples were split at the point of phosphopeptide enrichment. Ninety-five percent of the samples were subjected to phosphopeptide enrichment (titanium dioxide followed by nickel affinity chromatography; “enriched samples”). Five percent of the samples were reserved as a reference for the non-enriched proteome (“non-enriched samples”). To clarify this point, we have added the sentences “Approximately 95% of the proteomics sample was used for phosphopeptide enrichment” and “The remaining 5% of the sample was not subjected to the phosphopeptide enrichment protocol” to the Methods sections, after describing the multiplexing steps.

The samples were fractionated separately and run separately on an LC-MS system, which is described in the Methods section, for example lines 1130-1149 of the first submission. Raw files of the phosphopeptide-enriched and unenriched samples were analyzed separately, which is described in the Methods section, for example lines 1151-1158 of the first submission. To clarify this point, we have added the sentence “Raw files of the phosphopeptide-enriched and unenriched samples were analyzed separately” to the Methods sections. Many of the search parameters and descriptions of normalization and protein abundances were described in lines 1085-1093 of the first submission in reference to the 24h SPARK depletion proteome. We added this information to the description of the SPARK depletion time course phosphoproteome data analysis: “The allowed mass tolerance for precursor and fragment ions was 10 ppm and 0.02 Da, respectively. False discovery was assessed using Percolator with a concatenated target/decoy strategy using a strict FDR of 0.01, relaxed FDR of 0.05, and maximum Delta CN of 0.05. Only unique peptide quantification values were used. Co-isolation and signal-to-noise thresholds were set to 50% and 10, respectively. Normalization was performed according to total peptide amount. In the case of the unenriched samples, protein abundances were calculated from summation of non-phosphopeptide abundances.”

We hope that this clarifies how the unenriched sample protein-level abundances were calculated. When we discuss “protein abundance”, we are referencing the unenriched sample summed non-phosphopeptide abundance. Our phosphoproteome analysis was based only on phosphopeptides, as our phosphopeptide enrichment resulted in 99% efficiency, and peptides lacking phosphorylation sites were filtered out before subsequent analyses. We used “peptide” and “phosphopeptide” interchangeably because the only peptide-level analysis performed was based on phosphopeptide abundances. We have changed any mention of “peptide” to “phosphopeptide” in the main text. 

“The normalization step, computing the ratio of the phosphopeptide to the protein (peptide) intensity, appears omitted. It is the most critical step of the analysis.”:

Unlike common differential gene expression analysis pipelines, proteomics analysis pipelines are not settled. Many analyses do not perform peptide-to-parent-protein corrections; some normalize phosphopeptide abundances to parent protein abundances calculated from summing non-phosphopeptides or a combination of phosphopeptide and non-phosphopeptides on an ad hoc basis; some calculate global normalization factors based on regressions of protein and phosphopeptide abundances or other pairwise comparisons. A caveat of protein normalization of phosphopeptides is that it over-corrects cases in which protein abundance and phosphorylation are interdependent, as is the case for auto-phosphorylation and some activation loop phosphorylations (PMID: 37394063). We used the approach that retained the greatest complexity of the data, which is to not normalize abundances across different mass spectrometry experiments and discard information that was not in the overlap. We have updated Supplementary File 3.3 to include protein-level quantification values (from Supplementary File 3.2) if measured.

We clarified that the phosphopeptide abundances and protein-level abundances were derived from different datasets that were each internally normalized (globally centered by total peptide amount). Protein-level abundances were summed from non-phosphopeptide abundances. The calculated log2 changes are based on the globally centered data within each dataset. We analyzed the kinetic profiles of changing phosphopeptide abundances relative to a control using approaches similar to those described for several recent temporally resolved T. gondii phosphoproteomes (e.g. PMID: 37933960, 35976251, 36265000, 29141230) and as described in the Methods. The approach does not first correct for unenriched-sample parent protein abundance—in some applications, unenriched samples are not collected at all; instead, phosphopeptide ratios are median-normalized to non-phosphopeptide ratios (quantified due to inefficient phosphopeptide enrichment) and are individually tested against the null distribution of non-phosphopeptide ratios (e.g. PMID: 36265000, 29141230). We did not use this approach because our phosphopeptide enrichment was 99% efficient (18518 phosphopeptides of 18758 peptides with quantification values). In several cases using our approach, parent protein abundance is not quantified in the unenriched proteome dataset, but phosphopeptides are reliably quantified in the enriched proteome dataset. We note that phosphopeptide abundance changes can be difficult to interpret in such cases, e.g. in the first submission lines 178-186 and 193-194. We have added similar text to the results noting that in the case of PKA and PKG, both unenriched parent protein and enriched phosphopeptide abundances decreased (see below). We have also moved speculation about whether SPARK phosphorylates the activation loop of PKA and PKG, or whether the down-regulation of PKA and PKG arises from indirect effects, to the Discussion.

We have moved comparisons of protein and phosphopeptide abundances from the Results to the Discussion. We added the following sentences to the result section Clustering of phosphopeptide kinetics identifies seven response signatures: “Because non-phosphopeptide and phosphopeptide abundances were quantified in different mass spectrometry experiments, it is challenging to compare the rates of phosphopeptide and parent protein abundance changes, especially when phosphorylation status and protein stability are interconnected. In general, both PKA C1, PKA R, and PKG protein and phosphosite abundances decreased following SPARK depletion (Figure 3—figure supplement 1), as discussed further below. We also observed down-regulation of phosphosite and protein abundances of a MIF4G domain protein.” Figure 3—figure supplement 1E is a new panel that shows PKA C1, PKA R, and PKG phosphopeptide and parent protein abundances along with global changes in phosphopeptide and parent protein abundances in the cases which both were quantified. We changed lines 278-282 in the first submission to “The SPARK depletion time course phosphoproteome showed a reduction in the abundance of PKA C1 T190 and T341, which are located in the activation loop and C-terminal tail, respectively (Figure 4A). Several phosphosites residing in the N terminus of PKA R (e.g. S17, S27, and S94) also decreased following SPARK depletion (Figure 4B).” We changed lines 313-315 in the first submission to “The SPARK depletion time course phosphoproteome showed a reduction in the abundance of several phosphosites residing in the N terminus of PKG as well as T838, which corresponds to the activation loop (Figure 5A). By contrast, S105 did not greatly decrease, and S40 abundance slightly increased.”

The description of experiments should be more detailed. For example, the 3, 8, and 24 h treatments were used reversely; thus, they should be emphasized as time points before natural egress. Consequently, it seems that 3h treatment should be prioritized, given the SPARK/SPARKEL role in egress/invasion. Unexpectedly, the study draws more attention to a 24-hour treatment. If the AID-SPARK/SPARKEL is eliminated within 1h, parasites undoubtedly accumulate numerous secondary defects during a prolonged 23h deprivation. Since the SPARK pathway activates kinase/phosphatase cascades, the 24h data is likely overwhelmed with the consequences of the long-term complex degradation, making it a poor source of the putative SPARK substrates. Likewise, the downregulation of PKA observed in the 8 hours after SPARK depletion may be an indirect effect of the SPARK degradation. The direct effects and immediate substrates should be detectable within 2-3h of auxin treatment of the nearly egressing cultures.

The first submission described how parasites were harvested at 32 hours post-infection with 0, 3, 8, or 24 hours of IAA treatment (lines 157-160, 1097-1110, and Figure 3B). To reiterate this experimental detail, we have added “harvested 32 hours post-infection” to the sentence “...quantitative proteomics with tandem mass tag multiplexing that included samples with 0, 3, 8, and 24 hours of SPARK or SPARKEL depletion” and similarly in the figure legend. The time points are unrelated to natural egress because the experiment was terminated at 32 hours post-infection, which is earlier than the window typically used to study natural egress under these conditions (40-48 hours post-infection). We chose to terminate the experiment before natural egress to better localize phosphopeptide changes related to SPARK depletion. The phosphoproteome undergoes dramatic reorganization during egress due to the activity of myriad kinases and phosphatases (see PMID: 35976251, 37933960, and 36265000), which would have likely complicated the signal.

A pivotal result motivating time-course experiments and analysis was that SPARK/SPARKEL's role in egress and invasion emerges only after an extended depletion period (Figure 2E–J, first submission lines 126-145). The 24h depletion was used in the experimental system that first identified SPARK as a regulator of egress, which motivated our initial experiments, as stated in the first submission lines 126-144 and 149-151. We draw attention to the observation that SPARK and SPARKEL phenotypes develop over time in the first submission, lines 137-145. The role for SPARK/SPARKEL in egress/invasion does not manifest at 3h depletion; it manifests at 24h depletion. To ensure that this point is not overlooked by the reader, we have created a new heading in the Results section (SPARK and SPARKEL depletion phenotypes develop over time) for the paragraph that was previously lines 137-145. The remainder of the manuscript integrates data from proteomic, genetic, and cell-based assays across temporal dimensions to build a working model of how the phenotypes associated with SPARK depletion develop over time.

Underpinning this comment is an assumption that phosphopeptides that decrease the most rapidly following a kinase’s depletion are direct substrates, whereas phosphopeptides that decrease with slower kinetics are not. This is not always the case. Consider a kinase that phosphorylates sites on substrate A and substrate B. The site on substrate A is also the target of a phosphatase, whereas the site on substrate B is recalcitrant to phosphatase activity. If the kinase were inhibited, then the site on substrate A would be actively dephosphorylated. As measured by a phosphoproteomics experiment, the abundance of the substrate A phosphopeptide would drop rapidly due to the inactivity of the kinase and activity of the phosphatase. In the text, we called such sites “constitutively regulated” or dynamic—they are actively dephosphorylated and phosphorylated within a short timeframe. The phosphosite on substrate B is comparatively static; once it is phosphorylated by the kinase, it is unaffected by subsequent inhibition of the kinase. Only newly synthesized substrate B molecules would be affected by kinase inhibition. As measured by a phosphoproteomics experiment, the abundance of the substrate B phosphopeptide would drop more gradually after kinase inhibition, as the unphosphorylated peptide is found only on newly synthesized proteins that were not previously exposed to kinase activity. An example of the scenario described for substrate A would be that of yeast Cdk1 T14/Y15, which is phosphorylated by Wee1 and dephosphorylated by Cdc25 (e.g. PMID: 7880537). An example of the scenario described for substrate B would be that of the human PKA C activation loop T197, which is phosphorylated by PDK1 and is phosphatase-resistant under physiological conditions (e.g. PMID: 22493239, 15533936).

Both substrate A and B may be “direct” and functionally relevant targets of the kinase. Categorizing substrates as “immediate” is comparatively less informative in this context (although it may be relevant when studying fast, synchronized processes with high temporal resolution, such as induced Plasmodium spp. gametocyte activation or stimulation of T. gondii secretion). Furthermore, our earlier experiments had shown that the role for SPARK/SPARKEL in motility manifests after 3h depletion and is complete by 24h depletion. By this logic, we were most interested in the candidates showing differences at these time points. We conducted proximity labeling experiments to identify the overlap of proteins that exhibited SPARK-dependent decreases in the global proteomics and were also proximal to SPARK in space (first submission Figure 3I and lines 260-275), thus revealing a prioritized list of candidates, which included PKG and PKA. When technically feasible, we included a temporal dimension to follow-up experiments, rather than relying on a 24h terminal comparison (e.g. Figure 4E–H, Figure 5D–E, Figure 7D–F, Figure 7I–K; all first submission).

Fig2 (B and C). What antibodies had been used to detect tagged proteins? There is a concern regarding the use of multiple tags attached to the same protein to the point that it doubles the size of the studied protein. The switch of the mobility of the SPARK and SPARKEL on the WB due to a change in MW adds to the confusion. Furthermore, the study did not use all the fused epitopes (e.g., HA). At the same time, the same V5 tag was used to detect two factors in the same parasite. Although the controls are provided, it does not eliminate the possibility that the second band on the WB results from one protein degradation rather than the presence of two individual proteins. Different tags should be used to confirm the co-expression of two proteins. Panel E is missing the X-axis label.

Figure 2B was incorrectly labeled; the labels corresponding to SPARK and SPARKEL were switched. We corrected this error in the revised figures. The antibodies used were mouse monoclonal anti-V5 as described in the key resources table of the first submission. We added “V5” to Figure 2A and 2B. Regarding the effect of the tagging payload attached to the proteins, we have included in all assays a control relative to a parental strain (TIR1) without a tagging payload, and additionally included internal controls within tagged strains to calculate dependency of a phenotype on IAA treatment. The western blots in Figure 2B and 2C are from two different strains and experiments. The strains and experiments are described in the first submission main text (lines 113-124), the figure legend (lines 1847-1850), the key resources table, and the methods (lines 650-664, 872-891). A description of the SPARK-AID/SPARKEL-mNG strain was included in the key resources table but omitted in the methods. We therefore added the following section to the Methods:

“SPARKEL-V5-mNG-Ty/SPARK-V5-mAID-HA/RHΔku80Δhxgprt/TIR1

The HiT vector cutting unit gBlock for SPARKEL (P1) was cloned into the pALH193 HiT empty vector. The vector was linearized with BsaI and co-transfected with the pSS014 Cas9 expression plasmid into SPARK-V5-mAID-HA/RHΔku80Δhxgprt/TIR1 parasites. Clones were selected with 1 µM pyrimethamine and isolated via limiting dilution to generate the SPARKEL-V5-mNG-Ty/SPARK-V5-mAID-HA/RHΔku80Δhxgprt/TIR1 strain. Clones were verified by PCR amplification and sequencing of the junction between the 3′ end of SPARKEL (5’-GGGAGGCCACAACGGCGC-3’) and 5′ end of the protein tag (5’-gggggtcggtcatgttacgt-3’).”

To clarify the expected MW of each species, we have added the following text to the Methods:

“The expected molecular weight of SPARKEL-V5-HaloTag-mAID-Ty is 66 kDa, from the 42.7 kDa tagging payload and 23.3 kDa protein sequence. The expected molecular weight of SPARK-V5-mCherry-HA is 89.7 kDa, from the 31.9 kDa tagging payload and 57.8 kDa protein sequence. The expected molecular weight of SPARK-V5-mAID-HA is 71.3 kDa, from the 13.5 kDa tagging payload and 57.8 kDa protein sequence. The expected molecular weight of SPARKEL-V5-mNG-Ty is 55.2 kDa, from the 31.9 kDa tagging payload and 23.3 kDa protein sequence.”

SPARK and SPARKEL are lowly expressed, which may have been compounded by basal degradation due to the AID tag (see for example Figure 3—figure supplement 1D of the first submission). We attempted several immunoblot conditions and antibodies, and only the V5 antibody proved effective in recognizing these proteins above the limit of detection. For this reason, we included an additional single-tagged control in each immunoblot experiment. Uncropped images of the blots are included in the first submission as Figure 2—figure supplement 1D and E and as Figure 2 source data. We added the following statement to the results section of the text:

“However, SPARK and SPARKEL abundances are low and approach the limit of detection. We could only detect each protein by the V5 epitope. Although our experiments included single-tagged controls, we cannot formally eliminate the possibility that SPARK-AID yields degradation products that run at the expected molecular weight of SPARKEL. More sensitive methods, such as targeted mass spectrometry, may be required to measure the absolute abundance and stoichiometries of SPARK and SPARKEL.”

We added “h +IAA” to the x-axis of panel 2E.

Fig. 3. There is plentiful proteomic data on the factor-depleted parasites. Can it be used to confirm the co-degradation of the SPARK/SPARKEL complex components? This figure mainly includes quality control data that can be moved to Supplement. Did you detect SPARKEL in the TurboID experiment described in panel I? The plot shows only an AGC kinase.

SPARK and SPARKEL are lowly expressed, and we often do not detect SPARK or SPARKEL peptides with quantification values in complex samples (such as global depletion proteomes and phosphoproteomes; IPs and streptavidin pull-downs are comparatively less complex, with IPs being the least complex samples). We discussed this caveat in the first submission lines 178-186. To additionally clarify this point, we have added “We were unable to measure SPARK or SPARKEL abundances in this proteome” earlier in the text.

We consider the figure panels relevant to the discussion in the text.

SPARKEL was not quantified in the SPARK-TurboID experiment (Supplementary File 2). We have added “SPARKEL was not quantified in this experiment” to the text. “Not quantified” is a different outcome from “quantified but not enriched”. The interaction between SPARK and SPARKEL is supported by five other independent interaction experiments in which SPARKEL was quantified (Figure 1A, 1D, 1E; and Figure 1—figure supplement 1). The added insight from the SPARK proximity labeling experiments comes from integration with the global proteomics, which suggests that AGC kinases are in proximity to SPARK and exhibit SPARK-dependent stability and hence activity. The logic of the proximity labeling experiment is described in lines 258-275 of the first submission.

Fig. 6G is missing deltaBDF1 control for unbiased evaluation of the SPARK KD effect.

The logic of this experiment was to evaluate whether excess differentiation caused by SPARK and PKA C3 depletion (Figure 6A and 6B) was dependent on the BFD1 circuit. The ∆bfd1 phenotype is well-established under these experimental conditions: parasites lacking BFD1 do not differentiate under spontaneous or alkaline conditions (e.g. PMID: 31955846, 37081202, 37770433). Parasites lacking BFD1 do not differentiate when SPARK and PKA C3 are depleted, suggesting that differentiation caused by SPARK or PKA C3 depletion occurs through the BFD1 circuit. If differentiation caused by SPARK or PKA C3 depletion did not depend on the BFD1 circuit, we might have observed differentiation in the SPARK- and PKA C3-AID/∆bfd1 mutants.

To clarify this point, we have changed the first sentences of the last paragraph in the results section Depletion of SPARK, SPARKEL, or PKA C3 promotes chronic differentiation: “To assess whether excess differentiation caused by SPARK and PKA C3 depletion is dependent on a previously characterized transcriptional regulator of differentiation, BFD1 (Waldman et al., 2020), we knocked out the BFD1 CDS with a sortable dTomato cassette in the SPARK- and PKA C3-AID strains (Figure 6–figure supplement 1). The resulting SPARK- and PKA C3-AID/∆bfd1 mutants failed to undergo differentiation as measured by cyst wall staining (Figure 6G–H), suggesting that differentiation caused by depletion of these kinases depends on the BFD1 circuit.”

Lines 239-242. The logic behind the categories of "constitutively regulated sites" and "newly synthesized proteins dependent on SPARK activation" is odd. The former (3h treatment) represents the SPARK-specific events (even though it should be shortened to 1-2h), while an 8h treatment is already contaminated with secondary effects. Since Toxoplasma divides asynchronously, the "newly synthesized" proteins will be present at the time. Also, the protein phosphorylation does not always lead to substrate activation; it can be repressive, too.

We describe the logic in response to a comment above (substrate A vs. substrate B). It is correct that T. gondii divides asynchronously, with a cell cycle of approximately 8 hours, and 60% of parasites in G1 at a given time (PMID: 11420103). The proteomics experiments measure peptide and protein abundances at a population level. Newly synthesized proteins will be present at all time points; but the proportion of proteins synthesized after SPARK depletion relative to proteins synthesized before SPARK depletion will increase over time.

We moved lines 238-243 from the first submission to the Discussion.

It is accurate that phosphorylation does not always lead to substrate activation; it can also be repressive or not change substrate behavior. However, in the case of protein kinases, activation loop phosphorylation is highly correlated with activation (e.g. PMID: 15350212, 31521607).

Line 250-252: Because the SPARK degradation did not affect intracellular replication, SPARK is unlikely to affect cell cycle-specific phosphorylation.

To parallel the prior sentences describing different SPARK-dependent down-regulated clusters, we truncated this sentence to “The final cluster of depleted phosphopeptides, Cluster 4, only exhibits down-regulation at 8h of IAA treatment.”

SPARKEL depletion did not significantly affect intracellular replication under the time frames investigated here (approximately 25 hours post-invasion; Figure 2D). A prior study reported that SPARK depletion did not affect intracellular replication measured on a similar timescale (PMID: 35484233).

The opening sentence of the Discussion: Typically, we refer to the newly discovered proteins as the orthologs of the previously discovered counterparts and not the vice versa. Thus, calling Toxoplasma SPARK the ortholog of mammalian PDK1 would be more appropriate.

We changed the opening sentence of the Discussion to “SPARK is an ortholog of PDK1, which is considered a key regulator of AGC kinases”.

Reviewer #3 (Recommendations For The Authors):

(1) Authors should show alignment of SPARKEL with Elongin C. Are key residues conserved?

We have added an alignment of the SKP1/BTB/POZ domains of Homo sapiens elongin C, S. cerevisiae elongin C, and T. gondii SPARKEL as Figure 1—figure supplement 1B. This panel highlights elongin B interface, cullin binding sites, and target protein binding sites based on the human elongin C annotation. As discussed below, these interfaces may not be functionally conserved in T. gondii. Ultimately, future mechanistic and structural studies beyond the scope of the current work will be required to determine how SPARK and SPARKEL physically interact. The Discussion states, “further biochemical studies are required to discern the regulatory interactions between SPARK and SPARKEL” (lines 590-591).

(2) The failure to identify other Elongin B/C complex members should be addressed by direct IP analysis.

Indeed, elongin C has traditionally been characterized as a component of multisubunit complexes comprising Elongin A/B/C or Elongin BC/cullin/SOCS that regulate transcription or function as ubiquitin ligases, respectively (for a review, PMID: 22649776). We see two major issues when attempting to generalize these results to apicomplexan parasites. First, nearly all studies of the function of elongin C have been conducted in a single eukaryotic supergroup (the opisthokonts, including yeast and metazoans). The majority of eukaryotic diversity exists in other supergroups, including the SAR supergroup to which apicomplexans such as T. gondii belong (PMID: 31606140). Proteins with elongin C domains may serve alternative and unexplored functions in non-opisthokont unicellular eukaryotes. Second (in support of the first), we were unable to find orthologs of many of the opisthokont complex members in T. gondii, as systematically described below.

By BLAST, the most similar protein to SPARKEL in S. cerevisiae is ELC1 (YPL046C), with a BLAST E = 0.003. The next most similar protein was SCF ubiquitin ligase subunit SKP1 (YDR328C) with an E value of 0.62. ELC1 is 99 amino acids. The Elongin C (IPR039948) and SKP1/BTB/POZ superfamily domains (IPR011333) span most of this sequence. SPARKEL is 216 amino acids; the Elongin C and  SKP1/BTB/POZ superfamily domains occupy the C-terminal half of the protein. The N-terminal domain of SPARKEL may be important for its function; however, future work is required to address this hypothesis.

Elongin B: Elongin B is not found universally amongst even opisthokonts; fungi and choanoflagellates lack obvious orthologs. The most similar T. gondii protein to human Elongin B (Q15370) by BLAST is TGME49_223125 (E = 0.017), an apicoplast ubiquitin-like protein PUBL (PMID: 28655825, 33053376). TGME49_223125 has a C-terminal ubiquitin-like domain (IPR000626) but no ELOB domain (IPR039049); indeed, no T. gondii protein has an ELOB domain that can be identified by sequence searching. Given the lack of similarity between EloB and TGME49_223125, as well as this protein’s possible red algal endosymbiont origin, we consider it an unlikely ortholog of EloB and topologically unlikely to  interact with the SPARK/SPARKEL complex. We did not detect TGME49_223125 in SPARK or SPARKEL IPs (Supplementary File 1).

Elongin A: T. gondii appears to lack a human elongin A ortholog (Q14241) on the basis of sequence similarity. The most similar T. gondii protein to yeast Elongin A (O59671) by BLAST is TGME49_299230 (E = 0.022). Yeast EloA is 263 amino acids. TGME49_299230 is 1101 amino acids and does not have an EloA domain (IPR010684), suggesting it is not a true EloA ortholog.

Suppressor of cytokine signaling (SOCS): T. gondii appears to lack human SOCS1 or SOCS2 orthologs (O15524 and O14508) on the basis of sequence similarity. We were unable to identify T. gondii proteins with SOCS domains (PF07525, SM00253, SM00969, and SSF158235).

Von Hippel-Lindau tumor suppressor (VHL): T. gondii appears to lack a human VHL ortholog (P40337) on the basis of sequence similarity.  We were unable to identify T. gondii proteins with VHL domains (IPR024048, IPR024053, PF01847, and SSF49468).

Cul-2/5: Cullins appeared early in the eukaryotic radiation (PMID: 21554755), and thus T. gondii possesses several. Since the ELC complex has been best characterized with human cullin-2 (Q13617) and cullin-5 (Q93034), we searched for orthologs of these proteins and identified TGME49_289310, TGME49_289310, and TGME49_316660. TGME49_289310 functionally resembles cullin-1 of the SCF complex (PMID: 31348812). None of these proteins were enriched in the SPARK or SPARKEL IPs (Supplementary Table 1).

Rbx1: We searched for human Rbx1 orthologs (P62877) and identified TGME49_213690, which functionally resembles Rbx1 of the SCF complex (PMID: 31348812); as well as several other RING proteins (TGME49_267520, TGME49_277740, TGME49_261990, and TGME49_232160) that were not found in the SPARK or SPARKEL IPs (Supplementary File 1).

Rbx2: We searched for human Rbx2 orthologs (Q9UBF6) and identified several RING proteins (TGME49_285190, TGME49_254700, TGME49_292340, TGME49_226740, TGME49_244610, and TGME49_304460) that were not found in the SPARK or SPARKEL IPs (Supplementary File 1). No T. gondii protein has an Rbx2 domain (cd16466) that can be identified by sequence searching.

In conclusion, we conducted “direct IP analysis” (Figure 1A, 1D; Figure 1-supplement 1A) of the SPARK and SPARKEL complex in the first submission of the manuscript. The observation that SPARK and SPARKEL form strong interactions was validated in cellulo via proximity labeling (Figure 1E; Figure 1-supplement 1B) in the first submission of the manuscript. These results are described together in the results section SPARK complexes with an elongin-like protein, SPARKEL (lines 75-110, first submission of manuscript). The failure to identify an interaction between SPARKEL and Elongin B/C complex members in T. gondii may be due to the observation that Elongin B and several ELC complex members do not exist in most eukaryotes, including T. gondii. We added the sentences “The function of proteins with Elongin C-like domains has not been widely investigated in unicellular eukaryotes” to the Results and “However, the SPARK and SPARKEL IPs and proximity experiments failed to identify obvious components of ubiquitin ligase complexes” to the Discussion.

(3) PKA and PKG half-lives should be measured as well as their transcript abundances.

The finding that PKA C1 and PKG protein abundances decreased upon SPARK/SPARKEL depletion was internally consistent across experiments. This down-regulation may be due to transcriptional, translational, or post-translational mechanisms. We measured PKG and PKA C1 transcript abundances in SPARK-AID and TIR1 parasites after 24 hours of IAA treatment using RT-qPCR. We did not detect significant differences in transcript levels of the queried kinases. These findings suggest that SPARK depletion leads to PKG and PKA down-regulation through post-transcriptional mechanisms. Translational control is normally enacted globally, for example through regulation of eukaryotic translation factors (PMID: 15459663). The rapid and specific down-regulation of PKG and PKA C1 would suggest that the kinase abundance levels are regulated by non-global translational mechanisms (e.g. mRNA-specific) or rather post-translational mechanisms.

Substantial additional work is required to determine protein half-lives in eukaryotic parasites. In our discussion of possible mechanisms and models, we were agnostic as to the cause of reduced PKG and PKA abundances upon SPARK depletion. We note in the discussion, “The cause for reduction of PKA C1 and PKG levels requires further study” (lines 541-542).

Joint Public Review:

Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI oncogene. Ewing sarcoma cells are addicted to this chimeric transcription factor, which represents a strong therapeutic vulnerability. Unfortunately, targeting EWS-FLI has proven to be very difficult and better understanding how this chimeric transcription factor works is critical to achieving this goal. Towards this perspective, the group had previously identified a DBD-𝛼4 helix (DBD) in FLI that appears to be necessary to mediate EWS-FLI transcriptomic activity. Here, the authors used multi-omic approaches, including CUT&tag, RNAseq, and MicroC to investigate the impact of this DBD domain. Importantly, these experiments were performed in the A673 Ewing sarcoma model where endogenous EWS-FLI was silenced, and EWS-FLI-DBD proficient or deficient isoforms were re-expressed (isogenic context). The authors found that the DBD domain is key to mediate EWS-FLI cis activity (at msat) and to generate the formation of specific TADs. Furthermore, cells expressing DBD deficient EWS-FLI display very poor colony forming capacity, highlighting that targeting this domain may lead to therapeutic perspectives.

This new version of the study comprises as requested new data from an additional cell line. The new data has strengthened the manuscript. Nevertheless, some of the arguments of the authors pertaining to the limitations of immunoblots to assess stability of the DBD constructs or the poor reproducibility of the Micro C data remain problematic. While the effort to repeat MicroC in a different cell line is appreciated, the data are as heterogeneous as those in A673 and no real conclusion can be drawn. The authors should tone down their conclusions. If DBD has a strong effect on chromatin organization, it should be reproducible and detectable. The transcriptomic and cut and tag data are more consistent and provide robust evidence for their findings at these levels.

Concerning the issue of stability of the DBD and DBD+ constructs, a simple protein half-life assay (e.g. cycloheximide chase assay) could rule out any bias here and satisfactorily address the issue.

Suggestions:

The Reviewing Editor and a referee have considered the revised version and the responses of the referees. While the additional data included in the new version has consolidated many conclusions of the study, the MicroC data in the new cell line are also heterogeneous and as the authors argue, this may be an inherent limitation of the technique. In this situation, the best would be for the authors to avoid drawing robust conclusions from this data and to acknowledge its current limitations.

The referee and Reviewing Editor also felt that the arguments of the authors concerning a lack of firm conclusions on the stability of EWS-FLI1 under +/-DBD conditions could be better addressed. We would urge the authors to perform a cycloheximide chase type assay to assess protein half-life. These types of experiments are relatively simple to perform and should address this issue in a satisfactory manner.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

Ewing sarcoma is an aggressive pediatric cancer driven by the EWS-FLI oncogene. Ewing sarcoma cells are addicted to this chimeric transcription factor, which represents a strong therapeutic vulnerability. Unfortunately, targeting EWS-FLI has proven to be very difficult, and a better understanding of how this chimeric transcription factor works is critical to achieving this goal. Towards this perspective, the group had previously identified a DBD-𝛼𝛼4 helix (DBD) in FLI that appears to be necessary to mediate EWS-FLI transcriptomic activity. Here, the authors used multi-omic approaches, including CUT&tag, RNAseq, and MicroC to investigate the impact of this DBD domain. Importantly, these experiments were performed in the A673 Ewing sarcoma model where endogenous EWS-FLI was silenced, and EWS-FLI-DBD proficient or deficient isoforms were re-expressed (isogenic context). They found that the DBD domain is key to mediating EWS-FLI cis activity (at msat) and to generating the formation of specific TADs. Furthermore, cells expressing DBD-deficient EWS-FLI display very poor colony-forming capacity, highlighting that targeting this domain may lead to therapeutic perspectives.

We thank Reviewer 1 for their strong summary of Ewing sarcoma background and accurate description of our experimental approaches and findings.

Strengths:

The group has strong expertise in Ewing sarcoma genetics and epigenetics and also in using and analyzing this model (Theisen et al., 2019; Boone et al., 2021; Showpnil et al., 2022).

We thank the reviewer.  

They aim at better understanding how EWS-FLI mediated its oncogenic activity, which is critical to eventually identifying novel therapies against this aggressive cancer.

We are happy to see that our overall aim was also appreciated by Reviewer 1.

They use the most recent state-of-the-art omics methods to investigate transcriptome, epigenetics, and genome conformation methods. In particular, Micro-C enables achieving up to 1kb resolved 3D chromatin structures, making it possible to investigate a large number of TADs and sub-TADs structures where EWS-FLI1 mediates its oncogenic activity.

We thank Reviewer 1 for their acknowledgement of our approaches and the resolution achieved with our Micro-C experiments.  

They performed all their experiments in an Ewing sarcoma genetic background (A673 cells) which circumvents bias from previously reported approaches when working in non-orthologous cell models using similar approaches.

We agree with the reviewer about the importance of using model systems that accurately capture features of the disease being studied. As we have added an additional cell line in the revision we should note that this second model also represents a Ewing sarcoma genetic background while representing tumors expressing another oncogenic fusion found in this disease. 

Weaknesses:

The main weakness comes from the poor reproducibility of Micro-C data . Indeed, it appears that the distances/clustering observed between replicates are typically similar or even larger than between biological conditions. For instance, in Figure 1B, I do not see any clustering when considering DBD1, DBD2, DBD+1, DBD+2.

Lanes 80-83: "KD replicates clustered together with DBD replicate 1 on both axes and with DBD replicate 2 on the y-axis. DBD+ replicates, on the other hand, clustered away from both KD and DBD replicates. These observations suggest that the global chromatin structure of DBD replicates is more similar to KD than DBD+ replicates."

When replacing DBD replicate 1 with DBD replicate 2, their statement would not be true anymore.

Additional replicates to clarify this aspect seem absolutely necessary since those data are paving the way for the entire manuscript.

These are valid concerns and we thank the reviewers for highlighting this limitation of poor clustering of Micro-C replicates on MDS plot. We account for this variability between different replicates when identifying differentially interacting regions. By using an adjusted p-value < 0.05, we aim to ensure that repeating the experiments we will discover the same differentially interacting regions with a false discovery rate of 5%.

We also would like to note that the replicates cluster much closely on PCA plot of RNA-seq data (Supplementary Figure 1C) and as well as on PCA plot of H3K27ac CUT&Tag data (Figure 4A). Notably, the RNA-seq result has now reproduced when performed with different sets of hands across multiple studies (Boone, et. al., 2021 and this report), as well as in a second cell line (as reported in this manuscript revision). These observations suggest that the cells of these replicates are functionally similar to each other at a population level. Chromatin organization detected by Micro-C is a highly heterogenous within cells of a population (Misteli, et. al., 2020). Moreover, despite increased resolution with Micro-C over Hi-C, the conventional sequencing depth that Micro-C is performed at makes resolving finer scale 3D interactions, particularly between enhancers and promoters, challenging (Goel, et. al., 2023). Thus biologically relevant interactions driving EWSR1::ETS transcriptional regulation through de novo enhancers may have relatively weak signal in Micro-C. Both the strength of the signal and the heterogeneous chromatin state present in bulk samples could affect the average signal leading to poor clustering replicates (Hafner and Boettiger, 2022). 

Importantly, rather than add an additional replicate of a single cell line, we repeated our study in an additional cell line, TTC466, and largely reproduced our high-level findings for transcription, enhancer formation, and 3D chromatin. Specific limitations of the TTC466 study are addressed in the Discussion section (392-420). The reproduction of weak/moderate clustering in the MDS plot in both A673 and TTC466 cell lines suggests the α4 helix of EWSR1::ETS fusions are important for reshaping 3D chromatin. However, higher resolution analyses focused on specific EWSR1::ETS-bound loci are likely an important area of future study required to fully understand the role of the α4 helix in chromatin regulation in Ewing sarcoma.

Similarly:

- In Figure 1C, how would the result look when comparing DBD2/KD2/DBD+2? Same when comparing DBD 1 with KD1 and DBD+1. Would the difference go in the same direction?

This is a great point. We added distance decay plots of individual replicates in Supplementary Figure 2 and added discussion of these results in lines 88-89 of the text.

- Figure 1D-E. How would these plots look like when comparing each replicate to each other's? How much difference would be observed when comparing, for instance, DBD1/DBD2 ? or DBD1/DBD+1?

Unfortunately, separate replicates are required to conduct Differentially Interacting Region analysis as it determines statistically significant interactions. Therefore, we are unable to plot these analyses with individual replicates. 

- Figure 2: again, how would these analyses look like when performing the analysis with only DBD1/DBD+1/KD1 or DBD2/DBD+2/KD?

This is a good suggestion. It is possible to do such analysis. However, we will lose resolution as such that we may not accurately detect TADs, especially smaller TADs. Therefore, we decided to combine the biological replicates.   

Another major question is the stability of EWS-FLI DBD vs EWS-FLI DBD+ proteins. In the WB, FLAG intensities seem also higher (2/3 replicates) in DBD+ condition compared to the DBD condition (Figure S1B).

This is a valid concern with shRNA knock-down/rescue system and we regularly validate new constructs to ensure that they have similar expression levels as rescue with the wildtype fusion before proceeding to more exhaustive experimental workups. We would note that while we have not tested for differences in protein stability, for these constructs we largely see similar expression levels across multiple experiments, multiple cell lines, and multiple sets of hands. There may be some variations in expression level from experiment to experiment, but western blotting is a semiquantitative assay and it is also not possible to rule out that slight differences in band intensity may be a result of error in gel loading. For this reason, alongside western blotting for construct expression, we also validate construct function using RNA-seq and colony formation assays (as reported in this manuscript) and these show good agreement across biological replicates.  

Indeed, it seems that they have more FLAG (i.e., EWS-FLI) peaks in the DBD+ condition compared to the DBD condition (Figure 2B). 

We appreciate the comment since the legend of Figure 2B led to a misunderstanding. Figure 2B depicts the number of TADs detected in DBD and DBD+ conditions (height of the bar graphs) and the proportion of those TADs overlapped with FLAG, CTCF, both or neither peaks on y-axis. The number of FLAG peaks is actually lower in DBD+ as compared to DBD as shown in Figure 5A-B.  We clarified our Figure 2 legend to accurately describe the various proportions (color coded section) of TADs bound by DBD/DBD+ FLAG and CTCF.

Would it be possible that DBD+ is just more expressed or more stable than DBD? The higher stability of the re-expressed DBD+ could also partially explain their results independently of the 3D conformational change. In other words, can they exclude that DBD+ and DBD binding are not related to their respective protein stability or their global re-expression levels?

It is possible that DBD+ protein is overexpressed or more stable than DBD. With our current set of data, we cannot conclusively exclude if binding by DBD and DBD+ are not related to their expression level or stability. We would note, as above, that western blots, RNA-seq, and agar assays have largely reproduced across experiments, hands, and cell lines and that western blot is an imperfect assay for assessing protein stability.

Surprisingly, WB FLI bands in DBD+ conditions are systematically (3/3 replicates) fainter than in DBD conditions (Figure S1B). How do the authors explain these opposite results between FLI and FALG in the WB?

This is an excellent observation that highlights one of the intricacies of studying EWSR1::FLI1 in our KD/rescue system. Often the limiting factor for an experiment is whether or not the KD condition maintains KD through a second viral transduction for rescue and selection. We have observed over many years of working with this system that rescue conditions which are fully functional (i.e. wildtype EWSR1::FLI1, DBD+, etc.) tend to maintain better KD of endogenous EWSR1::FLI1. Constructs that don’t rescue EWSR1::FLI1 function sometimes maintain KD to a lesser degree, though frequently to a functional degree (i.e. cells are not transformed and EWSR1::FLI1 transcriptional regulation is not rescued). We suspect this observation, also raised by Reviewer 1 is resulted from a potential selection of cells with more endogenous EWSR1::FLI1 escaping KD in in DBD conditions due to selective pressures during expansion in tissue culture.

We should note that the antibody used for detecting FLI recognizes residues that are deleted in

DBD and DBD+ constructs, such that the FLI1 blot in Supplementary Figure 1B does not detect either construct. It only detects endogenous EWSR1::FLI1 and the 3X-FLAG-EWSR1::FLI1 construct in the middle lane that runs at a slightly higher molecular weight. The FLAG antibody is the only antibody that detects all three rescue constructs.    

Reviewer #2 (Public Review):

Summary:

The manuscript by Bayanjargal et al. entitled "The DBD-alpha4 helix of EWS::FLI is required for GGAA microsatellite binding that underlies genome regulation in Ewing sarcoma" reports on the critical role of a small alpha helix in the DNA binding domain (DBD) of the FLI1 portion of EWS::FLI1 that is critical for binding to repetitive stretches of GGAA-motifs, i.e. GGAA microsatellites, which serve as potent neoenhancers in Ewing sarcoma.

We thank Reviewer 2 for their succinct and accurate summary of our manuscript. 

Strengths:

The paper is generally well-written, and easy to follow and the data presented are of high quality, welldescribed and underpin the conclusions of the authors. The report sheds new light on how EWS::FLI1 mechanistically binds to and activates GGAA microsatellite enhancers, which is of importance to the field.

We appreciate the reviewer’s assessment of our work. 

Weaknesses:

While there are no major weaknesses in this paper, there are a few minor issues that the authors may wish to address before publication:

(1) While the official protein symbol for the gene EWSR1 is indeed EWS, the protein symbol for the gene FLI1 is identical, i.e. FLI1. The authors nominate the fusion oncoprotein EWS::FLI1 (even in the title) but it appears more adequate to use EWS::FLI1.

We appreciate the reviewer for bringing this to our attention. Indeed, the most recent guideline for fusion proteins nomenclature is to use the full gene symbols separated by double colons. Therefore, the accurate nomenclature is EWSR1::FLI1. We replaced instances of EWS::FLI with EWSR1::FLI1 and have used the EWSR1::ERG nomenclature in our revised manuscript.  

(2) The used cell lines should be spelled according to their official nomenclature (e.g. A-673 instead of A673).

Corrected, thanks!

(3) It appears as if the vast majority of results were generated in a single Ewing sarcoma cell line (A-673) which is an atypical Ewing sarcoma cell line harboring an activating BRAF mutation and may be genomically quite unstable as compared to other Ewing sarcoma cell lines (Kasan et al. 2023 preprint at bioRxiv https://www.biorxiv.org/content/10.1101/2023.11.20.567802v1). Hence, it may be supportive for the paper to recapitulate/cross-validate a few key results in other Ewing sarcoma cell lines, e.g. by using EWS::ERG-positive cell lines. Perhaps the authors could make use of available published data.

We thank Reviewer 2 for this helpful comment. We replicated the experiments in TTC-466 cells containing EWSR1::ERG fusion and found that as for A-673 cells the DBD-α4 helix is important for transcriptional, enhancer, and 3D chromatin regulation (Supplementary Figures 9-18).  

(4) Figure 6 and Supplementary Figure 5 are very interesting but focus on two selected target genes of the fusion (FCGRT and CCND1). It would be interesting to see whether these findings also extend to common EWS::ETS transcriptional signatures that have been reported. The authors could explore their data and map established consensus EWS::ETS signatures to investigate which other hubs might be affected at relevant target genes.

We expanded our analysis to other genes demonstrated to be regulated by EWSR1::FLI1 nucleated transcriptional hubs (Chong, et. al., 2018) and included NKX2-2 and GSTM4 gene regions in

Supplementary Figure 7-8 in A-673 cells. We also investigated the same gene regions of FCGRT, CCND1, NKX2-2, GSTM4 in TTC466 cells and report them in Supplementary Figures 14-17. For the purpose brevity, we decided to include the above examples. We may need to develop different tools to conduct further analysis to understand the gene regulatory networks driven by DBD and DBD+ in relation to hub formation. Although it is a great suggestion to map such network, this may be outside the scope of this manuscript. We thank the reviewer for bringing such a good point to our attention.  

(5) Table 1 is a bit hard to read. In my opinion, it is not necessary to display P-values with up to 8 decimal positions. The gene symbols should be displayed in italic font.

Suggestions are adapted, thanks!

Reviewing Editor (Recommendations For The Authors):

We would draw the authors' attention to the following issues that would best benefit from additional revision.

As indicated by Referee 1, an important issue concerns the apparent poor reproducibility of Micro-C data. In Figure 1B, the clustering of the DBD1, DBD2, DBD+1, and DBD+2 is poor.

It appears that the distances/clustering observed between replicates are typically similar or even larger than between biological conditions. Lines 80-83: "KD replicates clustered together with DBD replicate 1 on both axes and with DBD replicate 2 on the y-axis. DBD+ replicates, on the other hand, clustered away from both KD and DBD replicates. If one replaced DBD replicate 1 with DBD replicate 2, this statement would no longer be true. The referees believe that it is important to fully account for these potential discrepancies. Most of the study is based on analyses of these data sets, so if there are issues with them it has repercussions on the entire study. We note however that in Figure 4A the clustering of the H3K27ac data is much more convincing. The referees also feel that it is important to show immunoblots of the expression of DBD and DBD+ levels in the experiments performed here. While this was previously shown in the Boone et al publication in 2021, it could be illustrated again here.

We thank the editors for concisely summarizing the main weaknesses of the paper and underscoring the importance of the Micro-C data in the rest of the paper. While the Editors note tighter clustering of the H3K27ac (Figure 4A), we would like to note that the replicates cluster much closely on PCA plot of RNA-seq data (Supplementary Figure 1C). Notably, the RNA-seq result has now reproduced when performed with different sets of hands across multiple studies (Boone, et. al., 2021 and this report), as well as in a second cell line (as reported in this manuscript revision). Though not as tight, the H3K27ac CUT&Tag also reproduces in TTC466 cells. Thus, we interpret these findings to indicate that our replicates are functionally similar to each other. As discussed above in the response to Reviewer 1 in more detail, there are several factors that could affect how these functional similarities are represented in Micro-C data. Micro-C is ultimately a readout of the chromatin organization in a heterogeneous population of cells (Misteli et al., 2020). Additionally, sequencing depth limitations in conventional Micro-C experiments limit the ability to faithfully assess the enhancer-promoter interactions that may be relevant for our model system (Goel, et. al., 2023). Thus, both the strength of the biologically relevant signal and the heterogeneous chromatin state present in bulk samples could affect the average signal and lead to poorly clustering replicates (Hafner and Boettiger, 2022). 

To address these important concerns about rigor and reproducibility of the analyses, we repeated our study in an additional cell line, TTC466, and largely reproduced our high-level findings for transcription, enhancer formation, and 3D chromatin. These additional studies were not without their own limitations and these are addressed in the Discussion section (392-420). The reproduction of weak/moderate clustering in the MDS plot in both A673 and TTC466 cell lines suggests the α4 helix of EWSR1::ETS fusions are important for reshaping 3D chromatin. However, additional genomic analyses geared toward higher resolution at specific EWSR1::ETS-bound loci are likely an important area of future study required to fully understand the role of the α4 helix in chromatin regulation in Ewing sarcoma. Live cell imaging, as performed by Chong, et. al., 2018 and additional biochemical techniques may also be informative and are beyond the scope of this report.

With regards to concerns about construct expression, we have included immunoblots of the rescue constructs in both cell lines (Supplementary Figure 1B and 9A) and discussed Reviewer 1’s specific concerns in detail above.  

The referees also raise the issue of using an additional cell line to make a more general message. Although it would perhaps be asking too much to repeat the MicroC experiments, consolidation of the observations could be performed by focusing on specific loci such as FCGRT and CCND1 that were analyzed in this study. Could the authors use 4C-type experiments to reproduce the conclusions in an additional cell line? It would also be pertinent to consolidate the findings at these loci by 4C-type approaches even in the cell line used here. For the moment, all conclusions are based on the same set of data and a single technical approach.

We repeated the experiments in TTC466 cells and analyzed the data using same cut-offs used in A-673 cells. This allows us to compare between the two cell lines. We hope this new set of experiments and analyses address the reviewers’ concerns.  

Reviewer #1 (Recommendations For The Authors):

All the data are performed in A673 cells. Knowing the transcriptomic and epigenetic heterogeneity of Ewing sarcoma cells, some of the experiments supporting their findings should be replicated in at least another Ewing sarcoma model.

Per our discussion above, we have replicated our experiments in an additional cell line model of Ewing sarcoma. Importantly, the TTC466 cell line used expresses the EWSR1::ERG fusion found in 10-15% of Ewing sarcoma cases.  

Supplementary Figure 2B. Proportion of TAD boundaries bound by FLAG (i.e., EWS-FLI1) and CTCF. The number/proportion of FLAG (i.e., EWS-FLI) peaks observed at CTCF peak/TAD boundaries seems unexpectedly high. How do they explain this result since EWS-FLI peaks are rather intra-TAD to mediate their enhancer function?

In our previous study, we showed that EWSR1::FLI1 binding can be detected at boundaries of TADs (Showpnil, et. al., 2022). We think therefore it is likely that EWSR1::FLI1 binding is able to mediate enhancer function both inside TADs as well as at the borders of TADs and may, in some cases, function as an insulator between TADs.  

For the >50kb loop analysis, what was the low-range threshold? Up to 15-20 kp, contact frequency interactions may be caused by PFA crosslink (did they use a 5kb threshold ?). Were those excluded from that analysis?

We acknowledge that we did not use a lower threshold to exclude those short-range loop interactions. In our previous study, we observed that EWSR1::FLI1 binding reduces long-range interactions in favor of short-range interactions (Showpnil, et. al., 2022) and wanted to be able to capture short-range loops in our analysis.  

In Figure 2D, they observed that within TADs containing FLAG peaks at GGAA microsatellites, the intensity of the DBD+ FLAG peaks was higher compared to DBD FLAG peaks. How would this analysis look when considering the ETS FLAG peaks (i.e., EWS-FLI rather repressive peaks)? Could they compare TAD with GGAA msat vs TAD with ETS peaks?

We agree that this is an interesting observation. In our prior analyses we found no discernible relationship between EWSR1::FLI1 binding and changes in 3D chromatin associated with repression (Showpnil, et. al., Nucleic Acids Research, 2022). In contrast, EWSR1::FLI1-bound superenhancers had greater H3K27ac deposition when overlapping both a bound GGAA repeat and a non-microsatellite site. While there have been several additional reports about the relevance of EWSR1::FLI1 binding at nonmicrosatellite peaks, motifs at these loci have not yet been rigorously defined as GGAA repeats were by Johnson, et. al. in PLoS One, 2017. Each ETS factor binds different motifs containing the core 5’-GGAA-3’ with varying affinities depending on the flanking residues. There may be >100-fold difference in sequence-specific binding affinity for “high” vs. “low” affinity motifs. Better defining the types of ETS motifs bound by EWSR1::FLI1 and the functional changes associated with them thus represents an interesting area of future study.

Figure 1F: What is the biological meaning of these results (29.7, 39.5, and 54Mbp)? These distances are typically the size of a chromosome arm and clearly beyond classical chromatin loop/TAD structures in which EWS-FLI mediates its cis-activity.

We agree with referee here. This panel is now removed in our revised manuscript.  

How do DBD, KD, and DBD+ conditions compare with WT parental cells in the omics data? (Figures 1B, 4A). Do DBD+ conditions overlap with WT conditions? It would be nice to have these analyses also for Micro-C and Cut&Tag data. To be acknowledged here, the transcriptome data showing this aspect in Figure S1C are very convincing.

This is a fair point. We were not able to obtain similar sequencing depth of wtEF Micro-C libraries to that of KD, DBD and DBD+ due to disproportional use of wtEF libraries in troubleshooting. Therefore, we decided to exclude wtEF condition from these analysis. 

EWS-FLI cis-regulation at CCND1 also occurs through a much closer EWS-FLI peak (~-20kb msat upstream of CCND1 TSS) which was not taken into consideration. EWS-FLI peak intensity in both DBD and DBD+ at this msta seems similar. How would this fit into their model?

The referee is correct. The closest peak upstream of CCND1 TSS is about ~19kb away. We highlighted this peak with the dashed boxes near the CCND1 TSS (Supplementary Figure 6). Peak intensity of DBD+ FLAG is slightly higher compared to DBD. Nonetheless, we acknowledge that the difference is small. We suspect that the DBD-α4 helix is affecting binding dynamics at GGAA repeats, but these genomics approaches are not well suited to detect small, but significant, changes in binding affinity or dynamics. In this case a more biochemical approach may be needed. Even though, both protein can still bind the same microsatellites, it is possible that they might differ in their stability of binding or in the recruitment of additional proteins. These possibilities are discussed in the Discussion section (444-463).  

For the Micro-C, they sequenced only 7 to 8 million reads per condition. This coverage seems particularly low, especially for their analyses using 1-5kb bins. How does this compare with other published Micro-C data? Can this explain the variability observed between replicates?

We apologize for the inconsistent verbiage of sequencing coverage that may have caused confusion. 7 to 8 million reads were used for shallow sequencing and QC analysis. Once a sample passed QC, we then sequenced 300 million reads per sample. 300M is now changed to 300 million to prevent a misunderstanding at line 598.  

They mention:

"In our recent studies of EWS::FLI, we found a small alpha helix in the DNA binding domain DBD-𝛼𝛼4, to

be required for transcription and regulation by the fusion protein (Boone et al., 2021). Interestingly, this study did not find any change in chromatin accessibility (ATAC-Seq) and genome localization of EWS::FLI constructs (CUT&RUN) when DBD-𝛼𝛼4 helix was deleted leaving the mechanistic basis for the requirement of DBD-𝛼𝛼4 in transcription regulation unclear. "

And

"To assay the enhancer landscape, we collected H3K27ac CUT&Tag data from KD, DBD, and DBD+ cells. Principal component analysis of H3K27ac localization shows that the DBD replicates were clustered closer to the KD replicates while being in between the KD and the DBD+ replicates (Figure 4A), suggesting that DBD-𝛼𝛼4 helix is required to reshape the enhancer landscape."

But now H3K27ac CUT&Tag show strong differences which were not observed in ATAC seq. How to explain this discrepancy?

Though both H3K27ac and ATAC signal are associated with enhancers and promoters in euchromatin, they are not exactly measurements of the same thing. H3K4me2 is a mark more closely associated with ATAC signal than H3K27ac (Henikoff, et. al., 2020). Nonetheless, there are clear differences between the prior publication (Boone, et. al., 2021) and this work with regards to similar ATAC signal for each replicate and differences in H3K27ac. We suspect this may be related to a tighter association between H3K27ac and EWSR1::FLI1-mediated genome regulation and ATAC. Notably, there were very few differentially accessible regions between EWSR1::FLI1-depleted cells and conditions with EWSR1::FLI1 expression (either endogenous or wildtype rescue) using the A673 KD/Rescue system in Boone, et. al., 2021. In contrast, other A673 KD-rescue studies have reported differences in H3K27ac in EWSR1::FLI1 expressing conditions relative to EWSR1::FLI1-depleted conditions (Theisen, et. al., 2021). .  

The authors mention:

"Our study thus uncovered a surprising role for FLI DBD in the process of hub formation which is usually attributed to the EWS low complexity domain."

Not sure this can be claimed, hubs are composed of many other factors that are not investigated here. Furthermore, promoter enhancer hubs/loops often include combined ETS and mSat chains to generate transcriptional hubs which have not been considered here. None of these points were discussed here.

We replaced “uncovered” with “suggest” in our revised manuscript at line 476.  

What are the barcode patterns in Supp 5, are those frequently observed in their Micro-C data, likely mapping artifacts, do they have any impact on their analyses?

The barcode patterns in now Supplementary Figure 6 are blind spots in the hg19 genome assembly. Since they are few in numbers, we don’t expect these blind spots to impact our analysis.

Reviewer #1 (Public Review):

Summary:

This research article by Nath et al. from the Lee Lab addresses how lipolysis under starvation is achieved by a transient receptor potential channel, TRPγ, in the neuroendocrine neurons to help animals survive prolonged starvation. Through a series of genetic analyses, the authors identify that TRPγ mutations specifically lead to a failure in lipolytic processes under starvation, thereby reducing animals' starvation resistance. The conclusion was confirmed through total triacylglycerol levels in the animals and lipid droplet staining in the fat bodies. This study highlights the importance of transient receptor potential (TRP) channels in the fly brain to modulate energy homeostasis and combat metabolic stress. While the data is compelling and the message is easy to follow, several aspects require further clarification to improve the interpretation of the research and its visibility in the field.

Strengths:

This study identifies the biological meaning of TRPγ in promoting lipolysis during starvation, advancing our knowledge about TRP channels and the neural mechanisms to combat metabolic stress. Furthermore, this study demonstrates the potential of the TRP channel as a target to develop new therapeutic strategies for human metabolic disorders by showing that metformin and AMPK pathways are involved in its function in lipid metabolisms during starvation in Drosophila.

Weaknesses:

Some key results that might strengthen their conclusions were left out for discussion or careful explanation (see below). If the authors could improve the writing to address their findings and connect their findings with conclusions, the research would be much more appreciated and have a higher impact in the field.

Here, I listed the major issues and suggestions for the authors to improve their manuscript:

(1) Are the increased lipid droplet size and the upregulated total TAG level measured in the starved or sated mutant in Figure 1? This information might be crucial for readers to understand the physiological function of TRP in lipid metabolism. In other words, clarifying whether the upregulated lipid storage is observed only in the starved trp mutant will advance our knowledge of TRPγ. If the increase of total TAG level is only observed in the starved animals, TRP in the Dh44 neurons might serve as a sensor for the starvation state required to promote lipolysis in starvation conditions. On the other hand, if the total TAG level increases in both starved and sated animals, activation of Dh44 through TRPγ might be involved in the lipid metabolism process after food ingestion.

(2) It is unclear how AMPK activation in Dh44 neurons reduces the total triacylglycerol (TAG) levels in the animals (Figure 3G). As AMPK is activated in response to metabolic stress, the result in Figure 3G might suggest that Dh44 neurons sense metabolic stress through AMPK activation to promote lipolysis in other tissues. Do Dh44 neurons become more active during starvation? Is activation of Dh44 neurons sufficient to activate AMPK in the Dh44 neurons without starvation? Is activation of AMPK in the Dh44 neurons required for Dh44 release and lipolysis during starvation? These answers would provide more insights into the conclusion in Lines 192-193.

(3) It is unclear how the lipolytic gene brummer is further downregulated in the trpγ mutant during starvation while brummer is upregulated in the control group (Figure 6A). This result implies that the trpγ mutant was able to sense the starvation state but responded abnormally by inhibiting the lipolytic process rather than promoting lipolysis, which makes it more susceptible to starvation (Figure 3B).

(4) There is an inconsistency of total TAG levels and the lipid droplet size observed in the Dh44 mutant but not in the Dh44-R2 mutant (Figures 7A and 7F). This inconsistency raises a possibility that the signaling pathway from Dh44 release to its receptor Dh44-R2 only accounts for part of the lipid metabolic process under starvation. Adding discussion to address this inconsistency may be helpful for readers to appreciate the finding.

Reviewer #2 (Public Review):

Summary:

In this paper, the function of trpγ in lipid metabolism was investigated. The authors found that lipid accumulation levels were increased in trpγ mutants and remained high during starvation; the increased TAG levels in trpγ mutants were restored by the expression of active AMPK in DH44 neurons and oral administration of the anti-diabetic drug metformin. Furthermore, oral administration of lipase, TAG, and free fatty acids effectively restored the survival of trpγ mutants under starvation conditions. These results indicate that TRPv plays an important role in the maintenance of systemic lipid levels through the proper expression of lipase. Furthermore, authors have shown that this function is mediated by DH44R2. This study provides an interesting finding in that the neuropeptide DH44 released from the brain regulates lipid metabolism through a brain-gut axis, acting on the receptor DH44R2 presumably expressed in gut cells.

Strengths:

Using Drosophila genetics, careful analysis of which cells express trpγ regulates lipid metabolism is performed in this study. The study supports its conclusions from various angles, including not only TAG levels, but also fat droplet staining and survival rate under starved conditions, and oral administration of substances involved in lipid metabolism.

Weaknesses:

Lipid metabolism in the gut of DH44R2-expressing cells should be investigated for a better understanding of the mechanism. Fat accumulation in the gut is not mechanistically linked with fat accumulation in the fat body. The function of lipase in the gut (esp. R2 region) should be addressed, e.g. by manipulating gut-lipases such as magro or Lip3 in the gut in the contest of trpγ mutant. Also, it is not clarified which cell types in the gut DH44R2 is expressed. The study also mentioned only in the text that bmm expression in the gut cannot restore lipid droplet enlargement in the fat body, but this result might be presented as a figure.

Reviewer #3 (Public Review):

In this manuscript, the authors demonstrated the significance of the TRPγ channel in regulating internal TAG levels. They found high TAG levels in TRPγ mutant, which was ascribed to a deficit in the lipolysis process due to the downregulation of brummer (bmm). It was notable that the expression of TRPγ in DH44+ PI neurons, but not dILP2+ neurons, in the brain restored the internal TAG levels and that the knockdown of TRPγ in DH44+ PI neurons resulted in an increase in TAG levels. These results suggested a non-cell autonomous effect of Dh44+PI neurons. Additionally, the expression of the TRPγ channel in Dh44 R2-expressing cells restored the internal TAG levels. The authors, however, did not provide an explanation of how TRPγ might function in both presynaptic and postsynaptic cells in the non-cell autonomous manner to regulate the TAG storage. The authors further determined the effect of TRPγ mutation on the size of lipid droplets (LD) and the lifespan and found that TRPγ mutation caused an increase in the size of LD and a decrease in the lifespan, which were reverted by feeding lipase and metformin. These were creative endeavors, I thought. The finding that DH44+ PI neurons have non-cell autonomous functions in regulating bodily metabolism (mainly sugar/lipid) in addition to directing sugar nutrient sensing and consumption is likely correct, but the paper has many loose ends. I would like to see a revision that includes more experiments to tighten up the findings and appropriate interpretations of the results.

(1) The authors need to provide interpretations or speculations as to how DH44+ PI neurons have non-cell autonomous functions in regulating the internal TAG stores, and how both presynaptic DH44 neurons and postsynaptic DH44 R2 neurons require TRPγ for lipid homeostasis.

(2) The expression of TRPγ solely in DH44 R2 neurons of TRPγ mutant flies restored the TAG phenotype, suggesting an important function mediated by TRPγ in DH44 R2 neurons. However, the authors did not document the endogenous expression of TRPγ in the DH44R2+ gut cells. This needs to be shown.

(3) While Dh44 mutant flies displayed normal internal TAG levels, Dh44R2 mutant flies exhibited elevated TAG levels (Figure 7A). This suggested that the lipolysis phenotype could be facilitated by a neuropeptide other than Dh44. Alternatively, a Dh44 neuropeptide-independent pathway could mediate the lipolysis. In either case, an additional result is needed to substantiate either one of the hypotheses.

(4) While the authors observed an increased area of fat body lipid droplets (LD) in Dh44 mutant flies (Figure 7F), they did not specify the particular region of the fat body chosen for measuring the LD area.

(5) The LD area only accounts for TAG levels in the fat body, whereas TAG can be found in many other body parts, including the R2 area as demonstrated in Figure 5A-D using Nile red staining. As such, measuring the total internal TAG levels would provide a more accurate representation of TAG levels than the average fat body LD area.

(6) In Figure 5F-I, the authors should perform the similar experiment with Dh44, Dh44R1, and Dh44R2 mutant flies.

(7) The representative image in Figure 6B does not correspond to the GFP quantification results shown in Figure 6C. In trpr1;bmm::GFP flies, the GFP signal appears stronger in starved conditions than in satiated conditions.

(8) In Figure 6H-I, fat body-specific expression of bmm reversed the increased LD area in TRPγ mutants. The authors also showed that Dh44+PI neuron-specific expression of bmm yielded a similar result. The authors need to provide an interpretation as to how bmm acts in the fat body or DH44 neurons to regulate this.

(9) The authors should explain why the DH44 R1 mutant did not represent similar results as the wild type.

(10) It would be good to have a schematic that represents the working model proposed in this manuscript.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The main goal of the authors was to study the testis-specific role of the protein FBXO24 in the formation and function of the ribonucleoprotein granules (membraneless electron-dense structures rich in RNAs and proteins).

We appreciate the summary comment of reviewer #1.

Strengths:

The wide variety of methods used to support their conclusions (including transgenic models)

We appreciate the positive comment of reviewer #1.

Weaknesses:

The lack of specific antibodies against FBXO24. Some of the experiments showing a specific phenotype are descriptive and lack of logical explanation about the possible mechanism (i.e. AR or the tail structure).

Because we could not obtain specific antibodies against FBXO24, we generated Fbxo24-FLAG transgenic mice, which can be used to show the interaction between FBXO24 and IPO5. For the mechanism of impaired acrosome reaction, we added some results and discussion as written in the response to the question (1) of reviewer #1 (public review). For the mechanism of abnormal flagellar structure, we added new results and fixed the manuscript as written in the response to the major comments of reviewer #3 (recommendations for the authors).

Questions:

The paper is excellent and employs a wide variety of methods to substantiate the conclusions. I have very few questions to ask:

(1) KO mice cannot undergo acrosome reaction (AR) even spontaneously. How do you account for this, given that no visible defects were observed in the acrosome?

One possibility is that Fbxo24 KO spermatozoa cannot undergo capacitation; however, it is difficult to analyze the capacitation status such as tyrosine phosphorylation because most Fbxo24 KO spermatozoa are not alive (Figure S3A). Other possibility is that AR-related proteins are affected in Fbxo24 KO spermatozoa. Therefore, we analyzed the amounts of AR-related proteins with mass spectrometry (Figure S3C). Although previous studies indicate that the assembly of the SNARE complex is a key event prior to AR [Hutt et al., 2005 (PMID: 15774481); Katafuchi et al., 2000 (PMID: 11066067); Schulz et al., 1997 (PMID: 9356173); Tomes et al., 2002 (PMID: 11884041)], no clear differences were detected for SNARE proteins (Figure S3C and D). PLCD4 that is important for AR [Fukami et al., 2001 (PMID: 11340203)) was also detected in Fbxo24 KO spermatozoa (Figure S3C). Although we could not find differences in the amounts of AR-related proteins, it is still possible that FER1L5, another AR-related protein [Morohoshi et al., 2023 (PMID: 36696506)] not detected in the mass spectrometry analyses, or AR-related proteins not yet identified are affected in Fbxo24 KO spermatozoa. We added these results and discussion (line 160-166 and 305-312).

(2) KO sperm are unable to migrate in the female tract, and, more intriguingly, they do not pass through the utero-tubal junction (UTJ). The levels of ADAM3 are normal, suggesting that the phenotype is influenced by other factors. The authors should investigate the levels of Ly6K since mice also exhibit the same phenotype but with normal levels of ADAM3.

We detected LY6K in Fbxo24 KO spermatozoa with immunoblotting, but no difference was found.

We added the results (Figure S3E and line 172–175).

(3) In Figure 4A, the authors assert that "RBGS Tg mice revealed that mitochondria were abnormally segmented in Fbxo24 KO spermatozoa." I am unable to discern this from the picture shown in that panel. Could you please provide a more detailed explanation or display the information more explicitly?

We are sorry for the ambiguous explanation on the morphology of sperm mitochondria sheath. Fbxo24 KO cauda epidydimal spermatozoa shows disorganized mitochondria sheath rather than “segmented”. We fixed the sentence (line 190-192) and added white arrowheads that indicate the disorganized regions (Figure 4A).

Reviewer #2 (Public Review):

Summary:

The manuscript by Kaneda et al "FBXO24 ensures male fertility by preventing abnormal accumulation of membraneless granules in sperm flagella" is a significant paper on the role of FBXO24 in murine male germ cell development and sperm ultrastructure and function. The body of experimental evidence that the authors present is extraordinarily strong in both breadth and depth. The authors investigate the protein's functions in male germ cells and sperm using a wide variety of approaches but focusing predominantly on their novel mouse model featuring deletion of the Fbxo24 gene and its product. Using this mouse, and a cross of it with another model that expresses reporters in the head and midpiece, they logically build from one experiment to the next. Together, their data show that this protein is involved in the regulation of membraneless electron-dense structures; loss of FBXO24 led to an accumulation of these materials and defects in the sperm flagellum and fertilizing ability. Interestingly, the authors found that several of the best-known components of electron-dense ribonucleoprotein granules that are found in the intermitochondrial cement and chromatoid body were not disrupted in the Fbxo24 knockout, suggesting that the electron-dense material and these structures are not all the same, and the biology is more complicated than some might have thought. They found evidence for the most changes in IPO5 and KPNB1, and biochemical evidence that FBXO24 and IPO5 could interact.

We appreciate the summary comment of reviewer #2.

Strengths:

The authors are to be commended for the thoroughness of their experimental approaches and the extent to which they investigated impacts on sperm function and potential biochemical mechanisms. Very briefly, they start by showing that the Fbxo24 message is present in spermatids and that the protein can interact with SKP1, in a way that is dependent on its F-box domain. This points toward a potential function in protein degradation. To test this, they next made the knockout mouse, validated it, and found the males to be sterile, although capable of plugging a female. Looking at the sperm, they identified a number of ultrastructural and morphological abnormalities, which they looked at in high resolution using TEM. They also cross their model with RBGS mice so that they have reporters in both the acrosome and mitochondria. The authors test a variety of sperm functions, including motility parameters, ability to fertilize by IVF, cumulus-free IVF, zona-free-IVF, and ICSI. They found that ICSI could rescue the knockout but not other assisted reproductive technologies. Defects in male fertility likely resulted from motility disruption and failure to get through the utero-tubal junction but defects in acrosome exocytosis also were noted. The authors performed thorough investigations including both targeted and unbiased approaches such as mass spectrometry. These enabled them to show that although the loss of the FBXO24 protein led to more RNA and elevated levels of some proteins, it did not change others that were previously identified in the electron-dense RNP material.

The manuscript will be highly significant in the field because the exact functions of the electron-dense RNP materials have remained somewhat elusive for decades. Much progress has been made in the past 15 years but this work shows that the situation is more complex than previously recognized. The results show critical impacts of protein degradation in the differentiation process that enables sperm to change from non-descript round cells into highly polarized and compartmentalized mature sperm, with an equally highly compartmentalized flagellum. This manuscript also sets a high bar for the field in terms of how thorough it is, which reveals wide-ranging impacts on processes such as mitochondrial compaction and arrangement in the midpiece, the correct building of the major cytoskeletal elements in the flagellum, etc.

We appreciate the positive comment of reviewer #2.

Weaknesses:

There are no real weaknesses in the manuscript that result from anything in the control of the authors. They attempted to rescue the knockout by expressing a FLAG-tagged Fbxo24 transgene, but that did not rescue the phenotype, either because of inappropriate levels/timing/location of expression, or because of interference by the tag. They also could not make anti-FBXO24 that worked for coimmunoprecipitation experiments, so relied on the FLAG epitope, an approach that successfully showed co-IP with IPO5 and SKP1.

We could not rescue the phenotype with Fbxo24-FLAG transgene, but different Fbxo24 mutant mice show the same phenotypes (Figure S6G). Further, another group showed that Fbxo24 KO mice exhibited abnormal mitochondrial coiling [Li et al., 2024 (PMID: 38470475)], confirming that

FBXO24 is involved in the mitochondrial sheath formation.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors found that FBXO24, a testis-enriched F-box protein, is indispensable for male fertility. Fbxo24 KO mice exhibited malformed sperm flagellar and compromised sperm motility.

We appreciate the summary comment of reviewer #3.

Strengths:

The phenotype of Fbxo24 KO spermatozoa was well analyzed.

We appreciate the positive comment of reviewer #3.

Weaknesses:

The authors observed numerous membraneless electron-dense granules in the Fbxo24 KO spermatozoa. They also showed abnormal accumulation of two importins, IPO5 and KPNB1, in the Fbxo24 KO spermatozoa. However, the data presented in the manuscript do not support the conclusion that FBXO24 ensures male fertility by preventing the abnormal accumulation of membraneless granules in sperm flagella, as indicated in the manuscript title.

Fbxo24 KO mice showed abnormal accumulation of membraneless granules in sperm flagella and male infertility, suggesting that FBXO24 is involved in these processes, but there are no results that show the direct relationship as reviewer #3 mentioned. Therefore, we fixed the title.

Recommendations For The Authors:

Reviewer #2 (Recommendations For The Authors):

On page 4, lines 152-154, the authors introduce the RBGS mouse model and use it in their experiments.

However, they left out an obvious but helpful sentence that tells the reader that they crossed the Fbxo24-null mouse with the RBGS. As one continues reading it is clear, but best to avoid even slight confusion.

We revised the explanation in the result section (line 150-153).

Reviewer #3 (Recommendations For The Authors):

In this manuscript, the authors found that FBXO24, a testis-enriched F-box protein, is indispensable for male fertility. Fbxo24 KO mice exhibited malformed sperm flagellar and compromised sperm motility. The phenotype of Fbxo24 KO spermatozoa was well analyzed.

The authors observed numerous membraneless electron-dense granules in the Fbxo24 KO spermatozoa. They also showed abnormal accumulation of two importins, IPO5 and KPNB1, in the Fbxo24 KO spermatozoa. However, the data presented in the manuscript do not support the conclusion that FBXO24 ensures male fertility by preventing the abnormal accumulation of membraneless granules in sperm flagella, as indicated in the manuscript title.

Fbxo24 KO mice showed abnormal accumulation of membraneless granules in sperm flagella and male infertility, suggesting that FBXO24 is involved in these processes, but there are no results that show the direct relationship as reviewer #3 mentioned. Therefore, we fixed the title.

Major comments:

In the title, abstract, introduction, and some sections such as lines 275-276, the authors conclude that FBXO24 prevents the accumulation of importins and RNP granules during spermiogenesis. However, the provided data do not substantiate this claim. To provide conclusive evidence to support the current title, the authors need to present evidence supporting: 1) direct degradation of IPO5 and KPNB1 by FBXO24; 2) the direct requirement of IPO5 for the formation of the membraneless granules, and 3) infertility resulting from the presence of membraneless granules, rather than other issues such as abnormal ODF and AX.

(1) direct degradation of IPO5 and KPNB1 by FBXO24.

To examine if IPO5 can be degraded by FBXO24, we performed a ubiquitination assay using HEK293T cells. Ubiquitination of IPO5 was upregulated in the presence of WT FBXO24 but not with the mutant ΔF-box FBXO24, suggesting that IPO5 can be ubiquitinated by FBXO24. We did not examine the ubiquitination of KPNB1 because we failed to construct a plasmid vector expressing mouse KPNB1. We think that KPNB1 is not the substrate because we did not detect the interaction between FBXO24 and KPNB1 (Figure 5E). We added the results of the ubiquitination assay (Figure

5F and line 261-265) and mentioned it in the abstract (line 35).

(2) the direct requirement of IPO5 for the formation of the membraneless granules.

(3) infertility resulting from the presence of membraneless granules, rather than other issues such as abnormal ODF and AX.

We revealed that IPO5 aggregate under stress condition in COS7 cells (Figure 6C and D); however, we did not examine whether IPO5 is required for the formation of the membraneless granules. We consider that protein degradation systems such as PROTAC or Trim-Away to knockdown IPO5 at the protein level in Fbxo24 KO mice could be a good way to see if the membraneless granules are diminished and male fertility is rescued. However, it takes time to apply the degradation systems in vivo. Therefore, we would like to leave this rescue experiment for future studies. We fixed the title and  abstract (line 37-38), and removed the last sentence of the introduction.

Also, the other group reported the analyses of Fbxo24 KO mice [Li et al., 2024 (PMID: 38470475)] right after we submitted our manuscript to the eLife. They reported not only disorganized flagellar structures but also abnormal head morphology, which may lead to male infertility. The differences from our study may be due to different mouse genetic backgrounds. We mentioned it in the discussion section (line 348-353).

Minor comments:

(1) The authors claimed a significant increase in the total amount of RNAs in Fbxo24 KO spermatozoa (lines 259-261), suggesting that the ...contain RNAs. More direct evidence supporting this claim should be provided.

We show that the amounts of IPO5 and KBNB1 increased in Fbxo24 KO spermatozoa (Figure 5A and B), both of which could be incorporated into RNP granules in COS7 cells (Figure 6C and D), supporting the idea that membraneless electron-dense structures may be RNP granules. However, because we did not show direct evidence that electron-dense structures contain RNAs, we removed the sentences (line 259-261 of the 1st submission manuscript). 

(2) The author should provide an explanation for the absence of a FLAG band in the input Tg in Figure 5D and the larger size of the IPO5 band in the FLAG-IP group compared to the input. Similar observations are also noted in Figure 5E.

The FLAG band is weak because the protein amount is low. When we increase the contrast, we can see the FLAG band. We added an image with high contrast (Figure 5D). Sometimes, proteins run differently with SDS-PAGE after immunoprecipitation, likely due to varying protein composition in the sample. We explained it in the figure legend (line 868-869).

(3) In Line 526, clarify the procedure for sperm purification, and determine the potential for contamination from somatic cells.

We did not perform sperm purification, but when we observed spermatozoa obtained from cauda epididymis, we rarely observed either somatic cells or immature spermatogenic cells. We added  pictures in Figure S7. Further, we added detailed explanation about how to collect spermatozoa from the epididymis (line 549-550).

(4) Define the Y-axis in Figure 2E, F, and G.

We have revised the figures.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

We thank the reviewers for their positive and constructive criticism. We answer their points one by one below.

Reviewer #1

1.) In the baf-1 G12T mutants the authors find reduced levels of lamin in hypodermal nuclei. It would be good to also examine the dynamics of lamin in the second tissue that was subjected to DamID (intestinal cells).

We provide a complete analysis of GFP::LMN-1 and EMR-1::mCh in control and baf-1(G12T) day 1 adults in intestine and hypodermis and at 20°C and 25°C. These data demonstrate that GFP::LMN-1 expression is reduced in baf-1(G12T) mutants in both tissue and at both temperatures. In contrast, for EMR-1::mCh a significant reduction was only observed in hypodermal nuclei at 20°C.

The effects on GFP::LMN-1 and EMR-1::mCh in the hypodermis 20°C were reported in Figure 2E-F in the original version of our manuscript. We have moved these data to the new Supplementary Figure S5 and represent instead the data obtained for hypodermis at 25°C in Figure 2E-F for consistency with the data represented in Figure 2A-D. Data on intestine for both markers and both temperatures are also included in the new Supplementary Figure S5.

We have modified the text as follows:

“To test the impact of baf-1(G12T) on LMN-1, EMR-1, and BAF-1 localization in vivo, we quantified these factors at the NE of hypodermal and intestinal cells. We observed a significantly lower median GFP::LMN-1 signal at the NE in baf-1(G12T) mutants in both tissues at 20°C and 25°C (Figure ____2E; Supplementary Figure S____5A-C). In contrast, accumulation of EMR-1 at the NE was unaffected by the baf-1(G12T) mutation in both tissues at 25°C and reduced in the hypodermis at 20°C (Figure ____2F; Supplementary Figure S____5D-F). In human NGPS cells, emerin was observed to be delocalized to the ER (Janssen et al., 2022; Puente et al., 2011), but we detected no increase in cytoplasmic EMR-1::mCh signal in the mutant, indicating that this NGPS phenotype is not present in the C. elegans model. In agreement with these microscopy data, analysis of whole-worm mRNA levels by quantitative RT-PCR also revealed a significant reduction in lmn-1 expression whereas emr-1 was unaffected (Supplementary Figure S4E-F).”

2.) The authors make a statement that EMR-1 expression was reduced in the baf-1 G12T mutant, but do not comment on LMN-1 expression. Can a statement on this be made by RT PCR?

Our gene expression analysis by RAPID determined a significant reduction in emr-1 expression in the intestine of baf-1(G12T) mutants, using a fold change of 2 as threshold. In contrast, expression of emr-1 in hypodermis as well as baf-1 and lmn-1 expression in both tissues were not significantly different between wild type and baf-1(G12T) mutants in our RAPID data.

We performed qRT-PCR on bulk mRNA to compare the expression of baf-1, emr-1 and lmn-1 in control versus baf-1(G12T) mutants. No differences were detected for baf-1 and emr-1 (new Supplementary Figure S4E-F). Considering that the qRT-PCR is on bulk mRNA, the emr-1 result is compatible with the RAPID data that suggest deregulation of emr-1 only in intestine and unaffected expression in the hypodermis. For baf-1 there is agreement between qRT-PCR and RAPID data from both tissues (no difference in the mutant). For lmn-1, the qRT-PCR analysis suggests a modest reduction (23%; not reaching the threshold applied in the RAPID analysis) in baf-1(G12T) mutants, which is concordant with the reduction observed in GFP::LMN-1 intensity in hypodermis and intestine by confocal microscopy (e.g. 14% reduction in median GFP::LMN-1 intensity in hypodermis at 25C; Figure 2E).

The discordance between RAPID and live imaging for emr-1/EMR-1::mCh (a reduction in the intestine or the hypodermis according to RAPID or live imaging, respectively) is not surprising. Although mRNA and protein levels in general correlate well, often, variation in transcription can only explain We have added these two sentences to the manuscript:

“In agreement with these microscopy data, analysis of whole-worm mRNA levels by quantitative RT-PCR also revealed a significant reduction in lmn-1 expression whereas emr-1 was unaffected (Supplementary Figure S4E-F).”

“As described above, the amount of endogenously tagged EMR-1::mCh at the NE of intestinal cells was normal in baf-1(G12T) mutants (Supplementary Figure S5F), suggesting a cellular capacity to buffer the downregulation of emr-1 transcription (Vogel & Marcotte, 2012).”

3.) The authors find few alterations in gene regulation of the loci which have different occupancy WT BAF-1 versus BAF-1 G12T. It was surprising to see the DamID and RNA polymerase DamID experiments be done with worms grown at 20°C, because the more penetrant phenotypes at the organismal level were observed at 25°C. Could this be the reason for the little change of chromatin occupancy of BAF-1 and BAF-1 G12T or few changes in gene expression? Would it make sense to examine the expression of some selected BAF-1 bound loci by single molecule Fish at 25°C and compare expression wt versus baf-1 G12T?

We performed the DamID experiments at 20°C to avoid potential artifacts and/or toxicity by higher expression levels of Dam fusion proteins (Greil, Moorman, & van Steensel, 2006; Schuster et al., 2010). We note that altered UV and tert-butyl hydroperoxide was observed at 20°C, indicating that the baf-1(G12T) allele affects physiology at several temperatures. The original version of our manuscript described the expression of fluorescently tagged LMN-1 and EMR-1 in the hypodermis at 20°C (Figure 2E-F). As described above, in the revised version, we report the expression in the intestine at 20°C and in both tissues at 25°C. For GFP::LMN-1, a similar reduction in the baf-1(G12T) mutant was observed at the two temperatures in both tissues, whereas for EMR-1::mCh a reduction was only seen in the hypodermis at 20°C. Taken together, we conclude that 20°C is a suitable temperature for the DamID experiments.

We appreciate the suggestion to study expression of genes bound by BAF-1 by smFISH. However, we anticipate that because the hypodermis is composed mostly of large syncytia covering the round body of the animal, smFISH would be difficult to quantify. Regarding loci with different occupancy of WT BAF-1 versus BAF-1(G12T), the emr-1 locus was bound in the intestine by Dam::BAF-1 but not by Dam::BAF-1(G12T) (Figure 6B). As mentioned above, we observed that emr-1 expression was reduced in intestine of baf-1(G12T) mutants, suggesting that BAF-1 binding has a positive effect of transcription of this locus.

4.) The finding that BAF-1 nuclear envelope localization remains unchanged in the mutant stems from detection of the inserted GFP epitope. Given that the tag has an influence on the BAF-1 G12 12T mutant viability, this statement should be phrased with more care. The tag could influence the turnover of the protein for example. Maybe Western blots comparing the signal of WT BAF-1 worms and BAF-1 G12T mutant worms would be instructive to compare the levels of the protein (at 20oC and at 25oC, day 1 adults and day 8 adults).

We performed Western blot experiments to address this. As controls, we included strains expressing equal amounts of GFP::BAF-1 and GFP::BAF-1(G12T) strains (Figure 3E and Supplementary Figure 7 in original manuscript reported equal expression of the two proteins). Surprisingly, the polyclonal anti-BAF-1 serum raised against recombinant, full-length wild type BAF-1 (Gorjanacz et al., 2007) has significantly lower affinity for mutant GFP::BAF-1(G12T) than for GFP::BAF-1, which precludes the evaluation of untagged proteins:

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Figure 1. [png file provided to reviewers - not possible to include here for technical reasons] Western blot analyses with anti-BAF-1 serum (Gorjanacz et al, 2007). (A) Embryonic extracts. A band of the expected size is observed in wildtype embryos (*), but not in baf-1(G12T) embryos. (B) Extracts from young adults. A faint band of the expected size is observed in wildtype embryos (* in lane 1; longer exposure is shown below), whereas a more prominent band is present corresponding to endogenously tagged GFP::BAF-1 (** in lane 2). The intensity of the potential GFP::BAF-1(G12T) is reduced by >80% (lane 4; >90% reduction was observed in a second experiment).

We point out in the revised manuscript that the conclusion on equal BAF-1 and BAF-1(G12T) expression was based on endogenously tagged proteins: “Quantifying the intensity at the NE or in the nucleoplasm of hypodermal cells did not demonstrate any difference between endogenously GFP-tagged wild-type and mutant BAF-1 (Figure 3E). A small reduction in cytoplasmic signal was observed for BAF-1(G12T), however, no difference was detected in the ratio between nucleoplasmic/cytoplasmic signal (Figure 3E). Quantitative RT-PCR analysis of whole-worm RNA samples also indicated that baf-1 and baf-1(G12T) are expressed at identical levels (Supplementary Figure S4E-F).”

5.) Line 105: typo: remove "s"

Corrected.

6.) Line 154: A conclusion is missing for the fog-2 experiment.

We have modified the text as follows: “To test this possibility, we incubated baf-1(G12T) males with fog-2(q71) feminized worms that only produce oocytes and counted daily offspring. At 25°C, the fog-2(q71) allele prevents spermatogenesis specifically in XX hermaphrodites whereas X0 males are unaffected (Schedl & Kimble, 1988). We observed a reduction in brood size of approximately one third when sperm came from baf-1(G12T) males (Supplementary Figure S2B, C). Thus, we concluded that the baf-1(G12T) mutation has a negative impact on spermatogenesis. The male/female ratio in the progeny was ~1, suggesting that meiotic segregation of chromosomes was normal in baf-1(G12T) males.”

7). Would it make sense to discuss a possible influence of altered lamin binding to the nuclear envelope in the mutant in the context of the gene expression results?

We agree that this point is relevant, and we have added the following text to the Discussion: “At current, we can only speculate about how the NGPS mutation might affect gene expression. Proteomics analyses indicate that BAF interacts with several histones and transcription factors (Montes de Oca, Shoemaker, Gucek, Cole, & Wilson, 2009), and the differences between BAF-1 and BAF-1(G12T)’s chromatin binding profiles reported here might be accompanied by changes in the association of chromatin factors at the deregulated loci. A particularly interesting candidate is GCL-1/germ cell-less 1, a repressive factor involved in spermatogenesis (Holaska, Lee, Kowalski, & Wilson, 2003). Moreover, it is plausible that the diminished recruitment of LMN-1 to the NE in baf-1(G12T) mutants modifies its interaction with the genome and with chromatin factors.”

8). In a nutshell, the authors have established a convincing accessible model system for studying aging, ready for consecutive testing interventions to reduce the pace of premature aging.

We appreciate and share the opinion of the reviewer.

Reviewer #2

1). The value of this work is two-fold: First, it is a very robust characterization of NGPS worms. Second, this will be a very useful model for the study of NGPS. Overall, the study is well-designed, technically strong, and the results are carefully and thoughtfully interpreted, which is nicely exemplified by the discussion of the relatively small number of genes which are differentially bound by BAF1 and are also differentially expressed and the authors do a good job of not overinterpreting the data, but simply state them. The results are convincing and informative.

We thank the reviewer for her/his positive evaluation.

My only minor point that may make this paper marginally better is that it would be nice to have a paragraph in the Discussing elaborating on the potential and the limitations of using the worm model to understand human NGPS, for example, humans have multiple lamin proteins etc.

We agree with the reviewer and have added the following text to the Discussion: “We note that the simplicity of invertebrates also implies certain limitations. For instance, while both human and C. elegans genomes contain a single BAF gene, humans, but not C. elegans, express multiple lamin isoforms in tissue-specific ratios that regulate chromatin organization and nuclear mechanics (Swift et al., 2013). Thus, C. elegans is not suitable to explore potential differences in how wild type and NGPS BAF interacts differently with the various lamin isoforms.”

Reviewer #3

1). Overall, this manuscript strongly supports the major conclusion that this C. elegans line is a powerful model for human NGPS that complements a previously reported Drosophila model. Equally importantly, from the viewpoint of fundamental discovery, this manuscript also reports major advances in understanding how BAF influences gene expression at the molecular level.

We thank the reviewer for her/his positive evaluation.

2). DamID-Baf-1 access to chromatin was unaffected by the G12T mutation (Fig. S7), but they successfully identified subsets of genes 'occupied' by baf-1 in specific cell types, some of which were significantly affected in opposite ways by the NGPS mutation (Fig. 4, Fig. 5). However, these important new results are described too briefly, and discussion is inadequate. E.g., in hypodermal cells, the baf-1 G12T mutation dysregulated genes encoding proteins in five categories (ribosomal, proton transport, cuticle components, cell surface, lysine acetylation), by downregulating genes in three categories (ribosomal, proton transport, histone acetylation) and upregulating three other categories (cuticle components, cell surface, apical region). In intestinal cells, the mutation dysregulated genes in 8 categories (ribosomal, response to X-ray, proton transport, proteasome binding, mitochondrial protein import, endopeptidase activity, carboxy-lyase activity, ATP generation), by downregulating genes in 5 categories (ribosomal, proton transport, peptidase activity, NAD binding, metal cluster binding) and upregulating 3 categories (ribosomal, response to external stimulation, histone acetylation). Opposite results for "ribosomal genes" is confusing. Examples of genes in each affected category are shown in Fig. 6. To fully interpret this data, and address apparently-conflicting results, further analysis is needed to determine if any affected groups of genes have shared regulators. For example, Fig 5E shows "ribosomal protein genes" are both up- and down-regulated by the mutation. The authors should consider: (a) WHICH ribosomal genes are in each category, and (b) does either group of genes have known regulators that might be differentially affected by the baf-1 mutation? Similar consideration of other sets of differentially-affected genes might provide novel insight into specific chromatin-regulatory proteins (e.g., potential baf-1 partners; see next paragraph) affected by the NGPS mutation.

At first it may seem confusing that some ribosomal genes are downregulated while others are upregulated. However, the baf-1(G12T) mutant represents a disease situation and not a process of natural selection where one might expect “meaningful” groups of up- and down-regulated genes. We have looked closer at the individual deregulated ribosomal genes and found genes encoding structural components of large ribosomal subunits that are either upregulated (rpl-10, rpl-29, rpl-36) or downregulated (rpl-1, rpl-3, rpl-30) in the intestine. Although these opposite behaviors might seem confusing, we propose that they reflect deregulation of ribosome biosynthesis, which is in concordance with the observations in NGPS fibroblasts (Breusegem et al., 2022). We agree that it will be important to investigate how the NGPS mutation induces these oppositely directed effects on gene expression. We found a significant higher association of the 13 deregulated ribosomal genes to BAF-1(G12) than to BAF-1 in the intestine, but we believe it goes beyond the scope of this manuscript to focus on the underlying mechanisms.

3). The current manuscript is too strictly focused on establishing C. elegans as a model for NGPS, and neglects the novel discoveries. The authors did not consider or discuss HOW a baf-1 mutation might cause such complex gene expression outcomes- given that baf-1 binds dsDNA nonspecifically. One plausible molecular explanation is that the NGPS mutation might affect baf-1 interactions with: (a) transcription factors (Requiem, RBBP4, DDB1) or chromatin-regulators (PARP1; UV-regulated interactions with DDB2 and CUL4A) identified as BAF-associated in a proteomic study (Montes de Oca et al., 2009), or (b) histone modifiers such as SET/I2PP2A (blocks H3 dephosphorylation) or H3K9 methyltransferase 'G9a' (Montes de Oca et al., 2011), or (c) other regulators that control affected genes identified in this manuscript.

We agree that this point is very relevant, but at this point we do not have experimental support for any of these possibilities. As indicated in the response to Reviewer #2, we have added the following text to the Discussion: “At current, we can only speculate about how the NGPS mutation might affect gene expression. Proteomics analyses indicate that BAF interacts with several histones and transcription factors (Montes de Oca et al., 2009), and the differences between BAF-1 and BAF-1(G12T)’s chromatin binding profiles reported here might be accompanied by changes in the association of chromatin factors at the deregulated loci. A particularly interesting candidate is GCL-1/germ cell-less 1, a repressive factor involved in spermatogenesis (Holaska et al., 2003). Moreover, it is plausible that the diminished recruitment of LMN-1 to the NE in baf-1(G12T) mutants modifies its interaction with the genome and with chromatin factors.”

4). Figures 1, 2, 4, 5: the graphs in Fig 1A,B,D-F and Fig 2B,D and the colorscales in Fig 4F and Fig 5E are uninterpretable when printed in black-and-white. Please fix Figs 1 and 2 using black/light-gray/white/stippled for bar graphs, and black/light-gray/solid/dotted/dashed for line graphs. Fig 2B can be fixed by direct-labeling of class numbers within each bar (instead of 'color-coding' separately).

We thank the reviewer for this suggestion. We have modified the figures to enable better visualization when printed in BW.

5). Revise abstract lines 40-42 ("suggesting a direct relationship between BAF-1 binding [to what?] and gene expression") to reflect the deeper analysis.

We have rephrased this sentence, so it now reads: “Most genes deregulated by the baf-1(G12T) mutation were characterized by a change in BAF-1 association, suggesting a direct relation between association of a gene to BAF-1 and its expression.” However, we prefer to not extend into speculations in the abstract because of lack of experimental evidence.

6). Lines 132-155 (Figure 1): The impact on sperm production suggests the NGPS mutation might affect association with Germ cell-less (GCL), a transcription repressor that competes with BAF for binding to emerin in mammalian cells (Holaska et al., 2003 JBC).

This is indeed an interesting possibility and we have incorporated it into to Discussion (see answer to point 3 above).

7). Lines 151-154: Did not understand the fog-2 'feminized worm' experiments. Please briefly explain for non-worm experts.

Please see our response to Reviewer #1’s point 6.

8). Line 190: Clarify that nuclear shapes were categorized manually by single-blind observer.

We have amended the text: “Nuclei were manually classified by single-blind observer based on their morphology as previously described (Perez-Jimenez, Rodriguez-Palero, Rodenas, Askjaer, & Munoz, 2014), except that we introduced a fourth class to describe the most irregular nuclei (see Materials and Methods).”

9). Line 237-252: Abnormal chromosome segregation and postmitotic nuclear assembly in all gfp::baf-1(G12T) embryos is fully consistent (not 'presumably causative'; line 251) with the embryonic loss-of-function phenotype for baf-1 (Margalit et al., 2005, PNAS) and is consistent with mutational disruption of binding to lamin (Liu J et al., 2000, MBC) and/or LEM-domain proteins (Liu J, Lee KK et al., 2003, PNAS).

We thank the reviewer for pointing this out. We have added the following sentence: “These phenotypes are consistent with the effects of embryonic depletion of BAF-1 or LMN-1 (Liu et al., 2000; Margalit, Segura-Totten, Gruenbaum, & Wilson, 2005).”

10). Lines 530-533: Baf-1 localization (mobility) in intestinal cells is known to change profoundly in response to heat shock, caloric restriction or food deprivation (Bar et al., 2014, MBC). It would be worthwhile testing, in future, whether the NGPS mutation affects baf-1 localization in response to these stresses.

We appreciate this suggestion, and we agree with the reviewer that it would be important to test this in future studies.

Other changes:

Missing column in Table S3 added.

Mistake if column heading in Table S4 corrected.

Breusegem, S. Y., Houghton, J., Romero-Bueno, R., Fragoso-Luna, A., Kentistou, K. A., Ong, K. K., . . . Larrieu, D. (2022). A multiparametric anti-aging CRISPR screen uncovers a role for BAF in protein translation. bioRxiv. doi:10.1101/2022.10.07.509469

Gorjanacz, M., Klerkx, E. P., Galy, V., Santarella, R., Lopez-Iglesias, C., Askjaer, P., & Mattaj, I. W. (2007). Caenorhabditis elegans BAF-1 and its kinase VRK-1 participate directly in post-mitotic nuclear envelope assembly. Embo J, 26(1), 132-143. doi:10.1038/sj.emboj.7601470

Greil, F., Moorman, C., & van Steensel, B. (2006). DamID: mapping of in vivo protein-genome interactions using tethered DNA adenine methyltransferase. Methods Enzymol, 410, 342-359. doi:10.1016/S0076-6879(06)10016-6

Holaska, J. M., Lee, K. K., Kowalski, A. K., & Wilson, K. L. (2003). Transcriptional repressor germ cell-less (GCL) and barrier to autointegration factor (BAF) compete for binding to emerin in vitro. J Biol Chem, 278(9), 6969-6975.

Janssen, A., Marcelot, A., Breusegem, S., Legrand, P., Zinn-Justin, S., & Larrieu, D. (2022). The BAF A12T mutation disrupts lamin A/C interaction, impairing robust repair of nuclear envelope ruptures in Nestor-Guillermo progeria syndrome cells. Nucleic Acids Res. doi:10.1093/nar/gkac726

Liu, J., Rolef Ben-Shahar, T., Riemer, D., Treinin, M., Spann, P., Weber, K., . . . Gruenbaum, Y. (2000). Essential roles for Caenorhabditis elegans lamin gene in nuclear organization, cell cycle progression, and spatial organization of nuclear pore complexes. Mol Biol Cell, 11(11), 3937-3947.

Margalit, A., Segura-Totten, M., Gruenbaum, Y., & Wilson, K. L. (2005). Barrier-to-autointegration factor is required to segregate and enclose chromosomes within the nuclear envelope and assemble the nuclear lamina. Proc Natl Acad Sci U S A, 102(9), 3290-3295. doi:10.1073/pnas.0408364102

Montes de Oca, R., Shoemaker, C. J., Gucek, M., Cole, R. N., & Wilson, K. L. (2009). Barrier-to-autointegration factor proteome reveals chromatin-regulatory partners. PLoS ONE, 4(9), e7050. doi:10.1371/journal.pone.0007050

Perez-Jimenez, M. M., Rodriguez-Palero, M. J., Rodenas, E., Askjaer, P., & Munoz, M. J. (2014). Age-dependent changes of nuclear morphology are uncoupled from longevity in Caenorhabditis elegans IGF/insulin receptor daf-2 mutants. Biogerontology, 15(3), 279-288. doi:10.1007/s10522-014-9497-0

Puente, X. S., Quesada, V., Osorio, F. G., Cabanillas, R., Cadinanos, J., Fraile, J. M., . . . Lopez-Otin, C. (2011). Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome. Am J Hum Genet, 88(5), 650-656. doi:10.1016/j.ajhg.2011.04.010

Schedl, T., & Kimble, J. (1988). fog-2, a germ-line-specific sex determination gene required for hermaphrodite spermatogenesis in Caenorhabditis elegans. Genetics, 119(1), 43-61. doi:10.1093/genetics/119.1.43

Schuster, E., McElwee, J. J., Tullet, J. M., Doonan, R., Matthijssens, F., Reece-Hoyes, J. S., . . . Gems, D. (2010). DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO. Mol Syst Biol, 6, 399. doi:10.1038/msb.2010.54

Swift, J., Ivanovska, I. L., Buxboim, A., Harada, T., Dingal, P. C., Pinter, J., . . . Discher, D. E. (2013). Nuclear lamin-A scales with tissue stiffness and enhances matrix-directed differentiation. Science, 341(6149), 1240104. doi:10.1126/science.1240104

Vogel, C., & Marcotte, E. M. (2012). Insights into the regulation of protein abundance from proteomic and transcriptomic analyses. Nat Rev Genet, 13(4), 227-232. doi:10.1038/nrg3185

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Referee #3

Evidence, reproducibility and clarity

The mechanisms of rare human progeria syndromes caused by mutations in nuclear lamina proteins (lamins or BANF1) are still poorly understood, mainly because these proteins are complicated: they interact and are structurally essential for mitosis and nuclear assembly; hence, disrupting either protein can (and often does) disrupt the other. Lamins and BANF1 also have multiple interwoven roles with other partners involved in 3D genome organization, chromatin regulation, and tissue-specific gene regulation during interphase. To focus on Nestor-Guillermo progeria syndrome (NGPS), caused by the homozygous A12T missense mutation in human BANF1, the authors inserted the corresponding G12T mutation in C. elegans baf-1. They tested potential phenotypes at multiple levels (molecular, transcriptional, cellular and organismal) extensively and rigorously, and did careful controls to determine whether BAF, a tiny (89-aa) protein, was disrupted by fusion to proteins such as GFP or TurboID. Animals carrying the G12T mutation exhibited reduced lifespan (Fig. 1, S1), lower responses to UV irradiation and heat-stress (Fig 7B, 7C), and revealed unexpected germline-specific defects in male worms (Fig. S2, S3), and altered gene expression in two tissues affected by human HGPS (Figs. 4 and 5). Overall, this manuscript strongly supports the major conclusion that this C. elegans line is a powerful model for human NGPS that complements a previously reported Drosophila model.

Equally importantly, from the viewpoint of fundamental discovery, this manuscript also reports major advances in understanding how BAF influences gene expression at the molecular level. Through careful attention to controls, and experimental design, the authors overcome many complications that make BAF difficult to study: its essential roles in mitosis and early embryogenesis, the 'tag'-sensitivity of endogenous BAF, and the absolute necessity to study BAF in native cell types. The authors carefully compared the impacts of tagging either baf-1 or lamin, and compared wildtype versus G12T-mutated baf-1 interactions with lamin and emerin (Fig. S5, S6, S8, S9; videos S1 and S2). DamID-Baf-1 access to chromatin was unaffected by the G12T mutation (Fig. S7), but they successfully identified subsets of genes 'occupied' by baf-1 in specific cell types, some of which were significantly affected in opposite ways by the NGPS mutation (Fig. 4, Fig. 5). However, these important new results are described too briefly, and discussion is inadequate. E.g., in hypodermal cells, the baf-1 G12T mutation dysregulated genes encoding proteins in five categories (ribosomal, proton transport, cuticle components, cell surface, lysine acetylation), by downregulating genes in three categories (ribosomal, proton transport, histone acetylation) and upregulating three other categories (cuticle components, cell surface, apical region). In intestinal cells, the mutation dysregulated genes in 8 categories (ribosomal, response to X-ray, proton transport, proteasome binding, mitochondrial protein import, endopeptidase activity, carboxy-lyase activity, ATP generation), by downregulating genes in 5 categories (ribosomal, proton transport, peptidase activity, NAD binding, metal cluster binding) and upregulating 3 categories (ribosomal, response to external stimulation, histone acetylation). Opposite results for "ribosomal genes" is confusing. Examples of genes in each affected category are shown in Fig. 6. To fully interpret this data, and address apparently-conflicting results, further analysis is needed to determine if any affected groups of genes have shared regulators. For example, Fig 5E shows "ribosomal protein genes" are both up- and down-regulated by the mutation. The authors should consider: (a) WHICH ribosomal genes are in each category, and (b) does either group of genes have known regulators that might be differentially affected by the baf-1 mutation? Similar consideration of other sets of differentially-affected genes might provide novel insight into specific chromatin-regulatory proteins (e.g., potential baf-1 partners; see next paragraph) affected by the NGPS mutation.

The current manuscript is too strictly focused on establishing C. elegans as a model for NGPS, and neglects the novel discoveries. The authors did not consider or discuss HOW a baf-1 mutation might cause such complex gene expression outcomes- given that baf-1 binds dsDNA nonspecifically. One plausible molecular explanation is that the NGPS mutation might affect baf-1 interactions with: (a) transcription factors (Requiem, RBBP4, DDB1) or chromatin-regulators (PARP1; UV-regulated interactions with DDB2 and CUL4A) identified as BAF-associated in a proteomic study (Montes de Oca et al., 2009), or (b) histone modifiers such as SET/I2PP2A (blocks H3 dephosphorylation) or H3K9 methyltransferase 'G9a' (Montes de Oca et al., 2011), or (c) other regulators that control affected genes identified in this manuscript.

Other clarifications and revisions to improve the manuscript:

Figures 1, 2, 4, 5: the graphs in Fig 1A,B,D-F and Fig 2B,D and the colorscales in Fig 4F and Fig 5E are uninterpretable when printed in black-and-white. Please fix Figs 1 and 2 using black/light-gray/white/stippled for bar graphs, and black/light-gray/solid/dotted/dashed for line graphs. Fig 2B can be fixed by direct-labeling of class numbers within each bar (instead of 'color-coding' separately).

Revise abstract lines 40-42 ("suggesting a direct relationship between BAF-1 binding [to what?] and gene expression") to reflect the deeper analysis.

Lines 132-155 (Figure 1): The impact on sperm production suggests the NGPS mutation might affect association with Germ cell-less (GCL), a transcription repressor that competes with BAF for binding to emerin in mammalian cells (Holaska et al., 2003 JBC). Lines 151-154: Did not understand the fog-2 'feminized worm' experiments. Please briefly explain for non-worm experts.

Line 190: Clarify that nuclear shapes were categorized manually by single-blind observer.

Line 237-252: Abnormal chromosome segregation and postmitotic nuclear assembly in all gfp::baf-1(G12T) embryos is fully consistent (not 'presumably causative'; line 251) with the embryonic loss-of-function phenotype for baf-1 (Margalit et al., 2005, PNAS) and is consistent with mutational disruption of binding to lamin (Liu J et al., 2000, MBC) and/or LEM-domain proteins (Liu J, Lee KK et al., 2003, PNAS).

Line 424: Agree that this new C. elegans model is important and strongly complements the Drosophila NGPS model.

Lines 463-464: Agree that future suppressor analysis in this C. elegans model will be powerfully informative.

Lines 530-533: Baf-1 localization (mobility) in intestinal cells is known to change profoundly in response to heat shock, caloric restriction or food deprivation (Bar et al., 2014, MBC). It would be worthwhile testing, in future, whether the NGPS mutation affects baf-1 localization in response to these stresses.

Referees cross-commenting

I agree with the comments from both other reviewers.

Significance

Overall, this manuscript strongly supports the major conclusion that this C. elegans line is a powerful model for human NGPS that complements a previously reported Drosophila model.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Specific mutations in nuclear lamina proteins such as lamin A or BAF1 can cause premature aging syndromes (Huchinson Gilford progeria or Nestor Guillermo syndrome), which show age related deterioration of nuclear morphology as a hallmark and affected individuals have a severely shortened life span. In this study Romero-Bueno et al established an animal model system for the Nestor Guillermo syndrome, by generating C. elegans strains harboring homozygous baf-1::G12A mutations. They nicely recapitulate the expected cellular and organismal phenotypes: decreased life spans of the mutant animals and faster nuclear deterioration. In addition, the authors find reduced fertility in the mutant when BAF-1 was combined with a GFP tag and synthetic lethality when the baf-1::G12T mutant is introduced into strain carrying an epitope tagged Lamin allele. At the organismal level the authors report increased resistance to oxidative stress, but reduced thermotolerance and decreased UV resistance. By conducting tissue specific DamID together with tissue specific RNA polymerase DamID, the authors find that in the baf-1::G12T mutant the overall chromatin association of chromosome arms remains largely unchanged, however a few individual loci either lost or gained BAF-1 association. The authors report that loss of BAF-1 association with chromatin correlates with gene expression in some instances, however there was no strict uniform correlation. This is not surprising, because the changes in expression could be a secondary consequence of a BAF-1-mediated change of another locus or a consequence of altered lamin nucelar envelope association. The global pattern of BAF-1 and BAF-1 G12T binding to chromatin was very similar in both genotypes. The authors find unaltered localization of BAF-1 G 12T protein at the nuclear envelope, in contrast to reduced levels of lamin and emerin. Interestingly, BAF-1 is found on sperm, in contrast to the absence of other lamina proteins, like LMN-1 or Emerin.

Major comments:

1. In the baf-1 G12T mutants the authors find reduced levels of lamin in hypodermal nuclei. It would be good to also examine the dynamics of lamin in the second tissue that was subjected to DamID (intestinal cells).
2. The authors make a statement that EMR-1 expression was reduced in the baf-1 G12T mutant, but do not comment on LMN-1 expression. Can a statement on this be made by RT PCR?
3. The authors find few alterations in gene regulation of the loci which have different occupancy WT BAF-1 versus BAF-1 G12T. It was surprising to see the DamID and RNA polymerase DamID experiments be done with worms grown at 20oC, because the more penetrant phenotypes at the organismal level were observed at 25oC. Could this be the reason for the little change of chromatin occupancy of BAF-1 and BAF-1 G12T or few changes in gene expression? Would it make sense to examine the expression of some selected BAF-1 bound loci by single molecule Fish at 25oC and compare expression wt versus baf-1 G12T?

Minor comments:

The finding that BAF-1 nuclear envelope localization remains unchanged in the mutant stems from detection of the inserted GFP epitope. Given that the tag has an influence on the BAF-1 G12 12T mutant viability, this statement should be phrased with more care. The tag could influence the turnover of the protein for example. Maybe Western blots comparing the signal of WT BAF-1 worms and BAF-1 G12T mutant worms would be instructive to compare the levels of the protein (at 20oC and at 25oC, day 1 adults and day 8 adults)

Line 105: typo: remove "s"

Line 154: A conclusion is missing for the fog-2 experiment would it make sense to discuss a possible influence of altered lamin binding to the nuclear envelope in the mutant in the context of the gene expression results?

Referees cross-commenting I also see this as a valuable study. I regretted a bit that the analysis was not done at the higher temperature when the authors saw the most prominent phenotypes--but I suppose the analysis is very expensive and time consuming.

Significance

Since a long time, it has remained a matter of debate whether the progeria T to G transition in BAF-1 reduces binding of BAF-1 to lamin or whether the mutation affects the binding of BAF-1 to chromatin and thereby alters chromatin organization. Conflicting results emerged from the studies of BAF1 mutants in tissue culture cells. For this reason, this study-conducted in the context of a whole animal-is very important: it allowed the author to do their experiments with cells with unaltered ploidy, expression from the endogenous promoters and in the context of defined tissues. A second conflicting finding concerned the localization of BAF-1 G 12 to T mutant protein at the nuclear envelope: some labs find it reduced at the nuclear envelope, others find unchanged amounts at the nuclear envelope. With this work the authors contributed novel and interesting findings to those ongoing discussions, they found both altered affinity of BAF-1 with chromatin (not on a global scale, but on a local scale) and reduced affinity to lamin.

Furthermore, this is one of the first studies mapping BAF-1 association with individual gene loci in a specific tissue and the authors showed that in a given tissue BAF-1 tends to be associated with not expressed genes. In a nutshell, the authors have established a convincing accessible model system for studying aging, ready for consecutive testing interventions to reduce the pace of premature aging. Strengths: convincing presentation of a novel genetic model system to study progeria, first study where BAF-1 bound loci were shown from the analysis of a tissue (there is a correlation of BAF-1 bound loci, which are not expressed in the examined tissue), introduction of an easy-to-handle model to search for compounds suitable for clinical intervention for progeria patients or anti-aging drugs. This study adds some clarity to conflicting views in the filed: the NGS mutation in BAF-1 both reduces the amount of lamin at the nuclear periphery and affects the affinity of BAF-1 to chromatin.

Limitations: the observed transcriptional changes in the mutant can be either a direct consequence of BAF-1 chromatin association or a consequence of an altered lamina since lamin is less stable at the nuclear envelope. The transcriptomic analysis was not conducted at the temperature at which penetrant phenotypes at the organismal level were observed.

Advance: Previous studies presented conflicting results about the nuclear envelope localization of BAF-1 G12T protein: this study clearly shows that the localization of the protein remains unaltered. This study also clearly demonstrates that there is less lamin at the nuclear envelope in the mutant, lending support to the in vitro findings that the mutant is compromised in Lamin binding.

Audience: The study will be of interest to anyone who studies the nuclear lamina, the nuclear envelope, progeria, aging and stress response of an organism. Beyond this a convincing powerful novel genetic system is being presented to study progeria, which is of interest to clinicians. It is also of interest for translational research, because the system can be used to screen for compounds, which could be useful for therapeutic intervention for progeria patients or for the identification of compounds that combat aging in general. Some of the presented synthetic effects with tagged strains open the opportunity to conduct genetic suppressor screens, which would be a wonderful entry point to collect more mechanistic insights in the phenomena of aging and stress response. This genetic system is an awesome starting point for further studies and advancements of elucidating the molecular mechanism of progeria by genetic screens.

Expertise: I am a C. elegans geneticist and appreciate that all the conflicting results from tissue culture studies can now be compared to an analysis in a more physiological setting and the context of a real tissue and a living animal. I am not really competent to judge the sensitivity of RAPID assays.

Author response:

Reviewer #1 (Public Review):

This manuscript by Negi et al. investigates the effects of different ubiquitin and ubiquitin-like modifications on the stability of substrate proteins, seeking to provide mechanistic insights into known effects of these modifications on cellular protein abundance. The authors focus on comparative studies of two modifications, ubiquitin and FAT10 (a protein with two ubiquitin-like domains), on a panel of substrate proteins; prior work had established that FAT10-conjugated proteins had lower stability to proteosomal degradation than Ub-modified counterparts.

Strengths of the work include its integration of data across diverse approaches, including molecular dynamics simulations, solution NMR spectroscopy, and in vitro and cellular stability assays. From these, the authors provide provocative mechanistic insight into the lower stability of FAT10 on its own, and in FAT10-mediated destabilization of substrate proteins in computational and experimental findings. Notably, such destabilization impacts both the tag and tagged proteins, raising some provocative questions about mechanism. The data here are generally compelling, albeit with minor concerns on presentation in parts. Conclusions from this work will be interesting to scientists in several fields, particularly those interested in cellular proteostasis and in vitro protein design / long-range communication.

The most substantial weakness of this work from my perspective is the specificity of these destabilization effects. In particular, technical challenges of producing bona fide Ub- or FAT10-conjugated substrates with native linkages limits the ability to conduct in vitro studies on exactly the same molecules as being studied in cellular environments. Given some discussion in the manuscript about the importance of linkage location on the specificity of certain tag/substrate interactions, this raises an understandable but unfortunate caveat that needs to be considered more fully both in general and in light of data from other fields (e.g. single molecule pulling) showing site-dependence of comparable effects. I note that these concerns do not impact the caliber of the conclusions themselves, but perhaps suggest area for caution as to their potential impact at this time.

We thank the reviewer for positive assessment. The reviewer has pointed out the caveats regarding producing Ub- and Fat10-conjugated substrate, which we have now mentioned in the discussion in page 35 line 15.

Reviewer #2 (Public Review):

"Plasticity of the proteasome-targeting signal Fat10 enhances substrate degradation" is a nice study where the authors have shown the differences between two protein degradation tags namely, FAT10 and ubiquitin. Even though these tags are closely related in terms of folds, they have differential efficiency in degrading the substrates covalently attached to them. The authors have utilised extensive MD simulations combined with biophysics and cell biology to show the structural dynamics these tags provide for proteasomal degradation.

We thank the reviewer for positive assessment and suggestions to improve the manuscript quality.

eLife assessment

This manuscript probes the ways in which a protein tag might influence the structure, dynamics and stability of a covalently-attached substrate protein. Such findings are of important significance to several fields, particularly in understanding how these influences control the abundance of proteins within a cell. The evidence provided to support the authors' conclusions are, however, incomplete and further control experiments are necessary to fully support the proposed model.

Reviewer #1 (Public Review):

This manuscript by Negi et al. investigates the effects of different ubiquitin and ubiquitin-like modifications on the stability of substrate proteins, seeking to provide mechanistic insights into known effects of these modifications on cellular protein abundance. The authors focus on comparative studies of two modifications, ubiquitin and FAT10 (a protein with two ubiquitin-like domains), on a panel of substrate proteins; prior work had established that FAT10-conjugated proteins had lower stability to proteosomal degradation than Ub-modified counterparts.

Strengths of the work include its integration of data across diverse approaches, including molecular dynamics simulations, solution NMR spectroscopy, and in vitro and cellular stability assays. From these, the authors provide provocative mechanistic insight into the lower stability of FAT10 on its own, and in FAT10-mediated destabilization of substrate proteins in computational and experimental findings. Notably, such destabilization impacts both the tag and tagged proteins, raising some provocative questions about mechanism. The data here are generally compelling, albeit with minor concerns on presentation in parts. Conclusions from this work will be interesting to scientists in several fields, particularly those interested in cellular proteostasis and in vitro protein design / long-range communication.

The most substantial weakness of this work from my perspective is the specificity of these destabilization effects. In particular, technical challenges of producing bona fide Ub- or FAT10-conjugated substrates with native linkages limits the ability to conduct in vitro studies on exactly the same molecules as being studied in cellular environments. Given some discussion in the manuscript about the importance of linkage location on the specificity of certain tag/substrate interactions, this raises an understandable but unfortunate caveat that needs to be considered more fully both in general and in light of data from other fields (e.g. single molecule pulling) showing site-dependence of comparable effects. I note that these concerns do not impact the caliber of the conclusions themselves, but perhaps suggest area for caution as to their potential impact at this time.

Joint Public Review:

The present study explored the principles that allow cells to maintain complex subcellular proteinaceous structures despite the limited lifetimes of the individual protein components. This is particularly critical in the case of neurons, where the size and protein composition of synapses define synaptic strength and encode memory.

PSD95 is an abundant synapse protein that acts as a scaffold in the recruitment of transmitter receptors and other signaling proteins and is required for proper memory formation. The authors used super-resolution microscopy to study PSD95 super-complexes isolated from the brains of mice expressing tagged PSD variants (Halo-Tag, mEos, GFP). Their results show compellingly that a large fraction (~25%) of super-complexes contains two PSD95 copies about 13 nm apart, that there is substantial turnover of PSD95 proteins in super-complexes over a period of seven days, and that ~5-20% of the super-complexes contain new and old PSD95 molecules. This percentage is higher in synaptic fractions as compared to total brain lysates, and highest in isocortex samples (~20%). These important findings support the notion put forward by Crick that sequential subunit replacement gives synaptic super-complexes long lifetimes and thus aids in memory maintenance. Overall, this is very interesting, providing key insights into how synaptic protein complexes are formed and maintained. On the other hand, the actual role of these PSD95 super-complexes in long-term memory storage remains unknown.

Strengths

(1) The study employed an appropriate and validated methodology.

(2) Large numbers of PSD95 super-complexes from three different mouse models were imaged and analyzed, providing adequately powered sample sizes.

(3) State-of-the-art super-resolution imaging techniques (PALM and MINFLUX) were used, providing a robust, high-quality, cross-validated analysis of PSD95 protein complexes that is useful for the community.

(4) The result that PSD95 proteins in dimeric complexes are on average 12.7 nm apart is useful and has implications for studies on the nanoscale organization of PSD95 at synapses.

(5) The finding that postsynaptic protein complexes can continue to exist while individual components are being renewed is important for our understanding of synapse maintenance and stability.

(6) The data on the turnover rate of PSD95 in super-complexes from different brain regions provide a first indication of potentially meaningful differences in the lifetime of super-complexes between brain regions.

Weaknesses

(1) The manuscript emphasizes the hypothesis that stable super-complexes, maintained through sequential replacement of subunits, might underlie the long-term storage of memory. While an interesting idea, this notion requires considerably more research. The presented experimental data are indeed consistent with this notion, but there is no evidence that these complexes are causally related to memory storage.

(2) Much of the presented work is performed on biochemically isolated protein complexes. The biochemical isolation procedures rely on physical disruption and detergents that are known to alter the composition and structure of complexes in certain cases. Thus, it remains unclear how the protein complexes described in this study relate to PSD95 complexes in intact synapses.

(3) Because not all GFP molecules mature and fold correctly in vitro and the PSD95-mEos mice used were heterozygous, the interpretation of the corresponding quantifications is not straightforward.

(4) It was not tested whether different numbers of PSD95 molecules per super-complex might contribute to different retention times of PSD95, e.g. in synaptic vs. total-forebrain super-complexes.

(5) The conclusion that the population of 'mixed' synapses is higher in the isocortex than in other brain regions is not supported by statistical analysis.

(6) The validity of conclusions regarding PSD95 degradation based on relative changes in the occurrence of SiR-Halo-positive puncta is limited.

Author response:

The following is the authors’ response to the previous reviews.

Reviewer #1 (Public Review):

Key shortcomings include the unusual normalization strategies used for many experiments and the lack of quantification/statistical analyses for several experiments.

In the updated version of the paper, we have addressed all of this reviewer's criticisms. Most importantly, we have performed several additional experiments to address the concern that unusual normalization strategies were used in our paper and that quantification and statistical analyses were lacking for several experiments. We have now analyzed the full set of release conditions for Shh and engineered proteins from Disp-expressing n.t. control cells and Disp-/- cells both in the presence and absence of Scube2 (Figure 1A'-D', Figure 2E added to the paper, Figure 3B'-D', Figure 5C and Figure S2F-H). Previously, we had only quantified protein release from n.t. controls and Disp-/- cells in the presence but not in the absence of Scube2 under serum-depleted conditions. Quantifications of serum-free protein release and Shh release under conditions ranging from 0.05% FCS to 10% FCS were completely missing from the earlier versions of the manuscript, but have now been added to our paper. In addition, we have reanalyzed all of the data sets in the above figures, as well as Figures 2C and S1B, to address the issue of "unusual normalization strategies": unlike previous assays in which the highest amount of protein detected in the media was set to 100% and all other proteins in that experiment were expressed relative to that value, we now directly compare the relative amounts of cellular and corresponding solubilized proteins as a method to quantify release without the need for data normalization (Figs. 1A'-D', 2C,E, 3B'-D', E, 5C, Fig. S1B, S2F-H).

We have also repeated the qPCR analyses in C3H10T1/2 cells and now show that the same Shh/C25AShh activities can be observed when using another Shh responsive cell line, NIH3T3 cells (Fig. 4B, 6B, fig. S5B).

We would like to point out that if the criticism refers to the presentation of our RP-HPLC and SEC data, the normalization of the strongest eluted protein signal to 100% for all proteins tested is necessary to put their behavior in a clearer relationship. This is because only the relative positions of protein elution, and not their amounts, are important in these experiments.

The significance of the data provided is overstated because many of the presented experiments confirm/support previously published work.

To mitigate the first reviewer's comment that the significance of the data presented is overstated, we now clearly distinguish between our novel results and the known aspect of Hh release on lipoproteins throughout our paper. We now clearly describe what is new and important in our paper: First, contrary to the general perception in the field, Disp and Scube2 are not sufficient to solubilize Shh, casting doubt on the currently accepted model that Scube2 accepts dual-lipidated Shh from Disp and transports it to the receptor Ptch. Second, lipoproteins shift dual Shh processing to N-terminal peptide processing only to generate different soluble Hh forms with different activities (as shown in Figure 4C). Third, and again contrary to popular belief, this new release mode does not inactivate Shh, as we now show in two established cellular assays for Hh biofunction (Figures 4A-C, 5B'', 6B and S5C-G). Fourth, and most importantly, we show that spatiotemporally controlled, Disp-, Scube2- and HDL-mediated Shh release absolutely requires dual lipidation of the membrane-associated Shh precursor prior to its release. This finding (as shown in Figures 1 and S2) changes the interpretation of previously published in vivo data that have long been interpreted as evidence for the requirement of dual Shh lipidation for full receptor binding and activation.

The study provides a modest advance in our understanding of the complex issue of Shh membrane extraction.

Although we agree that our results integrate our novel observations into previously established concepts of Hh release and trafficking, we also hope that our data cast well-founded doubt on the current view that the issue of Hh release and trafficking is largely resolved by the model of Disp-mediated Shh hand-over to Scube2 and then to Ptch, which requires interactions with both Shh lipids. Our data show that this is clearly not the case in the presence of lipoproteins. Thus, the significance of our data is that models of Shh lipid-regulated signaling to Ptch obtained using the dual-lipidated Shh precursor prior to its Disp- and Scube2-mediated conversion into a delipidated or monolipidated, HDL-associated soluble ligand are likely to describe a non-physiological interaction. Instead, our work describes a highly bioactive soluble ligand with only one lipid still attached, which has not been described before in the literature. The in vivo endpoint analyses presented in Fig. S8 suggest that this new protein variant is likely to play an important role during development.

Reviewer #2 (Public Review):

The precise molecular identity (of the released Shh) remains to be defined.

We would like to respond that the direct comparison of soluble proteins and their well-defined double-lipidated precursors side-by-side in the same experiment, as shown in our paper, determines all relevant molecular changes in the Shh release process. Most importantly, we show by SDS-PAGE and RP-HPLC that HDL restricts Shh processing to the N-terminus and that the absence of HDL results in double processing of Shh during its release. We also show by SEC that the C-terminus binds the protein to HDL. In addition, the fly experiments confirm the requirement for N-terminal Hh processing, but not for processing of the C-terminal peptide, and suggest that the N-terminal Cardin-Weintraub sequence replaced by the functionally blocking tag represents the physiological cleavage site.

It would be important to demonstrate key findings in cells that secrete Shh endogenously.

We now confirm the key findings of our study in Panc1 cells that endogenously produce and secrete Shh: As shown in Fig. S1D, we find that soluble proteins are processed but retain the C-cholesterol, which we now directly confirm by RP-HPLC (Fig. S4F-H). The in vivo analyses shown in Fig. S8 suggest that the key finding - that N-terminal but not C-terminal Hh shedding is required for release - can be supported, at least in the fly: here, Hh variants impaired in their ability to be processed N-terminally strongly repress the endogenous protein, whereas the same protein impaired in its ability to be processed C-terminally does not.

The authors detect Shh variants that are expressed independently of Disp and Scube2 in secretion assays, but are excluded from interpretation as experimental artifacts.

We agree with the reviewer's criticism that the amounts of Shh released independently of Disp and Scube2 in secretion assays were not quantified and analyzed statistically to justify their proposed status as not physiologically relevant. We now show that these forms are indeed secretion artifacts (Fig. 3E and Fig. S2F-H show quantification of the lower electrophoretic mobility protein fraction (i.e., the "top" band representing the double-lipidated soluble protein fraction)) because this fraction is released independently of Disp and Scube2.

Use Templates

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved the majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study are solid and clear.

Author response:

The following is the authors’ response to the original reviews.

Reviewer 1:

(1) Peptides were synthesized with fluorescein isothiocyanate (FITC) and Tat tag, and then PEGylated with methoxy PEG Succinimidyl Succinate.

I have two concerns about the peptide design. First, FTIC was intended "for monitoring" (line 129), but was never used in the manuscript. Second, PEGylation targets the two lysine sidechains on the Tat, which would alter its penetration property.

We conducted an analysis of the cellular trafficking of FITC-tagged peptides following their permeabilization into cells.

Author response image 1.

However, we did not include it in the main text because it is a basic result.

(2) As can be seen in the figure above, after pegylation and permeabilization, the cells were stained with FITC. It appears that this does not affect the ability to penetrate into the cells.

(2) "Superdex 200 increase 10/300 GL column" (line 437) was used to isolate mono/di PEGylated PDZ and separate them from the residual PEG and PDZ peptide. "m-PEG-succinimidyl succinate with an average molecular weight of 5000 Da" (lines 133 and 134).

To my knowledge, the Superdex 200 increase 10/300 GL column is not suitable and is unlikely to produce traces shown in Figure 1B.

As Superdex 200 increase 10/300 GL featrues a fractionation range of 10,000 to 600,000 Da, we used it to fractionate PEGylated products including DiPEGylated PDZ (approx. 15 kDa) and MonoPEGylated PDZ (approx. 10 kDa) from residuals (PDZ and PEG), demonstrating successful isolation of PEGylated products (Figure 1C). Considering the molecular weights of PDZ and PEG are approximately 4.1 kDa and and 5.0 kDa, respectively, the late eluting peaks from SEC were likely to represent a mixed absorbance of PDZ and PEG at 215 nm.

However, as the reviewer pointed out, it could be unreasonable to annotate peaks representing PDZ and PEG, respectively, from mixed absorbance detected in a region (11-12 min) beyond the fractionation range.

In our revised manuscript, therefore, multiple peaks in the late eluting volume (11-12 min) were labeled as 'Residuals' all together. As a reference, the revised figure 1B includes a chromatogram of pure PDZ-WT under the same analytic condition.

Therefore, we changed Fig.1B to new results as followed:

(3) "the in vivo survival effect of LPS and PDZ co-administration was examined in mice. The pretreatment with WT PDZ peptide significantly increased survival and rescued compared to LPS only; these effects were not observed with the mut PDZ peptide (Figure 2a)." (lines 159-160).

Fig 2a is the weight curve only. The data is missing in the manuscript.

We added the survived curve into Fig. 2A as followed:

(4) Table 1, peptide treatment on ALT and AST appears minor.

In mice treated with LPS, levels of ALT and AGT in the blood are elevated, but these levels decrease upon treatment with WT PDZ. However, the use of mut PDZ does not result in significant changes. Figure 3A shows inflammatory cells within the central vein, yet no substantial hepatotoxicity is observed during the 5-day treatment with LPS. Normally, the ranges of ALT and AGT in C57BL6 mice are 16 ~ 200 U/L and 46 ~ 221 U/L, respectively, according to UCLA Diagnostic Labs. Therefore, the values in all experiments fall within these normal ranges. In summary, a 5-day treatment with LPS induces inflammation in the liver but is too short a duration to induce hepatotoxicity, resulting in lower values.

(5) MitoTraker Green FM shouldn't produce red images in Figure 6.

We changed new results (GREEN one) into Figs 6A and B as followed:

(6) Figure 5. Comparison of mRNA expression in PDZ-treated BEAS-2B cells. Needs a clearer and more detailed description both in the main text and figure legend. The current version is very hard to read.

We changed Fig. 5A to new one to understand much easier and added more detailed results and figure legend as followed:

Results Section in Figure 5:

“…we performed RNA sequencing analysis. The results of RNA-seq analysis showed the expression pattern of 24,424 genes according to each comparison combination, of which the results showed the similarity of 51 genes overlapping in 4 gene categories and the similarity between each comparison combination (Figure 5a). As a result, compared to the control group, it was confirmed that LPS alone, WT PDZ+LPS, and mut PDZ+LPS were all upregulated above the average value in each gene, and when LPS treatment alone was compared with WT PDZ+LPS, it was confirmed that they were averaged or downregulated. When comparing LPS treatment alone and mut PDZ+LPS, it was confirmed that about half of the genes were upregulated. Regarding the similarity between comparison combinations, the comparison combination with LPS…”

Figure 5 Legend Section:

“Figure 5. Comparison of mRNA expression in PDZ-treated BEAS-2B cells.

BEAS-2B cells were treated with wild-type PDZ or mutant PDZ peptide for 24 h and then incubated with LPS for 2 h, after which RNA sequencing analysis was performed. (a) The heat map shows the general regulation pattern of about 51 inflammation-related genes that are differentially expressed when WT PDZ and mut PDZ are treated with LPS, an inflammatory substance. All samples are RED = upregulated and BLUE = downregulated relative to the gene average. Each row represents a gene, and the columns represent the values of the control group treated only with LPS and the WT PDZ and mut PDZ groups with LPS. This was used by converting each log value into a fold change value. All genes were adjusted to have the same mean and standard deviation, the unit of change is the standard deviation from the mean, and the color value range of each row is the same. (b) Significant genes were selected using Gene category chat (Fold change value of 2.00 and normalized data (log2) value of 4.00). The above pie chart shows the distribution of four gene categories when comparing LPS versus control, WT PDZ+LPS/LPS, and mut PDZ+LPS/LPS. The bar graph below shows RED=upregulated, GREEN=downregulated for each gene category, and shows the number of upregulated and downregulated genes in each gene category. (c) The protein-protein interaction network constructed by the STRING database differentially displays commonly occurring genes by comparing WT PDZ+LPS/LPS, mut PDZ+LPS/LPS, and LPS. These nodes represent proteins associated with inflammation, and these connecting lines denote interactions between two proteins. Different line thicknesses indicate types of evidence used in predicting the associations.”

Reviewer 2:

(1) In this paper, the authors demonstrated the anti-inflammatory effect of PDZ peptide by inhibition of NF-kB signaling. Are there any results on the PDZ peptide-binding proteins (directly or indirectly) that can regulate LPS-induced inflammatory signaling pathway? Elucidation of the PDZ peptide-its binding partner protein and regulatory mechanisms will strengthen the author's hypothesis about the anti-inflammatory effects of PDZ peptide

As mentioned in the Discussion section, we believe it is crucial to identify proteins that directly interact with PDZ and regulate it. This direct interaction can modulate intracellular signaling pathways, so we plan to express GST-PDZ and induce binding with cellular lysates, then characterize it using the LC-Mass/Mass method. We intend to further research these findings and submit them for publication.

(2) The authors presented interesting insights into the therapeutic role of the PDZ motif peptide of ZO-1. PDZ domains are protein-protein interaction modules found in a variety of species. It has been thought that many cellular and biological functions, especially those involving signal transduction complexes, are affected by PDZ-mediated interactions. What is the rationale for selecting the core sequence that regulates inflammation among the PDZ motifs of ZO-1 shown in Figure 1A?

The rationale for selecting the core sequence that regulates inflammation among the PDZ motifs of ZO-1, as shown in Figure 1A, is grounded in the specific roles these motifs play in signal transduction pathways that are crucial for inflammatory processes. PDZ domains are recognized for their ability to function as scaffolding proteins that organize signal transduction complexes, crucial for modulating cellular and biological functions. The chosen core sequence is particularly important because it is conserved across ZO-1, ZO-2, and ZO-3, indicating a fundamental role in maintaining cellular integrity and signaling pathways. This conservation suggests that the sequence’s involvement in inflammatory regulation is not only significant in ZO-1 but also reflects a broader biological function across the ZO family.

(3) In Figure 3, the authors showed the representative images of IHC, please add the quantification analysis of Iba1 expression and PAS-positive cells using Image J or other software. To help understand the figure, an indication is needed to distinguish specifically stained cells (for example, a dotted line or an arrow).

We added the semi-quantitative results into Figs. 4d,e,f as followed:

Result section: “The specific physiological mechanism by which WT PDZ peptide decreases LPS-induced systemic inflammation in mice and the signal molecules involved remain unclear. These were confirmed by a semi-quantitative analysis of Iba-1 immunoreactivity and PAS staining in liver, kidney, and lung,respectively (Figures 4d, e, and f). To examine whether WT PDZ peptide can alter LPS-induced tissue damage in the kidney, cell toxicity assay was performed (Figure 3g). LPS induced cell damage in the kidney, however, WT PDZ peptide could significantly alleviate the toxicity, but mut PDZ peptide could not. Because cytotoxicity caused by LPS is frequently due to ROS production in the kidney (Su et al., 2023; Qiongyue et al., 2022), ROS production in the mitochondria was investigated in renal mitochondria cells harvested from kidney tissue (Figure 3h)....”

Figure legend section: “Indicated scale bars were 20 μm. (d,e,f) Semi-quantitative analysis of each are positive for Iba-1 in liver and kidney, and positive cells of PAS in lung, respectively. (g) After the kidneys were harvested, tissue lysates were used for MTT assay. (h) After...”

(4) In Figure 6G, H, the authors confirmed the change in expression of the M2 markers by PDZ peptide using the mouse monocyte cell line Raw264.7. It would be good to add an experiment on changes in M1 and M2 markers caused by PDZ peptides in human monocyte cells (for example, THP-1).

We thank you for your comments. To determine whether PDZ peptide regulates M1/M2 polarization in human monocytes, we examined changes in M1 and M2 gene expression in THP-1 cells. As a result, wild-type PDZ significantly suppressed the expression of M1 marker genes (hlL-1β, hIL-6, hIL-8, hTNF-ɑ), while increasing the expression of M2 marker genes (hlL-4, hIL-10, hMRC-1). However, mutant PDZ did not affect M1/M2 polarization. These results suggest that PDZ peptide can suppress inflammation by regulating M1/M2 polarization of human monocyte cells. These results are for the reviewer's reference only and will not be included in the main content.

Author response image 2.

Author response image 3.

Minor point:

The use of language is appropriate, with good writing skills. Nevertheless, a thorough proofread would eliminate small mistakes such as:

- line 254, " mut PDZ+LPS/LPS (45.75%) " → " mut PDZ+LPS/LPS (47.75%) "

- line 296, " Figure 6f " → " Figure 6h "

We changed these points into the manuscript.

https://www.derstandard.de/story/3000000226757/griechenland-kaempft-den-zweiten-tag-infolge-gegen-flaechenbrand-auf-kos

Rt

Reviewer #1 (Public Review):

Summary:

The manuscript by Zhou et al offers new high-resolution Cryo-EM structures of two human biotin-dependent enzymes: propionyl-CoA carboxylase (PCC) and methycrotonyl-CoA carboxylase (MCC). While X-ray crystal structures and Cryo-EM structures have previously been reported for bacterial and trypanosomal versions of MCC and for bacterial versions of PCC, this marks one of the first high resolution Cryo-EM structures of the human version of these enzymes. Using the biotin cofactor as an affinity tag, this team purified a group of four different human biotin-dependent carboxylases from cultured human Expi 293F (kidney) cells (PCC, MCC, acetyl-CoA carboxylase (ACC), and pyruvate carboxylase). Following further enrichment by size-exclusion chromatography, they were able to vitrify the sample and pick enough particles of MCC and PCC to separately refine the structures of both enzymes to relatively high average resolutions (the Cryo-EM structure of ACC also appears to have been determined from these same micrographs, though this is the subject of a separate publication). To determine the impact of substrate binding on the structure of these enzymes and to gain insights into substrate selectivity, they also separately incubated with propionyl-CoA and acetyl-CoA and vitrified the samples under active turnover conditions, yielding a set of cryo-EM structures for both MCC and PCC in the presence and absence of substrates and substrate analogues.

Strengths:

The manuscript has several strengths. It is clearly written, the figures are clear and the sample preparation methods appear to be well described. This study demonstrates that Cryo-EM is an ideal structural method to investigate the structure of these heterogeneous samples of large biotin-dependent enzymes. As a consequence, many new Cryo-EM structures of biotin-dependent enzymes are emerging, thanks to the natural inclusion of a built-in biotin affinity tag. While the authors report no major differences between the human and bacterial forms of these enzymes, it remains an important finding that they demonstrate how/if the structure of the human enzymes are or are not distinct from the bacterial enzymes. The MCC structures also provide evidence for a transition for BCCP-biotin from an exo-binding site to an endo-binding site in response to acetyl-CoA binding. This contributes to a growing number of biotin-dependent carboxylase structures that reveal BCCP-biotin binding at locations both inside (endo-) and outside (exo-) of the active site.

Weaknesses:

There are some minor weaknesses. Notably, there are not a lot of new insights coming from this paper. The structural comparisons between MCC and PCC have already been described in the literature and there were not a lot of significant changes (outside of the exo- to endo- transition) in the presence vs. absence of substrate analogues. There is not a great deal of depth of analysis in the discussion. For example, no new insights were gained with respect to the factors contributing to substrate selectivity (the factors contributing to selectivity for propionyl-CoA vs. acetyl-CoA in PCC). The authors state that the longer acyl group in propionyl-CoA may mediate stronger hydrophobic interactions that stabilize the alpha carbon of the acyl group at the proper position. This is not a particularly deep analysis and doesn't really require a cryo-EM structure to invoke. The authors did not take the opportunity to describe the specific interactions that may be responsible for the stronger hydrophobic interaction nor do they offer any plausible explanation for how these might account for an astounding difference in the selectivity for propionyl-CoA vs. acetyl-CoA. This suggests, perhaps, that these structures do not yet fully capture the proper conformational states. The authors also need to be careful with their over-interpretation of structure to invoke mechanisms of conformational change. A snapshot of the starting state (apo) and final state (ligand-bound) is insufficient to conclude *how* the enzyme transitioned between conformational states. I am constantly frustrated by structural reports in the biotin-dependent enzymes that invoke "induced conformational changes" with absolutely no experimental evidence to support such statements. Conformational changes that accompany ligand binding may occur through an induced conformational change or through conformational selection and structural snapshots of the starting point and the end point cannot offer any valid insight into which of these mechanisms is at play.

Some of these minor deficiencies aside, the overall aim of contributing new cryo-EM structures of the human MCC and PCC has been achieved. While I am not a cryo-EM expert, I see no flaws in the methodology or approach. While the contributions from these structures are somewhat incremental, it is nevertheless important to have these representative examples of the human enzymes and it is noteworthy to see a new example of the exo-binding site in a biotin-dependent enzyme.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this study, Jellinger et al. performed engram-specific sequencing and identified genes that were selectively regulated in positive/negative engram populations. In addition, they performed chronic activation of the negative engram population over 3 months and observed several effects on fear/anxiety behavior and cellular events such as upregulation of glial cells and decreased GABA levels.

Strengths:

They provide useful engram-specific GSEA data and the main concept of the study, linking negative valence/memory encoding to cellular level outcomes including upregulation of glial cells, is interesting and valuable.

Weaknesses:

A number of experimental shortcomings make the conclusion of the study largely unsupported. In addition, the observed differences in behavioral experiments are rather small, inconsistent, and the interpretation of the differences is not compelling.

Major points for improvement:

(1) Lack of essential control experiments

With the current set of experiments, it is not certain that the DREADD system they used was potent and stable throughout the 3 months of manipulations. Basic confirmatory experiments (e.g., slice physiology at 1m vs. 3m) to show that the DREADD effects on these vHP are stable would be an essential bottom line to make these manipulation experiments convincing.

In previous work from our lab performing long-term activation of Gq DREADD receptors in the vHPC, we quantify the presence of Gq receptor expression over 3-, 6- and 9-month timepoints and show that there is no decrease in receptor expression, as measured via fluorescence intensity (Suthard et al., 2023). In this study, we also address that even if our manipulation is only working for 1 month, rather than 3 months, we are observing the long-term effects of this shorter-term stimulation. This is still relevant, and only changes how we interpret these findings, as shorter-term stimulation or disruption of neuronal activity can still have detrimental effects on behavior.

Furthermore, although the authors use the mCherry vector as a control, they did not have a vehicle/saline control for the hM3Dq AAV. Thus, the long-term effects such as the increase in glial cells could simply be due to the toxicity of DREADD expression, rather than an induced activity of these cells.

For chemogenetic studies, our experimental rationale utilized a standard approach in the field, which includes one of two control options: 1) active receptor vs. control vector + ligand or 2) active receptor + ligand or saline control. We chose the first option, as this more properly controls for the potential off-target effects of the ligand itself, as shown in other previous work (Xia et al., 2017). This is particularly important for studies using CNO, as many off-target effects have been noted as a limitation (Manvich et al., 2018). We chose to use DCZ as it is closely related to CNO and newer ligands, but comes with added benefits of high specificity, low off-target effects, high potency and brain penetrance (Nagai et al., 2020), but any potential off-target effects of DCZ are yet to be completely investigated as this ligand is very new.

Evidence of DREADD toxicity has been shown at high titer levels of AAV2/7- CamKIIα-hM4D(Gi)-mCherry in the hippocampus at 5 weeks, as the reviewer pointed out in their above comment (Goossens et al., 2021). Our viral strategy is targeted to a much smaller number of cells using AAV9-DIO-Flex-hM3Dq-mCherry at a lower titer, unlike expression within a much larger population of CaMKII+ excitatory neurons in this study. Additionally, visual comparison of their viral load and expression with ours shows much more intense expression that spans a larger area of the hippocampus (Goossens et al, 2021; Figure 1D), whereas ours is isolated to a smaller region of vHPC (see Figure 1B).

Further, we attempted to quantify a decrease in neuronal health (Yousef et al., 2017) resulting from DREADD expression via NeuN counts within multiple hippocampal subregions for the 6- and 14-month groups across active Gq receptor and mCherry conditions and did not observe significant decreases in NeuN as a result (Supplemental Figure 1). However, immunohistochemistry of an individual marker may not be sufficient to capture the entire health profile of an individual neuron and future work should consider other markers of cell death or inflammation, which we have added to the Limitations & Future Work section of our Discussion.

(2) Figure 1 and the rest of the study are disconnected

The authors used the cFos-tTA system to label positive/negative engram populations, while the TRAP2 system was used for the chronic activation experiments. Although both genetic tools are based on the same IEG Fos, the sensitivity of the tools needs to be validated. In particular, the sensitivity of the TRAP2 system can be arbitrarily altered by the amount of tamoxifen (or 4OHT) and the administration protocols. The authors should at least compare and show the percentage of labeled cells in both methods and discuss that the two experiments target (at least slightly) different populations. In addition, the use of TRAP2 for vHP is relatively new; the authors should confirm that this method actually captures negative engram populations by checking for reactivation of these cells during recall by overlap analysis of Fos staining or by artificial activation.

We thank the reviewer for their comments and opportunity to discuss the marked differences between TRAP2 and DOX systems. In particular, we agree that while both systems rely on the the Fos promoter to drive an effector of interest, their efficacy and temporal resolution vary substantially depending on genetic cell-type, brain region, temporal parameters of Dox or 4-OHT delivery, subject-by-subject metabolic variability, and threshold to Fos induction given the promoter sequences inherent to each system. For example, recent studies have reported the following:

- The TRAP2 line labels a subset of endogenously activeCA1 pyramidal cells (e.g. 5-18%) while the DOX system labels 20-40% of CA1 pyramidal cells (DeNardo et al, 2019; Monasterio et al, BioRxiv 2024 ).

- The temporal windows for each range from hours in TRAP2 to 24-48 hours for DOX (DeNardo et al, 2019; Denny et al, 2014; Liu & Ramirez et al, 2012).

- The efficacy of “tagging” a population of cells with TRAP2 vs with DOX will constrain the number of possible cells that may overlap with cFos upon re-exposure to a given experience (e.g. see the observed overlaps in vCA1 - BLA circuits (Kim & Cho, 2020), compared to vCA1 in general (Ortega-de San Luis et al, 2023) and valence-specific vCA1 populations (Shpokayte et al, 2022).

- Tagging vCA1 cells with both the TRAP2 and DOX systems are nonetheless sufficient to drive corresponding behaviors (e.g. vCA1 terminal stimulation drives behavioral changes with the DOX and TRAP2 system (Shpokayte et al, 2022) and vCA1 stimulation of an updated fear-linked ensemble drives light-induced freezing in a neutral context utilizing the TRAP2 and DOX systems (Ortega-de San Luis et al, 2023)).

Finally, and promisingly, as more studies continue to link the in vivo physiological dynamics of these cell populations tagged using each system (e.g. compare Pettit et al, 2022 with Tanaka et al, 2018) and correlating their activity to behavioral phenotypes, our field is in the prime position to uncover deeper principles governing hippocampus-mediated engrams in the brain. Together, we believe a more comprehensive understanding of these systems is fully warranted, especially in the service of further cataloging cellular similarities and differences within such tagged populations.

(3)  Interpretation of the behavior data

In Figures 3a and b, the authors show that the experimental group showed higher anxiety based on time spent in the center/open area. However, there were no differences in distance traveled and center entries, which are often reduced in highly anxious mice. Thus, it is not clear what the exact effect of the manipulation is. The authors may want to visualize the trajectories of the mice's locomotion instead of just showing bar graphs.

Our findings show that our experimental group displays higher levels of anxiety-like behaviors as measured via time spent in center/open area, while there are no differences in distance traveled or center entries. For distance traveled, our interpretation is in line with complementary research (Jimenez et al, 2018; Kheirbek et al, 2013) that shows no changes in distance traveled/distance traveled in the center coupled with changes in anxiety levels as a result of manipulation within anxiety-related circuits. More broadly, any locomotion-related deficit could cause a change in distance traveled that is unrelated to anxiety-like behaviors alone. For example, a reduction in distance traveled could be coupled with a decrease in time spent in the center, but could also result only from motor or exploratory deficits. We hope that this explanation clarifies our interpretation of the open field and elevated plus maze findings in light of other literature.

In addition, the data shown in Figure 4b is somewhat surprising - the 14MO control showed more freezing than the 6MO control, which can be interpreted as "better memory in old". As this is highly counterintuitive, the authors may want to discuss this point. The authors stated that "Mice typically display increased freezing behavior as they age, so these effects during remote recall are expected" without any reference. This is nonsense, as just above in Figure 4a, older mice actually show less freezing than young mice. Overall, the behavioral effects are rather small and random. I would suggest that these data be interpreted more carefully.

In Figure 4B, we present our findings from remote recall and observe increased freezing levels in control mice with age, as mentioned by the reviewer, indicating increased memory. This is in line with previous work from Shoji & Miyakawa, 2019 which has been added as a reference for the quotation described above; we thank the reviewer for pointing this error out. As the reviewer has pointed out, above in Figure 4A, we measured freezing levels across all groups during contextual fear conditioning before the start of chronic stimulation, as this was the session we ‘tagged’ a negative memory in. Although it appears that there may be slightly lower levels of freezing in older (14-month old) mice, our findings do not determine statistical significance for difference between age group, only effects of time and subject which are expected as freezing increases within the session and animals display high levels of variability in freezing levels across many experiments (Figure 4A i-iii). We also find in previous work that control mice receiving 3-, 6- and 9-months of chronic DCZ stimulation in the vHPC with empty vector (mCherry) receptor show an increase in freezing with age (Suthard et al, 2023; Figure 2A ii).

(4) Lack of citation and discussion of relevant study

Khalaf et al. 2018 from Gräff lab showed that experimental activation of recall-induced populations leads to fear attenuation. Despite the differences in experimental details, the conceptual discrepancy should be discussed.

As mentioned by the reviewer, Khalaf et al. 2018 showed that experimental activation of recall-induced populations in the dentate gyrus leads to fear attenuation. Specifically, they pose that this fear attenuation occurs in these ensembles through updating or unlearning of the original memory trace via the engagement, rather than suppression, of an original traumatic experience. Despite the differences in experimental details with our current study and this work, we agree that the conceptual discrepancy should be discussed. First, one major difference is that we are reactivating an ensemble that was tagged during fear memory encoding, while Khalaf et al. are activating a remote recall-induced ensemble that was tagged one month after encoding. Although there is high overlap between the encoding and recall ensembles when mice are exposed to the conditioning context, these ensembles are not identical and may result in different behavioral phenotypes when chronically reactivated. Further, Khalaf et al rely on reactivation of the recall-induced ensemble during extinction to facilitate rapid fear attenuation. This differs from our current work, as their reactivation is occurring during the extinction process in the previously conditioned context, while we are reactivating chronically in the animal’s home cage over the course of a longer time period. It may be necessary that the memory is first reactivated, and thus, more liable to re-contextualization, in the original context compared to an unrelated homecage environment where there are presumably no related cues present. Importantly, this previous work tests the attenuation of fear shortly after an extinction process, while we are not traditionally extinguishing the context with aid of the memory reactivation. Finally, we are testing remote recall (3 months post-conditioning), while they are testing at a shorter time interval (28 days). In line with these ideas, future work may seek to tease out the mechanistic differences between recent and remote memory extinction both in terms of natural memory recall and chronically manipulated memory-bearing cells.

Reviewer #2 (Public Review):

Summary:

Jellinger, Suthard, et al. investigated the transcriptome of positive and negative valence engram cells in the ventral hippocampus, revealing anti- and pro-inflammatory signatures of these respective valences. The authors further reactivated the negative valence engram ensembles to assay the effects of chronic negative memory reactivation in young and old mice. This chronic re-activation resulted in differences in aspects of working memory, and fear memory, and caused morphological changes in glia. Such reactivation-associated changes are putatively linked to GABA changes and behavioral rumination.

Strengths:

Much of the content of this manuscript is of benefit to the community, such as the discovery of differential engram transcriptomes dependent on memory valence. The chronic activation of neurons, and the resultant effects on glial cells and behavior, also provide the community with important data. Laudable points of this manuscript include the comprehensiveness of behavioral experiments, as well as the cross-disciplinary approach.

Weaknesses:

There are several key claims made that are unsubstantiated by the data, particularly regarding the anthropomorphic framing of "rumination" on a mouse model and the role of GABA. The conclusions and inferences in these areas need to be carefully considered.

(1) There are many issues regarding the arguments for the behavioural data's human translation as "rumination." There is no definition of rumination provided in the manuscript, nor how rumination is similar/different to intrusive thoughts (which are psychologically distinct but used relatively interchangeably in the manuscript), nor how rumination could be modelled in the rodent. The authors mention that they are attempting to model rumination behaviours by chronically reactivating the negative engram ("To understand if our experimental model of negative rumination..."), but this occurs almost at the very end of the results section, and no concrete evidence from the literature is provided to attempt to link the behavioural results (decreased working memory, increased fear extinction times) to rumination-like behaviours. The arguments in the final paragraph of the Discussion section about human rumination appear to be unrelated to the data presented in the manuscript and contain some uncited statements. Finally, the rumination claims seem to be based largely upon a single data figure that needs to be further developed (Figure 6, see also point 2 below).

(2) The staining and analysis in Figure 6 are challenging to interpret, and require more evidence to substantiate the conclusions of these results. The histological images are zoomed out, and at this resolution, it appears that only the pyramidal cell layer is being stained. A GABA stain should also label the many sparsely spaced inhibitory interneurons existing across all hippocampal layers, yet this is not apparent here. Moreover, both example images in the treatment group appear to have lower overall fluorescence intensity in both DAPI and GABA. The analysis is also unclear: the authors mention "ROIs" used to measure normalized fluorescence intensity but do not specify what the ROI encapsulates. Presumably, the authors have segmented each DAPI-positive cell body and assessed fluorescence however, this is not explicated nor demonstrated, making the results difficult to interpret.

Based on the collective discussion from all reviewers on the completeness of our GABA quantification and its implications, we have decided to remove this figure and perform more substantive analysis of this E/I imbalance in future work.

(3) A smaller point, but more specific detail is needed for how genes were selected for GSEA analysis. As GSEA relies on genes to be specified a priori, to avoid a circular analysis, these genes need to be selected in a blind/unbiased manner to avoid biasing downstream results and conclusions. It's likely the authors have done this, but explicitly noting how genes were selected is an important context for this analysis.

As mentioned in our Methods section, gene sets were selected based on pre-existing biology and understanding of genes canonically involved in “neurodegeneration” such as those related to apoptotic pathways and neuroinflammation or “neuroprotection” such as brain-derived neurotrophic factor, to name a few. A limitation of this method is that we must avoid making strong claims about the actual function of these up- or down-regulated genes without performing proper knock-in or knock-out studies, but we hope that this provides an unbiased inventory for future experiments to perform causal manipulations.

Reviewer #3 (Public Review):

Summary:

The authors note that negative ruminations can lead to pathological brain states and mood/anxiety dysregulation. They test this idea by using mouse engram-tagging technology to label dentate gyrus ensembles activated during a negative experience (fear conditioning). They show that chronic chemogenetic activation of these ensembles leads to behavioral (increased anxiety, increased fear generalization, reduced fear extinction) and neural (increases in neuroinflammation, microglia, and astrocytes).

Strengths:

The question the authors ask here is an intriguing one, and the engram activation approach is a powerful way to address the question. Examination of a wide range of neural and behavioral dependent measures is also a strength.

Weaknesses:

The major weakness is that the authors have found a range of changes that are correlates of chronic negative engram reactivation. However, they do not manipulate these outcomes to test whether microglia, astrocytes, or neuroinflammation are causally linked to the dysregulated behaviors.

Recommendations For The Authors:

Reviewer #1 (Recommendations For The Authors):

- Figure 2c should include Month0, the BW before the start of the manipulation.

Regrettably, we do not have access to the Month 0 body weights at this time as this project changed hands over the course of the past year or so. This is an inherent limitation that we missed during analysis and we pose this as a limitation in the Results section after describing this finding. Therefore, it is possible that over the first month of stimulation (Month 0-1), there may have been a drop in body weight that rebounded by the first measurement at Month 1 that continued to increase normally through Months 2-3, as shown in our Figure 1. Thank you for this note.

- Figure 6a looks confusing - the background signal in the green channel is very different between control and experimental groups. Were representative images taken with different microscope settings?

The representative images were taken with the same microscope power settings, but were adjusted in brightness/contrast within FIJI for clarity in the Figure – we apologize that this was misleading in any way and thank the reviewer for their feedback. Further, based on the collective discussion from all reviewers on the completeness of our GABA quantification and its implications, we have decided to remove this figure and perform more substantive analysis of this E/I imbalance in future work.

- Typo mChe;try

This typo was fixed

- "During this contextual... mice in the 6- and 14- month groups..." Isn't it 3- and 11- month respectively at the time of fear conditioning? Throughout the manuscript, this point was written very confusingly.

Yes, we thank the reviewer for pointing this out. It has been corrected to 3- and 11-month old mice at the timing of fear conditioning and clarified throughout the manuscript where applicable.

- "GABAergic eYFP fluorescence" Where does the eYFP come from? The methods state that GABA quantification is based on IHC staining.

Based on the collective discussion from all reviewers on the completeness of our GABA quantification and its implications, we have decided to remove this figure and perform more substantive analysis of this

E/I imbalance in future work. We discuss this E/I balance not being directly assessed in the Limitations & Future Directions section of our Discussion, noting the importance of detailed quantification of both excitatory and inhibitory markers within the hippocampus.

Reviewer #2 (Recommendations For The Authors):

(1) There is a full methods section ("Analysis of RNA-seq data") that mostly describes RNA-seq analysis that seemingly does not appear in the paper. This section should be reviewed.

We have included this portion of the methods that explain the previous workflow from Shpokayte et al., 2022 where this dataset was generated and this has been noted in the “Analysis of RNA-seq data” section of the methods.

(2) Figure 6: GABA staining should be more critically analyzed, as discussed above, and validated with another GABA antibody for rigor. From the representative images provided in Figure 6, it looks possibly as though the hM3Dq images were simply not fully in the focal plane when being imaged or were over-washed, as DAPI staining also appears to be lower in these images.

Based on the collective discussion from all reviewers on the completeness of our GABA quantification and its implications, we have decided to remove this figure and perform more substantive analysis of this E/I imbalance in future work. Specifically, it will be necessary to rigorously investigate both excitatory and inhibitory markers within this region to ensure these claims are substantiated. Thank you for this suggestion.

(3) The first claim that human GABAergic interneurons cause rumination is uncited. (Page 19, first sentence beginning with: "Evidence from human studies suggests...").

Based on the collective discussion from all reviewers on the completeness of our GABA quantification and its implications, we have decided to remove this figure and perform more substantive analysis of this E/I imbalance in future work. Apologies for the lack of citation in-text, the proper citation for this finding is Schmitz et al, 2017.

(4) Gene names throughout the manuscript and figure are written in the wrong format for mice (eg: Page 13, second line: SPP1, TTR, and C1QB1 instead of Spp1, Ttr, C1qb1).

This was corrected throughout the manuscript.

(5) Tense on Page 15 third sentence of the second paragraph: "...spatial working memory was assessed...".

This was corrected throughout the manuscript.

(6) Supplemental Figure 1 would benefit from normalization of the NeuN+ cell counts. The inclusion of an excitatory and inhibitory neuron marker in this figure might benefit the argument that there is a change in the excitation/inhibition of the hippocampus - as the numbers of excitatory neurons outweigh the numbers of inhibitory neurons that would be assayed here.

In an effort to normalize the NeuN+ cell counts, for each of our ROIs (6-8 single tiles for each brain region (DG, vCA1, vSub) x 3-5 coronal slices = ~18 single tiles per mouse x 3-4 mice) we captured a 300 x 300 micrometer, single-tile z-stack at 20x magnification. These ROIs were matched for dimensions and brain regions across all groups for each hippocampal subregion quantified. We initially proposed to normalize these NeuN counts over DAPI, but because DAPI includes all nuclei (microglia, oligodendrocytes, astrocytes and neurons), we weren’t sure this was the most optimal tool. We do agree that further quantification of excitatory and inhibitory cell markers would be vital to more concrete interpretation of our findings and we have added this to our Limitations & Future Work section of the Discussion.

Reviewer #3 (Recommendations For The Authors):

(1) The DOX tagging window lacks temporal precision. I suggest the authors note this as a limitation.

We thank the reviewer for noting this, and we have added this limitation to the Methods section with the context of the 24-48 hour DOX window being longer than other methods like TRAP.

(2) Is there a homeostatic response to chronic engram stimulation? That is, is DCZ as effective in increasing neuronal excitability on day 90 as it is on day 1. This could be addressed with electrophysiology, or with IEG induction. Alternatively, the authors could refer to previous literature-- for example, Xia et al (2017) eLife-- that examined whether there was any blunting of the effects of DREADD ligands after sustained delivery via drinking water. There, of course, may be other papers as well.

As noted by the reviewer, it is important to determine if DCZ maintains its effects on neuronal excitability throughout the 3 month administration period. To address this, previous work has shown that CNO administration in drinking water over one month consistently inhibited hM4Di+ neurons without altering baseline neuronal excitability as measured by firing rate and potassium currents (Xia et al, 2017). Although this is only for one month, it is administered via the same oral route as our DCZ protocol and suggests that at least for that amount of time we are likely producing consistent effects. In our reply above to Reviewer #1’s comment, we also note that even if DCZ is only having an effect for one month, rather than 3 months, we are still observing enduring changes that resulted from this short-term disturbance.

(3) Please double check there is no group effect on weight in 6-month-old mice in Figure 2C.

Two-way RM ANOVA showed no main effect of Group within the 6-month-old control and hM3Dq groups.

Group: F(1,17) = 1.361, p=0.2594.

(4) The shock intensity is much higher than is typical for fear conditioning studies in mice. Why was this the case?

Yes, we do agree that this shock intensity is on the higher side of typical paradigms in mice, however, our lab has utilized 0.75mA to 1.5mA intensity foot shocks for contextual fear conditioning in the past (Suthard & Senne et al, 2023; 2024; Dorst & Senne et al, 2023; Grella et al., 2022; Finkelstein et al., 2022) and we maintained this protocol for internal consistency. However, it would be interesting to systematically investigate how differing intensities of foot shock, subsequent tagging of this ensemble and reactivation would uniquely impact behavioral state acutely and chronically in mice.

(5) Remote freezing is very low. The authors should comment on this-- perhaps repeated testing has led to some extinction?

A reviewer above suggested a similar phenomenon may be occuring, specifically fear attenuation as a result of chronic stimulation. They referenced previous work from Khalaf et al. 2018, where they reactivated a recall-induced ensemble, while we reactivated an ensemble tagged during encoding. We expand upon this work in light of our findings within the Limitations & Future Work section of our Discussion. However, we do appreciate the lower levels of freezing observed in remote recall and sought out other literature to understand the typical range of remote freezing levels. One thing that we note is that our remote recall is occurring 3 months after conditioning, which is much longer than typical 14-28 day protocols. However, we find that freezing levels at remote timepoints from 21-45 days results in contextual freezing levels of between 20-50% approximately (Kol et al., 2020), as well as 40-75% approximately in a variety of 28 day remote recall experiments (Lee et al., 2023). This information, together with our current experimental protocol demonstrates a wide range of remote freezing levels that may depend heavily on the foot shock intensity, duration of days after conditioning, and animal variability.

(6) "mice display increased freezing with age": please add a reference.

Apologies, we missed the citation for that claim and it has been added in-text and in the references list (Shoji & Miyakawa, 2019).

(7) Related to the low freezing levels for remote memory, why is generalization minimal? Many studies have shown that there is a time-dependent emergence of generalized fear, yet here this is not seen. Is it linked to extinction (as above)? Or genetic background?

Previous work has shown that rats receiving multiple foot shocks during conditioning displayed a time-dependent generalization of context memory, while those receiving less shocks did not (Poulos et al., 2016), as the reviewer noted in their comment. In our current study, we observe low levels of generalization in all of our groups compared to freezing levels displayed in the conditioned context at the remote timepoint, in opposition to this time-dependent enhancement of generalization. It is possible that the genetic background of our C57BL/6J mice compared to the Long-Evans rat strain in this previous work accounts for some of this difference. In addition, it is possible that the longer duration of time (3 months) compared to their remote timepoint (28 days) resulted in time-dependent decrease in generalization that decreases with greater durations of time from original conditioning. As noted above, it is indeed plausible that the reactivation of a contextual fear ensemble over time is attenuating freezing levels for both the original and similar contexts (Khalaf et al, 2018). We discuss the differences in our study and this 2018 work more comprehensively above.

(8) Morphological phenotypes of astrocytes/microglia. Would be great to do some transcriptomic profiling of microglia/astrocytes to couple with the morphological characterization (but appreciate this is beyond the scope of current work).

We thank the reviewer this suggestion, we agree that would be an incredibly informative future experiment and have added this to our Limitations & Future Experiments section of the Discussion.

(9) The authors could consider including a limitations section in their discussion which discusses potential future directions for this work:

- causal experiments.

- E/I balance is not assessed directly (interestingly, in this regard, expanded engrams are linked to increased generalization [e.g., Ramsaran et al 2023]).

Thank you for this suggestion, we have added a Limitations & Future Directions section to our Discussion and have expanded upon these suggested points.

(10) For Figure 10, consider adding an experimental design/timeline.

We are making the assumption that the reviewer meant Figure 1 instead of Figure 10 here, but note that there is a description of the viral expression duration (D0-D10), followed by an off Dox period of 48 hours (D10-D12), with subsequent engram tagging of a negative (foot shock) or positive (male-to-female exposure) on D12. In our experiments (Shpokayte et al., 2022), Dox was administered for 24 hours (D12-D13), which was followed by sacrificing the animal for cell suspension and sequencing of the positive and negative engram populations. This figure also shows the viral strategy for the Tet-tag system (Figure 1A), as well as representative viral expression in vHPC (Figure 1B). We are happy to add additional experimental design/timeline information to this figure that would be helpful to the reviewer.

Reviewer #2 (Public Review):

Summary:

The paper sought to determine the number of myosin 10 molecules per cell and localized to filopodia, where they are known to be involved in formation, transport within, and dynamics of these important actin-based protrusions. The authors used a novel method to determine the number of molecules per cell. First, they expressed HALO tagged Myo10 in U20S cells and generated cell lysates of a certain number of cells and detected Myo10 after SDS-PAGE, with fluorescence and a stained free method. They used a purified HALO tagged standard protein to generate a standard curve which allowed for determining Myo10 concentration in cell lysates and thus an estimate of the number of Myo10 molecules per cell. They also examined the fluorescence intensity in fixed cell images to determine the average fluorescence intensity per Myo10 molecule, which allowed the number of Myo10 molecules per region of the cell to be determined. They found a relatively small fraction of Myo10 (6%) localizes to filopodia. There are hundreds of Myo10 in each filopodia, which suggests some filopodia have more Myo10 than actin binding sites. Thus, there may be crowding of Myo10 at the tips, which could impact transport, the morphology at the tips, and dynamics of the protrusions themselves. Overall, the study forms the basis for a novel technique to estimate the number of molecules per cell and their localization to actin-based structures. The implications are broad also for being able to understand the role of myosins in actin protrusions, which is important for cancer metastasis and wound healing.

Strengths:

The paper addresses an important fundamental biological question about how many molecular motors are localized to a specific cellular compartment and how that may relate to other aspects of the compartment such as the actin cytoskeleton and the membrane. The paper demonstrates a method of estimating the number of myosin molecules per cell using the fluorescently labeled HALO tag and SDS-PAGE analysis. There are several important conclusions from this work in that it estimates the number of Myo10 molecules localized to different regions of the filopodia and the minimum number required for filopodia formation. The authors also establish a correlation between number of Myo10 molecules filopodia localized and the number of filopodia in the cell. There is only a small % of Myo10 that tip localized relative to the total amount in the cell, suggesting Myo10 have to be activated to enter the filopodia compartment. The localization of Myo10 is log-normal, which suggests a clustering of Myo10 is a feature of this motor.

One of the main critiques of the manuscript was that the results were derived from experiments with overexpressed Myo10 and therefore are hard to extrapolate to physiological conditions. The authors counter this critique with the argument that their results provide insight into a system in which Myo10 is a limiting factor for controlling filopodia formation. They demonstrate that U20S cells do not express detectable levels of Myo10 (supplementary Figure 1E) and thus introducing Myo10 expression demonstrates how triggering Myo10 expression impacts filopodia. An example is given how melanoma cells often heavily upregulation Myo10.

In addition, the revised manuscript addresses the concerns about the method to quantitate the number of Myo10 molecules per cell and therefore puncta in the cell. The authors have now made a good faith effort to correct for incomplete labeling of the HALO tag (Figure 2A-C, supplementary Figure 2D-E). The authors also address the concerns about variability in transfection efficiency (Figure 1D-E).

A very interesting addition to the revised manuscript was the quantitation of the number of Myo10 molecules present during an initiation event when a newly formed filopodia just starts to elongate from the plasma membrane. They conclude that 100s of Myo10 molecules are present during an initiation event. They also examined other live cell imaging events in which growth occurs from a stable filopodia tip and correlated with elongation rates.

Weaknesses:

The authors acknowledge that a limitation of the study is that all of the experiments were performed with overexpressed Myo10. They address this limitation in the discussion but also provide important comparisons for how their work relates to physiological conditions, such as melanoma cells that only express large amounts of Myo10 when they are metastatic. Also, the speculation about how fascin can outcompete Myo10 should include a mechanism for how the physiological levels of fascin can complete with the overabundance of Myo10 (page 10, lines 401-408).

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

This valuable study reports on the packing of molecules in cellular compartments, such as actin-based protrusions. The study provides solid evidence for parameters that enable the building of a biophysical model of filopodia, which is required to gain a complete understanding of these important actin-based structures. Some areas of the manuscript require further clarification.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript proposes an alternative method by SDS-PAGE calibration of Halo-Myo10 signals to quantify myosin molecules at specific subcellular locations, in this specific case filopodia, in epifluorescence datasets compared to the more laborious and troublesome single molecule approaches. Based on these preliminary estimates, the authors developed further their analysis and discussed different scenarios regarding myosin 10 working models to explain intracellular diffusion and targeting to filopodia.

Strengths:

Overall, the paper is elegantly written and the data analysis is appropriately presented.

Weaknesses:

While the methodology is intriguing in its descriptive potential and could be the beginning of an interesting story, a good portion of the paper is dedicated to the discussion of hypothetical working mechanisms to explain myosin diffusion, localization, and decoration of filopodial actin that is not accompanied by the mandatory gain/loss of function studies required to sustain these claims.

To be fair, the detailed mechanisms that we raise related to diffusion, localization, and decoration are based on extensive work by others. Many prior papers use domain deletions of Myo10 and fall in the category of gain/loss-of-function studies. It is true that we have not repeated those extensive studies, but it seems appropriate to connect with and cite their work where appropriate.

Reviewer #2 (Public Review):

Summary:

The paper sought to determine the number of myosin 10 molecules per cell and localized to filopodia, where they are known to be involved in formation, transport within, and dynamics of these important actin-based protrusions. The authors used a novel method to determine the number of molecules per cell. First, they expressed HALO tagged Myo10 in U20S cells and generated cell lysates of a certain number of cells and detected Myo10 after SDS-PAGE, with fluorescence and a stained free method. They used a purified HALO tagged standard protein to generate a standard curve which allowed for determining Myo10 concentration in cell lysates and thus an estimate of the number of Myo10 molecules per cell. They also examined the fluorescence intensity in fixed cell images to determine the average fluorescence intensity per Myo10 molecule, which allowed the number of Myo10 molecules per region of the cell to be determined. They found a relatively small fraction of Myo10 (6%) localizes to filopodia. There are hundreds of Myo10 in each filopodia, which suggests some filopodia have more Myo10 than actin binding sites. Thus, there may be crowding of Myo10 at the tips, which could impact transport, the morphology at the tips, and dynamics of the protrusions themselves. Overall, the study forms the basis for a novel technique to estimate the number of molecules per cell and their localization to actin-based structures. The implications are broad also for being able to understand the role of myosins in actin protrusions, which is important for cancer metastasis and wound healing.

Strengths:

The paper addresses an important fundamental biological question about how many molecular motors are localized to a specific cellular compartment and how that may relate to other aspects of the compartment such as the actin cytoskeleton and the membrane. The paper demonstrates a method of estimating the number of myosin molecules per cell using the fluorescently labeled HALO tag and SDS-PAGE analysis. There are several important conclusions from this work in that it estimates the number of Myo10 molecules localized to different regions of the filopodia and the minimum number required for filopodia formation. The authors also establish a correlation between number of Myo10 molecules filopodia localized and the number of filopodia in the cell. There is only a small % of Myo10 that tip localized relative to the total amount in the cell, suggesting Myo10 have to be activated to enter the filopodia compartment. The localization of Myo10 is log-normal, which suggest a clustering of Myo10 is a feature of this motor.

Weaknesses:

One main critique of this work is that the Myo10 was overexpressed. Thus, the amount in the cell body compared to the filopodia is difficult to compare to physiological conditions. The amount in the filopodia was relatively small - 100s of molecules per filopodia so this result is still interesting regardless of the overexpression. However, the overexpression should be addressed in the limitations.

This is a reasonable perspective and we now note this caveat in the Limitations section so that readers will take note. Our goal here was to understand a system in which Myo10 is the limiting reagent for filopodia, rather than a native system that expresses high Myo10 on its own. Because U2OS cells do not express detectable levels of Myo10 (see below), the natural perturbation here is overexpressing Myo10 to stimulate filopodial growth.

The authors have not addressed the potential for variability in transfection efficiency. The authors could examine the average fluorescence intensity per cell and if similar this may address this concern.

Indeed, cells are heterogenous and will naturally express different levels of Myo10 not only due to transfection efficiency, but also due to their state (cell cycle stage, motile behavior, and more). In fact, we measure the transfection efficiency of each bioreplicate and account for it in our calibration procedure. We also measure the fluorescence intensity per cell, which lets us calculate the total Myo10s per cell and the cell-to-cell variability. These Myo10 distributions across cells are shown in Fig. 1D-E.

We note here an error that we made in applying this transfection efficiency correction in the first submission. When we obtain the total Myo10 molecules by SDS-PAGE, we should divide by the total number of transfected cells. However, due to an operator precedence error, the transfection efficiency appeared in the numerator rather than the denominator. We have now corrected this error, which has the effect of increasing the number of molecules in all of our measurements. The effect of this correction has strengthened one of the paper’s main conclusions, that Myo10 is frequently overloaded at filopodial tips.

The SDS PAGE method of estimating the number of molecules is quite interesting. I really like this idea. However, I feel there are a few more things to consider. The fraction of HALO tag standard and Myo10 labeled with the HALO tagged ligand is not determined directly. It is suggested that since excess HALO tagged ligand was added we can assume nearly 100% labeling. If the HALO tag standard protein is purified it should be feasible to determine the fraction of HALO tagged standard that is labeled by examining the absorbance of the protein at 280 and fluorophore at its appropriate wavelength.

This is a fair point raised by the reviewer, and we have now measured a labeling efficiency of 90% in Supplementary Figure 2A-C. We have adjusted all values according to this labeling efficiency.

The fraction of HALO tagged Myo10 labeled may be more challenging to determine, since it is in a cell lysate, but there may be some potential approaches (e.g. mass spec, HPLC).

As noted, this value is considerably more challenging. Instead, we determined conditions under which labeling in cells is saturated. We have now stained with a concentration range for both fixed and live cell samples. Saturation occurs with ~0.5 μM HaloTag ligand-TMR in fixed/permeabilized cells and in live cells (Supplementary Figure 2D-E). This comparison of live cells vs. permeabilized cells allows us to say that the intact plasma membrane is not limiting labeling under these conditions.

In Figure 1B, the stain free gel bands look relatively clean. The Myo10 is from cell lysates so it is surprising that there are not more bands. I am not surprised that the bands in the TMR fluorescence gel are clean, and I agree the fluorescence is the best way to quantitate.

Figure 1B shows the focused view at high MW, and there is not much above Myo10. The full gel lanes shown in Supp. Fig. 1C show the expected number of bands from a cell lysate.

In Figure 3C, the number of Myo10 molecules needed to initiate a filopodium was estimated. I wonder if the authors could have looked at live cell movies to determine that these events started with a puncta of Myo10 at the edge of the cell, and then went on to form a filopodia that elongated from the cell. How was the number of Myo10 molecules that were involved in the initiation determined? Please clarify the assumptions in making this conclusion.

We thank the reviewer (and the other reviewers) for this excellent suggestion. We have now carried out these live cell experiments. These experiments were quite challenging, because we needed to collect snapshots of ~50 cells to measure the mean fluorescence intensity of transfected cells and then acquire movies of several cells for analysis. The U2OS cells were also highly temperature-sensitive and would retract their filopodia without objective heating.

We have now analyzed filopodial initiation events and measured considerably more Myo10 at the first signs of accumulation– in the 100s of molecules. The dimmer spots that we measured in the first draft were likely unrelated to filopodial initiation, and we have corrected the discussion on this point.

We now also track further growth from a stable filopodial tip (the phased-elongation mechanism from Ikebe and coworkers) and find approximately 500 molecules bud off in those events. We also track filopodial elongation rates as a function of Myo10 numbers. We have added additional live cell imaging sections that include these results.

It is stated in the discussion that the amount of Myo10 in the filopodia exceeds the number of actin binding sites. However, since Myo10 contains membrane binding motifs and has been shown to interact with the membrane it should be pointed that the excess Myo10 at the tips may be interacting with the membrane and not actin, which may prevent traffic jams.

This is also an excellent point to consider, and we have expanded the relevant discussion along these lines. We agree that the Myo10 at the filopodial tip is likely membrane-bound. We now estimate the 2D membrane area occupied by Myo10, and find that it reaches nearly full packing in many cases (under a number of assumptions that we spell out more fully in the manuscript).

Reviewer #3 (Public Review):

Summary:

The unconventional myosin Myo10 (aka myosin X) is essential for filopodia formation in a number of mammalian cells. There is a good deal of interest in its role in filopodia formation and function. The manuscript describes a careful, quantitative analysis of Myo10 molecules in U2OS cells, a widely used model for studying filopodia, how many are present in the cytosol versus filopodia and the distribution of filopodia and molecules along the cell edge. Rigorous quantification of Myo10 protein amounts in a cell and cellular compartment are critical for ultimately deciphering the cellular mechanism of Myo10 action as well as understand the molecular composition of a Myo10-generated filopodium.

Consistent with what is seen in images of Myo10 localization in many papers, the vast majority of Myo10 is in the cell body with only a small percentage (appr 5%) present in filopodia puncta. Interestingly, Myo10 is not uniformly distributed along the cell edge, but rather it is unevenly localized along the cell edge with one region preferentially extending filopodia, presumably via localized activation of Myo10 motors. Calculation of total molecules present in puncta based on measurement of puncta size and measured Halo-Myo10 signal intensity shows that the concentration of motor present can vary from 3 - 225 uM. Based on an estimation of available actin binding sites, it is possible that Myo10 can be present in excess over these binding sites.

Strengths:

The work represents an important first step towards defining the molecular stoichiometry of filopodial tip proteins. The observed range of Myo10 molecules at the tip suggests that it can accommodate a fairly wide range of Myo10 motors. There is great value in studies such as this and the approach taken by the authors gives one good confidence that the numbers obtained are in the right range.

Weaknesses:

One caveat (see below) is that these numbers are obtained for overexpressing cells and the relevance to native levels of Myo10 in a cell is unclear.

A similar concern was raised by Reviewer 2; please see above.

An interesting aspect of the work is quantification of the fraction of Myo10 molecules in the cytosol versus in filopodia tips showing that the vast majority of motors are inactive in the cytosol, as is seen in images of cells. This has implications for thinking about how cells maintain this large population in the off-state and what is the mechanism of motor activation. One question raised by this work is the distinction between cytosolic Myo10 and the population found at the ‘cell edge’ and the filopodia tip. The cortical population of Myo10 is partially activated, so to speak, as it is targeted to the cortex/membrane and presumably ready to go. Providing quantification of this population of motors, that one might think of as being in a waiting room, could provide additional insight into a potential step-by-step pathway where recruitment or binding to the cortical region/plasma membrane is not by itself sufficient for activation.

As mentioned in our response to Reviewer 2, we have now carried out quantitation in live cells to capture Myo10 transitions from cell body into filopodial movement. We attempted to identify this membrane-bound population of motors in our new live cell experiments but were unable to make convincing measurements. Notably, we see no noticeable enrichment of Myo10 at the cortex relative to the cytosol. Although we believe there is a membrane-bound waiting room (akin to the 3D-2D-1D mechanism of Molloy and Peckham), we suspect that the 2D population is diffusing too rapidly to be detected under our imaging conditions.

Specific comments:

(1) It is not obvious whether the analysis of numbers of Myo10 molecules in a cell that is ectopically overexpressing Myo10 is relevant for wild type cells. It would appear to be a significant excess based on the total protein stained blot shown in Fig S1E where a prominent band the size of tagged Myo10 seen in the transfected sample is almost absent in the WT control lane.

Even “wildtype” cells vary considerably in their Myo10 expression levels. For example, melanoma cells often heavily upregulate Myo10, while these U2OS cells produce nearly none (Supplementary Figure 1E). Thus, there is no single, widely acceptable target for Myo10 expression in wildtype cells.

Please note that the new Supplementary Figure 1E is a Myo10 Western blot, not total protein staining as before.

Ideally, and ultimately an important approach, would be to work with a cell line expressing endogenously tagged Myo10 via genome engineering. This can be complicated in transformed cells that often have chromosomal duplications.

Indeed, we chose U2OS cells for this work because they do not express detectable levels of Myo10, and thus we can avoid all of these complications. Here we can examine how Myo10 levels control filopodial production through ectopic expression.

However, even though there is an excess of Myo10 it would appear that activation is still under some type of control as the cytosolic pool is quite large and its localization to the cell edge is not uniform. But it is difficult to gauge whether the number of molecules in the filopodium is the same as would be seen in untransfected cells. Myo10 can readily walk up a filopodium and if excess numbers of this motor are activated they would accumulate in the tip in large numbers, possibly creating a bulge as and indeed it does appear that some tips are unusually large. Then how would that relate to the normal condition?

As noted above, the normal condition depends on the cellular system. However, endogenous Myo10 also accumulates in bulges at filopodial tips, so this is not a phenotype unique to Myo10 overexpression. For example, the images from Figure 1 of the Berg and Cheney (2002) citation show bulges from endogenous Myo10 in endothelial cells.

(2) Measurements of the localization of Myo10 focuses in large part on ‘Myo10 punctae’. While it seems reasonable to presume that these are filopodia tips, the authors should provide readers with a clear definition of a puncta. Is it only filopodia tips, which seems to be the case? Does it include initiation sites at the cell membrane that often appear as punctae?

We define puncta as any clusters/spots of Myo10 signal detected by segmentation, not limited to any location within the surface-attached filopodia. We exclude puncta that appear in the cell interior (~5 of which appear in Fig. 1A). These are likely dorsal filopodia, but there are few of these compared to the surface attached filopodia of U2OS cells. In Figure 2, “puncta” includes all Myo10 clusters along the filopodia shaft, though a majority happen to be tip-localized (please see Supplementary Figure 4B). We have edited the main text for clarification.

Along those lines, the position of dim punctae along the length of a filopodium is measured (Fig 3D). The findings suggest that a given filopodium can have more than one puncta which seems at odds if a puncta is a filopodia tip. How frequently is a filopodium with two puncta seen? It would be helpful if the authors provided an example image showing the dim puncta that are not present at the tip.

We have now provided an example image of dim puncta along filopodia in Supplementary Figure 4C.

(3) The concentration of actin available to Myo10 is calculated based on the deduction from Nagy et al (2010) that only 4/13 of the actin monomers in a helical turn are accessible to the Myo10 motor (discussion on pg 9; Fig S4). Subsequent work (Ropars et al, 2016) has shown that the heads of the antiparallel Myo10 dimer are flattened, but the neck is rather flexible, meaning that the motor can a variable reach (36 - 52 nm). Wouldn’t this mean that more actin could be accessible to the Myo10 motor than is calculated here?

Although we see why the reviewer might believe otherwise, the 4/13 fraction of accessible actin holds. This fraction is obtained from consideration of the fascin-actin bundle structure alone, independent of the reach of any particular myosin motor. Every repeating layer of 13 actin subunits (or 36 nm) has 4 accessible myosin binding-sites. The remaining 9 sites are rejected because a single myosin motor domain will have a steric clash with a neighboring actin filament in the bundle. A myosin with an exceptionally long reach might reach the next 13 subunit layer, but that layer also has only 4 binding sites. Thus, we can calculate the number of binding sites per unit length along the filopodium. This number would hold for a dimeric myosin with any reach, including myosin-5 or myosin-2.

(4) Quantification of numbers of Myo10 molecules in filopodial puncta (Fig 3C) leads the authors to conclude that ‘only ten or fewer Myo10 molecules are necessary for filopodia initiation’ (pg 7, top). While this is a reasonable based on the assumption that the formation of a puncta ultimately results from an initiation event, little is known about initiation events and without direct observation of coalescence of Myo10 at the cell edge that leads to formation of a filopodium, this seems rather speculative.

As noted above, we have now performed the necessary live cell imaging of filopodial nucleation events and have updated our conclusions accordingly.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

I have made a series of comments that might help the authors improve their manuscript:

- A full calibration of the methodology would require testing a wider range of protein amounts, to exhaustively detect the dynamic range of the technique. The authors acknowledge in the discussion that “Furthermore, our estimates of molecules are predicated on the calibration curve of the Halo Standard Protein on the SDS-PAGE gels, which is likely the highest source of error on our molecule counts”. A good way of convincing a nasty reviewer is to provide a calibration with more than 3 reference points. At least this will help exclude from the analysis cells where Myo10 estimates are not in the linear regime of detection.

We completely agree with the reviewer’s suggestion to build a robust calibration curve. The SDS gel shown in Figure 1C originally contained 4 reference points, but the highest HaloTag standard protein point oversaturated the detector at the set exposure in the TMR channel and was omitted. We have now re-run the SDS gel to include a HaloTag standard protein curve comprising 5 points, alongside all three bioreplicates from the fixed cell experiments and all three bioreplicates from the live cell experiments (updated in Figure 1B-C). We had saved frozen lysates from the original fixed cell work, so we were able to reanalyze our data with the new set of standards. The Myo10 quantities are consistent, but with much tighter CIs from the standard curve.

- As already said this methodology is intriguing, however, a correlative validation with a conventional SMLM approach to address the bona-fide of the method would be ideal.

Unfortunately, single molecule approaches for validation are impractical for us. Due to the relatively high magnification of our TIRF microscope and the large spread area of the U2OS cells, single cells typically extend beyond the field of view. We acknowledge the benefits of SMLM quantitative techniques and other approaches cited in the introduction section. To avoid use of special tools/instruments, we offer our methodology, based off Pollard group’s quantitative Western blotting of GFP, as a simpler alternative accessible to anyone.

- TMR is a small ligand likely interacting also with Halo in its denatured state. However, to clear any doubts a parallel Native-PAGE investigation should be included, or if existing a specific reference should be provided.

Perhaps there is a misunderstanding here. One of the key advantages of the HaloTag labeling system is that the engineered dehalogenase is covalently modified by the ligand (the TMR-ligand is a suicide substrate). This means that the TMR remains bound even under denaturing conditions, which allows its detection in SDS-PAGE. Native gels are unnecessary here.

- Moreover, SDS-PAGE is run at alkaline pH, have the authors considered these points when designing the methodology? Fluorescence images were taken in PBS, which has a different pH. Could the authors, or the literature, exclude these aspects as potential pitfalls in the methodology? Also temperature is affecting fluorescence emission, but it is easier to control with certain tolerance in the room-temperature regime.

Our method does not compare fluorescence values that cross the experimental systems (SDS-PAGE vs. microscopy). Cellular proteins and HaloTag protein standards are compared in a single setting of SDS-PAGE to obtain the average number of Myo10s per transfected cell. Likewise, all measurements on intact (live or fixed) cells are conducted in that single setting to obtain average fluorescence per cell. Thus, there is no issue with the different buffers or temperatures affecting fluorescence emission.

- The authors should test their approach also with truncation variants of Myosin10 (for instance lacking the PH or motor domain). This is a classical approach that might prove the potential of the technique when altering the capacity of the protein to interact with a main binding partner. Also, treatments that induced filopodia formation might prove useful (i.e., hypotonic media induce filopodia formation in some fibroblast cell lines in our hands).

The reviewer raises interesting suggestions that we aim to address in future experiments, but truncation variants and environmental perturbations are beyond the focus of the current manuscript. Here, we report on the otherwise unperturbed state when we add exogenous full-length Myo10 to the U2OS cells. But indeed, experiments with Myo10 domain truncations, PI3K and PTEN inhibition, and cargo protein / activating cofactor knock-downs (among others) are on our drawing board.

- Most of the mechanisms hypothesized in the discussion are sound and plausible. However, the authors have chosen an experimental model where transient transfection of exogenous Myo10 in U2OS is performed. This approach poses two main and fundamental questions that are not resolved by the data provided:

A) how do different expression levels affect the Myo10 counting?

Our counting procedure does not assume uniform expression across a population of cells– quite the opposite, in fact. We directly measure Myo10 expression levels on a cell-by-cell basis with microscopy, once we know the number of molecules in our total pool (see the Methods for details). As an example of the final output, Figs. 1D and 1E show the total number of Myo10 molecules per cell for fixed and live cells, respectively.

B) how does endogenous and unlabeled Myo10 hamper the bonafide of counts? The authors claimed “U2OS cells express low levels of Myo10, so there is a small population of unlabeled endogenous Myo10 unaddressed by this paper”. As presented, the low levels of endogenous Myo10 sound an arbitrary parameter, and there are no data presented that can limit if not exclude this bias in the analysis. To produce data in a genetically modified cell line with Halo-tag on the endogenous protein will represent a much cleaner system. Alternatively, the authors should look for Myo10 KO cell lines where they can back-transfect their Halo-Tagged Myo10 construct in a more consistent framework, focusing on cells with low-to-mid levels of expression.

We agree, this is an important point to nail down (and is often neglected in the literature). We have now measured the endogenous Myo10 levels in U2OS cells by Western blotting and found that it is undetectable compared to our HaloTagged construct expression. Please see Supp. Fig 1E. Thus, for all intents and purposes, every Myo10 molecule in these experiments came from our expression plasmid. Accordingly, we have removed this caveat from the paper.

Minor points

- Figure 1B. To help the reader SDS-PAGE gels annotations should be clearer already from the figure.

We have updated the annotations for clarity.

- Methods should be organized in sessions. As it stands, it is hard for the reader to look for technical details.

We have expanded and added subsections to the Methods as requested.

- The good practice of indicating the gene and transcript entry numbers and the primer used to amplify and clone into the backbone vectors is getting lost in many papers. I would strongly encourage the authors to add this information to the methods.

We have included the gene entries to the methods and will include a full FASTA file of the coding sequence as supplementary information to avoid any ambiguity here.

The authors write “It is unclear how myosins navigate to the right place at the right time, but our results support an important interplay between Myo10 and the actin network.” It is a bit scholastic to say that Myo10 and actin have an important interplay, they are major binding partners. What is the new knowledge contained in this sentence?

Agreed– we have deleted the sentence in question.

Reviewer #2 (Recommendations For The Authors):

The authors should address all the weaknesses indicated in the public review.

There were a few other places that require clarification.

On page 4, the last paragraph. It is stated that the targeting of Myo10 was reported/proposed based on previous work (ref 31). The next few sentences are not referenced and thus likely refer to ref 31. The authors did not measure the parameters discussed in these sentences, so it is important to clarify that they are referring to previous work and not the current study.

Indeed, the next few sentences still refer to old reference 31, so we have now edited the paragraph for clarity.

On page 7, the reference to Figure 3A indicates that the trend of higher Myo10 correlating with more filopodia. However, the reference to Figure 3B indicates total intracellular Myo10 weakly correlates with more filopodia. However, the x-axis on Figure 3B is filopodia molecules not the intracellular Myo10. Please clarify.

We appreciate the reviewer for catching our mistake. Those plots are now in Fig. 2 and have been edited accordingly.

Reviewer #3 (Recommendations For The Authors):

The Discussion of results at the end of each section is rather brief and could be expanded on a bit more.

Before we were operating under the constraints of an eLife Short Report. We have now expanded the discussion for a full article.

The authors mention that actin filaments at the tips of filopodia could be frayed, citing Medalia et al, 2007 (ref 40). That paper describes an early cryoEM analysis of filopodia from the amoeba Dictyostelium. EM images of mammalian filopodia tips, e.g. Svitkina et al, 2003, JCB, do not show quite the same organization of actin as seen in the Dictyostelium filopodia tips. However, recent work from the Bershadsky lab, Li et al, 2023, presents a few cryoEM images of tips of left-bent filopodia that are tightly adhered to a substrate and there it looks like actin filaments become disorganized in tips, along with membrane bulging. The authors should consider expanding discussion of the filopodia tips to take into account what is known for mammalian filopodia.

We thank the reviewer for bringing these enlightening papers to our attention. We have now included these citations in the discussion.

Fig 1D - The x-axis is a bit odd, it goes from 0 then to 2.5e+06 with no indication of the bin size. Can this be re-labelled or the scale displayed a bit differently?

We have double-checked the axis breaks, which are large because the underlying values are large. We have also provided the bin size as requested for all histograms.

Fig 4A - What is the bin size for the histogram?

As above, we have now updated the figure legends (now in Fig. 3) to include the bin size.

Methods -

- Please provide an accession number for the Myo10 nucleotide sequence used for this work as there are at least two known isoforms.

Thank you for noting this. We are using the full-length, not the headless isoform. We have now updated the Methods accordingly.

- No mention is made of the SDS sample buffer used, was that also added to the sample?

We have now updated the Methods accordingly.

- How are samples boiled at 70 deg C? Do the authors actually mean ‘heated’?

Indeed. We have now corrected “boiled” to “heated.”

- Could the authors please briefly explain the connected component analysis used to identify filopodia?

We have now updated the Methods accordingly.

- The intensity of filopodia was determined by dividing tip intensity by the total bioreplicate sum of intensities then multiplying it by the total pool, if this reviewer understands correctly. It sounds like intensities are being averaged across a whole cell population instead of cell-by-cell. Is that correct? If so, can the authors please provide the underlying rationale for this? If not, then please better describe what was actually done.

We apologize for the confusion. Intensities are being averaged (summed) across a whole cell population, but importantly that step is only used to obtain a scale factor that converts the fluorescence signal at the microscope to the number of molecules. We then use that scale factor for all cells imaged in the bioreplicate, to both 1) find the total Myo10 in that cell, and 2) find the total amount of that Myo10 in any given location within that cell.

To further clarify, each bioreplicate has a known total number of Myo10 molecules associated with the number of cells loaded onto the SDS gel. From the SDS gel, we have an average number of Myo10 molecules per positively transfected cell. If 50 cell images are analyzed, then there is a Myo10 ‘total pool’ of (50 cells) * (average Myo10 molecules/cell). The fluorescence signal intensities in microscopy were summed for all cells within the bioreplicate (50 cells in this example). However, due to variation in expression, not every cell has the same signal intensity when imaged under the same conditions. It would be inaccurate to assume each cell contains the average Myo10 molecules/cell. Therefore, to get the number of molecules within a given Myo10 cell (or punctum), the summed cell (punctum) intensity was divided by the bioreplicate fluorescence signal intensity sum and multiplied by ‘total pool.’

- The authors quantify Myo10 protein amounts by western blotting using Halo tag fluorescence, a method that should provide good accuracy. The results depend on the transfection efficiency and it is rarely the case that it is 100%. The authors state that they use a ‘value correction for positively transfected cells’ (pg 11). It is likely that there was a range of expression levels in the cells, how was a cut-off for classifying a cell as non-expressing determined or set?

As described in the Methods, “microscopy was used to count the percentage of transfected cells from ~105-190 randomly surveyed cells per bioreplicate.” Cells were labeled and located with DAPI. If no TMR signal could be visually detected by microscopy, then the cell was deemed to be non-Myo10 expressing. We did not set a cutoff fluorescence value, as untransfected cells have no detectable signal. Please see Supplementary Figure 1F for examples.

- “In-house Python scripts” are used for image analysis. Will these be made publicly available?

Yes, we will package these up on GitHub.

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Public Review):

Summary:

In this manuscript, Chowdhury and co-workers provide interesting data to support the role of G4-structures in promoting chromatin looping and long-range DNA interactions. The authors achieve this by artificially inserting a G4-containing sequence in an isolated region of the genome using CRISPR-Cas9 and comparing it to a control sequence that does not contain G4 structures. Based on the data provided, the authors can conclude that G4-insertion promotes long-range interactions (measured by Hi-C) and affects gene expression (measured by qPCR) as well as chromatin remodelling (measured by ChIP of specific histone markers).

Whilst the data presented is promising and partially supports the authors' conclusion, this reviewer feels that some key controls are missing to fully support the narrative. Specifically, validation of actual G4-formation in chromatin by ChIP-qPCR (at least) is essential to support the association between G4-formation and looping. Moreover, this study is limited to a genomic location and an individual G4-sequence used, so the findings reported cannot yet be considered to reflect a general mechanism/effect of G4-formation in chromatin looping.

Strengths:

This is the first attempt to connect genomics datasets of G4s and HiC with gene expression. The use of Cas9 to artificially insert a G4 is also very elegant.

Weaknesses:

Lack of controls, especially to validate G4-formation after insertion with Cas9. The work is limited to a single G4-sequence and a single G4-site, which limits the generalisation of the findings.

In the revised version we validated G4 formation inside cells at the insertion site using the reported G4-selective antibody BG4. Significant BG4 binding (by ChIP-qPCR) was clear in the G4-array insert, and not in the G4-mutated insert, supporting formation of G4s by the inserted G4-array (included as Figure S4).

To directly address the second point, we inserted the G4-sequence, or the mutated control, at a second relatively isolated locus (at the 10 millionth position on Chr12, denoted as 10M site in text). First, BG4 ChIP was done to confirm intracellular G4 formation by the inserted array. BG4 ChIP-qPCR binding was significant within the inserted region, and not in the negative control region (Figure S8), consistent with the 79M locus. Together these demonstrate intracellular G4 formation by inserted sequences at two different loci.

We next checked the state of chromatin of the G4-array inserted at the 10M locus, or its negative control. Histone marks H3K4Me1, H3K27Ac, H3K27Me3, H3K9me3 and H3K4Me3 were tested at the G4-array, or the negative control locus. Relative increase in the enhancer histone marks was evident, relative to the control sequence. This was largely similar to the 79M locus, supporting an enhancer-like state. Interestingly, here we further noted presence of the H3K27me3 histone mark. The presence of the H3K27Me3 repressor histone mark, along with H3K4Me1/H3K27Ac enhancer histone marks, support a poised enhancer-like status of the inserted G4 region, as has been observed earlier in other studies. Together, although data from the two distinct G4 insertion sites support the enhancer-like state, there are contextual differences likely due to the sequence/chromatin of the sites adjacent to the inserted sequence.

Effect of the 10M G4-insertion on activation of surrounding genes (10 Mb window), and not the G4-mutant insert, was evident for most genes. Consistent with the enhancer-like state of the G4-array insert; in line with the 79M G4-array insert.

These results have been added as the final section in the revised version, data is shown in Figure 7.

Reviewer #2 (Public Review):

Summary:

Roy et al. investigated the role of non-canonical DNA structures called G-quadruplexes (G4s) in long-range chromatin interactions and gene regulation. Introducing a G4 array into chromatin significantly increased the number of long-range interactions, both within the same chromosome (cis) and between different chromosomes (trans). G4s functioned as enhancer elements, recruiting p300 and boosting gene expression even 5 megabases away. The study proposes a mechanism where G4s directly influence 3D chromatin organization, facilitating communication between regulatory elements and genes.

Strength:

The findings are valuable for understanding the role of G4-DNA in 3D genome organization and gene transcription.

Weaknesses:

The study would benefit from more robust and comprehensive data, which would add depth and clarity.

(1) Lack of G4 Structure Confirmation: The absence of direct evidence for G4 formation within cells undermines the study's foundation. Relying solely on in vitro data and successful gene insertion is insufficient.

Using the reported G4-specific antibody, BG4, we performed BG4 ChIP-qPCR at the 79M locus. In addition, a second G4-insertion site was created and BG4 ChIP-qPCR was used to validate intracellular G4 formation. Briefed below, more details in the response above.

In the revised version we validated G4 formation inside cells at the insertion site using the reported G4-selective antibody BG4. Significant BG4 binding (by ChIP-qPCR) was clear in the G4-array insert, and not in the G4-mutated insert, supporting formation of G4s by the inserted G4-array (included as Figure S4).

Further, we inserted the G4-sequence, or the mutated control, at a second relatively isolated locus (at the 10 millionth position on Chr12, denoted as 10M site in text). First, BG4 ChIP was done to confirm intracellular G4 formation by the inserted array. BG4-ChIP-qPCR was significant within the G4-array inserted region, and not in the negative control region (Figure S8), consistent with the 79M locus. Together these demonstrate intracellular G4 formation by inserted sequences at two different loci. Added in revised text in the second and the final sections of results, data shown in Figures 7, S4 and S8.

(2) Alternative Explanations: The study does not sufficiently address alternative explanations for the observed results. The inserted sequences may not form G4s or other factors like G4-RNA hybrids may be involved.

As mentioned in response to the previous comment, we confirmed that the inserted sequence indeed forms G4s inside the cells. RNA-DNA hybrid G4s can form within R-loops with two or more tandem G-tracks (G-rich sequences) on the nascent RNA transcript as well as the non-template DNA strand (Fay et al., 2017, 28554731). A recent study has observed that R-loop-associated G4 formation can enhance chromatin looping by strengthening CTCF binding (Wulfridge et al., 2023, 37552993). As pointed out by the reviewer, the possibility of G4-RNA hybrids remains, we have mentioned this possibility for readers in the second last paragraph of the Discussion.

(3) Limited Data Depth and Clarity: ChIP-qPCR offers limited scope and considerable variation in some data makes conclusions difficult.

We noted variation with one of the primers in a few ChIP-qPCR experiments (in Figures 2 and 3D). The changes however were statistically significant across replicates, and consistent with the overall trend of the experiments (Figures 2, 3 and 4). Enhancer function, in addition to ChIP, was also confirmed using complementary assays like 3C and RNA expression.

(4) Statistical Significance and Interpretation: The study could be more careful in evaluating the statistical significance and magnitude of the effects to avoid overinterpreting the results.

We reconfirmed our statistical calculations from biological replicate experiments. We carefully looked at potential overinterpretations, and made appropriate changes in the manuscript (details of the changes given below in response to comment to authors).

Reviewer #3 (Public Review):

Summary:

This paper aims to demonstrate the role of G-quadruplex DNA structures in the establishment of chromosome loops. The authors introduced an array of G4s spanning 275 bp, naturally found within a very well-characterized promoter region of the hTERT promoter, in an ectopic region devoid of G-quadruplex and annotated gene. As a negative control, they used a mutant version of the same sequence in which G4 folding is impaired. Due to the complexity of the region, 3 G4s on the same strand and one on the opposite strand, 12 point mutations were made simultaneously (G to T and C to A). Analysis of the 3D genome organization shows that the WT array establishes more contact within the TAD and throughout the genome than the control array. Additionally, a slight enrichment of H3K4me1 and p300, both enhancer markers, was observed locally near the insertion site. The authors tested whether the expression of genes located either nearby or up to 5 Mb away was up-regulated based on this observation. They found that four genes were up-regulated from 1.5 to 3-fold. An increased interaction between the G4 array compared to the mutant was confirmed by the 3C assay. For in-depth analysis of the long-range changes, they also performed Hi-C experiments and showed a genome-wide increase in interactions of the WT array versus the mutated form.

Strengths:

The experiments were well-executed and the results indicate a statistical difference between the G4 array inserted cell line and the mutated modified cell line.

Weaknesses:

The control non-G4 sequence contains 12 point mutations, making it difficult to draw clear conclusions. These mutations not only alter the formation of G4, but also affect at least three Sp1 binding sites that have been shown to be essential for the function of the hTERT promoter, from which the sequence is derived. The strong intermingling of G4 and Sp1 binding sites makes it impossible to determine whether all the observations made are dependent on G4 or Sp1 binding. As a control, the authors used Locked Nucleic Acid probes to prevent the formation of G4. As for mutations, these probes also interfere with two Sp1 binding sites. Therefore, using this alternative method has the same drawback as point mutations. This major issue should be discussed in the paper. It is also possible that other unidentified transcription factor binding sites are affected in the presented point mutants.

Since the sequence we used to test the effects of G4 structure formation is highly G-rich, we had to introduce at least 12 mutations to be sure that a stable G4 structure would not form in the mutated control sequence. Sp1 has been reported to bind to G4 structures (Raiber et al., 2012). Therefore, Sp1 binding is likely to be associated with the G4-dependent enhancer functions observed here. We also appreciate that apart from Sp1, other unidentified transcription factor binding sites might be affected by the mutations we introduced. We have discussed these possibilities in the fourth paragraph of the Discussion section in the revised manuscript.

Reviewer #1 (Recommendations For The Authors):

Whilst the data presented is promising and partially supports the authors' conclusion, this reviewer feels that some key controls are missing to fully support the narrative used. Below are my main concerns:

(1) The main thing missing in the current manuscript is to validate the actual formation of G4 in chromatin context for the repeat inserted by CRISPR-Cas. Whilst I appreciate this will form promptly a G4 in vitro, to fully support the conclusions proposed the authors would need to demonstrate actual G4-formation in cells after insertion. This could be done by ChIP-qPCR using the G4-selective antibody BG4 for example. This is an essential piece of evidence to be added to link with confidence G4-formation to chromatin looping.

To address the concern regarding whether the inserted G4 sequence forms G4s in cells, as suggested, we used the G4-selective antibody BG4. PCR primers in the study were designed keeping multiple points in mind: Primers should not bind to any site of G/C alteration in the mutated control insert; either the forward/reverse primer is from the adjacent region for specificity; covers adjacent regions for studying any effects on chromatin; and, PCRs optimized keeping in mind the repeats within the inserted sequence. Given these, primer pairs R1-R4 were chosen for further work following optimizations (Figure 2, top panel). For BG4 ChIP-qPCR we used primer pairs R2, which covered >100 bases of the inserted G4-array, or the G4-mutated control. Significant BG4 binding was clear in the G4-array insert, and not in the G4-mutated insert, demonstrating formation of G4s by the inserted G4-array (Figure S4).

In response to comment #3 below, we inserted the G4-forming sequence (or its mutated control) at a second locus. This insertion was near the 10 millionth position of chromosome 12 (10M insertion locus in text). Here also, BG4 binding was significant within the G4-array inserted region, and not in the negative control region (Figure S8). Together these demonstrate G4 formation by the inserted sequence at two different loci.

(2) I found the LNA experiment very elegant. However, what would be the effect of LNA treatment on the control sequence that does not form G4s? This control is essential to disentangle the effect of LNA pairing to the sequence itself vs disrupting the G4-structure.

As per the reviewer’s suggestion, we performed a control experiment where we treated the G4-mutated insert (control) cells with the G4-disrupting LNA probes. The changes in the expression of the surrounding genes in this case were not significant, indicating that the effects observed in the G4-array insert cells were possibly due to disruption of the inserted G4 structures. This data is presented in Figure S5.

(3) The authors describe their work and present its conclusion as if this were a genome-wide study, whilst the work is focused on a specific genomic location, and the looping, along with the effect on histone acetylation and gene expression, is limited to this. The authors cannot conclude, therefore, that this is a generic effect and the discussion should be more focused on the specific G4s used and the genomic location investigated. Ideally, insertion of a different G4-forming sequence or of the same in a different genomic location is recommended to really claim a generic effect.

To address this we inserted the G4-array sequence, or the G4-mutated control sequence, at another relatively isolated locus – at the 10 millionth position of chromosome 12 – denoted as 10M. Using BG4 ChIP-qPCR intracellular G4 formation was confirmed. We observed that the enhancer-like features in terms of enhancer histone marks and increase in the expression of surrounding genes were largely reproduced at the 10M locus on G4 insertion (Figure 7). These results are added as the final section under Results.

Reviewer #2 (Recommendations For The Authors):

The study proposes a mechanism where G4s directly influence 3D chromatin organization, facilitating communication between regulatory elements and genes.

While the present manuscript presents an interesting hypothesis, it would benefit from enhanced novelty and more robust data. The study complements existing G4 research (e.g., PMID: 31177910). While the conclusions hold biological relevance, they largely reiterate established knowledge. Furthermore, the presented data appear preliminary and still lack depth and clarity.

Hou et al., 2019 (PMID: 31177910) showed presence of potential G4-forming sequences correlated with TAD boundaries, along with enrichment of architectural proteins and transcription factor binding sites. Also, other studies noted enrichment of potential G4-forming sequences at enhancers along with nucleosome depletion and higher transcription factor binding (Hou et al., 2021; Williams et al., 2020). These studies proposed the role of G4s in chromatin/TAD states based on analysis of potential G4-forming sequences using correlative bioinformatics analyses. Here we sought to directly test causality. Insertion of G4 sequence, and formation of intracellular G4s in an isolated, G4-depleted region resulted in altered characteristics of chromatin, and not in the negative control insertion that does not form G4s. These, in contrast to earlier studies, directly demonstrates the causal role of G4s as functional elements that impact local and distant chromatin.

Major concerns:

(1) Lack of G4 Structure Confirmation: Implement G4-specific antibodies or fluorescent probes to verify G4 structures inside the cells.

Detailed response given above. Briefly, in the revised version we validated G4 formation inside cells at the insertion site using the reported G4-selective antibody BG4. Significant BG4 binding (by ChIP-qPCR) was clear in the G4-array insert, and not in the G4-mutated insert, supporting formation of G4s by the inserted G4-array (included as Figure S4).

Further, we inserted the G4-sequence, or the mutated control, at a second relatively isolated locus (at the 10 millionth position on Chr12, denoted as 10M site in text). First, BG4 ChIP was done to confirm intracellular G4 formation by the inserted array. BG4 ChIP-qPCR binding was significant within the G4-array inserted region, and not in the negative control region (Figure S8), consistent with the 79M locus. Together these demonstrate intracellular G4 formation by inserted sequences at two different loci. Added in revised text in the second and the final sections of results, data shown in Figures 7, S4 and S8.

(2) Alternative Explanations: Explore the possibility that the sequences may not form G4s or that other factors like G4-RNA hybrids are involved.

Response provided in the public reviews section.

(3) Limited Data Depth and Clarity: ChIP-qPCR offers limited scope. Consider employing G4 ChIP-seq for genome-wide analysis of G4 association with histone modifications. Address inconsistencies in data like H3K27me3 variation and incomplete H3K9me3 data sets.

A recent study performed G4 CUT&Tag (Lyu et al., 2022, 34792172) and observed G4 formation at both active promoters and active and poised enhancers. We have discussed this in the sixth paragraph of the Discussion. The H3K27Me3 occupancy at the 79M locus insertions did not have any significant G4-dependent changes, however, at the second insertion site at the 10M locus (introduced in the revised manuscript, Figure 7) there was significant G4-dependent increase in H3K27Me3 occupancy along with the H3K4Me1 and H3K27Ac enhancer histone marks, indicating formation of a poised enhancer-like element.

We completed the H3K9me3 data sets for both insertion sites.

(4) Statistical Significance and Interpretation: Re-evaluate the statistical significance of results and interpret them in the context of relevant biological knowledge. Avoid overstating the impact of minor changes.     

We revised several lines to avoid overstating results. Some of the changes are as below (changes underline/strikethrough)

- There was an a relatively modest increase in the recruitment of both p300 and a substantial increase in the recruitment of the more functionally active acetylated p300/CBP to the G4-array when compared against the mutated control.

- As expected, although modest, a decrease in the H3K4Me1 and H3K27Ac enhancer histone modifications was evident within the insert upon the LNAs treatment.

- Moreover, the enhancer marks were relatively reduced, although not markedly, when the inserted G4s were specifically disrupted.

(5) Unexplored Aspects: Investigate the relationship between G4 DNA and R-loops, and consider the role of CTCF and cohesin proteins in mediating long-range interactions. Integrate existing research to build a more comprehensive framework and draw more robust conclusions.

As mentioned in response to one of the earlier comments, a recent publication extensively studied the association between G4s, R-loops, and CTCF binding (Wulfridge et al., 2023). While, here we focused on the primary features of a potential enhancer, further work will be necessary to establish how G4s influence the coordinated action between cohesin and CTCF and consequent chromatin looping. We have described this for readers in the second last paragraph of the Discussion in the revised version.

Minor Concern:

(1) Enhancer Definition: The term "enhancer" requires specific criteria. Modify the section heading or provide evidence demonstrating the G4 sequence fulfills all conditions for being an enhancer, such as position independence and long-range effects.

Although we checked some of the primary features of a potential enhancer: Like expression of surrounding genes, enhancer histone marks, chromosomal looping interactions, and recruitment of transcriptional coactivators, further aspects may need to be validated. As suggested, in the revised manuscript the section heading has been modified to ‘Enhancer-like features emerged upon insertion of G4s.’

Reviewer #3 (Recommendations For The Authors):

In addition to the points in my public review, I would like to mention some less significant points.

The authors mention that "the array of G4-forming sequences used for insertion was previously reported to form stable G4s in human cells" (Lim et al., 2010; Monsen et al., 2020; Palumbo et al., 2009). However, upon reading the publications, I found that these observations were made in vitro. I may have missed something, but there are now several mappings of folded-G4 in human cells based on different approaches. It would be beneficial to investigate whether the hTERT promoter is a site of G-quadruplex formation in vivo. If confirmed, a similar analysis should be conducted on the 275 bp region inserted into the ectopic region to determine if it also has the ability to form a structured G4.

We performed BG4 ChIP to confirm in vivo G4 formation by the inserted G4-array as suggested (Figures S4, S8). Detail response given above. Briefly, in the revised version we validated G4 formation inside cells at the insertion site using the reported G4-selective antibody BG4. Significant BG4 binding (by ChIP-qPCR) was clear in the G4-array insert, and not in the G4-mutated insert, supporting formation of G4s by the inserted G4-array (included as Figure S4).

Further, we inserted the G4-sequence, or the mutated control, at a second relatively isolated locus (at the 10 millionth position on Chr12, denoted as 10M site in text). First, BG4 ChIP was done to confirm intracellular G4 formation by the inserted array. BG4-ChIP-qPCR was significant within the inserted region, and not in the negative control region (Figure S8). Consistent with the 79M locus. Together these demonstrate intracellular G4 formation by inserted sequences at two different loci. Added in revised text in the second and the final sections of results, data shown in Figures 7, S4 and S8.

The inserted sequence originates from a well-characterized promoter. The authors suggest that placing it in an ectopic position creates an enhancer-like region, based on the observation of increased levels of H3K27Ac and H3K4me1 on the WT array. To provide a control that it is not a promoter, it would be useful to also analyze a specific mark of promoter activity, such as H3K4me3.

As suggested by reviewer, we also analysed the H3K4Me3 promoter activation mark at both the 79M and 10M (introduced in the revised manuscript, Figure 7) insertion loci. We did not observe any significant G4-dependent changes in the recruitment of H3K4Me3 (Figures 2, 7).

In the discussion, the authors mention "it was proposed that inter-molecular G4 formation between distant stretches of Gs may lead to DNA looping". To investigate this further, it would be worthwhile to examine whether the promoter regions of activated genes (PAWR, PPP1R12A, NAV3, and SLC6A15) contain potentially forming G-quadruplexes (pG4). Additionally, sites that establish more contact with the G4 array described in Figure 6F could be analyzed for enrichment in pG4.

Thank you for pointing this out. We found promoters of the four genes (PAWR, PPP1R12A, NAV3, and SLC6A15) harbour potential G4-forming sequences (pG4s). Also as suggested, we analysed the contact regions in Fig 6F, along with the whole locus, for pG4s. Relative enrichment in pG4 was seen, particularly within the significantly enhanced interacting regions, which at times spreads beyond the interacting regions also. This is shown in the lower panel of Figure 6F in the revised version. We have described this in Discussion for readers.

Comment by onewheeljoe:

For example, we might simply ask that each participant refrain from using hashtags as a final thought because that is a form of sarcasm or punchline that can be misconstrued or shut down honest debate or agreeable disagreement.

We could ask respondents to reply to any comment that they read twice because of tone to use "ouch" as a tag or a textual response. The offending respondent could respond with "oops" in order to preserve good will in an exchange of ideas.

Finally, the first part of a flash mob might occur here, in the page notes, where norms could be quickly negotiated and agreed upon with a form of protocol.

Reviewer #1 (Public Review):

Summary:

Guo, Hue et al. focused on understanding the epigenetic activity and functional dependencies for two different fusions found in infantile rhabdomyosarcoma, VGLL2::NCOA2, and TEAD1::NCOA2. They use a variety of models and methods; specifically, ectopic expression of the fusions in human 293T cells to perform RNAseq (both fusions), CUT&RUN (VGLL2::NCOA2), and BioID mass spec (both fusions). These data identify that the VGLL2::NCOA2 fusion has peaks that are enriched for TEAD motifs. Further, CPB/p300 CUT&RUN support an enrichment of binding sites and three TEAD targets in VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells. They also functionally evaluated genetic and chemical dependencies (TEAD inhibition), and found this was only effective for the VGLL2::NCOA2 fusion, and not for TEAD1::NCOA2. Using complementary biochemical approaches they suggest (with other supporting data) that the fusions regulate TEAD transcriptional outputs via a YAP/TAZ independent mechanism. Further, they expand into a C2C12 myoblast model and show that TEAD1::NCOA2 is transforming in colony formation assays and in mouse allografts. This is consistent with previously published strategies using VGLL2::NCOA2. Importantly, they show that a CBP/p300 (a binding partner found in their BioID mass spec) small molecule inhibitor suppresses tumor formation using this mouse allograft model, that the tumors are less proliferative, and have a reduction in transcriptional of three TEAD target genes. Generally, the data is interesting and suggests new biology for these fusion-oncogenes. However, the choice of 293T for the majority of the transcriptional, epigenetic, and proteomic studies makes the findings difficult to interpret in the context of the human disease, and the rationale for the choice of an epithelial-like kidney cell line is not discussed. Further, details are missing from the figures, figure legends, and methods that make the data difficult to interpret, and should be added to improve the reader's understanding. Overall, the breadth of methods used in this study, and the comparison of the two fusion-oncogene's biology is of interest to the fusion-oncogene, pediatric sarcoma, and epigenetic therapeutic targeting fields.

Strengths:

(1) Multiple experimental approaches were used to understand the biology of the fusion-oncogenes, including genomic, proteomic, chemical, and genetic inhibition. These approaches identify potential new mechanisms of convergent fusion-oncogene activity, around TEAD transcriptional targeting (that is YAP/TAZ independent) and reveal CBP/p300 as a functional dependency.

(2) Complementary models were used, including cell-based assays and mouse allograft models to show the dependency on CBP/P300.

(3) Co-IPs were clear and convincing and showed direct interaction of the fusion-oncogene with ectopic and endogenous TEAD1/pan-TEAD, but not YAP/TAZ.

(4) Potential to follow-up on additional targets/mechanisms of tumorigenesis. For example, in the BioID proteomics screen, a unique VGLL2::NCOA2 and TEAD::NCOA2 interactor is P53, which also is an enriched pathway in Figure 4C in the p300 CUT&RUN peaks in the VGLL2::NCOA2 and TEAD1::NCOA2 expressing cells - is this indicative of the toxicity of the fusion-oncogenes or do you think this informs potential mechanisms for transformation.

Weaknesses:

(1) The rationale for performing genomics, transcriptional, and proteomics work in 293T cells is not discussed. Further, there are no functional readouts mentioned in the 293T cells with expression of the fusion-oncogenes. Did these cells have any phenotypes associated with fusion-oncogene expression (proliferation differences, morphological changes, colony formation capacity)? Further, how similar are the gene expression signatures from RNA-seq to rhabdomyosarcoma? This would help the reader interpret how similar these cell models are to human disease.

(2) TEAD1::NCOA2 fusion-oncogene model was not credentialed past H&E, and expression of Desmin. Is the transcriptional signature in C2C12 or 293T similar to a rhabdomyosarcoma gene signature?

(3) For the fusion-oncogenes, did the HA, FLAG, or V5 tag impact fusion-oncogene activity? Was the tag on the 3' or 5' of the fusion? This was not discussed in the methods.

(4) Generally, the lack of details in the figures, figure legends, and methods make the data difficult to interpret. A few examples are below:

a. Individual data points are not shown for figure bar plots (how many technical or biological replicates are present and how many times was the experiment repeated?).<br /> b. What exons were included in the fusion-oncogenes from VGLL2 and NCOA2 or TEAD1 and NCOA2?<br /> c. For how long were the colony formation experiments performed? Two weeks?<br /> d. In Figure 2D, what concentration of CP1 was used and for how long?<br /> e. How was A485 resuspended for cell culture and mouse experiments, what is the percentage of DMSO?<br /> f. How many replicates were done for RNA-seq, CUT&RUN, and ATACseq experiments?

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

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Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity (Required):

The majority of the conclusions are well supported by strong experimental evidence. The only area where that is not fully the case is the role of Pak1 as a downstream effector of FoxG1-FoxO6 and its effects on macropinocytosis. To further strengthen this claim, the authors should demonstrate that ablation of Pak1 can rescue the functional consequences of forced FoxO6 expression and whether overexpression of Pak1 rescues quiescence exit in FoxO6 knockout. Thank you to the reviewer for these helpful suggestions. To investigate the effects of Pak1 ablation, and therefore more directly the link between FOXG1 and FoxO6 and macropinocytosis, we tested the published Pak1 inhibitor IPA-3. Unfortunately, to distinguish the role of Pak1 in quiescence exit and macropinocytosis, we would need a dosage of IPA-3 that is efficacious but does not affect cell proliferation. It was not possible to optimise such a dosage (a dosage of 10uM is shown to be efficacious at inhibiting Pak1 (Verma et al, 2020; Wong et al, 2013) however even at 2.5uM we see significant cell death in our cells. Indeed, this is potentially due to pleiotropic roles for Pak1.

Also, it is not feasible to overexpress Pak1 in the FoxO6 KO cells with inducible FOXG1. To ensure we are investigating quiescence exit this would need to be in an inducible manner; however, re-transfecting cells using the PiggyBac system would potentially alter FOXG1 transgene levels by excising the existing transgene.

As shown in Figure S3, we do not observe clear vacuole formation in F6 (FOXG1-inducible) cells upon Dox addition. As detailed in the discussion, we hypothesise that FoxO6-induced macropinocytosis could represent a stalled state, with other pathways downstream of FOXG1 necessary to be activated concomitantly to ensure cell cycle re-entry, e.g., through increased pinocytic flux that cannot be assessed within our experimental timeframes. Indeed, active Pak1 has been found to modulate pinocytic cycling, enhancing both FITC-dextran uptake and efflux (Dharmawardhane et al, 2000). We therefore would not hypothesise that high Pak1 levels alone would be sufficient to drive quiescence exit.

Alternatively, the macropinocytosis observed may be a metabolic stress response because of the hyperactivation of signalling pathways upon FoxO6 overexpression. Hyperactivation of Ras signalling, canonical Wnt and PI3K signalling have all been shown to play roles in inducing macropinocytosis (Overmeyer et al, 2008; Tejeda-Muñoz et al, 2019; Recouvreux & Commisso, 2017).

We believe the observed macropinocytosis phenotype upon Foxo6 overexpression, and the changes in Pak1 expression upon Foxo6 loss or FOXG1 induction provide interesting insights into the function of this underexplored FoxO family member. However, currently we are unable to demonstrate a direct link between these processes and have therefore modified the text to reflect this (see lines 292-4, 330-3, 365-8).

• The manuscript stresses the role of NSC quiescence exit in GBM and demonstrates that FoxG1 KO reduces FoxO6 levels in a murine GBM cell line but a BMP4-mediated quiescence and dox-induced FoxG1 over-expression or an abolishment of cell cycle re-entry thereof by reduced FoxO6 levels in the case of FoxG1 KO is lacking. But this would significantly substantiate the relevance of the findings. *

Mouse GBM cells have elevated levels of FoxG1 and have been shown to be refractory to BMP4-mediated quiescence entry, maintaining colony formation following BMP treatment (Bulstrode et al, 2017). It is therefore challenging to specifically investigate cell cycle re-entry/ quiescence exit using these mouse GBM cells, or indeed any GBM cell line due to their inability to respond fully to BMP cues (Caren et al, 2015). It has also been shown by Bulstrode et al, 2017 that Foxg1 null mouse neural stem cells show an increased propensity to exit cycle in response to BMP treatment, and reduced colony formation on return to EGF/FGF-2 growth factors. FOXG1 null cell lines therefore show a reduced response to BMP cues, making it difficult to explore quiescence exit per se.To navigate this, instead we investigated Dox-induced FOXG1 overexpression in FoxO6 WT and KO mouse NS cells, which display similar quiescence characteristics upon BMP treatment (Figure 4).

• In the introduction and discussion, FoxO6 is mentioned for its oncogenic roles in various cancers but no reference to GBM specifically is cited. It feels like a missed opportunity to not show evidence of this in the IENS cell line that has reduced levels of FoxO6; is there an effect in their proliferative capacity? What are the expression levels of Pak1 following FoxG1 KO in IENS cells? *

Thank you for the helpful suggestion. It is indeed true the literature on FoxO6 in GBM is lacking, explaining the absence of citations on this. On investigation of expression of the proliferation marker Ki67 in these cells we found no significant difference in expression, now shown in Figure 1H. This is in fitting with previous findings of our lab (Bulstrode et al, 2017) which show that FOXG1 is dispensable for the maintenance of continued NSC or GSC proliferation in vitro. We investigated the expression levels of Pak1 following FOXG1 KO in IENS and found a decrease in both KO lines compared to parental cells (updated Figure 6F).

As explained in our discussion, these data suggest that Foxg1/FoxO6/Pak1 are not functionally important in sustaining GSC/NSC proliferation, as shown by the lack of proliferation defects upon Foxg1 or FoxO6 deletion (Bulstrode et al, 2017), but impact regulatory transitions, as cells prepare to exit quiescence into the proliferative radial-glia like state.

*Minor comments *

- Fig1A shows 4 and 2-fold respectively for the two mouse NSC lines, not 17 and 4-fold increase as written on manuscript, please adjust accordingly.

The qRT-PCR data are presented as log2(fold change) or - ddCt, where this value equals zero for the calibrator sample, as indicated in the figure legends and axes. The data are presented in this way to enable accurate visualisation of up- and down-regulation of gene expression. Data are stated as ‘fold increase’ in the text for ease of reading, which we have clarified in the text and figure legends (e.g. lines 154 and 176).

• • Fig2G manuscript reports a 235-fold upregulation, but graph looks more like a 7 or 8-fold as shown on Fig1A for the F6 NSC line. I would recommend checking the fold changes reported throughout the paper. *

See previous comment above. The qRT-PCR data are presented as log2(fold change) or - ddCt, where this value equals zero for the calibrator, as indicated in the figure legends and axes. The data are presented in this way to enable accurate visualisation of up- and down-regulation of gene expression. Data are stated as ‘fold increase’ in the text for ease of reading, which we have clarified in the text and figure legends (e.g. lines 154 and 176).

• The manuscript describes the increase of FOXG1 after BMP4-induced cell cycle exit as compared to non-BMP4 treated cells (p.8 first paragraph), but I am wondering if this expression is rather compared to dox negative and not vs BMP4 negative treatment. *

Data are presented relative to the non-BMP treated (EGF/FGF-2) control throughout the manuscript for consistency. This is to enable changes in expression between -Dox and +Dox to be visualised throughout the quiescence-exit time course relative to the initial starting population in EGF/FGF-2 growth media, prior to BMP treatment.

• 1. In Fig2G it is interesting that FoxO6 is upregulated in BMP4 treated throughout the experiment with highest values at day10 post treatment. At the same time, non-BMP4 treated cells keep decreasing their FoxO6 levels dramatically but there is no mention or reference to this effect.*

In Figure 2G, all cells have been treated with BMP4, prior to return to growth media (EGF/FGF) with or without Dox. It is true that in the +Dox condition with FOXG1 induction, FoxO6 levels continue to increase up to Day 10, perhaps reflective of the expansion of a highly proliferative radial glia-like population.

• 1. Fig2 would benefit from a western blot like Fig1D where FoxG1 and FoxO6-HA protein levels are also shown in dox-treated comparing BMP4-treated vs non-treated. *

Due to the lack of specific FoxO6 antibodies and the absence of a FoxO6-HA tag in this cell line, it is not possible to perform protein analysis of FoxO6 levels in this figure as for Figure 1D.

• The colonies in Fig3E should be quantified, as their ability to form neurospheres seems somewhat compromised upon FoxO6 KO. Fig3B and 3F could perhaps be consolidated into one panel in the interest of space and presentation. *

Good suggestion. We have now consolidated Fig 3B and 3F into one panel (now Figure 3F) as suggested by the reviewer. We performed additional replicates for Figure 3E to quantify the colony formation efficiency. This showed a small but insignificant decrease in colony forming ability in the KO cells (Figure 3E). Importantly the FoxO6 null cells do form colonies, and our results show that FoxO6 is not essential for proliferation or colony formation of NSCs in EGF/FGF-2 – this therefore does not account for the complete loss in colony formation we see the in the FoxO6 KO cells upon FOXG1 induction.

• Fig4A shows vs "parental" non-BMP on y axis but wouldn't this show fold change of dox+ parental vs parental. The authors should clarify this. *

All samples in Figure 4A are compared to parental cells in EGF/FGF-2, i.e. non-BMP treated, as the calibrator sample where log2(fold change) equals zero. We chose to set a single calibrator sample for all data (parental and FoxO6 KO cells included) to allow us to compare changes in FOXG1 transgene across the entire experiment.

• Perhaps the authors can add a non-BMP4 treated count of % FOXG1 positive cells to Fig4C for reference. *

As shown in Figure 4A, both parental and FoxO6 KO cells show similar, i.e. negligible, FOXG1 transgene expression without Dox, compared to the parental non-BMP4 treated control, therefore negligible FOXG1-V5 positive cells are seen by ICC. We have edited Figure 4A to include a non-BMP treated and BMP-treated control to show the negligible FOXG1-V5 expression by qPCR as controls.

• The sentence mentioning Fig5D for the first time (p.10 third paragraph) needs rephrasing for clarity and should also call out Fig5C for the mCherry expression live cell imaging data where appropriate. Fig5D does not appear to be live imaging as implied by the text. If vacuole formation is observed already as early as 10-11h after Dox induction, then it should be shown somewhere in Fig5. Vacuole formation is shown with a higher magnification image inset only in the 22h timepoint image. I think Fig5E should be more substantiated with some sort of quantification, e.g. % of vacuoles positive for EEA1 and/or LAMP1. *

We apologise for this. The first reference to Figure 5D one line 234 should refer to Figure 5C, this has now been corrected in the text. Vacuoles are visible in Figure 5C panel 10 h 30 min, however, to make this clearer we have also supplied an accompanying movie of the live imaging (Movie 1). The imaging in Fig 5E has not been quantified as this imaging was performed with the purpose of confirming the vacuole structures seen are not simply enlarged lysosomes, due to their similarity in appearance to those published elsewhere (Ramosaj et al, 2021; Leeman et al, 2018). Instead, we have provided Western blotting data in Figure S5E to support this conclusion that there is no clear increase in EEA1 or LAMP1 (early endosomal or lysosomal) expression upon FoxO6-HA induction.

*- Could the authors comment on the lack of proliferative advantage of the FoxO6 overexpression. FigS3 shows Edu staining, but there is no proliferation assay in either Fig5 or S3. What would be the effect of FoxO6 overexpression on BMP4-mediated quiescence with or without FoxG1 over-expression? *

Induction of FoxO6-HA overexpression does not provide a proliferative advantage to the cells. Looking at individual cells, those with high FoxO6-HA levels seem to associate with EdU negativity. In Figure S3 we provide quantitative EdU incorporation assay as a proliferation assay (quantification of the number of cells cycling, therefore incorporating EdU, within a 24h pulse period). Quantification of the EdU staining in Figure S3G is provided in Figure S3H. We have now clarified this in the text on page 11, lines 263-4.

Unfortunately, due to transgene overexpression using the PiggyBac transposon method, it is not feasible to overexpress FoxO6 and FOXG1 in the same cell line, as re-transfecting cells using the PiggyBac system would potentially alter FOXG1 transgene levels and make results difficult to interpret. Given the association of vacuolated cells with EdU negativity, we predict that FoxO6 overexpression would not give an advantage for quiescence exit. Indeed, BMP-treated cells with FoxO6 overexpression show a decrease in EdU positivity, as shown in Figure S3H. As discussed in the text, we hypothesise that cells with FoxO6 overexpression are in a stalled state, potentially due to signalling hyperactivation. While this may not be physiological, it gives us clues as to the function and downstream targets of FoxO6, which remain uncharacterised.

*- Can the authors clarify if there is a proliferation change in F6 cells in Fig6F as in Fig2F? Fig6F shows Pak1 is already upregulated in quiescent NSCs, what are the expression levels of Pak1 in FoxO6 -/- ANS4 cells upon FoxG1-mediated quiescence exit as shown in Fig4? Is there a particular reason why the F6 cell line data is shown only up to day2 post Dox-induction rather than d4 or d10? For consistency with the rest of similar experimental data this timeline should be extended. Does Pak1 remain elevated, plateaus or keeps reducing further post day2? *

The data is (previous) Figure 6F is the same assay and cell line as presented in Figure 2, but at an early timepoint (Day 2) during the quiescence exit assay. We have provided in the panel qRT-PCR analysis of Ki67 to show that cells begin to show increased proliferation at this timepoint. Due to our hypothesis that Pak1 is required at an early transition point, we decided to analyse this expression at an earlier timepoint than Figure 2. We have also repeated this at D10 (data below), showing Pak1 levels continue to increase with time, along with FoxO6 and the proliferative marker Ki67. Due to technical issues with variable FOXG1 transgene levels we were unable to analyse Pak1 expression levels in FoxO6+/- ANS4 cells upon FOXG1-mediated quiescence exit.

*15 . Reviewer #1 (Significance (Required)): *

The study provides a conceptual advance for exit from stem cell quiescence. There is strong evidence provided for murine neural stem cells, but the link to GBM cancer stem cells is less developed (but perhaps this is the subject of a separate manuscript).

While FoxG1 is a known regulator of neurodevelopment and glioblastoma, the functions of FoxO6 have not been studied in the context of neural stem cells. In my view, this study should be of high interest to audiences in both neurodevelopment and cancer research. * Expertise: glioblastoma, cancer stem cells, neurodevelopment *

We have edited the text and title to clarify that neural stem cells are used here as a model for GSCs with high levels of FOXG1 (e.g. lines 36 and 69).

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Reviewer #2 (Evidence, reproducibility and clarity (Required)):

*Major comments: *

-The choice of NSCs as a main experimental model to understand the effects of FoxG1 and FoxO6 is not fully justified. The authors had previously shown that FoxG1 is expressed at very low levels in NSCs (Fig. 1A in Bulstrode et al. 2017). FoxO6 also seems to be barely expressed in NSCs (Fig. 1 of the current manuscript) and, in addition, its levels seem to go further down as cells exit quiescence (-Dox line in Fig. 2H). Therefore, these two genes do not seem to play an important role in the normal exit from quiescence of NSCs, with FoxO6 only affecting FoxG1 overexpression-induced exit from quiescence. * * *If the aim is to mimic a GBM-like state by FoxG1 overexpression, this should be made much clearer in the text, including title and abstract. In that case, the authors should also show a direct comparison of the levels of FoxG1 in GBM and upon Dox-induced overexpression in NSCs. *

We agree with this criticism and suggestion to fix this. It is indeed our aim to mimic a GBM-like state by inducing FOXG1 overexpression and we should have made that more explicit. All experiments are performed in the context of high FOXG1 level. Like Foxg1, FoxO6’s homeostatic roles may be subtle in adulthood, and mostly involved in neural plasticity (Yu et al, 2019). This is in keeping with our finding that basal FoxO6 levels are low in adult NSCs and not required for sustained proliferation but are important for cell state transitions. If the FoxO6 levels activated by elevated FOXG1 represent an acquired dependency of GBM, there may be a therapeutic window to target this pathway. However, given the poorly understood roles of FoxO6, further work is needed to determine its specific value as a therapeutic target. We have modified the title and the text to make this clearer. This is also stated in the first paragraph of the results section on page 7 (line 148).

We have provided below a Western Blot (Bulstrode, 2016) in which FOXG1 levels in F6 cells induced with Dox (1000 ng/ml the dosage used) with the GBM cell lines G7 and G144, and the normal NS cell line U5. This shows that the FOXG1 levels induced are significantly higher than found in normal neural stem cells (mouse or human). This model has been previously used and published in Bulstrode et al, 2017, upon which this manuscript expands.

*-While the authors state that they aim to study NSC quiescence, they use a protocol that is closer to modelling astrocytic differentiation. In fact, in their previous work, they use this very same protocol (removal of growth factors and addition of BMP) to study the role of FoxG1 and Sox2 on astrocyte de-differentiation (Bulstrode et al. 2017). While there is arguably no perfect in vitro model of NSC quiescence, the current standard in the field is treatment with both BMP and FGF for 48 to 72 hours (e.g.: Mira et al., 2010, Martynoga et al., 2013, Knobloch et al., 2017, Leeman et al., 2020). BMP alone is regarded as a pro-astrocytic differentiation cue, and 24 hours might not be enough for NSCs to fully commit to either differentiation or quiescence. Therefore, either the claims in the paper are changed to match the astrocytic differentiation model, or a standard quiescence protocol should be used throughout to confirm the findings also apply to the exit from quiescence of NSCs. *

We agree with the reviewer that there is indeed no perfect in vitro model of NSC quiescence and thank the reviewer for this useful discussion. Coincident with this project, this was an active area of research from our laboratory as explored by Marques-Torrejon et al, 2021 (Nature Comms). After 24 h BMP4 treatment, we found that adult mouse NS cells: exit cell cycle, are growth factor unresponsive, obtain an astrocytic morphology, upregulate astrocytic markers such as Gfap and Aqp4, and downregulate radial glia/NS cell markers such as Nestin and Olig2 (Figure 3).

We therefore initially viewed them as terminally differentiated. However, the exact state of these cells is difficult to define due to the lack of definitive markers and transcriptional differences that can distinguish terminally differentiated GFAP-expressing astrocytes from quiescent type B SVZ NS cells (which also express GFAP) (Bulstrode et al, 2017; Doetsch et al, 1999; Codega et al, 2014). Findings from our laboratory later suggested some NS cell markers are maintained following BMP4 treatment and these cells can be forced back into cycle with combined Wnt/EGF signalling, or FGF/BMP signalling (Marques-Torrejon et al 2021). This suggests in vitro NS cells may lie along a continuous spectrum of states from dormant quiescent, activated quiescent (primed for cell cycle re-entry) to actively proliferating, similar to that observed in vivo in the mouse SVZ (Dulken et al, 2017). Indeed, after 24 h BMP4 treatment, we observe a minimal level of colony formation in no Dox controls following 10 days of exposure to the growth factors EGF/FGF-2 (Figure 2D-F).

These non-cycling BMP4-induced astrocytic cells might therefore be better viewed as dormant quiescent NSCs, hence our reference as quiescent NSCs. The assay conditions used in this manuscript differ to those of Marques-Torrejon et al, in terms of density and length of BMP4 treatment; it is therefore likely that our BMP-treated cells are at different stages along the continuum between dormancy and primed quiescent states. Importantly, regardless of the exact cell type induced by 24 h BMP4 treatment, we have considered the changes induced by FOXG1 overexpression, in comparison to the effect of NS cell media alone.

*-The FoxO6-induced vacuole formation in NSCs is a very interesting finding. However, so far it was only observed upon FoxO6 overexpression. To claim vacuolization is required for quiescence exit, the authors should show whether this phenomenon is also observed upon normal exit from quiescence and FoxG1-induced reactivation of NSCs. From the author's own data, Pak1 (which induces vacuolization) is unlikely to reactivate NSCs, as its expression is highest in BMP-treated cells (Figure 6F). The authors should show whether some vacuolization is present at these stage in NSCs and if not, discuss the possible interplay between Pak1 and FoxO6 in vacuole formation and quiescence exit. *

As detailed in the discussion, we hypothesise that FoxO6- induced macropinocytosis could represent a stalled state, with other pathways downstream of FOXG1 necessary to be activated concomitantly to ensure cell cycle re-entry, e.g., through increased pinocytic flux that cannot be assessed within our experimental timeframes. Indeed, active Pak1 has been found to modulate pinocytic cycling, enhancing both FITC-dextran uptake and efflux (Dharmawardhane et al, 2000). Alternatively, the macropinocytosis observed may be a metabolic stress response because of hyperactivation of signalling pathways upon FoxO6 overexpression Hyperactivation of Ras signalling, canonical Wnt and PI3K signalling have all been shown to play roles in inducing macropinocytosis (Overmeyer et al, 2008; Tejeda-Muñoz et al, 2019; Recouvreux & Commisso, 2017).

We do not see clear evidence of vacuoles in FOXG1-induced reactivation of NSCs – this supports that the macropinocytosis seen upon FoxO6 overexpression is a stalled state or due to hyperactivation. While this may not be physical, it gives us clues as to the function and downstream targets of FoxO6, which remain uncharacterised (such as a link of FoxO6 and FOXG1 with Pak1-related pathways). Demonstrating a requirement for vacuolisation in quiescence exit is outwidth this manuscript and therefore we are careful not to claim this. We have modified the text to clarify this.

As the reviewer noted, it is interesting that Pak1 is highest in BMP-treated cells; it seems that BMP signalling itself is triggering elevated Pak1 levels, likely as cells undergo extensive cell shape changes during the transition from proliferation to quiescence. However, in EGF/FGF-2, Pak1 levels decrease, and our data suggests that FOXG1/FoxO6 are required to increase or maintain Pak1, potentially to again enable the cell shape/metabolic changes required on quiescence exit. We have added to the text to expand upon this observation on page 14 (lines 330-333). -Finally, the data on the regulation of Pak1 expression by FoxO6 is insufficient to draw any strong conclusions. Downregulation of Pak1 in FoxO6 cells is not enough evidence to claim a direct regulation. The authors should show whether Pak1 levels are increased after FoxO6 overexpression and whether FoxG1 is downregulated in FoxO6 KO NSCs (indirectly affecting Pak1 expression).

We have performed qRT-PCR analysis of Foxg1 expression in FoxO6 KO NSCs and see no consistent difference in expression, indicating this is not indirectly affecting Pak1 expression (see below, 1). We have also investigated Pak1 levels upon FoxO6 overexpression, over a time course following Dox addition (see below, 2). Interestingly, when FoxO6 is overexpressed, Pak1 is not clearly upregulated at any time-point. It may be that as Pak1 is already expressed in the -Dox controls, due to its roles in a variety of cellular functions, that the levels are saturated already. It is clear that Pak1 expression decreases upon FoxO6 loss in EGF/FGF (without coincident Foxg1 downregulation) and in F6 cells, higher FOXG1 correlates with higher Pak1 in EGF/FGF. Together with the induction of macropinocytosis upon FoxO6 overexpression, these data provide interesting insights into the potential pathways downstream of Foxo6 in controlling quiescence exit, directly or indirectly related to Pak1 signalling. We have modified the text to reflect this on page 14 (lines 330-333).

Minor comments: * Please state in the main text that NSCs are derived from the SVZ. *

This has been added to the text on page 7 (line 149) and is in the methods ‘Cell Culture’ section.

Reviewer #2 (Significance (Required)):

As I said before, I find this work tackles a very important question, how is the exit from quiescence controlled in NSCs. This manuscript will be of interest to researchers in the fields of adult stem cell biology and adult neurogenesis. While my expertise lies mostly on NSC biology, this work is of potential great interest for the cancer field, particularly for brain cancer research. Elucidating the mechanisms GBM cells use to exit quiescence is crucial in order to avoid the relapse of this aggressive form of brain cancer. To increase the relevance of the work to the cancer community, some of the key findings should be reproduced with GBM cells. It would be particularly important to show whether Pak1 induced vacuolization and macropinocytosis can be observed in GBM cells.

As detailed in the discussion, we hypothesise that FoxO6- induced macropinocytosis could represent a stalled state, with other pathways downstream of FOXG1 necessary to be activated concomitantly to ensure cell cycle re-entry, e.g., through increased pinocytic flux that cannot be assessed within our experimental timeframes. Alternatively, the macropinocytosis observed may be a metabolic stress response because of hyperactivation of signalling pathways upon FoxO6 overexpression Hyperactivation of Ras signalling, canonical Wnt and PI3K signalling have all been shown to play roles in inducing macropinocytosis (Overmeyer et al, 2008; Tejeda-Muñoz et al, 2019; Recouvreux & Commisso, 2017). We do not see clear evidence of vacuoles in FOXG1-indued reactivation of NSCs– this supports that the macropinocytosis seen upon FoxO6 overexpression is a stalled state or due to hyperactivation. We do not therefore think macropinocytosis per se would be observed in quiescence exit of GBM cells – indeed a normal form of macropinocytosis-induced cell death called methuosis has been observed in GBM cells with hyperactivated Ras signalling (Overmeyer et al, 2008). However, this phenotype still gives us clues as to the function of FoxO6 in quiescence exit in GSCs and the downstream signalling pathways it may regulate, such as Pak1-related signalling (discussed on lines 330-3 and 366-9).

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary: * The overall objective of the paper is to investigate the mechanisms by which co-option of the activity of developmental master lineage regulators by cancer cells allows them to gain fitness. To answer this question, they focus on FOXG1. This TF acts during the specification of the telecephalon. Its expression can be increased in Glioblastoma (GBM) and, more importantly for the paper, FOXG1 has previously been shown to promote exit from quiescence of glioblastoma stem cells (GSCs) and non-transformed neural stem cells (NSCs). In a previous screen, the authors identified FoxO6 as a potential direct target gene of FOXG1. In this paper, they showed that with the gain of expression for FOXG1 in NSCs and loss of FOXG1 in GSCs, FoxO6 is increased or decreased, respectively. Loss of FoxO6 in NSCs does not alter their cell cycle or cell shape and specification. Yet, loss of FoxO6 in NSCs blocks FOXG1-mediated exit from quiescence. To understand the mechanisms, they decided to overexpress FoxO6 in NSCs and demonstrated that the cells undergo macropinocytosis, a process by which cells can engulf large amount of nutriments from the external medium. It remains to be determined whether this macropinocytosis occurs in cells overexpressing FOXG1 and GSCs. The authors provide a first answer by showing that overexpression of FOXG1 induces not only FoxO6 but also the expression of PAK1, one of the key kinases that regulates the membrane engulfment of macropinocytosis in NSCs. In GSC lines, the decrease of FOXO6 decreases PAK1 levels. *

Major comments: * The paper describes interesting and convincing results (number of cell lines, repeated experiments seems sufficient) but it is difficult to reconcile them all in a single model, and this diminishes the impact of the study. Epistatic interactions between FoxG1, FoxO6, PAK1 and macropinocytosis are not always studied in the same cell models. Whether FOXG1-induced exit from quiescence of NSCs is dependent on a FOXG1-->FOXO6-->PAK1-->Macropinocytosis axis remains to be demonstrated. Also does such an axis operate in tumor cells remains to be fully assessed? In particular, if FoxO6 overexpression in NSCs can induce macropinocytosis, is this cellular process induced by FoxO6 downstream of FOXG1 activity during NSC quiescence exit? Is PAK1 a relay of FoxO6? Experiments looking at macropinocytosis and the involvement of PAK1 in the cell models of Figure 4 will definitely help to bridge the different results all together. *

We thank the reviewer for this useful insight and discussion for future work.

To directly investigate the effects of Pak1 ablation, and therefore more directly the link between FOXG1 and FoxO6 and macropinocytosis, we tested the published Pak1 inhibitor IPA-3. Unfortunately, to distinguish the role of Pak1 in quiescence exit and macropinocytosis, we would need a dosage of IPA-3 that is efficacious but does not affect cell proliferation. It was not possible to optimise such a dosage (a dosage of 10uM is shown to be efficacious at inhibiting Pak1 (Verma et al, 2020; Wong et al, 2013) however even at 2.5uM we see significant cell death in our cells. Indeed, this is potentially due to the variety of cellular functions Pak1 is involved in. Conversely, it is not feasible to overexpress Pak1 in the FoxO6 KO cells with inducible FOXG1. To ensure we are investigating quiescence exit this would need to be in an inducible manner; however, re-transfecting cells using the PiggyBac system would potentially alter FOXG1 transgene levels (through excision of the existing transgene) and therefore make results difficult to interpret.

We hypothesise that FoxO6- induced macropinocytosis could represent a stalled state, with other pathways downstream of FOXG1 necessary to be activated concomitantly to ensure cell cycle re-entry, e.g., through increased pinocytic flux that cannot be assessed within our experimental timeframes (as detailed in the text discussion). Alternatively, the macropinocytosis observed may be a metabolic stress response because of hyperactivation of signalling pathways upon FoxO6 overexpression Hyperactivation of Ras signalling, canonical Wnt and PI3K signalling have all been shown to play roles in inducing macropinocytosis (Overmeyer et al, 2008; Tejeda-Muñoz et al, 2019; Recouvreux & Commisso, 2017). We do not see clear evidence of vacuoles in FOXG1-induced reactivation of NSCs– this supports that the macropinocytosis seen upon FoxO6 overexpression is a stalled state or due to hyperactivation and therefore not a physiological process in quiescence exit. We do not therefore think macropinocytosis per se would be observed in quiescence exit of GBM cells – indeed a normal form of macropinocytosis-induced cell death called methuosis has been observed in GBM cells with hyperactivated Ras signalling (Overmeyer et al, 2008).

However, we believe the observed macropinocytosis phenotype upon Foxo6 overexpression, and the changes in Pak1 expression upon Foxo6 loss or FOXG1 induction provide interesting insights into the function of this underexplored FoxO family member, in GSCs and the downstream signalling pathways it may control, such as Pak1-related signalling. We have modified the text to reflect the limitations of our current data and discuss this (lines 330-3 and 366-9).

I only have a few very specific memes indexed in my box: https://boffosocko.com/tag/zettelkasten-memes/ and a few more at https://hypothes.is/users/chrisaldrich?q=zettelkasten+meme

Historically, Aby Warburg had a large image-based zettelkasten for his work on art which predated Richard Dawkins' conception of meme, but I think qualifies. See: https://boffosocko.com/tag/aby-warburg/ or his Bilderatlas Mnemosyne project: https://warburg.sas.ac.uk/archive/bilderatlas-mnemosyne

It's digital in nature, but Shawn Gilmore has a large collection of images of string walls, Anacapa charts, walls and floors littered with paperwork by obsessives, etc. for his cultural research. It also includes some popular memes. https://www.vaultofculture.com/nst

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replyy to u/a2jc4life at https://www.reddit.com/r/Zettelkasten/comments/1ddhn9n/memes/

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In this manuscript, Eaton et al. examine the regulation of transcription directionality using a powerful genomic approach (more about the methodology below). Their data challenge the notion that the polyadenylation signal-reading Cleavage and Polyadenylation (CPA) complex is responsible for controlling promoter directionality by terminating antisense transcription. Namely, depletion of the required CPA factor RBBP6 has little effect on antisense transcription measured by POINT. They find instead that initiation is intrinsically preferential in the sense direction and additionally maintained by the activities of an alternative processing complex called Integrator, together with the kinase CDK9. In the presence of CDK9 activity, depletion of Integrator endoribonuclease INTS11 leads to globally increased transcription in the antisense direction, and minor effects in the sense direction. However, CDK9 inhibition reveals that sense transcription is also sensitive to INS11 depletion. The authors suggest that CDK9 activity is stronger in the sense direction, preventing INTS11-mediated premature termination of sense transcrpts.

Strengths:

The combination of acute depletion of the studied factors using degron approaches (important to limit possible secondary effects), together with novel and very sensitive nascent transcriptomics methods POINT and sPOINT is very powerful. The applied spike-in normalization means the analysis is more rigorous than most. Using this methodology allowed the authors to revisit the interesting question of how promoter/transcription directionality is determined.

The data quality appears very good and the fact that both global analysis as well as numerous gene-specific examples are shown makes it convincing.

The manuscript is well written and hence a pleasure to read.

We appreciate this positive assessment.

Weaknesses:

I am slightly worried about the reproducibility of the data - it is unclear to me from the manuscript if and which experiments were performed in replicate (lack of table with genomic experiments and GEO access, mentioned in more detail in below recommendations to authors), and the methods could be more detailed.

All sequencing data was deposited with GEO. Multiple biological replicates were performed for each sequencing experiment.  Bigwig files are presented as a table in the GEO submissions. This data has now been made public.

A separate discussion section would be useful, particularly since the data provided challenge some concepts in the field. How do the authors interpret U1 data from the Dreyfuss lab in light of their results? How about the known PAS-density directionality bias (more PAS present in antisense direction than in sense) - could the differential PAS density be still relevant to transcription directionality?

As suggested, we have expanded our discussion to relate our findings to existing data. We think the results from the Dreyfuss lab are very important and highlight the role of U1 snRNA in enforcing transcriptional elongation.  It does this in part by shielding PAS sequences.  Recent work from our lab also shows that U1 snRNA opposes the Restrictor complex and PNUTS, which otherwise suppress transcription (Estell et al., Mol Cell 2023).  Most recently, the Adelman lab has demonstrated that U1 snRNA generally enhances transcription elongation (Mimoso and Adelman., Mol Cell 2023).  Our work does not challenge and is not inconsistent with these studies.

The role of U1 in opposing PAS-dependent termination inspired the idea that antisense transcriptional termination may utilise PASs.  This was because such regions are rich in AAUAAA and comparatively poor in U1 binding sites. However, our RBBP6 depletion and POINT-seq data suggest that PAS-dependent termination is uncommon in the antisense direction. As such, other mechanisms suppress antisense transcription and influence promoter directionality. In our paper, we propose a major role for the Integrator complex.

We do not completely rule out antisense PAS activity and discuss the prior work that identified polyadenylated antisense transcripts. Nevertheless, this was detected by oligo-dT primed RT-PCR/Northern blotting, which cannot determine the fraction of non-polyadenylated RNA that could result from PAS-independent termination (e.g. by Integrator).  To do that requires an analysis of total nascent transcription as achieved by our POINT-seq.  Based on these experiments, Integrator depletion has a greater impact on antisense transcription than RBBP6 depletion. 

I find that the provided evidence for promoter directionality to be for the most part due to preferential initiation in the sense direction should be stressed more. This is in my eyes the strongest effect and is somehow brushed under the rug.

We agree that this is an important finding and incorporated it into the title and abstract.  As the reviewer recommends, we now highlight it further in the new discussion.

References 12-17 report an effect of Integrator on 5' of protein-coding genes, while data in Figure 2 appears contradictory. Then, experiments in Figure 4 show a global effect of INST11 depletion on promoter-proximal sense transcription. In my opinion, data from the 2.5h time-point of depletion should be shown alongside 1.5h in Figure 2 so that it is clear that the authors found an effect similar to the above references. I find the current presentation somehow misleading.

We are grateful for this suggestion and present new analyses demonstrating that our experiment in Figure 2 concurs with previous findings (Supplemental Figures 2A and B). Our original heatmap (Figure 2E) shows a very strong and general antisense effect of INTS11 loss. On the same scale, the effects in the sense direction are not as apparent, which is also the case using metaplots.  New supplemental figure 2A now shows sense transcription from this experiment in isolation and on a lower scale, demonstrating that a subset of genes shows promoter-proximal increases in transcription following INTS11 depletion.  This is smaller and less general than the antisense effect but consistent with previous findings.  Indeed, our new analysis in supplemental figure 2B shows that affected protein-coding genes are lowly expressed, in line with Hu et al., Mol Cell 2023. This explains why a sense effect is not as apparent by metaplot, for which highly expressed genes contribute the most signal.

As a result of our analyses, we are confident that the apparently larger effect at the 2.5hr timepoint (Figure 4) that we initially reported is due to experimental variability and not greater effects of extended INTS11 depletion. Overlaying the 1.5h and 2.5h datasets (Supplemental Figure 4B) revealed a similar number of affected protein-coding genes with a strong (83%) overlap between the affected genes.  To support this, we performed qPCR on four affected protein-coding transcripts which revealed no significant difference in the level of INTS11 effect after 2.5h vs 1.5h (Supplemental Figure 4C).

We now present data for merged replicates in Figures 2 and 4 which reveal very similar average profiles for -INTS11 vs +INTS11 at both timepoints. Overall, we believe that we have resolved this discrepancy by showing that it amounts to experimental variability and because the most acutely affected protein-coding genes are lowly expressed. As detailed above, we show this in multiple ways (and validate by qPCR) We have revised the text accordingly and removed our original speculation that differences reflected the timeframe of INTS11 loss.

Conclusion/assessment:

This important work substantially advances our understanding of the mechanisms governing the directionality of human promoters. The evidence supporting the claims of the authors is compelling, with among others the use of advanced nascent transcriptomics including spike-in normalization controls and acute protein depletion using degron approaches.

In my opinion, the authors' conclusions are in general well supported.

Not only the manuscript but also the data generated will be useful to the wide community of researchers studying transcriptional regulation. Also, the POINT-derived novel sPOINT method described here is very valuable and can positively impact work in the field.

We are grateful for the reviewers' positive assessment of our study.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper that uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full-length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

We appreciate this positive assessment.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figures 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

We agree that other (so far, unknown) factors promote sense transcription over antisense, which was demonstrated by our short POINT.  We have provided an expanded discussion on this in the revision. In our opinion, demonstrating that sense transcription is driven by preferential initiation in that direction is a key finding and we agree that the identification of the underlying mechanism constitutes an interesting avenue for future study.

Reviewer #3 (Public Review):

Summary:

Using a protein degradation approach, Eaton et al show that INST11 can terminate the sense and anti-sense transcription but higher activity of CDK9 in the sense direction protects it from INS11-dependent termination. They developed sPOINT-seq that detects nascent 5'-capped RNA. The technique allowed them to reveal robust transcription initiation of sense-RNA as compared to anti-sense.

Strengths:

The strength of the paper is the acute degradation of proteins, eliminating the off-target effects. Further, the paper uses elegant approaches such as POINT and sPOINT-seq to measure nascent RNA and 5'-capped short RNA. Together, the combination of these three allowed the authors to make clean interpretations of data.

We appreciate this positive assessment.

Weaknesses:

While the manuscript is well written, the details on the panel are not sufficient. The methods could be elaborated to aid understanding. Additional discussion on how the authors' findings contradict the existing model of anti-sense transcription termination should be added.

We have added more detail to the figure panels, which we hope will help readers to navigate the paper more easily. Specifically, the assay employed for each experiment is indicated in each figure panel. As requested, we provide a new and separate discussion section in the revision.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Congratulations on this important piece of work!

Some specific suggestions.

MAJOR

-The data are not available (Accession "GSE243266" is currently private and is scheduled to be released on Sep 01, 2026.) This should be corrected and as a minimum, the raw sequencing files as well as the spike-in scaled bigwig files should be provided in GEO.

We have made the data public. Raw and bigwig files are provided as part of the GEO upload.

MINOR

- It would be useful for readers if you could include catalog numbers of the reagents used in the study.

We have included this information in our revision.

- A table in experimental procedures summarizing the genomic experiments performed in this study as well as published ones reanalyzed here would be helpful.

This is now provided as part of the resources table.

- It would be easier for reviewers to evaluate the manuscript if the figure legends were included together with the figures on one page. This is now allowed by most journals.

We have used this formatting in the revision.

- Providing some captions for the results sections would be helpful.

We have included subheadings as suggested.

Reviewer #2 (Recommendations For The Authors):

Generally, I would suggest writing the experiment-type above panels where it is not immediately obvious what they are so a reader can appreciate the figures without referencing the legend. E.g. write POINT-seq on Figure 1B just to make it obvious to someone looking at the figures what methodology they are looking at. Likewise, you could write RNAPII ChIP-seq for Supplementary Figures 3D and 3E.

We have carried out this recommendation.

Can a y-axis be indicated on POINT-seq genome browser tracks? This could make them easier to interpret.

Y-axis scales are provided as RPKM as stated in the figure legends.

The authors could address/speculate in the text why there is less POINT-seq signal for the antisense transcript in the treatment condition in Figure 1B? Or could consider including a different example locus where this is not the case for clarity.

Acute depletion of poly(A) factors (like RBBP6) results in a strong read-through beyond the poly(A) signal of protein-coding genes as Figure 1 shows.  However, it also causes a reduction in transcription levels, which can be seen in the figure and is correctly noted by the reviewer in this comment.  We see this with other poly(A) factor depletions (e.g. CPSF73 and CPSF30 – Eaton et al., 2020 and Estell et al., 2021) and other labs have observed this too (e.g for CPSF73-dTAG depletion (Cugusi et al., Mol Cell 2022)).  Plausible reasons include a limited pool of free RNAPII due to impaired transcriptional termination or limited nucleotide availability due to their incorporation within long read-through transcripts. For these reasons, we have retained the example in Figure 1B as a typical representation of the effect. Moreover, the heatmap in Figure 1D fairly represents the spectrum of effects following RBBP6 loss – highlighting the strong read-through beyond poly(A) signals and the marginal antisense effects.

"The established effect of INTS11 at snRNAs was detected in our POINT-seq data and demonstrates the efficacy of this approach (Figure 2B)." The authors could explain this point more clearly in the text and describe the data - e.g. As expected, depletion of INTS11 leads to increased POINT-seq signal at the 3' end of snRNAs, consistent with defects in transcriptional termination. This is highlighted by the RNU5A-1 and RNU5B-1 loci (Figure 2B).

We agree and have added more context to clarify this.

I would suggest adjusting the scale of the heatmap in Figure 2E - I think it would be easier to interpret if the value of 0 was white - with >0 a gradient of orange and <0 a gradient of blue (as is done in Figure 1C). I think making this change would make the point as written in the text clearer i.e. "heatmap analysis demonstrates the dominant impact of INTS11 on antisense versus sense transcription at most promoters (Figure 2E)." I'm assuming most of the sense transcription would be white (more clearly unchanging) when the scale is adjusted.

We agree and have done this. The reviewer is correct that most sense transcription is unchanged by INTS11 loss.  However, as we alluded to in the original submission, a subset of transcripts shows a promoter-proximal increase after INTS11 depletion. We have expanded the analyses of this effect (see responses to other comments) but stress that it is neither as general nor as large as the antisense effect.

The authors make the point that there is mildly increased transcription over the 5' end of some genes upon INST11 depletion and show a track (Supplementary Fig 2A). It is not immediately obvious from the presentation of the meta-analysis in Figure 2D how generalisable this statement is. Perhaps the size of the panel or thickness of the lines in Figure 2D could be adjusted so that the peak of the control (in blue) could be seen. Perhaps an arrow indicating the peak could be added? I'm assuming the peak at the TSS is slightly lower in the control compared to INST11 depletion based on the authors' statement.

We have provided multiple new analyses of this data to highlight where there are promoter-proximal effects of INTS11 loss in the sense direction.  Please see our response to the public review of reviewer 1 and new supplemental figures 2A, 2B, 4A and 4B which highlight the sense transcription increased in the absence of INTS11.

The authors label Figure 4 "Promoters lose their directionality when CDK9 is inhibited" - but in INST11 depleted cells treated with CDK9i they find that there still is a bias towards sense transcription. Suggested edit "Some promoter directionality is lost when CDK9 is inhibited" or similar.

We agree and have made this change.

The authors conclude that INTS11-mediated effects are the result of perturbation of the catalytic activities of Integrator, the authors should perform rescue experiments with the catalytically dead E203Q-INTS11 mutant.

This is a very good suggestion and something we had intended to pursue.  However, as we will describe below (and shown in Supplemental Figure 4G), there were confounding issues with this experiment.

The E203Q mutant of INTS11 is widely used in the literature to test for catalytic functions of INTS11.  However, we have found that this mutation impairs the ability of INTS11 to bind other Integrator modules in cells. Based on co-immunoprecipitation of flag-tagged WT and E203Q derivatives, INTS1 (backbone module), 10 (tail module), and 8 (phosphatase module) all show reduced binding to E203Q vs. WT. Because E203Q INTS11 is defective in forming Integrator complexes, rescue experiments might not fully distinguish the effects of INTS11 activity from those caused by defects in complex assembly. While this may at first seem unexpected, in the analogous 3’ end processing complex, catalytic mutants of CPSF73 (which is highly related to INTS11) negatively affect its interaction with other complex members (Kolev and Steitz, EMBO Reports 2005).

We hypothesise that INTS11 activity is most likely involved in attenuating promoter-proximal transcription, but we cannot formally rule out other explanations and discuss this in our revision. Regardless of how INTS11 attenuates transcription, our main conclusion is on its requirement to terminate antisense transcription whether this involves its cleavage activity or not.

The authors suggest that CDK9 modulates INTS11 activity/assembly and suggest this may be related to SPT5. Is there an effect of CDK9 inhibition on the snRNA's highlighted in Figure 2B?

We believe that snRNAs are different from protein-coding genes concerning CDK9 function. Shona Murphy’s lab previously showed that, unlike protein-coding genes, snRNA transcription is insensitive to CDK9 inhibition, and that snRNA processing is impaired by CDK9 inhibition (Medlin et al., EMBO 2003 and EMBO 2005).  We reproduce these findings by metaanalysis of 15 highly expressed and well-separated snRNAs and by qRT-PCR of unprocessed RNU1-1, RNU5A-1 and RNU7-1 snRNA following CDK9 inhibition. We observe snRNA read-through by POINT-seq following INTS11 loss whether CDK9 is inhibited or not (left panel, below). Note the higher TES proximal signal in CDK9i conditions, which likely reflects the accumulation of unprocessed snRNA as validated by qPCR for three example snRNAs (right panel, below).

Author response image 1.

For Figure 4, would similar results be observed using inhibitors targeting other transcriptional CDKs such as CDK7,12/13?

In response to this suggestion, we analysed four selected protein-coding transcripts (the same 4 that we used to validate the CDK9i results) by qRT-PCR in a background of CDK7 inhibition using the THZ2 compound (new Supplemental Figure 4E).  THZ2 suppresses transcription from these genes as expected.  Interestingly, expression is restored by co-depleting Integrator, recapitulating our findings with CDK9 inhibition.  As CDK7 is the CDK-activating kinase for CDK9, its inhibition will also inhibit CDK9 so THZ2 may simply hit this pathway upstream of where CDK9 inhibitors.  Second, CDK7 may independently shield transcription from INTS11.  We allude to both interesting possibilities.

What happens to the phosphorylation state of anti-sense engaged RNAPII when INTS11 is acutely depleted and/or CDK9 is inhibited? This could be measured by including Ser5 and Ser2 antibodies in the sPOINT-seq assay and complemented with Western Blot analysis.

We have performed the western blot for Ser5 and Ser2 phosphorylation as suggested.  Both signals are mildly enhanced by INTS11 loss, which is consistent with generally increased transcription.  Ser2p is strongly reduced by CDK9 inhibition, which is consistent with the loss of nascent transcription in this condition.  Interestingly, both modifications are partly recovered when INTS11 is depleted in conjunction with CDK9 inhibition. This is consistent with the effects that we see on POINT-seq and shows that the recovered transcription is associated with some phosphorylation of RNAPII CTD.  This presumably reflects the action(s) of kinases that can act redundantly with CDK9.

We have not performed POINT-seq with Ser5p and Ser2p antibodies under these various conditions.  Our rationale is that our existing data uses an antibody that captures all RNAPII (regardless of its phosphorylation status), which we feel most comprehensively assays transcription in either direction. Moreover, the lab of Fei Chen (Hu et al., Mol Cell 2023) recently published Ser5p and Ser2p ChIP-seq following INTS11 loss. By ChIP-seq, they observe a bigger increase in antisense RNAPII occupancy vs. sense providing independent and orthogonal support for our POINT-seq data.  Interestingly, this antisense increase is not paralleled by proportional increases in Ser5p or Ser2p signals.  This suggests that the unattenuated antisense transcription resulting from INTS11 loss does not have high Ser5p or Ser2p.  Since CDK7 and 9 are major Ser5 and 2 kinases, this supports our model that their activity is less prevalent for antisense transcription.  We now discuss these data in our revision.   

The HIV reporter RNA experiments should be performed with the CDK9 inhibitor added to the experimental conditions. Presumably CDK9 inhibition would result in no upregulation of the reporter upon addition of TAT and/or dTAG. Perhaps the amount of TAT should be reduced to still have a dynamic window in which changes can be detected. It is possible that reporter activation is simply at a maximum. Can anti-sense transcription be measured from the reporter?

We have performed the requested CDK9 inhibitor experiment to confirm that TAT-activated transcription from the HIV promoter is CDK9-dependent (new supplemental figure 4F).  Consistent with previous literature on HIV transcription, CDK9 inhibition attenuates TAT-activated transcription.  Importantly, and in line with our other experiments, depletion of INTS11 results in significant restoration of transcription from the HIV promoter when CDK9 is inhibited. Thus, TAT-activated transcription is CDK9-dependent and, as for endogenous genes, CDK9 prevents attenuation by INTS11.

While TAT-activated transcription is high, we do not think that the plasmid is saturated. When considering this question, we revisited previous experiments using this system to study RNA processing (Dye et al., Mol Cell 1999, Cell 2001, Mol Cell 2006). In these cases, mutations in splice sites or polyadenylation sites have a strong effect on RNA processing and transcription around HIV reporter plasmids. Effects on transcription and RNA processing are; therefore, apparent in the appropriate context. In contrast, we find that the complete elimination of INTS11 has no impact on RNA output from the HIV reporter. Our original experiment assessing the impact of INTS11 loss in +TAT conditions used total RNA.  One possibility is that this allows non-nascent RNA to accumulate which might confound our interpretation of INTS11 effects on ongoing transcription.  However, the new experiment described in the paragraph above was performed on chromatin-associated (nascent) RNA to rule this out.  This again shows no impact of INTS11 loss on HIV promoter-derived transcription in the presence of TAT.

To our knowledge, antisense transcription is not routinely assayed from plasmids. They generally employ very strong promoters (e.g. CMV, HIV) to drive sense transcription.  Crucially, their circular nature means that RNAPII going around the plasmid could interfere with antisense transcription coming the other way which does not happen in a linear genomic context. This is why we restricted our use of plasmids to looking at the effects of stimulated CDK9 recruitment (via TAT) on transcription rather than promoter directionality.   

The authors should clearly state how many replicates were performed for the genomics experiments. Ideally, a signal should be quantified and compared statistically rather than relying on average profiles only.

We have stated the replicate numbers for sequencing experiments in the relevant figure legends. All sequencing experiments were performed in at least two biological replicates, but often three. In addition, we validated their key conclusions by qPCR or with orthogonal sequencing approaches.

Reviewer #3 (Recommendations For The Authors):

The authors provide strong evidence in support of their claims.

ChIP-seq of pol2S5 and S2 upon INST11 and CDK9 inhibition will strengthen the observation that transcription in the sense direction is more efficient.

We view the analysis of total RNAPII as the most unbiased way of establishing how much RNAPII is going one way or the other. Importantly, ChIP-seq was very recently performed for Ser2p and Ser5p RNAPII derivatives in the lab of Fei Chen (Hu et al., Mol Cell 2023). Their data shows that loss of INTS11 increases the occupancy of total RNAPII in the antisense direction more than in the sense direction, which is consistent with our finding. Interestingly, the increased antisense RNAPII was not paralleled with an increase in Ser2p or Ser5p. This suggests that, following INTS11 loss, the unattenuated antisense transcription is not associated with full/normal Ser2p or Ser5p. These modifications are normally established by CDK7 and 9; therefore, this published ChIP-seq suggests that they are not fully active on antisense transcription when INTS11 is lost. This supports our overall model that CDK9 (and potentially CDK7 as suggested for a small number of genes in new Supplemental Figure 4E) is more active in the sense direction to prevent INTS11-dependent attenuation. We now discuss these data in our revision.

In Supplementary Figure 2, the eRNA expression increases upon INST11 degradation, I wonder if the effects of this will be appreciated on cognate promoters? Can the authors test some enhancer:promoter pairs?

We noticed that some genes (e.g. MYC) that are regulated by enhancers show reduced transcription in the absence of INTS11. Whilst this could suggest a correlation, the transcription of other genes (e.g. ACTB and GAPDH) is also reduced by INTS11 loss although they are not regulated by enhancers.  A detailed and extensive analysis would be required to establish any link between INTS11-regulated enhancer transcription and the transcription of genes from their cognate promoters.  We agree that this would be interesting, but it seems beyond the scope of our short report on promoter directionality.

Line 111, meta plot was done of 1316 genes. Details on this number should be provided. Overall, the details of methods and analysis need improvement. The layout of panels and labelling on graphs can be improved.

We have now explained the 1316 gene set.  In essence, these are the genes separated from an expressed neighbour by at least 10kb.  This distance was selected because depletion of RBBP6 induces extensive read-through transcription beyond the polyadenylation site of protein-coding genes.  To avoid including genes affected by transcriptional read-through from nearby transcription units we selected those with a 10kb gap between them. This was the only selection criteria so is unlikely to induce any unintended biases. Finally, we have added more information to the figure panels and their legends, which we hope will make our manuscript more accessible.

Reviewer #2 (Public Review):

Summary:

Eaton and colleagues use targeted protein degradation coupled with nascent transcription mapping to highlight a role for the integrator component INST11 in terminating antisense transcription. They find that upon inhibition of CDK9, INST11 can terminate both antisense and sense transcription - leading to a model whereby INST11 can terminate antisense transcription and the activity of CDK9 protects sense transcription from INST11-mediated termination. They further develop a new method called sPOINT which selectively amplifies nascent 5' capped RNAs and find that transcription initiation is more efficient in the sense direction than in the antisense direction. This is an excellent paper which uses elegant experimental design and innovative technologies to uncover a novel regulatory step in the control of transcriptional directionality.

Strengths:

One of the major strengths of this work is that the authors endogenously tag two of their proteins of interest - RBBP6 and INST11. This tag allows them to rapidly degrade these proteins - increasing the likelihood that any effects they see are primary effects of protein depletion rather than secondary effects. Another strength of this work is that the authors immunoprecipitate RNAPII and sequence extracted full length RNA (POINT-seq) allowing them to map nascent transcription. A technical advance from this work is the development of sPOINT which allows the selective amplification of 5' capped RNAs < 150 nucleotides, allowing the direction of transcription initiation to be resolved.

Weaknesses:

While the authors provide strong evidence that INST11 and CDK9 play important roles in determining promoter directionality, their data suggests that when INST11 is degraded and CDK9 is inhibited there remains a bias in favour of sense transcription (Figure 4B and C). This suggests that there are other unknown factors that promote sense transcription over antisense transcription and future work could look to identify these.

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Reply to the reviewers

We would like to thank all reviewers for their detailed and constructive feedback, which substantially helped improve the manuscript. We apologise for the time taken for the revisions, which was partially due to the first author (successfully) writing and defending her PhD thesis in the same time frame. We would like to point out already here that, based on reviewers' feedback, main figure 6 is completely redone and the conclusions of this figure have changed substantially. We no longer suggest RNA chaperoning activity (it was identified as being due to the high concentration of TEV protease, in a control suggested by the reviewers). Instead, our refined assay conditions with lower TEV protease concentration identified ribonuclease activity of membrane-bound full-length 2C, which is consistent with a publication from 2022 (PMID: 35947700).

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Reviewer #1 (Evidence, reproducibility and clarity (Required)):

Evidence, reproducibility, and clarity

Summary:

In this study by Shankar and colleagues, the authors aim to understand the structure and function of the enterovirus 2C protein, a putative viral helicase with AAA+ ATPase activity. Using poliovirus (as a model enterovirus) 2C, the author's propose the protein contains two amphipathic helices (AH1 and AH2) at the N-terminus that are divided by a conserved glycine. Using purified MBP-tagged 2C and N-terminal 2C truncations, their data suggests AH1 is primarily responsible for clustering at membranes, whilst AH2 is the main mediator of 2C oligmerisation and membrane binding. Furthermore, 2C was suggested to be able to recruit RNA to membranes, with a preference for dsRNA, and the author's data implies that the helicase activity of 2C is ATP-independent. Instead, the ATP activity appears to be required for 2C hexamer formation or chaperone activity. The manuscript is generally well written /presented and the author's present very interesting data which raises several questions, some of which require additional experimentation to help support the author's conclusions. Specific comments are as follows.

We thanks the reviewer for the overall positive assessment, as well as the specific comments below.

Major Comments:

1. The authors use four main constructs throughout the paper: full-length 2C, 2C with deletion of AH1 (ΔAH1), 2C with both AH1 and AH2 deleted (ΔMBP) and 2C with an extended N-terminal deletion. From this, the author's draw conclusions on the function of both AH1 and AH2. One of the author's main conclusions is that AH2 is the main mediator of 2C membrane association (e.g., in line 169). However, is it possible to conclude the relative importance of AH1 vs AH2 without testing a construct containing the deletion of AH2 only (ΔAH2)? This should be generated and used alongside this data to fully define the relative importance of AH1 and AH2 in these assay and remove the possibility that the deletion of AH1 changes the structure and/or function of AH2, which could also result in the observed differences.

This was a very good suggestion. We expressed and purified the ΔAH2 protein requested by the reviewer and characterized its oligomeric state as well as its membrane binding. It turns out, as suspected, that the ΔAH2 protein behaves very similarly to the ΔMBD protein (i.e. it does not form higher order oligomers and does not bind membranes). The changes in the manuscript due to this addition are many but can primarily be found in main figures 2-3 and their associated supplementary figures.

Previous structural predictions of 2C do not appear to have two separate AHs at the N-terminus. Are the AH1 and AH2 structures predicted to be formed in the context of the entire 2C protein, 2BC precursors and polyprotein? Are there structural approaches that could provide experimental evidence for two separate AH at the N-terminus?

This is a good point. Previous predictions were not that detailed, partially since they were done in the pre-alphafold era. Unfortunately, we cannot think of a tractable experimental method that could verify the split nature of the amphipathic helix in the only context that would matter: the protein bound to a membrane. A long-term goal would be in situ structures of full-length 2C on membranes using cryo-electron tomography, but our current sample and data sets are not sufficient for this. We added a mention of the long-term need for experimental structures of full-length 2C on lines 315-318 in the discussion.

Why are the 2C dimers (lines 137-138) not apparent on the mass photometry data presented (figure 2)?

Different constructs were measured by mas photometry and SEC-MALS. Also, the required concentration is 100-1000x lower for mass photometry which will affect a dynamic equilibrium in case the same construct were measured by the two methods.

It appeared that binding of ΔMBD-2C was better when POPS is in the membrane (line 174). What is the explanation for this and was this finding significant?

Well spotted. It may mean that 2C has a second, lower affinity membrane-binding site which is charge-dependent somewhere outside the MBD. We now added a mention of this in the discussion, lines 321-323.

From the author's data on lipid drop clustering they conclude ΔAH1 is more effective for clustering, however, the ΔAH1 construct produces pentamers not hexamers (from Figure 2). Is formation of hexamers related to or required for membrane clustering?

ΔAH1 is LESS effective at clustering, not more. As for the mention of pentamers in the original submission: we now think this was an unfortunate choice of words. The mass photometry data for 2C(ΔAH1) could more parsimoniously be interpreted as a mix of hexamers and other (unknown to us) smaller oligomers such as trimers. We have removed all mentions of pentamers.

The replicon data presented in Figure 7 should include a replication-defective control (e.g., polymerase mutant), in order to compare how defective in replication ΔAH1 and ΔMBP deletions are compared to a fully-defective construct. Likewise, deletion of ΔAH1 in this construct is likely to affect processing of the viral polyprotein where several previous studies with picornaviruses have demonstrated that the residues in the P2'-P4' positions can change cleavage efficiency (e.g., PMID: 2542331), or the structure of 2C, leading to the reduction of replication.

Thanks for these good comments. We made the polymerase-dead (GDD-to-GAA) replicon and remeasured it side by side with the 2C replicons. It has a similar luciferase activity indicating that no replication takes place in the 2C deletion replicons. This is shown in the new figure 7. As for the possibility or processing defects, we mentioned this in the original discussion and have now cited the reference suggested by the reviewer in this context (line 324).

How does the author's model of ATPase-independent helicase activity and an APT-dependent required RNA chaperone activity fit with 2 step model for RNA binding and ATPase activity suggested by Yeager et al (PMID: 36399514)?

Acting upon comments from other reviewers, we completely redid the "helicase assay" in the revised manuscript. It turns out that the ATP-independent unwinding activity in the original submission was an artefact of the assay conditions (specifically, of the TEV protease at the higher concentration we used in the old assay). In our improved assay we neither see helicase activity nor ATP-independent RNA chaperoning activity.

Optional major comments that would increase the significance of the work:

All of the optional comments below are exceptionally interesting. But given the long time needed for the several major changes to this manuscript (e.g. the ΔAH2 protein characterization and reoptimisation of the helicase assay) we believe it is more sensible to address them in future studies, for which the 2C reconstitution system can be used.

The preference for dsRNA over ssRNA appears to be quite small (Figure 5d). In the context of a viral infection where ssRNA is likely to outnumber dsRNA at different times during infection is this preference physiologically relevant? In relation to this, what size stretch of dsRNA is required for preference, and could this correspond to cis-acting RNA structural elements, dsRNA as it escapes 3D polymerase or as part of the RF and RI forms (PMID: 9343205)? What is the proposed mechanism of how dsRNA outcompetes membrane tethering of 2C? OPTIONAL The author's study has been conducted in the absence of other viral non-structural proteins. What is the physiological importance of the observations, such as membrane interaction/clustering or RNA binding when presented in the context of the other replication machinery. OPTIONAL Do 2C monomers, dimers and hexamers have different functions in viral replication perhaps at different stages of replication and which of these forms are relevant during viral infection or can they all be detected during infection? Can any suggested separate functional arrangements be separated by genetic complementation experiments? OPTIONAL

Minor comments:

1. The author's appear to interchange between naming/nomenclature of the constructs which makes it confusing to follow (for example, ΔMBD is the same as 2C(41-329) likewise, 2C(Δ115) is sometimes called 2C(116-329)). It would be much easier to follow if the naming of constructs was consistent throughout (unless I am misunderstanding some subtlety in the difference between such constructs).

Thanks very much for spotting this. We have fixed it.

The author's suggest a pentamer arrangement for the ΔAH1 construct, however in the mass photometry data (figure 2D), a hexamer is indicated with the arrow. It would be helpful to change the label to indicate the size of the pentamer where this is being generated, not the hexamer.

As mentioned above, we think the "pentamer" designation of the original manuscript was unfortunate. It is more parsimonious to interpret this as a mix of states, hexamer and undefined snaller.

In most figures, data for full-length 2C, ΔAH1 and ΔMBP is shown. However data for ΔMBP is missing in Figure 4. Using ΔMBP may demonstrate even lower clustering, hinting that AH2 is also involved in this process.

Thanks for this comment. In our view, it can be derived from figure 3 (which shows lack of binding to PC/PE membranes) that the ΔMBD construct would not cluster membranes under the conditions of the assay (clustering requires concomitant binding to two membranes). We now describe our rationale for this on lines 220-222. However, we did include the ΔMBD protein in the new negative staining TEM supplementary figure where it and ΔAH2 show no signs of clustering (figure S10).

I think it would be better for normalise the data in the flotation experiments such that the percentage of 2C in the upper faction is presented as relative to the amount of lipid in the upper fraction (presented in Figure S4).

The change suggested by the reviewer would make it impossible to show the important no-liposome control (leftmost bar in Fig. 3C) in the same plot as the other measurements. We believe that would unnecessarily complicate the figure. Thus, we opted to keep the measurement that are normalised by lipid fluorescence in the supplementary figure. Instead, we now added another mention of this supplementary figure in the legend to main figure 3.

At several places (e.g., lines 232 and 272) the author's refer to "realistic systems". I think the term "physiologically relevant" might be more appropriate.

Agreed and changed throughout.

Line 237: I think "y" is a typo and should read "by".

Thanks. This text was reworked due to the major changes to figure 6.

Reviewer #1 (Significance (Required)):

Significance

I have limited expertise with structural biology but specialise my research on positive-sense RNA virus replication, structure and function. This research is of interest to a broad audience of researchers investigating many positive-sense RNA viruses, which extends beyond the viral family studied here. The work utilises novel techniques to begin to understand the specific roles of 2C in poliovirus replication. The author's data add important incremental new insight into recent studies on viral helicase proteins as referenced in the study, however, a key limitation is understanding the importance/relevance of their observations during a viral infection.

We thanks the reviewer for this positive and nuanced appraisal of our work.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors present an alternative assay system to investigate picornavirus 2C, a protein that is tricky to analyze biochemically in its full length form because of an amphipathic helix at the N-terminus. Poliovirus 2C is expressed with an N-terminal MBP tag, a 50kD protein that helps with solubility as is commonly used for 2C investigations. A difference here is that liposomes are included to mimic membranes for 2C attachment. The key findings are that 2C induces clustering of of liposomes, that double stranded RNA binding by 2C impacts this clustering effect and that a free N-terminus (after cleavage of MBP by TEV protease) is needed for RNA binding and an ATP independent (ie non helicase) RNA duplex separation activity.

Major:

In the floatation assays in figure 3 the authors use a system where MBP-2C is fluorophore-labeled with ATTO488 on exposed cysteines. Poliovirus and other enterovirus 2C has a very well characterized zinc finger domain that has cysteines coordinating a zinc ion. Mutation experiments previously showed that these cysteines are necessary for viral replication and 2C stability. Have the authors controlled for disruption of the zinc finger domain by the labelling of cysteines with ATT0488 and checked if the protein remains folded?

We completely agree with the reviewer and apologise for the omission in the original submission. We have now included a Zn content measurement, which shows unchanged levels between labelled and unlabelled 2C protein (Figure S7). Also, we now in the revised manuscript explicitly describe our original reasoning for labelling on native cysteines: the presence of two cysteines which are not necessary for viral replication and which are more solvent exposed-exposed (and thus more likely to be labelled) in the crystal structure of the soluble fragment of 2C (lines 176-181).

In the analysis of the amphipathic helix, did the authors include membranes in their structural predictions o just the free helix? How does inclusion of membranes impact the predictions? In the predictions in Figure D, only 2 of 4 show a kink and there doesn't seem to be a correlation between those that predict a kink or not and whether the hydrophobic side is aligned in Figure S1.

Unfortunately, predicting a protein structure with the interacting membrane is beyond what is currently doable with protein prediction methods (one would have to combine protein structure predictions with molecular dynamics simulations including a membrane). Based on general principles of protein structure, it is likely that there is some flexibility around G17. Thus there may not be a single "kink angle" for any given virus, but we believe that the presence of the kink (and offset hydrophobic surfaces) for a number of viruses lends credibility and robustness to the observation. We added some descriptions of this thinking on lines 126-127.

Based on previous structures of 2C from different viruses the N-terminal amphipathic helix containing region is predicted to localize on one face of the predicted hexametric structure tethering 2C to the membrane. How does the authors hypothesized model explain 2C dependent clustering? is there evidence that 2C hexamers can oligomerize further into dodecamers for example, maintaining separate faces to enable N-terminal interaction with different membranes? What is the distance between the liposomes in figure 4 at the points of density attributed to 2C? How does this compare to the size of 2C determined in previous structural studies? Is it consistent with one hexamer/2 hexamers sitting on top of one another?

These are very interesting questions but we believe it is prudent to limit our speculation at this point. Eventually, we hope that larger data sets of cryo-electron tomography, coupled to subtomogram averaging, may provide a more definitive answer. What we managed to do with our current cryo-electron tomography data set is to estimate the volume of individual protein densities, and from the volume calculate an estimated molecular mass of the individual complexes seen in the tomograms. This correlates very well with 2C hexamers (new figure 4D).

In the Discussion lines 278-285 the authors suggest that having MBP attached may reflect the polyprotein condition. Can they make a construct with MBP-2B2C to examine interaction with liposomes and assess 2C function?

This is a highly relevant question, but the biochemistry of 2BC is even more challenging than 2C, and we are unfortunately nowhere near being able to work with purified 2BC at the moment.

Discussion lines 293-296, the possibility of two different populations of 2C, binding RNA or membranes cannot be excluded, there is much more 2C around late in infection that present in early infection- the model in figure 8 doesn't acknowledge/capture this.

We have changed the model figure such that more 2C is seen later, and the clustering function is also seen late in infection. The original discussion text referred to (which is unchanged) talks about a "preferential role in RNA replication and particle assembly at later time points" specifically for this reason. We hope the new figure 8 is better at conveying this message.

Discussion lines 313-317, the authors don't reference a study where a mutant of foot-and-mouth disease virus 2C lacking the n-terminal amphipathic helix that could bind but not hydrolyze ATP, hexamerized in the presence of RNA that seems pertinent here (PMID: 20507978).

Thanks for the suggestion. However, after the extensive changes we made to the revised to figure 6 based on excellent reviewer comments (essentially: the RNA chaperoning activity turned out to be an artefact, the improved assay shows no sign of RNA unwinding but instead of 2C-mediated ribonuclease activity), these sentence of the original discussion lost most of their context and we opted to remove them.

Some evidence of MBP-2C cleavage by TEV in the different assays used should be presented as this is a major focus of discussion and currently no gels show TEV cleavage is happening.

Thanks for the suggestion - we agree. We now show these in the new supplementary figures S5 and S12.

Reviewer #2 (Significance (Required)):

The work presents an additional methodology to investigate a a protein that has previously been difficult to study. The authors acknowledge that there is still a lot of 2C biology that remains to be discovered.

Thanks, we agree.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

The manuscript provides insights into the role of the N-terminus in membrane binding and its importance in the various functions of 2C.

Major issues

Line 103-119. Is this novel? I thought people had done a lot of bioinformatic analysis of PV 2C (especially Wimmer) who also did mutational work to analyse the importance of various amino acids in the N-terminal helix. I feel like the paper in general, and this section in particular, underplays the large body of work that has been done on the amphipathic helix by various groups.

We apologise if our original manuscript didn't sufficiently acknowledge previous work in the field. In the first sentence of the mentioned paragraph (now lines 112-113) , we did however cite several papers that have previously addressed the amphipathic nature of the N-terminus of 2C. We have now added two more references along the same line, and changed the wording in a way that we hope better bring across that the amphipathic nature per se has been studies before. We would be happy to add more specific references if the reviewer has any suggestions. However, the rest of our analysis IS indeed novel for the following reasons: (i) we show that the amphipathic region is not a simple, single amphipathic helix, but instead has a conserved glycine (helix breaker/destabiliser residue) and two distinct amphipathic stretches before and after this region, (ii) we use alphafold2 (not available at the time of the earlier work) to provide the first reliable structural models of the membrane-binding domain. These models consistently, across several enterovirus 2C proteins, reveal that the hydrophobic surfaces of the first and second amphipathic regions, on either side of the conserved glycine 17, are offset from one another. This lends additional credibility to the distinct nature of these regions which have not previously been identified as such and which we also show in the biochemical assays to be functionally distinct. We have now also added a clarification to the Discussion that the N-terminus of 2C had previously been identified as its membrane-binding domain and we cite references for this. We hope that these changes will sufficiently acknowledge earlier work in the field while clearly pointing out the advance that our paper makes.

Line 132. Did you validate your column with known MW standards? The peak for full length and deltaAH1 look fairly standard for 2C, in that you have a mixture of species. Not sure you can say it is a hexamer when it is such a broad peak. C doesn't really help you too much since the counts at 400 (pentamer) and 480 (hexamer) are almost the same with quite large error bars. Like most people that have worked with 2C I think the best you can say is that you are making some kind of oligomerized 2C that includes hexamer, pentamer, etc. Why no dimer for MBP-2C and MBP-2C(delta AH1) when compared to the other constructs?

We did not calibrate the gel filtration column since the outcome would anyway be a more crude estimate of molecular mass than the mass photometry and SEC-MALS measurements. But we do agree with the reviewer on the broad mass photometry peaks. To address this experimentally, we compared the existing MBP-2C spectra to new recordings on apoferritin, a highly stable homomultimeric protein complex of a similar mass to aa MBP-2C hexamer. The apoferritin mass estimate is overlayed with the full-length MBP-2C in the new figure 2D and the corresponding supplementary figure S3. This indeed shows that the MBP-2C peak is broader, i.e. consistent with a mix of species which are predominantly but not only hexamers. We describe and discuss this on lines 145-149. As for the mention of pentamers in the original submission: we now think this was an unfortunate choice of words. The mass photometry data for 2C(ΔAH1) could more parsimoniously be interpreted as a mix of hexamers and other (unknown to us) smaller oligomers such as trimers. We have removed all mentions of pentamers.

Line 143. Does your data show that there are two amphipathic helices? Bioinformatics suggests it but your experiments just show the importance of the two areas in oligomerization, not that it is forming two helices.

We agree that the choice of words was not idea and have now changed it to "structure predictions indicate" (lines 162).

Figure S2. Your preps are still relatively dirty, which isn't ideal for biochemical assays. Especially lane 3, where you are looking at 50-60% purity. I don't want you to re-run experiments but I think you need to comment on the purity of the protein you are working with. Also I don't like that you removed the top and bottom of the SDS-PAGE. How much protein never entered the gel. Is there a big fat band at 20 kDa? You need to have the full gel here. Did you measure 260 nm of the preps as well to see if you had bound RNA to the 2C?

Thanks for the comment, we agree that our original submission lacked detail in the description of the protein purification. This is now addressed with the new figure S2 which shows size exclusion chromatograms of the fluorophore-labelled proteins (same chromatograms as in figure 2) and the corresponding uncropped gels imaged both in the stain-free channel (showing all proteins) and in the fluorescence channel. The A260/A280 ratio measured for all proteins shows that they are free of nucleic acids at the point of imaging. The protein preps are not 100% homogeneous but we do believe that they are more than 50-60% pure.

Lines 170. Wasn't this done in the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles"? I don't see it referenced. What is novel about the current work when compared to that paper? Any differences?

Thanks for pointing this out. The referenced study worked with a synthesized, isolated peptide corresponding to AH2 (i.e. not with full protein). An amphipathic peptide outside the context of its protein cannot be expected to recapitulate the properties of the entire protein, e.g. since it is not spatially constrained in how it interactis with membranes. As one example (relating to the title of that paper) we don't see full-length 2C protein tubulating membranes the way the isolated peptide does. As for the reviewer's question about novelty, the paper mentioned does not identify the split nature of the amphipathic region, does not consider the role of AH1, does not characterise the membrane-binding properties of full-length 2C with respect to liposome membrane composition and size, does not identify and characterise the membrane clustering properties of 2C, nor its interactions with nucleic acid when bound to a membrane. However, we do agree that we should have cited the paper in our manuscript. We now cite it in the discussion, lines 320-321.

I'm surprised by the lack of electron microscopy (negative stain mostly) of both the oligomerized 2C and the various liposomes. I know the Carlson group is a microscopy group so why the lack of validation using electron microscopy of the various DLS experiments? I know you did cryo-ET for one of the constructs but I think negative stain electron microscopy of other constructs would be useful.

Thanks for the suggestion. As suggested, we have now expanded the analysis with negative staining EM of several more constructs studied by DLS. It can be found in the new supplementary figure S10.

Figure 4C. What evidence is there that this is 2C apart from you added it to the liposomes? It also comes back to the relative impurity of your protein prep. Could this be E.coli contamination?

Thanks for this comment. We have now added a new supplementary figure (S5) showing SDS-PAGE gels of the reactions used for flotation and DLS assays - which are identical to the cryo-ET samples. In addition, we estimated the molecular mass of the individual, putative 2C desities in the cryo-electron tomograms by measuring their volume. This analysis, which can be found in the new figure 4D, shows that the estimated mass of individual protein densities is consistent with a hexamer of full-length 2C. In addition, we mention in the discussion the long-term need to determine high-resolution structures of membrane-bound 2C using cryo-ET and subtomogram averaging (lines 315-318).

Figure 8. Is this model supported by the data in this paper? Your cryo-ET says that 2C is there but that isn't supported by any other data. How is the dsRNA protected from the innate immune system in this model? is it just sat out in the cytosol? How is the nascent ssRNA packeged into the capsid? Is there competition between the dsRNA and capsid for 2C binding (which your model suggests)? I know it sounds like I am being overly critical of the model but in my opinion there are still too many unanswered questions in the field to come up with a half decent model.

Thanks for this comment. We are the first to agree that our understanding of the roles of 2C is far from complete! We should have been more clear that the model figure represents some of the roles of 2C identified to date, and does not claim to be complete. However we do feel that a model figure serves a purpose of putting our findings into a context, and also providing testable hypotheses for future research . As for the question, some of the roles of 2C shown in the model figure (in particular, particle assembly) are rather supported but earlier work of ourselves and others. We have now produced a new model figure and changed the figure legend to better reflect the incompleteness of the current understanding, and the origin of the different parts of the model figure. In addition, we extended the final paragraph of the discussion (which lists still-unknown aspects of 2C) with the reviewer's mention of dsRNA shielding from innate immunity (lines 374-375). The other aspects mentioned by the reviewer as not yet fully understood are already mentioned in that paragraph.

Minor issues

Lines 43-45: I feel like you underplay the success of the poliovirus vaccination program. Approximately 30 of WPV1 in 2022 and the full eradication of WPV2 and 3. Vaccine derived polio is still an issue but even that is relatively low compared to where the world was in the 1950s.

We agree that the previous wording was not ideal. We replaced it and added another recent reference - related to the type 2 vaccine switch (lines 47-49).

Line 66. I agree there are 11 individual proteins but I feel like this leaves out the fact that some of the uncleaved precursors appear to have some functions, for example 2BC.

Good point. We have now added a mention of 2BC and the fact that it has distinct functions to the introduction (lines 70-71). 2BC is also mentioned in the legend of the model figure (figure 8).

Line 56: LD needs to be defined.

Well spotted thanks. Since the abbreviation was not used anywhere else we opted to spell it out instead (line 59).

Line 75. I think you have misrepresented Xia et al here. They clearly say that in their study that they show helicase and chaperone activity. I never managed to repeat that work but you should still report what they claim. One major thing is that they used insect expressed protein, whereas most people (including myself and in the paper under review) use E.coli expressed protein. Do post translational modifications play an important role in function?

You are right that the reference to their paper for this statement was incorrect. We have now made this part of the introduction more explicit (lines 82-83) and we also in the new discussion mention the possibility of e.g. post-translational modifications affecting 2C helicase activity, under reference to Xia et al (lines 359-361)

Line 103. Need to make it clear here it is poliovirus 2C.

Thanks, we added it (line 112).

Line 135. I assume you mean kDa instead of uM?

It should actually be μM. It is the solution concentration at which the assay was performed. We added some words to clarify this (line 154).

Figure 3. What do you mean by "Only 2C"? Is that MBP-2C? Maybe I am reading the data wrong but adding TEV does nothing? How do you know TEV is removing the MBP? It looks like MBP-2C binds to the liposomes just the same as cleaved MBP-2C. I see in line 165 you acknowledge this. Could an alternative conclusion for line 168 be that MBP isn't being cleaved off but that AH2 is too small to be exposed in that construct? Did you do that construct without MBP being cleaved? I think you need to confirm that MBP is being cleaved off.

Thanks for spotting this mistake. It should indeed be MBP-2C (in the absence of liposomes). We corrected figure 3. Also, in response to this comment and similar ones, we have now added a new supplementary figure showing SDS-PAGE gels of the reaction loaded onto flotation assays and DLS (figure S5). It shows that MBP-2C is cleaved.

Line 184. Is there a reason you use the 2019 paper as a reference instead of the far earlier Bienz et al papers? I'd suggest they are the seminal papers on 2C membrane association. Once again how is this work different from the recent "An Amphipathic Alpha-Helix Domain from Poliovirus 2C Protein Tubulate Lipid Vesicles" paper?

See our response above of the paper mentioned here (which we have now cited). As for why we cite the 2019 paper here: our statement pertains specifically to the contact sites between lipid droplets and replication organelles, not to the membrane binding of 2C per se. We have now added a more general mention of membrane remodelling by non-structural proteins in the introduction, where we cite on of the Bienz papers (lines 75-77).

Figure 5D. So only 1-3% of RNA is found in the upper fraction? Is that significant enough to say that dsRNA was recruited significantly more than ssRNA? How confident are you in your quantification of the starting amounts of RNA?

We agree that the fraction is low, however, the fluorescence signal is very clearly above background. We are thus confident in the measurement. The low percentage at the end of the experiment likely has a simple physico-chemical explanation: in a dynamic equilibrium in a density gradient, whatever RNA dissociates during the run will migrate away from the 2C-vesicle fraction and not be able to rebind. We still tried to address this concern by a complementary experiment where we used fluorescence anisotropy to measure binding of RNA to 2C on vesicles. While the measurements showed the same tendency, they curves were not clean enough to be published, which we think is due to the complex system with 2C bound to vesicles and clusters of vesicles. Still, in view of the relatively low percentage of measured recruitment we opted to adjust the paper title and the title of figure 5 (including the subheading related to figure 5) to put less emphasis on the dsRNA recruitment.

Line 223. Any idea why the MBP needs to be cleaved off? Clearly the MDB is accessible or it would not bind to the liposomes.

Since we have no data directly supporting this we prefer not to speculate in the paper. But one guess would be that the NTD of 2C, as implicated by previous publications, has a dual role in membrane binding and RNA binding. It may be that it can bind membrane while conjugated to MBP, but needs MBP to be removed in order to simultaneously bind membrane and RNA.

Line 237: missing "b" in "by"

Thanks. This paragraph was rewritten in the light of the changes to figure 6.

Figure 6. I don't fully understand the results here. Earlier you showed that the delta MBD didn't really bind SUV. So presumably it isn't really membrane bound. Why does it have similar activity to full-length MBP in your helicase assay if membrane is important? Did you do SUV and TEV protease only control?

We are very grateful to this reviewer (and others) for pointing out the need for a TEV control. When performing the control, we found that the TEV protease, at the high concentrations initially used, surprisingly had an artefactual RNA chaperone-like effect on its own. We then proceeded to titrate down the TEV protease concentration to the point where it no longer interfered. At this TEV protease concentration, although 2C was substantially cleaved (see the new supplementary figure S12), we could no longer detect an RNA chaperone activity. Thus, the contents of the new figure 6, and its conclusions, have been substantially changed. We now focused our attention on the remaining effect that 2C has on RNA: single-strand ribonuclease activity. These experiments were all conducted in the presence of RNase inhibitors, and the presence of Mg2+-dependent ribonuclease activity parallels a recent publication that found this for truncated 2C from hepatitis A and several enteroviruses.

Line 257: "staring"?

Thanks, corrected. A staring glycine would indeed be something strange.

Line 336. Need to change the u to mu.

Thanks, corrected.

Any discussion on your observation in Figure 1D that EV71 and CVB3 don't appear to have AH1 and AH2 or do you think that the domains are conserved across the different viruses?

Thanks for bringing this up. Based on this and a comment from another reviewer, we have now clarified our thinking around this. Since the glycine will introduce some flexibility between AH1 and AH2, we cannot say from the single alphafold predictions that this is THE kink angle. The presence of the kink in the predictions of several MBDs lends more credibility to the robustness of the observation, but most importantly the hydrophobic surfaces in AH1 and AH2 are non-aligned for ALL sequences we looked at. This is now described on lines 126-128.

Table 1 (and possibly elsewhere): an apostrophe is not the prime symbol. 5' compared to 5′.

Thanks, we corrected this throughout.

Line 702 "and" should be "an".

Thanks, corrected.

I couldn't open one of the movies (140844_0_supp_2820374_a2g272.avi).

Sorry to hear this, we will check the movie again.

Reviewer #3 (Significance (Required)):

Overall I liked the paper and is worth publishing. One of the issues in the 2C field is the difficulty in making pure 2C and carrying out in vitro assays that correlate with what is observed in the natural infection. I think this paper suffers from similar struggles with a 2C preparation that doesn't appear that pure. I think it also suffers from not having 2C from a wild-type infection. I don't think that it is feasible to get that kind of 2C but by once again using a recombinant protein from E.coli we are left with another manuscript that provides conflicting evidence of the functions of 2C without a definitive answer. The experiments are well done, although are missing some controls and the manuscript is laid out in a logical manner and is relatively easy to follow.

We thanks the reviewer for these comments. We believe that we have now provided better information regarding the purification of the recombinant 2C protein, and we do think that the controls present in the original manuscript and the revised manuscript alleviate the concerns about lack of specificity. Of course, isolating 2C vesicles from wildtype infection would be another interesting way of approaching its function, but such an approach would come with its own set of challenges related e.g. to the presence of confounding host factors.

Reviewer #4 (Evidence, reproducibility and clarity (Required)):

This is an interesting manuscript that reports the development of an in vitro membrane assay for probing the biochemical functions of the enterovirus 2C protein. The technique is interesting because it can be applied to 2C proteins from other members of the picornavirus family, an important group of mammalian pathogens. It has the capacity to probe different functions (e.g. membrane clustering, ATPase activity, RNA-binding and manipulation activities).

Overall, the manuscript is well written and gives a clear account of the work undertaken. It adds insight to previous studies of enteroviral (and picornaviral) 2C proteins, providing confirmation of some earlier work in a more physiological context and some new insights, particularly into the membrane and RNA binding aspects of 2C.

That said, there are a number of places where some amendment of the claims made is required to provide a more precise statement of the findings of this work. These are listed below.

We thank the reviewer for this positive feedback on our work, as well as for the specific comments below.

Line 21 (Abstract) - The authors claim to have shown that a conserved glycine divides the N-terminal membrane-binding domain into 2 helices. I would suggest instead what they have produced are computational predictions that this is the case - some way short of an experimental demonstration. Sequence analysis predicts helical secondary structure in the N-terminus and indeed Alphafold2 also predicts a helical structure, but these predictions require experimental verification. The authors should therefore rewrite sections that claim to have shown the presence of 2 helices. In doing so, they should perhaps also comment on the fact that Alphafold2 does not predict 2 helices in this region for all enteroviruses (see Fig 1D). Moreover, the sequence analysis in Fig. S1 shows the presence of two Lys residues in the segment 17-38; it would be interesting for the reader to have these indicated in the figures showing the Alphafold2 prediction - do they in any way interrupt the hydrophobic face of the predicted helix?

Thanks very much for this comment, which is in line with what other reviewers also wrote. We agree, and changed the abstract sentence. We have also rewritten the manuscripts in several places to address the limits of structure predictions and the eventual need for an experimental structure of full-length membrane-bound 2C (lines 126-128 and 315-318).

Line 82 (Introduction) - The authors write that the membrane binding domain (MBD) of poliovirus has been shown to mediate hexamerisation, citing Adams et al (2009) - reference 43. However, that is not what this paper shows. Rather it provides evidence of aggregation of an MBP-2C fusion protein into forms that ranged from tetramer to octamer, but no evidence that these aggregates assume functional forms (e.g. the presumed hexameric ring structure characteristic of the AAA+ ATPase family to which 2C belongs). As far as I am aware the first demonstration of hexameric ring formation by a picornaviral 2C protein was for the 2C of foot-and-mouth disease virus (see Sweeney et al, JBC, 2010). Although this is not an enterovirus, this finding was later confirmed for Echovirus 30 (ref 51). I should declare an interest here: the Sweeney paper is from my lab. I will leave it to the editor and the authors to determine how to write a more precise account of the early observations of hexamerisation in picornaviral and enteroviral 2C proteins.

Thanks very much for this insightful comment. As a response to this and other similar comments, we are much more cautious about our wording in the revised manuscript (see also response to comment below. In the part of the introduction discussed here (now lines 89-91) we now use the original wording of the Adams paper ("oligomerization"). In the context of that new text we didn't feel that Sweeney et al paper was a suitable reference, but we now cite it in the later mention of 2C's oligomeric/hexameric state in the first part of the Results (lines 137-138 ).

Line 132 - the authors used mass photometry to investigate oligomeric forms of their MBP-2C constructs and state that for the full length 2C protein "the high-mass peak closely corresponds to a hexamer". While it is true that the peak shown in Fig 2C aligns with the expected MW for an MBP-2C hexamer, the peak is very broad, indicative of the presence of other oligomeric states with lower and higher numbers of monomers. This should be commented on. Indeed, the finding seems to echo the early findings of Adams et al (ref 43) with poliovirus MBP-2C.

Thanks for this comment, which was also made by another reviewer. We cite here what we replied to that reviewer

...we do agree with the reviewer on the broad mass photometry peaks. To address this experimentally, we compared the existing MBP-2C spectra to new recordings on apoferritin, a highly stable homomultimeric protein complex of a similar mass to aa MBP-2C hexamer. The apoferritin mass estimate is overlayed with the full-length MBP-2C in the new figure 2D and the corresponding supplementary figure S3. This indeed shows that the MBP-2C peak is broader, i.e. consistent with a mix of species which are predominantly but not only hexamers. We describe and discuss this on lines 145-149.

Line 143 - for the reasons given above, this summary paragraph represents too strong a statement of what has been observed.

We agree, and changed the paragraph. It now only refers to "oligomerization" (lines 162-164).

Line 197 - I note that the authors did not test the membrane clustering capabilities of the 2C(41-329) construct. Although the 2C(deltaAH1) construct had already shown a significant loss of activity, the shorter construct could still have been a useful control. I don't think it is necessary for this experiment to be done, but if the authors have a rationale for not performing the experiment, perhaps they could include it in a revised manuscript.

Thanks for the suggestion. The rationale is that a protein that doesn't bind a membrane in the first place will also not cluster them (an action that requires binding TWO membranes). We now describe our reasoning on lines 220-222. Nevertheless, we did test these constructs in the new supplementary figure showing negative staining TEM (figure S10).

Line 223 - typo. I think you mean MBD.

Thanks! Corrected (now line 257).

Line 215 - the authors observed that the presence of ssDNA reduced membrane clustering and conclude that "nucleic acid binding partially outcompetes membrane tethering activity". Two things: (1) although I agree is it likely that this effect is due to binding of DNA to 2C, binding has not been demonstrated experimentally so the authors should be more careful in how they describe their result; (2) there is no data presented to show that RNA binding reduces membrane tethering so at best I think the conclusion has to be that the data are consistent with the notion that DNA binding reduces membrane tethering. It would of course be interesting to see the effects of RNA and I'm curious to know why the assay was not performed.

Thanks for the comment. The honest answer is that previous publications (primarily Yeager et al, NAR 2022) convinced us that the outcome should be near-identical with DNA, so we chose DNA oligos because they are cheaper and easier to work with. But we agree with the reviewer that RNA is of course more relevant. We now present a comparison at 5 μM of ssDNA and ssRNA, which in fact shows a slightly stronger effect on membrane clustering by RNA (figure 5C). In the light of this additional experiment, we feel that some of the text changes suggested by the reviewer may no longer be necessary.

Line 237 - typo: by, not y

Thanks. In the light of the extensive changes to figure 6 this text was removed.

Line 284 - the authors claim that 2C may only bind RNA after the N-terminus is liberated from 2B in infected cells, since cleavage of the MBP tag from their construct was needed for 2C to bind RNA in their in vitro assay. However, this does not automatically follow given the large structural differences between MBP and 2B and the fact that the authors have not tested the RNA binding capacity of a 2BC fusion protein. Their claim here is too strong and should be re-written.

We agree, and have added a discussion along the lines suggested by the reviewer (line 330-332).

Line 293 - The authors speculate that RNA binding might cause a shift between the membrane clustering activities and the role of the protein in RNA replication. However, since they have not shown that RNA binding reduces membrane clustering, this is too speculative.

In our revised manuscript we have studied the effect of RNA on membrane binding, thus we feel that this text is relevant in the context of the extended experiments.

Line 299-317 - within this discussion is the assumption that in their assay system enterovirus 2C adopts the ring-like hexameric structure typical of AAA+ ATPases. While I agree this may well be the case, it has not been demonstrated in this study so the authors should make clear they are making this assumption. The same applies to the legend of Fig 8.

This part of the discussion was extensively rewritten after our changes to figure 6. We now only refer to "hexamer" once in the corresponding part of the discussion, where we talk about structural models of hexamers produced by other groups who have crystallised fragments of 2C. There we believe we should refer to hexamers to accurately cite their work.

We are not sure what the reviewer is referring to when it comes to the legend for figure 8: the original legend had no reference to the oligomeric state of 2C. We have substantially changed figure 8 and its legend and the new figure and legend make no references to hexamers/oligomers.

Line 302 - the authors claim to have shown that 2C is 'selective' for dsRNA. I think at best they have shown a preference for binding dsRNA over ssRNA.

We changed the wording (line 349). We have also changed the title of the paper where we removed "double-stranded".

Line 313 - The sentence starting "A recent study..." needs a reference.

The revised discussion no longer contains this sentence.

Line 332 - the full sequence of the synthetic gene used in this study should be made available (e.g. as supplementary information or a deposited sequence with an accession number). This is a critical point before the paper can be published.

We will of course submit the sequences as supplementary data. Thanks for the reminder.

Line 362 - the authors should describe the likely points of attachment of fluorophores and comment on how this labelling might affect 2C function.

Thanks for the comment. In response to this and a similar comment from another reviewer, we discuss the likely conjugation site of the fluorophore (lines 175-181), and also (due to the proximity to the Zn finger) provide a new measurement showing that equal amounts of Zn can be detected in the labelled and unlabelled protein (figure S7).

Line 372 - Is a single protein standard (BSA) sufficient to calibrate the SEC-MALS system?

Yes, it is the recommended procedure (note that SEC-MALS is only dependent on scattering, not elution volumes etc).

Reviewer #4 (Significance (Required)):

As stated above this is an interesting study that presents findings from a novel assay. It will be of interest to picornavirologists and the wider community interested in the mechanisms of AAA+ ATPases.

We thanks the reviewer for this positive appraisal of our work.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #4

Evidence, reproducibility and clarity

This is an interesting manuscript that reports the development of an in vitro membrane assay for probing the biochemical functions of the enterovirus 2C protein. The technique is interesting because it can be applied to 2C proteins from other members of the picornavirus family, an important group of mammalian pathogens. It has the capacity to probe different functions (e.g. membrane clustering, ATPase activity, RNA-binding and manipulation activities).

Overall, the manuscript is well written and gives a clear account of the work undertaken. It adds insight to previous studies of enteroviral (and picornaviral) 2C proteins, providing confirmation of some earlier work in a more physiological context and some new insights, particularly into the membrane and RNA binding aspects of 2C.

That said, there are a number of places where some amendment of the claims made is required to provide a more precise statement of the findings of this work. These are listed below.

Line 21 (Abstract) - The authors claim to have shown that a conserved glycine divides the N-terminal membrane-binding domain into 2 helices. I would suggest instead what they have produced are computational predictions that this is the case - some way short of an experimental demonstration. Sequence analysis predicts helical secondary structure in the N-terminus and indeed Alphafold2 also predicts a helical structure, but these predictions require experimental verification. The authors should therefore rewrite sections that claim to have shown the presence of 2 helices. In doing so, they should perhaps also comment on the fact that Alphafold2 does not predict 2 helices in this region for all enteroviruses (see Fig 1D). Moreover, the sequence analysis in Fig. S1 shows the presence of two Lys residues in the segment 17-38; it would be interesting for the reader to have these indicated in the figures showing the Alphafold2 prediction - do they in any way interrupt the hydrophobic face of the predicted helix?

Line 82 (Introduction) - The authors write that the membrane binding domain (MBD) of poliovirus has been shown to mediate hexamerisation, citing Adams et al (2009) - reference 43. However, that is not what this paper shows. Rather it provides evidence of aggregation of an MBP-2C fusion protein into forms that ranged from tetramer to octamer, but no evidence that these aggregates assume functional forms (e.g. the presumed hexameric ring structure characteristic of the AAA+ ATPase family to which 2C belongs). As far as I am aware the first demonstration of hexameric ring formation by a picornaviral 2C protein was for the 2C of foot-and-mouth disease virus (see Sweeney et al, JBC, 2010). Although this is not an enterovirus, this finding was later confirmed for Echovirus 30 (ref 51). I should declare an interest here: the Sweeney paper is from my lab. I will leave it to the editor and the authors to determine how to write a more precise account of the early observations of hexamerisation in picornaviral and enteroviral 2C proteins. Line 132 - the authors used mass photometry to investigate oligomeric forms of their MBP-2C constructs and state that for the full length 2C protein "the high-mass peak closely corresponds to a hexamer". While it is true that the peak shown in Fig 2C aligns with the expected MW for an MBP-2C hexamer, the peak is very broad, indicative of the presence of other oligomeric states with lower and higher numbers of monomers. This should be commented on. Indeed, the finding seems to echo the early findings of Adams et al (ref 43) with poliovirus MBP-2C.

Line 143 - for the reasons given above, this summary paragraph represents too strong a statement of what has been observed.

Line 197 - I note that the authors did not test the membrane clustering capabilities of the 2C(41-329) construct. Although the 2C(deltaAH1) construct had already shown a significant loss of activity, the shorter construct could still have been a useful control. I don't think it is necessary for this experiment to be done, but if the authors have a rationale for not performing the experiment, perhaps they could include it in a revised manuscript.

Line 223 - typo. I think you mean MBD.

Line 215 - the authors observed that the presence of ssDNA reduced membrane clustering and conclude that "nucleic acid binding partially outcompetes membrane tethering activity". Two things: (1) although I agree is it likely that this effect is due to binding of DNA to 2C, binding has not been demonstrated experimentally so the authors should be more careful in how they describe their result; (2) there is no data presented to show that RNA binding reduces membrane tethering so at best I think the conclusion has to be that the data are consistent with the notion that DNA binding reduces membrane tethering. It would of course be interesting to see the effects of RNA and I'm curious to know why the assay was not performed.

Line 237 - typo: by, not y

Line 284 - the authors claim that 2C may only bind RNA after the N-terminus is liberated from 2B in infected cells, since cleavage of the MBP tag from their construct was needed for 2C to bind RNA in their in vitro assay. However, this does not automatically follow given the large structural differences between MBP and 2B and the fact that the authors have not tested the RNA binding capacity of a 2BC fusion protein. Their claim here is too strong and should be re-written.

Line 293 - The authors speculate that RNA binding might cause a shift between the membrane clustering activities and the role of the protein in RNA replication. However, since they have not shown that RNA binding reduces membrane clustering, this is too speculative.

Line 299-317 - within this discussion is the assumption that in their assay system enterovirus 2C adopts the ring-like hexameric structure typical of AAA+ ATPases. While I agree this may well be the case, it has not been demonstrated in this study so the authors should make clear they are making this assumption. The same applies to the legend of Fig 8.

Line 302 - the authors claim to have shown that 2C is 'selective' for dsRNA. I think at best they have shown a preference for binding dsRNA over ssRNA.

Line 313 - The sentence starting "A recent study..." needs a reference.

Line 332 - the full sequence of the synthetic gene used in this study should be made available (e.g. as supplementary information or a deposited sequence with an accession number). This is a critical point before the paper can be published.

Line 362 - the authors should describe the likely points of attachment of fluorophores and comment on how this labelling might affect 2C function.

Line 372 - Is a single protein standard (BSA) sufficient to calibrate the SEC-MALS system?

Significance

As stated above this is an interesting study that presents findings from a novel assay. It will be of interest to picornavirologists and the wider community interested in the mechanisms of AAA+ ATPases.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

The authors present an alternative assay system to investigate picornavirus 2C, a protein that is tricky to analyze biochemically in its full length form because of an amphipathic helix at the N-terminus. Poliovirus 2C is expressed with an N-terminal MBP tag, a 50kD protein that helps with solubility as is commonly used for 2C investigations. A difference here is that liposomes are included to mimic membranes for 2C attachment. The key findings are that 2C induces clustering of of liposomes, that double stranded RNA binding by 2C impacts this clustering effect and that a free N-terminus (after cleavage of MBP by TEV protease) is needed for RNA binding and an ATP independent (ie non helicase) RNA duplex separation activity.

Major:

In the floatation assays in figure 3 the authors use a system where MBP-2C is fluorophore-labeled with ATTO488 on exposed cysteines. Poliovirus and other enterovirus 2C has a very well characterized zinc finger domain that has cysteines coordinating a zinc ion. Mutation experiments previously showed that these cysteines are necessary for viral replication and 2C stability. Have the authors controlled for disruption of the zinc finger domain by the labelling of cysteines with ATT0488 and checked if the protein remains folded?

In the analysis of the amphipathic helix, did the authors include membranes in their structural predictions o just the free helix? How does inclusion of membranes impact the predictions? In the predictions in Figure D, only 2 of 4 show a kink and there doesn't seem to be a correlation between those that predict a kink or not and whether the hydrophobic side is aligned in Figure S1.

Based on previous structures of 2C from different viruses the N-terminal amphipathic helix containing region is predicted to localize on one face of the predicted hexametric structure tethering 2C to the membrane. How does the authors hypothesized model explain 2C dependent clustering? is there evidence that 2C hexamers can oligomerize further into dodecamers for example, maintaining separate faces to enable N-terminal interaction with different membranes? What is the distance between the liposomes in figure 4 at the points of density attributed to 2C? How does this compare to the size of 2C determined in previous structural studies? Is it consistent with one hexamer/2 hexamers sitting on top of one another?

In the Discussion lines 278-285 the authors suggest that having MBP attached may reflect the polyprotein condition. Can they make a construct with MBP-2B2C to examine interaction with liposomes and assess 2C function?

Discussion lines 293-296, the possibility of two different populations of 2C, binding RNA or membranes cannot be excluded, there is much more 2C around late in infection that present in early infection- the model in figure 8 doesn't acknowledge/capture this.

Discussion lines 313-317, the authors don't reference a study where a mutant of foot-and-mouth disease virus 2C lacking the n-terminal amphipathic helix that could bind but not hydrolyze ATP, hexamerized in the presence of RNA that seems pertinent here (PMID: 20507978).

Some evidence of MBP-2C cleavage by TEV in the different assays used should be presented as this is a major focus of discussion and currently no gels show TEV cleavage is happening.

Significance

The work presents an additional methodology to investigate a a protein that has previously been difficult to study. The authors acknowledge that there is still a lot of 2C biology that remains to be discovered.

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Reply to the reviewers

1. General Statements [optional]

We thank all the reviewers for their constructive and critical comments. We provide a point-by-point response to the reviewers' comments, as detailed below. By responding to them, we believe that our revised manuscript will significantly improve so that it will be of interest for researchers in the field of cell biology, signaling pathways, physiology and nutrition.

Reply to the reviewers

Reviewer #1 (Evidence, reproducibility and clarity):

Summary: The manuscript by Yusuke Toyoda and co-workers describes that the phosphorylation of the a-arrestin Aly3 downstream of TORC2 and GAD8 (AKT) negatively regulates endocytosis of the hexose transporter Ght5 in S.pombe under glucose limiting growth conditions.

To arrive at these conclusions, the researchers define a set of redundant c-terminal phosphorylation sites in Aly3 that are downstream by GAD8. Phosphorylation of these sites reduces Ght5 ubiquitination and endocytosis. For ubiquitination, Aly3 interacts with the ubiquitin ligases Pub1/3.

We thank the reviewer for his/her time and reporting advantages and issues of this study.

Major points:

Figure 3B: it would be interesting to compare Aly3 migration pattern (and hence potential phosphorylation) under glucose replete or limiting growth conditions. Can the authors provide direct evidence that Aly3 phosphorylation changes in response to glucose availability? Also please explain the 'smear' in lanes aly3(4th Ala), aly3(4th Ala, A584S), aly3(4th Ala, A586T).

While it is an interesting possibility that the Aly3 migration pattern changes in response to glucose concentrations in medium, we think that this is unlikely and that examining this possibility is beyond the scope of this study. Because a phospho-proteomics study reported by Dr. Paul Nurse's lab showed Tor1-dependent phosphorylation of Aly3 at S584 under high glucose (2%) conditions (Mak et al, EMBO J, 2021), the Aly3 phosphorylation (migration) pattern is likely to be constant regardless of glucose conditions. Glucose conditions affect the mRNA and protein levels of Ght5, but supposedly not its endocytosis to vacuoles (Saitoh et al, Mol Biol Cell, 2015; Toyoda et al, J Cell Sci, 2021).

As for the smear in Aly3(4th A), Aly3(4th A;A584S), Aly3(4th A; A586T), we suspect that some posttranslational modification occurs on these mutant Aly3 proteins, but the identity of the modification is unclear. We did not mention the smear signals in the original manuscript, because the presence or absence of the smear did not necessarily correlate with cell proliferation in low glucose and thus vacuolar localization of Ght5, which is the main topic of this study. In the revised manuscript, we will mention this point more clearly.

Figure 4: Ght5 localization should be analyzed + / - thiamine and in media with different glucose levels. Also, a co-localization with a vacuolar marker (FM4-64) would be nice (but not necessary). Ideally, the authors should add WB analysis of Ght5 turnover to complement the imaging data. Also, would it be possible to measure directly the effects on glucose uptake (using eg: 2-NBDG).

In this revision, we plan to observe Ght5 localization under the conditions indicated by the reviewer (+/- thiamine and high/low glucose levels) to unambiguously show that the vacuolar localization of Ght5 occurs in a manner dependent solely on expression of the mutant Aly3 protein.

We thank the reviewer for the suggestion of co-staining with FM4-64. Indeed, because we previously reported that the cytoplasmic Ght5 signals were surrounded by FM4-64 signals in the TORC2-deficient tor1Δ mutant cells (Toyoda et al, J Cell Sci, 2021), the cytoplasmic Ght5-GFP signals in Figure 4 are very likely to co-localize with vacuoles. We will modify the text to clarify this point.

As suggested, we plan to add Western blot analysis of Ght5 turnover in Aly3-expressing cells, to complement the imaging data (Figure 4) in the revised manuscript. Persistent appearance of GFP in Western blot would be a good support for vacuolar transport of Ght5-GFP.

While regulation of glucose uptake is an important issue, measurement of Ght5-dependent glucose uptake using 2-NBDG was very difficult in our hands. Another reviewer (Reviewer #2) also mentioned the difficulty of this measurement in the Referees cross-commenting section.

Figure 5: Given the localization of Ght5 shown in Figure 4, I'm surprised that it is possible in to detect full length Ght5, and its ubiquitination in the phospho-mutants of Aly3. I expected that the majority of Ght5 would be constitutively degraded, and that one would need to prevent endocytosis and/or vacuolar degradation to detect full length Ght5 and ubiquitination. Please explain the discrepancy. Also it seems that the quantification in B was performed on a single experiment.

As the aim of Figure 5 is to compare the ubiquitinated species of Ght5 among the samples expressing different species of Aly3, the loading amount of each sample was adjusted so that the abundance of immunoprecipitated Ght5 is same across them. Therefore, as the reviewer points out, before the adjustment, abundance of the full-length Ght5 might be different in these samples. In the revised manuscript, we will add explanation on this point; why the anti-GFP blot of Figure 5A has the similar intensities in those samples.

In the revised manuscript, we will add two additional replicates of the same experiment as Figure 5 in Supplementary material to show reproducibility of the result.

Figure 6: Which PPxY motif of Aly3 is used for interaction with Pub1/3 and does their interaction depend on (de)phosphorylation?

In the revised manuscript, we will discuss that "both PY motifs of Aly3 might be required for full interaction with Pub1/3," by citing the following published knowledge:

(a) Mutation of both PPxY motif of budding yeast Rod1 and Rog3 (Aly3 homologs) diminished their interaction with the ubiquitin ligase Rsp5 (Andoh et al, FEBS Lett, 2002).

(b) Mutating either one of two PPxY motifs of budding yeast Cvs7/Art1 greatly decreased interaction with WW domain, and mutating both abolished the interaction (Lin et al, Cell, 2008).

Our preliminary results indicated that Pub3 interacted with Aly3, Aly3(4th A) and phospho-mimetic Aly3(4th D), and thus suggested that the Aly3-Pub1/3 interaction does not depend on the phosphorylation status of Aly3. Consistently, budding yeast Rod1 reportedly interacts with Rsp5 regardless of its phosphorylation status (e.g. Becuwe et al, J Cell Biol, 2012). While we have partially mentioned this point in the original manuscript (L499-503), we will discuss this point more clearly in the revised manuscript.

Reviewer #1 (Significance):

The results are well presented and clear cut (with few exceptions, please see major points). They provide further evidence that metabolic cues instruct the phosphorylation of a-arrestins. Phosphorylation then negatively regulates a-arrestin function in selective endocytosis and is essential to adjust nutrient uptake across the plasma membrane to the given biological context.

We thank the reviewer for finding significance of our study. We believe that adding new results of the requested experiments and responding to the raised comments will clarify the significance of our revised manuscript.

Reviewer #2 (Evidence, reproducibility and clarity):

**Summary / background. This paper focuses on the regulation of endocytosis of the hexose transporter, Ght5, in S. pombe by nutrient limitation through the arrestin-like protein Aly3. Ght5 is induced when glucose is limiting and is required for growth and proliferation in these conditions. ght5+ encodes the only high-affinity glc transporter from fission yeast. ght5+ is induced in low glucose conditions at the transcriptional level and is translocated to the plasma membrane to allow glc import. Ght5 is targeted to the vacuole in conditions of N limitation. Mutations in the TORC2 pathway lead to the same process, thus preventing growth on low glucose medium, as shown in the gad8ts mutant, mutated for the Gad8 kinase acting downstream of TORC2. Previously, the authors demonstrated that the vacuolar delivery of Ght5 in the gad8ts mutant is suppressed by mutation of the arrestin-like protein Aly3. Arrestin-like proteins are in charge of recognising and ubiquitinating plasma membrane proteins to direct their vacuolar targeting by the endocytosis pathway. This suggested that Aly3 is hyperactive in TORC2 mutants, and accordingly, Ght5 ubiquitination was increased in gad8ts.

**Overall statement This study aims at deepening our understanding of the regulation of endocytosis by signalling pathways through arrestin-like proteins. Ght5 is a nice model to study a physiological regulation, and the authors have a great set of tools at hand. However, I think the conclusions are not always rigorous and the conclusions are sometimes far-reaching. The main problem is that much of the conclusions concern a potential phosphorylation of Aly3 which is not experimentally addressed. An additional issue is the fact that they look at Ght5 ubiquitination by co-immunoprecipitation in native conditions (or at least, it seems to me) which cannot be conclusive. Overall, I think some experiments should be performed to address (at least) these 2 points before the manuscript can be published, see detailed comments below.

We thank the reviewer for pointing both advantages and issues of our manuscript.

We admit that phosphorylation of Aly3 was not experimentally shown in our manuscript, although its phosphorylation has already been shown in phospho-proteomic studies by other groups. For this issue, we plan to add an experiment and modify the text, as explained below.

The other major issue raised by this reviewer is that detection of Ght5 ubiquitination by immunoprecipitation in a native condition cannot be conclusive. Although we noticed that many studies perform affinity purification after denaturing and precipitating proteins with TCA or acetone to detect ubiquitination of the affinity-purified protein (e.g. Lin et al, Cell, 2008), we disagree with this opinion of the reviewer #2. In a review article describing methods to study ubiquitination by immunoblotting (Emmerich and Cohen, Biochem Biophys Res Comm, 2015), affinity purification of the protein of interest in a native condition is mentioned as one major choice. Moreover, a denaturing condition was not applicable to detect ubiquitinated Ght5 because the Ght5 protein that is once denatured and precipitated with TCA cannot be re-solubilized for immune-purification and -blotting. As the reviewer points out, a pitfall of detection of ubiquitinated Ght5 in a native condition is the presence of co-immunoprecipitated proteins. In our previous study (Toyoda et al, J Cell Sci, 2021), we purified GFP-tagged Ght5 and showed that a 110 kDa band detected in an anti-Ub immunoblot was also recognized by an anti-GFP antibody, confirming that the detected 110 kDa band corresponded to an ubiquitinated species of Ght5, but not a co-immunoprecipitated protein. Similarly, in the revised manuscript, we will add a panel of high-contrast (over-exposed) anti-GFP immunoblot, in which the indicated 110 kDa band was clearly detected by an anti-GFP antibody, in Figure 5A.

We appreciate these issues raised by the reviewer #2. By responding to them, we believe that conclusions of our study will be more rigorous and undoubtful in the revised manuscript.

**Major statements and criticism.

*Fig 1. Based on the hypothesis that TORC2-mediated phosphorylation regulate Ght5 endocytosis, the authors first considered a possible phosphorylation of Ght5. They mutagenised 11 **possible** phosphorylation sites on the Ct of Ght5, but none affected the growth on low glucose in the absence of thiamine, suggesting that they don't contribute to the observed TORC2-mediated regulation. However, I disagree with the statement that "phosphorylation of Ght5 is dispensable for cell proliferation in low glucose", given that the authors do not show 1- that Ght5 is phosphorylated and 2-that this is abolished by these mutations. They should either provide data on this or tone down and say that these residues are not involved in the regulation, without implying phosphorylation which is not proven.

Although we did not experimentally test whether these 11 residues of Ght5 was phosphorylated in our hand, these residues have been shown to be phosphorylated in phospho-proteomics studies by other groups (Kettenbach et al, Mol Cell Proteomics, 2015; Swaffer et al, Cell Rep, 2018; Tay et al, Cell Rep, 2019; Halova et al, Open Biol, 2021; Mak et al, EMBO J, 2021). In the revised manuscript, we plan to be more precise by replacing this conclusion with the following statement: "11 Ser/Thr residues of Ght5, which are reportedly phosphorylated, are not essential for cell proliferation in low glucose."

In the presence of Thiamine (Supp fig 1), it seems that the ST/A mutant grows better in low glucose, and this is not explained nor commented. Since the transporter is not expressed, could the authors provide an explanation to this? If the promoter is leaky and some ght5-ST/A is expressed, it may be more stable and allow better growth than the WT, which would tend to indicate that impairing phosphorylation prevents endocytosis (which is classical for many transporters, see the body of work on CK1-mediated phosphorylation of transporters). Have the authors tried to decrease glc concentration lower than 0.14% in the absence of thiamine to see if this also true when the transporters is strongly expressed? (OPTIONAL)

Improved growth of Ght5(ST11A)-expressing cells in the presence of thiamine was mentioned in the legend of Supplementary Figure 1A. In the revised manuscript, we will mention this observation also in the main text for better description of the results.

Adding thiamine to medium does not completely shut off transcription from the nmt1 promoter but allows some transcription, as previously reported (Maundrell, J Biol Chem, 1990; Forsburg, Nuc Acid Res, 1993). In the revised manuscript, we will mention this "leakiness" of the nmt1 promoter and, by citing the suggested studies, will discuss a possibility that the ST11A mutations might prevent endocytosis of Ght5 and consequently promote cell proliferation in low glucose conditions.

We found that, in the absence of thiamine, cells expressing ght5+ and ght5(ST11A) proliferated to the comparable extent on medium containing 0.08% glucose. This result will be added to the revised manuscript.

*Fig 2. The authors then follow the hypothesis that TORC2 exerts its Ght5-dependent regulation through the phosphorylation of Aly3. They mutagenised 18 **possible** phosphorylation sites on Aly3. This led to a strong defect in growth in low-glc medium. Mutation of the possible Gad8 site (S460) did not recapitulate this phenotype, suggesting that it is not sufficient, however, mutations of 4 ST residues in a CT cluster (582-586) mimicked the full 18ST/A mutation, suggesting these are the important residues for Ght5 endocytosis.

We thank the reviewer for appreciating the results in Fig. 2. As we explain below, we plan to perform an additional experiment to show that the Aly3 C-terminus is phosphorylated. With this result, our model will gain another experimental support.

*Fig 3A. Further dissection did not allow to pinpoint this regulation to a specific residue, beyond the dispensability of the T586 residue. Fig 3B. The authors look at the effects of mutation of Aly3 on these sites at the protein level. They had to develop an antibody because HA-epitope tagging did not lead to a functional protein (Supp fig 2). Whereas I agree that the mutations causing a phenotype lead to a change in the migration pattern, I disagree with the statement that "This observation indicated that slower migrating bands were phosphorylated species of Aly3" (p.9 l.271). First, lack of phosphorylation usually causes a slower mobility on gel, which is not clear to spot here. Second, a smear appears on top of the mutated proteins (eg. 4th Ala) which is possibly caused by another modification. There are many precedents in the literature about arrestins being ubiquitinated when they are not phosphorylated (see the work on Bul1, Rod1, Csr2 in baker's yeast from various labs). My gut feeling is that lack of phosphorylation unleashes Aly3 ubiquitination leading to change in pattern. All in all, it is impossible to state about the phosphorylation of a protein without addressing its phosphorylation properly by phosphatase treatment + change in migration, or MS/MS. Thus, whereas the data looks promising, this hypothesis that Aly3 is phosphorylated at the indicated sites is not properly demonstrated.

We disagree with the reviewer's opinion that a lack of phosphorylation usually causes slower mobility on gel. There are many examples in which phosphorylation causes slower mobility on gel, including budding yeast Rod1 (Alvaro et al, Genetics, 2016), and mammalian TXNIP (Wu et al, Mol Cell, 2013). In the revised manuscript, we will cite these reports to support our interpretation that the slower migrating bands are likely phosphorylated species of Aly3 (L270-271).

Smear-like signals in Aly3(4th Ala), Aly3(4th A;A584S) and Aly3(4th A;A586T) might result from some modification, but identity of the modification is unknown. As the reviewer #2 mentioned, phosphorylation on Aly3 might negatively regulate another modification. The precedent studies revealed that budding yeast Rod1 and Rog3 arrestins tend to be ubiquitinated in snf1/AMPK-deficient cells (Becuwe et al, J Cell Biol, 2012; O'Donnell et al, Mol Cell Biol, 2015), and that Bul1 arrestin is dephosphorylated and ubiquitinated in budding yeast cells deficient in Npr1 kinase (Merhi and Andre, Mol Cell Biol, 2012). Also, budding yeast Csr2 arrestin is deubiquitinated and phosphorylated upon glucose replenishment, while non-phosphorylated Csr2 is ubiquitinated and activated by Rsp5 (Hovsepian et al, J Cell Biol, 2012). While the smear-like signals are interesting, we noticed that the smear-like signals did not necessarily correlate with cell proliferation defects in low glucose. We therefore think that clarifying the identity of the smear-like signals is beyond the scope of this study. We will discuss the smear-like signals only briefly in the revised manuscript, and would address this issue in our future work, hopefully.

While the 4 S/T residues at the C-terminus of Aly3 as well as the other 14 S/T residues have been already shown to be phosphorylated in the precedent studies (Kettenbach et al, Mol Cell Proteomics, 2015; Tay et al, Cell Rep, 2019; Halova et al, Open Biol, 2021), we will confirm that the slower migrating Aly3 is indeed phosphorylated by phosphatase treatment in the revised manuscript. This planned experiment will further strengthen our study and support our conclusion and model.

*Fig 4. The authors now look at the functional consequences of these mutations on ALy3 on Ght5 localisation. The data clearly shows that mutation of the 4 identified S/T residues (Aly3-4th A) causes aberrant localisation of the transporter to the vacuole, likely to cause the observed growth defect on low glucose. There is a nice correlation between the vacuolar localisation and growth in low-glucose for the various aly3 mutants. (A final proof could be to express this in the context of an endocytic mutant, which should restore membrane localisation and suppress the aly3-4thA phenotype - OPTIONAL). However, I still disagree with the statement that "These results indicate that phosphorylation of Aly3 at the C-terminal 582nd, 584th, and/or 585th serine residues is required for cell-surface localization of Ght5." given that phosphorylation was not properly demonstrated.

While phosphorylation of the 582nd, 584th and/or 585th serine residues of Aly3 is not experimentally demonstrated in our hands, they have been shown to be phosphorylated in phospho-proteomics studies by other groups (Kettenbach et al, Mol Cell Proteomics, 2015; Tay et al, Cell Rep, 2019; Halova et al, Open Biol, 2021; Mak et al, EMBO J, 2021). Among them, the 584th serine residue (S584) was reported to be phosphorylated in a TORC2-dependent manner (Mak et al, EMBO J, 2021), consistent with our model. To explicitly demonstrate that S584 is phosphorylated, we plan to make a strain expressing a mutant Aly3 protein in which all the possible phosphorylation sites except S584 are replaced with alanine, namely Aly3(ST17A;S584). Hopefully, we can properly show the phosphorylation of S584 by measuring the mobility of the Aly3(ST17A;S584) on gel with/without phosphatase treatment or gad8 mutation.

We thank the reviewer for suggestion of the experiment using an endocytic mutant. Previously we reported that vacuolar localization of Ght5 in gad8 mutant cells was suppressed by mutations in not only aly3 but also genes encoding ESCRT complexes (Toyoda et al, J Cell Sci, 2021). We therefore think that in cells expressing Aly3(ST18A) or Aly3(4th Ala), Ght5 is subject to endocytosis and ensuing selective transport to vacuoles via endosome-localized ESCRT complexes. We will discuss this point in the revised manuscript.

*Fig 5. Here, the authors question the role of Aly3 mutations on Ght5 ubiquitination. They immunoprecipitate Ght5 and address its ubiquitination status in various Aly3 mutants. The data is encouraging for a role in Aly3 phosphorylation (?) in the negative control of Ght5 ubiquitination. My main problem with this experiment is that it seems that Ght5 immunoprecipitations were made in non-denaturing conditions, which leads to the question of what is the anti-ubiquitin revealing here (Ght5 or a co-immunoprecipitated protein, for example Aly3 itself, or the Pub ligases, or an unknown protein). It seems that this protocol was previously used in their previous paper, but I stand by my conclusion that ubiquitination of a given protein can only be looked in denaturing conditions. The experiments should be repeated in buffers classical for the study of protein ubiquitination to be able to conclude unambiguously that we are looking at Ght5 ubiquitination itself, especially in the absence of a non-ubiquitinable form of Ght5 as a negative control. Could the authors comment on the fact that S-A or S-D mutations display the same phenotype regarding the possible Ght5 ubiquitination?

As mentioned above, immunoprecipitation of Ght5 in denaturating conditions is not feasible. Ght5 can be affinity-purified only in a non-denaturing condition. In addition, affinity purification in a native condition is considered as a major choice to examine its ubiquitination according to a literature by Emmerich and Cohen (Emmerich and Cohen, Biochem Biophys Res Comm, 2015). A drawback of native condition is, as the reviewer points out, that the affinity-purified fraction might include non-bait (non-Ght5) proteins. The 110 kDa band indicated by an arrow in Fig. 5A was confirmed to be Ght5, not a non-bait protein, as a band at the identical position was detected in the immunoblot with anti-GFP antibody. Because this band in the anti-GFP immunoblot was too faint to be visible in Fig. 5A of the original manuscript, we will add an additional panel showing the contrast-enhanced anti-GFP immunoblot in which the 110 kDa band is clearly visible.

As for the result that "S-A or S-D mutations display the same phenotype regarding the possible Ght5 ubiquitination," we are afraid that the reviewer #2 misunderstood the labels of the samples. We apologize for confusing notational system of the sample name. Full description of samples is as follows; In Aly3(4th A), all of S582, S584, S585 and T586 are replaced with A; In Aly3(4th A;A584S), S582, S585 and T586 are replaced with A, whereas S584 remains intact; In Aly3(4th A;A584D), S582, S585 and T586 are replaced with A, and S584 is replaced with phospho-mimetic D. Because cells expressing Aly3(4th A;A584S) and Aly3(4th A;A584D) exhibited similarly low levels of Ght5 ubiquitination, we speculated that phosphorylation at S584 of Aly3 negatively regulates ubiquitination of Ght5.

In the revised manuscript, we plan to add a table showing amino acid sequence of each species of Aly3 (just like Figure 3A) to avoid confusion.

*Fig 6. The authors want to document the model whereby Aly3 may interact with some of the Nedd4 ligases (Pub1/2/3) to mediate its Ght5-ubiquitination function. They actually use the Aly3-4thA mutant, it should have been better with the WT protein. But the results indicate a clear interaction with at least Pub1 and Pub3. By the way, are the Pub1/2/3 fusions functional? Nedd4 proteins are notoriously affected in their function by C-terminal tagging and are usually tagged at their N-terminus (See Dunn et al. J Cell Biol 2004).

We plan to test whether Pub1-myc is functional by comparing proliferation of the Pub1-myc-expressing strain and pub1Δ strain, as pub1Δ cells reportedly show proliferation defects at a high temperature (Tamai and Shimoda, J Cell Sci, 2002). As deletion of pub2 or pub3 reportedly exhibited no obvious defects (Tamai and Shimoda, J Cell Sci, 2002; Hayles et al, Open Biol, 2013), it is not easy to assess functionality of the myc-tagged genes.

Please note that C-terminally tagged Pub1/2/3 proteins have been widely used in studies with fission yeast. Both Pub1-HA and non-tagged Pub1 were reported to be ubiquitinated (Nefsky and Beach, EMBO J, 1996; Strachan et al, J Cell Sci, 2023). Pub1-GFP, which complemented the high temperature sensitivity of pub1Δ, localized to cell surface and cytoplasmic bodies (Tamai and Shimoda, J Cell Sci, 2002). Pub2-GFP, overexpression of which arrested cell growth just like overexpression of non-tagged Pub2, localized to cell surface, and consistently Pub2-HA was detected in membrane-enriched pellet fractions after ultracentrifugation (Tamai and Shimoda, J Cell Sci, 2002). They also reported ubiquitin conjugation of the HECT domain of Pub2 fused with myc epitope at its C-terminus. Pub3-GFP localized to cell surface (Matsuyama et al, Nat Biotech, 2006).

Regardless of functionality of the myc-tagged Pub1/2/3, we believe that results of this experiment (Figure 6) support our model, because the aim of this experiment, which is to identify the HECT-type and WW-domain containing ubiquitin ligase(s) that interact with Aly3, is irrelevant to functionality of the myc-tagged Pub proteins.

*Fig 7. The authors want to provide genetic interaction between the Pub ligases and the growth defects in low glc due to alterations in Ght5 trafficking. It is unclear how the gad8ts pub1∆ mutant was generated since it doesn't seem to grow on regular glc concentration (Supp fig 5), could the authors provide some information about this? It is also not clear whether it can be stated thatches mutant is "more sensitive" to glc depletion because of the low level of growth to begin with (even at 3%). Altogether, the data show that deletion of pub3+ is able to suppress the growth defect of the gad8ts mutant on low glc medium, suggesting it is the relevant ligase for Ght5 endocytosis. This is confirmed by microscopy observations of Ght5 localisation. However, I would again tone down the main conclusion, which I feel is far-reaching: "Combined with physical interaction data, these results strongly suggest that Aly3 recruits Pub3, but not Pub2, for ubiquitination of Ght5." Work on Rsp5 in baker's yeast has shown that Rsp5 function goes beyond cargo ubiquitination, including ubiquitination of arrestins (which is often required for their function as mentioned in the introduction) or other endocytic proteins (epsins, amphyphysin etc). I agree that the data are compatible with this model but there are other possible explanations. Anything that would block endocytosis would supposedly suppress the gad8ts phenotype.

gad8ts pub1Δ was produced at 26 {degree sign}C, a permissive temperature of the gad8ts mutant. While this is described in the Methods section of the original manuscript, we will mention this more clearly in the Results section of the revised manuscript.

We did not conclude low glucose sensitivity of gad8ts pub1Δ cells in the indicated part (L376-377). Rather, we compared proliferation of gad8ts single mutant and pub1Δ single mutant cells in low glucose, and we found that the pub1Δ single mutant exhibited the higher sensitivity. In the revised manuscript we will correct the text to clarify that we compared proliferation of two single mutants (but not gad8ts pub1Δ mutant).

We agree with the opinion that the recruited Pub3 may ubiquitinate proteins other than Ght5. In the revised manuscript, we will correct our conclusion of the Figure 7 experiment (L388-390), not to limit the possible ubiquitination target(s) to Ght5.

In a genetic screen, we found that mutations in aly3+ and genes encoding ESCRT complexes suppressed low-glucose sensitivity and vacuolar transport of Ght5 of gad8ts mutant cells (Toyoda et al, J Cell Sci, 2021). This finding appears consistent with the reviewer's opinion that blocking endocytosis would supposedly suppress the gad8ts phenotype. We will mention this point in the revised manuscript.

*Discussion Some analogy with the regulation of the Bul arrestins by TORC1/Npr1 and PP2A/Sit4 could be mentioned (Mehri et al. 2012), at the discretion of the authors. The possibility that phosphorylation may neutralise a basic patch on Aly3 Ct, possibly involved in electrostatic interactions with Ght5 is very interesting. Regarding the effect of the mutations on Aly3 localisation (p.15 l.498), did the authors tag Aly3 with GFP? There are examples where proteins tagged with HA are not functional whereas tagging with GFP does not alter their function (eg. Rod1, Laussel et al. 2022) - and here Supp Fig 2 only relates to HA-tagging. Proof of a change in Aly3 localisation upon mutation would definitely be a plus (OPTIONAL).

We thank the reviewer for the suggestion of a reference. In the revised manuscript, we will cite the indicated report in the corresponding part for an additional support of TORC1-mediated control of Aly3 (de)phosphorylation.

While examining localization of Aly3 by GFP-tagging is interesting, we do not believe that it is necessary in this study. We would like to produce Aly3-GFP and to examine its functionality and localization in our future study. We thank the reviewer's insightful suggestion.

**Minor comments.

*Introduction: - I believe the text corresponding to the work on TXNIP is incorrect (p.5 l.127). TXNIP is degraded after its phosphorylation, not "rectracted" from the surface.

In the revised manuscript, we will correct the text accordingly.

• For the sake of completion, the authors could add other references concerning the regulation of Rod1 in budding yeast such as Becuwe et al. 2012 J Cell Biol and O'Donnell et al. 2015 Mol Cell Biol, in addition to Llopis-Torregrosa et al. 2016.

In the revised manuscript, we will add the suggested references and correct the text in the corresponding part of the Introduction (L123-138).

• Other examples of the requirement for arrestin ubiquitination beyond Art1 (p.5 l.136-137) are listed in the ref cited: Kahlhofer et al. 2021.

We will cite the indicated review to navigate readers for more examples of arrestin ubiquitination (and transporter ubiquitination).

*Figures: In general, I think it would be clearer if the authors showed on the figures that the background strain in which the XXX gene is added (or its mutant forms) is a xxx∆ strain.

We will modify the figures to clearly show the genetic background of the strains used.

**Referees cross-commenting**

Cross review of Reviewer 1 - *I don't believe that the authors "define a set of redundant c-terminal phosphorylation sites in Aly3", because phosphorylation is not proven. *I thinks the points raised for Fig 3B are valid but the authors should focus on making their story conclusive before expanding to other data (except for the explanation of the smear, see my review). Also, I don't think 2NBDG actually works to measure Glc uptake. * same for Fig 6 - not sure the interaction site mapping between Aly3 and Pubs would bring much value since there are more urgent things to do to make the story solid.

As mentioned above, we will experimentally show phosphorylation of the Aly3 C-terminus in the revised manuscript. Such experiments would make our story more solid and conclusive. We truly appreciate the comments and suggestions.

We agree with the comments on difficulty of measuring glucose uptake using 2-NBDG. In fact, we tried and failed measuring Ght5-mediated glucose uptake using 2-NBDG.

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Cros review of Reviewer 3 - we have many overlaps, so briefly : *I agree that the bibliography is incomplete (mentioned in my review) *I agree that there is no demonstration of the phospho-status of Aly3, and it is a problem *I agree that the results can be better quantified, esp. in the light of the points raised by this referee concerning the variability of expression of ST18A Other specific comments : *I agree that the statement that dephosphorylation activates alpha-arresting should be toned down - this was observed in several instances but there are examples of arrestin-mediated endocytosis which does not require their prior dephosphorylation. *I fully agree that efforts could be made regarding the classification/nomenclature of arrestins in S. pombe, this had escaped my attention

As detailed in the individual point raised by the reviewers, we will add the suggested references and accordingly correct the text in the revised manuscript.

In addition to experimentally showing Aly3 phosphorylation, we will quantify the immunoblot result.

Our statement that dephosphorylation activates alpha-arrestins might be too generalized. We will mention reports in which arrestin-mediated endocytosis does not require prior dephosphorylation (e.g. O'Donnell et al, Mol Biol Cell, 2010; Gournas et al, Mol Biol Cell, 2017; Savocco et al, PLoS Biol, 2019), and modify the text precisely.

Reviewer #2 (Significance):

*strengths and limitations This study aims at deepening our understanding of the regulation of endocytosis by signalling pathways through arrestin-like proteins in S. pombe. Ght5 is a nice model to study a physiological regulation, and the authors have a great set of tools at hand, including the discovery of Aly3 as the main arrestin for this regulation, and a signalling pathway (TORC2/Gad8) acting upstream. The main question is now to understand at the mechanistic level how TORC2 signaling impinges on the regulation of this arrestin.

Overall, the authors nicely demonstrate that C-terminal Ser/Thr residues are crucial for the function of Aly3 in Ght5 endocytosis. They propose a model whereby Aly3 phosphorylation by an unknownn kinase inhibits its function on Ght5 ubiquitination, which would favour its endocytosis. However, I think the conclusions are not always rigorous and the conclusions are sometimes far-reaching. The main problem is that much of the conclusions concern a potential phosphorylation of Aly3 which is not experimentally addressed. An additional issue is the fact that they look at Ght5 ubiquitination by co-immunoprecipitation in native conditions (or at least, it seems to me) which cannot be conclusive. Overall, I think some experiments should be performed to address (at least) these 2 points before the manuscript can be published, see detailed comments above.

*Advance

This study, if completed carefully, would provide among the first examples of mapping of phosphorylation sites on arrestins, which are usually phosphorylated at many sites and are thus difficult to study. Few studies went down to this level in this respect (see Ivshov et al. eLife 2020). There are no changes in paradigms or new conceptual insights, but this work is a nice example of the conservation of these regulatory mechanisms.

We appreciate that this study is highly evaluated by this reviewer. We understand the main problems raised by the reviewer, and as we detailed above, we plan to perform an experiment and make explanation to respond to the problems. With the raised issues answered, we believe that conclusions of the revised manuscript will be more rigorous.

Our study reveals mechanisms regulating vacuolar transport of the Ght5 hexose transporter via the TORC2 pathway in fission yeast. The serine residues at the Aly3 C-terminus (582nd, 584th and 585th serine residues), which are probably phosphorylated in a manner dependent on the TORC2 pathway, are required for sustained Ght5 localization to cell surface and cellular adaptation to low glucose. To our knowledge, there is no such study, and thus we think that this study is novel. By responding to the reviewers' comments and adding new data as explained above, the revised manuscript will be able to present novelty of our study more clearly. Comparison of our study in fission yeast to related studies in other model organisms may reveal the conservation and diversity of these regulatory mechanisms.

*Audience Should be of interest for people studying basic research in the field of cell biology, signalling pathways, transporter regulation by physiology. Reviewer background is on the regulation of transporter endocytosis by signalling pathways and arrestin-like proteins.

Reviewer #3 (Evidence, reproducibility and clarity): (Authors' response in blue)

In this manuscript, the authors work to address how phospho-regulation of a-arrestin Aly3 in S. pombe regulates the glucose transporter Ght5. The authors use a series of phospho-mutants in Aly3 and assess function of these mutants using growth assays and localization of Ght5. My main concerns with the manuscript are that 1) there is a lack of appreciation for the similar work that has been done in S. cerevisiae to define a-arrestin phospho-regulation, which is evidenced by the severe lack of referencing throughout the document, 2) the sites mutated on Aly3 are not demonstrated to change phospho-status of Aly3 and so all interpretations of these mutants need to be better contextualized and 3) almost none of the findings are quantified (imaging or immunoblots) making it difficult to assess the rigor of the outcomes. More detailed comments are provided below.

We thank the reviewer for thorough reading of the manuscript and the detailed comments. As explained below, we will respond to the points raised by the reviewer and accordingly modify the manuscript.

Minor Comments

Immunoblotting or immunostaining to define the levels and localization of phospho-mutants - In Figure 1, an immunoblot or immunostaining to define the abundance/localization of WT Ght5 vs its ST11A mutant would be appreciated. It is very difficult to know if ST11A is as functional as WT or not without an assessment of the levels and localization of the WT and mutant proteins to accompany the spot assays. Perhaps a version of Ght5 that is a phospho-mimetic would be more useful here as well since that version should not be dephosphorylated and then presumably would be internalized and not allow for growth on low glucose medium.

We plan to add fluorescence microscopy data of WT Ght5 and Ght5(ST11A) in the revised manuscript, to compare the localization and abundance of these two Ght5 species. In our preliminary observation, those of two Ght5 species seemed to be indistinguishable.

We'd like to emphasize that the primary aim of this study is to reveal mechanisms regulating Ght5 localization and consequently ensuring cell proliferation in low glucose. While analyzing a phospho-mimetic Ght5 mutant (e.g. Ght5(ST11D)) is interesting in terms of understanding of the nature of Ght5, we believe that such an analysis is out of the scope on this study. As Ght5(ST11A)-expressing cells proliferated comparably to Ght5(WT)-expressing cells and WT and ST11A Ght5 indistinguishably localize on the cell surface, phosphorylation of the ST residues of Ght5 is not likely to be the primary mechanism regulating Ght5 localization and function. We would like to assess a phospho-mimetic Ght5 mutant protein in our future studies.

For the Aly3 mutants where the abundance of Aly3 appears lower via immunoblotting (i.e., 4thA-A582S or S582A) how is the near perfect functional readout explained when the levels of the protein are much lower than WT? For the ST18A mutant, this is a particularly important point since the authors indicate on lines 194-197 that based on the functional data for ST18A, some of these ST residues are needed for phospho-regulation of Aly3. However, in Figure 3B the authors clearly show that there is very little ST18A protein in cells, and so these mutations have impacted Aly3 stability, which may or may not be linked to its phospho-status. The authors should be upfront about this finding on lines 194-197 and should not present this phospho-model as the only reason for why ST18A may not be functional. On lines 265-276 for the authors indicate that ST18A is expressed equivalently to WT Aly3, which is just not the case in Figure 3B. Perhaps quantification of replicate data would help clarify this issue. Further, if the authors wish to conclude that the upper MW bands in Figure 3B are due to phosphorylation, perhaps they should perform phosphatase treatments of their extracts to collapse these bands. However, most certainly the overall abundance of the single band for ST18A is reduced compared to the total bands of WT Aly3.

We disagree with the opinion that the levels of the mutant Aly3 are much lower than WT. For semi-quantitative measurement of the protein abundance, 2-fold dilution series of the WT Aly3 sample were loaded in the leftmost 3 lanes of Figure 3B. Although the levels of Aly3(4th A;A582S), Aly3(S582A) and Aly3(ST18A) were lower than that of WT Aly3, those are 50% or more of the WT, judging from the intensities of the serially-diluted WT samples. To clearly show that the expression of these Aly3 proteins is within comparable levels, we plan to add a column chart of the quantified expression levels and to mention abundances of the Aly3 proteins more quantitatively in the revised text. We do not think that replicate data (of Western blots as in Figure 3B) helps clarify this issue, because nmt1 promoter-driven gene transcription is induced with a small variation (Forsburg, Nuc Acid Res, 1993). We will cite this report and mention this point in the revised text.

We are afraid that this reviewer seems to consider that Aly3(ST18A) is not functional, but it is not a case and we do not intend to claim so. While deletion of aly3 did not interfere with cell proliferation in low glucose (see vector controls in Figures 2B, 2C and 3A, -Thiamine), expression of the ST18A mutant clearly hinders cell proliferation in low glucose, indicating that the ST18A performs dominant negative function to inhibit cell proliferation. That is, even though the expression level and/or stability of the ST18A is reduced, it is still sufficiently abundant to perform the dominant negative function. We propose the phospho-model not due to dysfunctionality of ST18A, but its dominant negative functionality. The 18 S/T residues of Aly3, which are shown to be phosphorylated in precedent phospho-proteomics studies, seem to be required to down-regulate Aly3's function to inhibit cell proliferation in low glucose. We apologize for this confusion, and we will modify the text and figures to clarify these points in the revised manuscripts.

To obtain an experimental support for our description that the slower migrating bands in Figure 3B are due to phosphorylation, we plan to perform a phosphatase treatment experiment as suggested.

Figure 2A - how do the phosphorylation sites identified in Aly3 compare to those identified in Rod1 from S. cerevisiae? See PMID 26920760 or SGD for more information. I am confused as to why the Aly3 protein has an arrowhead at the C-terminus. What does this denote?

We will mention reported phosphorylation sites of Aly3 and budding yeast Rod1/Art4 in the revised manuscript, by referring to the indicated report and database. It should be noted that similarity between amino acid sequences of Aly3 and S. cerevisiae Rod1 is not so high and limited in Arrestin-N and -C domains. The C-terminal half of Aly3, in which most of the potential phosphorylation sites are found, is not similar to Rod1. Thus, these sites are unlikely to be conserved between them.

An arrowhead indicates the direction of transcription (from N to C-terminus). We will describe it explicitly in the revised figure legend.

Figure 2 - The WT and Aly3-ST18A are expressed in S. pombe from a non-endogenous locus under the control of the Nmt1 promoter. However, are these mutants present in cells that contain WT copies of Aly3 at other genomic loci? If so, this would surely muddy the interpretations of these data as a- and b-arrestins are capable of multimerizing and the effect of multimerization on their activities can vary.

As mentioned in L188, an aly3 deletion mutant strain (aly3Δ) was used as a host, and thus all strains harboring an nmt1-driven aly3 gene lack the endogenous aly3 gene. We will add an illustration clearly showing that the host strain lacks the endogenous aly3+ gene and modify the legend of Figure 2.

Functional readouts for Aly3 using Ght5 localization - The reduced surface levels of Ght5 does correspond to the spot assay growth in low glucose for the various Aly3 mutants used. However, it would be useful if these assays incorporated an endocytosis inhibitor to help prevent the activities of these Aly3 plasmids to see if the transporter is retained at the PM. At the end of these mutational analyses, the authors conclude that phosphorylation of Aly3 at any of 3 sites is required for Ght5 trafficking to the vacuole in low glucose, however no experiment is done to demonstrate that these sites are phosphorylated residues. A phosphatase assay would be useful to help demonstrate that the modifications in 3B really are phosphorylation and a quantification of the phosphorylated bands in these WBs would also be useful to solidify the statement made on lines 306-309.

We thank the reviewer for suggestion of the experiment using an endocytosis inhibitor. Previously we reported that vacuolar localization of Ght5 in gad8ts mutant cells was suppressed by mutations in not only aly3 but also genes encoding ESCRT complexes (Toyoda et al, J Cell Sci, 2021). We therefore think that, in cells expressing Aly3(ST18A) or Aly3(4th Ala), Ght5 is subject to endocytosis and subsequent selective transport to vacuoles via ESCRT complexes. We will mention these previous findings in the revised manuscript.

As mentioned in responses to the comments above and other reviewer's, we will perform a phosphatase treatment experiment and its quantification in the revised manuscript. Here, we'd like to emphasize that these 3 sites have been shown to be phosphorylated in phospho-proteomic studies by other researchers (Kettenbach et al, Mol Cell Proteomics, 2015; Tay et al, Cell Rep, 2019; Halova et al, Open Biol, 2021; Mak et al, EMBO J, 2021), although we do not show it directly in this study.

Phosphorylation assessments - in general, it would be good to not only build the non-phosphorylatable versions of Aly3 but also the phospho-mimetic forms.

We produced a phospho-mimetic mutant Aly3 (i.e. Aly3(4th A;A584D)), and showed the result in Figure 5A; cells expressing Aly3(4th A;A584D) exhibited a low ubiquitination of Ght5, similarly to Aly3(WT)- and Aly3(4th A;A584S)-expressing cells. According to our experiences, replacing S/T with D/E does not necessarily mimic phosphorylation. Thus, we do not believe that systematic production of phospho-mimetic Aly3 mutants would help achieve the aim of this study.

Pub1, 2, and 3 - It would be helpful if the authors indicated what genes Pubs 1-3 correspond to in S. cerevisiae, where Rsp5 is the predominant Ub ligase interacting with a-arrestins. Is there no ortholog of Rsp5 in S. pombe?

Pub1, Pub2 and Pub3 are regarded as orthologs of budding yeast Rsp5, according to the fission yeast database PomBase. We will perform a homology search for these E3 proteins, and based on the result, we will add a description in the revised manuscript.

Pub-Aly3 interactions - could the authors please comment on the reason why so very little Aly3 is copurified with Pub1 or Pub2? Can any clear conclusion be drawn about pub2 given how very little Pub2 is present in the IPs? Based on my understanding of these data I do not think that this can be cleanly interpreted. What is is the identity of the ~50kDa MW band in Figure 6 in the upper MYC detection panel?

We do not have an accurate answer for the result that a small amount of Aly3 is copurified with Pub1 or Pub3. The Pub1/3-Aly3 interaction may be weak or transient. We will discuss this point in the revised manuscript.

Regarding whether Aly3 interacts with Pub2, we agree with the reviewer. As described in the Results (L360-362), we could not conclude anything about Aly3-Pub2 interaction by this immunoprecipitation experiment alone. On the other hand, the genetic interaction experiment (Figure 7A) suggests that pub2+ is not involved in defects caused by the gad8ts mutation (while pub3+ and aly3+ are). By this experiment, we think that Pub2 is not a partner of Aly3.

In the revised manuscript, we will describe that Pub2 is not a partner of Aly3 in a paragraph describing the Figure 7A experiment.

Because the 50 kDa band found in the IP fraction of all the samples appears even in "beads only" (Figure 6), those are supposedly derived from mouse IgG dissociated from the beads used for immunoprecipitation. We will mention this in the legend of Figure 6.

Phosphorylation and ubiquitination of a-arrestins - The paragraph from lines 123-138 is very superficial in addressing what is known about phosphorylation and ubiquitination of a-arrestins. The way this section is written, it feels misleading to the reader as it omits many of the details for regulation that would help place the current study in context. The discussion of Rod1 phosphorylation by AMPK for example, which is directly relevant to this study, is underdeveloped. I would recommend splitting this into two paragraphs and providing a more in depth, and accurate, view of the literature on this topic, with a focus on the regulation that is relevant for the ortholog of Aly3 in S. cerevisiae. For example, Rod1 phosphorylation by AMPK is greatly expanded upon in the following papers (PMID 22249293 and 25547292) and AMPK regulation of C-tail phosphorylation of a-arrestins is defined further in PMID 26920760. These references are each particularly important to compare with the current findings presented in this manuscript. Torc2 regulation ofa-arrestins is also reviewed in PMID 36149412 and references therein should be considered.

Because the primary aim of this study is to reveal mechanisms regulating Ght5 localization in fission yeast, but not to dissect modification and regulation of α-arrestins, we decided not to get into the details of phosphorylation and ubiquitination of α-arrestins. Furthermore, although budding yeast Rod1 and Rog3 are found to be downstream of the TORC2-Ypk1 signaling in the context of internalization of the Ste2 pheromone receptor, it is not clear whether TORC2-Ypk1 signaling also regulate α-arrestin-mediated internalization of hexose transporters in budding yeast. For these reasons, we focused on limited literatures essential for interpretation of the results and omitted many references describing the details of α-arrestin regulation. However, as this reviewer commented, we realize that our decision makes the discussion superficial and misleading to the reader. We sincerely apologize for this inconvenience.

In the revised manuscript, we will reorganize the paragraphs in the discussion and include the suggested references. Regarding budding yeast Rod1, we will cite the study reporting Ypk1-mediated phosphorylation on Rod1 in mating pheromone response via regulation of Ste2 endocytosis (Alvaro et al, Genetics, 2016). We will also mention other reports (Becuwe et al, J Cell Biol, 2012; O'Donnell et al, Mol Cell Biol, 2015) about AMPK-dependent phosphorylation of Rod1 in the corresponding part (e.g. L129-130). In addition, we will mention that Aly2, Rod1 and Rog3 α-arrestins were found downstream of the TORC2-Ypk1 signaling (Muir et al, eLife, 2014; Thorner, Biochem J, 2022).

As a further detailed example, there is far more work done on ubiquitination of a-arrestins in S. cerevisiae than the single citation provided by the authors on line 137. The way this section is written it feels misleading. Considerable effort has been spent on defining how mono- and poly-ubiquitination regulate a-arrestins and the authors should consider the data provided in the following citations and revise the two sentences they provide in this introduction to better reflect the breadth of our understanding rather than simply indicate that the 'mechanisms that regulate functions of a-arrestisn are not fully understood'. (PMIDs 23824189; 22249293; 17028178; 28298493)

Ubiquitination of α-arrestin itself is not the topic of this study, and physiological consequences of ubiquitination of Aly3 remain unknown. Because of these reasons, we did not describe the details of ubiquitination of α-arrestins in the original manuscript. However, we never intend to mislead the reader, and thus to avoid it, we will revise the indicated sentences and cite the suggested literatures (O'Donnell et al, J Biol Chem, 2013; Becuwe et al, J Cell Biol, 2012; Kee et al, J Biol Chem, 2006; Ho et al, Mol Biol Cell, 2017) in the revised manuscript.

Context of the findings and lack of citations - The referencing in this manuscript is very poor as many of the key papers that report analogous findings in the budding yeast Saccharomyces cerevisiae are not cited. This oversight in citing the appropriate literature must be remedied before this manuscript can be considered further for publication. Examples of these omissions occur at the following places:

We will modify the text and carefully cite more literatures describing analogous finding in budding yeast and other organisms in the revised manuscript. We appreciate the insightful suggestions by this reviewer. It should be noted, however, that it is not evident whether budding yeast Rod1 and Rog3 are orthologous to fission yeast Aly3. Although Rod1 and Aly3 share overlapping roles, amino acid sequence similarity of them is not high and limited only in domains which are generally conserved among α-arrestin-family proteins.

Line 90 - The Puca and Brou citations is one example of this but the first examples come from Daniela Rotin's work looking at Rsp5 interactions in budding yeast, which is where the association between HECT-domain Ub ligases and a-arrestins is also documented by Scott Emr and Hugh Pelham's labs. Here are some PMID numbers to improve the citations of this section (PMID 17551511; 18976803; 19912579) and each of these references long predates the Puca and Brou publication.

In the revised manuscript, we will improve the citations by including the suggested studies (Gupta et al, Mol Syst Biol, 2007; Lin et al, Cell, 2008; Nikko and Pelham, Traffic, 2009).

Lines 123-126 - Phosphorylation can also increase vacuole-dependent degradation of alpha-arrestins as demonstrated in PMID 35454122. The interaction with 14-3-3 proteins that is driven by phosphorylation of a-arrestins was first demonstrated by the Leon group in PMID 22249293). Lines 129-132 - Here again the Leon reference that helps demonstrate the 14-3-3 inhibition of Rod1 is lacking (PMID 22249293).

We will cite the suggested studies in description of these topics (Bowman et al, Biomolecules, 2022; Becuwe et al, J Cell Biol, 2012).

Lines 130-132 - Please include references for the statement that dephosphorylation activates a-arrestin activity. There are no citations on this statement and there are many to choose from and I would urge the authors to cite the primary literature on these points.

We will cite studies for the statement "Conversely, dephosphorylation is thought to activate α-arrestins and to promote selective endocytosis of transporter proteins" (L130-132).

These are just a few examples from the Introduction, but the Discussion is similarly wrought with issues in referencing and framing the experimental results within the context of the larger field, including what is known about Rod1/Rog3 regulation in S. cerevisiae. For example, the Llopis-Torregrosa et al reference and statement on lines 508-510 is incorrect. There are other phosphorylation sites defined in the C-terminus of Rod1, as described in Alvaro et al. PMID: 26920760.

We will carefully correct Discussion by citing the suggested references (e.g. Alvaro et al, Genetics, 2016) and framing the obtained results within the context of the larger field.

Of note, a combination of α-arrestin, upstream kinase(s) and distinct phosphorylation sites appears to determine the target transporter (Kahlhofer et al, Biol Cell, 2021; Thorner, Biochem J, 2022), and it has not been explicitly proved that TORC2-Ypk1 signaling also regulate α-arrestin-mediated internalization of hexose transporters in budding yeast. For these reasons, we stated "S. cerevisiae Rod1 and Rog3 are phosphorylated solely by Snf1p/AMPK" in the context of internalization of hexose transporters. We will also discuss this point in the revised manuscript.

Minor Comments Clarification needed - Lines 107-121 - The relationship between the S. pombe arrestins and those in other organisms is somewhat unclear. Frist, all the arrestins in humans and S. cerevisiae can be sorted into the alpha, beta and Vps26 classes. However, the authors indicate that the S. pombe genome has 11 arrestin-like proteins but only 4 of these are a-arrestins. What classes do the other 7 arrestins belong to? It would be appreciated if this point was clarified.

To our knowledge, fission yeast arrestins are not well classified yet. We will perform a phylogenetic tree analysis to classify them, and modify the description of the indicated part accordingly. We will also cite our previous report (Toyoda et al, J Cell Sci, 2021), in which the overall protein structure and domains of 11 fission yeast arrestin-like proteins were reported.

Next, for the 4 a-arrestins identified in S. pombe the authors indicate that Aly3 is the homolog of Rod1/Art4 and Rog3/Art7 from S. cerevisiae. What is the relationship of Rod1 in S. pombe to Rod1 in S. cerevisiae? Are these also homologs? You can see how the nomenclature is confusing and, given the functional overlap of S. cerevisiae Rod1/Rog3 proteins it is important to know if Aly3 is the only version of these a-arrestins or if there is an additional counterpart in S. pombe. This point becomes somewhat more confusing when on lines 134-136 the authors talk about Arn1/Any1 as an arrestin related protein in S. pombe yet this protein was not included on the list of a-arrestins in the preceding section. What class of arrestin is this protein?

According to PomBase, both Aly3 and Rod1 are assigned as the orthologue of budding yeast Rod1 and Rog3. However, as mentioned in responses above, it is unclear whether Aly3 is really orthologous to budding yeast Rod1/Rod3. In the revised manuscript, we will perform a homology search for these 4 proteins, and add information on how much these arrestins share homology.

Arn1/Any1 is regarded as a β-arrestin (Nakase et al, J Cell Sci, 2013). We will also mention this in the revised manuscript.

Alpha-arrestin homology - On lines 127-129 the authors indicate that TXNIP is the mammalian homolog of Aly3. To my knowledge, there are no evolutionary analyses that can draw these lines of homology between the a-arrestins in humans and those in yeasts. It would be appreciated if the authors could cite the work that leads to this conclusion or revise the sentence to more accurately reflect what is known on this topic. It certainly appears that, given their functional overlap in regulating glucose transporters, Txnip and Rod1/Rog3 in humans and S. cerevisiae are functionally connected. I urge the authors to use more caution when describing this protein family.

Among human α-arrestins, ARRDC2 (22%) but not TXNIP (20%) has the highest amino acid identity to Aly3 (Toyoda et al, J Cell Sci, 2021). However, as TXNIP has been reported to regulate endocytosis of hexose transporters, GLUT1 and 4 (Wu et al, Mol Cell, 2013; Waldhart et al, Cell Rep, 2017), we think that TXNIP and Aly3 share physiological roles. We will revise the sentence (L127-129) more accurately.

Text editing - The text could use editing as there are awkward and grammatically incorrect sentences in several places. Here are a few examples to help the authors:

Please note that the original manuscript is edited by a professional editor, who is a native English (American) speaker and has edited thousands of research papers, before initial submission. We will ask an editor to check the revised draft again before submission.

Lines 57-60 - the protein is not expressed over the entire cell surface, but is localized to the entire cell surface.

We will correct this wording.

Lines 80-83 - this sentence is very confusing

We will correct this part by changing the phrase "Unlike TORC1," into a clause.

Line 86 - Is there more than one gene encoding Aly3 in S. pombe?

No, there is only one gene encoding Aly3. We will correct this part so as to avoid being misunderstood.

Line 88, 109, - these sentences need to start with a capitol so either capitalize the A in arrestin or write out Alpha with a capitol A.

We will correct the sentence as suggested.

Lines 145-148 - unclear as written

We will clarify the meaning of the sentence by changing the voice.

Line 224 - why are these amino acids being referred to as hydroxylated? Perhaps hydroxyl-containing amino acids or 18 amino acids with hydroxyl side chains would be better choices?

We will correct the word as suggested.

Line 300 - very confusing sentence structure

We will correct this part by simplifying the structure of the sentence.

And elsewhere....

We will carefully check the revised text before submission.

Reviewer #3 (Significance):

The authors provide some information as to the residues needed in the Aly3 C-tail for Ght5 trafficking in S. Pombe. These results are not places in the context of similar phosphor-regulatory work done for a-arrestins in S. cerevisiae, and this is needed for appreciation of the significance of the study.

Overall, it appears that the model put forth is very similar to the one already proposed in S. cerevisiae where phosphorylation impedes a-arrestin-mediated trafficking of glucose transporters. It is interesting to see this similarity hold in S. Pombe, but it does not dramatically alter our appreciation of a-arrestin biology.

The significance of the findings are somewhat underscored by the fact that very little quantification of data are presented, making the rigor of the work difficult to assess.

We thank the reviewer for careful reading and evaluation of our study. As the reviewer states, the results are not placed in the context of similar phospho-regulatory works done for α-arrestins in S. cerevisiae. This may partly come from the fact that it remains unclear whether internalization of hexose transporters is regulated by TORC2-dependent phosphorylation in S. cerevisiae. We believe that our study is novel and significant for this reason. By performing the additional experiments/quantification and revising the text as suggested by the reviewers, the manuscript will be further strengthened, and we will be able to clearly conclude that TORC2-dependent phosphorylation of Aly3 regulates localization of the Ght5 hexose transporter and cellular responses to glucose shortage stress.

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Referee #2

Evidence, reproducibility and clarity

Summary/background.

This paper focuses on the regulation of endocytosis of the hexose transporter, Ght5, in S. pombe by nutrient limitation through the arrestin-like protein Aly3. Ght5 is induced when glucose is limiting and is required for growth and proliferation in these conditions. ght5+ encodes the only high-affinity glc transporter from fission yeast. ght5+ is induced in low glucose conditions at the transcriptional level and is translocated to the plasma membrane to allow glc import. Ght5 is targeted to the vacuole in conditions of N limitation. Mutations in the TORC2 pathway lead to the same process, thus preventing growth on low glucose medium, as shown in the gad8ts mutant, mutated for the Gad8 kinase acting downstream of TORC2. Previously, the authors demonstrated that the vacuolar delivery of Ght5 in the gad8ts mutant is suppressed by mutation of the arrestin-like protein Aly3. Arrestin-like proteins are in charge of recognising and ubiquitinating plasma membrane proteins to direct their vacuolar targeting by the endocytosis pathway. This suggested that Aly3 is hyperactive in TORC2 mutants, and accordingly, Ght5 ubiquitination was increased in gad8ts.

Overall statement

This study aims at deepening our understanding of the regulation of endocytosis by signalling pathways through arrestin-like proteins. Ght5 is a nice model to study a physiological regulation, and the authors have a great set of tools at hand. However, I think the conclusions are not always rigorous and the conclusions are sometimes far-reaching. The main problem is that much of the conclusions concern a potential phosphorylation of Aly3 which is not experimentally addressed. An additional issue is the fact that they look at Ght5 ubiquitination by co-immunoprecipitation in native conditions (or at least, it seems to me) which cannot be conclusive. Overall, I think some experiments should be performed to address (at least) these 2 points before the manuscript can be published, see detailed comments below.

Major statements and criticism.

• Fig 1. Based on the hypothesis that TORC2-mediated phosphorylation regulate Ght5 endocytosis, the authors first considered a possible phosphorylation of Ght5. They mutagenised 11 possible phosphorylation sites on the Ct of Ght5, but none affected the growth on low glucose in the absence of thiamine, suggesting that they don't contribute to the observed TORC2-mediated regulation. However, I disagree with the statement that "phosphorylation of Ght5 is dispensable for cell proliferation in low glucose", given that the authors do not show 1- that Ght5 is phosphorylated and 2-that this is abolished by these mutations. They should either provide data on this or tone down and say that these residues are not involved in the regulation, without implying phosphorylation which is not proven. In the presence of Thiamine (Supp fig 1), it seems that the ST/A mutant grows better in low glucose, and this is not explained nor commented. Since the transporter is not expressed, could the authors provide an explanation to this? If the promoter is leaky and some ght5-ST/A is expressed, it may be more stable and allow better growth than the WT, which would tend to indicate that impairing phosphorylation prevents endocytosis (which is classical for many transporters, see the body of work on CK1-mediated phosphorylation of transporters). Have the authors tried to decrease glc concentration lower than 0.14% in the absence of thiamine to see if this also true when the transporters is strongly expressed? (OPTIONAL)
• Fig 2. The authors then follow the hypothesis that TORC2 exerts its Ght5-dependent regulation through the phosphorylation of Aly3. They mutagenised 18 possible phosphorylation sites on Aly3. This led to a strong defect in growth in low-glc medium. Mutation of the possible Gad8 site (S460) did not recapitulate this phenotype, suggesting that it is not sufficient, however, mutations of 4 ST residues in a CT cluster (582-586) mimicked the full 18ST/A mutation, suggesting these are the important residues for Ght5 endocytosis.
• Fig 3A. Further dissection did not allow to pinpoint this regulation to a specific residue, beyond the dispensability of the T586 residue. Fig 3B. The authors look at the effects of mutation of Aly3 on these sites at the protein level. They had to develop an antibody because HA-epitope tagging did not lead to a functional protein (Supp fig 2). Whereas I agree that the mutations causing a phenotype lead to a change in the migration pattern, I disagree with the statement that "This observation indicated that slower migrating bands were phosphorylated species of Aly3" (p.9 l.271). First, lack of phosphorylation usually causes a slower mobility on gel, which is not clear to spot here. Second, a smear appears on top of the mutated proteins (eg. 4th Ala) which is possibly caused by another modification. There are many precedents in the literature about arrestins being ubiquitinated when they are not phosphorylated (see the work on Bul1, Rod1, Csr2 in baker's yeast from various labs). My gut feeling is that lack of phosphorylation unleashes Aly3 ubiquitination leading to change in pattern. All in all, it is impossible to state about the phosphorylation of a protein without addressing its phosphorylation properly by phosphatase treatment + change in migration, or MS/MS. Thus, whereas the data looks promising, this hypothesis that Aly3 is phosphorylated at the indicated sites is not properly demonstrated.
• Fig 4. The authors now look at the functional consequences of these mutations on ALy3 on Ght5 localisation. The data clearly shows that mutation of the 4 identified S/T residues (Aly3-4th A) causes aberrant localisation of the transporter to the vacuole, likely to cause the observed growth defect on low glucose. There is a nice correlation between the vacuolar localisation and growth in low-glucose for the various aly3 mutants. (A final proof could be to express this in the context of an endocytic mutant, which should restore membrane localisation and suppress the aly3-4thA phenotype - OPTIONAL). However, I still disagree with the statement that "These results indicate that phosphorylation of Aly3 at the C-terminal 582nd, 584th, and/or 585th serine residues is required for cell-surface localization of Ght5." given that phosphorylation was not properly demonstrated.
• Fig 5. Here, the authors question the role of Aly3 mutations on Ght5 ubiquitination. They immunoprecipitate Ght5 and address its ubiquitination status in various Aly3 mutants. The data is encouraging for a role in Aly3 phosphorylation (?) in the negative control of Ght5 ubiquitination. My main problem with this experiment is that it seems that Ght5 immunoprecipitations were made in non-denaturing conditions, which leads to the question of what is the anti-ubiquitin revealing here (Ght5 or a co-immunoprecipitated protein, for example Aly3 itself, or the Pub ligases, or an unknown protein). It seems that this protocol was previously used in their previous paper, but I stand by my conclusion that ubiquitination of a given protein can only be looked in denaturing conditions. The experiments should be repeated in buffers classical for the study of protein ubiquitination to be able to conclude unambiguously that we are looking at Ght5 ubiquitination itself, especially in the absence of a non-ubiquitinable form of Ght5 as a negative control. Could the authors comment on the fact that S-A or S-D mutations display the same phenotype regarding the possible Ght5 ubiquitination?
• Fig 6. The authors want to document the model whereby Aly3 may interact with some of the Nedd4 ligases (Pub1/2/3) to mediate its Ght5-ubiquitination function. They actually use the Aly3-4thA mutant, it should have been better with the WT protein. But the results indicate a clear interaction with at least Pub1 and Pub3. By the way, are the Pub1/2/3 fusions functional? Nedd4 proteins are notoriously affected in their function by C-terminal tagging and are usually tagged at their N-terminus (See Dunn et al. J Cell Biol 2004).
• Fig 7. The authors want to provide genetic interaction between the Pub ligases and the growth defects in low glc due to alterations in Ght5 trafficking. It is unclear how the gad8ts pub1∆ mutant was generated since it doesn't seem to grow on regular glc concentration (Supp fig 5), could the authors provide some information about this? It is also not clear whether it can be stated thatches mutant is "more sensitive" to glc depletion because of the low level of growth to begin with (even at 3%). Altogether, the data show that deletion of pub3+ is able to suppress the growth defect of the gad8ts mutant on low glc medium, suggesting it is the relevant ligase for Ght5 endocytosis. This is confirmed by microscopy observations of Ght5 localisation. However, I would again tone down the main conclusion, which I feel is far-reaching: "Combined with physical interaction data, these results strongly suggest that Aly3 recruits Pub3, but not Pub2, for ubiquitination of Ght5." Work on Rsp5 in baker's yeast has shown that Rsp5 function goes beyond cargo ubiquitination, including ubiquitination of arrestins (which is often required for their function as mentioned in the introduction) or other endocytic proteins (epsins, amphyphysin etc). I agree that the data are compatible with this model but there are other possible explanations. Anything that would block endocytosis would supposedly suppress the gad8ts phenotype.

Discussion

Some analogy with the regulation of the Bul arrestins by TORC1/Npr1 and PP2A/Sit4 could be mentioned (Mehri et al. 2012), at the discretion of the authors. The possibility that phosphorylation may neutralise a basic patch on Aly3 Ct, possibly involved in electrostatic interactions with Ght5 is very interesting. Regarding the effect of the mutations on Aly3 localisation (p.15 l.498), did the authors tag Aly3 with GFP? There are examples where proteins tagged with HA are not functional whereas tagging with GFP does not alter their function (eg. Rod1, Laussel et al. 2022) - and here Supp Fig 2 only relates to HA-tagging. Proof of a change in Aly3 localisation upon mutation would definitely be a plus (OPTIONAL).

Minor comments.

Introduction:

• I believe the text corresponding to the work on TXNIP is incorrect (p.5 l.127). TXNIP is degraded after its phosphorylation, not "rectracted" from the surface.
• For the sake of completion, the authors could add other references concerning the regulation of Rod1 in budding yeast such as Becuwe et al. 2012 J Cell Biol and O'Donnell et al. 2015 Mol Cell Biol, in addition to Llopis-Torregrosa et al. 2016.
• Other examples of the requirement for arrestin ubiquitination beyond Art1 (p.5 l.136-137) are listed in the ref cited: Kahlhofer et al. 2021.

Figures: In general, I think it would be clearer if the authors showed on the figures that the background strain in which the XXX gene is added (or its mutant forms) is a xxx∆ strain.

Referees cross-commenting

Cross review of Reviewer 1

• I don't believe that the authors "define a set of redundant c-terminal phosphorylation sites in Aly3", because phosphorylation is not proven.
• I thinks the points raised for Fig 3B are valid but the authors should focus on making their story conclusive before expanding to other data (except for the explanation of the smear, see my review). Also, I don't think 2NBDG actually works to measure Glc uptake.
• same for Fig 6 - not sure the interaction site mapping between Aly3 and Pubs would bring much value since there are more urgent things to do to make the story solid.

Cros review of Reviewer 3 - we have many overlaps, so briefly :

• I agree that the bibliography is incomplete (mentioned in my review)
• I agree that there is no demonstration of the phospho-status of Aly3, and it is a problem
• I agree that the results can be better quantified, esp. in the light of the points raised by this referee concerning the variability of expression of ST18A

Other specific comments :

• I agree that the statement that dephosphorylation activates alpha-arresting should be toned down - this was observed in several instances but there are examples of arrestin-mediated endocytosis which does not require their prior dephosphorylation.
• I fully agree that efforts could be made regarding the classification/nomenclature of arrestins in S. pombe, this had escaped my attention

Significance

strengths and limitations

This study aims at deepening our understanding of the regulation of endocytosis by signalling pathways through arrestin-like proteins in S. pombe. Ght5 is a nice model to study a physiological regulation, and the authors have a great set of tools at hand, including the discovery of Aly3 as the main arrestin for this regulation, and a signalling pathway (TORC2/Gad8) acting upstream. The main question is now to understand at the mechanistic level how TORC2 signaling impinges on the regulation of this arrestin.

Overall, the authors nicely demonstrate that C-terminal Ser/Thr residues are crucial for the function of Aly3 in Ght5 endocytosis. They propose a model whereby Aly3 phosphorylation by an unknownn kinase inhibits its function on Ght5 ubiquitination, which would favour its endocytosis. However, I think the conclusions are not always rigorous and the conclusions are sometimes far-reaching. The main problem is that much of the conclusions concern a potential phosphorylation of Aly3 which is not experimentally addressed. An additional issue is the fact that they look at Ght5 ubiquitination by co-immunoprecipitation in native conditions (or at least, it seems to me) which cannot be conclusive. Overall, I think some experiments should be performed to address (at least) these 2 points before the manuscript can be published, see detailed comments above.

Advance

This study, if completed carefully, would provide among the first examples of mapping of phosphorylation sites on arrestins, which are usually phosphorylated at many sites and are thus difficult to study. Few studies went down to this level in this respect (see Ivshov et al. eLife 2020). There are no changes in paradigms or new conceptual insights, but this work is a nice example of the conservation of these regulatory mechanisms.

Audience

Should be of interest for people studying basic research in the field of cell biology, signalling pathways, transporter regulation by physiology. Reviewer background is on the regulation of transporter endocytosis by signalling pathways and arrestin-like proteins.

Author response:

The following is the authors’ response to the previous reviews.

Public Reviews:

Reviewer #2 (Public Review):

Although the study by Xiaolin Yu et al is largely limited to in vitro data, the results of this study convincingly improve our current understanding of leukocyte migration.

(1) The conclusions of the paper are mostly supported by the data and in the revised manuscript clarification is provided concerning the exact CCL5 forms (without or with a fluorescent label or His-tag) and amounts/concentrations that were used in the individual experiments. This is important since it is known that modification of CCL5 at the N-terminus affects the interactions of CCL5 with the GPCRs CCR1, CCR3 and CCR5 and random labeling using monosuccinimidyl esters (as done by the authors with Cy-3) is targeting lysines. The revised manuscript more clearly indicates for each individual experiment which form is used. However, a discussion on the potential effects of the modifications on CCL5 in the results and discussion sections is still missing.

Many thanks for the reviewer's suggestion. We fully agree it is important to clarify the potential issue of Cy3 labeling, and believe it is more suitable in the Materials and Methods section (line 312-314).

(2) In general, authors used high concentrations of CCL5 in their experiments. In their reply to the comments they indicate that at lower CCL5 concentrations no LLPS is detected. This is important information since it may indicate the need for chemokine oligomerization for LLPS. This info should be added to the manuscript and comparison with for instance the obligate monomer CCL7 and another chemokine such as CXCL4 that easily forms oligomers may clarify whether LLPS is controlled by oligomerization.

We are pleased by the help of the reviewers and accordingly inserted a brief discussion as suggested (line 240-246).

(3) Statistical analyses have been improved in the revised manuscript.

Thanks to the reviewer for his/her comment.

Worth considering Brooks's distinction of essential versus incidental complexity. It's especially worth considering the instances where the "incidental" part comes from being incidental to the fact that if it were easier, there it would make a lot of people unhappy for reasons that I call "the consultant effect".

Reviewer #1 (Public Review):

The study identifies the epigenetic reader SntB as a crucial transcriptional regulator of growth, development, and secondary metabolite synthesis in Aspergillus flavus, although the precise molecular mechanisms remain elusive. Using homologous recombination, researchers constructed sntB gene deletion (ΔsntB), complementary (Com-sntB), and HA tag-fused sntB (sntB-HA) strains. Results indicated that deletion of the sntB gene impaired mycelial growth, conidial production, sclerotia formation, aflatoxin synthesis, and host colonization compared to the wild type (WT). The defects in the ΔsntB strain were reversible in the Com-sntB strain.

Further experiments involving ChIP-seq and RNA-seq analyses of sntB-HA and WT, as well as ΔsntB and WT strains, highlighted SntB's significant role in the oxidative stress response. Analysis of the catalase-encoding catC gene, which was upregulated in the ΔsntB strain, and a secretory lipase gene, which was downregulated, underpinned the functional disruptions observed. Under oxidative stress induced by menadione sodium bisulfite (MSB), the deletion of sntB reduced catC expression significantly. Additionally, deleting the catC gene curtailed mycelial growth, conidial production, and sclerotia formation, but elevated reactive oxygen species (ROS) levels and aflatoxin production. The ΔcatC strain also showed reduced susceptibility to MSB and decreased aflatoxin production compared to the WT.

This study outlines a pathway by which SntB regulates fungal morphogenesis, mycotoxin synthesis, and virulence through a sequence of H3K36me3 modification to peroxisomes and lipid hydrolysis, impacting fungal virulence and mycotoxin biosynthesis.

The authors have achieved majority of their aims at the beginning of the study, finding target genes, which led to catC mediated regulation of development, growth and aflatoxin metabolism. Overall most parts of the study is solid and clear.

Reviewer #1 (Public Review):

Summary:

In this manuscript by Beardslee and Schmitz, the authors undertook a screen for potential degrons - short peptide sequences at the C-terminus that would target the toxin VapC for degradation. The authors randomly mutagenized 5 amino acids appended to the C-terminus of VapC and transformed this library into E. coli to look for surviving cells when the VapC gene was expressed. The authors found an enrichment for tags ending Ala-Ala, and found that this enrichment was dependent on the presence of the ClpXP protease, since this sequence was not similarly enriched in a mutant lacking this protease. Moreover, the authors identify the sequence FKLVA as the tag with the highest fold enrichment in the screen and confirm that GFP tagged with this sequence is degraded by ClpXP with similar kinetics to GFP tagged with the ssrA-derived tag.

Strengths:

This study has two major implications for understanding the nature of degrons in E. coli. First, peptides ending Ala-Ala, and especially degrons resembling the ssrA degron are likely the most degradation-promoting sequences in E. coli. Second, these findings suggest that ClpXP is the most central protease, at least for this particular protein with a randomized C-terminus under the particular conditions of this screen. It is also notable that the ribosome quality control protein RqcH tags truncated proteins with an alanine tag in a template-free manner when the large ribosomal subunit is obstructed. Although E. coli doesn't encode RqcH, the utility of alanine-tagging for protein degradation likely extends to other organisms.

Weaknesses:

The authors remark and show that mutations that inactivate the VapC protein are enriched potentially more than the proteolysis tags. This is a limitation of the study and the authors have done well to describe this as it will inform future screens. Perhaps using a protein with more intermediate toxicity in future screens would help to prioritize C-terminal mutations instead of toxin-inactivating mutations.

For clarity, the authors should explain why the NNK structure of the random codons was used. Why is it important that the codon end with a G or T?

Authors state on page 7 that by determining enrichment of individual tags they can rank the relative Km for proteolysis of the individual tags. This statement is not accurate since the tag could variously impact its association with any of the proteases in the cell. Since Km is specific to each particular protease, these can't be ranked in vivo when all proteases are present.

Reviewer #2 (Public Review):

Summary:

The authors studied the sequence determinants of C-terminal tags that govern protein degradation in bacteria. They introduce a new strategy to determine degron sequences: Detox (Degron Enrichment by toxin). This unbiased approach links degron efficiency to cell growth as degrons are C-terminally fused to the toxin VapC, which inhibits protein translation. Selecting for bacterial growth and thus toxin degradation enabled the identification of potent degron derived from a randomized library of pentapeptides. Remarkably, most degrons show sequence similarity to the SsrA-tag, which is fused to incomplete polypeptides at stalled ribosomes by the tmRNA-tagging system. These findings underline the extraordinary efficiency of the SsrA-tag and the ClpXP protease in removing incomplete polypeptides and demonstrate that most proteins are spared from degradation by harboring different C-termini. The introduced method will be highly useful to determine degron sequences in other positions and other bacterial species.

Strengths:

The work introduces an innovative and powerful strategy to identify degron sequences in bacteria. The study is well-controlled and results have been thoroughly analyzed. It will now become important to broaden the technology, making it also accessible for more complex degrons.

Weaknesses:

The approach is efficient in identifying strong degron sequences that are predominantly recognized by the ClpXP protease. The sequence specificity of other proteolytic systems, however, is not efficiently addressed, pointing to a potential limitation of this technology. The GS-rich linker sequence connecting the degron and the toxin might also impact proteolysis and thus outcome.

Author response:

The following is the authors’ response to the original reviews.

Key Considerations:

There seem to be two inconsistencies related to some results depicted in Figures 1, 2, 3 and 5.

Firstly, Figure 1 shows the effect on C_Las infection (_C_Las+) compared to the control (_C_Las-), where results show an increase of TAG, Glycogen, lipid droplet size, oviposition period, and fecundity. In Figures 2, 3, and 5, the authors establish the involvement of the genes _DcAKH, DcAKHR, and miR34 in this process, by showing that by preventing the function of these three factors the effects of _C_Las+ are lost. However, while Figure 1 shows the increase of TAG and lipid droplet size in _C_Las+, Figures 2, 3, and 5 do not show a significant elevation in TAG when comparing _C_Las- and _C_Las+.

Secondly, in addition to the absence of statistical difference in TAG and lipid droplet size observed in Figure 1, Figures 2, 3, and 5 show an increase in TAG and lipid droplet size after ds_DcAKH_ (Figure 2), ds_DcAKHR_ (Figure 3) and agomiR34 (Figure 5) treatments. Considering that AKH, AKHR, and miR34 are important factors to _C_Las-induce increase in TAG and lipid droplet size, one might expect a reduction in TAG and lipid droplet size when _C_Las+ insects are silenced for these factors, contrary to the observed results.

Thanks for your excellent suggestion. Lipid droplets are cellular organelles responsible for storing lipids within cells, playing a crucial role in fat metabolism and energy homeostasis. The formation and breakdown of lipid droplets involve a complex interplay of genes and enzymes, including DGAT (for synthesis), ATGL and HSL (for breakdown). In C_Las-negative _D. citri, there is a delicate balance between creasing and breaking down of lipid droplets. The enlargement of lipid droplet size following C_Las infection may result from a significantly higher synthesis rate compared to breakdown, as more energy is required during early ovarian development. The hormone AKH, a key player in fat metabolism, primarily stimulates fat breakdown. Therefore, when _DcAKH and DcAKHR are silenced without affecting fat synthesis, there is no enhancement of fat breakdown; instead, there is an accumulation of lipid droplets, resulting in their enlargement. This suggests that _C_Las infection affects both the breakdown and synthesis of lipid droplets, while AKH and AKHR primarily impact the breakdown, leading to similar outcomes. However, the underlying physiological mechanisms warrant further in-depth exploration.

Reviewer #1 (Recommendations For The Authors):

(1) Line 25: change "In addition" to "Additionally".

Thanks for your wonderful suggestion. We have changed “In addition” to “Additionally” in our revised manuscript (Line 26).

(2) Lines 60-72: Have there been any previous reports on the interaction between host AKH hormones and microorganisms in insects or animals? If yes, please add more background.

Thanks for your wonderful suggestion. We have added the interactions between host AKH hormones and microorganisms in insects (Line 74-81).

(3) Lines 82-95: add the following reference about the miR-275 of Diaphorina citri in the background. Nian, X., Luo, Y., He, X., Wu, S., Li, J., Wang, D., Holford, P., Beattie, G. A. C., Cen, Y., Zhang, S., & He, Y. (2024). Infection with 'Candidatus Liberibacter asiaticus' improves the fecundity of Diaphorina citri aiding its proliferation: A win-win strategy. Molecular Ecology, 33, e17214.

Thanks for your wonderful suggestion. We have added the sentence “in D. citri-C_Las interaction, _C_Las hijacks the JH signaling pathway and host miR-275 that targets the _vitellogenin receptor (DcVgR) to improve D. citri fecundity, while simultaneously increasing the replication of C_Las itself, suggesting a mutualistic interaction in _D. citri ovaries with _C_Las” in our revised manuscript (Line 97-100).

(4) In the figures of Nile red staining, the digit of the scale bar should be added.

Thanks for your wonderful suggestion. We have added the digit of the scale bar for Nile red staining in the Figure 1C, 2E, 3E, 5C.

(5) In Figures 2G-H, 3G-H, 5E-F, the presentation of data should be consistent with Figure 1D-E.

Thanks for your wonderful suggestion. We have changed figure 1D-E in our revised manuscript.

(6) In the discussion part, more information should be added about miR-275 and DcVgR from the above reference.

Thanks for your wonderful suggestion. We have added the information “In D. citri-C_Las interaction, _C_Las operates host hormone signaling and miRNA to mediate the mutualistic interaction between _D. citri fecundity and its replication” in Line 350-353.

(7) For the primer specific, please add the melting curves for qPCR primers of DcAKH, DcAKHR, Dcβ-ACT, U6, and miR-34 in the supplementary material.

Thanks for your wonderful suggestion. We have added the melting curves for qPCR primers of DcAKH, DcAKHR, Dcβ-ACT, U6 and miR-34 in the supplementary material of Figure S6.

(8) Line 476: Dcβ-ACT was indicated as a gene and should be Italic.

Thanks for your wonderful suggestion. We have changed “DcβACT” to “Dcβ-ACT” in our revised manuscript (Line 491).

(9) Reference style should be consistent and correct. Like [5], [10], [37], [47].

Thanks for your wonderful suggestion. We have revised them in our revised manuscript.

Reviewer #2 (Recommendations For The Authors):

(1) In order to better engage readers, I suggest emphasizing the "enhanced fecundity" in the title. A suggestion for the revised title is: Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'.

Thanks for your wonderful suggestion. We have changed the title to “Adipokinetic hormone signaling mediates the enhanced fecundity of Diaphorina citri infected by 'Candidatus Liberibacter asiaticus'” in our revised manuscript.

(2) For the abstract, in lines 14-15, please change the first sentence to "Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus' (C_Las), the bacterium associated with the severe Asian form of huanglongbing." In line 18, delete "present". In line 19, change "increased" to "increasing". In line 21, change "triacylglycerol accumulation" to "the accumulation of triacylglycerol". In line 33, change "in _D. citri ovaries with C_Las" to "between _C_Las and _D. citri ovaries".

Thanks for your wonderful suggestion. We have revised them following your suggestion in our revised manuscript, including changed “Diaphorina citri is the primary vector of the bacterium, ‘Candidatus Liberibacter asiaticus’ (C_Las) associated with the severe Asian form of huanglongbing” to “_Diaphorina citri serves as the primary vector for 'Candidatus Liberibacter asiaticus' (C_Las), the bacterium associated with the severe Asian form of huanglongbing” in Line 15-16; deleted "present" in Line 19; changed "increased" to "increasing" in Line 20; changed "triacylglycerol accumulation" to "the accumulation of triacylglycerol" in Line 22; changed "in _D. citri ovaries with C_Las" to "between _C_Las and _D. citri ovaries" in Line 34.

(3) In lines 57-59, change "How D. citri maintains a balance between lipid metabolism and increased fecundity after infection with C_Las is not known." to "However, the mechanism of how _D. citri maintains a balance between lipid metabolism and increased fecundity after infection with _C_Las remains unknown.".

Thanks for your wonderful suggestion. We have changed " How D. citri maintains a balance between lipid metabolism and increased fecundity after infection with C_Las is not known" to "However, the mechanism of how _D. citri maintains a balance between lipid metabolism and increased fecundity after infection with _C_Las remains unknown" in our revised manuscript (Line 58-60).

(4) In Figure 1, "n.s" should be changed to "n.s.", "n.s." should be added in 13 DAE of Figure 1A, and the specific numerical value of the scale bar should be indicated on Figures 1C, 2E, 3E, and 5C.

Thanks for your wonderful suggestion. We have revised them in our revised manuscript.

(5) In all the figure legends, the "**P < 0.01,***P < 0.001" should be changed to "**p < 0.01,***p < 0.001".

Thanks for your wonderful suggestion. We have revised them in our revised manuscript.

(6) In Figures 1D-E, the preoviposition period and oviposition period were presented using a box diagram, but in other figures (including Figure 2G-H, Figure 3G-H, Figure 5E-F) these were shown using a column chart. Please keep the method of presentation consistent.

Thanks for your wonderful suggestion. We have revised the figure 1D-E in our revised manuscript.

(7) For discussion, in line 333, change "Increasing numbers" to "An increasing number". In line 334, change "vertically transmitted" to "transmitted vertically".

Thanks for  your wonderful suggestion. We have changed "Increasing numbers" to "An increasing number" in Line 345; changed "vertically transmitted" to "transmitted vertically" in Line 346 in our revised manuscript.

(8) In lines 338-342, change "There are few studies on the mechanisms underlying vector-bacteria interactions. However, Singh and Linksvayer (2020) [38] found that Wolbachia-infected colonies of Monomorium pharaonis had increased colony-level growth, accelerated colony reproduction, and shortened colony life cycles compared to those that were uninfected." to "Although there is limited research on the mechanisms underlying vectorbacteria interactions, Singh and Linksvayer (2020) [38] found that Wolbachia_infected colonies of _Monomorium pharaonis exhibited increased colony-level growth, accelerated colony reproduction, and shortened colony life cycles compared to uninfected colonies.".

Thanks for your wonderful suggestion. We have revised it in our revised manuscript (Line 350-355) .

(9) In line 370, delete "present". In lines 386-387, change "More and more miRNAs have been reported to be involved in the metabolic processes of insects including reproduction." to "There is increasing evidence implicating miRNAs in the metabolic processes of insects, particularly in relation to reproduction.".

Thanks for your wonderful suggestion. We have revised them in our revised manuscript, including deleted "present" in Line 383 and changed "More and more miRNAs have been reported to be involved in the metabolic processes of insects including reproduction" to "There is increasing evidence implicating miRNAs in the metabolic processes of insects, particularly in relation to reproduction" in Line 399-400.

(10) In line 423, change "After infection with C_Las, _D. citri are more fecund than their uninfected counterparts." to "Upon infection with C_Las, _D. citri exhibits enhanced fecundity compared to uninfected individuals.". In lines 424-425 and 439-440, change "the more offspring of D. citri, the more C_Las in the field" to "the increased offspring of _D. citri contributes to a higher presence of _C_Las in the field.". In Line 429, change " information" to "insights".

Thanks for your wonderful suggestion. We have revised them in our revised manuscript, including changed "After infection with C_Las, _D. citri are more fecund than their uninfected counterparts" to "Upon infection with C_Las, _D. citri exhibits enhanced fecundity compared to uninfected individuals" in Line 436-437; changed "the more offspring of D. citri, the more C_Las in the field" to "the increased offspring of _D. citri contributes to a higher presence of _C_Las in the field" in Line 438-439; changed "information" to "insights" in Line 443.

(11) In lines 446-447, change "The _C_Las-infected lemon plants and psyllids were monitored to detect _C_Las infection monthly using the quantitative polymerase chain reaction (qPCR)" to "Monthly monitoring of the _C_Las infection in both the lemon plants and psyllids was conducted using quantitative polymerase chain reaction (qPCR)".

Thanks for your wonderful suggestion. We have revised it in our revised manuscript (Line 460-461).

(12) In lines 452-458, how did the authors identify homologous sequences of AKH and AKHR for phylogenetic tree analysis and alignment of the amino acid sequences? From NCBI or other databases? The methodological details should be added.

Thanks for your wonderful suggestion. We have added the methodological details in our revised manuscript (Line 469-470).

(13) In line 476, Dcβ-ACT should be italic.

Thanks for your wonderful suggestion. We have changed “DcβACT” to italic in our revised manuscript (Line 491).

(14) In line 538, the manufacturer should be provided for Nile Red.

Thanks for your wonderful suggestion. We have provided the manufacturer of Nile Red in our revised manuscript (Line 553).

(15) Does miR-34 have any other target genes? If yes, whether they have any function in the fecundity improvement of D. citri after infected by CLas.

Thanks for your insightful suggestion. In addition to DcAKHR, we predicted three other genes have binding sites in 3’UTR with miR-34, including Innexin, T-box transcription factor TBX1, and fatty acid synthase. Despite this, the mRNA expression levels of all three genes remained unchanged between _C_Las-negative and _C_Las-postive females. Therefore, we believe that these genes are not implicated in the fecundity improvement.

(16) The reference format should be unified. Please revise references 10, 28, 43, 47, and 53.

Thanks for your wonderful suggestion. We have revised them in the revised manuscript.

Reviewer #1 (Public Review):

Summary:

This manuscript by Shea and Villeda furnishes the field with a valuable scRNAseq data set detailing microglial aging in the mouse hippocampus. They provide clear evidence that changes in microglial attributes begin in mid-life, well before time points when mice are traditionally considered to be "aging." It also adds to a growing body of data in the field demonstrating that there is substantial heterogeneity in microglial responses to aging. Using in vitro experiments and transgenic manipulations in mice, the authors show that transforming growth factor beta (TGFb1)-based signaling can potently impact microglial state, consistent with previous findings in the field. They also demonstrate that manipulation of microglial TGFb1-based signaling can impact hippocampus-dependent behaviors.

Limitations of the study lie primarily in reaching too far with interpretations of the data. The authors argue that changes in microglial transcriptome during midlife represent a type of "checkpoint," after which microglial aging can progress along distinct trajectories depending on the status of TGFb1 signaling. They also posit that a specific intermediate "stress response" state in midlife is mechanistically linked to a translational burst that drives the subsequent progression of microglia to an "inflammatory state." Unequivocal data to support these causal links is lacking, however. similarly, key additional experiments would be needed to demonstrate that TGFb1 signaling and microglial progression through these identified intermediate states are causally linked to cognitive decline.

Guidance for readers along with study strengths and caveats:

The present manuscript provides valuable strengthening and expansion to a growing body of data showing prominent changes in the microglial state during aging. Microarray(1), bulkRNAseq(2-5), scRNAseq(6,7), snRNAseq(8,9), and spatial transcriptomic(10) approaches have been leveraged to map changes in microglial transcriptome during aging in rodents, non-human primates, and humans. A number of these studies include the hippocampus (1,8,9,11) and have highlighted variation across brain regions in microglial transcriptomic changes during aging (1,11). They have also revealed differences across sex (7) as well as increased cell-to-cell heterogeneity (6-10), consistent with the idea that individual microglia can follow distinct aging trajectories. Several of these studies revealed that changes in microglial attributes begin in middle age (1,7,11), supporting similar observations from studies that did not use omics (12-14). The present manuscript utilizes scRNAseq of hippocampal microglia at adulthood (6mo), middle age (12mo), late middle age (18mo) and aging (24mo) to show that aging-induced changes in microglia begin in middle age and that microglia exhibit ample phenotypic heterogeneity during the progression of aging.

To gain further insight into the dynamics of microglial aging in the hippocampus, the authors used a bioinformatics method known as "pseudotime" or "trajectory inference" to understand how cells may progress through different functional states, as defined by cellular transcriptome (15,16). These bioinformatics approaches can reveal key patterns in scRNAseq / snRNAseq datasets and, in the present study, the authors conclude that a "stress response" module characterized by expression of TGFb1 represents a key "checkpoint" in microglial aging in midlife, after which the cells can move along distinct transcriptional trajectories as aging progresses. This is an intriguing possibility. However, pseudotime analyses need to be validated via additional bioinformatics as well as follow-up experiments. Indeed, Heumos et al, in their Nature Genetics "Expert Guidelines" Review, emphasize that "inferred trajectories might not necessarily have biological meaning." They recommend that "when the expected topology is unknown, trajectories and downstream hypotheses should be confirmed by multiple trajectory inference methods using different underlying assumptions."(15) Numerous algorithms are available for trajectory inference (e.g. Monocle, PAGA, Sligshot, RaceID/StemID, among many others) and their performance and suitability depends on the individual dataset and nature of the trajectories that are to be inferred. It is recommended to use dynGuidelines(16) for the selection of optimal pseudotime analysis methods. In the present manuscript, the authors do not provide any justification for their use of Monocle 3 over other trajectory inference approaches, nor do they employ a secondary trajectory inference method to confirm observations made with Monocle 3. Finally, follow-up validation experiments that the authors carry out have their own limitations and caveats (see below). Hence, while the microglial aging trajectories identified by this study are intriguing, they remain hypothetical trajectories that need to be proven with additional follow-up experiments.

To follow up on the idea that TGFb1 signaling in microglia plays a key role in determining microglial aging trajectories, the authors use RNAscope to show that TGFb1 levels in microglia peak in middle age. They also treat primary LPS-activated microglia with TGFb1 and show that this restores expression of microglial homeostatic gene expression and dampens expression of stress response and, potentially, inflammatory genes. Finally, they utilize transgenic approaches to delete TGFb1 from microglia around 8-10mo of age and scRNAseq to show that homeostatic signatures are lost and inflammatory signatures are gained. Hence, findings in this study support the idea that TGFb1 can strongly regulate microglial phenotype. Loss of TGFb1 signaling to microglia in adulthood has already been shown to cause decreased microglial morphological complexity and upregulation of genes typically associated with microglial responses to CNS insults(17-19). TGFb1 signaling to microglia has also been implicated in microglial responses to disease and manipulations to increase this signaling can improve disease progression in some cases(19). In this light, the findings in the present study are largely confirmatory of previous findings in the literature. They also fall short of unequivocally demonstrating that TGFb1 signaling acts as a "checkpoint" for determining subsequent microglial aging trajectory. To show this clearly, one would need to perturb TGFb1 signaling around 12mo of age and carry out sequencing (bulkRNAseq or scRNAseq) of microglia at 18mo and 24mo. Such experiments could directly demonstrate whether the whole microglial population has been diverted to the TGFb1-low aging trajectory (that progresses through a translational burst state to an inflammation state as proposed). Future development of tools to tag TGFb1 high or low microglia could also enable fate tracing type experiments to directly show whether the TGFb1 state in middle age predicts cell state at later phases of aging.

The present study would also like to draw links between features of microglial aging in the hippocampus and a decline in hippocampal-dependent cognition during aging. To this end, they carry out behavioral testing in 8-10mo old mice that have undergone microglial-specific TGFb1 deletion and find deficits in novel object recognition and contextual fear conditioning. While this provides compelling evidence that TGFb1 signaling in microglia can impact hippocampus-dependent cognition in midlife, it does not demonstrate that this signaling accelerates or modulates cognitive decline (see below). Age-associated cognitive decline refers to cognitive deficits that emerge as a result of the normative brain aging process(20-21). For a cognitive deficit to be considered age-associated cognitive decline, it must be shown that the cognitive operation under study was intact at some point earlier in the adult lifespan. This requires longitudinal study designs that determine whether a manipulation impacts the relationship between brain status and cognition as animals age (22-24). Alternatively, cross-sectional studies with adequate sample sizes can be used to sample the variability in cognitive outcomes at different points of the adult lifespan(22-24) and show that this is altered by a particular manipulation. For this specific study, one would ideally demonstrate that hippocampal-based learning/memory was intact at some point in the lifespan of mice with microglial TGFb1 KO but that this manipulation accelerated or exacerbated the emergence of deficits in hippocampal-dependent learning/memory during aging. In the absence of these types of data, the authors should tone down their claims that they have identified a cellular and molecular mechanism that contributes to cognitive decline.

A final point of clarification for the reader pertains to the mining of previously generated data sets within this study. The language in the results section, methods, and figure legends causes confusion about which experiments were actually carried out in this study versus previous studies. Some of the language makes it sound as though parabiosis experiments and experiments using mouse models of Alzheimer's Disease were carried out in this study. However, parabiosis and AD mouse model experiments were executed in previous studies (25,26), and in the present study, RNAseq datasets were accessed for targeted data mining. It is fantastic to see further mining of datasets that already exist in the field. However, descriptions in the results and methods sections need to make it crystal clear that this is what was done.

References:

(1) Grabert, K. et al. Microglial brain region-dependent diversity and selective regional sensitivities to aging. Nat. Neurosci. (2016). doi:10.1038/nn.4222<br /> (2) Hickman, S. E. et al. The microglial sensome revealed by direct RNA sequencing. Nat. Neurosci. (2013). doi:10.1038/nn.3554<br /> (3) Deczkowska, A. et al. Mef2C restrains microglial inflammatory response and is lost in brain ageing in an IFN-I-dependent manner. Nat. Commun. (2017). doi:10.1038/s41467-017-00769-0<br /> (4) O'Neil, S. M., Witcher, K. G., McKim, D. B. & Godbout, J. P. Forced turnover of aged microglia induces an intermediate phenotype but does not rebalance CNS environmental cues driving priming to immune challenge. Acta Neuropathol. Commun. (2018). doi:10.1186/s40478-018-0636-8<br /> (5) Olah, M. et al. A transcriptomic atlas of aged human microglia. Nat. Commun. (2018). doi:10.1038/s41467-018-02926-5<br /> (6) Hammond, T. R. et al. Single-Cell RNA Sequencing of Microglia throughout the Mouse Lifespan and in the Injured Brain Reveals Complex Cell-State Changes. Immunity 50, 253-271 (2019).<br /> (7) Li, X. et al. Transcriptional and epigenetic decoding of the microglial aging process. Nat. aging 3, 1288-1311 (2023).<br /> (8) Zhang, H. et al. Single-nucleus transcriptomic landscape of primate hippocampal aging. Protein Cell 12, 695-716 (2021).<br /> (9) Su, Y. et al. A single-cell transcriptome atlas of glial diversity in the human hippocampus across the postnatal lifespan. Cell Stem Cell 29, 1594-1610.e8 (2022).<br /> (10) Allen, W. E., Blosser, T. R., Sullivan, Z. A., Dulac, C. & Zhuang, X. Molecular and spatial signatures of mouse brain aging at single-cell resolution. Cell 186, 194-208.e18 (2023).<br /> (11) Soreq, L. et al. Major Shifts in Glial Regional Identity Are a Transcriptional Hallmark of Human Brain Aging. Cell Rep. 18, 557-570 (2017).<br /> (12) Hefendehl, J. K. et al. Homeostatic and injury-induced microglia behavior in the aging brain. Aging Cell (2014). doi:10.1111/acel.12149<br /> (13) Nikodemova, M. et al. Microglial numbers attain adult levels after undergoing a rapid decrease in cell number in the third postnatal week. J. Neuroimmunol. 0, 280-288 (2015).<br /> (14) Moca, E. N. et al. Microglia Drive Pockets of Neuroinflammation in Middle Age. J. Neurosci. 42, 3896-3918 (2022).<br /> (15) Heumos, L. et al. Best practices for single-cell analysis across modalities. Nat. Rev. Genet. 24, 550-572 (2023).<br /> (16) Saelens, W., Cannoodt, R., Todorov, H. & Saeys, Y. A comparison of single-cell trajectory inference methods: towards more accurate and robust tools. (2018). doi:10.1101/276907<br /> (17) Zöller, T. et al. Silencing of TGFβ signalling in microglia results in impaired homeostasis. Nat. Commun. 9, (2018).<br /> (18) Bedolla, A. et al. Microglia-derived TGF-β1 ligand maintains microglia homeostasis via autocrine mechanism and is critical for normal cognitive function in adult mouse brain. bioRxiv Prepr. Serv. Biol. (2023). doi:10.1101/2023.07.05.547814<br /> (19) Spittau, B., Dokalis, N. & Prinz, M. The Role of TGFβ Signaling in Microglia Maturation and Activation. Trends Immunol. 41, 836-848 (2020).<br /> (20) L. Nyberg, M. Lövdén, K. Riklund, U. Lindenberger, L. Bäckman, Memory aging and brain maintenance. Trends Cogn. Sci. 16, 292-305 (2012).<br /> (21) L. Luo, F. I. M. Craik, Aging and memory: A cognitive approach. Can. J. Psychiatry 53, 346-353 (2008).<br /> (22) Y. Stern, M. Albert, C. Barnes, R. Cabeza, A. Pascual-Leone, P. Rapp.<br /> A framework for concepts of reserve and resilience in aging. Neurobiol. Aging, 124 (2022), pp. 100-103, 10.1016/j.neurobiolaging.2022.10.015<br /> (23) Y. Stern, C.A. Barnes, C. Grady, R.N. Jones, N. Raz. Brain reserve, cognitive reserve, compensation, and maintenance: operationalization, validity, and mechanisms of cognitive resilience. Neurobiol. Aging, 83 (2019), pp. 124-129, 10.1016/j.neurobiolaging.2019.03.022<br /> (24) R. Cabeza, M. Albert, S. Belleville, F.I.M. Craik, A. Duarte, C.L. Grady, U. Lindenberger, L. Nyberg, D.C. Park, P.A. Reuter-Lorenz, M.D. Rugg, J. Steffener, M.N. Rajah. Maintenance, reserve and compensation: the cognitive neuroscience of healthy ageing. Nat. Rev. Neurosci., 19 (11) (2018), Article 11, 10.1038/s41583-018-0068-2<br /> (25) Palovics, R. et al molecular hallmarks of heterochronic parabiosis at single-cell resolution. Nature 603, 309-314 (2022)<br /> (26) Sala Frigerio, C. et al. The major risk factors for Alzheimer's Disease: age, sex, and genes modulate the microglial response to Abeta plaques. Cell Rep, 27, 1293-1306 (2019)

Reviewer #1 (Public Review):

Summary:

In this manuscript, "PAbFold: Linear Antibody Epitope Prediction using AlphaFold2", the authors generate a python wrapper for the screening of antibody-peptide interactions using AlphaFold, and test the performance of AlphaFold on 3 antibody-peptide complexes. In line with previous observations regarding the ability of AlphaFold to predict antibody structures and antigen binding, the results are mixed. While the authors are able to use AlphaFold to identify and experimentally validate a previously characterized broad binding epitope with impressive precision, they are unable to consistently identify the proper binding registers for their control [Myc-tag, HA-tag] peptides. Further, it appears that the reproducibility and generality of these results are low, with new versions of AlphaFold negatively impacting the predictive power. However, if this reproducibility issue is solved, and the test set is greatly increased, this manuscript could contribute strongly towards our ability to predict antibody-antigen interactions.

Strengths:

Due to the high significance, but difficulty, of the prediction of antibody-antigen interactions, any attempts to break down these predictions into more tractable problems should be applauded. The authors' approach of focusing on linear epitopes (peptides) is clever, reducing some of the complexities inherent to antibody binding. Further, the ability of AlphaFold to narrow down a previously broadly identified experimental epitope is impressive. The subsequent experimental validation of this more precisely identified epitope makes for a nice data point in the assessment of AlphaFold's ability to predict antibody-antigen interactions.

Weaknesses:

Without a larger set of test antibody-peptide interactions, it is unclear whether or not AlphaFold can precisely identify the binding register of a given antibody to a given peptide antigen. Even within the small test set of 3 antibody-peptide complexes, performance is variable and depends upon the scFv scaffold used for unclear reasons. Lastly, the apparent poor reproducibility is concerning, and it is not clear why the results should rely so strongly on which multi-sequence alignment (MSA) version is used, when neither the antibody CDR loops nor the peptide are likely to strongly rely on these MSAs for contact prediction.

Major Point-by-Point Comments:

(1) The central concern for this manuscript is the apparent lack of reproducibility. The way the authors discuss the issue (lines 523-554) it sounds as though they are unable to reproduce their initial results (which are reported in the main text), even when previous versions of AlphaFold2 are used. If this is the case, it does not seem that AlphaFold can be a reliable tool for predicting antibody-peptide interactions.

(2) Aside from the fundamental issue of reproducibility, the number of validating tests is insufficient to assess the ability of AlphaFold to predict antibody-peptide interactions. Given the authors' use of AlphaFold to identify antibody binding to a linear epitope within a whole protein (in the mBG17:SARS-Cov-2 nucleocapsid protein interaction), they should expand their test set well beyond Myc- and HA-tags using antibody-antigen interactions from existing large structural databases.

(3) As discussed in lines 358-361, the authors are unsure if their primary control tests (antibody binding to Myc-tag and HA-tag) are included in the training data. Lines 324-330 suggest that even if the peptides are not included in the AlphaFold training data because they contain fewer than 10 amino acids, the antibody structures may very well be included, with an obvious "void" that would be best filled by a peptide. The authors must confirm that their tests are not included in the AlphaFold training data, or re-run the analysis with these templates removed.

(4) The ability of AlphaFold to refine the linear epitope of antibody mBG17 is quite impressive and robust to the reproducibility issues the authors have run into. However, Figure 4 seems to suggest that the target epitope adopts an alpha-helical structure. This may be why the score is so high and the prediction is so robust. It would be very useful to see along with the pLDDT by residue plots a structure prediction by residue plot. This would help to see if the high confidence pLDDT is coming more from confidence in the docking of the peptide or confidence in the structure of the peptide.

(5) Related to the above comment, pLDDT is insufficient as a metric for assessing antibody-antigen interactions. There is a chance (as is nicely shown in Figure S3C) that AlphaFold can be confident and wrong. Here we see two orange-yellow dots (fairly high confidence) that place the peptide COM far from the true binding region. While running the recommended larger validation above, the authors should also include a peptide RMSD or COM distance metric, to show that the peptide identity is confident, and the peptide placement is roughly correct. These predictions are not nearly as valuable if AlphaFold is getting the right answer for the wrong reasons (i.e. high pLDDT but peptide binding to a non-CDR loop region). Eventual users of the software will likely want to make point mutations or perturb the binding regions identified by the structural predictions (as the authors do in Figure 4).

Author response:

Reviewer #1 (Public Review):

This manuscript presents an exciting new method for separating insulin secretory granules using insulator-based dielectrophoresis (iDEP) of immunolabeled vesicles. The method has the advantage of being able to separate vesicles by subtle biophysical differences that do not need to be known by the experimenter, and hence could in principle be used to separate any type of organelle in an unbiased way. Any individual organelle ("particle") will have a characteristic ratio of electrokinetic to dielectrophoretic mobilities (EKMr) that will determine where it migrates in the presence of an electric field. Particles with different EKMr will migrate differently and thus can be separated. The present manuscript is primarily a methods paper to show the feasibility of the iDEP technique applied to insulin vesicles. Experiments are performed on cultured cells in low or high glucose, with the conclusion that there are several distinct subpopulations of insulin vesicles in both conditions, but that the distributions in the two conditions are different. As it is already known that glucose induces release of mature insulin vesicles and stimulates new vesicle biosynthesis and maturation, this finding is not necessarily new, but is intended as a proof of principle experiment to show that the technique works. This is a promising new technology based on solid theory that has the possibility to transform the study of insulin vesicle subpopulations, itself an emerging field. The technique development is a major strength of the paper. Also, cellular fractionation and iDEP experiments are performed well, and it is clear that the distribution of vesicle populations is different in the low and high glucose conditions. However, more work is needed to characterize the vesicle populations being separated, leaving open the possibility that the separated populations are not only insulin vesicles, but might consist of other compartments as well. It is also unclear whether the populations might represent immature and mature vesicles, distinct pools of mature vesicles such as the readily releasable pool and the reserve pool, or vesicles of different age. Without a better characterization of these populations, it is not possible to assess how well the iDEP technique is doing what is claimed.

Major comments:

1) There is no attempt to relate the separated populations of vesicles to known subpopulations of insulin vesicles such as immature and mature vesicles, or the more recently characterized Syt9 and Syt7 vesicle subpopulations that differ in protein and lipid composition (Kreutzberger et al. 2020). Given that it is unclear exactly what populations of vesicles will be immunolabeled (see point #2 below), it is also possible that some of the "subpopulations" are other compartments being separated in addition to insulin vesicles. It will be important to examine other markers on these separated populations or to perform EM to show that they look like insulin vesicles.

We thank the reviewer for this comment and have added the following to the discussion:

“The intensity peaks we observed at specific EKMr values likely correspond to some of the previously described insulin vesicle subpopulations34,54-57. Larger particles are expected to have a smaller EKMr value compared to smaller particles50. Subpopulations containing larger insulin vesicles, such as a mature pool34,54, synaptotagmin IX-positive vesicles57, or docked vesicles near the plasma membrane34 may have lower EKMr values than smaller immature vesicles. Additionally, phosphatidylcholine lipids increase the zeta potential of tristearoylglycerol crystals58. This effect may extend to insulin vesicle subpopulations containing more phosphatidylcholine, such as young insulin vesicles55 which could lead to higher EKMr values. Taken together, these two properties may be used to predict the EKMr values of known insulin vesicle subpopulations. For example, insulin vesicles with EKMr values of 1-2×109 V/m2 (Fig. 4C) may represent a synaptotagmin IX-positive subpopulation due to their larger radii and depletion under glucose stimulation. Additionally, young insulin vesicles may have EKMr values between 5 and 7.5×109 V/m2 (Fig. 4C) due to higher amounts of phosphatidylcholine present in this subpopulation55. In this EKMr range, we observed a higher intensity for glucose-treated cells which may suggest biosynthesis of new vesicles. Immature insulin vesicles are likely to have higher EKMr values due to their smaller size34, such as an EKMr value between 1.5-1.6×1010 V/m2 (Fig. 4C). Here we demonstrated the capabilities of DC-iDEP to separate insulin vesicle subpopulations in an unbiased manner. Future experiments using chemical probes to label subpopulations will be useful to accurately define the EKMr values associated with specific subpopulations.” pages 7-8, lines 176-191

Furthermore, we have conducted additional experiments using a modified INS-1 cell line with a GFP-tagged C-peptide (hPro-CpepSfGFP, GRINCH cells RRID:CVCL_WH61) in order to visualize a more complete population of insulin vesicles. By using this cell line, we have performed confocal microscopy, transmission electron microscopy, and cryo-electron microscopy experiments, demonstrating that the isolated vesicles resemble insulin vesicles and contain GFP-tagged C-peptide (Fig. 1-S3). While we acknowledge that further investigation using a more detailed labeling strategy of known insulin vesicle populations with DC-iDEP would be informative, we believe it is beyond the scope of our initial proof-of-concept experiments.

The following text was added to the results section to describe our additional microscopy analysis:

“To verify that the insulin vesicles were intact prior to DC-iDEP, we imaged a modified INS-1E cell line that contains a human insulin and green fluorescent protein-tagged C peptide (hPro-CpepSfGFP).49 This GFP tag allowed for quick visual verification of intact vesicles using fluorescence confocal microscopy. We observed distinct puncta rather than a diffuse GFP signal which indicated that the vesicles were intact and not ruptured. Further analysis of isolated vesicles was done using EM. We observed intact vesicles with the expected size and shape using both transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM) (Fig. 1—figure supplement 3).” Page 5, lines 104 – 109.

2) An antibody to synaptotagmin V is used to immunolabel vesicles, but there has been confusion between synaptotagmins V and IX in the literature and it isn't clear what exactly is being recognized by this antibody (this reviewer actually thinks it is Syt 9). If it is indeed recognizing Syt 9, it might already be labeling a restricted population of insulin vesicles (Kreutzberger et al. 2020). The specificity of this antibody should be clarified. Furthermore, Figure 2 is not convincing at showing that this synaptotagmin antibody specifically labels insulin vesicles nor is there convincing colocalization of this synaptotagmin antibody with insulin vesicles. In the image shown, several cells show very weak or no staining of both insulin and the synaptotagmin. The highlighted cell appears to show insulin mainly in a perinuclear structure (probably the Golgi) rather than in mature vesicles (which should be punctate), and insulin is not particularly well-colocalized with the synaptotagmin. Other cells in the image appear to have even less colocalization of insulin and synaptotagmin, and there is no quantification of colocalization. It seems possible that this antibody is recognizing other compartments in the cell, which would change the interpretation of the populations measured in the iDEP experiments. It would also be good to perform synaptotagmin staining under glucose-stimulating conditions, in case this alters the localization.

We thank the reviewer for bringing this issue to our attention. The antibody originally used in Figure 2 recognizes the 386 aa isoform of synaptotagmin, which is called Syt 9 in the paper mentioned above (Kreutzberger et al. 2020). We have edited our manuscript to label this antibody as “Synaptotagmin IX” to match the existing literature. This antibody, therefore, likely labels only a subset of insulin vesicles. We believe that populations measured in the iDEP experiments consist solely of insulin vesicles, as supported by Western blot and dynamic light scattering results (Fig. 1—figure supplement 2B-C), as well as EM images (Fig. 1—figure supplement 3). Even with a subset of insulin vesicles, these results show the potential of this method, as iDEP analysis reveals heterogeneity within the population of Syt 9-positive insulin vesicles. We have replaced the original immunofluorescence images in Figure 2 with images that are more representative of INS-1E cells. We recognize that immuno-labeling did not yield perfect co-localization, which was expected. However, these experiments do provide valuable insights into the promise of using DC-iDEP for more in-depth separation analysis. Future work will use a modified INS-1 cell line or mouse model with a GFP-tagged C-peptide (hPro-CpepSfGFP, GRINCH cells RRID:CVCL_WH61) in order to visualize a less restricted set of insulin vesicles, avoiding the limitations associated with antibodies confined to a specific insulin vesicle subpopulation.

3) The EKMr values of the vesicle populations between the low and high glucose conditions don't seem to precisely match. It is unclear if this just a technical limitation in comparing between experiments or instead suggests that glucose stimulation does not just change the proportion of vesicles in the subpopulations (i.e. the relative fluorescent intensities measured), but rather the nature of the subpopulations (i.e. they have distinct biophysical characteristics). This again gets to the issue of what these vesicle subpopulations represent. If glucose stimulation is simply converting immature to mature vesicles, one might expect it to change the proportion of vesicles, but not the biophysical properties of each subpopulation.

We thank the reviewer for this question. We agree that glucose likely shifts the proportion of vesicles within a specific EKMr value rather than impacting the overall biophysical characteristics of all vesicles. We have performed new statistical analysis as suggested and rewritten this section to better explain the differences between conditions.

“Visual inspection of the collected data revealed generally similar patterns of vesicles collected at specific EKMr values (Fig. 4). However, at 1200 V we achieved adequate separation of vesicle populations to discern unique populations of vesicles from cells treated with glucose compared to no treatment. Using a two-way ANOVA, we found a statistically significant interaction between the effect of treatment on vesicles collected at each EKMr value for data collected only at 1200 V [F (8, 45) = 3.61, p= 0.003]. A Bonferroni post hoc test revealed a significant difference in the intensity or quantity of vesicles collected between treated and untreated samples at 1.10x109 V/m2 (p=0.0249), 5.35x109 V/m2 (p=0.0469), 7.45x109 V/m2 (p=0.0369). These differences reflect a shift in the populations of insulin vesicles upon glucose stimulation.” Page 7, lines 158-165

We have also now directly addressed the potential identities of the different populations in the discussion section. This was addressed in major comment #1 and on page 7 lines, 176-191 of the manuscript.

4) The title of the paper promises "isolation" of insulin vesicles, but the manuscript only presents separation and no isolation of the separated populations. Isolation of the separated populations is important to be able to better define what these populations are (see point #1 above). Isolation is also critical if this is to be a valuable technique in the future. Yet the paper is unclear on whether it is actually technically feasible to isolate the populations separated by iDEP. In line 367, it states "this method provides a mechanism for the isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis (imaging, proteomics, lipidomics, etc.)." However, in line 361 it says "developing the capability to port the collected individual boluses will enable downstream analyses such as mass spectrometry or electron microscopy," suggesting that true isolation of these populations is not yet feasible. This should be clarified.

We thank the reviewer for pointing this out. We have modified the text and title to put more focus on our ability to separate vesicles rather than isolate. We agree that the isolation and further biophysical characterization of these subpopulations will be critical to understanding them. However, this capability is still in development. We have made the following change to clarify that a way to isolate these subpopulations once iDEP-assisted separation has occurred is currently being developed.

Title: “Insulator-based dielectrophoresis-assisted separation of insulin secretory vesicles”

“this method serves as a stepping stone towards isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis…” page 9, line 230-232.

Reviewer #2 (Public Review):

This manuscript used DC-iDEP, a technology previously used on other organelle preparations to isolate insulin secretory granules from INS1 cells based on differences in dielectrophoretic and electrokinetic properties of synaptotagmin V positive insulin granules.

The major motivation presented for this work is to provide a methodology to allow for more sensitive isolation of subpopulations of granules allowing better understanding of the biochemical composition of these populations. This manuscript clearly demonstrates the ability of this technology to separate these subpopulations which will allow for future biochemical characterizations of insulin granules in future studies.

After proving these subpopulations can be observed, this method was then utilized to show there are shifts in these subpopulations when granules are isolated from glucose stimulated cells. Overall the method of isolation is novel and could provide a tool for further characterization of purified secretory granules.

The observation of glucose stimulation causing shifts in subpopulations is unsurprising. Glucose stimulation could cause a depletion of insulin and other secretory content from a subset of granules. It would be expected that this loss of content would cause a shift in electrochemical properties of the granules, but this is a nice confirmation that the isolation method has the sensitivity to delineate these changes.

Major comments:

1) It is unclear what Synaptotagmin isoform is being looked at. Synaptotagmin V and IX have been repetitively interchanged in the literature. See note in syt IX section of "Moghadam and Jackson 2013 Front. Endocrinology" or read "Fukuda and Sagi- Eisenberg Calcium Bind Proteins 2008".

The 386 aa. isoform that is abundant in PC12 cells has been robustly observed in INS1 cells in multiple studies and has been frequently referred to as syt IX. The sequence the antibody was raised against should be determined from the company where this was purchased and then this should be mapped to to which isoform of Synaptotagmin by sequence and clarified in the text.

We thank the reviewer for this comment. The supplier (Thermo Fisher Scientific) calls this antibody “Synaptotagmin V.” As it recognizes the 386 aa synaptotagmin isoform, we have changed references to this antibody to call it “Synaptotagmin IX” to match the existing literature.

2) Immunofluorescence of insulin and syt V is confusing. The example images do not appear to show robust punctate structures that are characteristic of secretory granules (in both the insulin and syt V stain).

We appreciate the reviewer bringing this point to our attention. We agree that the immunofluorescence images in Figure 2 are not representative of typical INS-1E cells and have replaced the original image for Figure 2 with new images that show punctate structures that are more characteristic of secretory granules. These images also have better colocalization of insulin and synaptotagmin V (now labeled synaptotagmin IX) than the original image, with Pearson’s R values of 0.66 and 0.64.

3) In the discussion it says, "Finally, this method provides a mechanism for the isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis (imaging, proteomics, lipidomics, etc.) that otherwise would not be possible given the low-abundance components of these subpopulations."

It would help to elaborate more on the yield and concentrations of isolated granules. This would give a better sense of what level of biochemical characterization could be performed on sub- populations of granules.

We thank the reviewer for this comment. This line has been changed to clarify the current capabilities of iDEP, as subpopulations cannot presently be removed from the channel.

“this method serves as a stepping stone towards isolation and concentration of fractions which show the largest difference between the two population patterns for further bioanalysis…” page 9 line 230-232.

Once it is possible to isolate subpopulations from the channel, we expect to obtain sufficient sample for further characterization. We anticipate that biophysical characterization such as imaging will be highly feasible, and small-scale proteomics could also be possible. However, currently we have not measured the concentration of isolated vesicles due to complications in the isolation steps. If the quantity of isolated subpopulations proves inadequate for proteomic analysis, we plan to scale up our cell culture to generate enough insulin vesicles for further biochemical characterization. However, these experiments are out of scope for our current work, so we removed details on this idea in the Introduction and Discussion.

Reviewer #3 (Public Review):

The manuscript from Barekatain et al. is investigating heterogeneity within the population of insulin vesicles from an insulinoma cell line (INS-1E) in response to glucose stimulation. Prevailing dogma in the beta-cell field suggests that there are distinct pools of mature insulin granules, such as ready-releasable and a reserve pool, which contribute to distinct phases of insulin release in response to glucose stimulation. Whether these pools (and others) are distinct in protein/lipid composition or other aspects is not known, but has been suggested. In this manuscript, the authors use density gradient sedimentation to enrich for insulin vesicles, noting the existence of a number of co-purifying contaminants (ER and mitochondrial markers). Following immunolabeling with synaptotagmin V and fluorescent-conjugated secondary antibodies, insulin vesicles were applied to a microfluidic device and separated by dielectrophoretic and electrokinetic forces following an applied voltage. The equilibrium between these opposing forces was used to physically separate insulin granules. Here some differences were observed in the insulin (Syt V positive) granule populations, when isolated from cells that were either non-stimulated or stimulated with glucose, which has been suggested previously by other studies as noted by the authors; however in the current manuscript, the inclusion of a number of control experiments may provide a better context for what the data reveal about these changes.

The major strength of the paper is in the use of the novel, highly sophisticated methodology to examine physical attributes of insulin granules and thus begin to provide some insight into the existence of distinct insulin granule populations within a beta-cell -these include insulin granules that are maturing, membrane- docked (i.e. readily releasable), in reserve, newly-synthesized, aged, etc. Whether physical differences exist between these various granule pools is not known. In this capacity, the technical abilities of the current manuscript may begin to offer some insight into whether these perceived distinctions are physical.

The major weakness of the manuscript is that the study falls short in terms of linking the biology to the sophisticated changes observed and primarily focuses on differences in response to glucose. Without knowing what the various populations of granules are, it is challenging to understand what the changes in response to glucose mean.

Specific concerns are as follows:

1) There is confusion on what the DC-iDEP separation between stimulated and stimulated cells reveals. Do these changes reflect maturation state of granules, nascent vs. old granules? Ready- releasable vs. reserve pool? The comments in the text seem to offer all possibilities.

We thank the reviewer for this comment. Additional experiments will be useful to concretely define the physical nature of these subpopulations. Our primary goal in this study is to assess the utility of DC-iDEP in reproducibly separating these subpopulations. Our current results reflect variations in the amounts of subpopulations described in the literature and/or in currently uncharacterized subpopulations. As addressed in Reviewer #1 question #1, we have added to the discussion to review these possibilities (Page 7-8, lines 176-191).

2) It is unclear what we can infer regarding the physical changes of granules between the stimulated states of the cells. Without an understanding of the magnitude of the effect, it is unclear how biologically significant these changes are. For example, what degree of lipid or protein remodeling would be necessary to give a similar change?

We thank the reviewer for this question. Separation by iDEP is sufficiently sensitive to distinguish particles with minimal differences between them. For example, we could successfully separate wild type GFP from a point mutation variant of GFP. We anticipate that this method is capable of distinguishing vesicles with greater physical differences between them resulting in more distinct EKMr values. However, significant future experiments are likely necessary to determine the extent of lipid and protein remodeling between each subpopulation to define the biological significance of each subpopulation.

3) The reliance on a single vesicle marker, Syt V, is concerning given that granule remodeling is the focus.

We appreciate the reviewer’s concern. The current manuscript focuses on synaptotagmin V (IX)-positive insulin vesicles. The results of these experiments demonstrate the capabilities of iDEP to reveal heterogeneity in a seemingly similar set of particles. In future experiments we plan to use the modified INS-1 cell line with a GFP-tagged C-peptide (hPro-CpepSfGFP, GRINCH cells RRID:CVCL_WH61). All insulin vesicles from this cell line contain GFP-tagged C-peptide, and therefore would allow for the detection of a more complete set of insulin vesicles. The results from the current manuscript provide the proof-of-concept validation that this method is promising for understanding vesicle remodeling in more detail in the future.

4) Additional confirmation that the isolated vesicles are in fact insulin granules would be helpful. As noted, granules were gradient enriched, but did carry contaminants. Note that the microscopy image provided does not provide any real validation for this marker.

Further confirmation that the immune-isolated vesicles are in fact insulin granules should be included. EM with immunogold labeling post-SytV enrichment would be a potential methodology to confirm.

We thank the reviewer for this comment. We have performed new immunofluorescence imaging to demonstrate the overlap of insulin and synaptotagmin (Fig 2). Additionally, we have performed microscopy experiments with a modified INS-1 cell line with a GFP-tagged C-peptide (hPro-CpepSfGFP, GRINCH cells RRID:CVCL_WH61) in order to provide evidence of these granules’ identity. Fluorescence microscopy revealed that the isolated granules contain GFP-tagged C-peptide (Fig. 1—figure supplement 3A), while transmission electron microscopy and cryo-electron microscopy confirmed that these vesicles have radii within the correct range to be considered insulin vesicles (Fig 1—figure supplement 3B-C). We added the following text in the results section to describe the new results included:

“To verify that the insulin vesicles were intact prior to DC-iDEP, we imaged a modified INS-1E cell line that contains a human insulin and green fluorescent protein-tagged C peptide (hPro-CpepSfGFP).49 This GFP tag allowed for quick visual verification of intact vesicles using fluorescence confocal microscopy. We observed distinct puncta rather than a diffuse GFP signal which indicated that the vesicles were intact and not ruptured. Further analysis of isolated vesicles was done using EM. We observed intact vesicles with the expected size and shape using both transmission electron microscopy (TEM) and cryo-electron microscopy (cryo-EM) (Fig. 1—figure supplement 3). Page 5, lines 104 – 109.

5) It would be useful to understand if the observed effects are specific to the INS-1E cell line or are a more universal effect of glucose on beta-cells.

We agree with the reviewer that it would be interesting to study these effects in primary beta cells. While we expect to see similar results in these cells, there may be differences in the population variations or EKMr values. However, working with beta cells is currently beyond the scope of this study, as our primary focus is on validating this approach.

Author response:

Reviewer #1 (Public Review):

Authors propose a mechanism where actin polymerization in the dendritic shaft plays a key role in trapping AMPAR vesicles around the stimulated site, promoting the preferential insertion of AMPAR into the potentiated synapse. This dendritic mechanism is novel and may be important for phenomena. Authors also developed a sophisticated method to observe the endogenous behavior of AMPAR using the HITI system.

However, there are some major issues that need to be addressed to support the authors' claims. Also, overall, it is hard to follow. It could be better written.

We thank the reviewer for carefully reading our text and for the helpful recommendations. We have performed additional experiments and analysis to address the raised issues (detailed below). In addition, we have streamlined and shortened the text to improve its clarity and focus on the biological story.

Reviewer #2 (Public Review):

In this study, Wong and colleagues investigate mechanisms leading to input-specificity of LTP. They focus on the trafficking of AMPA receptors as the surface accumulation of AMPARs is one of the key features of potentiated synapses. They employ an elegant strategy to label endogenous GluA1 with a HaloTag using CRISPR-based technology and succeed to find targeting site which does not interfere with receptor's trafficking or function. This allowed them to visualize and track single receptors in endosomes as well as at the plasma membrane of primary rat hippocampal neurons. They develop and extend particle tracking and molecule counting algorithms to analyze active transport and diffusion of AMPARs and, as expected find that neuronal activation leads to increased surface expression of labelled AMPARs. Interestingly, they also observe a strong decrease in long-range motion of AMPAR-containing vesicles upon induction of chemical LTP. From this point, the manuscript focuses on explaining this observation. The authors switch from a global activation protocol to glutamate uncaging to induce LTP at individual synapses. Also, in these settings, they measure the reduction in mobile vesicle fraction within about 30 µm long dendritic segment containing the activated spine. In search of an explanation, they investigate activity-dependent actin polymerization as a possible confinement factor that could change the motility of organelles in dendrites. Their hypotheses is based on pre-existing literature demonstrating the role of F-actin in trapping and stalling dendritic endolysosomes as well similar role of F-actin in non-neuronal cells. Indeed, the authors convincingly show that pharmacological depolymerization or stabilization of F-actin bidirectionally impacts the trafficking behavior of AMPAR-containing vesicles in the dendritic shaft. To directly visualize effects of structural LTP at individual synapses on dendritic actin cytoskeleton, they employ a F-actin-binding probe Tractin. Here they find that cLTP results in the formation of dendritic F-actin fibers and bundles arranged in a network. The spatial extent of such a network correlates with an area where AMPAR vesicles exhibit decreased motility. Although this makes sense, I have some concerns about these experiments.

Tractin has been previously published as F-actin marker but like several other binding probes (i.e. lifeact), it affects F-actin structure and dynamics. The large number of F-actin bundles is not very typical for dendrites of hippocampal neurons and might be an artifact of Tractin overexpression. It is difficult to judge whether this is a case because there is no comparison with the endogenous situation where F-actin is labelled directly. The final series of experiments focus on the role of processive myosins in stalling and exocytosis of AMPAR vesicles. To address this point, the authors employ a mixture of three different myosin inhibitors and show that although myosins are not responsible for increased vesicle confinement they facilitate exocytosis of AMPARs. What I find somewhat missing are data and examples of AMPAR trafficking into dendritic spines. Also here, stronger experimental support could benefit the conclusions.

Overall, the authors achieved the aims of their study. They demonstrated that synapse-specific potentiation results in signaling which triggers actin polymerization in dendritic shaft beneath the activated input. This leads to trapping and accumulation of AMPAR-containing endosomes which then have higher probability to be delivered and secreted at activated dendritic spines. In addition to conceptual advance of this work, several state-of-the-art labeling and analysis techniques where developed in this project and they will likely be used by other groups.

We thank the reviewer for raising these important issues with regards to the use of tractin as a marker for actin polymerization. We have performed additional experiments (detailed below) using phalloidin and also dominant negative inhibitors of myosin Va, Vb, and VI in order to strengthen our conclusions. We find that inducing synaptic activity with cLTP increases phalloidin labeling and the appearance of F-actin fibers. Moreover, inhibition of myosin Va and Vb (but not VI) using their dominant negative c-terminal domains recapitulates the effects of pharmacological inhibition on both the motion states and directional bias of GluA1-HT vesicles in response to cLTP.

With regards to AMPAR trafficking into spines, we and others have found that GluA1-containing vesicles rarely enter dendritic spines (see response to Reviewer #2, comment 3). Furthermore, exocytic events occur largely at extrasynaptic sites, such as on the dendritic shaft (Figure 5-video 1-3; Lin et al., 2007; Makino et al., 2009; Patterson et al., 2010). Consequently, we believe vesicles are concentrated proximal to synaptic activity in the dendritic shaft rather than in the dendritic spine itself, creating a larger reservoir of intracellular AMPARs that can exocytose during synaptic activity. Others have demonstrated that surface bound AMPARs diffuse across the cell membrane into stimulated synapses where they are captured (Choquet and Opazo, 2022).

We also thank the reviewers for acknowledging the conceptual and technical advances in this work.

Reviewer #3 (Public Review):

Wong et al. developed a new versatile approach with a robust signal to track protein dynamics by inserting a tag into the endogenous loci and different properties of fluorescent dyes for conjugation. Using this approach, the authors monitor the trafficking of Fluorescent dye and Halo-tagged GluA1 with time-lapse imaging and found that neuronal stimulation induces GluA1 accumulation surrounding stimulated synapses on dendritic shafts and actin polymerization at synapses and dendrites. Furthermore, combining with pharmacological manipulations of actin polymerization or myosin activity, the authors found that actin polymerization facilitates exocytosis of GluA1 near activated synapses. The new approach may provide broad impacts upon appropriate control experiments, and the practical application of this approach to GluA1 trafficking upon neuronal activation is significant. However, there are several weaknesses, including confirmation of activity of the tagged receptors and receptor specificity mimicking endogenous LTP machinery. If the receptor tagged by the new robust approach reflects endogenous machinery, this approach will provide a big opportunity to the community as a versatile method to visualize a protein not visualized previously.

Although we use methods previously demonstrated to stimulate LTP, we do not ourselves demonstrate LTP using electrophysiological methods, and consequently we have changed the text to focus on synaptic plasticity (specifically structural plasticity). Furthermore, we confirm the activity of HaloTag knock-in receptors by expressing GluA1-HT and GluA1-HT-SEP in HEK293T cells and performing whole-cell patch clamp experiments. We find that GluA1-HT and GluA1-HT-SEP responds to glutamate in a similar manner to untagged GluA1.

We also thank the reviewer for acknowledging the novelty of our strategy.

Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

Learn more at Review Commons

---

Reply to the reviewers

Reply to reviewers

First, we would like to extend our gratitude to all reviewers for their supportive and enthusiastic feedback, which acknowledges our study as an interesting, well-executed, and well-documented contribution to the field. We are also pleased that the novelty and significance of our work have been recognized and appreciated.

As highlighted by reviewers 2, 3, and 4, our research represents a substantial advancement in understanding the mechanisms that coordinate the development of different cell types. Our findings have broader implications for developmental biology. We also thank the reviewers for their valuable insights, which have significantly improved the overall readability of our manuscript. We have carefully considered all minor corrections and text modifications they had suggested and made amendments accordingly.

The reviewers proposed several complementary experiments to enhance and clarify our points. We have conducted most of these experiments, with one exception (detailed below), and incorporated the corresponding results into this revised version of the manuscript.

Additionally, reviewers agreed that higher resolution images depicting the interactions between tendon and myoblast membranes would strengthen our manuscript. In response, we are pleased to present new high-resolution images of Ama::EGFP localization with respect to tendon and muscle cells, obtained using Zeiss Airyscan technology. We also provide new images using newly generated flies that allow simultaneous observation of both myoblast and tendon membranes.

We believe these modifications substantially enhance the quality and interest of our results, as already highlighted by the reviewers.

__Referees cross-commenting: __

All reviewers agreed* that "this is an interesting study that is well done and well documented. I agree with reviewer 1 that the study would further benefit from better imaging of the cellular extensions of tendons and myoblasts to see how both cell types interact." *

Reply: We agree with this point. To address it, we analyzed leg discs from Sr-Gal4>UAS-myrGFP line (labelling tendon membranes) crossed with a newly generated line R32D05::CD4TdTomato (myoblast specific expression of membrane tagged Tomato protein). Using confocal Zeiss Airy Scan technology, we generated high resolution images for which both tendon cell extensions and myoblast membranes are simultaneously visualized. These images are included in the new Figure 2 (O, P and O', P'). To be noticed: as we also provide new high resolution images of Ama::EGFP and Nrt localizations (Fig. 2 M, N and M', N'), we removed images of zoom in of 5h APF leg disc.

REVIEWER 1

Moucaud et al carried out single cell sequencing on myoblasts from the developing drosophila leg muscles, focusing on gene expressions overlapping with tendon and muscle cells. This study proposes that neuronal cell adhesion molecules Ama and Nrt interact in myoblast and tendon adhesion to support tendon and in proliferation of muscle progenitors. This study traces Ama and Nrt expression with various drosophila mutant strains and provides evidence to support its claims using single cell sequencing, immuno-fluoresence and in situ hybridisation. *The authors report novel markers to study the interactions between muscle and tendon progenitors in the Drosophila leg provide convincing evidence of their functions in muscle and muscle and tendon formation. *

• The authors report novel markers to study the interactions between muscle and tendon progenitors in the Drosophila leg provide convincing evidence of their functions in muscle and muscle and tendon formation. *

__ Reply:__ We are grateful to the Reviewer 1 for his/her supportive comments on the quality of our work.

Page 2 "cell types*..." might be worth including other cell types such as vascular/endothelial if listing all cell types in the limb, as the sentence is suggesting. *

__ Reply__: "blood vessels" have been added as components of the limb musculoskeletal system.

Reviewer's comment: The authors discuss the interactions between the myoblasts and tendon cells but do not show any cellular resolution of the interaction between the cells and the secreted adhesion proteins. It would enhance the manuscript if the authors could show high resolution images of these cellular interactions with the secreted protein in vivo.

__ Reply__: see reply to referees cross-commenting about newly generated high-resolution images shown in Fig. 2.

Lots of examples of definite article (the) missing throughout the text.

Reply: The text has been edited and missing articles added

Second line of Abstract does not flow: Ama encodes secreted proteins to "Ama encodes a secreted protein"

__ Reply__ the correction has been made accordingly

2nd para Intro- this para is essentially discussing vertebrate limb muscle/tendon precursors, although includes a non-vertebrate citation. It could be helpful to (briefly) compare/contrast the non-vertebrate vs vertebrate literature on this topic.

__ Reply__: Indeed, this paragraph is primarily focused on the development of the musculoskeletal system in vertebrates. The comparison (from a molecular standpoint) with the muscle system of the Drosophila leg appears in the following paragraph. For clarity, we have included a brief, more general description (end of second paragraph) about the muscle/tendon system in Drosophila to highlight certain divergences between vertebrate and invertebrate systems and to introduce the subsequent paragraph.

"in limb of chick embryo add "the limb"

__ Reply:__ the correction has been made accordingly

p6 because these two antibodies were raised in rabbit, as the Twist antibody, needs some additional explanatory text.

__ Reply__: We have modified the text to give a more accurate explanation: "Because these two antibodies were raised in rabbit, as was the Twist antibody, we could not use this latter to visualize the myoblasts"

P9 discussion creeping into results section-with some speculation on Ama forming homophilic adhesions which has not been experimentally tested.

__ Reply:__ Because we chose to submit this work as a short format paper, Results and Discussion sections are indeed combined. However, we agree that homophilic adhesion properties of Ama have been shown only in cell culture and not tested in physiological context. To clarify this point, we have modified the corresponding part of the text and only suggest that Ama could directly bind to membranes through its putative GPI modification as proposed by Seeger et al.

Ama depletion affects both viability and the proliferation rate of leg disc myoblasts (in a Nrt-independent way) Does it have similar role in tendon precursors? Could the authors provide any evidence of apoptosis given proposed role of Ama in glial cells?

__ Reply: __As asked by Reviewer 1, we have tested these two points and included the results in suppl figure 3 (A-C). As expected, the proliferation rate of tendon cells is not affected as we have previously showed that tendon cells are post-mitotic cells (Laurichesse et al. 2021). Moreover, we now show that Ama depletion does not lead to apoptosis of tendon cells. See Supp. Figure 3 (A-C), the main text has also been modified accordingly.

REVIEWER 2:

In this well-written, comprehensive, and interesting manuscript, the authors study the molecular circuitry that supports the coordinated activity of tendon cells and myoblasts during development. As the authors themselves point out in the introduction, the assembly of tissues within the musculoskeletal system provides a particularly attractive system in which to study how different cell types coordinate their behaviours to form higher-order structures. Using single-cell transcriptomics, the authors first identify the cell adhesion molecule Ama and transmembrane protein Nrt as enriched in Drosophila myoblasts and tendon cells. Their transcriptomic data suggest that Nrt is specifically expressed in the tendon cells while Ama is expressed in both. They support these data with a variety of in situ, antibody, and endogenous stainings. Using a series of genetic manipulations, they then convincingly show that Ama controls the total number of myoblasts during the larval stages: Ama knockdown is associated with both decreased proliferation and increased apoptosis of myoblasts. Ama's role in regulating myoblast number is shown to be independent of Nrt and likely under the control of the FGF/RTK pathway. Finally, the authors show that the loss of either Nrt or Ama activity is associated with a loss of adhesion between myoblasts and tendon cells and with the stunted growth of the long tendon. Thus, their data point to Ama playing dual roles in muscle development by regulating both myoblast number and cell adhesion.

* I very much enjoyed reading the paper, which I think makes an important contribution to our understanding of both the developing musculature and inter-cell-type coordination during development more broadly. I have only a handful of grammatical errors to point out.*

__Reply: __We appreciate these enthusiastic and supportive comments, and we would like to thank the Reviewer for highlighting the broader contribution of our work to the understanding of the mechanisms of coordination between different cell types.

*- Grammar: 'This prompted us to use Drosophila model to search' should read 'This prompted us to use the Drosophila model to search' - Grammar: '...identify Neurotactin (Nrt) and its binding partner, Amalgam (Ama) as candidates...' should read '...identify Neurotactin (Nrt) and its binding partner, Amalgam (Ama), as candidates...' - Grammar: 'As tendon precursors in leg disc' should read 'As tendon precursors in the leg disc'. - Grammar : '...we performed a series of in situ hybridization...' should read '...we performed a series of in situ hybridizations...' - Grammar: 'Because these two antibodies were raised in rabbit, as the Twist antibody' should read 'Because these two antibodies were raised in rabbit, as was the Twist antibody' - Grammar: 'Statistical analysis reveals an increase myoblast total number when overexpressing an activated ERK' should read 'Statistical analysis reveals an increase in the total number of myoblasts when overexpressing an activated ERK' - Grammar: The following section header needs rephrasing: 'Ama, potential downstream effector of FGF pathway in the regulation of myoblast number'. Maybe 'Ama is a potential downstream effector of the FGF pathway in the regulation of myoblast number' or Ama: a potential downstream effector of the FGF pathway in the regulation of myoblast number. - Grammar: 'whereas its reduction (UAS-StyRNAi) lead to more myoblasts' should read 'whereas its reduction (UAS-StyRNAi) leads to more myoblasts' - For clarity 'The expression of the constitutively active form of ERK could rescue the phenotype of Ama depletion in glial cells (Ariss et al. 2020)' might read better as 'Previous work has shown that the expression of the constitutively active form of ERK can rescue the phenotype of Ama depletion in glial cells (Ariss et al. 2020). Therefore, we tried...' - Grammar: 'showed a significant higher number of myoblasts compared' should read 'showed a significantly higher number of myoblasts compared' - Grammar: 'Another, not exclusive, possibility' should read Another, non-mutually exclusive, possibility'. - Grammar: 'We measured the length of the tilt relatively to the length' should read '. We measured the length of the tilt relative to the length' *

Reply: All the modifications suggested above by Reviewer 2 are now included in the text.

REVIEWER 3:

*Myoblast and tendon precursors stem from different developmental origins. Hence, they need to find each other to build a functional muscle-skeleton. How they do so is an exciting biological problem, not only for this reviewer who is working on Drosophila muscle development, too. As we currently understand little about how myoblasts communicate with tendons during development, I find this manuscript a generally interesting contribution unravelling a new mechanism of cell-cell communication between these two cell types. It proposes a role for 2 interesting proteins that are little studied. Furthermore, Drosophila leg muscle-tendon development is complex and results in an intricate final architecture. Thus, a better understanding of its molecular mechanisms of development is exciting to this reviewer and to the muscle and tendon fields. *

Reply: We express our gratitude to Reviewer 3 for his/her keen interest in our work and for emphasizing its significance within the field of developmental biology.

*While some Ama mRNA expression in myoblasts was confirmed with in situ hybridisation, it was also shown that Ama mRNA is expressed in other sources including tendon precursors. As the interesting AmaGFP protein overlapping with the developing tendon cells is found at some distance from the myoblasts, the source for this Ama protein population is not entirely clear. To identify if it is secreted from myoblasts I suggest to stain for Ama-GFP in the muscle-specific Ama knock-down discs at 5h APF. This could use the late knock-down condition. *

__ Reply:__ In Suppl Fig.2 in a close-up view of the femur region, we show that at 5h APF Ama is transcribed in addition to myoblasts also in the developing tilt tendon. This tendon associated Ama expression is specific as it is detected after myoblast specific Ama knockdown. Thus, at 5h APF, the Ama-GFP signal detected at the interface of muscle and tendon precursors could in part correspond to Ama secreted by the tilt tendon cells. However, we also observed clear Ama-GFP signal at the interface of myoblasts and tendon precursors at 0h APF when Ama is not yet transcriptionally activated in tilt tendon precursors (not shown). Thus, we are confident that the myoblasts are the main source of secreted Ama protein that ensure close proximity of myoblast and tendon precursor cells. A view supported by the loss of myoblast-tendon cell proximity in myoblast-specific Ama knockdown. However, to clarify this point, we immunostained myoblast-specific Ama knock-down discs for the Ama protein in at 5h APF as suggested. As stated in the text, we were concerned that GFP tag could influence the life-time of the Ama protein, as GFP itself is pretty stable. This is why we used anti-Ama antibody kindly provided by Dr. Silman to determine whether myoblast-specific Ama knockdown (using R32D05-Gal4 driver) would completely abolish Ama protein at 5h APF. We indeed observed a strong reduction of Ama protein at this stage indicating that the contribution of Ama protein from tendon cells is minimal (but cannot be completely excluded), with myoblasts remaining the major source at this stage. This new result is now presented in Suppl Fig. 2M-P. This result is also in accordance with our new result showing that tendon-specific AmaKD has no effect on tendon growth (see reply to the comment below regarding tendon length). In light of this new result, we have modified the text accordingly in the corresponding paragraph (p5-6).

Generally, it might be useful to move the part of Figure 2 that shows the Ama-GFP Nrt co-staining to the later part in the text that addresses the interaction of both cell types and keep the autonomous Ama role in muscle for the start of paper only.

__ Reply: __We have indeed debated extensively about this possibility before submitting this work. While such a presentation would have some logical coherence, it also has the disadvantage of having to resume, at least partially, the expression of Ama, leading to certain redundancies. Additionally, we chose to begin with a comparison of the new myoblast transcriptomic data with pre-established tendon data to highlight the presence of ligand-receptor pairs. In this context, it seemed to us more pertinent to present the expression patterns of Ama and Nrt together in the initial figures.

To quantify the interaction of the myoblast cell membranes and the tendon cells better it would be useful to combine sr>CAAXmCherry with a myoblast membrane maker (possibly Him-CD8-GFP or use R15B03-Gal4 with R79D08-lexA). This could also improve the "mean distance" measurements. As currently presented, it is not so clear how the mean distance was measured. It could be helpful to indicate some examples in zoom-in vies on Figure 5. Does a distance of 4 µm in wild type mean that the myoblast is not touching the tendon precursors, or is only the myoblast nucleus that is Twi positive at this distance?

__ Reply: We are grateful to this reviewer for its relevant suggestion. Thus, as stated above (referees cross-commenting), we provide new high-resolution images with labelled membranes of both tendon cells and myoblasts (fig 2 O-P). As shown here, myoblast membranes are very closed to each other, and nuclei occupy an important part of myoblast volumes. So, we found more accurate to use the myoblast nucleus (stained with Twist antibody) to detect individual myoblasts using Imaris Spot function rather than myoblast membranes. We also believe that the distances between the center of myoblast nuclei and the tendon surface are representative of the distance between these two cell types as nucleus myoblast occupies most of the cell volume. We addressed this point in the new Suppl. Fig.5. __Regarding the distance of 4____ µm: As mentioned in the original text, the 4 µm distance represents the average distance between myoblasts and the tendon surface in wild type discs. We do not perceive this distance as indicative of a threshold distinguishing myoblasts that interact physically with tendons from those that do not. We rather use this mean distance to quantify the distribution of myoblasts around the tendon and their dispersion/mis-distribution in Ama and Nrt knockdown leg discs. To clarify this point, we have modified the corresponding paragraph: "This result indicates that AmaKD leads to myoblasts mis-distribution around the tilt, suggesting that the reduction of Ama level could affect myoblast-tendon adhesion".

For a better understanding of how the mean distance was measured, we added a new Supplementary Figure 5 (rather than a zoom-in in the main figure as suggested by this reviewer), with a corresponding description of how this distance was measured in addition to the explanations in the material and method section.

Is the tendon elongation phenotype seen after Ama RNAi in muscle and in Nrt mutants due to the fact that myoblasts are further away from tendons or is it an Ama/Nrt role that is autonomous to tendons? This could be tested by assaying tendon elongation after tendon-specific Ama knock-down as shown in Figure S2.

__ Reply: __As asked by this reviewer, we have performed this experiment using Sr-Gal4 driver to induce tendon-specific Ama knockdown and assessed tendon elongation using R79D08-lexA>lexAop-GFP marker. Overall statistical analysis is now included in Fig 5F and G graphs. This analysis shows that tendon-specific Ama knockdown does not affect tendon elongation. This is in concordance with the fact that Ama knockdown in myoblasts leads to tendon defects similar to that of Nrt loss of function in tendon clearly indicating that the observed phenotypes are due to Ama's role in myoblasts. This does not exclude an additional subsequent Ama function in growing tendon precursors in later development.

Minor : Is Figure 2Q a zoom-in from Figure 2P? If yes, it would be helpful to indicate the rough position of it in the lower magnification image.

Reply: Figure 2Q was not a zoom-in from Figure 2P in the previous version of the paper. As stated above Fig. 2 has now been modified.

Minor page 6 - w[1118] is with small "w".

Reply: modifications have been made accordingly in main and figure texts.

REVIEWER 4:

*The manuscript is well-organized, with clear descriptions of methods and results. The use of transcriptomic datasets and gene expression analyses provides insights into the molecular mechanisms underlying the interaction between muscle and tendon precursors. *

*The immunostaining and in situ hybridization experiments well illustrate the expression patterns of Ama and Nrt in muscle and tendon cells during leg disc development in Drosophila. *

*The functional analyses, including knockdown experiments, support the conclusion that Ama plays a crucial role in maintaining the pool of leg muscle precursor cells and coordinating tendon and muscle precursor growth. *

The manuscript significantly enhances our understanding of cell-cell interactions in the musculoskeletal system of Drosophila. The findings have broader implications for the field of developmental biology. In general, this manuscript provides valuable insights into the molecular processes governing leg muscle and tendon development.

Reply: We are indebted to Reviewer 4 for highlighting that our manuscript is well-organized and well-illustrated. We are also grateful to Reviewer 4 for highlighting the valuable insights of our work.

some aspects in the manuscript, for example how Ama regulates myoblast number and its interaction with the FGFR pathway, could be further explored or clarified.

__ Reply:__ Regarding Ama's contribution for maintaining the myoblast pool through its interaction with the FGF pathway, we demonstrate here that, contrary to what has been proposed for glial cells, Ama acts downstream of this pathway, although we emphasize that there is a synergistic effect with the MAPK pathway inhibitor, Sprouty. These findings thus reveal complex and variable regulatory mechanisms between Ama and the FGF pathway that would require specific investigation, the entirety of which appears challenging to integrate into this same publication.

the organization of the abstract could be improved to provide a clearer and more comprehensive overview of the study. The abstract currently lacks a structured presentation of essential components such as methods, results, and conclusions. It would greatly benefit from a more systematic arrangement.

__ Reply:__ We have made modifications to propose a more structured abstract.

I don't have time to proof this entire play, but hopefully someone else already has or will do so? I've been comparing it to the Dial publication, but probably better to look at Sara Crangle's edited version, reprinted in the Essays and Stories of Mina Loy. This is also the authoritative addition of Sacred Prostitute.

indranet

we have

FORK LYFT

https://philosophybreak.com/articles/if-a-tree-falls-in-the-forest-and-theres-no-one-around-to-hear-it-does-it-make-a-sound/#:~:text=So%2C%20the%20answer%20to%20this%20age-old%20question%20seems,lonesome%20falling%20tree%20does%20not%20make%20a%20sound.

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If a Tree Falls in the Forest, and There's No One Around to Hear It, Does It Make a Sound?

The age-old question of whether a falling tree makes a sound when there's no one around to hear it exploits the tension between perception and reality. This article explores possible answers and their consequences.

By Jack Maden  |  September 2022

3-MIN BREAK  

If a tree falls in the forest, and there's no one around to hear it, does it make a sound? Well, if by 'sound' we mean vibrating air, then yes, when the tree falls, it vibrates the air around it.

However, if by 'sound' we mean the conscious noise we hear when our sensory apparatus interacts with the vibrating air, then if no one is around to hear the tree when it falls, there'd be no sensory apparatus for the vibrating air to interact with, and thus no conscious noise would be heard.

So, the answer to this age-old question seems to be simple: it depends on how we define 'sound'. If we define it as 'vibrating air', the falling tree makes a sound. If we define it as a conscious experience, the lonesome falling tree does not make a sound.

There, problem solved.

The point of asking this question, however, is not so that it can be answered quickly and put aside.

Rather, its point is to draw out the rather strange tension between our two very different definitions of the word 'sound'.

On the one hand, we classify sound as a mechanistic process that exists without us, 'out there' in the world. On the other, we regard it as a private conscious experience, its existence entirely dependent on us.

And when you dwell on this latter definition, you realize it doesn't just extend to sounds. Everything we experience --- everything we see, hear, smell, touch, taste --- all of it depends on our sensory apparatus, on us. Without us, our experiences would not exist.

As the great 16th-century astronomer Galileo Galilei put it:

Tastes, odors, colors, and so on... reside only in consciousness. If the living creature were removed, all these qualities would be wiped away and annihilated.

Take away our senses, and the world of our experience would be replaced by a colorless, soundless, odorless, tasteless nothingness. Without us, what remains?

The reason our original question --- When a tree falls in the forest, and there's no one around to hear it, does it make a sound? --- is such a teaser, is because it hits on a deeper question. Namely:

If there was no conscious life, would the physical universe exist?

Our kneejerk reaction to this question might be, 'of course it would'. But let's think about it again: if there was nothing conscious, then nothing would be experienced. There would be nothing resembling anything we call 'existence'. No colors, no sounds, no smells, no tastes, no touch, no sense of time, no sense of space.

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Is consciousness more fundamental than matter?

Reflecting on this strange state of affairs, numerous great thinkers have concluded that consciousness must be more fundamental than the 'stuff' that consciousness experiences.

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For instance, in his 1710 work, A Treatise Concerning the Principles of Human Knowledge, the philosopher George Berkeley discusses the absurdity of a world existing independently of our conscious minds:

It is indeed an opinion strangely prevailing amongst people that houses, mountains, rivers, and in a word all sensible objects, have an existence natural or real, distinct from their being perceived by the understanding... for what are the forementioned objects but things we perceive by sense? And what do we perceive besides our own ideas or sensations? And is it not plainly repugnant that any one of these or any combination of them should exist unperceived?

On this view, it is absurd to say a lonesome falling tree makes a sound. For Berkeley, it is absurd to say the tree, without a conscious mind there perceiving it, even exists. (You can learn more about his mind-bending arguments for this position in our short explainer piece on Berkeley's subjective idealism, his theory that the world is in our minds).

But to conclude this brief reflection on the tension between perception and reality, consider a comment from the Nobel Prize-winning quantum physicist Max Planck in a 1931 interview (italics added):

I regard consciousness as fundamental. I regard matter as derivative from consciousness. We cannot get behind consciousness. Everything that we talk about, everything that we regard as existing, postulates consciousness.

What do you think? Can we get behind consciousness?

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Jack MadenFounder\ Philosophy Break

Having received great value from studying philosophy for 15+ years (picking up a master's degree along the way), I founded Philosophy Break in 2018 as an online social enterprise dedicated to making the subject's wisdom accessible to all. Learn more about me and the project here.

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https://www.poetryfoundation.org/poems/44272/the-road-not-taken

The Road Not Taken

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BY ROBERT FROST

Two roads diverged in a yellow wood,

And sorry I could not travel both

And be one traveler, long I stood

And looked down one as far as I could

To where it bent in the undergrowth;

Then took the other, as just as fair,

And having perhaps the better claim,

Because it was grassy and wanted wear;

Though as for that the passing there

Had worn them really about the same,

And both that morning equally lay

In leaves no step had trodden black.

Oh, I kept the first for another day!

Yet knowing how way leads on to way,

I doubted if I should ever come back.

I shall be telling this with a sigh

Somewhere ages and ages hence:

Two roads diverged in a wood, and I---

I took the one less traveled by,

And that has made all the difference.

n/a

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note the "honey of the ancients" description here with a tangential nod to the commonplace tradition

see: <br /> - https://hypothes.is/a/mCsl9voQEeuP3t8jNOyAvw<br /> - https://hypothes.is/users/chrisaldrich?q=tag%3A%22jonathan+swift%22+tag%3A%22commonplace+books%22

Author response:

The following is the authors’ response to the original reviews.

Reviewer #1 (Recommendations For The Authors):

Major concerns:

(1) It is not clear about the biological significance of the inhibitory effects of human Abeta42 on gammasecretase activity. As the authors mentioned in the Discussion, it is plausible that Abeta42 may concentrate up to microM level in endosomes. However, subsets of FAD mutations in APP and presenilin 1 and 2 increase Abeta42/Abeta40 ratio and lead to Abeta42 deposition in brain. APP knock-in mice NLF and NLGF also develop Abeta42 deposition in age-dependent manner, although they produce more human Abeta42 than human Abeta40. 

If the production of Abeta42 is attenuated, which results in less Abeta42 deposition in brain. So, it is unlikely that human Abeta42 interferes gamma-secretase activity in physiological conditions. This reviewer has an impression that inhibition of gamma-secretase by human Abeta42 is an interesting artifact in high Abeta42 concentration. If the authors disagree with this reviewer's comment, this manuscript needs more discussion in this point of view. 

We thank the Reviewer for raising this key conceptual point, we acknowledge that it was insufficiently discussed in the original manuscript. In response to this point, we introduced the following paragraph in the discussion section of the revised manuscript:

“From a mechanistic standpoint, the competitive nature of the Aβ42-mediated inhibition implies

that it is partial, reversible, and regulated by the relative concentrations of the Aβ42 peptide (inhibitor) and the endogenous substrates (Figure 10C and 10D). The model that we put forward is that cellular uptake, as well as endosomal production of Aβ, result in increased intracellular concentration of Aβ42, facilitating γ-secretase inhibition and leading to the buildup of APP-CTFs (and γ-secretase substrates in general). As Aβ42 levels fall, the augmented concentration of substrates shifts the equilibrium towards their processing and subsequent Aβ production. As Aβ42 levels rise again, the equilibrium is shifted back towards inhibition. This cyclic inhibitory mechanism will translate into pulses of (partial) γsecretase inhibition, which will alter γ-secretase mediated-signaling (arising from increased CTF levels at the membrane or decreased release of soluble intracellular domains from substrates). These alterations may affect the dynamics of systems oscillating in the brain, such as NOTCH signaling, implicated in memory formation, and potentially others (related to e.g. cadherins, p75 or neuregulins). It is worth noting that oscillations in γ-secretase activity induced by treatment with a γ-secretase inhibitor semagacestat have been proposed to have contributed to the cognitive alterations observed in semagacestat treated patients in the failed Phase-3 IDENTITY clinical trial (7) and that semagacestat, like Aβ42, acts as a high affinity competitor of substrates (85).

The convergence of Aβ42 and tau at the synapse has been proposed to underlie synaptic dysfunction in AD (86-89), and recent assessment of APP-CTF levels in synaptosome-enriched fractions from healthy control, SAD and FAD brains (temporal cortices) has shown that APP fragments concentrate at higher levels in the synapse in AD-affected than in control individuals (90).  Our analysis adds that endogenous Aβ42 concentrates in synaptosomes derived from end-stage AD brains to reach ~10 nM, a concentration that in CM from human neurons inhibits γ-secretase in PC12 cells (Figure 7). Furthermore, the restricted localization of Aβ in endolysosomal vesicles, within synaptosomes, likely increases the local peptide concentration to the levels that inhibit γ-secretase-mediated processing of substrates in this compartment. In addition, we argue that the deposition of Aβ42 in plaques may be preceded a critical increase in the levels of Aβ present in endosomes and the cyclical inhibition of γsecretase activity that we propose. Under this view, reductions in γ-secretase activity may be a (transient) downstream consequence of increases in Aβ due to failed clearance, as represented by plaque deposition, contributing to AD pathogenesis.“

We have also added figures 10C and 10D, presented here for convenience.

Author response image 1.

(2) It is not clear whether the FRET-based assay in living cells really reflects gamma-secretase activity.

This reviewer thinks that the authors need at least biochemical data, such as levels of Abeta. 

We have established a novel, HiBiT tag based assay reporting on the global γ-secretase activity in cells, using as a proxy the total levels of secreted HiBiT-tagged Aβ peptides. The assay and findings are presented in the revised manuscript as follows:

In the result section, in the “Aβ42 treatment leads to the accumulation of APP C-terminal fragments in neuronal cell lines and human neuron” subsection:

“The increments in the APP-CTF/FL ratio suggested that Aβ42 (partially) inhibits the global γ-

secretase activity. To further investigate this, we measured the direct products of the γ-secretase mediated proteolysis of APP. Since the detection of the endogenous Aβ products via standard ELISA methods was precluded by the presence of exogenous human Aβ42 (treatment), we used an N-terminally tagged version of APPC99 and quantified the amount of total secreted Aβ, which is a proxy for the global γsecretase activity. Briefly, we overexpressed human APPC99 N-terminally tagged with a short 11 amino acid long HiBiT tag in human embryonic kidney (HEK) cells, treated these cultures with human Aβ42 or p3 17-42 peptides at 1 μM or DAPT (GSI) at 10 µM, and determined total HiBiT-Aβ levels in conditioned media (CM). DAPT was considered to result in full γ-secretase inhibition, and hence the values recorded in DAPT treated conditions were used for the background subtraction. We found a ~50% reduction in luminescence signal, directly linked to HiBiT-Aβ levels, in CM of cells treated with human Aβ42 and no effect of p3 peptide treatment, relative to the DMSO control (Figure 3D). The observed reduction in the total Aβ products is consistent with the partial inhibition of γ -secretase by Aβ42.”

In Methods:

“Analysis of γ-secretase substrate proteolysis in cultured cells using secreted HiBiT-Aβ or -Aβ-like peptide levels as a proxy for the global γ-secretase endopeptidase activity

HEK293 stably expressing APP-CTF (C99) or a NOTCH1-based substrate (similar in size as

APP- C99) both N-terminally tagged with the HiBiT tag were plated at the density of 10000 cells per 96-well, and 24h after plating treated with Aβ or p3 peptides diluted in OPTIMEM (Thermo Fisher Scientific) supplemented with 5% FBS (Gibco). Conditioned media was collected and subjected to analysis using Nano-Glo® HiBiT Extracellular Detection System (Promega). Briefly, 50 µl of the medium was mixed with 50 µl of the reaction mixture containing LgBiT Protein (1:100) and Nano-Glo HiBiT Extracellular Substrate (1:50) in Nano-Glo HiBiT Extracellular Buffer, and the reaction was incubated for 10 minutes at room temperature. Luminescence signal corresponding to the amount of the extracellular HiBiT-Aβ or -Aβ-like peptides was measured using victor plate reader with default luminescence measurement settings.”

As the direct substrate of γ -secretase was used in this analysis, the observed reduction (~50%) in the levels of N-terminally-tagged (HiBiT) Aβ peptides in the presence of 1 µM Aβ42, relative to control conditions, demonstrates a selective inhibition of γ-secretase by Aβ42 (not by the p3). These data complement the FRET-based findings presented in Figure 5.

(3) Processing of APP-CTF in living cells is not only the cleavage by gamma-secretase. This reviewer thinks that the authors need at least biochemical data, such as levels of Abeta in Figures 4, 5 and 7.

We tried to measure the levels of Aβ peptides secreted by cells into the culture medium directly by ELISA (using different protocols) or MS (using established methods, as reported in Koch et al, 2023), but exogenous Aβ42 (treatment) present at relatively high levels interfered with the readout and rendered the analysis inconclusive. 

However, we were successful in the determination of total secreted (HiBiT-tagged) Aβ peptides from the HiBiT tagged APP-C99 substrate, as indicated in the previous point. The quantification of the levels of these peptides showed that Aβ42 treatment resulted in ~50% reduction in the γ -secretase mediated processing of the tagged substrate.    

In addition, we would like to highlight that our analysis of the contribution of other APP-CTF degradation pathways, using cycloheximide-based assays in the constant presence of γ-secretase inhibitor, failed to reveal significant differences between Aβ42 treated cells and controls (Figure 6B & C). The lack of a significant impact of Aβ42 on the half-life of APP-CTFs under the conditions of γsecretase inhibition maintained by inhibitor treatment is consistent with the proposed Aβ42-mediated inhibitory mechanism.

(4) Similar to comment #3. Processing of Pancad-CTF and p75 in living cells may be not only the cleavage by gamma-secretase. This reviewer thinks that the authors need at least biochemical data, such as levels of ICDs in Figures 6C and E. 

To address this comment we have now performed additional experiments where we measured Nterminal Aβ-like peptides derived from NOTCH1-based substrate using the HiBiT-based assay. These experiments showed a reduction in the aforementioned peptides in the cells treated with Aβ42 relative to the vehicle control, and hence further confirmed the inhibitory action of Aβ42. These new data have been included as Figure 8D in the revised manuscript and described as follow:

Finally, we measured the direct N-terminal products generated by γ-secretase proteolysis from a HiBiT-tagged NOTCH1-based substrate, an estimate of the global γ-secretase activity. We quantified the Aβ-like peptides secreted by HEK 293 cells stably expressing this HiBiT-tagged substrate upon treatment with 1 µM Aβ1-42,  p3 17-42 peptide or  DAPT (GSI) (Figure 8D). DAPT treatment was considered to result in a complete γ-secretase inhibition, and hence the values recorded in the DAPT condition were used for background subtraction. A ~20% significant reduction in the amount of secreted

N-terminal HiBiT-tagged peptides derived from the NOTCH1-based substrates in cells treated with Aβ1-

42 supports the inhibitory action of Aβ1-42 on γ-secretase mediated proteolysis.

Minor concerns:

(1) Murine Abeta42 may be converted to murine Abeta38 easily, compared to human Abeta42. This may be a reason why murine Abeta42 exhibits no inhibitory effect on gamma-secretase activity. 

In order to address this question, we performed additional experiments where we assessed the processing of murine Aβ42 into Aβ38. Analogous to human Aβ42, the murine Aβ42 peptide was not processed to Aβ38 in the assay conditions. These new data have been integrated in the manuscript and added as a Supplementary figure 1B.

(2) It is curious to know the levels of C99 and C83 in cells in supplementary figure 3.  

The conditions used in these assays were analogous to the conditions used in the figure 3 (i.e. treatment with Aβ peptides at 1 µM concentrations). Such conditions were associated with profound and consistent APP-CTF accumulation in this model system.

Reviewer #2 (Recommendations For The Authors):

In the current study, the authors show that Aβs with low affinity for γ-secretase, but when present at relatively high concentrations, can compete with the longer, higher affinity APPC99 substrate for binding and processing. They also performed kinetic analyses and demonstrate that human Aβ1-42 inhibits γ-secretase-mediated processing of APP C99 and other substrates. Interestingly, neither murine Aβ1-42 nor human p3 (17-42 amino acids in Aβ) peptides exerted inhibition under similar conditions. The authors also show that human Aβ1-42-mediated inhibition of γ-secretase activity results in the accumulation of unprocessed, which leads to p75-dependent activation of caspase 3 in basal forebrain cholinergic neurons (BFCNs) and PC12 cells. 

These analyses demonstrate that, as seen for γ-secretase inhibitors, Aβ1-42 potentiates this marker of apoptosis. However, these are no any in vivo data to support the physiological significance of the current finding. The author should show in APP KO mice whether gamma-secretase enzymatic activity is elevated or not, and putting back Aβ42 peptide will abolish these in vivo effects. 

The findings presented in this manuscript form the basis for further in vitro and in vivo research to investigate the mechanisms of inhibition and its contribution to brain pathophysiology. Here, we used well-controlled model systems to investigate a novel mechanism of Aβ42 toxicity. Multiple mechanisms regulate the local concentration of Aβ42 in vivo, making the dissection of the biochemical mechanisms of the inhibition more complex. Nevertheless, beyond the scope of this report, we consider these very reasonable comments as a motivation for further research activities. 

The experimental concentrations for Aβ42 peptide in the assay are too high, which are far beyond the physiological concentrations or pathological levels. The artificial observations are not supported by any in vivo experimental evidence.

It is correct that in the majority of the experiments we used low μM concentrations of Aβ42. However, we would like to note that we have also performed experiments where conditioned medium collected from human APP.Swe expressing neurons was used as a source of Aβ. In these experiments total Aβ concentration was in low nM range (0.5-1 nM) (Figure 7). Treatment with this conditioned medium  led to the increase APP-CTF levels, supporting  that low nM concentrations of Aβ are sufficient for partial inhibition of  γ-secretase. 

In addition, we highlight that analyses of the brains of the AD affected individuals have shown that APPCTFs accumulate in both sporadic and genetic forms of the disease (Pera et al. 2013, Vaillant-Beuchot et al. 2021); and recently, Ferrer-Raventós et al. 2023 have revealed a correlation between APP-CTFs and Aβ levels at the synapse (Ferrer-Raventós et al. 2023). We therefore assessed the concentration of Aβ42 in synaptosomes derived from frontal cortices of post-mortem AD and age-matched non-demented (ND) control individuals. Our findings and conclusions are included in the revised version as follows: 

In the results section:

“We next investigated the levels of Aβ42 in synaptosomes derived from frontal cortices of post-mortem AD and age-matched non-demented (ND) control individuals (Figure 10B). Towards this, we prepared synaptosomes from frozen brain tissues using Percoll gradient procedure (62, 63). Intact synaptosomes were spun to obtain a pellet which was resuspended in minimum amount of PBS, allowing us to estimate the volume containing the resuspended synaptosome sample. This is likely an overestimate of the actual synaptosome volume. Finally, synaptosomes were lysed in RIPA buffer and Aβ peptide concentrations measured using ELISA (MSD). We observed that the concentration of Aβ42 in the synaptosomes from (end-stage) AD tissues was significantly higher (10.7 nM)  than those isolated from non-demented tissues (0.7 nM), p<0.0005***. These data provide evidence for accumulation at nM concentrations of endogenous Aβ42 in synaptosomes in end-stage AD brains. Given that we measured Aβ42 concentration in synaptosomes, we speculate that even higher concentrations of this peptide may be present in the endolysosome vesicle system, and therein inhibit the endogenous processing of APP-CTF at the synapse. Of note treatment of PC12 cells with conditioned medium containing even lower amounts of Aβ (low nanomolar range (0.5-1 nM)) resulted in the accumulation of APP-CTFs.” 

In the discussion: 

“The convergence of Aβ42 and tau at the synapse has been proposed to underlie synaptic dysfunction in AD (86-89), and recent assessment of APP-CTF levels in synaptosome-enriched fractions from healthy control, SAD and FAD brains (temporal cortices) has shown that APP fragments concentrate at higher levels in the synapse in AD-affected than in control individuals (90).  Our analysis adds that endogenous Aβ42 concentrates in synaptosomes derived from end-stage AD brains to reach ~10 nM, a concentration that in CM from human neurons inhibits γ-secretase in PC12 cells (Figure 7). Furthermore, the restricted localization of Aβ in endolysosomal vesicles, within synaptosomes, likely increases the local peptide concentration to the levels that inhibit γ-secretase-mediated processing of substrates in this compartment. In addition, we argue that the deposition of Aβ42 in plaques may be preceded by a critical increase in the levels of Aβ present in endosomes and the cyclical inhibition of γ-secretase activity that we propose. Under this view, reductions in γ-secretase activity may be a (transient) downstream consequence of increases in Aβ due to failed clearance, as represented by plaque deposition, contributing to AD pathogenesis. ”

Tubbo knows exactly what to do to bring out Ranboo's genuine self

Author response:

The following is the authors’ response to the original reviews.

eLife assessment

The authors present evidence suggesting that MDA5 can substitute as a sensor for triphosphate RNA in a species that naturally lacks RIG-I. The key findings are potentially important for our understanding of the evolution of innate immune responses, but the evidence is incomplete, as additional biochemical and functional experiments are needed to unambiguously assign MDA5 as a bona fide sensor of triphosphate RNA in this model. This also leaves the title as overstating its case.

We would like to thank the editorial team for these positive comments on our manuscript and the constructive suggestions to improve our manuscript. According to the suggestions and valuable comments of the referees, we have added substantial amounts of new data and analysis to substantiate our claims, and the manuscript, including the title, has been carefully revised to better reflect our conclusions. We are now happy to send you our revised manuscript, we hope the modified manuscript addresses your and the reviewers’ concerns satisfactorily and is suitable for publication in eLife now.

Reviewer #1 (Public Review):

This study offers valuable insights into host-virus interactions, emphasizing the adaptability of the immune system. Readers should recognize the significance of MDA5 in potentially replacing RIG-I and the adversarial strategy employed by 5'ppp-RNA SCRV in degrading MDA5 mediated by m6A modification in different species, further indicating that m6A is a conservational process in the antiviral immune response.

However, caution is warranted in extrapolating these findings universally, given the dynamic nature of host-virus dynamics. The study provides a snapshot into the complexity of these interactions, but further research is needed to validate and extend these insights, considering potential variations across viral species and environmental contexts.

We concur with the viewpoint that virus-host coevolution complicates the derivation of universal conclusions. To address this challenge, incorporated additional experiments and data based on the suggestions of the reviewers. These experiments were carried out across diverse models, including two distinct vertebrate species (M. miiuy and G. gallus), two different viruses (SCRV and VSV), and the synthesis of corresponding 5’ppp-RNA probes. We believe that these supplementary data bolster the evidence supporting the immune replacement role of MDA5 in the recognition of 5'ppp-RNA in RIG-I deficient species (Figure 1C-1E, Figure 2O and 2P, Figure 4). Moreover, we have duly incorporated references in both the introduction and discussion sections to further support our conclusion that MDA5 in T. belangeri, a mammal lacking RIG-I, possesses the ability to detect RNA viruses posed as RIG-I agonists (doi: 10.1073/pnas.1604939113). Lastly, meticulous revisions have been undertaken in the manuscript, including adjustments to the title, to ensure harmonization with our research outcomes.

Reviewer#2 (Public Review):

This manuscript by Geng et al. aims to demonstrate that MDA5 compensates for the loss of RIG-I in certain species, such as teleost fish miiuy croaker. The authors use siniperca cheats rhabdovirus (SCRV) and poly(I:C) to demonstrate that these RNA ligands induce an IFN response in an MDA5-dependent manner in M. miiuy derived cells. Furthermore, they show that MDA5 requires its RD domain to directly bind to SCRV RNA and to induce an IFN response. They use in vitro synthesized RNA with a 5'triphosphate (or lacking a 5'triphosphate as a control) to demonstrate that MDA5 can directly bind to 5'-triphosphorylated RNA. The second part of the paper is devoted to m6A modification of MDA5 transcripts by SCRV as an immune evasion strategy. The authors demonstrate that the modification of MDA5 with m6A is increased upon infection and that this causes increased decay of MDA5 and consequently a decreased IFN response.

The key message of this paper, i.e. MDA5 can sense 5'-triphosphorylated RNA and thereby compensate for the loss of RIG-I, is novel and interesting, yet there is insufficient evidence provided to prove this hypothesis. Most importantly, it is crucial to test the capacity of in vitro synthesized 5'-triphosphorylated RNA to induce an IFN response in MDA5-sufficient and -deficient cells. In addition, a number of important controls are missing, as detailed below.

To further support the notion that MDA5 is capable of detecting 5'ppp-RNA in species lacking RIG-I, we conducted additional experiments. Initially, we isolated the RNA from SCRV and VSV viruses. Subsequently, we synthesized 5'ppp-RNA probes that corresponded to the genome termini of SCRV and VSV in vitro. Then, these RNAs were treated with Calf intestinal phosphatase (CIAP) to generate dephosphorylated derivatives. Next, we separately tested the activation ability of various RNAs on IRF3 dimer and IFN response in MKC (M. miiuy kidney cell line) and DF-1 (G. gallus fibroblast cell line) cells, and determined that the immune activation ability of SCRV/VSV viruses depends on their triphosphate structure (Figure 1C-1E, Figure 4C and 4J). In addition, the knockdown of MDA5 inhibited the immune response mediated by SCRV RNA (Figure 2P and 2Q). Finally, we incorporated essential experimental controls (Figure 4B and 4I). We think that the inclusion of these supplementary experimental data significantly enhances the credibility and further substantiates our hypothesis.

The authors describe an interaction between MDA5 and STING which, if true, is very interesting. However, the functional implications of this interaction are not further investigated in the manuscript. Is STING required to relay signaling downstream of MDA5?

To better explore the role of STING in MDA5 signal transduction, we constructed a STING expression plasmid and synthesized specific siRNA targeting STING. Next, we found that co-expression of STING and MDA5 significantly enhance MDA5-mediated IFN-1 response during SCRV virus infection (Figure 2N). Conversely, silencing of STING expression restored the MDA5-mediated IFN-1 response (Figure 2O). These findings provide important evidence for the critical involvement of STING in the immune signaling cascade mediated by MDA5 in response to 5'ppp-RNA viruses.

The second part of the paper is quite distinct from the first part. The fact that MDA5 is an interferon-stimulated gene is not mentioned and complicates the analyses (i.e. is there truly more m6A modification of MDA5 on a per molecule basis, or is there simply more total MDA5 and therefore more total m6A modification of MDA5).

For the experimental data analysis in Figure 5E and 5F, we first compared the m6A-IP group to the input group, and then normalized the control group (IgG group of 5E and Mock group of 5F) to a value of “1”. Given the observed variability in MDA5 expression levels within the input group of Mock and SCRV virus-infected cells, our analysis represents the actual m6A content of each MDA5 molecule. To enhance clarity, we have updated the label on the Y-axis in Figure 5E and 5F.

Finally, it should be pointed out that several figures require additional labels, markings, or information in the figure itself or in the accompanying legend to increase the overall clarity of the manuscript. There are frequently details missing from figures that make them difficult to interpret and not self-explanatory. These details are sometimes not even found in the legend, only in the materials and methods section. The manuscript also requires extensive language editing by the editorial team or the authors.

We acknowledge the valuable feedback from the reviewer and have made significant improvements to our manuscript based on the recommendations provided in the "Recommendation for the authors" section. Furthermore, we have conducted a thorough review of the entire article, resulting in substantial enhancements to the format, clarity, and overall readability of our manuscript.

Reviewer#3 (Public Review):

Summary: In this manuscript, the authors investigated the interaction between the pattern recognition receptor MDA5 and 5'ppp-RNA in a teleost fish called Miiuy croaker. They claimed that MDA5 can replace RIG-I in sensing 5'ppp-RNA of Siniperca cheats rhabdovirus (SCRV) in the absence of RIG-I in Miiuy croaker. The recognition of MDA5 to 5'ppp-RNA was also observed in the chicken (Gallus gallus), a bird species that lacks RIG-I. Additionally, they reported that the function of MDA5 can be impaired through m6A-mediated methylation and degradation of MDA5 mRNA by the METTL3/14-YTHDF2/3 regulatory network in Miiuy croaker under SCRV infection. This impairment weakens the innate antiviral immunity of fish and promotes the immune evasion of SCRV.

Strengths:<br /> These findings provide insights into the adaptation and functional diversity of innate antiviral activity in vertebrates.

Weaknesses:<br /> However, there are some major and minor concerns that need to be further addressed. Addressing these concerns will help the authors improve the quality of their manuscript.One significant issue with the manuscript is that the authors claim to be investigating the role of MDA5 as a substitute for RIG-I in recognizing 5'ppp-RNA, but their study extends beyond this specific scenario. Based on my understanding, it appears that sections 2.2, 2.3, 2.5, 2.6, and 2.7 do not strictly adhere to this particular scenario. Instead, these sections tend to investigate the functional involvement of Miiuy croaker MDA5 in the innate immune response to viral infection. Furthermore, the majority of the data is focused on Miiuy croaker MDA5, with only a limited and insufficient study on chicken MDA5. Consequently, the authors cannot make broad claims that their research represents events in all RIG-I deficient species, considering the limited scope of the species studied.

We agree with the reviewer's perspective that functional analysis of MDA5 in M. miiuy may not adequately represent all species lacking RIG-I. To address this concern, we have incorporated additional experimental data utilizing different model systems, including two different vertebrate species (M. miiuy and G. gallus), two distinct viruses (SCRV and VSV), and the synthesis of two corresponding 5’ppp-RNA probes. While the functional characterization of G. gallus MDA5 remains relatively limited compared to M. miiuy, our current experimental findings provide support for two key observations. Firstly, the triphosphate structure of the VSV virus is pivotal in activating the innate immune response in G. gallus against the virus (Figure 1D and 4J). Secondly, G. gallus MDA5 can recognize 5’ppp-RNA (Figure 4I, 4K and 4L). Consequently, although we cannot definitively establish the immune surrogate function of MDA5 in all RIG-I-deficient species, our research data further substantiates this hypothesis. Moreover, we have adopted a more cautious attitude in summarizing our experimental conclusions, thereby enhancing the rigor of our manuscript language.

The current title of the article does not align well with its actual content. It is recommended that the focus of the research be redirected to the recognition function and molecular mechanism of MDA5 in the absence of RIG-I concerning 5'ppp-RNA. This can be achieved through bolstering experimental analysis in the fields of biochemistry and molecular biology, as well as enhancing theoretical research on the molecular evolution of MDA5. It is advisable to decrease or eliminate content related to m6A modification.

Following the reviewer's recommendations, we have revised the title to emphasize that our main research focus is a teleost fish devoid of RIG-I. Furthermore, we have conducted additional molecular experiments to further elucidate the 5'ppp-RNA recognition function of MDA5 in RIG-I-deficient species. In an attempt to analyze the potential molecular evolution of MDA5 resulting from RIG-I deficiency, we collected MDA5 coding sequences from diverse vertebrates. However, due to multiple independent loss events of RIG-I in fish, fish with or without RIG-I genes in the phylogenetic tree cannot be effectively clustered separately, making it extremely difficult to perform this aspect of analysis. Consequently, we have regrettably opted to forgo the molecular evolution analysis of MDA5.

Our article topic is to reveal an antagonistic phenomenon between fish receptor and RNA viruses. The MDA5 of RIG-I-lost fish has evolved the ability to recognize 5’ppp-RNA virus and mediate IFN response to resist SCRV infection. Conversely, the m6A methylation mechanism endows the SCRV virus with a means to weaken the immune capacity of MDA5. Therefore, we believe that the latter part is an important part of the arms race between the virus and its host, and should be retained.

Additionally, the main body of the writing contains several aspects that lack rigor and tend to exaggerate, necessitating significant improvement.

We appreciate the reviewer’s comment and have improved the manuscript addressing the points raised in the “Recommendation for the authors”. We have added corresponding experiments to strengthen the verification of the conclusions, and in addition, we are more cautious in summarizing the language of the conclusions.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The evidential foundation within the Result 1 section appears somewhat tenuous.

Firstly, the author derives conclusions regarding the phenomenon of RIG-I loss in lower vertebrates by referencing external literature and conducting bioinformatics analyses. It is pertinent to inquire whether the author considered fortifying these findings through additional WB/PCR experiments, particularly for evaluating RIG-I expression levels across diverse vertebrates, encompassing both lower and higher orders.

Firstly, the species we analyzed are mostly model species with excellent genomic sequence information in the database. Secondly, the RIG-I protein sequences (at least some domain sequences) are relatively conserved in vertebrates. Therefore, the credibility of evaluating the existence of RIG-I in these species through homology comparison is high. Therefore, we do not intend to conduct additional PCR/WB experiments to confirm this.

Additionally, following the identification of RIG-I loss, the author postulates MDA5 as a substitute of RIG-I, grounding this speculation in the analysis of MDA5 and LGP2 protein structures. It is imperative to address whether the author could enhance the manuscript by supplying expression data for MDA5 and LGP2 across different vertebrates and elucidating further why MDA5 is posited as the compensatory mechanism for RIGI loss.

Like MDA5, LGP2 is also an interferon-stimulating gene, so they both likely exhibit high sensitivity to viral infections. Therefore, we think that comparing the expression data of these two genes is difficult to evaluate their function. In mammals, the regulatory mechanisms of LGP2 to RIG-I and MDA5 were complicated and ambiguous. To evaluate the potential function of LGP2 in M. miiuy, we further constructed LGP2 plasmid and synthesized siRNA targeting LGP2. Then, our results indicate that mmiLGP2 can enhance the antiviral immune response mediated by mmiMDA5 (Figure 1H and 1I), further indicating the regulatory role of mmiLGP2 in RLR signaling, rather than acting as a compensatory receptor for RIG-I.

Also, is it conceivable that other receptors contribute to this compensatory effect in lower vertebrates?

5’ triphosphate short blunt-end double-strand RNA is the ligand of RIG-I as contained in the panhandle of negative-strand viral genomes. We mainly focus on the immune recognition and compensatory effects of other receptors on RIG-I loss, and MDA5, as the protein with the most similar structure, first attracted our attention. In addition, IFIT proteins have been reported to recognize triphosphate single-stranded RNA (doi: 10.1038/nature11783). However, we used SCRV and VSV RNA as viral models, both of which have negative stranded genomes and meet the ligand standards of RIG-I, rather than IFIT. Therefore, we excluded the IFIT protein from our research scope.

(2) The article exclusively employs a singular type of 5'PPP-RNA virus and one specific lower vertebrate species, thereby potentially compromising the robustness of the assertion that this phenomenon is prevalent in lower vertebrates. To bolster this claim, could the author consider incorporating data from an alternative 5'PPP-RNA virus and a different lower vertebrate species?

To address this concern, we have incorporated additional experimental data utilizing different model systems, including two different vertebrate species (M. miiuy and G. gallus) and two distinct viruses (SCRV and VSV). While the functional characterization of G. gallus MDA5 remains relatively limited compared to M. miiuy, our current experimental findings provide support for two key observations. Firstly, the triphosphate structure of the VSV virus is pivotal in activating the innate immune response in G. gallus against the virus (Figure 1D and 4J). Secondly, G. gallus MDA5 can recognize 5’ppp-RNA (Figure 4I, 4K and 4L). Consequently, these experimental results further confirmed the conservatism of this immune compensation mechanism.

(3) A nuanced consideration of the statement in Result 5 is warranted. Examination of the results under SCRV infection conditions suggests dynamic fluctuations in MDA5 expression levels, challenging the veracity of the statement implying "increased expression", which contradicts the proposed working model of this article.

Because MDA5 acts as a receptor and plays a recognition immune role in the early stages of virus infection, the expression of MDA5 in the early stage of SCRV infection rapidly increases. In the later stage of infection, the expression of MDA5 may gradually decrease again due to the negative feedback mechanism in the host body to prevent excessive inflammation. However, compared to the uninfected group, the expression of MDA5 was significantly increased in the SCRV-infected group, so we believe that the term "increased expression" is not a problem. In addition, the m6A mechanism can weaken the function of MDA5, but it still cannot prevent the overall increase of MDA5 expression, which is not contradictory to the working model in this article.

Additionally, the alterations in m6A levels in miiuy croaker under SCRV infection conditions warrant clarification. Could the author employ m6A dot blotting to supplement the findings related to total m6A levels?

Our previous studies (doi: 10.4049/jimmunol.2200618) have suggested that the total m6A level is increased after SCRV infection in miiuy croaker. We cited this conclusion in the discussion of our manuscript.

(4) It would be beneficial if the editors could assist the author in enhancing the language of the manuscript.

We have carefully checked the full article and modified it with Grammarly tools, and we believe that the grammar, format, and readability of our articles have been greatly improved.

Reviewer #2 (Recommendations For The Authors):

Figure 1

(1) Figure 1B - some clarification needs to be added about this figure in the text. It is unclear what the main point is that the authors would like to convey.

What we want to emphasize is that some species with RIG-I, such as zebrafish, have also experienced RIG-I loss events, but have undergone whole genome replication events before the loss, thus preserving a copy of RIG-I. This indicates that loss events of RIG-I are very common in vertebrates and do not occur randomly. We have elaborated on this point in the results and discussion.

(2) Figure 1C - is not very informative other than showing Mm MDA5 and LGP2 side-by-side. It would be more useful to show a comparison of human RIG-I/MDA5 alongside Mm and Gg MDA5. Are there any conserved/shared key residues between hRIG-I/hMDA5 versus mmMDA5?

Homologous proteins are often known to adopt the same or similar structure and function. We have added human RIG-I domain information to this figure (Figure 1F). By comparing the domain information of human RIG-I with M. miiuy MDA5 and LGP2, M. miiuy MDA5 has a similar structure to human RIG-I, making it most likely to compensate for the missing RIG-I. While M. miiuy LGP2 lacks the CARD domain, which is crucial for signal transduction, so we will shift our focus to M. miiuy MDA5. In addition, we collected protein sequences of MDA5 and RIG-I from various vertebrates to identify key residues evolved in recognizing 5'ppp-RNA by M. miiuy MDA5. However, unfortunately, no potential residues were found during the comparison process.

Figure 2

(1) Figure 2B - It would be important to demonstrate MDA5-Flag expression by immunoblot and compare MDA5-Flag overexpression to endogenous MDA5 expression using the anti-MDA5 antibody from panel 2A. If IF is used, more cells need to be visible in the field.

After transfecting the MDA5 plasmid into MKC, endogenous MDA5 expression was detected using MDA5 antibodies. The results showed a significant increase in MDA5 protein levels, indicating that MDA5 antibodies can specifically recognize MDA5 protein. In addition, we retained the original immunofluorescence images to better demonstrate the subcellular localization of MDA5.

(2) Figure 2C - The 1:1 stoichiometry of MDA5:MAVS (in the absence of any stimulus) is quite surprising. How does the interaction between MDA5 and MAVS change upon stimulation with an RNA ligand (SCRV, poly(I:C))?

We do not believe that the actual stoichiometry between MDA5 and MAVS is what you described as 1:1. In fact, the proportion of proteins in the complex depends on many factors in the experimental results with Co-IP. Firstly, the MDA5 plasmid in this study has a 3 × Flag tag, while the MAVS only has a 1x Myc tag, which makes the antibody more sensitive for detecting MDA5-Flag. In addition, the Co-IP results are also affected by multiple factors such as the type of antibody and the number of recoveries, making it difficult to estimate the actual ratio of MDA5 to MAVS. Based on the above reasons and the fact that the detection of the interaction strength between MDA5 and MAVS after infection seems to be off-topic, we did not continue to explore this point.

(3) Figure 2D - The interaction between MDA5 and STING is a very interesting finding but is not elaborated on in the paper (even though the interaction between MDA5 and STING is mentioned in the abstract). The manuscript would be strengthened if the interaction between MDA5 and STING is further investigated. For example, does the IFN response that is reported in panels 2E to 2H require the presence of STING? Does mmMDA5 signal via STING in response to a DNA ligand?

We appreciate the referee's suggestion to study the mutual influence between MDA5 and STING. We found that co-expression of STING and MDA5 can enhance MDA5-mediated IFN-1 response during SCRV virus infection, while knocking down STING can restore MDA5-mediated IFN-1 (Figure 2N and 2O). This indicates that STING plays an important signaling role in the immune response of MDA5 to RNA viruses. We understand the importance of cGAS/STING pathways in identifying exogenous DNA, so exploring the MDA5 pathway for DNA ligand recognition is an interesting and meaningful perspective. But this seems to be detached from the theme of our article, so we didn't continue to explore this point.

(4) Figures 2F and 2H - the authors demonstrate that SCRV induces a type I IFN response in an MDA5-dependent manner. While SCRV is a single-stranded negative-sense RNA virus that contains 5'ppp-RNA, it cannot be excluded that MDA5 is activated here in response to a double-stranded RNA intermediate of viral origin or even a host-derived RNA whose expression or modification is altered during infection. To demonstrate in an unambiguous manner that MDA5 senses 5'ppp-RNA, it is crucial to use the in vitro synthesized 5'ppp-RNA (and its dephosphorylated derivative as a control) from Fig. 4 in these experiments.

We transfected 5 'ppp SCRV and 5' ppp VSV (and their dephosphorylated derivatives) synthesized in vitro into MKC cells and DF-1 cells, respectively. The results showed that 5’ppp-RNAs significantly promoted the formation of IRF3 dimers, while their dephosphorylated derivatives did not (Figure 4C and 4J). In addition, we extracted virus RNA from the SCRV and VSV viruses and dephosphorylated them with Calf intestinal phosphatase (CIAP). These RNAs were transfected into MKC and DF-1 cells and found that the immune response mediated by virus RNAs was much higher than the dephosphorylated form (Figure 1C-1E). The above results indicate that the immune response activated by SCRV and VSV is indeed dependent on their triphosphate structure. Finally, the IRF3 dimer and IFN induction activated by SCRV RNA can be inhibited by si-MDA5 (Figure 2P and 2Q), further demonstrating the involvement of MDA5 in the immune response mediated by 5’ppp-RNA ligands.

(5) In mice and humans, MDA5 is known to collaborate with LGP2 to jointly induce an IFN response. Does M.miiuy express LGP2? If so, it would be informative to include a siRNA targeting LGP2 in the experiments in panel F. In mammals, LGP2 potentiates the response via MDA5 while it may inhibit RIG-I activation.

M.miiuy express LGP2. We constructed an LGP2 plasmid and synthesized si-LGP2 to investigate the impact of LGP2 on MDA5-mediated immune processes (Figure 1G-1I). The results showed that LGP2 can enhance the IFN response mediated by MDA5 during SCRV virus infection, similar to that in mammals.

(6) Minor comment - Is the poly(I:C) used in this figure high or low molecular weight poly(I:C)? HMW poly(I:C) preferentially stimulates MDA5, while LMW poly(I:C) preferentially stimulates RIG-I.

We used poly(I:C)-HMW as a positive control for activating MDA5. We have modified the relevant information in Figure 2 and its legend.

Figure 3

(1) Figure 3F/G - The normalization in this Figure is difficult to interpret. It would be better to split Figure 3G into 4 separate graphs and include the mock-infected cells alongside the infected samples (as done in Figure 2).

To better demonstrate the function of the RD domain of MDA5 in M. miiuy, we have changed the experimental plan, as shown in figure 3F. We detected the induction of antiviral factors by overexpression of MDA5 and MDA5-△RD under poly (I:C)-HMW stimulation. This can indicate that the RD domain of MDA5 has a conserved function in the recognition of poly(I:C)-HMW in M. miiuy, and can serve as a positive control for the recognition of SCRV virus by the RD domain.

Figure 4

(1) Figure 4B - A number of important controls are missing. Was the immunoprecipitation of RNA successful? This could be shown by running a fraction of the immunoprecipitated material on an RNA gel and/or by showing that the input RNA was depleted after IP. In addition, a control IP (Streptavidin beads without biotinylated RNA) is missing to ensure that MDA5 does not stick non-specifically to the Streptavidin resin.

We appreciate the referee's suggestions. We rerun this experiment and added a non-biomarker RNA IP control group, and the results showed that MDA5 did not adsorb non-specific onto the beads (Figure 4B). In addition, based on the referee's suggestion, we tested the consumption of RNA before and after immunoprecipitation, and the results showed that biotin-labeled RNA, rather than non-biotin-labeled RNA, could be adsorbed by beads, indicating the success of RNA precipitation. However, we think that this is not necessary for the final presentation of the experimental results, so we did not show this in the figure.

(2) Figure 4B - It is unclear why there is such a large molecular weight difference between endogenous MDA5 and MDA5-Flag (110 kDa versus 130/140 kDa). Why is there less MDA5-Flag retrieved than endogenous MDA5?

After careful analysis, we believe that the significant difference in molecular weight between endogenous MDA5 and MDA5 Flag may be due to three reasons. Firstly, MDA5 flag has a 3× Flag tag. Secondly, as shown in the primer table, we constructed MDA5 between the NotI and XbaI cleavage sites in the pcDNA3.1 vector, which are located at the posterior position in the vector. This means that the Flag tag has a certain distance from the starting codon of MDA5, and these sequences on the vector can also be translated and increase the molecular weight of the exogenous MDA5 protein. Finally, in order to facilitate the amplification of the primers, the F-terminal primers of MDA5 contain a small portion of the 3'UTR sequence (excluding the stop codon). These above reasons may have led to significant differences in molecular weight. In addition, in order to supplement important experimental controls, we have conducted a new RNA pull-down experiment as shown in Figure 4B.

(3) Minor point: Figure 4B - please clarify in the figure whether RNA or protein is immunoprecipitated and via which tags.

We have conducted a new RNA pull-down experiment as shown in Fig 4B, and we have clearly labeled the relevant information in the figure.

(4) Figure 4E - the fraction of MDA5 that binds 5'ppp-RNA seems incredibly minor. And why is this experiment done using 5'OH-RNA as a competitor, rather than simply incubating MDA5 and 5'OH-RNA together and demonstrating that these do not form a complex?

The proportion of MDA5 combined with 5’ppp-RNA is influenced by many conditions, including the concentration and purity of the probe and purified protein. In addition, the dosage ratio between the RNA probe and MDA5 protein in the EMSA experiment can also have a significant impact on the results. Therefore, it is not possible to accurately determine the actual binding force between MDA5 and RNA. In the EMSA experimental program, both cold probes (5’ppp-RNA) and mutated cold probes (5’OH-RNA and 5’pppGG-RNA) are crucial for demonstrating the specific binding between MDA5 and 5’ppp-RNA, as they can exclude false positive errors caused by factors such as the presence of biotin in the purified MDA5 protein itself.

(5) Figure 4B/4C/4F - These experiments would be strengthened by including an MDA5 mutant that cannot bind to RNA. These mutants are well-described in mammals. If these residues are conserved, it is straightforward to generate this mutant.

As shown in Figure 3, the MDA5 of M. miiuy has an RD domain that can recognize the SCRV virus. We constructed MDA5-△RD mutant plasmids with 6x His-tags and purified them for EMSA experiments (Figure 4E). The experimental results further indicate that MDA5, rather than MDA5-△RD, can bind to 5’ppp-SCRV (Figure 4G). This further confirms the crucial role of the RD domain in recognizing the 5'ppp-RNA virus.

(6) Minor point: Figure 4E: please clarify in which lanes MDA5 has been added.

Thank you for the referee's suggestion. We have synthesized new 5'ppp-RNA probes (5’ppp-SCRV and their dephosphate derivatives) and rerun this experiment, and relevant information has been added in the Figure (Figure 4F).

Figure 5

(1) Figure 5C - As MDA5 is an interferon-stimulated gene (as shown in panel G/H/I)) the increased MDA5 expression could simply explain the increase in the amount of m6A-MDA5 that is immunoprecipitated after infection. Could this figure be improved by doing a fold change between input vs m6A-IP OR uninfected vs SCRV-infected conditions? This would reveal whether the modification of MDA5 with m6A is really increased after infection.

As shown in Figure 5F below, our data indicates that the proportion of m6A-modified MDA5 does indeed increase after SCRV infection, rather than solely due to the increased expression of MDA5 itself.

(2) Figure. 5E/F - The y-axis is unclear: relative MDA5 m6A levels. Relative to what? Input? Mock infected?

For experiments in Figure 5E/F, we first compared the m6A-IP group with the input group, and then normalized the control group (IgG group of 5E and Mock group of 5F) to “1”. We have replaced the Y-axis name with a clearer one (Figure 5E and 5F).

(3) General comment - It is not mentioned in the text that MDA5 is an interferon-stimulated gene. This would account for the increase in expression (qPCR) after viral infection or poly(I:C) transfection, hence there is no novelty in this finding. In addition, the authors suggest that MDA5 increases at the protein level (by immunoblot) but the increase on these blots is not convincing (figure 5H/5I).

We understand that the increase in expression of MDA5 as an interferon-stimulated gene after viral infection is a common phenomenon. We present this to further validate the m6A sequencing transcriptome data, and to demonstrate that although m6A modification interferes with MDA5 expression during viral infection, it cannot prevent the increase of mRNA level of MDA5. In addition, we rerun the experiment and the results showed that the expression of MDA5 protein can indeed be specifically activated by the SCRV virus and poly(I:C)-HMW.

Figure 6

(1) Figure 6E - What was the MOI of the virus used in this experiment? It is not mentioned in the figure legend.

MOI=5, we have added this point in the figure legend.

Figure 7

(1) Figure 7J - This graphic is somewhat misleading and should be altered to better reflect the conclusions that are drawn in the manuscript. The graphic suggests that MAVS and STING interact, but this is not demonstrated in the paper. In addition, the paper does not demonstrate whether MAVS or STING (or both) are needed downstream of MDA5 to relay signalling. Finally, please draw an arrow from type I IFNs to increased expression of MDA5 to illustrate that MDA5 is an ISG.

Thank you for the referee's suggestion. We have revised the images to more accurately match the conclusions of the manuscript (Figure 7J). Firstly, we have separated the STING protein from the MAVS protein. Secondly, arrows have been used to indicate that MDA5 is an IFN-stimulated gene. Finally, as we have added relevant experiments to demonstrate the importance of MITA protein in the signaling process of MDA5-activated IFN response. In addition, the function of MAVS binding to MDA5 protein and promoting its signal transduction is very conserved, and there is a good research background even in fish with RIG-I deficiency (10.1016/j.dci.2021.104235). Therefore, in Figure 7J, we still chose to bind MAVS to MDA5 protein and use it as a downstream signal transducer of MDA5.

Discussion<br /> (1) There is very little discussion about METTL and YTHDF proteins in the discussion despite the fact that the last 2 figures are entirely devoted to these proteins.

Based on the referee's suggestion, we have added relevant content about METTL and YTHDF proteins in the discussion. In addition, the basic mechanism and function of METTL and YTHDF proteins were briefly described in the introduction.

Reviewer #3 (Recommendations For The Authors):

Please refer to the specific suggestions and recommendations. They include proposals for experimental additions, improved methodologies, and suggestions to resolve writing-related concerns.

Major concerns

(1) I suggest changing the article title to "Functional Replacement of RIG-I with MDA5 in Fish Miiuy Croaker", or a similar title, to make it more focused and closely aligned with the content of the article.

Following the reviewer's recommendations, we have revised the title to emphasize our primary research subject is a teleost fish that lacks RIG-I. In addition, we have changed “5’ppp-RNA” to “5’ppp-RNA virus” to emphasize the interaction between the virus and the receptor. We believe that the revised title is more in line with the content of the article.

(2) Due to the inherent limitations in genome sequencing, assembly, and annotation for the Miiuy croaker, comprehensive annotation of immune-related genes remains incomplete. To address this critical gap, it is recommended that authors establish experimental protocols, such as Fluorescence In Situ Hybridization (FISH), to confirm the absence of RIG-I in the Miiuy croaker. They should simultaneously employ MDA5 probes as a positive control for validation purposes.

The miiuy croaker has good genomic information at the chromosomal level (doi: 10.1016/j.aaf.2021.06.001). In addition, studies have shown that RIG-I is absent in the orders of Perciformes (doi: 10.1016/j.fsirep.2021.100012), while miiuy croaker belongs to the order Perciformes, so it does indeed lose the RIG-I gene. Therefore, we do not intend to use FISH technology to prove this.

(3) Similarly, it is recommended that the authors first provide evidence of the presence of 5'ppp at the 5' terminus of the genome RNA of SCRV, as demonstrated in the study by Goubau et al. (doi: 10.1038/nature13590, Supplementary figure 1). This evidence is crucial before drawing conclusions about the compensatory role of MDA5 in recognizing 5'ppp RNA viruses, using SCRV as the viral model.

As suggested by the referee, we extracted SCRV RNA from SCRV virus particles and assessed the 5’-phosphate-dependence of stimulation by SCRV RNA. Calf intestinal phosphatase (CIAP) treatment substantially reduced the stimulatory activity of SCRV RNA in MKC cells of M. miiuy (Figure 1C and 1E). In addition, similar results were obtained by transfecting VSV-RNA isolated from VSV virus into DF-1 cells of G. gallus (Figure 1D). The above evidences confirm the presence of triphosphate molecular features between SCRV and VSV viruses, and indicating that birds and fish lacking RIG-I have other receptors that can recognize 5’ppp-RNA.

(4) The 62-nucleotide (nt) 5'ppp-RNA utilized in this study was obtained from Vesicular Stomatitis Virus (VSV). In order to provide direct evidence, it is necessary to include a 62-nt 5'ppp-RNA that is directly derived from SCRV itself.

We adopted this suggestion and synthesized a 67-nucleotide 5’ppp-SCRV RNA probe. We found that 5’ppp-SCRV activates dimerization of IRF3 and binds to MDA5 of M. miiuy in a 5’-triphosphate-dependent manner (Figure 4A-4F).

(5) Given that RNAs with uncapped diphosphate (PP) groups at the 5′ end also activate RIG-I, similar to RNAs with 5′-PPP moieties, and the 5′-terminal nucleotide must remain unmethylated at its 2′-O position to allow RNA recognition by RIG-I, it is necessary for the authors to conduct additional experiments to supplement and validate these two distinguishing features of RIG-I in RNA recognition. This will provide more reliable evidence for the replacement of RIG-I by MDA5 in RNA recognition.

Thank you for the reviewer's professional suggestions. We understand that exploring the combination of 5’pp-RNA and 2′-O-methylated RNA with MDA5 can further demonstrate the alternative function of MDA5. But we think that the use of 5’ppp-RNA and their dephosphorylation derivatives can fully demonstrate that the MDA5 of M. miiuy and G. gallus have evolved to recognize 5’triphosphate structure like human RIG-I. Therefore, we do not intend to conduct any additional experiments

(6) In section 2.3, the authors assert that Miiuy croaker recognizes SCRV through its RD domain. This claim is supported by their data showing that cells overexpressed with the MDA5 ΔRD mutant lost the ability to inhibit SCRV replication. As a result, the authors draw the conclusion that "these findings provide evidence that MDA5 may recognize 5'-triphosphate-dependent RNA (5'ppp-RNA) through its RD domain." However, to strengthen their argument, the authors should first demonstrate that during SCRV infection, MDA5-mediated antiviral immune response is indeed initiated by recognizing the 5'ppp part of the SCRV RNA, rather than the double-strand part (which can exist in ssRNA virus) of the viral RNA, as this is naturally a ligand for MDA5. Additionally, the authors should treat the isolated SCRV RNA with CIP to remove the phosphate group and examine the binding of MDA5 with SCRV RNA before and after treatment. They should also transfect CIP-treated or untreated SCRV RNA into MDA5 knockdown and wild-type MKC cells to investigate the induction of antiviral signaling and levels of viral replication. Finally, the authors should verify the binding ability of the mutants with isolated SCRV RNA, with or without CIP treatment, to determine which domain of MDA5 is responsible for SCRV 5'ppp-RNA recognition.

We understand the reviewer's concern that MDA5 may be identified by binding to dsRNA in the SCRV virus. Based on the reviewer's suggestion, we extracted SCRV RNA and obtained its dephosphorylated RNA using Calf intestinal phosphatase (CIAP). Next, we transfected them into MDA5-knockdown and wild-type MKC cells, and detected the dimerization of IRF3 and IFN reaction. The results indicate that SCRV RNA does indeed activate immunity in a triphosphate-dependent manner, and knockdown of MDA5 prevents immune activation of SCRV RNA (Figure 1C and 1E, Figure 2P and 2Q). Finally, we synthesized a 5'ppp-SCRV RNA probe and demonstrated that MDA5 binds to 5'ppp-SCRV through the RD domain (Figure 4E-4G). We believe that these results can better demonstrate that MDA5 recognizes 5’ppp-RNA through its RD domain and addresses the concerns of the reviewers.

(7) Similarly, merely presenting Co-IP data demonstrating the interaction between Miiuy croaker MDA5 and STING in overexpressed EPC cells does not justify the claim that "in vertebrates lacking RIG-I, MDA5 can utilize STING to facilitate signal transduction in the antiviral response". This is because interactions observed through overexpression may not accurately reflect the events occurring during viral infection or their actual antiviral functions. To provide more robust evidence, it is essential to conduct functional experiments after STING knockout (or at least knockdown). Furthermore, it is important to note that Miiuy Croaker alone cannot adequately represent all "vertebrates lacking RIG-I".

We found that co-expression of STING and MDA5 can enhance MDA5-mediated IFN-1 response during SCRV virus infection, while knocking down STING can restore MDA5-mediated IFN-1 response (Figure 2N and 2O). This indicates that STING plays an important signaling role in the immune response of MDA5 to RNA viruses. In addition, loss of RIG-I is a common phenomenon in vertebrates, and STING of birds such as chickens (doi: 10.4049/jimmunol.1500638) and mammalian tree shrews (doi: 10.1073/pnas.1604939113) can also bind to MDA5, indicating that STING can indeed play a crucial role in MDA5 signaling in species with RIG-I deficiency. We have added this section to our discussion and elaborated on our observations in more cautious language.

(8) In the manuscript, a series of experiments were conducted using an antibody (Beyotime Cat# AF7164) against endogenous MDA5. The corresponding immunogen for this MDA5 antibody is a recombinant fusion protein containing amino acids 1-205 of human IFIH1/MDA5 (NP_071451.2). However, the amino acid sequences of IFIH1/MDA5 differ substantially between humans and Miiuy croaker, which could introduce errors in the results. Therefore, it is essential to employ antibodies specifically designed for targeting Miiuy croaker's own MDA5 in the experiments.

As shown in Figure 2B, endogenous MDA5 antibodies can detect the MDA5 portion that is forcibly overexpressed by plasmids, suggesting that the MDA5 antibody can indeed specifically recognize the MDA5 protein of M. miiuy.

(9) It is recommended to investigate the phosphorylation of IRF3 in order to confirm the downstream signaling pathway during viral infection when MDA5 is knocked down or overexpressed.

Due to the lack of available phosphorylation antibodies for fish IRF3, we used IRF3 dimer experiments to detect downstream signaling (Figure 1C and 1D, Figure 2P, Figure 4C and 4J).

(10) The use of poly I:C as a mimic for dsRNA to investigate MDA5's recognition of 5'ppp-RNA in hosts lacking RIG-I, as well as the examination of the regulatory role of MDA5 m6A methylation upon activation by 5'ppp-RNA, may be inappropriate. Poly I:C does not possess 5'ppp, and while it has been identified as a ligand for MDA5 in various studies, MDA5 cannot serve as a substitute for RIG-I in recognizing poly (I:C). Therefore, the authors should utilize 5'ppp-dsRNA as the mimic and include the corresponding 5'ppp-dsRNA control without a 5'triphosphate as the negative control (both available from InvivoGen). This approach will specifically elucidate the mechanisms involved when MDA5 functions similarly to RIG-I in the recognition of 5'ppp-RNA.

In our study, we used poly(I:C)-HMW, a known dsRNA mimetic that can be preferentially recognized by MDA5 rather than RIG-I, as a positive control for activating MDA5. What we want to demonstrate is that, like poly(I:C)-HMW (positive control), SCRV can also promote MDA5-mediated IFN immunity, further indicating the important role of MDA5 in 5’ppp-RNA virus invasion. We have clearly labeled the type of poly(I:C) in the figures and legends to avoid misunderstandings for readers.

(11) In Figure 2, Figure 3, and Figure 6, the appearance of virus plaques is not readily apparent, and it is necessary to replace these images with clearer photographs. It appears that MKC or MPC cells are not appropriate for conducting plaque assays. To accurately assess viral proliferation, the authors should measure key indicators throughout the process, such as the production of positive-strand RNAs (+RNAs), replication intermediates (RF), and transcription of subgenomic RNAs. This approach is preferable to solely measuring the M and G protein genes from the virus genome as positive results can still be observed in contaminated cells.

As pointed out by the reviewer, we also think that the virus plaque images in Figure 2K and Figure 3D are not clear enough, so we have replaced them with new clear images (Figure 2J and Figure 3D). But we think that other images can clearly display the proliferation of the SCRV virus, so we did not replace them. In addition, the primers we currently use do measure +RNA, so the replication level of the SCRV virus can be accurately evaluated without being affected by virus contamination. Because the regions where the two pairs of primers are located belong to the SCRV-M and SCRV-G protein genes, we label them as SCRV-M and SCRV-G to distinguish between the two pairs of genes. To avoid reader misunderstanding, we have modified the Y-axis label in the figures (Figure 2I and 2K, Figure 3E, Figure 6E and 6O).

(12) There is a substantial disparity in the molecular size of M. miiuy MDA5 between endogenous and exogenously expressed proteins, as shown in Figure 2A and 2C-D. Please provide clarification.

Please refer to the response to Reviewer 2's question regarding Figure 4B above.

(13) The manuscript incorporates the evolutionary perspective, but lacks specific evolutionary analysis. Thus, it is essential to include relevant analysis to comprehend the evolutionary dynamics and positive selection on MDA5 and LGP2 in the absence of RIG-I in Miiuy croaker. This can be achieved through theoretical calculations using appropriate algorithms, such as the branch models and branch-site models based on the maximum-likelihood method implemented in the phylogenetic analysis by maximum likelihood (PAML) package.

In fact, we have analyzed the molecular evolution of MDA5 and LGP2. Unfortunately, even when analyzing only the MDA5/LGP2 CDS sequences in fish, we found that the topologies of gene trees of MDA5/LGP2 were largely consistent with the species tree. Thus, species with or without RIG-I in the gene trees cannot effectively separate clusters, making it extremely difficult to analyze the molecular evolution of MDA5/LGP2 caused by RIG-I deficiency. Consequently, we gave up this aspect of analysis.

(14) If the narrative regarding m6A methylation goes beyond the activation of MDA5 through recognition of 5'ppp-RNA and represents a regulatory mechanism for all MDA5 activation events, it is not relevant to the theme of "An arms race under RIG-I loss: 5'ppp-RNA and its alternative recognition receptor MDA5." Therefore, all investigations in this paper should focus solely on events when MDA5 recognizes 5'ppp-RNA. Any data associated with the broader regulatory mechanisms and m6A methylation of MDA5 should be excluded from this manuscript and instead be included in a separate study dedicated to exploring this specific topic.

Our theme aims to showcase RNA viruses, rather than an interaction between 5'ppp-RNA and host virus receptors, which our current topic cannot accurately express. Therefore, we made two main changes: firstly, we limited the study species to M. miiuy, although some studies on the functional substitution of MDA5 for RIG-I involved birds. Secondly, change “5’ppp-RNA” to “5’ppp-RNA virus”. We believe that the revised title is more in line with our current research contents.

(15) The running title appears to be hastily done.

We modified it to “MDA5 recognizes 5’ppp-RNA virus in species lacking RIG-I”.

(16) There are many descriptions that are not strongly related to the main theme of the article in the introduction section, making it lengthy and fragmented. Please focus on the research background of RIG-I and MDA5, including their structures, functions, and regulatory mechanisms, as well as the research progress on the compensatory effect of MDA5 in the absence of RIG-I and its evolutionary adaptation mechanism in other species.

Based on the suggestions of the reviewers, we have removed some of the less relevant content in the introduction and added research progress on the compensatory effect of MDA5 in the evolutionary adaptation mechanism of tree shrews in the absence of RIG-I.

(17) Lines 149-156 in the "Results" section include content that resembles an "Introduction" It is important to avoid duplicating information in the results section. Therefore, the authors are encouraged to revise this paragraph to ensure conciseness in the article.

We have streamlined this section to enhance the article's conciseness and clarity.

(18) In the "Results" section, at line 177, the authors assert, "As depicted in Figure 1F-1H," which should be corrected to Figure 2F-2H. Furthermore, the y-axis of the two figures on the right-hand side of Figure 2H represents the ISG15 genes. At line 182, "as demonstrated in Figure 1I-1L," should be revised as "as illustrated in Figure 2I-2L". The authors demonstrated a lack of attention to detail.

Thank you to the reviewer for pointing out our errors, and we have made the necessary corrections.

(19) In lines 197-198, the authors stated that "MDA5-ΔRD showed an inability to interact with SCRV." However, Figure 3D did not reveal any significant difference, thus it is advisable to repeat this experiment at least once.

We have replaced this virus spot image with a new one (Figure 3D).

(20) In lines 200-201 of the "2.3 RD domain is required for MDA5 to recognize SCRV" section, the authors report that the expression of antiviral genes was induced by the overexpression of both MDA5 and MDA5-ΔRD, even in the absence of infection (Figure 3F). Why does the expression of antiviral genes increase in the absence of viral RNA stimulation? Please provide a reasonable explanation.

In the absence of viral infection, overexpression of viral receptor proteins may still transmit erroneous signaling, affecting the body's immunity. We speculate that due to the preservation of the CARD domain by MDA5 and MDA5-ΔRD, they can still induce the expression of antiviral factors without ligands, although this induction effect is much smaller than that of viral infection. However, in order to better demonstrate the function of the RD domain of MDA5 in M. miiuy, we have changed the experimental plan, as shown in the figure 3F. We detected the induction of antiviral factors by overexpression of MDA5 and MDA5-△RD under poly (I:C)-HMW stimulation. This can indicate that the RD domain has a conserved function in the recognition of poly(I:C)-HMW in M. miiuy, and can serve as a positive control for the recognition of SCRV virus invasion by the RD domain of MDA5.

(21) Please provide the GeneBank accession number of M. miiuy MDA5.

The GeneBank accession number of M. miiuy MDA5 was added in the section 4.5 plasmids construction.

(22) The content of lines 228-233 in the "Results" section bears resemblance to that of the "Introduction." To ensure the avoidance of information duplication, it is recommended to remove this paragraph from the results section.

This section has been streamlined.

(23) The bands of mmiMDA5 in the 5'ppp-RNA and dsRNA lanes in Figure 4B are weak and almost unobservable. Please replace them with clear images.

We have rerun this experiment and replaced the images (Figure 4B).

(24) In Figure 5G and at line 253, there are only results presented for the SCRV infection group, while no results are shown for the control group. This raises the question of why the control group results are missing. It is necessary to provide a reasonable explanation or correction for this issue.

The "0 h" infection time point of the SCRV virus is the control group, and we have replaced it with a more intuitive image (Figure 5G).

(25) In Figure 7C, it would be necessary to include the western blot result of YTHDF protein expression in order to verify the efficiency of YTHDF siRNA.

In fact, we have attempted to detect the endogenous expression of YTHDF protein using available commercial antibodies. Unfortunately, only the YTHDF2 antibody can specifically recognize the endogenous protein expression of YTHDF2 in M. miiuy. In addition, the knockdown effect of si-YTHDF2 has been validated by YTHDF2 antibody (doi: 10.4049/jimmunol.2200618).

(26) In line 422 of the "4.3 Cell culture and treatment" section, the paragraph raises a question regarding the nature of Miiuy croaker kidney cells (MKCs) and spleen cells (MPCs) - whether they are cell lines or freshly isolated cells (or primary cultures) derived from kidney and spleen tissues. If these cells are indeed cell lines, it is requested to provide detailed information about the sources and properties of the cells (such as whether they are epithelial cells or other mixed cell types) and the generations of propagation. Alternatively, if the cells were freshly isolated or primary cultures obtained from fish, the method for cell isolation should be provided. The source and stability of cells are extremely important for ensuring the repeatability and reliability of experimental outcomes.

M. miiuy kidney cells (MKCs) and spleen cells (MPCs) are cell lines derived from the kidney and spleen tissues of M. miiuy, with passages ranging from 20 to 40 times. These details have been incorporated into section 4.3.

(27) There are many inaccurate descriptions in the text, which employ concepts that are too broad. These descriptions need to be narrowed down to specific species or objects. Here are a few examples, along with the necessary revisions. Other similar instances should also be revised accordingly. For instance, in line 119, "fish MDA5" should be changed to "Miiuy croaker MDA5." Similarly, in line 166, "fish MDA5-mediated signaling pathway" should be changed to "Miiuy croaker MDA5-mediated signaling pathway." In line 174, "fish MDA5" should be revised to "Miiuy croaker MDA5." Additionally, in line 185, "antiviral responses of teleost" should be changed to "antiviral responses of Miiuy croaker." In line 197, "interact with SCRV" should be revised to "interact with 5'ppp-RNA of SCRV." In line 337, "loss of RIG-I in the vertebrate" should be modified to "loss of RIG-I in Miiuy croaker and chicken." Similarly, in line 338, "MDA5 of fish" should be changed to "MDA5 of Miiuy croaker." Lastly, in line 348, "RIG-I deficient vertebrates" should be revised to "RIG-I deficient Miichthys miiuy and Gallus gallus."

Thank you for the reviewer's suggestions. We have made revisions to these inaccurate descriptions and reviewed the entire manuscript to address similar statements with broad concepts.

(28) Finally, it should be noted that a similar discovery has already been reported in tree shrews (Ling Xu, et al., Proc Natl Acad Sci., 2016, 113(39):10950-10955). This article shares similarities with that research report, therefore it is necessary to discuss in detail the relationship between the two in the discussion and compare and analyze the evolutionary patterns of MDA5 from it.

Based on the reviewer's suggestions, we have compared the similarities and differences between these two reports during the discussion and analyzed the evolutionary dynamics of MDA5 in these vertebrates lacking RIG-I.

Minor concerns:

Thank you to the reviewer for their meticulous examination to our manuscript, we have made revisions to the following suggestions.

(1) At line 120, the sentence "SCRV(one 5'ppp-RNA virus)" should have a space between "SCRV" and "(one 5'ppp-RNA virus)". Please make this correction.

Corrected.

(2) At lines 147-148, the sentence "However, the downstream gene of TOPORSa is missing a RIG-I" is not accurate and needs modification.

We have modified this sentence.

(3) At line 184, "findings indicate" should be corrected to "findings indicated".

Corrected.

(4) At line 189, "a 5'ppp-RNA virus" should be deleted and the text seems redundant.

Deleted.

(5) At line 198, "replication. (Figure 3C-3E)", please remove the punctuation between "replication" and "(Figure 3C-3E)".

Corrected.

(6) At line 416 in "Materials and methods" section, "4.2 Sample and challenge" should be corrected to "4.2 Fish and challenge".

Corrected.

(7) At line 419, the authors state that "The experimental procedure for SCRV infection was performed as described", please briefly describe the SCRV infection method and the infectious dose.

Based on the reviewer's suggestions, we have added relevant descriptions of SCRV infection in section 4.2.

(8) There are several formatting issues in the "Materials and Methods" section. For instance, in line 424, there is no space between the number and letter in "100 μg/ml" and "26 ℃" should be corrected to "26℃". Additionally, in line 430, "Cells" should be corrected to "cells".

Corrected.

(9) At line 446, "50 ng/ul" and "100 mU/ul" should be corrected to "50 ng/μl" and "100 mU/μl".

Corrected.

(10) At line 459, "primers 1)" should be corrected to "primers".

Corrected.

(11) At lines 461-464, the description "For protein purification, MDA5 plasmids with 6× His tag was constructed based on pcDNA3" seems to be no direct logical connection between protein purification and the plasmid construction. Please make the necessary corrections.

Corrected.

(12) At line 548, "cytoplasmic" should be corrected to "Cytoplasmic".

Corrected.

(13) At line 549, "5× 107" should be corrected to "5 × 107".

Corrected.

(14) At line 557, "MgCl2" should be corrected to "MgCl2".

Corrected.

(15) At line 558, "6 %" should be corrected to "6%".

Corrected.

(16) At line 565, "50μg" should be corrected to "50 μg".

Corrected.

(17) At line 571, "300{plus minus}50 bp." should be corrected to "300 {plus minus} 50 bp."

Corrected.

(18) At lines 592-593, the sentence "After several incubations, the m6A level was quantified colorimetrically at a wavelength of 450 nm" does not read smoothly, please improve it.

Revised.

(19) At line 786, "MDA5 recognize" should be corrected to "MDA5 recognized".

Corrected.

(20) At lines 788 and 798, "Pulldown" should be corrected to "Pull-down".

Corrected.

(21) At lines 790 and 796, "bluestaining" should be corrected to "blue staining".

Deleted.

(22) At line 825, "SCRV and infection" should be corrected to "SCRV infection".

Corrected.

(23) At lines 826-827, "SCRV (H) and poly(I:C) (I) infection" should be corrected to "SCRV infection (H) and poly(I:C) stimulation (I)".

Corrected.