long list of cool humans interested in hypermaps

Der Artikel beschäftigt sich mit den gesundheitlichen Folgen der der globalen Erhitzung. Anlass ist, das bei der COP28 zum ersten Mal bei einer COP ein ganzer Tag der Gesundheit gewitdmet war. Gefordert wird ein interdisziplinärer Zugang an der der Stelle des biher gängigen Totschweigens, https://www.liberation.fr/idees-et-debats/tribunes/le-rechauffement-de-la-terre-est-une-urgence-de-sante-publique-20240105_A27LZFF2B5EYJD25FOPJ22CYCI/

Author response:

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

Reviewer #1 (Public Review): 

Though the Norrin protein is structurally unrelated to the Wnt ligands, it can activate the Wnt/βcatenin pathway by binding to the canonical Wnt receptors Fzd4 and Lrp5/6, as well as the tetraspanin Tspan12 co-receptor. Understanding the biochemical mechanisms by which Norrin engages Tspan12 to initiate signaling is important, as this pathway plays an important role in regulating retinal angiogenesis and maintaining the blood-retina-barrier. Numerous mutations in this signaling pathway have also been found in human patients with ocular diseases. The overarching goal of the study is to define the biochemical mechanisms by which Tspan12 mediates Norrin signaling. Using purified Tspan12 reconstituted in lipid nanodiscs, the authors conducted detailed binding experiments to document the direct, high-affinity interactions between purified Tspan12 and Norrin. To further model this binding event, they used AlphaFold to dock Norrin and Tspan12 and identified four putative binding sites. They went on to validate these sites through mutagenesis experiments. Using the information obtained from the AlphaFold modeling and through additional binding competition experiments, it was further demonstrated that Tspan12 and Fzd4 can bind Norrin simultaneously, but Tspan12 binding to Norrin is competitive with other known co-receptors, such as HSPGs and Lrp5/6. Collectively, the authors proposed that the main function of Tspan12 is to capture low concentrations of Norrin at the early stage of signaling, and then "hand over" Norrin to Fzd4 and Lrp5/6 for further signal propagation. Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data. 

Strengths: 

• Biochemical reconstitution of Tspan12 and Fzd4 in lipid nanodiscs is an elegant approach for testing the direct binding interaction between Norrin and its co-receptors. The proteins used for the study seem to be of high purity and quality. 

• The various binding experiments presented throughout the study were carried out rigorously. In particular, BLI allows accurate measurement of equilibrium binding constants as well as on and off rates. 

• It is nice to see that the authors followed up on their AlphaFold modeling with an extensive series of mutagenesis studies to experimentally validate the potential binding sites. This adds credence to the AlphaFold models. 

• Table S1 is a further testament to the rigor of the study. 

• Overall, the study is comprehensive and compelling, and the conclusions are well supported by the experimental and modeling data. 

Suggestions for improvement: 

• It would be helpful to show Coomassie-stained gels of the key mutant Norrin and Tspan12 proteins presented in Figures 2E and 2F. 

We have included Stain-Free SDS-PAGE gels from the purification of the Norrin and Tspan12 mutants in a new Figure S4.

• Many Norrin and Tspan12 mutations have been identified in human patients with FEVR. It would be interesting to comment on whether any of the mutations might affect the NorrinTspan12 binding sites described in this study. 

Thank you for this suggestion. We have inspected human mutation databases gnomAD, ClinVar, and HGMD for known mutations in the predicted Tspan12-Norrin binding interface and their occurrence in human patients with FEVR or Norrie disease.

While a number of Tspan12 residues that we predict to interact with Norrin are impacted by rare mutations in humans (e.g., L169M, E170V, E173K, D175N, E196G, S199C, as found in the gnomAD database), these alleles are of unknown clinical significance (as found in ClinVar or HGMD databases). It is possible that mutations that slightly weaken the Norrin-Tspan12 interface may not produce a strong phenotype, especially given the avidity we expect from this system. By our examination, the missense variants of clinical significance that have been found in the Tspan12 LEL would be expected to destabilize the protein (i.e., mutations to or from cysteine or proline, or mutations to residues involved in packing interactions within the LEL fold), and therefore these mutations may produce a disease phenotype by impacting Tspan12 protein expression levels.  

Several Norrin mutations that are associated with Norrie disease, FEVR, or other diseases of the retinal vasculature have been found in the predicted Tspan12 binding site. For example, Norrin mutations at positions L103 (L103Q, L103V), K104 (K104N, K104Q), and A105 (A105T, A105P, A105E, A105S, A105T, A105V) have been found in patients, all of which may disrupt binding to Tspan12. However, the deleterious effect of K104 mutations on Norrin-stimulated signaling could also be explained by a weakened Norrin-Fzd4 binding interface. Norrin mutations at R115 (R115L and R115Q), as well as R121 (R121L, R121G, R121Q, and R121W) have also been found in patients with various diseases of the retinal vasculature. Additionally, the Norrin mutation T119P has been found in patients with Norrie disease, but we would expect this mutation to destabilize Norrin in addition to disrupting the Tspan12 binding site. 

While we commented briefly on mutations R115L and R121W in the original draft (page 5, paragraphs 4 and 1, respectively), we have updated the manuscript with more comments on disease-associated mutations to the predicted Tspan12 binding site on Norrin (page 5, first partial paragraph; page 9, first partial paragraph). 

• Some of the negative conclusions (e.g. the lack of involvement of Tspan12 in the formation of the Norrin-Lrp5/6-Fzd4-Dvl signaling complex) can be difficult to interpret. There are many possible reasons as to why certain biological effects are not recapitulated in a reconstitution experiment. For instance, the recombinant proteins used in the experiment may not be presented in the correct configurations, and certain biochemical modifications, such as phosphorylation, may also be missing. 

We agree that different Tspan12 and Fzd4 stoichiometries, lipid compositions, and posttranslational modifications could impact the results of our study, and that it is important to mention these possibilities. We have added these caveats to the discussion section (page 10, last paragraph).  

Reviewer #2 (Public Review): 

This is an interesting study of high quality with important and novel findings. Bruguera et al. report a biochemical and structural analysis of the Tspan12 co-receptor for norrin. Major findings are that Norrin directly binds Tspan12 with high affinity (this is consistent with a report on BioRxiv: Antibody Display of cell surface receptor Tetraspanin12 and SARS-CoV-2 spike protein) and a predicted structure of Tspan12 alone or in complex with Norrin. The

Norrin/Tspan12 binding interface is largely verified by mutational analysis. An interaction of the Tspan12 large extracellular loop (LEL) with Fzd4 cannot be detected and interactions of fulllength Tspan12 and Fzd4 cannot be tested using nano-disc based BLI, however, Fzd4/Tspan12 heterodimers can be purified and inserted into nanodiscs when aided by split GFP tags. An analysis of a potential composite binding site of a Fzd4/Tspan12 complex is somewhat inconclusive, as no major increase in affinity is detected for the complex compared to the individual components. A caveat to this data is that affinity measurements were performed for complexes with approximately 1 molecule Tspan12 and FZD4 per nanodisc, while the composite binding site could potentially be formed only in higher order complexes, e.g., 2:2 Fzd4/Tspan12 complexes. Interestingly, the authors find that the Norrin/Tspan12 binding site and the Norrin/Lrp6 binding site partially overlap and that the Lrp6 ectodomain competes with Tspan12 for Norrin binding. This result leads the authors to propose a model according to which Tspan12 captures Norrin and then has to "hand it off" to allow for Fzd4/Lrp6 formation. By increasing the local concentration of Norrin, Tspan12 would enhance the formation of the Fzd4/Lrp5 or Fzd4/Lrp6 complex. 

Thank you for pointing out the BioRxiv report showing Norrin-Tspan12 LEL binding. We have cited this in the introduction of our revised manuscript (page 2, paragraph 3).

The experiments based on membrane proteins inserted into nano-discs and the structure prediction using AlphaFold yield important new insights into a protein complex that has critical roles in normal CNS vascular biology, retinal vascular disease, and is a target for therapeutic intervention. However, it remains unclear how Norrin would be "handed off" from Tspan12 or Tspan12/Fzd4 complexes to Fzd4/Lrp6 complexes, as the relatively high affinity of Norrin to Fzd4/Tspan12 dimers likely does not favor the "handing off" to Fzd4/Lrp6 complexes. 

While the Fzd4-Tspan12 interaction is strong, our data suggest that Fzd4 and Tspan12 bind Norrin with negative cooperativity, suggesting that Fzd4 binding may enhance Norrin-Tspan12 dissociation to facilitate handoff. This model is based on 1) the dissociation of Norrin from beadbound Tspan12 in the presence of saturating Fzd4 CRD (Figure 3D), and 2) a weaker measured affinity of Norrin-Tspan12LEL in the presence of saturating Fzd4 CRD (Figure 3F). We have now added wording to emphasize this in the discussion section (page 9, end of first full paragraph).

However, as you note, the Norrin-Tspan12 affinity that we measured in the presence of Fzd CRD (tens of nM) is still much stronger than the known Norrin-LRP6 affinity (0.5-1µM), which predicts that the efficiency of this handoff may be low. We have now commented on this in the discussion section and mentioned an alternative model in which Tspan12 presents the second Norrin protomer to LRP5/6 for signaling, instead of dissociating (page 9, paragraph 2). However, the handoff efficiency could also be impacted by other factors such as the relative abundance and surface distribution of Tspan12, Fzd4, LRP6 and HSPGs.  

Areas that would benefit from further experiments, or a discussion, include: 

-  The authors test a potential composite binding site of Fzd4/Tspan12 heterodimers for norrin using nanodiscs that contain on average about 1 molecule Fzd4 and 1 molecule Tspan12. The Fzd4/Tspan12 heterodimer is co-inserted into the nanodiscs supported by split-GFP tags on Fzd4 and Tspan12. The authors find no major increase in affinity, although they find changes to the Hill slope, reflecting better binding of norrin at low norrin concentrations. In 293F cells overexpressing Fzd4 and Tspan12 (which may result in a different stoichiometry) they find more pronounced effects of norrin binding to Fzd4/Tspan12. This raises the possibility that the formation of a composite binding requires Fzd4/Tspan12 complexes of higher order, for example, 2:2 Fzd4/Tspan12 complexes, where the composite binding site may involve residues of each Fzd4 and Tspan12 molecule in the complex. This could be tested in nanodiscs in which Fzd4 and Tspan12 are inserted at higher concentrations or using Fzd4 and Tspan12 that contain additional tags for oligomerization. 

It is quite possible that Tspan12 and Fzd4 cluster into complexes with a stoichiometry greater than 1:1 in cells (this is supported by e.g., BRET experiments in (Ke et al., 2013)), and we mention in the discussion that that receptor clustering may be an additional mechanism by which Tspan12 exerts its function (page 10, paragraph 4). We would be quite interested to know the stoichiometry of Fzd4 and Tspan12 complexes in cells at endogenous expression levels, both in the presence and absence of Norrin, and to biochemically characterize these putative larger complexes in the future. We have amended the discussion to mention the caveat that our reconstitution experiments do not test higher-stoichiometry Fzd4/Tspan12 complexes (page 10, last paragraph).

- While Tspan12 LEL does not bind to Fzd4, the successful reconstitution of GFP from Tspan12 and Fzd4 tagged with split GFP components provides evidence for Fzd4/Tspan12 complex formation. As a negative control, e.g., Fzd5, or Tspan11 with split GFP tags (Fzd5/Tspan12 or Fzd4/Tspan11) would clarify if FZD4/Tspan12 heterodimers are an artefact of the split GFP system. 

The split-GFP system allows us to co-purify receptors that do not normally co-localize (for example, as we have shown with Fzd4 and LRP6 in the absence of ligand (Bruguera et al., 2022)) so we do not mean to claim that it provides evidence for Fzd4/Tspan12 complex formation. In fact, we were unable to co-purify co-expressed Fzd4 and Tspan12 unless they were tethered with the split GFP system, and separately-purified Fzd4 and Tspan12 did not incorporate into nanodiscs together unless they were tethered by split GFP. Based on these experiments, we expect that the purported Fzd4-Tspan12 interaction that others have found by co-IP or co-localization is easily disrupted by detergent, may require a specific lipid, and/or may not be direct.

To clarify this point, we have noted in the results section that without the split GFP tags, Tspan12 and Fzd4 did not co-purify or co-reconstitute into nanodiscs, and that co-reconstitution was enabled by the split GFP system (page 6, first full paragraph).   

- Fzd4/Tspan12 heterodimers stabilized by split GFP may be locked into an unfavorable orientation that does not allow for the formation of a composite binding site of FZD4 and Tspan12, this is another caveat for the interpretation that Fzd4/Tspan12 do not form a composite binding site. This is not discussed. 

While the split GFP does enforce a Fzd4/Tspan12 dimer, the split GFP is removed by protease cleavage during the final step of the purification process, after the dimer is contained in a nanodisc. This should allow Fzd4 and Tspan12 to freely adopt any pose and to diffuse within the confines of the nanodisc lipid bilayer. However, it has been shown that the phospholipid bilayer in small nanodiscs is not as fluid as the physiological plasma membrane, and although we used the slightly larger belt protein (MSP1E3D1, 13 nm diameter nanodiscs), perhaps the receptors are indeed locked in some unfavorable state for this reason. Additionally, the nanodiscs are planar, so if the formation of a composite binding site requires membrane curvature, this would not be recapitulated in our system. We have cited these caveats in the discussion section (page 10, last paragraph).  

- Mutations that affect the affinity of norrin/fzd4 are not used to further test if Fzd4 and Tspan12 form a composite binding site. Norrin R41E or Fzd4 M105V were previously reported to reduce norrin/frizzled4 interactions and signaling, and both interaction and signaling were restored by Tspan12 (Lai et al. 2017). Whether a Fzd4/Tspan12 heterodimer has increased affinity for Norrin R41E was not tested. Similarly, affinity of FZD4 M105V vs a Fzd4 M105V/Tspan12 heterodimer were not tested. 

Since the high affinity of Norrin for both Fzd4 and Tspan12 may have obscured any enhancement of Norrin affinity for Fzd4/Tspan12 compared to either receptor alone, we did consider weakening Fzd-Norrin affinity to sensitize this experiment, inspired by the experiments you mention in (Lai et al., 2017). However, we suspected that the slight increase in Norrin affinity for the Fzd4/Tspan12 dimer compared to Fzd4 alone was driven mainly by increased avidity that enhanced binding of low Norrin concentrations, and this avidity effect would likely confound the interpretation of any experiment monitoring 2:2 complex formation. Additionally, on the basis that soluble Fzd4 extracellular domain and Tspan12 bind Norrin with negative cooperativity (Figures 3D and 3F), we concluded that this composite binding site was unlikely.

- An important conclusion of the study is that Tspan12 or Lrp6 binding to Norrin is mutually exclusive. This could be corroborated by an experiment in which LRP5/6 is inserted into nanodiscs for BLI binding tests with Norrin, or Tspan12 LEL, or a combination of both. Soluble LRP6 may remove norrin from equilibrium binding/unbinding to Tspan12, therefore presenting LRP6 in a non-soluble form may yield different results. 

We agree that testing this conclusion in an orthogonal experiment would be a valuable addition to this study. We have now performed a similar experiment to the one you described, but with Norrin immobilized on biosensors, and with LRP6 in detergent competing with Tspan12 LEL for Norrin binding (Figure S12, discussed on page 8, first full paragraph). The results of this experiment show that biosensor-immobilized Norrin will bind LRP6, and that soluble Tspan12 inhibits LRP6 binding in a concentration-dependent manner. The LRP6 construct we use (residues 20-1439) includes the transmembrane domain but has a truncated C terminus, since LRP6 constructs containing the full C terminus tend to aggregate during purification. We chose to immobilize Norrin to make the experiment as interpretable as possible, since immobilizing LRP6 and competing Norrin off with the LEL could result in an increase in signal (from the LEL binding the second available Norrin protomer) as well as a decrease (from Norrin being competed off of the immobilized LRP6). We conducted the experiment in detergent (DDM) instead of nanodiscs to be able to test higher concentrations of LRP6.

- The authors use LRP6 instead of LRP5 for their experiments. Tspan12 is less effective in increasing the Norrin/Fzd4/Lrp6 signaling amplitude compared to Norrin/Fzd4/Lrp5 signaling, and human genetic evidence (FEVR) implicates LRP5, not LRP6, in Norrin/Frizzled4 signaling. The authors find that Norrin binding to LRP6 and Tspan12 is mutually exclusive, however this may not be the case for Lrp5. 

This is an important point which we have now addressed in the text (page 8, end of first full paragraph). LRP5 is indeed the receptor implicated in FEVR and expressed in the relevant tissues for Tspan12/Norrin signaling. Unfortunately, LRP5 expresses poorly and we are unable to purify sufficient quantities to perform these experiments. However, LRP5 and LRP6 both transduce Tspan12-enhanced Norrin signaling in TOPFLASH assays (as you mention and as shown by (Zhou and Nathans, 2014)), bind Norrin, and are highly similar (they share 71% sequence identity overall and 73% sequence identity in the extracellular domain), so we expect their Norrin-binding sites to be conserved.

- The biochemical data are largely not correlated with functional data. The authors suggest that the Norrin R115L FEVR mutation could be due to reduced norrin binding to tspan12, but do not test if Tspan12-mediated enhancement of the norrin signaling amplitude is reduced by the R115L mutation. Similarly, the impressive restoration of binding by charge reversal mutations in site 3 is not corroborated in signaling assays. 

We agree that testing the impact of Norrin mutations in cell-based signaling assays would be an informative way to further test our model. However, the Norrin mutants we tested generated poor TopFlash signals in all conditions tested. This may be due to general protein instability, weakened affinity for LRP, or weaker interactions with HSPGs. Whatever the cause, the low signal made it challenging to conclusively say whether the Norrin mutations affected Tspan12mediated signaling enhancement.

When expressed for purification, Tspan12 mutants generally expressed poorly compared to WT Tspan12, so we were concerned that differences in protein stability or trafficking would lead to lower cell-surface levels of mutant Tspan12 relative to WT in TopFlash signaling assays, which would confound interpretation of mutant Tspan’s ability to enhance Norrin signaling.

Because of these challenges, follow-up experiments to investigate the signaling capabilities of Norrin and Tspan12 mutants were not informative and we have not included them in the revised manuscript.

Reviewer #3 (Public Review): 

Brugeuera et al present an impressive series of biochemical experiments that address the question of how Tspan12 acts to promote signaling by Norrin, a highly divergent TGF-beta family member that serves as a ligand for Fzd4 and Lrp5/6 to promote canonical Wnt signaling during CNS (and especially retinal) vascular development. The present study is distinguished from those of the past 15 years by its quantitative precision and its high-quality analyses of concentration dependencies, its use of well-characterized nano-disc-incorporated membrane proteins and various soluble binding partners, and its use of structure prediction (by AlphaFold) to guide experiments. The authors start by measuring the binding affinity of Norrin to Tspan12 in nanodiscs (~10 nM), and they then model this interaction with AlphaFold and test the predicted interface with various charge and size swap mutations. The test suggests that the prediction is approximately correct, but in one region (site 1) the experimental data do not support the model. [As noted by the authors, a failure of swap mutations to support a docking model is open to various interpretations. As AlphFold docking predictions come increasingly into common use, the compendium of mutational tests and their interpretations will become an important object of study.] Next, the authors show that Tspan12 and Fzd4 can simultaneously bind Norrin, with modest negative cooperativity, and that together they enhance Norrin capture by cells expressing both Tspan12 and Fzd4 compared to Fzd4 alone, an effect that is most pronounced at low Norrin concentration. Similarly, at low Norrin concentration (~1 nM), signaling is substantially enhanced by Tspan12. By contrast, the authors show that LRP6 competes with Tspan12 for Norrin binding, implying a hand-off of Norrin from a Tspan12+Fzd4+Norrin complex to a LRP5/6+Fzd4+Norrin complex. Thanks to the authors' careful dose-response analyses, they observed that Norrin-induced signaling and Tspan12 enhancement of signaling both have bell-shaped dose-response curves, with strong inhibition at higher levels of Norrin or Tspan12. The implication is that the signaling system has been built for optimal detection of low concentrations of Norrin (most likely the situation in vivo), and that excess Tspan12 can titrate Norrin at the expense of LRP5/6 binding (i.e., reduction in the formation of the LRP5/6+Fzd4+Norrin signaling complex). In the view of this reviewer, the present work represents a foundational advance in understanding Norrin signaling and the role of Tspan12. It will also serve as an important point of comparison for thinking about signaling complexes in other ligand-receptor systems. 

Recommendations for the authors: 

Reviewer #2 (Recommendations For The Authors):   

- In Figure 5F high concentrations of transfected Tspan12 plasmid inhibit signaling, which the authors interpret to support the model that Tspan12/Norrin binding prevents Norrin/LRP6/FZD4 complex formation. Alternatively, the cells do not tolerate the expression of the tetraspanin at high levels, for example, due to misfolding and aggregate formation. To distinguish these possibilities: Do high levels of Tspan12 overexpression also inhibit signaling induced by Wnt3a and appropriate Frizzled receptors, even though Tspan12 has no influence on Wnt/LRP6 binding? 

We thank the reviewer for suggesting this important control experiment. We have added the Wnt-simulated TOPFLASH values to the figure in 5F for all conditions. In repeating this experiment, we noticed that high levels of transfected Tspan12 may decrease cell viability and therefore have adjusted the range of transfected Tspan12 in the new Figure 5F (discussed on page 8, second full paragraph). Under this new protocol, both Norrin- and Wnt-stimulated signaling were inhibited by the highest amount of transfected Tspan12. However, Norrinstimulated signaling is inhibited by lower amounts of transfected Tspan12 than Wnt-stimulated signaling, and to a greater extent, supporting our proposed model that Tspan12 competes with LRP for Norrin binding.

- Is Tspan12 with c-terminal rho-tag (the form incorporated into nanodiscs) also used for functional luciferase assays, or was untagged Tspan12 used for the luciferase assays in Fig 4D and 5F? Does the c-terminal tag interfere with Tspan12-mediated enhancement of Norrin signaling? 

For the luciferase assays included in this manuscript, wildtype, full-length, untagged Tspan12 is used. We have clarified this in our methods section. When we tested the wildtype vs Cterminally rho1D4-tagged version of Tspan12 in TOPFLASH assays, we saw that the enhancement of Norrin signaling by Tspan12-1D4 was weaker than enhancement by untagged Tspan12. This is consistent with the finding reported in Cell Reports (Lai et al., 2017) that a chimeric Tspan12 receptor with its C-terminus replaced with that of Tspan11 was still capable of enhancing Norrin signaling, though to a lesser extent than WT Tspan12. The deficiency of signaling by our rho1D4-tagged Tspan12 could be due to a difference in receptor expression level or trafficking, but in the absence of a reliable antibody against Tspan12, we were unable to assess the expression levels or localization of the untagged Tspan12 to compare it to the rho1D4-tagged version. (For binding experiments, we reasoned that the C-terminal tag should not affect Tspan12’s ability to bind Norrin extracellularly, especially as we found that purified fulllength Tspan12 and Tspan12∆C (residues 1-252) bound Norrin equally well; we have added this comparison to table S1.)  

Reviewer #3 (Recommendations For The Authors): 

Minor comments. 

Based on the Fzd4-Dvl binding experiment, the authors might state explicitly the possibility that Tspan12's relevance is entirely accounted for by extracellular ligand capture. 

We have stated this possibility explicitly in the discussion section (page 9, last paragraph). 

Page 4, 3rd paragraph. I suggest "To experimentally test this structural prediction..." rather than "validate". 

Thank you for this suggestion; we have replaced this wording. 

This next item is optional, but I hope that the authors will consider it. This manuscript provides an opportunity for the authors to be more expansive in their thinking, and to put their work into the larger context of ligand+receptor+accessory protein interactions. The authors describe the Wnt7a/7b-Gpr124-RECK system and the role of HSPs in Norrin and Wnt signaling, but perhaps they can also comment on non-Wnt ligand-receptor systems where accessory proteins are found. They might add a figure (or supplemental figure) with a schematic showing the roles of HSP and Gpr124-RECK, and some non-Wnt ligand-receptor systems. This would help to make the present work more widely influential.

Thank you for this suggestion. We have added a figure (Figure 6, discussed on page 10, paragraphs 2 and 3) and expanded our discussion to include other co-receptor systems. We have specifically focused on co-receptors that both capture ligands and interact with their primary receptor(s), thus delivering ligands to their receptors, as we have proposed for Tspan12. Within Wnt signaling, other co-receptor systems with this mechanism are RECK/Gpr124 (for Wnt7a/b) and Glypican-3. We found it interesting that this mechanism is also shared by several growth factor pathways with cystine knot ligands (like Norrin), so we have illustrated and mentioned three of these examples.

Reviewer #1 (Public review):

Summary:

Wang et al. identify Hamlet, a PR-containing transcription factor, as a master regulator of reproductive development in Drosophila. Specifically, the fusion between the gonad and genital disc is necessary for the development of continuous testes and seminal vesicle tissue essential for fertility. To do this, the authors generate novel Hamlet null mutants by CRISPR/Cas9 gene editing and characterize the morphological, physiological, and gene expression changes of the mutants using immunofluorescence, RNA-seq, cut-tag, and in-situ analysis. Thus, Hamlet is discovered to regulate a unique expression program, which includes Wnt2 and Tl, that is necessary for testis development and fertility.

Strengths:

This is a rigorous and comprehensive study that identifies the Hamlet-dependent gene expression program mediating reproductive development in Drosophila. The Hamlet transcription targets are further characterized by Gal4/UAS-RNAi confirming their role in reproductive development. Finally, the study points to a role for Wnt2 and Tl as well as other Hamlet transcriptionally regulated genes in epithelial tissue fusion.

Weaknesses:

The image resolution and presentation of figures is a major issue in this study. As a non-expert, it is nearly impossible to see the morphological changes as described in the results. Quantification of all cell biological phenotypes is also lacking therefore reducing the impact of this study to those familiar with tissue fusion events in Drosophila development.

Reviewer #2 (Public review):

Strengths:

Wang and colleagues successfully uncovered an important function of the Drosophila PRDM16/PRDM3 homolog Hamlet (Ham) - a PR domain-containing transcription factor with known roles in the nervous system in Drosophila. To do so, they generated and analyzed new mutants lacking the PR domain, and also employed diverse preexisting tools. In doing so, they made a fascinating discovery: They found that PR-domain containing isoforms of ham are crucial in the intriguing development of the fly genital tract. Wang and colleagues found three distinct roles of Ham: (1) specifying the position of the testis terminal epithelium within the testis, (2) allowing normal shaping and growth of the anlagen of the seminal vesicles and paragonia and (3) enabling the crucial epithelial fusion between the seminal vesicle and the testis terminal epithelium. The mutant blocks fusion even if the parts are positioned correctly. The last finding is especially important, as there are few models allowing one to dissect the molecular underpinnings of heterotypic epithelial fusion in development. Their data suggest that they found a master regulator of this collective cell behavior. Further, they identified some of the cell biological players downstream of Ham, like for example E-Cadherin and Crumbs. In a holistic approach, they performed RNAseq and intersected them with the CUT&TAG-method, to find a comprehensive list of downstream factors directly regulated by Ham. Their function in the fusion process was validated by a tissue-specific RNAi screen. Meticulously, Wang and colleagues performed multiplexed in situ hybridization and analyzed different mutants, to gain a first understanding of the most important downstream pathways they characterized, which are Wnt2 and Toll.

This study pioneers a completely new system. It is a model for exploring a process crucial in morphogenesis across animal species, yet not well understood. Wang and colleagues not only identified a crucial regulator of heterotypic epithelial fusion but took on the considerable effort of meticulously pinning down functionally important downstream effectors by using many state-of-the-art methods. This is especially impressive, as the dissection of pupal genital discs before epithelial fusion is a time-consuming and difficult task. This promising work will be the foundation future studies build on, to further elucidate how this epithelial fusion works, for example on a cell biological and biomechanical level.

Weaknesses:

The developing testis-genital disc system has many moving parts. Myotube migration was previously shown to be crucial for testis shape. This means, that there is the potential of non-tissue autonomous defects upon knockdown of genes in the genital disc or the terminal epithelium, affecting myotube behavior which in turn affects fusion, as myotubes might create the first "bridge" bringing the epithelia together. The authors clearly showed that their driver tools do not cause expression in myoblasts/myotubes, but this does not exclude non-tissue autonomous defects in their RNAi screen. Nevertheless, this is outside the scope of this work.

However, one point that could be addressed in this study: the RNAseq and CUT&TAG experiments would profit from adding principal component analyses, elucidating similarities and differences of the diverse biological and technical replicates.

Author response:

Thank you for reviewing our manuscript and providing constructive feedback. We are grateful that you recognize the importance of our work and find the evidences presented compelling. We will revise our manuscripts in accordance with reviewers’ recommendations. Below is our plan.

(1) As recommended by Reviewer 1, we will improve the image resolution and presentation in the figures, by adjusting dark colors into brighter ones, including single-channel images, and incorporating schematic illustrations to dipict morphological changes.

(2) Following the suggestions of reviewer 2, we will provide explanations and speculative insights into potential non-tissue autonomous effects.

(3) As suggested by reviewer 2, we will perform principal component analyses on our RNA-seq and Cut&Tag data. 

(2) Once we have addressed all the major and minor points raised by the reviewers, we will provide a detailed point-to-point response and submit the revised version of the manuscript.

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Reviewer #1 (Public review):

Summary:

The manuscript by Bindu et al. created an AAV-based tool (GEARAOCS) to perform in vivo genome editing of mouse astrocytes. The authors engineered a versatile AAV vector that allows for gene deletion through NHNJ, site-specific knock-in by HDR, and gene trap. By utilizing this tool, the authors deleted Sparcl1 virally in subsets of astrocytes and showed that thalamocortical synapses in cortical layer IV are indeed reduced during a critical period of ocular dominance plasticity and in adulthood, whereas there is no change in excitatory synapse number in cortical layer II/III. Furthermore, the authors made a VAMP2 gene-trap AAV vector and showed that astrocyte-derived VAMP2 is required for the maintenance of both excitatory and inhibitory synapses.

Strengths:

This AAV-based tool is versatile for astrocytic gene manipulation in vivo. The work is innovative and exciting, given the paucity of tools available to probe astrocytes in vivo.

Weaknesses:

Several important considerations need to be made for the validation and usage of this tool, including:

Major points:

(1) Efficiency and specificity of spCas9-sgRNA mediated gene knockout in astrocytes. In Figure 3, the authors utilized Sparcl1 gene deletion as the proof-of-principle experiment. The readout for Sparcl1 KO efficiency is solely the immunoreactivity using an antibody raised against Sparcl1. As the method is based on NHEJ, the indels can be diverse and can occur in one allele or two. For the tool and proof-of-principle experiment, it will be important to know the percentage of editing near the PAM site, as well as the actual sequences of indels. This can be done by single-cell PCR of edited astrocytes, similar to the published work (Ye... Chen, Nature Biotechnology 2019).

(2) Along the same line, the authors showed that GEARBOCS TagIn of Sparcl1 resulted in 12.49% efficiency based on the immunohistochemistry of mCherry tag. It is understandable that the knock-in efficiency is much reduced as compared to gene knockout. However, it remains unclear if those 12.49% knock-in cells represent sequence-correct ones, as spCas9-mediated HDR is also an error-prone process, and it may accidentally alter nucleotides near the PAM site without causing the frameshift. The author will need to consider the related evidence or make comments in the discussion.

(3) What are the efficiencies of Sparcl1 GEARBOCS GeneTrap (Figure 3V) and Vamp2 GeneTrap and HA TagIn (Figure 5)?

Minor points:

(1) Figure 3H-J. The authors only showed the representative images of Sparcl1 KO. Please consider including the control (without gRNA), given that there are still many Sparcl1+ signals in Figure 3I (likely because of its expression in other cell types?).

(2) In figure 3Q-T, it appears that some Cas9-EGFP+ astrocytes (Q) do not express Sparcl1 (R). Is Sparcl1 expressed in subsets of astrocytes? Does Cas9-EGFP or Sparcl1-TagIn alter Sparcl1 endogenous expression?

(3) On Page 8, for the explanation of the design of the GEARBOCS construct, the authors have made a self-citation (#43). That was a BioRxiv paper that is being reviewed currently.

(4) For Figures 4 and 6, the graphs seem to be made in R with the x-axis labeled as "Condition". The y-axis labels are too small to read properly, especially in print. It would be better to make the graphs clearer like Figure 2 and Figure 3.

(5) On Page 13, "Figures 3V-Y" were referred to. However, there are no Figures 3W, X, and Y.

(6) There are a few typos in the manuscript, including line 900 "immunofluorescence microscopy images of a Cas9-EGFP-positive astrocytes (green)".

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

Manuscript number: RC-2024-02465

Corresponding author(s): Saravanan, Palani

1. General Statements

We would like to thank the Review Commons Team for handling our manuscript and the Reviewers for their constructive feedback and suggestions. In our revised manuscript, we have addressed and incorporated all the major suggestions of the reviewers, and we have also added new significant data on the role of Tropomyosin in regulation of endocytosis through its control over actin monomer pool maintenance and actin network homeostasis. We believe that with all these additions, our study has significantly gained in quality, strength of conclusions made, and scope for future work.

2. Point-by-point description of the revisions

Reviewer #1

Evidence, reproducibility and clarity

There are 2 Major issues -

Having an -ala-ser- linker between the GFP and tropomyosin mimics acetylation. This is not the case, and more likely the this linker acts as a spacer that allows tropomyosin polymers to form on the actin, and without it there is steric hindrance. A similar result would be seen with a simple flexible uncharged linker. It has been shown in a number of labs that the GFP itself masks the effect of the charge on the amino terminal methionine. This is consistent with NMR, crystallographic and cryo structural studies. Biochemical studies should be presented to demonstrate that the impact of a linker for the conclusions stated to be made, which provide the basis of a major part of this study.

Response: We would like to clarify that all mNG-Tpm constructs used in our study contain a 40 amino-acid (aa) flexible linker between the N-terminal mNG fluorescent protein and the Tpm protein as per our earlier published study (Hatano et al., 2022). During initial optimization, we have also experimented with linker length and the 40aa-linker length works optimally for clear visualization of Tpm onto actin cable structures in budding yeast, fission yeast (both S. pombe and S. japonicus), and mammalian cells (Hatano et al., 2022). These constructs have also been used since in other studies (Wirshing et al., 2023; Wirshing and Goode, 2024) and currently represents the best possible strategy to visualize Tpm isoforms in live cells. In our study, we characterized these proteins for functionality and found that both mNG-Tpm1 and mNG-Tpm2 were functional and can rescue the synthetic lethality observed in Dtpm1Dtpm2 cells. During our study, we observed that mNG-Tpm1 expression from a single-copy integration vector did not restore full length actin cables in Dtpm1 cells (Fig. 1B, 1C). We hypothesized that this could be a result of reduced binding affinity of the tagged tropomyosin due to lack of normal N-terminal acetylation which stabilizes the N-terminus. The 40aa linker is unstructured and may not be able to neutralize the charge on the N-terminal Methionine, thus, we tried to insert -Ala-Ser- dipeptide which has been routinely used in vitro biochemical studies to stabilize the N-terminal helix and impart a similar effect as the N-terminal acetylation (Alioto et al., 2016; Palani et al., 2019; Christensen et al., 2017) by restoring normal binding affinity of Tpm to F-actin (Monteiro et al., 1994; Greenfield et al., 1994). We observed that addition of the -Ala-Ser- dipeptide to mNG-Tpm fusion, indeed, restored full length actin cables when expressed in Dtpm1 cells, performing significantly better in our in vivo experiments (Fig. 1B, 1C). We agree with the reviewer that the -AS- dipeptide addition may not mimic N-terminal acetylation structurally but as per previous studies, it may stabilize the N-terminus of Tpm and allow normal head-to-tail dimer formation (Greenfield et al., 1994; Monteiro et al., 1994; Frye et al., 2010). We have discussed this in our new Discussion section (Lines 350-372). Since, the addition of -AS- dipeptide was referred to as "acetyl-mimic (am)" in a previous study (Alioto et al., 2016), we continued to use the same nomenclature in our study. Now as per your suggestions and to be more accurate, we have renamed "mNG-amTpm" constructs as "mNG-ASTpm" throughout the study to not confuse or claim that -AS- addition mimics acetylation. In any case, we have not seen any other ill effect of -AS- dipeptide introduction in addition to our 40 amino acid linker suggesting that it can also be considered part of the linker. Although, we agree with the reviewer that biochemical characterization of the effect of linker would be important to determine, we strongly believe that it is currently outside the scope of this study and should be taken up for future work with these proteins. Our study has majorly aimed to understand the functionality and utility of these mNG-Tpm fusion proteins for cell biological experiments in vivo, which was not done earlier in any other model system.

My major issue however is making the conclusions stated here, using an amino-terminal fluorescent protein tag that s likely to impact any type of isoform selection at the end of the actin polymer. Carboxyl terminal tagging may have a reduced effect, but modifying the ends of the tropomyosin, which are integral in stabilising end to end interactions with itself on the actin filament, never mind any section systems that may/maynot be present in the cell, is not appropriate.

Response: __ We agree with the reviewer that N-terminal tagging of tropomyosin may have effects on its function, but these constructs represent the only fluorescently tagged functional tropomyosin constructs available currently while C-terminal fusions are either non-functional (we were unable to construct strains with endogenous Tpm1 gene fused C-terminally to GFP) or do not localize clearly to actin structures (See __Figure R1 showing endogenous C-terminally tagged Tpm2-yeGFP that shows almost no localization to actin cables). To our knowledge, our study represents a first effort to understand the question of spatial sorting of Tpm isoforms, Tpm1 and Tpm2, in S. cerevisiae and any future developments with better visualization strategies for Tpm isoforms without compromising native N-terminal modifications and function will help improve our understanding of these proteins in vivo. We have also discussed these possibilities in our new Discussion section (Lines 391-396).

Significance

This paper explores the role of formin in determining the localisation of different tropomyosins to different actin polymers and cellular locations within budding yeast. Previous studies have indicated a role for the actin nucleating proteins in recruiting different forms of tropomyosin within fission yeast. In mammalian cells there is variation in the role of formins in affiecting tropomyosin localisation - variation between cell type. There is also evidence that other actin binding proteins, and tropomyosin abundance play roles in regulating the tropomyosin-actin association according to cell type. Biochemical studies have previously been undertaken using budding yeast and fission yeast that the core actin polymerisation domain of formins do not interact with tropomyosin directly. The significance of this study, given the above, and the concerns raised is not clear to this reviewer.

Response: __Our study explores multiple facets of Tropomyosin (Tpm) biology. The lack of functional tagged Tpm has been a major bottleneck in understanding Tpm isoform diversity and function across eukaryotes. In our study, we characterize the first functional tagged Tpm proteins (Fig. 1, Fig. S1) and use them to answer long-standing questions about localization and spatial sorting of Tpm isoforms in the model organism S. cerevisiae (Fig. 2, Fig. 3, Fig. S2, Fig. S3). We also discover that the dual Tpm isoforms, Tpm1 and Tpm2, are functionally redundant for actin cable organization and function, while having gained divergent functions in Retrograde Actin Cable Flow (RACF) (Fig. 4, Fig. 5A-D, Fig. S4, Fig. S5, Fig. S6). We have now added new data on role of global Tpm levels controlling endocytosis via maintenance of normal linear-to-branched actin network homeostasis in S. cerevisiae (Fig. 5E-G)__. We respectfully differ with the reviewer on their assessment of our study and request the reviewer to read our revised manuscript which discusses the significance, limitations, and future perspectives of our study in detail.

Reviewer #2

Evidence, reproducibility and clarity

This manuscript by Dhar, Bagyashree, Palani and colleagues examines the function of the two tropomyosins, Tpm1 and Tpm2, in the budding yeast S. cerevisiae. Previous work had shown that deletion of tpm1 and tpm2 causes synthetic lethality, indicating overlapping function, but also proposed that the two tropomyosins have distinct functions, based on the observation that strong overexpression of Tpm2 causes defects in bud placement and fails to rescue tpm1∆ phenotypes (Drees et al, JCB 1995). The manuscript first describes very functional mNeonGreen tagged version of Tpm1 and Tpm2, where an alanine-serine dipeptide is inserted before the first methionine to mimic acetylation. It then proposes that the Tpm1 and Tpm2 exhibit indistinguishable localization and that low level overexpression (?) of Tpm2 can replace Tpm1 for stabilization of actin cables and cell polarization, suggesting almost completely redundant functions. They also propose on specific function of Tpm2 in regulating retrograde actin cable flow.

Overall, the data are very clean, well presented and quantified, but in several places are not fully convincing of the claims. Because the claims that Tpm1 and Tpm2 have largely overlapping function and localization are in contradiction to previous publication in S. cerevisiae and also different from data published in other organisms, it is important to consolidate them. There are fairly simple experiments that should be done to consolidate the claims of indistinguishable localization, and levels of expression, for which the authors have excellent reagents at their disposal.

1. Functionality of the acetyl-mimic tagged tropomyosin constructs: The overall very good functionality of the tagged Tpm constructs is convincing, but the authors should be more accurate in their description, as their data show that they are not perfectly functional. For instance, the use of "completely functional" in the discussion is excessive. In the results, the statement that mNG-Tpm1 expression restores normal growth (page 3, line 69) is inaccurate. Fig S1C shows that tpm1∆ cells expressing mNG-Tpm1 grow more slowly than WT cells. (The next part of the same sentence, stating it only partially restores length of actin cables should cite only Fig S1E, not S1F.) Similarly, the growth curve in Fig S1C suggests that mNG-amTpm1, while better than mNG-Tpm1 does not fully restore the growth defect observed in tpm1∆ (in contrast to what is stated on p. 4 line 81). A more stringent test of functionality would be to probe whether mNG-amTpm1 can rescue the synthetic lethality of the tpm1∆ tpm2∆ double mutant, which would also allow to test the functionality of mNG-amTpm2.

__Response: __We would like to thank the reviewer for his feedback and suggestions. Based on the suggestions, we have now more accurately described the growth rescue observed by expression of mNG-ASTpm1 in Dtpm1 cells in the revised text. We have also removed the use of "completely functional" to describe mNG-Tpm functionality and corrected any errors in Figure citations in the revised manuscript.

As per reviewers' suggestion, we have now tested rescue of synthetic lethality of Dtpm1Dtpm2 cells by expression of all mNG-Tpm variants and we find that all of them are capable of restoring the viability of Dtpm1Dtpm2 cells when expressed under their native promoters via a high-copy plasmid (pRS425) (Fig. S1E) but only mNG-Tpm1 and mNG-ASTpm1 restored viability of Dtpm1Dtpm2 cells when expressed under their native promoters via an integration plasmid (pRS305) (Fig. S1F). These results clearly suggest that while both mNG-Tpm1 and mNG-Tpm2 constructs are functional, Tpm1 tolerates the presence of the N-terminal fluorescent tag better than Tpm2. These observations now enhance our understanding of the functionality of these mNG-Tpm fusion proteins and will be a useful resource for their usage and experimental design in future studies in vivo.

It would also be nice to comment on whether the mNG-amTpm constructs really mimicking acetylation. Given the Ala-Ser peptide ahead of the starting Met is linked N-terminally to mNG, it is not immediately clear it will have the same effect as a free acetyl group decorating the N-terminal Met.

Response: __We agree with the reviewer's observation and for the sake of clarity and accuracy, we have now renamed "mNG-amTpm" with "mNG-ASTpm". The use of -AS- dipeptide is very routine in studies with Tpm (Alioto et al., 2016; Palani et al., 2019; Christensen et al., 2017) and its addition restores normal binding affinities to Tpm proteins purified from E. coli (Monteiro et al., 1994). We agree with the reviewer that the -AS- dipeptide addition may not mimic N-terminal acetylation structurally but as per previous studies, it may help neutralize the impact of a freely protonated Met on the alpha-helical structure and stabilize the N-terminus helix of Tpm and allow normal head-to-tail dimer formation (Monteiro et al., 1994; Frye et al., 2010; Greenfield et al., 1994). Consistent with this, we also observe a highly significant improvement in actin cable length when expressing mNG-ASTpm as compared to mNG-Tpm in Dtpm1 cells, suggesting an improvement in function probably due to increased binding affinity (Fig. 1B, 1C). We have also discussed this in our answer to Question 1 of Reviewer 1 and the revised manuscript (Lines 350-372)__.

__ Localization of Tpm1 and Tpm2:__Given the claimed full functionality of mNG-amTpm constructs and the conclusion from this section of the paper that relative local concentrations may be the major factor in determining tropomyosin localization to actin filament networks, I am concerned that the analysis of localization was done in strains expressing the mNG-amTpm construct in addition to the endogenous untagged genes. (This is not expressly stated in the manuscript, but it is my understanding from reading the strain list.) This means that there is a roughly two-fold overexpression of either tropomyosin, which may affect localization. A comparison of localization in strains where the tagged copy is the sole Tpm1 (respectively Tpm2) source would be much more conclusive. This is important as the results are making a claim in opposition to previous work and observation in other organisms.

Response: __We thank the reviewer for this observation and their suggestions. We agree that relative concentrations of functional Tpm1 and Tpm2 in cells may influence the extent of their localizations. As per the reviewer's suggestion, we have now conducted our quantitative analysis in cells lacking endogenous Tpm1 and only expressing mNG-ASTpm1 from an integrated plasmid copy at the leu2 locus and the data is presented in new __Figure S3. We compared Tpm-bound cable length (Fig. S3A, S3B) __and Tpm-bound cable number (Fig. S3A, S3C) along with actin cable length (Fig. S3D, S3E) and actin cable number (Fig. S3D, S3F) in wildtype, Dbnr1, and Dbni1 cells. Our analysis revealed that mNG-ASTpm1 localized to actin cable structures in wildtype, Dbnr1, and Dbni1 cells and the decrease observed in Tpm-bound cable length and number upon loss of either Bnr1 or Bni1, was accompanied by a corresponding decrease in actin cable length and number upon loss of either Bnr1 or Bni1. Thus, this analysis reached the same conclusion as our earlier analysis (Fig. 2) that mNG-ASTpm1 does not show preference between Bnr1 and Bni1-made actin cables. mNG-ASTpm2 did not restore functionality, when expressed as single integrated copy, in Dtpm1Dtpm2 cells (new results in __Fig. S1E, S1F, S5A) thus, we could not conduct a similar analysis for mNG-ASTpm2. This suggests that use of mNG-ASTpm2 would be more meaningful in the presence of endogenous Tpm2 as previously done in Fig. 2D-F.

We have now also performed additional yeast mating experiments with cells lacking bnr1 gene and expressing either mNG-ASTpm1 or mNG-ASTpm2 and the data is shown in new Figure 3. From these observations, we observe that both mNG-ASTpm1 and mNG-ASTpm2 localize to the mating fusion focus in a Bnr1-independent manner (Fig. 3B, 3D) and suggests that they bind to Bni1-made actin cables that are involved in polarized growth of the mating projection. These results also add strength to our conclusion that Tpm1 and Tpm2 localize to actin cables irrespective of which formin nucleates them. Overall, these new results highlight and reiterate our model of formin-isoform independent binding of Tpm1 and Tpm2 in S. cerevisiae.

In fact, although the authors conclude that the tropomyosins do not exhibit preference for certain actin structures, in the images shown in Fig 2A and 2D, there seems to be a clear bias for Tpm1 to decorate cables preferentially in the bud, while Tpm2 appears to decorate them more in the mother cell. Is that a bias of these chosen images, or does this reflect a more general trend? A quantification of relative fluorescence levels in bud/mother may be indicative.

Response: __We thank the reviewer for pointing this out. Our data and analysis do not suggest that Tpm1 and Tpm2 show any preference for decoration of cables in either mother or bud compartment. As per the reviewer's suggestion, we have now quantified the ratio of mean mNG fluorescence in the bud to the mother (Bud/Mother) and the data is shown in __Figure. S2G. The bud-to-mother ratio was similar for mNG-ASTpm1 and mNG-ASTpm2 in wildtype cells, and the ratio increased in Dbnr1 cells and decreased in Dbni1 cells for both mNG-ASTpm1 and mNG-ASTpm2 (Fig. S2G). __This is consistent with the decreased actin cable signal in the mother compartment in Dbnr1 cells and decreased actin cable signal in the bud compartment in Dbni1 cells (Fig. S2A-D). Thus, our new analysis shows that both mNG-ASTpm1 and mNG-ASTpm2 have similar changes in their concentration (mean fluorescence) upon loss of either formins Bnr1 and Bni1 and show similar ratios in wildtype cells as well, suggesting no preference for binding to actin cables in either bud or mother compartment. The preference inferred by the reviewer seems to be a bias of the current representative images and thus, we have replaced the images in __Fig. 2A, 2D to more accurately represent the population.

The difficulty in preserving mNG-amTpm after fixation means that authors could not quantify relative Tpm/actin cable directly in single fixed cells. Did they try to label actin cables with Lifeact instead of using phalloidin, and thus perform the analysis in live cells?

__Response: __We did not use LifeAct for our analysis as LifeAct is known to cause expression-dependent artefacts in cells (Courtemanche et al., 2016; Flores et al., 2019; Xu and Du, 2021) and it also competes with proteins that regulate normal cable organization like cofilin. Use of LifeAct would necessitate standardization of expression to avoid such artefacts in vivo. Also, phalloidin staining provides the best staining of actin cables and allows for better quantitative results in our experiments. The use of LifeAct along with mNG-Tpm would also require optimization with a red fluorescent protein which usually tend to have lower brightness and photostability. However, during the revision of our study, a new study from Prof. Goode's lab has developed and optimized expression of new LifeAct-3xmNeonGreen constructs for use in S. cerevisiae (Wirshing and Goode, 2024). Thus, a similar strategy of using tandem copies of bright and photostable red fluorescent proteins can be explored for use in combination with mNG-Tpm in the future studies.

__ Complementation of tpm1∆ by Tpm2:__

I am confused about the quantification of Tpm2 expression by RT-PCR shown in Fig S3F. This figure shows that tpm2 mRNA expression levels are identical in cells with an empty plasmid or with a tpm2-encoding plasmid. In both strains (which lack tpm1), as well as in the WT control, one tpm2 copy is in the genome, but only one strain has a second tpm2 copy expressed from a centromeric plasmid, yet the results of the RT-PCR are not significantly different. (If anything, the levels are lower in the tpm2 plasmid-containing strain.) The methods state that the primers were chosen in the gene, so likely do not distinguish the genomic from the plasmid allele. However, the text claims a 1-fold increase in expression, and functional experiments show a near-complete rescue of the tpm1∆ phenotype. This is surprising and confusing and should be resolved to understand whether higher levels of Tpm2 are really the cause of the observed phenotypic rescue.

The authors could for instance probe for protein levels. I believe they have specific nanobodies against tropomyosin. If not, they could use expression of functional mNG-amTpm2 to rescue tpm1∆. Here, the expression of the protein can be directly visualized.

Response: __We thank the reviewer for pointing this out. We would like to clarify that in our RT-qPCR experiments, the primers were chosen within the Tpm1 and Tpm2 gene and do not distinguish between transcripts from endogenous or plasmid copy. We have now mentioned this in the Materials and Methods section of the revised manuscript. So, they represent a relative estimate of the total mRNA of these genes present in cells. We were consistently able to detect ~19 fold increase in Tpm2 total mRNA levels as compared to wildtype and ∆tpm1 cells (Fig. S4D) when tpm2 was expressed from a high-copy plasmid (pRS425). This increase in Tpm2 mRNA levels was accompanied by a rescue in growth (Fig. S4A) and actin cable organization (Fig. S4B) of ∆tpm1 cells containing pRS425-ptpm2TPM2. When tpm2 was expressed from a low-copy number centromeric plasmid (pRS316), we detected a ~2 fold increase in Tpm2 transcript levels when using the tpm1 promoter and no significant change was detected when using tpm2 promoter (Fig. S4E)__. We have made sure that these results are accurately described in the revised manuscript.

As per the reviewer's suggestion, we have now conducted a more extensive analysis to ascertain the expression levels of Tpm2 in our experiments and the data is now presented in new Figure S5. We used mNG-ASTpm1 and mNG-ASTpm2 to rescue growth of ∆tpm1 (Fig. S5A) and correlated growth rescue with protein levels using quantified fluorescence intensity (Fig. S5B, S5C) and western blotting (anti-mNG) (Fig. S5D, S5E). We find that ∆tpm1 cells containing pRS425-ptpm1mNG-ASTpm1 had the highest protein level followed by pRS425-ptpm2 mNG-ASTpm2, pRS305-ptpm1mNG-ASTpm1, and the least protein levels were found in pRS305-ptpm2 mNG-ASTpm2 containing ∆tpm1 cells in both fluorescence intensity and western blotting quantifications (Fig. S5C, S5E). Surprisingly, we were not able to detect any protein levels in ∆tpm1 cells containing pRS305-ptpm2 mNG-ASTpm2 with western blotting (Fig. S5D) which was also accompanied by a lack of growth rescue (Fig. S5A). This most likely due to weak expression from the native Tpm2 promoter which is consistent with previous literature (Drees et al., 1995). Taken together, this data clearly shows that the rescue observed in ∆tpm1 cells is caused due to increased expression of mNG-ASTpm2 in cells and supports our conclusion that increase in Tpm2 expression leads to restoration of normal growth and actin cables in ∆tpm1 cells.

__ Specific function of Tpm2:__

The data about the retrograde actin flow is interpreted as a specific function of Tpm2, but there is no evidence that Tpm1 does not also share this function. To reach this conclusion one would have to investigate retrograde actin flow in tpm1∆ (difficult as cables are weak) or for instance test whether Tpm1 expression restores normal retrograde flow to tpm2∆ cells.

Response: __We agree with the reviewer and as per the reviewer's suggestion, we have performed another experiment which include wildtype, ∆tpm2 cells containing empty pRS316 vector or pRS316-ptpm2TPM1 or pRS316-ptpm1TPM1. We find that RACF rate increased in ∆tpm2 cells as compared to wildtype and was restored to wildtype levels by exogenous expression of Tpm2 but not Tpm1 (Fig. S6E, S6F). Since, actin cables were not detectable in ∆tpm1 cells, we measured RACF rates in ∆tpm1 cells expressing Tpm1 or Tpm2 from a plasmid copy, which restored actin cables as shown previously in __Fig. 5A-C. We observed that RACF rates were similar to wildtype in ∆tpm1 cells expressing either Tpm1 or Tpm2 (Fig. S6E, S6F), suggesting that Tpm1 is not involved in RACF regulation. Taken together, these results suggest a specific role for Tpm2, but not Tpm1, in RACF regulation in S. cerevisiae, consistent with previous literature (Huckaba et al., 2006).

Minor comments: __1.__The growth of tpm1∆ with empty plasmid in Fig S3A is strangely strong (different from other figures).

Response: __ We thank the reviewer for pointing this out. We have now repeated the drop test multiple times (__Fig. R2), but we see similar growth rates as the drop test already presented in Fig. S4A. __At this point, it would be difficult to ascertain the basis of this difference observed at 23{degree sign}C and 30{degree sign}C, but a recent study that links leucine levels to actin cable stability (Sing et al., 2022) might explain the faster growth of these ∆tpm1 cells containing a leu2 gene carrying high-copy plasmid. However, there is no effect on growth rate at 37{degree sign}C which is consistent with other spot assays shown in __Fig. S1D, S4F, S5A.

Significance

I am a cell biologist with expertise in both yeast and actin cytoskeleton.

The question of how tropomyosin localizes to specific actin networks is still open and a current avenue of study. Studies in other organisms have shown that different tropomyosin isoforms, or their acetylated vs non-acetylated versions, localize to distinct actin structures. Proposed mechanisms include competition with other ABPs and preference imposed by the formin nucleator. The current study re-examines the function and localization of the two tropomyosin proteins from the budding yeast and reaches the conclusion that they co-decorate all formin-assembled structures and also share most functions, leading to the simple conclusion that the more important contribution of Tpm1 is simply linked to its higher expression. Once consolidated, the study will appeal to researchers working on the actin cytoskeleton.

We thank the reviewer for their positive assessment of our work and the constructive feedback that has greatly improved the quality of our study. After addressing the points raised by the reviewer, we believe that our study has significantly gained in consolidating the major conclusions of our work.

**Referees cross-commenting**

Having read the other reviewers' comments, I do agree with reviewer 1 that it is not clear whether the Ala-Ser linker really mimics acetylation. I am less convinced than reviewer 3 that the key conclusions of the study are well supported, notably the issue of Tpm2 expression levels is not convincing to me.

Response: __We acknowledge the reviewer's point about the effect of Ala-Ser dipeptide and would request the reviewer to refer to our response to Reviewer 1 (Question 1) for a more detailed discussion on this. We have also extensively addressed the question of Tpm2 expression levels as suggested by the reviewer (new data in __Figure S5) which has further strengthened the conclusions of our study.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)):

Summary:__ The study presents the first fully functional fluorescently tagged Tpm proteins, enabling detailed probing of Tpm isoform localization and functions in live cells. The authors created a modified fusion protein, mNG-amTpm, which mimicked native N-terminal acetylation and restored both normal growth and full-length actin cables in yeast cells lacking native Tpm proteins, demonstrating the constructs' full functionality. They also show that Tpm1 and Tpm2 do not have a preference for actin cables nucleated by different formins (Bnr1 and Bni1). Contrary to previous reports, the study found that overexpressing Tpm2 in Δtpm1 cells could restore growth rates and actin cable formation. Furthermore, it is shown that despite its evolutionary divergence, Tpm2 retains actin-protective functions and can compensate for the loss of Tpm1, contributing to cellular robustness.

Major and Minor Comments: 1. The key conclusions of this paper are convincing. However, I suggest that more detail be provided regarding the image analysis used in this study. Specifically, since threshold settings can impact the quality of the generated data and, therefore, its interpretation, it would be useful to see a representative example of the quantification methods used for actin cable length/number (as in refs. 80 and 81) and mitochondria morphology. These could be presented as Supplemental Figures. Additionally, it would help to interpret the results if the authors could be more specific about the statistical tests that were used.

Response: __We agree with the reviewer's suggestions and have now updated our Materials and Methods section to describe the image analysis pipelines used in more detail. We have also added examples of quantification procedure for actin cable length/number and mitochondrial morphology as an additional Supplementary __Figure S7. Briefly, the following pipelines were used:

• Actin cable length and number analysis: This was done exactly as mentioned in McInally et al., 2021, McInally et al., 2022. Actin cables were manually traced in Fiji as shown in __ S7A__, and then the traces files for each cell were run through a Python script (adapted from McInally et al., 2022) that outputs mean actin cable length and number per cell.
• Mitochondria morphology: Mitochondria Analyzer plug-in in Fiji was used to segment out the mitochondrial fragments. The parameters used for 2D segmentation of mitochondria were first optimized using "2D Threshold Optimize" to find the most accurate segmentation and then the same parameters were run on all images. After segmentation of the mitochondrial network, measurements of fragment number were done using "Analyze Particles" function in Fiji. An example of the overall process is shown in __ S7B.__ As per the reviewer's suggestion, we have now included the description of the statistical test used in the Figure Legends of each Figure in the revised manuscript. We have used One-Way Anova with Tukey's Multiple Comparison test, Kruskal-Wallis test with Dunn's Multiple Comparisons, and Unpaired Two-tailed t-test using the in-built functions in GraphPad Prism (v.6.04).

**Referees cross-commenting**

I agree with both reviewers 1 and 2 regarding the issues with the Ala-Ser acetylation mimic and Tpm2 expression levels, respectively. I think the authors should be more careful in how they frame the results, but I consider that these issues do not invalidate the main conclusions of this study.

Response: __We acknowledge the reviewer's concern about the Ala-Ser dipeptide and would request them to refer our earlier discussion on this in response to Reviewer 1 (Question 1) and Reviewer 2 (Question 2). We would also request the reviewer to refer to our answer to Reviewer 2 (Question 6) where we have extensively addressed the question of Tpm2 expression levels and their effect on rescue of Dtpm1 cells. This data is now presented as new __Figure S5 in our revised manuscript.

Reviewer#3 (Significance (Required)):

The finding that Tpm2 can compensate for the loss of Tpm1, restoring actin cable organization and normal growth rates, challenges previous assumptions about the non-redundant functions of these isoforms in Saccharomyces cerevisiae (ref. 16). It also supports a concentration-dependent and formin-independent localization of Tpm isoforms to actin cables in this species. The development of fully functional fluorescently tagged Tpm proteins is a significant methodological advancement. This advancement overcomes previous visualization challenges and allows for accurate in vivo studies of Tpm function and regulation in S. cerevisiae.

The findings will be of particular interest to researchers in the field of cellular and molecular biology who study actin cytoskeleton dynamics. Additionally, it will be relevant for those utilizing advanced microscopy and live-cell imaging techniques.

As a researcher, my experience lies in cytoskeleton dynamics and protein interactions, though I do not have specific experience related to tropomyosin. I use different yeast species as models and routinely employ live-cell imaging as a tool.

We thank the reviewer for their positive outlook and assessment of our study. We have incorporated all their suggestions, and we are confident that the revised manuscript has significantly improved in quality due to these additions.

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Referee #1

Evidence, reproducibility and clarity

There are 2 Major issues:

1. Having an -ala-ser- linker between the GFP and tropomyosin mimics acetylation. This is not the case, and more likely the this linker acts as a spacer that allows tropomyosin polymers to form on the actin, and without it there is steric hindrance. A similar result would be seen with a simple flexible uncharged linker. It has been shown in a number of labs that the GFP itself masks the effect of the charge on the amino terminal methionine. This is consistent with NMR, crystallographic and cryo structural studies. Biochemical studies should be presented to demonstrate that the impact of a linker for the conclusions stated to be made, which provide the basis of a major part of this study.
2. My major issue however is making the conclusions stated here, using an amino-terminal fluorescent protein tag that s likely to impact any type of isoform selection at the end of the actin polymer. Carboxyl terminal tagging may have a reduced effect, but modifying the ends of the tropomyosin, which are integral in stabilising end to end interactions with itself on the actin filament, never mind any section systems that may/maynot be present in the cell, is not appropriate.

Significance

This paper explores the role of formin in determining the localisation of different tropomyosins to different actin polymers and cellular locations within budding yeast. Previous studies have indicated a role for the actin nucleating proteins in recruiting different forms of tropomyosin within fission yeast. In mammalian cells there is variation in the role of formins in affiecting tropomyosin localisation - variation between cell type. There is also evidence that other actin binding proteins, and tropomyosin abundance play roles in regulating the tropomyosin-actin association according to cell type. Biochemical studies have previously been undertaken using budding yeast and fission yeast that the core actin polymerisation domain of formins do not interact with tropomyosin directly.

The significance of this study, given the above, and the concerns raised is not clear to this reviewer.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

This manuscript by Bai et al concerns the expression of Scleraxis (Scx) by muscle satellite cells (SCs) and the role of that gene in regenerative myogenesis. The authors report the expression of this gene associated with tendon development in satellite cells. Genetic deletion of Scx in SCs impairs muscle regeneration, and the authors provide evidence that SCs deficient in Scx are impaired in terms of population growth and cellular differentiation. Overall, this report provides evidence of the role of this gene, unexpectedly, in SC function and adult regenerative myogenesis.

We appreciate the comments and thank her/him for the support.

There are a few minor points of concern.

(1) From the data in Figure 1, it appears that all of the SCs, assessed both in vitro and in vivo, express Scx. The authors refer to a scRNA-seq dataset from their lab and one report from mdx mouse muscle that also reveals this unexpected gene expression pattern. Has this been observed in many other scRNA-seq datasets? If not, it would be important to discuss potential explanations as to why this has not been reported previously.

Thanks for this question regarding data in Fig.1. We did initially use immunofluorescence staining of Pax7 and GFP on muscle sections and primary myoblast cultures prepared from Tg-ScxGFP mice to conclude that Scx was expressed in satellite cells (SCs). In addition to the cited mdx RNA-seq data, we have included a re-analysis of a published scRNA-seq data set in Fig.2E (Dell'Orso et al., Development, 2019), and our own scRNA-seq data (Fig.S5D, F). We have now re-examined an additional scRNA-seq data set of TA muscles at various regeneration time points (De Micheli et al., Cell Rep. 2020), in which Scx expression was detected in MuSC progenitors and mature muscle cells. We have added the De Micheli et al. reference and the re-analysis of that scRNA-seq data set for Scx expression as an additional panel in Fig. 2E, with accompanying text (p. 7, ln. 4-6). Thus, our immunostaining results are consistent with scRNA-seq data from our and two other independent scRNA-seq data sets.

We think that Scx expression in the adult myogenic lineage was not previously reported mainly because its expression level was low, and might be dismissed as spurious detection. Additionally, detecting such low expression levels requires sophisticated detection methods with high capture efficiency. Previous studies have noted limitations in transcript capture or transcription factor dropout in 10x Genomics-based datasets (Lambert et al., Cell, 2018; Pokhilko et al., Genome Res., 2021). The most likely and straightforward reason is that Scx was simply not a focus in prior studies amid so many other genes of interest. We have now added this last explanation in the text (p.7, ln. 8-9), following the re-analyses of Scx expression in published scRNA-seq data sets.

(2) A major point of the paper, as illustrated in Fig. 3, is that Scx-neg SCs fail to produce normal myofibers and renewed SCs following injury/regeneration. They mention in the text that there was no increased PCD by Caspase staining at 5 DPI. A failure of cell survival during the process of SC activation, proliferation, and cell fate determination (differentiation versus self-renewal) would explain most of the in vivo data. As such, this conclusion would seem to warrant a more detailed analysis in terms of at least one or two other time points and an independent method for detecting dead/dying cells (the in vitro data in Fig. 4F is also based on an assessment of activated Caspase to assess cell death). The in vitro data presented later in Fig. S4G, H do suggest an increase in cell loss during proliferative expansion of Scx-neg SCs. To what extent does cell loss (by whatever mechanism of cell death) explain both the in vivo findings of impaired regeneration and even the in vitro studies showing slower population expansion in the absence of Scx?

We appreciate these constructive suggestions. Based on the number of available control and cKO animals, we were limited to one additional time point at 3 dpi to assess PCD by TUNEL in vivo. We were disappointed again to find no appreciable levels of PCD at 3 dpi by TUNEL (new Fig.S4I), thus no quantifications were included. We also re-did the in vitro experiment using purified SCs and monitored PCD by staining for cleaved Caspase-3 using a validated tube of antibodies (positive staining after 6 h of treatment by 1 mM staurosporine of control and ScxcKO cells; included as new Fig. S4J and legend). We were pleased to find an increase of cleaved Caspase3 stained cells, i.e. PCD, of Scx-cKO SCs at day 4 in culture, compared to that of the control. We have now replaced the old Fig. 4F with new Fig.4F and 4G to document PCD. We also provided new text/legend for these new data (p.10. ln. 2-10; new legend for Fig. 4F and 4G).

(3) I'm not sure I understand the description of the data or the conclusions in the section titled "Basement membrane-myofiber interaction in control and Scx cKO mice". Is there something specific to the regeneration from Scx-neg myogenic progenitors, or would these findings be expected in any experimental condition in which myogenesis was significantly delayed, with much smaller fibers in the experimental group at 5 DPI?

We very much appreciate this comment. We agree that there is unlikely anything specific about the regeneration from Scx-negative myogenic progenitors. Unfilled or empty ghost fibers (basement membrane remnant) are expected due to small fiber and poor regeneration in the ScxcKO mice at 5 dpi. We have removed the subtitle and changed the content to an expected consequence rather than something special (p. 8, ln. 19-22).

(4) The data presented in Fig. 4B showing differences in the purity of SC populations isolated by FACS depending on the reporter used are interesting and important for the field. The authors offer the explanation of exosomal transfer of Tdt from SCs to non-SCs. The data are consistent with this explanation, but no data are presented to support this. Are there any other explanations that the authors have considered and that could be readily tested?

Thanks for highlighting this phenomenon. We struggled with the SC purity issue for a long time. The project started with using the R26RtdT reporter for tdT’s paraformaldehyde  resistant strong fluorescence (fixation) to aid visualization in vivo. Later, when we used the tdT signal to purify SCs by FACS, we found that only 80% sorted tdT+ cells are Pax7+. We then switched to the R26RYFP reporter, from which we achieved much higher purity (95%) of SCs (Pax7+) by FACS. As such, we also repeated and confirmed many in vivo experimental results using the R26RYFP reporter (included in the manuscript). Due to the low purity of tdT+SCs by FACS, we discontinued that mouse colony after we confirmed the superior utility of the R26RYFP reporter for SC isolation.

We sincerely apologize for not being able to conduct further testable experiments on this intriguing phenomenon. However, this issue has since been addressed and published by Murach et al., iScience, (2021). Like our experience, they found non-satellite mononuclear cells with tdT fluorescence after TMX treatment when SCs were isolated via FACS. To determine this was not due to off-target recombination or a technical artifact from tissue processing, they conducted extensive analyses. They found that the tdT+ mononuclear cells included fibrogenic cells (fibroblasts and FAPs), immune cells/macrophages, and endothelial cells. Additionally, they confirmed the significant potential of extracellular vesicle (EV)-mediated cargo transfer, which facilitates the transfer of full-length tdT transcript from lineage-marked Pax7+ cells to those mononuclear cells. We have modified the text to emphasize and acknowledge their contribution to this important point, and explained the difference between YFP and tdT reporter alleles in more detail (p.9, ln. 11-17).

(5) The Cut&Run data of Fig. 6 certainly provide evidence of direct Scx targets, especially since the authors used a novel knock-in strain for analyses. The enrichment of E-box motifs provides support for the 207 intersecting genes (scRNA-seq and Cut&Run) being direct targets. However, the rationale elaborated in the final paragraph of the Results section proposing how 4 of these genes account for the phenotypes on the Scx-neg cells and tissues is just speculation, however reasonable. These are not data, and these considerations would be more appropriate in the Discussion in the absence of any validation studies.

We agree with this comment and have moved speculations into the Discussion (p. 15, ln. 4-15, and from p. 18, ln. 4 to p. 19, ln. 4).

Reviewer #2 (Public Review):

Summary:

Scx is a well-established marker for tenocytes, but the expression in myogenic-lineage cells was unexplored. In this study, the authors performed lineage-trace and scRNA-seq analyses and demonstrated that Scx is expressed in activated SCs. Further, the authors showed that Scx is essential for muscle regeneration using conditional KO mice and identified the target genes of Scx in myogenic cells, which differ from those of tendons.

Strengths:

Sometimes, lineage-trace experiments cause mis-expression and do not reflect the endogenous expression of the target gene. In this study, the authors carefully analyzed the unexpected expression of Scx in myogenic cells using some mouse lines and scRNA-seq data.

We appreciate the comments and thank her/him for noting the strengths of our manuscript.

Weaknesses:

Scx protein expression has not been verified.

We are aware of this weakness. We had previously used Western blotting (WB) using cultured SCs from control and ScxcKO mice, but did not detect endogenous Scx protein even in the control. In response to this comment, we have re-done several WB experiments using new lysates from control and ScxcKO SCs and two commercial antibodies: anti-Scx antibody 1 from Abcam (ab58655) and anti-Scx antibody 2 from Invitrogen (PA5-23943). These antibodies have been reported to detect endogenous Scx protein in tendon cells in Spang et al., BMC Musculoskelet Disord (2016) and  Bochon et al., Int J Stem Cells (2021). Despite our best efforts, we were not able to detect a reliable Scx band. We have also conducted immunofluorescence using these two antibodies. Still, we failed to detect a difference of staining signals between control and cKO SCs using these antibodies. Lastly, we conducted immunofluorescence using the ScxTy1 myoblasts and we did not find the staining signal coinciding with the Ty1 signal (by double staining). We have been very frustrated by not knowing what caused this technical difficulty in our hands. Given that these were negative data, we did not include them. However, we do hope that the combined data from scRNA-seq, ScxCreERT2 lineage-tracing, Tg-ScxGFP expression, and ScxTy1 knock-in together are deemed sufficient to make up for the deficiency of data for endogenous Scx protein in regenerative myogenic cells.

Response to Recommendations for the Authors:

Reviewer #1 (Recommendations For The Authors):

p. 8: The text refers to Fig. 3I, but this should be Fig. 3H.

We apologize for the confusion. Please note that by keeping all 14 dpi data in the same row, we placed Fig.3I at an unconventional/unexpected position, i.e., next to 3D &3E, and above 3F-H. We were aware that this unconventional placement could cause confusion, and it did. With that said, we have now re-arranged the subfigures (same data content) so that the updated Fig.3 contains subfigures in the expected and proper spatial order. We double-checked the figure referral in the text (p. 8, ln. 16-17) and the text is correct – just that the original Fig.3I should have been at the original Fig.3H position and that is now corrected.

Reviewer #2 (Recommendations For The Authors):

(1) Given that Scx binds to the E-box and regulates gene expression, it is of interest to know the relevance between MyoD and Scx. If possible, the reviewer recommends to include some discussions.

Thanks for the comment. MyoD1 is a well-known transcript factor regulating myogenesis, whereas Scx is primarily studied in tenocytes and other connective tissues. We agree that our new findings deserve a discussion regarding the relevance between MyoD1 and Scx.  We have added a description of their differences in the discussion and two new references (p.19, ln. 7-17).

(2) Considering that Scx is a transcriptional factor, it is interesting that Scx-GFP was not detected in the nuclei of regenerated myofibers. Could the subcellular localization of Scx-GFP provide some insights into the function of Scx as a transcription factor during muscle regeneration?

Tg-ScxGFP is a transgenic line generated by random insertion into the genome (Pryce et al., 2007; cited). The plasmid used for transgenesis was constructed by replacing most of Scx’s first exon with GFP, and including ~ 9Kb flanking regulatory sequences. As such, the ScxGFP is not a fusion gene, but rather that the GFP expression is regulated by Scx promoter and enhancer(s). This GFP reporter lacks a nuclear localization signal (NLS), hence it is mainly detected in the cytoplasm; some nuclear signal is detected, presumably due to GFP’s small size permitting passive diffusion into the nucleus. Thus, the GFP signal is used as a reporter for Scx expression, but GFP subcellular localization does not provide insight into Scx function per se. Conversely, ScxTy1/Ty1 is a knock-in allele created by fusing a triple-Ty1 tag (3XTy1) to the C-terminus of Scx, and we observed that Ty1 is located in the nucleus by the immunofluorescent staining. We used the Ty1 epitope to carry out CUT&RUN experiments to gain insight to the function of Scx as a transcription factor.

(3) Fig1D The number of arrows in the Merge image is not matched with others. In addition, the star mark in the Pax7 image is likely an error.

Apologies. We have now corrected these errors in the revised Fig.1D.

(4) FigS1A Is there only one myofiber shown in the dashed line in this image? It is unclear why only this myofiber is surrounded by the dashed line.

The dashed line encircles a single fiber because it was not visible in the provided image. However, there are 3 fibers in this image. Because we did not immuno-stain for myofibers here, we circled one fiber for illustration. For clarity, we brightened the background (of the entire original images) so the background signals from myofiber boundaries are discernable without outlines.

(5) FigS1B There was no overlapped DAPI staining in the Myogenin+ cell. DAPI-staining should be present in Myogenin+ cells because myogenin is located in the nucleus.

Fig.S1B is immuno-staining for MyoD , and we marked one MyoD+DAPI+GFP+ cell/nucleus. Fig.S1C is immune-staining for Myogenin, and we also marked one (cell/nucleus) that is triple positive.

(6) The position of the asterisk for the ScxGFP in FigS1D is misaligned. In addition, the position is not matched with Fig1C. Because all myofibers are Scx-positive, it is strange that only one myofiber has an asterisk. The reviewer suggests removing the mark.

Thank you for pointing out these errors. We have now corrected the misalignment and removed the unnecessary asterisk.

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Reply to the reviewers

Response to Reviewers

We thank all three reviewers for their time and engagement, for their generally supportive comments, and for raising some important concerns. We are pleased to submit a significantly revised manuscript where we tried to accommodate all suggested changes and extensions. Importantly, we have included additional experiments that support the relevance of FACT for the overall stability of the inner kinetochore. Below is a detailed point-to-point response. Changes to the manuscript relative to the original submission have been highlighted at the end of this response.

__Reviewer #1 (Evidence, reproducibility and clarity (Required)): __

Summary: The authors investigated molecular interactions between CCAN and FACT complexes. They revealed contact domains in FACT and the cognate subcomplexes of CCAN by in vitro reconstitution from recombinant proteins followed by SEC and pull-down assay.

They also revealed a couple of potential means to control interactions between FACT and the CCAN. They conclude that phosphorylation of FACT by CK2 is essential for binding to the CCAN; and CENP-A nucleosomes or DNA prevent CCAN from interacting with FACT.

Major comments:

The authors show that phosphorylation of FACT is essential for interaction with CCAN.

They argue that this phosphorylation is partly catalysed by CK2.

My concerns are:

-1- The authors assume that the sites phosphorylated in insect cell are also phosphorylated in human cells. However, it is not demonstrated which residues are phosphorylated in human cells and whether they match those from insect cells. Whether phosphorylation of recombinant proteins in insect cells is physiologically relevant to mammalian is uncertain. Kinetochore components are not very well conserved evolutionarily, thus their regulation may be different.

We thank the reviewer for these remarks, which we answer together with point 2 below.

-2- They identify several residues which are phosphorylated by CK2 in vitro. However, these are not necessarily the same sites as those phosphorylated in insect cells or more importantly in human cells. The in vitro phosphorylation by CK2 did not restore binding affinity in full, suggesting phosphorylation at other sites may be critical for interaction with CCAN. Further evidence is required to support the claim that those sites are phosphorylated in vivo and important for integrity of kinetochores in mitosis.

Our analysis of FACT phosphorylation represents a relatively small part of a very data-rich paper, and was not meant to be exhaustive. Nonetheless, the reviewer's comments are important and well received. We agree that we have no definitive evidence that the same sites are phosphorylated in insect cells, in vitro, and in human cells. However, it is quite remarkable, and supports specificity, that the interaction with FACT, lost after dephosphorylation in vitro, is restored with CK2 and not with three additional mitotic kinases (CDK1, Aurora B, and PLK1 - Figure S8D). We also note that S437, S444 and S667 of SSRP1, which were phosphorylated by CK2 in vitro, were also detected as phosphorylated sites on recombinant FACT purified from insect cells (Table S1). So collectively, while we agree with the reviewer that the analysis of FACT phosphorylation is not complete, it does significantly add to the manuscript and more generally to the FACT field.

Minor comments:

Figure 1H

I am confused with 4 stars shown at the top of the right plot. If the 4 stars are meant to show a significant difference, then the statement in the text (line 123) is not correct.

"SSRP1 localization was also largely unaffected ..."

Similar discrepancies are found in Figures 3H (line 212), Figures S2 (line 122), S5I (line 197), and S6I (line209). Figure S6H is not referred to anywhere.

There is no description for the numbers at the top. Are they mean values? Do red bars represent S.D.?

We thank the reviewer for these comments. In this revised version of the manuscript, we have substantially improved the quantification and statistical analysis. The main problem with the previous automated analysis is that the non-circular shape of the CREST-staining led to inconsistencies with the statistical analysis and the statement. In contrast, the same analysis works well when the CENP-C signal was used for KT identification (e.g. in Figure 3), as CENP-C staining yields well separated circular signals ideally suited for our automated identification of individual KTs and subsequent retrieval of fluorescence intensities. We have therefore modified our analysis macro for all experiments where CREST was used as a reference. We used Othsu-thresholding of the DAPI signal for generating a segmentation mask per each cell. Then, integrated cell intensities were calculated for each fluorescence channel based on the DAPI reference mask. With these adjustments, the statistical analyses (Figures 1, S2, S3) support the claim presented. We have updated the Methods and Results sections to reflect the revised analysis.

The numbers on top of the graphs are median values, bars represent interquartile ranges. We have now included the description in figure legends.

We appreciate your feedback, which prompted us to clarify and enhance the rigor of our approach.

We are now referring to Fig. S6H in the text.

Figure 1D

There is no description of R* to the right of gels.

We have added a description of R* to the relevant figure legend.

Figure S2

A 4 hour nocodazole treatment is too short to drive all cells into mitosis. Is the data taken from mitotic cells only?

Yes, the data are taken only from the mitotic population. We have now clarified this in the figure legend.

Reviewer #1 (Significance (Required)):

The interaction of FACT with kinetochore components has been known for several years. However how FACT contributes to architecture or function of kinetochore is not very well understood. How the FACT complex, which is known for its established role as a histone chaperone, is involved in kinetochore assembly/architecture will attract interest in several fields of basic research including epigenetics, mitosis, structural biology.

We are grateful to the reviewer for this supportive statement that recognizes the broad potential interest of the manuscript.

Identification of CCAN subunits that interact with FACT is important for future analysis to understand the kinetochore function of FACT. The authors identified OPQRU and CHIKM subcomplex in addition to TW as FACT-interacting domains. These subcomplexes are geographically scattered in a 3D model of CCAN holocomplex. Stoichiometry of CCAN and FACT might be informative whether a single or multiple FACT binds to the multiple sites of CCAN. The authors do not address whether these multiple sites are occupied simultaneously, separately or sequentially.

We thank the reviewer for raising this point. As mentioned in the discussion, we have not yet been able to perform a structural analysis of the FACT/CCAN complex to determine its stoichiometry. However, we have now added a newexperiment (Figure S1B,C) where we quantified in-gel tryptophan fluorescence after analytical size-exclusion chromatography. This strongly suggests that FACT and CCAN form a complex with a 1:1 stoichiometry. Nevertheless, we cannot comment on which sites are occupied.

The statement at the end of Abstract (lines 23-25) is a speculative hypothesis without evidence for "a pool of CCAN that is not stably integrated into chromatin", "chaperoning CCAN", and "stabilisation of CCAN".

We agree with the reviewer that this is speculative, and have therefore modified the Abstract to clearly indicate this point.

__Reviewer #2 (Evidence, reproducibility and clarity (Required)): __

FACT is a histone chaperone and is involved in various events on chromatin such as transcription and replication. In addition, FACT interacts with various kinetochore components, suggesting potential functions at the kinetochore. However, it is largely unclear how FACT functions in the kinetochore. Authors of this MS took the biochemical approach to understand roles of FACT in the kinetochore.

Authors demonstrated that FACT forms a complex with the constitutive centromere associated network (CCAN), which contains 16 subunits on centromeric chromatin, using multiple binding sites. They also showed that casein kinase II (CK2) phosphorylated FACT and dephosphorylated FACT did not bind to CCAN. Furthermore, they displayed that DNA addition disrupt the stable FACT-CCAN complex.

Overall, while authors have done solid and high-quality biochemical analyses (these are elegant), it is still unclear how FACT plays its roles in the kinetochore. Simple knockout or knockdown study on FACT might be complicated, because FACT has multi-functions. If authors can identify specific regions of FACT for interaction with CCAN, they would put specific mutations into FACT to analyze phenotype. Although they did not reach a high-resolution structure for the FACT-CCAN complex, they can utilize AlphaFold and test specific interaction regions, biochemically. Then, using such information, significance of FACT-CCAN interaction might be testable in cells. Such a kind of study would be expected. In summary, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.

We thank the reviewer for praising the biochemical work in our manuscript. The reviewer, however, also underscored the limits of our functional analysis. The reviewer proposes generating separation-of-function mutants in a minimal kinetochore-binding region. Indeed, we have identified the minimal domain for the interaction of FACT with kinetochores. However, this information is insufficient for a reliable functional analysis at this stage, as the region we identified encompasses the AIDs and the phosphorylation-rich region, both of which have been previously shown to be important for transcription and other functions. Furthermore, any suitable mutant should be tested in cells devoid of endogenous FACT, raising the concern that the resulting phenotype may be indirect.

Nonetheless, as we wanted to provide at least an initial answer to the reviewer's concern, we enriched the manuscript by adding experiments in a recently published cell line (K562-SSRP1-dTAG) where FACT levels can be controlled with a small molecule (Žumer et al. Mol Cell., 2024) and that the authors generously shared with us. In this line, which grows in suspension and that we had to adapt to grow on a substrate for imaging, we were able to deplete FACT while cells were arrested in mitosis. We are glad to report that we found a significant reduction in the kinetochore levels of CENP-TW after this treatment, which is consistent with other conclusions from our study. These experiments add an initial functional characterization of the interaction of FACT with kinetochores, and extend the significance of the manuscript. We refer to these results again below in response to specific point 5.

Specific point

Authors showed nice mitotic localization of FACT. Can they observe this localization by a usual IF? Using GFP fusion, do they observe kinetochore localization like IF experiments?

The localization of FACT was observed using pre-extraction and fixation followed by antibody staining. We have now added a panel demonstrating mitotic localization of GFP-SSRP1 at the kinetochore in transiently transfected RPE-1 cells (Fig. S2A).

On page 7, authors tested CENP-C binding to FACT and they conclude that C-teminal region of CENP-C preferentially binds to FACT. However, they used N-terminal region of CENP-C (2-545) for CCAN-FACT complex formation in entire MS. therefore, this is complicated, and story on CENP-C N-terminal region can be removed from this MS.

We were only able to purify full-length CENP-C with tags at the N- and C-terminus, including an MBP tag with a stabilizing effect. At the time of our first successful purification of full-length CENP-C, we had already established the solid phase assay using MBPFACT as a bait on amylose beads and CENP-C2-545HIKM as one of the preys. As we cannot obtain stable full-length CENP-C without MBP, this form of CENP-C is incompatible with our pull-down assay. Nevertheless, CENP-C2-545 still has low affinity for FACT, influencing the FACT/CCAN interaction independent of the PEST-rich region. We, therefore, opted for keeping this information in the manuscript.

On page 9, authors suddenly focus on N-terminal tails of CENP-Q and CENP-U. Why did they focus on this region. They should explain this. If they perform a structural prediction, they should describe this point.

Thanks for raising this point. We focused on the N-terminal tails of CENP-QU because they are known interaction hubs. We have now added a sentence to introduce this concept and citing the appropriate literature.

I agree the fact that FACT phosphorylation is required for FACT-CCAN interaction. They may explain how the phosphorylation contributes to stable FACT-CCAN interaction.

We have added a sentence explaining that FACT is known to mimic DNA, and negative charges due to phosphorylation could drive this effect. A more detailed mechanistic understanding will require identifying specific phosphorylation sites required for the interaction.

Readers really want to know phenotype, if FACT-CCAN interaction was compromised without disruption pf CCAN assembly in cells. Although I agree that FACT has some functions in the kinetochore, it is still unclear what FACT does in the kinetochore.

We wholeheartedly agree with the reviewer. As depletion of FACT by RNAi required 48 h, an unreasonably long time for this multifunctional protein. We therefore turned to engineering RPE-1 cells for rapid degradation of SSRP1. While these attempts were unsucessful, earlier this year, Žumer et al. Mol Cell., 2024 reported generating a K562-SSRP1-dTAG cell line growing in suspension. As already reported, this cell line now allowed to demonstrate a significant effect on the kinetochore stability of CENP-TW upon mitotic depletion of FACT.

Reviewer #2 (Significance (Required)):

As mentioned above, biochemical parts are beautiful, but the paper did not address significance of FACT-CCAN interaction.

We thank the reviewer for this positive assessment. In this revision, we have obtained initial evidence that FACT contributes to kinetochore stability.

__Reviewer #3 (Evidence, reproducibility and clarity (Required)): __

Main findings:

The major findings of this paper are:

Detailed dissection of CCAN subunit interactions and requirements to bind the FACT complex using in vitro reconstituted components Binding of FACT and nucleosomes to CENP-C are mutually exclusive FACT phosphorylation by CK2 enhances interaction with CCAN FACT localization in mitosis depends on the CCAN CCAN binding to FACT is outcompeted by DNA and CENP-A nucleosomes The claims and conclusions of the paper are supported by the data and do not require additional experiments. All experiments include biological replicates and appropriate controls.

We are thankful to the reviewer for this very positive assessment of our work.

Minor comments

Intro: • Line 81: In humans [...], here it is worth mentioning that in Drosophila, FACT subunits have been shown to interact directly with the CENP-A assembly factor CAL1 (Ref 61). This paper is perfunctorily cited once in the context of its implication of FACT in CENP-A deposition, but it merits more consideration when setting up the foundational context for the present work.

We have extended the Introduction and discuss the specified paper more thoroughly.

Figure 1:

1F: Add insets.

Done.

1G and all other figures containing IFs: Avoid red/green color scheme (red-green colorblindness is fairly common, affecting about 8% of men).

Done.

1E: Please add a table summarizing interactions.

We have included this table as Fig. S1E.

Results: • It's fine to direct readers to previous work in which you reconstituted the CCAN, but the text should mention how proteins are exogenously expressed and purified (as done for FACT in line 247).

Done.

Line 113: FACT has been shown to localize to the mitotic kinetochore also in Drosophila (Ref 61).

We have included this information now.

Line 132: The authors should cite work from the Drosophila system as well when they mention centromere transcriptional activity in mitosis (e.g. https://doi.org/10.1083/jcb.201404097; https://doi.org/10.1083/jcb.201611087; and Ref 61).

We have added these citations.

Figure 2F: The authors could use a line to mark the region interacting with FACT and that interacting with CENP-A to help summarize the data in this diagram.

Done.

Figure 4: Highlight constructs n.2 (FACT^TRUNC) since these are sufficient for interaction (e.g., use a box around them).

Done.

Line 276: "CCAN decodes CENP-A^NCP..." What do the authors mean by "decodes"? This whole sentence would benefit from clearer language.

We thank the reviewer for this suggestion and have aimed for clearer language.

Figure 6: There's a lot of information in these experiments that would benefit from two schematics, one showing the mechanism of FACT + CCAN binding with DNA and one with CENP-A nucleosomes.

Done.

Discussion: The authors discuss FACT localization at kinetochores in mitosis. In Drosophila Schneider cells, FACT is observed enriched at the centromeres in both mitosis and interphase (Ref 61). The authors mention their inability to detect FACT in interphase in the discussion, but I did not find this mentioned in the results. The authors state that FACT "redistributes to the entire chromosome" upon entry into interphase. They cite Figure 1F in reference to this statement, but the staining in the early G1 panel is difficult to interpret with the low signal/noise scaling of CENP-C and the lack of zoom insets. Their protocol uses a pre-extraction step with Triton prior to fixation. Apparently, this was not enough to reveal FACT in interphase, but better images and a brief description are warranted.

We have now added a staining of SSRP1 in interphase in the panel.

It is unlikely that FACT would change its localization pattern in mitosis. A more likely possibility is that in mitosis FACT is not redistributed, but rather more tightly bound (and thus less easily extracted by Triton treatment) at kinetochores, while along the arms FACT is more readily removed by extraction because at this time transcription is repressed and FACT is likely less engaged in transcription-mediated histone destabilization.

We thank the reviewer for these remarks and have updated the Discussion.

Given the well-known function of FACT in transcription and the many studies linking transcription to centromere maintenance, including with the involvement of FACT, the model that "the localization of FACT at the kinetochore coincides with active centromeric transcription in mitosis and interphase" is very tempting. A speculative model would go a long way to help the reader visualize all these complex aspects of FACT's interactions and possible functions.

We agree with the reviewer that such a model is tempting. However, we also feel that it would be rather speculative at this stage and we feel that the manuscript does not provide sufficient data to support the model.

Reviewer #3 (Significance (Required)):

The strongest aspect of the study is the detailed characterization of protein-protein interactions, as well as competition with DNA and CENP-A nucleosomes. The siRNA experiments in cells complement this largely in vitro study. However, a limitation of the study is that it does not shed light on what FACT might be doing at the centromere. Additionally, it does not sufficiently provide context for these findings in relation to previous studies that have demonstrated the roles of FACT at the centromere in budding yeast, fission yeast, and Drosophila. Nonetheless, this study provides valuable insights into the details of FACT interactions at the kinetochore and will be of interest to readers interested in centromeres and kinetochore. I am a centromere biologist with molecular and cell biology expertise.

We are very grateful to the reviewer for his/her support.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

(1) The technology requires a halo-tagged derivation of the active compound, and the linked position will have a huge impact on the potential "target hits" of the molecules. Given the fact that most of the active molecules lack of structure-activity relationship information, it is very challenging to identify the optimal position of the halo tag linkage.

We appreciate your insightful comment. While finding the optimal position to attach a chemical linker to a small molecule of interest is indeed a challenging but necessary step, this is a common difficulty across all target-ID methods, except for those that are modification-free, as we described in Discussion. However, modification-free approaches such as DARTS, CETSA, and TPP have their own limitations, such as low sensitivity and a high false-positive rate. Additionally, DARTS and SPROX are limited to use with cell lysates. Please refer to the introduction in our manuscript for more details on these approaches. On the other hand, synthesizing HTL derivatives is relatively straightforward compared to other modifications, and we provide helpful guidelines for chemical linker design, provided the optimal chemical moiety has been identified, which is crucial for target identification. We selected dasatinib and HCQ/CQ as model compounds because previous studies offered insights into their derivative synthesis. Our data also show that DH5 retains strong kinase inhibitory activity (Figure 4—figure supplement 2), and DC661-H1 demonstrates potent inhibition of autophagy (Figure 6—figure supplement 1). For novel compounds, conducting a thorough structure-activity relationship (SAR) study is essential to determine the optimal position for HTL derivative synthesis.

(2) Although POST-IT works in zebrafish embryos, there is still a long way to go for the broad application of the technology in other animal models.

Thank you for your constructive comment. Yes, there is still a long way to go in developing the POST-IT system for broader applications in other animal models, especially in mice. However, we hope that our study provides valuable insights and inspiration to scientists and experts for applying the POST-IT system in various models. We are also committed to further improving its applicability.

(3) The authors identified SEPHS2 as a new potential target of dasatinib and further validated the direct binding of dasatinib with this protein. However, considering the super strong activity of dasatinib against c-Src (sub nanomolar IC50 value), it is hard to conclude the contribution of SEPHS2 binding (micromolar potency) to its antitumor activity.

Thank you for your insightful comment. We agree that the anticancer activity of dasatinib primarily results from inhibiting tyrosine kinases such as SRC and ABL. However, SEPHS2 contains an “opal" termination codon, UGA, at the 60th amino acid residue, which codes for selenocysteine. Due to the technical challenge of expressing selenoproteins in E. coli, we mutated it to cysteine for expression in E. coli to avoid premature translation termination, as described in the Materials and Methods section. Although the purified recombinant SEPHS2 shows a Kd of about 10 µM for dasatinib, the binding affinity to endogenous SEPHS2 may be higher since selenocysteine is larger and more electronegative than cysteine. This presents an interesting area for future investigation. Furthermore, our study of dasatinib’s binding to SEPHS2 could help facilitate the development of new SEPHS2 inhibitors, potentially targeting the active site of SEPHS2.

Reviewer #3 (Public review):

(1) Target Specificity: It is crucial for the authors to differentiate between the primary targets of the POST-IT system and those identified as side effects. This distinction is essential for assessing the specificity and utility of the technology.

Thank you for your insightful comment. Drugs inevitably bind to various proteins with differing affinities, which can contribute to both side effects and beneficial outcomes. Typically, the primary targets exhibit high affinities. In this manuscript, we ranked the identified protein targets of DH5 based on affinity from mass spectrometry and p-values (Fig. 5A), and for DC661-H1, we used the SILAC ratio (Fig. 6A). We also individually assessed many drug-protein binding affinities using the MST assay, as well as in vitro and in cellulo assays, demonstrating their specificity. Moreover, we believe it is essential to identify as many protein targets as possible at physiological drug concentrations to better understand the drug’s side effects. Of course, further investigation is required to assess the roles and effects of these target proteins.

(2) In Vivo Target Identification: The manuscript lacks detailed clarity on which specific targets were successfully identified in the in vivo experiments. Expanding on this information would provide a clearer view of the system's effectiveness and scope in complex biological settings.

Thank you for your insightful comment regarding in vivo target identification. In this manuscript, we utilized a cell line as the primary method for in vivo target identification and validation after optimizing our system in test tubes. We successfully validated many of the targets identified using our POST-IT system (Figure 6—figure supplement 3). To demonstrate the proof of principle for in vivo application, we employed zebrafish embryos as an in vivo model, showing that endogenous SRC can be effectively pulled down by DH5 treatment (Fig. 7). While we could have explored the entire proteome to identify endogenous target proteins in zebrafish that bind to DH5 or dasatinib, we felt this would extend beyond our original scope, given that we have already demonstrated POST-IT’s ability to identify target proteins for dasatinib. Specific target identification and validation are crucial when using zebrafish for drug discovery. Additionally, we acknowledge that drugs likely interact with a range of protein targets in living organisms and may undergo metabolism and interactions within the circulatory system, which we address in our discussion.

(3) Reproducibility and Scalability: Discussion on the reproducibility of the POST-IT system across various experimental setups and biological models, as well as its scalability for larger-scale drug discovery programs, would be beneficial.

Thank you for the suggestion. While our system has shown  high reproducibility in our experiments, further improving both reproducibility and scalability would be advantageous. One potential approach to address this is through the generation of stable-expressing cell lines and transgenic zebrafish lines, which we have discussed in the revised manuscript. Establishing stable cell lines with robust POST-IT expression could enhance scalability for drug discovery applications.

(4) Quantitative Analysis: A more detailed quantitative analysis of the protein interactions identified by POST-IT, including statistical significance and comparative data against other technologies, would enhance the manuscript.

Thank you for your suggestion. In our assessment of drug-protein affinity, we included Kd values as quantitative measures using MST assays. The protein targets of dasatinib identified through mass spectrometry are also accompanied by p-values for quantitative analysis (Fig. 5A), and the detailed procedures are described in the Material and methods section. While it is challenging to provide direct comparative data against other technologies, our system successfully identified many known target proteins for dasatinib, as well as SEPHS2 and VPS37C as new targets for dasatinib and for HCQ/CQ, respectively, which were not detected by other methods.

(5) Technological Limitations: The authors should discuss any limitations or potential pitfalls of the POST-IT system, which would be crucial for future users and for guiding subsequent improvements.

Thank you for your insightful suggestion We agree that clearly defining the technological limitations is important. Therefore, we have expanded our original discussion on the limitations of our POST-IT system (Discussion section, paragraph 6).

(6) Long-Term Stability and Activity: Information on the long-term stability and activity of the POST-IT components in different biological environments would ensure the reliability of the system in prolonged experiments.

Yes, this is an important question. We did not notice any stability or toxicity issues with Halo-PafA and Pup substrates in HEK293T cells or zebrafish, which is an important factor for stable cell lines and transgenic zebrafish lines. However, HTL derivatives of the drug could be toxic or unstable due to the nature of the drug or its metabolism, which needs to be taken into account when designing experiments, and we have included this in the Discussion.

(7) Comparison with Existing Technologies: A detailed comparison with existing proximity tagging and target identification technologies would help position POST-IT within the current landscape, highlighting its unique advantages and potential drawbacks.

We appreciate your valuable feedback and agree that such comparisons are crucial. We have included a detailed overview and comparison of existing proximity-tagging systems and their related target identification technologies in the Introduction (lines 78-100) and Discussion (lines 391-412), highlighting their respective pros and cons. Additionally, we have expanded the discussion to further compare these technologies with our POST-IT system, addressing its advantages and limitations (lines 378-390, lines 448-467). We hope this provides sufficient context and information to effectively position POST-IT among the landscape of proximity-tagging target identification technologies.

(8) Concerns Regarding Overexposed Bands: Several figures in the manuscript, specifically Figure 3A, 3B, 3C, 3F, 3G, Figure 4D, and the second panels in Figure 7C as well as some figures in the supplementary file, exhibit overexposed bands.

We appreciate your astute observation regarding the overexposed bands and apologize for any confusion. The “overexposed” bands represent the unpupylated proteins, while the bands above them correspond to the pupylated proteins. We intended to clearly show both pupylated and unpupylated bands, although the latter are generally much weaker. We are currently working on further improving our POST-IT system to enhance pupylation efficiency.

(9) Innovation Concern: There is a previous paper describing a similar approach: Liu Q, Zheng J, Sun W, Huo Y, Zhang L, Hao P, Wang H, Zhuang M. A proximity-tagging system to identify membrane protein-protein interactions. Nat Methods. 2018 Sep;15(9):715-722. doi: 10.1038/s41592-018-0100-5. Epub 2018 Aug 13. PMID: 30104635. It is crucial to explicitly address the novel aspects of POST-IT in contrast to this earlier work.

Thank you for bringing this to our attention. Proximity-tagging systems like BioID, TurboID, NEDDylator, and PafA (Lui Q et al., Nat Methods 2018) were initially developed to study protein-protein interactions or identify protein interactomes, as these applications are of broader interest and generally easier to implement. However, applying proximity-tagging systems for small molecule target identification requires significant optimization. As described in the introduction (lines 78-100), target protein identification systems have since been developed using TurboID and NEDDylator (Tao AJ et al., Nat Commun 2023; Hill ZB et al., J Am Chem Soc 2016). It is conceivable that a PafA-based proximity-tagging system could also be adapted for target-ID, and other groups may pursue this approach in the future. Although the PafA-Pup system shows great promise for target-ID applications, extensive optimization was needed to enable its use for this purpose. Finally, we demonstrate that POST-IT offers distinct advantages over other proximity-tagging-based target-ID systems. For more details, please refer to the introduction and discussion sections.

Recommendations for the authors:

Reviewer #2 (Recommendations for the authors):

(1) Figure 1- Figure Supplement 1A: The Pup substrate "HB-Pup" is mentioned, but the main text or figure legend provides no introduction or description.

We appreciate your astute observation. We have added a description in the main text and figure legend as follows: “…and used HB-Pup as a control, which contains 6´His and BCCP at the N terminus of Pup” in the main text (line 142) and “HB, TS, and SBP refer to 6´His and BCCP, twin-STII (Strep-tag II), and streptavidin binding peptide, respectively.” in the Figure 1-figure supplement 1A.

(2) Figure 1 - Figure Supplement 3B: The authors used TS-sPupK61R as a substrate but did not explain why. The main text mentions that mutating sPup alone did not affect polypupylation, raising the question of why TS-sPupK61R was used in this figure. Furthermore, while the authors state that polypupylation becomes evident after 1 hour of incubation (more pronounced after 2 or 3 hours), the reactions here were conducted for only 30 minutes.

Thank you for your question. Figure 1 - Figure Supplement 3B was conducted to test self-pupylation levels in the different Halo-PafA derivatives. For this purpose, we could use any Pup substrate such as SBP-sPup and SBPK4R-sPupK61R, instead of Ts-sPup and TS-sPupK61R, as they do not show any differences in pupylation activity. We chose Ts-sPup and TS-sPupK61R simply because any Pup substrates could be used for this purpose. Similarly, we did not need to incubate the reaction for a longer time to detect polypupylation, as our intention was to test “self-pupylation”. We demonstrated in Figure 1 – figure supplement 2 that polypupylation is dependent on the number or position of lysine residues in Pup substrate or tags. The results clearly showed that self-pupylation was almost completely abolished by the Halo8KR mutation. To clarify this, we added the following description in lines 168-169: “Ts-sPup and TS-sPupK61R were chosen as sPup substrates for this experiment, although any Pup substrates could have been used. The levels of self-pupylation were assessed.”

(3) Line 156: The statement that "the TS-tag completely abolished polypupylation in TS-sPup" is inaccurate. Using TSK8R-sPupK61R as the substrate, several bands appear, which likely represent Halo-PafA with varying degrees of polypupylation. Some bands also appear to correspond to those seen when using TS-sPup as a substrate. The authors should clarify how they distinguish between multipupylation and polypupylation in this case.

We sincerely appreciate your insight into clarifying the distinction between multipupylation and polypupylation. Polypupylation refers to the addition of a new Pup onto a previously linked Pup on the target protein, akin to polyubiquitination. In contrast, multipupylation involves multiple single pupylations at different positions on the target proteins. Since pupylation occurs exclusively at lysine residues in tag-Pup substrates, mutating all lysine residues to arginine, as in TSK48R-sPupK61R, prevents the mutant tag-Pup from linking to another Pup. This means that only single pupylation can proceed with this type of mutant Pup substrate. If multiple pupylated bands are observed with this mutant substrate, it indicates “multipupylation” rather than “polypupylation”, as shown in Figure 1-figure supplement 2D. The same applies to the pupylation bands in Figure 1-figure supplement 2E and F, as sSBP-sPupK61R and SBPK4R-sPupK61R lack lysine residues. By comparing these multipupylation bands, it is also possible to distinguish them from polypupylation bands, which are marked by yellow arrows. However, after 2-3 pupylation bands, higher-order bands become increasingly difficult to distinguish.

To clarify the mutation in the TS-tag, we revised the sentence in line 156 from “However, further mutations within the TS-tag completely abolished polypupylation in TS-sPup” to “However, further mutations of two lysine residues within the TS-tag, creating TSK8R-sPupK61R, completely abolished polypupylation in TS-sPup”. Additionally, we have inserted sentences in line 152 to define polypupylation and multipupylation, as described here.

(4) Line 160: Similar to the above concern about line 156, the claim that SBPK4R and sSBP completely prevented polypupylation is unconvincing and requires more supporting evidence.

Thank you for raising this concern. As mentioned above, both SBPK4R and sSBP lack lysine residues required for pupylation. As a result, these mutants can only undergo multiple single pupylations on the lysine residues of the target protein, which leads to “multipupylation”. In Figure 1-figure supplement 2E and F, pupylation bands by sSBP-sPupK61R or SBPK4R-sPupK61R do not display doublet bands (one from multipupylation and the other from polypupylation), as seen with SBP-sPup, marked by yellow arrows. Notably, Halo-PafA containing polypupylated branches migrates more slowly than one with an equal number of multipupylation events. To clarify this point, we have added the phrase “as shown in sSBP-sPupK61R and SBP4KR-sPupK61R” at the end of the sentence in line 160.

(5) Lines 176-177: The authors claim that PafAS126A exhibited reduced polypupylation compared to PafA, but given that PafAS126A may reduce depupylase activity, how could it reduce polypupylation levels? Moreover, it is hard to find any data supporting this conclusion in Figure 1 - Figure Supplement 3B.

We appreciate your insightful comment. At this point, we do not fully understand how the mutation that reduces depupylase activity also decreases polypupylation. It is possible that PafAS126A has a lower preference for pupylated Pup as a prey, which is required for polypupylation, since depupylase activity depends on recognizing pupylated Pup as a prey to remove it. Nonetheless, Halo-PafAS126A shows reduced levels of higher molecular weight bands compared to Halo-PafA, as shown in Figure 1-figure supplement 3B, while exhibiting increased pupylation in lower molecular weight bands, which represent either multipupylation or low-degree polypupylation. Since higher molecular weight bands (> 150 kD) are likely due to polypupylation, this result suggests reduced polypupylation and increased multipupylation in Halo-PafAS126A. To clarify this in the main text, we have added the following description in line 177: “as evidenced by the decreased levels of high molecular weight bands and an increase in low molecular weight bands”

(6) POST-IT system in cellulo validation: The system was developed using the Halo-tag, yet the in-cell validation uses FRB and FKBP instead, without explaining this switch. This inconsistency makes the logic of the experiment unclear.

We appreciate your insightful comment. The interaction between rapamycin and FRB or FKBP is known to be highly specific and robust, making this system useful in various biological contexts. Due to this property, rapamycin can induce interaction between two proteins when one is fused with FRB and the other with FKBP. Before testing or optimizing the POST-IT system in cells, we hypothesized that using the rapamycin-induced interaction between FRB and FKBP could introduce pupylation of the target protein, provided that PafA is fused with FRB or FKBP and the target protein is fused with the other. The results demonstrate that PafA can introduce pupylation of the target protein in a proximity-dependent manner via this chemically induced interaction. To further clarify this in the main text, we modified the original sentence in lines 214-216 as follows: “To mimic drug-target interaction-induced pupylation in live cells and assess the potential of PafA as a proximity-tagging system for target-ID, we incorporated the rapamycin-induced interaction between FRB and FKBP into our PL system, as this interaction between a small molecule and a protein is known to be highly specific and robust (Figure 3—figure supplement 1A).”

(7) Line 209: The authors decided to use the SBP-tag for further studies due to better performance, but in Figure 3 - Figure supplement 1, they still used the unintroduced HB-Pup as the substrate, which is confusing and lacks explanation.

Thank you for raising your question. The SBP-tag is not superior to the TS-tag in terms of pupylation activity. However, the TSK8R mutant cannot bind to Strep-Tactin beads, while the SBP mutants, SBPK4R and sSBP, can bind to streptavidin. Therefore, we chose the SBP-tag instead of the TS-tag for further studies as a Pup substrate in POST-IT system, as we needed to pull down the target proteins. HB-Pup is consistently used as a control throughout various experiments, as it is the original Pup substrate. In Figure 3-figure supplement 1B and C, HB-Pup was used to test chemically induced pupylation by PafA. In these cases, it was not so critical which Pup substrate was chosen. Furthermore, we compared HB-Pup and different SBP-sPup substrates in Figure 3-figure supplement 1D, where HB-Pup was used as a control or for comparison. Although pupylation bands with HB-Pup appear more robust, this substrate contains multiple lysine residues, leading to high levels of polypupylation. To make it clear, we modified the sentence in line 209 to “Therefore, we decided to use the SBP-tag as a Pup substrate in the POST-IT system for further studies.”.

(8) Line 220: Both SBP-sPup and SBPK4R-sPupK61R are described as exhibiting efficient pupylation, but the data show mostly self-pupylation and little to no pupylation of the target protein.

Thank you for your concern. However, pupylation of the target protein is actually quite substantial, as the intensities of the free form and pupylated proteins are relatively similar, as shown in the upper panel of Figure 3-figure supplement 1D. Self-pupylation is always much higher than target pupylation, because PafA constantly pupylates itself, whereas pupylation of the target protein occurs only through interaction. Furthermore, V5-FRB-mKate2-PafA contains many lysine residues, which increases the levels of self-pupylation.

(9) Lines 222-224: The authors chose SBPK4R-sPupK61R to avoid polypupylation, although SBP-sPup did not cause detectable polypupylation. Neither substrate caused pupylation of the target protein, so the rationale behind this choice is unclear.

Thank you for raising your question. Similar to the above comment (#8), please refer to the pupylation bands of the target protein, as shown in the upper panel of Figure 3-figure supplement 1D. The pupylation band of the target protein is quite remarkable, as the intensities of the free form and pupylated proteins are comparable. Additionally, there are no multiple pupylation bands in either case, except for one additional weak multipupylation band, indicating no polypupylation by SBP-sPup, which does not have K-to-R mutations. Of course, SBPK4R-sPupK61R can only undergo single pupylation, as it does not contain lysine residues. Although we did not observe polypupylation by SBP-sPup in this experimental condition, it is possible that SBP-sPup may cause polypupylation under different experimental conditions or with other target proteins. Since SBPK4R-sPupK61R exhibits comparable pupylation of the target protein at least in this experiment setting as SBP-sPup, we selected SBPK4R-sPupK61R as the Pup substrate for POST-IT system to avoid any potential polypupylation that could be caused by SBP-sPup in other cases. We believe that polypupylation can introduce bias into the analysis and hinder the comprehensive discovery of additional target proteins for small molecules.

(10) Line 224: The authors conclude that rapamycin greatly reduced self-pupylation, but the supporting data are unclear.

Thank you for your constructive comments on our manuscript. Please refer to the lower panel of Figure 3-figure supplement 1D. When using either SBPK4R-sPupK61R or SBP-sPup, rapamycin treatment results in reduced levels of self-pupylation compared to the no-treatment control. However, we did not observe this reduction with HB-Pup and do not know the reason. To clarify this in the main text, we added the following description to the end of the sentence: “when using either SBPK4R-sPupK61R or SBP-sPup, as shown in the lower panel of Figure 3—figure supplement 1D”

(11) Line 234: The authors selected an 18-amino acid linker, but given that linkers longer than 10 amino acids enhance labeling, this choice should be explained.

Thank you for raising your question. In fact, a linker of 10 amino acids (aa) or longer is likely to behave similarly. We chose an 18 aa linker instead of a 40 aa linker primarily for the convenience of cloning and to reduce the potential for DNA sequence recombination associated with longer repeats. Additionally, a longer, flexible linker may behave like an intrinsically disordered protein (Harmon et al., 2017), which can lead to unwanted protein-protein interactions or phase separation. To elaborate on this, we added the following sentences after the sentence in line 233-235: “We chose the 18-amino acid linker instead of the 40-amino acid linker for easier cloning and to lower the risk of DNA recombination from longer repeats. Additionally, a longer, flexible linker may behave like an intrinsically disordered protein (Harmon et al., 2017), an unwanted feature for target-ID.”

(12) S126A and K172R mutations: The authors claim that these mutations additively enhanced pupylation under cellular conditions, but in Figure 3B, the band intensities appear similar for the wild-type and mutant versions.

Thank you for raising your concern. Although a single pupylation band appears similar among the three different Halo-PafA proteins, multipupylation bands are slightly but noticeably increased by the S126A and K172R mutations compared to Halo8KR-PafA. Since we used SBPK4R-sPupK61R as a Pup substrate, all higher molecular weight bands result from multipupylation rather than polypupylation. This illustrates why it is preferable to use SBPK4R-sPupK61R over SBP-sPup, as the pupylation bands with SBP-sPup are mixtures of poly- and multipupylation, making it difficult to assess levels of target labeling. To clarify this in the main text, we added the following description after the sentence in line 236: “as the higher molecular weight multipupylation bands are slightly but noticeably increased with these mutations compared to Halo8KR-PafA”

(13) Line 263: The authors selected DH5 for further experiments due to its efficiency, but the data suggest that the performance of DH1 to DH5 is similar.

We appreciate your question about the different dasatinib HTL derivatives. However, our data clearly show that DH2-5 derivatives bind significantly more effectively to Halo-PafA in vitro and in live cells compared to DH1 (Figure 4A and B). Additionally, the DH2-5 derivatives result in dramatically increased pupylation of the target protein in vitro and noticeable enhancement in live cells (Figure 4C and D). Among DH2 to DH5, there is no obvious difference in binding to Halo-PafA or pupylation of the target protein. Therefore, we chose DH5, as we believe that the longer linker in DH5 may facilitate the binding of a more diverse range of target proteins to dasatinib, enabling the discovery of additional target proteins.

(14) Line 309: The authors introduce HCQ and CQ as important drugs but then investigate the mechanism using DC661 without introducing or justifying the choice of this compound.

Thank you for your point. We explained the reason to choose DC661, a dimer form of CQ, instead of CQ for the synthesis of an HTL derivative in line 310. “assuming that a dimer would enhance binding affinity as previously described.” As the dimer forms of a drug or a small molecule such as testosterone dimers, estrogen dimers, and numerous anticancer drug dimers have been often developed to enhance drug effects (Paquin A et., Molecules 2021). Similarly, dimer forms of HCQ/CQ have been introduced and shown to be more potent (Hrycyna CA et al., ACS Chem Biol 2014; Rebecca VW et al., Cancer Discovery 2019). We expected that using a dimer form might offer higher probability to identify target proteins for HCQ/CQ.

(15) The authors suggest that multipupylation levels were enhanced but do not explain whether this might benefit the system or introduce other issues. Clarifying this point would provide valuable insight for potential users of this system.

Thank you for your thoughtful suggestion. Polypupylation likely leads to biased enrichment of a limited set of target proteins, and its levels may not correlate with the binding affinity of target proteins to the small molecule of interest, features that can negatively impact target-ID. In contrast, multipupylation may be correlated with binding affinity or interaction frequency, as we observed increased levels of multipupylation with higher Pup concentrations and longer incubation times. This suggests that target proteins with multiple lysines in proximity to PafA can be sequentially pupylated, starting with the most accessible lysine. However, if a target protein has only one accessible lysine, pupylation will occur only once, regardless of the protein’s affinity to the small molecule. In summary, while polypupylation may be a drawback for target-ID, multipupylation could be useful for both target-ID and understanding binding mode. To elaborate on this, we added the following additional explanation after the sentence in line 152: “, whereas multipupylation is more likely correlated with binding affinity or interaction frequency.”

(16) The author should address whether the Halotag ligand modification of the drug alters the binding properties between the drug and targets. That may be causing artifact binding of the drug and other proteins.

Thank you for your insightful comment. Yes, it is true that chemical modifications of the small molecule of interest, such as linker derivatization (e.g., HTL) or photo-affinity labeling, generally lead to reduced activity or affinity compared to the original molecule. Synthesizing a derivative is a common challenge across all target-ID methods, except for modification-free approaches, as we mentioned in the Discussion. However, modification-free methods like DARTS, CETSA, and TPP have their own limitations, including low sensitivity or high false positive rates. Identifying the optimal position for chemical modification on the small molecule of interest is critical. We chose dasatinib and HCQ/CQ as model compounds, because previous studies provided insights into their derivative synthesis. In addition, our data show that DH5 retains robust kinase inhibitory activity (Figure 4-figure supplement 2), and DC661-H1 exhibits potent autophagy inhibition (Figure 6-figure supplement 1). For novel compounds, a thorough structure-activity relationship study is essential to identify the optimal position for HTL derivative synthesis.

(17) The author stated there is no observable toxicity in zebrafish without providing a detailed analysis or enough data. Further analysis of the expression of Halo-PafA and its substrate sPup influence on toxicity or side effects to the living cells or animals would be needed. It is important for in vivo applications.

Thank you for your constructive suggestion. We have now included additional experimental data in Figure 7-figure supplement 1, showing no toxicity in zebrafish embryos expressing the POST-IT system. We assessed toxicity in two ways: by injecting the POST-IT DNA plasmid into one-cell-stage embryos for acute expression, and by using embryos from transgenic zebrafish expressing POST-IT under a heat-shock inducible promoter. Neither the injection nor the heat-shock activation of POST-IT expression resulted in any noticeable toxicity.

Author response:

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

Reviewer #1 (Public Review):

This study provides compelling evidence that RAR, rather than its obligate dimerization partner RXR, is functionally limiting for chromatin binding. This manuscript provides a paradigm for how to dissect the complicated regulatory networks formed by dimerizing transcription factor families.

Dahal and colleagues use advanced SMT techniques to revisit the role of RXR in DNA-binding of the type-2 nuclear receptor (T2NR) RAR. The dominant consensus model for regulated DNA binding of T2NRs posits that they compete for a limited pool of RXR to form an obligate T2NR-RXR dimer. Using advanced SMT and proximity-assisted photoactivation technologies, Dahal et al. now test the effect of manipulating the endogenous pool size of RAR and RXR on heterodimerization and DNA-binding in live U2OS cells. Surprisingly, it turns out that RAR, rather than RXR, is functionally limiting for heterodimerization and chromatin binding. By inference, the relative pool size of various T2NRs expressed in a given cell, rather than RXR, is likely to determine chromatin binding and transcriptional output.

The conclusions of this study are well supported by the experimental results and provide unexpected novel insights into the functioning of the clinically important class of T2NR TFs. Moreover, the presented results show how the use of novel technologies can put long-standing theories on how transcription factors work upside down. This manuscript provides a paradigm for how to further dissect the complicated regulatory networks formed by T2NRs or other dimerizing TFs. I found this to be a complete story that does not require additional experimental work. However, I do have some suggestions for the authors to consider.

Reviewer #1 (Recommendations For The Authors):

(1) Does the increased chromatin binding measured when the RAR levels are increased reflect a higher occupancy of a similar set of loci, or are additional loci bound? The authors could discuss this issue in the context of the published literature. Obviously, this could be addressed experimentally by ChIP-seq or a similar analysis, but this would extend beyond the main topic of this manuscript.

We attempted to explore this experimentally using ChIP-seq with multiple RAR- and RXR-specific antibodies. Unfortunately, our results were inconclusive, as the antibody enrichment relative to the IgG control was insufficient for reliable interpretation. Specifically, our ChIP-seq enrichment levels were only around 1.5fold, while the accepted standard for meaningful ChIP enrichment is typically at least 2-fold. Due to these technical limitations, we decided to defer these experiments for now.

However, we agree with the reviewer that understanding whether the increased chromatin binding of RAR reflects higher occupancy at the same set of loci or binding to additional loci is a key question. In similar experiments involving the transcription factor TFEB (Esbin et al., 2024, Genes Dev, doi: 10.1101/gad.351633.124) where an increase in the SMT bound fraction occurred, both scenarios—higher occupancy at known loci and binding to additional loci in ChIP-seq was observed. So, addressing this intriguing possibility in future studies focused on RAR and RXR would be interesting.

(2) The results presented suggest convincingly that endogenous RXR is normally in excess to its binding partners (in U2OS cells). This point could be strengthened further by reducing RXR levels, e.g., by knocking out 1 allele or the use of shRNAs (although the latter method might be too hard to control). Overexpression of another T2NR might also help determine the buffer capacity of RXR.

We appreciate the reviewers’ acknowledgment that our results convincingly demonstrate that endogenous RXR is typically in excess relative to its binding partners in U2OS cells. We agree that this conclusion could be further reinforced by experiments such as overexpression of another T2NR to test RXR's buffering capacity. We are actively pursuing follow-up experiments involving overexpression of additional T2NRs to address this question in more detail. These studies are ongoing, and we plan to explore the buffer capacity of RXR more extensively in a future manuscript.

(3) The ~10% difference in fbound of RAR and RXR (in Figs 1 and 2), while they should be 1:1 dimers, is explained by invoking the expression of RXR isoforms. Can the authors be more specific concerning the nature of these isoforms?

We have provided detailed information about different T2NRs expressed in U2OS cells according to the Expression Atlas and the Human Protein Atlas Database in Supplementary Table S1. Table S1 specifically shows that both isoforms of RXRα and RXRβ are expressed in U2OS cells. Additionally, the caption of Table S1 explicitly notes the presence of isoform RXRβ in U2OS cells. In the main text, we reference Table S1 when discussing the 10% difference in fbound between RARα and RXRα, and we have now suggested that the expression of RXRβ likely accounts for the observed discrepancy.

Reviewer #2 (Public Review):

Summary:

In the manuscript "Surprising Features of Nuclear Receptor Interaction Networks Revealed by Live Cell Single Molecule Imaging", Dahal et al combine fast single molecule tracking (SMT) with proximity-assisted photoactivation (PAPA) to study the interaction between RARa and RXRa. The prevalent model in the nuclear receptor field suggests that type II nuclear receptors compete for a limiting pool of their partner RXRa. Contrary to this, the authors find that over-expression of RARa but not RXRa increases the fraction of RXRa molecules bound to chromatin, which leads them to conclude that the limiting factor is the abundance of RARa and not RXRa. The authors also perform experiments with a known RARa agonist, all trans retinoic acid (atRA) which has little effect on the bound fraction. Using PAPA, they show that chromatin binding increases upon dimerization of RARa and RXRa.

Strengths:

In my view, the biggest strength of this study is the use of endogenously tagged RARa and RXRa cell lines. As the authors point out, most previous studies used either in vitro assays or over-expression. I commend the authors on the generation of single-cell clones of knock-in RARa-Halo and Halo-RXRa. The authors then carefully measure the abundance of each protein using FACS, which is very helpful when comparing across conditions. The manuscript is generally well written and figures are easy to follow. The consistent color-scheme used throughout the manuscript is very helpful.

Weaknesses:

(1) Agonist treatment:

The authors test the effect of all trans retinoic acid (atRA) on the bound fraction of RARa and RXRa and find that "These results are consistent with the classic model in which dimerization and chromatin binding of T2NRs are ligand independent." However, all the agonist treatments are done in media containing FBS. FBS is not chemically defined and has been found to have between 10 and 50 nM atRA (see references in PMID 32359651 for example). The addition of 1 nM or 100 nM atRA is unlikely to result in a strong effect since the medium already contains comparable or higher levels of agonist. To test their hypothesis of ligand-independent dimerization, the authors should deplete the media of atRA by growing the cells in a medium containing charcoal-stripped FBS for at least 24 hours before adding agonist.

We acknowledge the reviewer's concern regarding the presence of atRA in FBS and agree that it may introduce baseline levels of agonist. However, in our experiments, both the 1 nM and 100 nM atRA treatments resulted in observable changes in RAR expression levels (Figure S3C). Additionally, the luciferase assays demonstrated that 100 nM atRA significantly increased retinoic acid-responsive promoter activity (Figure S1C). Given these clear responses to atRA, we believe the observed lack of effect on the chromatin-bound fraction cannot be attributed to the presence of comparable or higher levels of atRA in the FBS, as the reviewer suggests. Moreover, since our results align with the established literature and do not impact the core findings of our study, we decided not to pursue the suggested experiments with charcoal-stripped FBS in this manuscript.  

(2) Photobleaching and its effect on bound fraction measurements:

The authors discard the first 500 to 1000 frames due to the high localization density in the initial frames. This will preferentially discard bound molecules that will bleach in the initial frames of the movie and lead to an over-estimation of the unbound fraction.

For experiments with over-expression of RAR-Halo and Halo-RXR, the authors state that the cells were pre-bleached and that these frames were used to calculate the mean intensity of the nuclei. When pre-bleaching, bound molecules will preferentially bleach before the diffusing population. This will again lead to an over-representation of the unbound fraction since this is the population that will remain relatively unaffected by the pre-bleaching. Indeed, the bound fraction for over-expressed RARa and RXRa is significantly lower than that for the corresponding knock in lines. To confirm whether this is a biological result, I suggest that the authors either reduce the amount of dye they use so that this pre-bleaching is not necessary or use the direct reactivation strategy they use for their PAPA experiments to eliminate the pre-bleaching step.

As for the measurement of the nuclear intensity, since the authors have access to multiple HaloTag dyes, they can saturate the HaloTagged proteins with a high concentration of JF646 or JFX650 to measure the mean intensity of the protein while still using the PA-JFX549 for SMT. Together, these will eliminate the need to prebleach or discard any frames.

The Janelia Fluor dyes used in our experiments are known for their high photostability (Grimm et al., 2021, JACS Au, doi: 10.1021/jacsau.1c00006). During the initial 80 ms imaging to calculate the mean nuclear intensity, the laser power was kept at very low intensity (~3%) for a brief duration (~10 seconds), in contrast to the high-intensity (~100%) used during the tracking experiments, which span around 3 minutes. This low-power illumination does not induce significant photobleaching but merely puts the dyes in a temporary dark state. Therefore, this pre-bleaching step closely resembles the direct reactivation strategy employed in our PAPA experiments.

To further address the reviewer's concern, we performed a frame cut-off analysis for our SMT movies of endogenous RARα-Halo and over-expressed RARα-Halo (Figure S9B). The analysis shows no significant change in the bound fraction of either endogenous or over-expressed RARα-Halo when discarding the initial 1000 frames. Based on these results, we conclude that the pre-bleaching does not lead to an overestimation of the unbound fraction, and that our experimental approach is robust.

(3) Heterogeneous expression of the SNAP fusion proteins:

The cell lines expressing SNAP tagged transgenes shown in Fig S6 have very heterogeneous expression of the SNAP proteins. While the bulk measurements done by Western blotting are useful, while doing single-cell experiments (especially with small numbers - ~20 - of cells), it is important to control for expression levels. Since these transgenic stable lines were not FACS sorted, it would be helpful for the reader to know the spread in the distribution of mean intensities of the SNAP proteins for the cells that the SMT data are presented for. This step is crucial while claiming the absence of an effect upon over-expression and can easily be done with a SNAPTag ligand such as SF650 using the procedure outlined for the over-expressed HaloTag proteins.

We agree with the reviewer that there is heterogeneity in SNAP protein expression across the transgenic lines. In response to the reviewer’s suggestion, we performed the proposed experiment to assess the distribution of mean intensities for two key experimental conditions: Halo-RXRα with overexpressed RARα-SNAP and HaloRXRα with overexpressed RARαRR-SNAP. These results again confirm that the increase in chromatin-bound fraction of Halo-RXRα is observed only in the presence of RARα capable of heterodimerizing with RXRα, supporting our main conclusion (Figure S9).

For these experiments, we followed the same labelling procedure described in the methods section for tracking endogenous Halo-tagged proteins alongside transgenic SNAP proteins. As shown in Figure S9, for ~ 70 cell nuclei, the distribution of mean intensities is similar for both conditions, with the bound fraction of Halo-RXRα significantly increasing in the presence of RARα-SNAP compared to RARαRR-SNAP. This analysis underscores that the observed effects are indeed due to the functional differences between the two RARα variants rather than variability in expression levels.

(4) Definition of bound molecules:

The authors state that molecules with a diffusion coefficient less than 0.15 um2/s are considered bound and those between 1-15 um2/s are considered unbound. Clarification is needed on how this threshold was determined. In previous publications using saSPT, the authors have used a cutoff of 0.1 um2/s (for example, PMID 36066004, 36322456). Do the results rely on a specific cutoff? A diffusion coefficient by itself is only a useful measure of normal diffusion. Bound molecules are unlikely to be undergoing Brownian motion, but the state array method implemented here does not seem to account for non-normal diffusive modes. How valid is this assumption here?

We acknowledge the inconsistency in the diffusion coefficient thresholds for defining the chromatin-bound fraction used across our group’s publications. The choice of threshold or cutoff (0.1 µm²/s vs 0.15 µm²/s) is largely arbitrary and does not significantly impact the results. To validate this, we tested the effect of different cutoffs on fbound (%) for endogenously expressed Halo-tagged RARα and RXRα (Figure S10). As shown in Figure S10, there was no substantial difference in fbound (%) calculated using a 0.1 µm²/s versus 0.15 µm²/s cutoff (e.g., RARα clone c156: 47±1% vs 49±1%; RXRα clone D6: 34±1% vs 35±1%). 

Since we have consistently applied the 0.15 µm²/s cutoff throughout this manuscript across all experimental conditions, the comparative analysis of fbound (%) remains valid. While we agree that a Brownian diffusion model may not fully capture the motion of bound molecules, our state array model accounts for localization error, which likely incorporates some of the chromatin motion features. Moreover, the distinction between bound (<0.15 µm²/s) and unbound (1-15 µm²/s) populations is sufficiently large that using a normal diffusion model is reasonable for our analysis.

(5) Movies:

Since this is an imaging manuscript, I request the authors to provide representative movies for all the presented conditions. This is an essential component for a reader to evaluate the data and for them to benchmark their own images if they are to try to reproduce these findings.

We have now included representative movies for all the SMT experimental conditions presented in the manuscript. Please see data availability section of the manuscript.

(6) Definition of an ROI:

The authors state that "ROI of random size but with maximum possible area was selected to fit into the interior of the nuclei" while imaging. However, the readout speed of the Andor iXon Ultra 897 depends on the size of the defined ROI. If the ROI was variable for every movie, how do the authors ensure the same sampling rate?

We used the frame transfer mode on the Andor iXon Ultra 897 camera for our acquisitions, which allows for fast frame rate measurements without altering the exposure time between frames. Additionally, we verified the metadata of all our movies to ensure a consistent frame interval of 7.4 ms across all conditions. This confirms that the sampling rate was maintained uniformly, despite the variability in ROI size. 

Reviewer #2 (Recommendations For The Authors):

(1) 'Hoechst' is mis-spelled.

We have now corrected this typo in the manuscript.

(2) Cos7 appears in several places throughout the text. I assume this is a typo. If so, please correct it. If not, please explain if some experiments were done in Cos7 cells and kindly provide a justification for that.

The use of Cos7 cells is intentional and not a typo. Cos7 cells have been previously utilized in studies investigating the interaction between T2NRs (Kliewer et al., 1992, Nature, doi: 10.1038/355446a0). In our study, due to technical issues with antibodies for coIP in U2OS cells, we initially used Cos7 cells for control experiments to verify that Halo-tagging of RARα and RXRα did not disrupt their interaction, by transiently expressing the constructs in Cos7 cells. Following these control experiments, we confirmed the direct interaction of endogenously expressed RAR and RXR in U2OS cells with their respective binding partners using the SMT-PAPA assay. Since these results confirmed that Halo-tagging did not interfere with RAR-RXR interactions, we chose not to repeat the coIP experiments in U2OS cells.

Reviewer #3 (Public Review):

Summary:

This study aims to investigate the stoichiometric effect between core factors and partners forming the heterodimeric transcription factor network in living cells at endogenous expression levels. Using state-of-the-art single-molecule analysis techniques, the authors tracked individual RARα and RXRα molecules labeled by HALO-tag knock-in. They discovered an asymmetric response to the overexpression of counter-partners. Specifically, the fact that an increase in RARα did not lead to an increase in RXRα chromatin binding is incompatible with the previous competitive core model. Furthermore, by using a technique that visualizes only molecules proximal to partners, they directly linked transcription factor heterodimerization to chromatin binding.

Strengths:

The carefully designed experiments, from knock-in cell constructions to singlemolecule imaging analysis, strengthen the evidence of the stoichiometric perturbation response of endogenous proteins. The novel finding that RXR, previously thought to be a target of competition among partners, is in excess provides new insight into key factors in dimerization network regulation. By combining the cutting-edge single-molecule imaging analysis with the technique for detecting interactions developed by the authors' group, they have directly illustrated the relationship between the physical interactions of dimeric transcription factors and chromatin binding. This has enabled interaction analysis in live cells that was challenging in single-molecule imaging, proving it is a powerful tool for studying endogenous proteins.

Weaknesses:

As the authors have mentioned, they have not investigated the effects of other T2NRs or RXR isoforms. These invisible factors leave room for interpretation regarding the origin of chromatin binding of endogenous proteins (Recommendations 4). In the PAPA experiments, overexpressed factors are visualized, but changes in chromatin binding of endogenous proteins due to interactions with the overexpressed proteins have not been investigated. This might be tested by reversing the fluorescent ligands for the Sender and Receiver. Additionally, the PAPA experiments are likely to be strengthened by control experiments (Recommendations 5).

We agree that this would be an interesting experiment. However, there are three technical challenges that complicate its implementation: First, as demonstrated in our original PAPA paper, dark state formation is less efficient when dyes are conjugated to Halo compared to SNAPf, making the reverse configuration less optimal. Second, SNAPf-tagged proteins have slower labeling kinetics than Halotagged proteins, often resulting in under-labeling of SNAPf. Third, our SNAPf transgenes were integrated polyclonally. Since background PAPA scales with the concentration of the sender-labeled protein, variable concentrations of the senderlabeled SNAPf proteins would introduce significant variability, complicating the interpretation of the background PAPA signal. Due to these concerns, we believe that performing reciprocal measurements with reversed fluorescent ligands may not yield reliable results. 

Reviewer #3 (Recommendations For The Authors):

(1) The term "Surprising features" in the title is ambiguous and may force readers to search for what it specifically refers to. Including a word that evokes specific features might be helpful.

Our findings contradict previous work, which suggested that chromatin binding of T2NRs is regulated by competition for a limited pool of RXR. In contrast, we found that RAR expression can limit RXR chromatin binding, but not the other way around, which challenges the existing model. This unexpected result is what we refer to as a "surprising feature" in our title, and we believe it accurately reflects the novel insights our study provides. We also think that this is clearly conveyed in our manuscript abstract, supporting the use of "Surprising features" in the title. 

(2) p.3, line 11 - The threshold of 0.15 μm2s-1 seems to be a crucial value directly linked to the value of fbound. What is the rationale for choosing this specific value? If consistent conclusions can be obtained using threshold values that are similar but different, it would strengthen the robustness of the results.

Please refer to our response to Reviewer #2’s Public Review point 4. The threshold choice is arbitrary and doesn’t affect the overall conclusions. To test this, we compared fbound (%) values calculated using both 0.1 μm²s-1 and 0.15 μm²s-1 cutoffs. For example, with endogenously expressed Halo-tagged RARα (clone c156), we observed fbound values of 47±1% vs 49±1%, and for RXRα (clone D6), 34±1% vs 35±1%, respectively (Figure S10). Since we have consistently applied the 0.15 μm²s-1 cutoff across all experimental conditions in this manuscript, the comparisons of fbound (%) between different conditions are robust and valid.

(3) p.4, line 13 - "the fbound of endogenous RARα-Halo (47{plus minus}1%) was largely unchanged upon expression of SNAP (47{plus minus}1%)" part of the sentence is not surprising. It would make more sense if it were expressed as "the fbound of endogenous RARα-Halo (47{plus minus}1%) was largely unchanged upon expression of RXRα-SNAP (49{plus minus}1%), consistent with the control SNAP (47{plus minus}1%).".

We understand how the original phrasing may be confusing to the readers and have restructured the sentence as suggested by the reviewer for clarity.

(4) p.6, line 26 - The discussion that "most chromatin binding of endogenous RXRα in U2OS cells depends on heterodimerization partners other than RARα" seems to contradict the top right figure in Figure 4. If that's the case, the binding partner for the bound red molecule might be yellow rather than blue. Given a decrease in the number of RARα molecules with an unchanged binding ratio, the total number of binding molecules has decreased. Could it be interpreted that the potential reduction in RXRα chromatin binding, accompanying the decrease in binding RARα, is compensated for by other partners?

We agree with the reviewer that both the yellow and blue molecules in Figure 4 represent T2NRs that can heterodimerize with RXR. For simplicity, we chose to omit the depiction of RXR dimerization with other T2NRs (represented in yellow) in Figure 4. We have now included a note in the figure caption to clarify this. We plan to follow up on the buffer capacity of RXR with other T2NRs in a separate manuscript and will discuss this aspect in more detail once we have data from those experiments.

(5) Fig. 3 - I expected that DR localizations always appear more frequently than PAPA localizations by the difference in the number of distal molecules. Why does the linear line for SNAP-RXRα in Fig. 3 B have a slope exceeding 1? Also, although the sublinearity is attributed to binding saturation, is there any possibility that this sublinearity originates from the PAPA system like the saturation of PAPA reactivation? Control samples like Halo-SNAPf-3xNLS might address these concerns.

The number of DR and PAPA localizations depends on the arbitrarily chosen intensity and duration of green and violet light pulses. For any given protein pair, different experimental settings can result in PAPA localizations being greater than, less than, or equal to the number of DR localizations. Therefore, the informative metric is not the absolute number of DR and PAPA localizations, but rather how the ratio of PAPA to DR localizations changes between different conditions—such as between interacting pairs and non-interacting controls.

Regarding the sublinearity, we agree that it is essential to consider whether the observed sublinearity might stem from saturation of the PAPA signal. We know of two ways in which this could occur:

First, PAPA can be saturated as the duration of the green light pulse increases and dark-state complexes are depleted. However, this cannot explain the nonlinearity that we observe, because the duration of the green light pulse is constant, and thus the probability that a given complex is reactivated by PAPA is also constant. Likewise, holding the violet pulse duration constant yields a constant probability that a given molecule is reactivated by DR. PAPA localizations are expected to scale linearly with the number of complexes, while DR localizations are expected to scale linearly with the total number of molecules. Sublinear scaling of PAPA localizations with DR localizations thus implies that the number of complexes scales sublinearly with the total concentration of the protein.

Second, saturation could occur if PAPA localizations are undercounted compared to DR localizations. While this is a valid concern, we consider it unlikely in this case because 1) our localization density is below the level at which our tracking algorithm typically undercounts localizations, and 2) we observe sublinearity for RXR → RAR PAPA even though the number of PAPA localizations is lower than the DR localizations; undercounting due to excessive localization density would be expected to introduce the opposite bias in this case.

(6) Fig. 4 - The differences between A, B, and C on the right side of the model are subtle, making it difficult to discern where to see. Emphasizing the difference in molecule numbers or grouping free molecules at the top might help clarify these distinctions.

We appreciate the reviewer’s feedback. In response, we have revised Figure 4 by grouping the free molecules on the top right side for panels A, B and C, as suggested.

(7) While the main results are obtained through single-molecule imaging, no singlemolecule fluorescence images or trajectory plots are provided. Even just for representative conditions, these could serve as a guide for readers trying to reproduce the experiments with different custom-build microscope setups. Also, considering data availability, depositing the source data might be necessary, at least for the diffusion spectra.

We have now included representative movies for all the presented SMT conditions as source data. Please see data availability section of the manuscript.

(8) Tick lines are not visible on many of the graph axes. 

We have revised the figures to ensure that the tick lines are now clearly visible on all graph axes.

(9) Inconsistencies in the formatting are present in the methods, such as "hrs" vs. "hours", spacing between numbers and units, and "MgCl2". "u" should be "μ" and "x" should be "×". 

We have corrected the formatting errors.

(10) Table S4, rows 16 and 17 - Are "RAR"s typos for "RXR"s? 

We have corrected this in the manuscript.

(11) p.10~12 - Are three "Hoestch"s typos for "Hoechst"s? 

This is now corrected in the manuscript.

(12) p.11, line 17 - According to the referenced paper, the abbreviation should be "HILO" in all capital letters, not "HiLO". 

This is now corrected in the manuscript.

(13) "%" on p.3, line 18, and "." on p.6, line 27 are missing. 

This missing “%”  and “.” are now added.

Reviewer #3 (Public review):

Summary:

This study aims to investigate the stoichiometric effect between core factors and partners forming the heterodimeric transcription factor network in living cells at endogenous expression levels. Using state-of-the-art single-molecule analysis techniques, the authors tracked individual RARα and RXRα molecules labeled by HALO-tag knock-in. They discovered an asymmetric response to the overexpression of counter-partners. Specifically, the fact that an increase in RARα did not lead to an increase in RXRα chromatin binding is incompatible with the previous competitive core model. Furthermore, by using a technique that visualizes only molecules proximal to partners, they directly linked transcription factor heterodimerization to chromatin binding.

Strengths:

The carefully designed experiments, from knock-in cell constructions to single-molecule imaging analysis, strengthen the evidence of the stoichiometric perturbation response of endogenous proteins. The novel finding that RXR, previously thought to be a target of competition among partners, is in excess provides new insight into key factors in dimerization network regulation. By combining the cutting-edge single-molecule imaging analysis with the technique for detecting interactions developed by the authors' group, they have directly illustrated the relationship between the physical interactions of dimeric transcription factors and chromatin binding. This has enabled interaction analysis in live cells that was challenging in single-molecule imaging, proving it is a powerful tool for studying endogenous proteins.

Weaknesses:

None noted.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Exploiting synthetic lethality based on functional correlations has the potential to significantly improve the survival of cancer patients by reducing resistance to targeted therapies and increasing anti-tumour efficacy when combined with other treatment modalities. Schreuder et al., aim to identifying novel vulnerabilities of patient-derived mutations that could improve patient stratification based on a specific genetic background. Precisely, they established a model system to perform a genome-wide CRISPR-Cas9 KO screen to identify genomic vulnerabilities of BRCA1 variants with reported hypomorphic phenotypes, namely BRCA1 R1699Q and BRCA1 I26A in engineered RPE1 hTERT cells with AID tag. Using this system the authors were able to confirm known synthetic lethal genes reported in literature (e.g. APEX2, PARP1, POLQ) comparing cells with acute BRCA loss and BRCA1 deficiency. Moreover, the screen identified two genes, CSA and GPX4 that were not previously described as synthetic lethal with BRCA1 loss. What is potentially interesting, but marginally explored, is the identification of a unique synthetic vulnerability of cells expressing BRCA1 R1699Q mutant and NDE1 gene encoding for a dynamic scaffold protein essential in neocortical neurogenesis and heterochromatin patterning by H4K20me3, whose loss of function results in nuclear architecture aberrations and DNA double-strand breaks (Chomiak et al., iScience 2022). Accordingly, cells ablated of NDE1 and expressing BRCA1 R1699Q mutant show less proliferation of cells expressing either BRCA1 WT or BRCA1-depleted. Furthermore, cells lacking NDE1 show increased genomic instability by means of increased micronuclei and anaphase bridges compared to BRCA1 proficient and BRCA1 R1699Q mutant.

Major comments:

1. The authors claim that cells expressing BRCA1-I26A are largely HR-proficient, based on a milder effect on Olaparib sensitivity compared to cells expressing BRCA1-R1699A (Fig. 1C). However, I26A mutant cells are defective in RAD51 IRIF (Fig. 1B), indicative of an HR defect. Recently it has been shown that BRCA1 RING mutations that do not impact BARD1 binding, including I26A, render BRCA1 unable to accumulate to DNA damage sites and unable to form RAD51 foci when such mutation is combined with mutations that disable RAP80-BRCA1 interaction (Sherker et al., 2021). How do the authors explain this discrepancy with the literature?
2. The reduction in survival following CSA depletion in BRCA1-proficient vs. -deficient cells is only 20% (Figure 2 and S2B). In my opinion, such a minor difference is not supporting the notion of a SL interaction between BRCA1 and CSA. To substantiate CSA as synthetic lethal hit, I would recommend the authors comparing the effect of CSA loss to that of EXO1 or BLM loss, both genes recently identified by the same group as SL partners of BRCA1 using the same experimental screening set up (van de Kooij et al, 2024). Moreover, validation data for GPX4 is missing.
3. Similar to the minor effect observed for CSA, DOT1L and OTUD5 depletions caused rather mild and/or divergent phenotypes between the two sgRNAs used (Figure 4B), rather arguing against robust SL interactions between those genes and BRCA1 deficiency that could be therapeutically exploited.
4. To strengthen their conclusion in Figure 4C the authors should perform complementation experiments with NDE1 WT and, ideally, with NDE1 mutant(s). On a related note, are NDE1 knock-out cells expressing BRCA1-R1699A more sensitive to PARPi?
5. Graphs shown in Fig. 1A-C, Fig. 4B, S2D, S3B, S3E and S3F are lacking proper statistical analysis of the differences. Some experiments have only been repeated twice (e.g. Figure 1C), precluding running statistical tests.

Minor comments:

1. The authors should include representative images for results shown in Fig.1 A-C
2. The authors should add immunoblots for BRCA1 in Fig. S2C to indicate successful BRCA1 cDNA complementation in HCC1937 cells.
3. Most numbers in the Venn diagram shown in Figure 3A cannot be read when printed.
4. In the western blots shown in Supplemental Figure 1A, the electrophoretic mobility of BRCA1 variants expressed in RPE1 is quite variable. Could the authors indicate in the Figure (e.g. with arrowheads) which bands represent endogenous and which transgenic BRCA1. Moreover, in BRCA-wt complemented cells there are two bands following auxin/DOX addition, whereas there is one band observed in cells expressing BRCA1 hypomorphic variants
5. Line 229 please correct "BRCA1-proficient" to "BRCA1-depleted".

Significance

General assessment:

This manuscript starts with an attractive hypothesis, which is the generation of a cellular system to study patient-derived hypomorphic BRCA1 missense mutations rather than using BRCA1 knockout cells. Performing CRISPR/Cas9-mediated genome-wide synthetic lethal screens in this system allowed uncovering genetic vulnerabilities of cells expressing BRCA1-R1699A, a pathogenic mutant identified in several cancer patients. The data are of good quality and the manuscript is coherent and generally well written (few typos). However, some data describe mainly negative results (e.g. BRCA1-I26A mutant) or weak phenotypes while other more interesting aspects are not rigorously exploited (e.g. NDE1 SL) and therefore need to be interpreted with more caution and extended by additional experiments.

Advance:

BRCA1-R1699Q is classified as a pathogenic variant despite its low penetrance and intermediate cancer risk in breast and ovarian cancer compared to other variants. A recent case report highlighted the unique clinical outcome of a patient with the BRCA1 R1699Q variant, suggesting that this variant may differ from others in terms of cancer risk and drug response (Saito et al., Cancer Treatment and Research Communications 2022). These findings underscore the need for further studies to confirm these observations and to elucidate the specific mechanisms underlying the response to platinum agents and PARP inhibitors in patients with the BRCA1 R1699Q variant. The manuscript proposed by the authors has the potential to help understanding how BRCA1 missense mutations can contribute to determine treatment sensitivity and pave the way to patient stratification.

Audience:

This manuscript is suitable for a specialized, basic research audience.

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Reply to the reviewers

1. Point-by-point description of the revisions

• *Reviewer #1 (Evidence, reproducibility and clarity (Required)):

The authors present the use of previously identified biosensors in a single-molecule concentration regime to address lipid effector recruitment. Using controlled and careful single-cell based analysis, the study investigates how expression of the commonly used PIP3 sensor based on Akt-PH domain interferes with the native detection of PIP3. Predominantly live-cell fluorescence microscopy coupled to image analysis drives their studies.

Conceptually, this manuscript carefully and quantitatively describes the influence of lipid biosensor overexpression and presents a means to overcome the inherent and long-recognized problems therein. This solution, namely employing low expression of the lipid biosensor, should be generally applicable. The work is of general interest to cell biologists focused on answering questions at membranes and organelles, including especially those interested in lipid-mediated signaling transductions.

Reviewer 1 Major:

#1.1 The terminology "single molecule biosensor" is not really appropriate. A protein is not "single-molecule". An enzyme does not "single molecule". Better is biosensors at single-molecule expression levels. In most cases, this should be changed. Single-molecule vs single-cell vs. bulk measurements are often poorly defined in quantifications and conflating these does not help the case, which is already supported by generally clear data.

We appreciate the reviewer’s thoughtful critique of our grammatically incorrect use of jargon; we saw this as soon as they mentioned it! We have amended the manuscript where appropriate as detailed:

• Title is now changed to “Lipid Biosensors Expressed at Single Molecule Levels Mitigates Inhibition of Endogenous Effector Proteins”
• Last paragraph of the introduction on __ 2__ now reads “As well as alleviating inhibition of PI3K signaling, biosensors expressed at these low levels show improved dynamic range and report more accurate kinetics than their over-expressed counterparts."
• The title of the results section on __ 6__ is now: Mitigating PIP3 competition using biosensors expressed at single molecule levels
• Last paragraph of the results section on 6 now reads: “this showed that when expressed at single molecule levels, the biosensor has substantially better dynamic range”. #1.2 Figure 1D-F, images not as clearly describing quantitation as one would hope. Untransfected cells in 1E should demonstrate more translocated Akt-pS473 than transfected, but it is difficult for this reviewer to find. Consider inset images in addition to the wider field. Consider also moving the "negative" data of Fig 1B-C to Supplement.

We regret not making this figure easier to interpret; we have substantially updated the figure, as comprehensively detailed in our point-by-point response to reviewer 2’s point 2.3. To specifically address this reviewer’s concerns:

The older figure used non-confocal, low-resolution images that were used for quantification. Such an approach was employed to enable fluorescence from the entire cellular volume to be captured, which produces more robust quantification. However, to the reviewer’s point, it is not possible to see the translocation of PH-AKT1 nor translocated AKT-pS473 in these images. Fortunately, we had in parallel captured high resolution confocal images for some experiments. These are now shown in Fig 1D-E, which clearly shows translocated AKT-pS473 and PH-AKT-EGFP

#1.3 The cell line being used is not clearly specified after the initial development of the NG1 followed by CRISPRed NG2 onto Akt. For example, for the Figure 3C experiments, the text states "complete ablation of endogenous AKT1-NG2" but this information is not apparent from the figure legend or figure. Throughout the cell line used and the aspects transfected need to be made explicitly clear.

We are grateful to the reviewer for highlighting this ambiguity. We have now defined the gene-edited cells used throughout as “AKT1-NG2 cells” and expressly used this term when referring to experiments in figures 2-5.

#1.4 Fig. 5 shows single cells. It is therefore unclear if broken promoters have resulted in decreased expression. This point is important because the expression plasmids should be made publicly available, and for their use to be understood properly, this must be clarified. The details of the plasmids are unclear. Perhaps listed in the table? - unclear. This aspect would be important for the field to effectively use the reagents.

Thank you for drawing our attention to the lack of adequate detail here. We have now updated the results text to expressly reference Morita et al., 2022 where the origins of the truncated CMV promoters are detailed. We have also updated the plasmids table 1 to add pertinent details for these constructs: *pCMVd3 plasmids are based on the pEGFP-C1 backbone, with the CMV promoter truncated to remove 18 of the 26 putative transcription factor binding sites in the human Cytomegalovirus Major Intermediate Enhancer/Promoter (pCMV∆3 as described in Morita et al., 2012). The full sequences will be deposited with the plasmids on Addgene.

We did not perform a formal comparison of full vs truncated promoters. Our only observation is that the truncated promoters greatly help in increasing the number of expressing cells presenting single-molecule resolvable expression levels (though the approach can still work with full promoters).

#1.5 This manuscript speculates several times that with more abundant PIs like PI45P2, the observed saturation effect is probably not happening. This should be removed. While the back of envelope calculations may reflect an ideal scenario, the heterogeneity of distribution and multiple key cellular structures involved would seem to corral increased PI45P2 levels in certain regions. These factors amid multivalency and electrostatic mechanisms of lipid effector recruitment (e.g. MARCKS) suggest that speculation may be too strong. Moreover, Maib et al JCB 2024 demonstrated PI4P probe overexpression could directly mask the ability to detect PI4P post-fixation - not fully, but partially. Repeating the titration experiments of this manuscript for multiple PIs is entirely beyond the scope of reasonable, and hence, such experiments are not requested, in favor of adopting more conscientious speculation.

The reviewer’s point is well taken. Whilst we still believe the overall argument for lipids is sounds (for example, PS or cholesterol are far too abundant for any expressed, stoichiometric binding protein to bind the majority of the population) even abundant phosphoinositides like PI4P and PI(4,5)P2 are an edge case. We have therefore undated the first paragraph of the introduction on __p. 1 __to be less explicit: One of the most prominent is the fact that lipid engagement by a biosensor occludes the lipid’s headgroup, blocking its interaction with proteins that mediate biological function. It follows that large fractions of lipid may be effectively outcompeted by the biosensor, inhibiting the associated physiology. We have argued that, in most cases, this is unlikely because the total number of lipid molecules outnumbers expressed biosensors by one to two orders of magnitude (Wills et al., 2018). However, for less abundant lipids, total molecule copy numbers may be in the order of tens to hundreds of thousands, making competition by biosensors a real possibility.

We also removed the explicit discussion of PI(4,5)P2 from the introduction, and focus now solely on the PI3K lipids.

Reviewer 1 Minor:

1.6 Schematics throughout need simplification, enabling their enlargement.

We have now enlarged the size of all schematics

#1.7 Numerous spelling (Fig. 4 schemas) and capitalizations need fixing.

Thank you for drawing our attention to these. We have thoroughly proof-read the figure panels and corrected errors.

#1.8 Pg 1 Famous is not appropriate wording

We respectfully beg to differ with the reviewer here. We believe it is perfectly accurate to state that PIP3 is a second messenger molecule that is known about by many people; we see this as the dictionary definition of the word “famous”.

#1.9 Fig. 1A statistical testing of microscopy quantifications absent (generally, throughout) and should be included.

This was indeed an oversight on our part. We have now added appropriate multiple comparisons tests to the data presented in figures 1F, 3F, 4C, 4F and 5C.

#1.10 Fig.1. In a transient transfection, the protein expression is not uniform. Please explain how you normalized the quantification.

We hope this is now clarified by the expanded “Image Analysis” part of the methods section on pp. 10-11 (relevant sentence is underlined): For immunofluorescence, we identified individual cells by auto thresholding the DAPI channel using the “Huang” method, followed by the Watershed function to segment bunched cells that appeared to touch. We then used the Voronoi function to generate boundary lines for the segmentation of the cells. To identify cytoplasm, auto thresholding of the CellMask channel using the “Huang” function was employed, with the cells segmented by adding the nuclear Voronoi boundaries. The “analyze particles” function was then used to identify individual cellular ROIs that were greater than 10 µm2 and were not touching the image periphery. These ROIs were used to measure the raw 12-bit intensity of the EGFP and AKT-pS473 channels. A cutoff of EGFP > 100 was used to define EGFP-positive cells, since this value was greater than the mean ± 3 standard deviations of the non-transfected cells’ EGFP intensity. Background intensity of AKT-pS473 was estimated from control cells subject to immunofluorescence in the absence of AKT-pS473 antibody; this value was subtracted from the measured values of all other conditions.

#1.11 Fig. 1D. EGFP expression levels increased with EGF stimulation. How is this possible?

There appeared to be a difference due to the presence of 5 strongly expressing cells in the chosen field in the original field for the EGF stimulated, EGFP cells. However, this arose just by chance. The new set of high-resolution images in the new figure 1 were selected to be more representative.

#1.12 Fig. 1D. The images have pS473 whereas the y-axis label on box plots has p473. Can these box plots be labelled separately for consistency?

Thank you. This has now been corrected in the revised Figure 1.

#1.13 Fig.1. T308 phosphorylation is mentioned in Figure 1, but only pS473 data is shown.

Both T308 and S473 phosphorylation are indicative of AKT activation. However, antibodies suitable for immunofluorescence are only available for pS473, hence why our experiments are restricted to this moiety.

#1.14 Fig.1 legend. 'Over-expression of PH-AKT is hypothesised to outcompete the endogenous AKT's PH domain'. Why do you need to state a hypothesis in the legend?

We included this statement for the benefit of the casual reader – i.e. one who looks at the pictures, but doesn’t read the main text!

#1.15 Fig.1E You stated that the PH-AKT R25C-EGFP is stimulated by EGF addition. However, the GFP signal looks the same in both unstimulated and stimulated. Could you please clarify? Are you sure that the stimulation worked?

We have clarified the second paragraph of the results section “Inhibition of AKT activation by PIP3 biosensor”__on __p. 4 as follows: In the non PIP3 binding PH-AKT1R25C-EGFP positive cells, we still observed an increase in pS473 intensity.

The revised figure 1 images also show that PH-AKT1R25C does not translocate to the membrane with EGF stimulation.

#1.16 You mention...that the AKT enzyme is activated by PDK1 and TORC2, which phosphorylate at residues T308 and S473, respectively. Phosphorylation is also known to occur on T450 at c-tail. Does this phosphorylation also contribute to its activation?

Yes and no. Threonine 450 phosphorylation is thought to occur co-translationally and is important for AKT stability (see Truebestein et al as cited in the manuscript). It is not really relevant in the context for T308 and S473, which are phosphorylated acutely to activate the protein.

#1.17 Fig. 1 scale bar in all images equivalent?

We have now added scale bars to panels in both figure 1D and E to clarify.

__#1.18 __Pg. 1 paragraph 1 "we have argued..." vs. paragraph 3"...consider that an..." feels like arguing with themselves.

We believe the re-write we have done in response to major point #1.5 clarifies this point also.

#1.19 Pg. 1 para 3 what is RFC score - must explain

We have now defined this more clearly in third __paragraph of the __introduction on p. 1: PH domain containing PIP3effector proteins can be predicted based on sequence comparison to known PIP3 effectors vs non effectors using a recursive functional classification matrix for each amino acid (Park et al., 2008).

#1.20 Discussion of numbers of PIP3 vs. effectors etc may not be appropriate for the introduction, as the points made by these calculations are already made in the previous paragraphs. May fit better in pg 6 Mitigating PIP3 titration... with an accompanying schematic.

Respectfully, we prefer to keep this discussion of molecular concentrations, as this adds details and specifics to the pathway that is core to the paper.

#1.21 Pg 2 "a neonGreen" not well defined, needs accurate description.

We have clarified this in the sentence in the first paragraph of the results section “Genomic tagging of AKT1…” __on __p. 4, which includes the citation to the full description of the tag: To that end, we used gene editing to incorporate a bright, photostable neonGreen fluorescent protein to the C-terminus of AKT1 via gene editing using a split fluorescent protein approach (Kamiyama et al., 2016).

#1.22 Fig 2C should give a unstimulated trajectory of puncta/100 um2 to compare with the stimulated

Unfortunately, we did not record a full 5.5-minute video-rate time-lapse with unstimulated cells. However, we do not believe this control is essential for this experiment, since this example data is included to illustrate (1) the problem of photobleaching, which is clear in the 30-s pre-stimulus and (2) the variability in the raw molecule counts.

#1.23 Fig 2C and F and G should be systematized for easier comparison. E.g. min vs seconds, 0 timepoint of EGF/rapa addition

We have made the adjustment to figure 2C to be consistent with 2F and G:

#1.24 Pg 5 "...and calibrated them..." unclear what is being calibrated, as the text later states that the histograms are fit to monomer/dimer/multimer model resulting in 98.1% in monomer. Minor point.

We have clarified this point in the second paragraph of the results section “__Genomic tagging of AKT1…” __on __p. 4 __as follows: We analyzed the intensity of these spots and compared them to intensity distributions from a known monomeric protein localized to the plasma membrane (PM) and expressed at single molecule levels

#1.25 Explain why baselines in Fig2CFG are different

We did not comment on figure 2C; it is a single cell measurement, as opposed to the mean of 20 cells reported in F. However, we do now clarify the difference between figure 2F and G as the very end of the “Genomic tagging of AKT1…” results section on p 4: Notably, baseline AKT-NG2 localization increased from ~5 to ~15 per 100 µm2 in iSH2 cells, perhaps because the iSH2 construct does not contain the inhibitory SH2 domains of p85 regulatory subunits, producing higher basal PI3K activity.

#1.26 Fig. 2 has quantification with images; Fig. 3 has it separate. Make consistent.

We sometimes combine images with quantification, and other times separate the panel containing graphs. This is done deliberately, depending on whether the reader is directed to both together, or whether we consider the data separately in the results section.

#1.27 Fig. 3B comes before images? Where are the images? Also, y-axis = Intensity (a.u.). Is intensity just full image field? Or per cell? All very unclear.

We have modified both the graph y-axis label and the figure legend to clarify: (C) TIRF imaging of AKT1-NG2 cells from (B) stimulated with 10 ng/ml EGF

#1.28 Fig. 3C missing images

We believe the reviewer is referring to the mCherry channel for the “0 ng cDNA” condition. These images are missing because they do not exist. Since these cells were transfected with pUC19, there was no mCherry fluorescence to image.

#1.29 Fig 3 C needs brightness/contrast adjusted as images are nearly entirely black (zero values).

We believe the addition of insets addresses this concern. To the reviewer’s specific suggestion, we found that further increases in the brightness and contrast will bring up the camera noise, but this then occludes the signal from single molecules, such as those found after EGF stimulation of the 0 ng condition.

#1.30 Fig 3C needs scale bar systemization

We believe that the incorporation of scaled 6 µm insets addresses this point.

#1.31 Fig 4 needs 4 panels A-D

We have now added these individual panel labels to figure 4.

#1.32 Pg 6 5-OH phosphatases needs reference

We have added a citation to Trésaugues at the very end of the “Sequestration of PIP3 by lipid biosensors” results section on p. 6, which describes the activity of the whole 5-OH phosphatase activity against PIP3, not just the SHIP phosphatases.

#1.33 Fig 5B, make images bigger

Again, we trust that the addition of insets to all single molecule images has addressed this point.

Reviewer 1 Referees cross-commenting**

I have read the other reviews and find them entirely reasonable. My impression is we landed on similar general content that needs work, none of which is out of line. The importance and care taken in the author's work is uniformly lauded.

We agree. At the risk of restoring to alliteration, we have been delighted to receive a trio of clear, concise and consistent comments on the manuscript! We believe it is now much improved.

Reviewer #1 (Significance (Required)):

This manuscript clearly and reasonably demonstrates that the commonly used PIP3 sensor can be titrated to low concentrations, at which it does not interfere with Akt translocation and activation. This work is a good technical reference for the field. Signal transduction and membrane biologists should be especially interested in the data. The reviewer/s have core expertise in phosphoinositides, protein biochemistry, cell biology, and membrane biophysics.

Reviewer #2 (Evidence, reproducibility and clarity (Required)):

The authors characterize the inhibition of lipid second messenger mediated cell signaling through lipid biosensors that outcompete endogenous effector proteins. This is a very important study that as it quantitatively assesses an issue that many people suspected to exit, yet never properly characterized. This paper is therefore as much a service to the community as a research study in its own right and should be published without undue delay. I am glad that the authors decided to carry out this study & really appreciate their work.

I do however, have a number of suggestions that I think will make the manuscript stronger and can be readily implemented, mostly by reformulating and/or re-analysis of exiting datasets. I've structured my comments by the datasets in the respective figures to follow the logic of the paper.

Reviewer 2 Major:

#2.1 Throughout the manuscript, statistical tests are missing, e.g. in figures 1C-F. This must be amended in the revised version. The authors are making a very quantitative point about buffering, data should be treated accordingly.

We have now added appropriate multiple comparisons tests to figures 1F, 3F, 4C, 4F and 5C.

#2.2 I do not think that "PIP3 titration" is the best term to describe the observed effect. "Titration" usually implies the controlled modulation of a concentration, e. g. in analytical chemistry. I think either "competitive binding of PIP3" or "buffering of free PIP3" are more adequate.

This point is well taken. We have now replaced the word “titration” throughout, replacing it with either “competitive binding” or “sequestration”.

#2.3 Specific comments: Figure 1

#2.3a Why are data in 1D-Ff shown as median, with interquartile ranges and 10-90 percentile distance when everything else in the paper is mean +/- se? There might be a good reason for it, but I did not find it mentioned everywhere

For consistency’s sake, we have changed figure 1F to show a bar graph, though as noted in the figure legend: Graphs show medians ± 95% confidence interval of the median from 82-160 cells pooled from three experiments (medians are reported since the data are not normally distributed).

#2.3b The authors should test, whether the difference between the +EGF conditions in 1D (EGFP) and 1F (PH-AktR25C-EGFP) is indeed statistically significant. If this observation holds up, what does it mean? Is the mutant still competing with endogenous Akt despite the much-reduced binding affinity? The authors should discuss.

We have re-analyzed the data in figure 1, with the quantitative data presented in figure 1F combined with statistical analysis. The new data shows no significant effect of the PH-AKT1R25C mutant in either resting or EGF stimulated condition

There results are also described in the__ second paragraph__ of the first results section on pp. 3-4: This analysis showed that the R25C mutant had no substantial effect on pS473 levels, whereas wild-type PH-AKT greatly inhibited pS473 staining in EGF-stimulated cells as well as reducing basal levels in serum starved cells (Fig. 1F).

#2.3c How were biosensor/GFP positive cells chosen? Did the authors choose a defined fluorescence intensity cut-off? I think that a pure manual selection is problematic from a methodological point of view as this may introduce biases. Since the authors use Fiji, they can also simply use the "Analyze particles" function, which allows to automatically segment cells from a thresholded image. By choosing the same threshold for all images, it would be ensured that all images are treated exactly the same way.

We had initially opted for manual outlining of cells since automatic segmentation of irregularly-shaped HEK293a cells is imperfect. However, we agree with André that this opens the possibility of bias. We have therefore re-run the analysis with an automated segmentation and thresholding approach, as suggested. This is detailed in the__ second paragraph__ of the first results section on pp. 3-4: In parallel, we imaged cells with a low resolution 0.75 NA air objective to capture fluorescence from the cells’ entire volume, then quantified these images using an automatically determined threshold for GFP-positive cells (see Materials and Methods). This analysis showed that the R25C mutant had no substantial effect on pS473 levels, whereas wild-type PH-AKT greatly inhibited pS473 staining in EGF-stimulated cells as well as reducing basal levels in serum starved cells (Fig. 1F).

Further detail is provided in the first paragraph of the “Image analysis” subsection of the methods on pp. 10-11: For immunofluorescence, we identified individual cells by auto thresholding the DAPI channel using the “Huang” method, followed by the Watershed function to segment bunched cells that appeared to touch. We then used the Voronoi function to generate boundary lines for the segmentation of the cells’ cytoplasm. To identify cytoplasm, auto thresholding of the CellMask channel using the “Huang” function was employed, with the images segmented by adding the nuclear Voronoi boundaries. The “analyze particles” function was then used to identify individual cellular ROIs that were greater than 10 µm2 and were not touching the image periphery. These ROIs were used to measure the raw 12-bit intensity of the EGFP and AKT-pS473 channels. A cutoff of EGFP > 100 was used to define EGFP-positive cells, since this value was greater than the mean ± 3 standard deviations of the untransfected cells’ EGFP intensity. Background intensity of AKT-pS473 was estimated from control cells subject to immunofluorescence with the AKT-pS473 antibody omitted; this value was subtracted from the measured values of all other conditions.

#2.3d I am missing a statement in the methods section that all images were acquired using the same settings.

This was indeed an important oversight on our part – thanks for spotting the omission of this crucial detail. This is now included at the end of the “Immunofluorescence” section of the Methods on pp. 9-10: Identical laser excitation power, scan speeds and photomultiplier gains were used across experiments to enable direct comparison.

#2.3e I recommend that the authors include a single cell correlation plot of EGFP fluorescence intensity vs AktpS473 intensity in Figure 1 D-F. This should be rather informative & make the concentration dependence clear.

We did not observe a strong correlation between PH-AKT1-EGFP intensity and pS473 staining, likely driven by both the imprecision of the cell segmentation and the fact that very low concentrations of PH domain effectively inhibit endogenous AKT1 (as we show in the later figures with the more precise, live cell AKT-NG2 recruitment experiments: see response to #2.5).

#2.3f I further recommend that the authors look at alterations of baseline Akt activity in the presence of the biosensor. In the images it looks like there might be an effect, but this is then lost in the analysis due to the normalization.

As covered in our response to #2.3b, there is indeed an inhibition of baseline pS473 in PH-AKT1-EGFP expressing cells, now explicitly quantified and documented in results.

#2.3g Please include zoomed image insets in Fig. 1D-F, in the current magnification one needs to zoom in quite a bit to see the effect in the raw data. It is a clear effect, but having a zoomed version would make for much easier reading.

We now include high-resolution confocal images instead of low power, low NA volumes as shown in the last version of the manuscript, which we believe addresses this point and also reviewer #1.2.

2.3h Up to the authors: I wonder whether it is possible to extract an IC50 value for the competitive inhibition of Akt by the respective biosensors. The transient expression gives the authors access to a wide range of expression levels at the single cell level, which could be quantified by counterstaining with a EGFP-nanobody at a different color (since the EGFP fluorophore went through the fixation process, it is likely unsuitable for quantification) and microscope calibration. Activity could be quantified as the ratio of observed and expected Akt-pS473 fluorescence (derived from the mean FI per cell from the EGFP control). This is not strictly necessary, but would be a beautiful quantitative experiment, give an easy-to-understand number & make the paper much stronger.

This is a great suggestion, but does not produce precise enough data to work out, as we detail in response to #2.3e. From our data in new figure 3F and figure 5, it seems we have not explored the appropriate expression range to see intermediate levels of inhibition necessary to estimate IC50. This would be a cool experiment though!

__#2.4 __Specific comments: Figure 2. Overall, compelling data. However, 25 molecules/100 um^2 at maximal recruitment feels low. Assuming a total cell surface area of appr. 2000 um^2 per cell and taking a baseline of 5 molecules/100 um^2 into account, this would mean that only about 400 copies of Akt are recruited in response to a pretty robust stimulus. Is it possible that the association reaction of the split GFP is not complete under these conditions? I think that a direct measurement of intracellular endogenous Akt concentration is required to put these numbers into context.

This is an excellent point that we had missed. We now specifically address this point in the third paragraph of the “Genomic tagging of AKT…” section on p. 4: __Accumulation of AKT-NG2 was ~25 molecules per 100 µm2, which assuming a surface area of ~1,500 µm2 per cell corresponds to ~375 molecules total. It should be noted that tagging likely only occurred at a single allele in each cell, and the population still exhibited expression of non-edited AKT1 (__Fig. 2B). Given that HEK293 are known to be pseudotriploid (Bylund et al., 2004), the true number of AKT1 molecules would be at least 1,125. However, given an estimated total copy number of 23,000 AKT1 in these cells (Cho et al., 2022), this is still only about 5%. However, we do not interpret these raw numbers due to uncertainties in the efficiency of NG2 complementation under these conditions, as well as potential for reduced expression from the edited allele.

We also removed the specific comment on molecule density from the abstract.

#2.5 Specific comments: Figure 3 I think that the classification by plasmid dose does not make a lot of sense, as the resulting expression levels are rather similar. I suggest to pool all traces and calculate mean curves by actual expression levels using a binning approach (e.g. 0-50 au, 50-100 au and so on in raw intensity from Figure 3b). If there is an effect in the realized concentration regime, this should pick it up.

This is an excellent suggestion, and we have done just that: thank you! The data is now included as a new panel Fig. 3F. The result is described in the results section, “Sequestration of PIP3 by lipid biosensors”, end of the first paragraph on pp. 4-6: To observe the concentration-dependence of AKT1-PH-mCherry inhibition, we pooled the single cell data from these experiments and split transfected cells into cohorts based on raw expression level (excitation and gain were consistent between experiments, allowing direct comparison). This analysis showed profound inhibition of AKT1-NG2 recruitment at all expression levels, with a slightly reduced effect only visible in the lowest expressing cohort (Fig. 2F).

#2.6 Specific comments: Figure 5 These are very interesting data, in particular with regard to the underlying PIP3 dynamics. I agree with the conclusion of the authors that shielding of PIP3 from degradation is the likely culprit. What I would like to see here is actual kinetic fits - and different terms. On- and off-rate imply biosensor binding, but these are likely rather fast and not on the minute-timescale. The detected processes are much more likely to reflect production and degradation of PIP3 and that should be reflected in the terminology. For the fit: I think that a simple rate law for subsequent reactions ([PIP3]=C(e^-k1t-e^k2t)) will give good results and yield effective rate constants for PIP3 generation and degradation. This implies the quasi-steady state assumption for biosensor binding and implies that [PIP3] is proportional to the biosensor bound [PIP3], but these are reasonable assumptions to make.

The is an excellent suggestion, which we have added. Specifically, fits are now present on Figs. 5G and 5I; we describe these in the last paragraph of results on p. 8: Normalizing data from both expression modes to their maximum response (Fig. 5G) and fitting kinetic profiles for cooperative synthesis and degradation reactionsrevealed the rate of synthesis is remarkably similar: 1.09 min–1 (95% C.I. 1.02-1.17) for single molecule expression vs 1.02 min-1 (95% C.I. 0.98-1.06) for over-expression. On the other hand, degradation slowed with over expression from 0.34 min–1 (95% C.I. 0.24-0.58) to 0.13 min–1 (95% C.I. 0.12-0.15). This is expected, since synthesis of PIP3molecules would not be prevented by biosensor. On the other hand, PIP3 degradation could be slowed by the over-expressed biosensor competing with PTEN and 5-OH phosphatases that degrade PIP3. An even more exaggerated result is achieved with the cPHx1 PI(3,4)P2 biosensor; this shows an increase in fold-change over baseline of 600% for single molecule expression levels, compared to only 100% in over-expressed cells (Fig. 5H). Again, the degradation rate of the signal is substantially slowed by the over-expressed sensor, reducing from 0.27 min–1 (95% C.I. 0.22-0.39) to 0.16 min–1 (95% C.I. 0.14-0.19), whereas synthesis remains only minorly impacted, changing from 0.61 min–1 (95% C.I. 0.57-0.64) to 0.54 min–1 (95% C.I. 0.52-0.56) with over-expression (Fig. 5I). Collectively, these data show that single molecule based PI3K biosensors show improved dynamic range and kinetic fidelity compared to the same sensors over-expressed.

Details of the fits are given in a new methods section on p. 11:

Fitting of reaction kinetics

Curve fitting was performed in Graphpad Prism 9 or later. For the data presented in Figs. 5G and 5I, both synthesis and degradation phases displayed clear “s” shaped profiles not well fit by simple first order kinetics. Since activation of the PI3K pathway involves many multiplicative interactions between adapters and allosteric activation of the enzymes themselves, we assumed cooperativity and fit reactions with the two phase reaction as follows:

Where Ft denotes ∆Ft/∆FMAX, nsyn and ndeg are the Hill coefficients of the respective synthesis and degradation reactions, and the rate constants for the reactions are derived from ksyn = 1/τsyn and kdeg = 1/τdeg.

André Nadler

Reviewer #2 (Significance (Required)):

This is an important paper, analyses the effects of over-expressed lipid biosensors on cell signalling in some detail and will be of significant interest to a broad readership.

Reviewer #3 (Evidence, reproducibility and clarity (Required)):

This is essentially a methods paper in which the authors provide a detailed and highly quantitative analysis of the potentially deleterious effects of expressing phosphoinositide-binding domains as biosensors. Specifically, they study the effects on PIP3 signalling, using biosensors that are widely used in the field.

They show that the most-commonly used method of expressing PIP3 biosensors using transient transfection with viral promotors has clear deleterious effects on downstream signalling due to out-competing the endogenous effectors. Importantly, they also describe a new approach to overcome this by developing new plasmids and methodology to express these reporters at low levels.

Reviewer 3 Major comments:

The work in this paper is thorough and very nicely done. I particularly appreciate the efforts to quantitate or estimate actual numbers and densities of molecules, which significantly strengthen their arguments. The data are excellent and strongly support all their conclusions. I would therefore be happy to see this work published in its current form.

Reviewer 3 Minor comments:

I only have some minor and optional suggestions for improvement.

#3.1 In figure 1D-F they show that PH-Atk-EGFP expression can suppress downstream Akt activation by quantifying P-Akt signal my microscopy. In these panels they say tgey selectively measure this in GFP-expressing cells, but it is not clear how they define which cells are expressing GFP - was a threshold used? Also, it would be nice to also measure both PH-Akt-GFP and P-Akt staining by flow cytometry to look for a correlation. Is there a threshold of biosensor expression that blocks downstream signalling, or is there a linear relationship? This might help specifically measure how much biosensor is too much.

This is an important comment, also raised by reviewer 2. We provide a detailed explanation and outline revisions that address this in our response to reviewer #2.3c; essentially, we replaced the analysis with an automated segmentation and quantification, estimating GFP-positive cells from a fraction of non transfected cells. We have not performed a FACS analysis, but as we note in our response to #2.3e __and #2.3h, the correlation between EGFP and pAKT staining is imprecise in these experiments. The new __Fig. 3C does address this point for AKT1-NG2 recruitment, as described in our response to #2.5.

#3.2 Some of their microscopy images (e.g. Fig 1D-F, Fig 5) are very small and would benefit from a zoom box - especially when they are trying to demonstrate single molecule detection.

This is a fair point raised by all of the reviewers in one form or another. We have added zoomed insets to all of the single molecule images in Figs 2-5, and added higher magnification, confocal section images to Fig. 1.

Reviewer #3 (Significance (Required)):

This is both a methods paper and cautionary tale for cell biologists working in this field. Whilst everyone who uses these probes should be aware of the potential risk of biosensors titrating our effectors, this is often not sufficiently acknowledged. This paper is a very nice and clear demonstration of these risks, exemplified with probably the most highly-used biosensor and key downstream signalling pathway.

Whilst the concepts presented are not especially novel, this paper nonetheless makes an important contribution to the community and hopefully will make others more cautious in how they use these biosensors.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The work from Petazzi et al. aimed at identifying novel factors supporting the differentiation of human hematopoietic progenitors from induced pluripotent stem cells (iPSCs). The authors developed an inducible CRISPR-mediated activation strategy (iCRISPRa) to test the impact of newly identified candidate factors on the generation of hematopoietic progenitors in vitro. They first compared previously published transcriptomic data of iPSCderived hemato-endothelial populations with cells isolated ex vivo from the aorta-gonadmesonephros (AGM) region of the human embryo and they identified 9 transcription factors expressed in the aortic hemogenic endothelium that were poorly expressed in the in vitro differentiated cells. They then tested the activation of these candidate factors in an iPSCbased culture system supporting the differentiation of hematopoietic progenitors in vitro. They found that the IGF binding protein 2 (IGFBP2) was the most upregulated gene in arterial endothelium after activation and they demonstrated that IGFBP2 promotes the generation of functional hematopoietic progenitors in vitro.

Strengths:

The authors developed an extremely useful doxycycline-inducible system to activate the expression of specific candidate genes in human iPSC. This approach allows us to simultaneously test the impact of 9 different transcription factors on in vitro differentiation of hematopoietic cells, and the system appears to be very versatile and applicable to a broad variety of studies.

The system was extensively validated for the expression of 1 transcription factor (RUNX1) in both HeLa cells and human iPSC, and a detailed characterization of this test experiment was provided.

The authors exhaustively demonstrated the role of IGFBP2 in promoting the generation of functional hematopoietic progenitors in vitro from iPSCs. Even though the use of IGFBP2interacting proteins IGF1 and IGF2 have been previously reported in human iPSC-derived hematopoietic differentiation in vitro (Ditadi and Sturgeon, Methods 2016; Ng et al., Nature Biotechnology 2016), and IGFBP-2 itself has been shown to promote adult HSC expansion ex vivo (Zhang et al., Blood 2008), its role on supporting in vitro hematopoiesis was demonstrated here for the first time.

Weaknesses:

Although the authors performed a very thorough characterization of the system in proof-ofprinciple experiments activating a single transcription factor, the data provided when 9 independent factors were used is not sufficient to fully validate the experimental strategy. Indeed, in the current version of the manuscript, it is not clear whether the results presented in both the scRNAseq analysis and the functional assays are the consequence of the simultaneous activation of all 9 TF or just a subset of them. This is essential to establish whether all the proposed factors play a role during embryonic hematopoiesis, and a more complete analysis of the scRNAseq dataset could help clarify this aspect.

Similarly, the data presented in the manuscript are not sufficient to clarify at what stage of the endothelial-to-hematopoietic transition (EHT) the TF activation has an impact. Indeed, even though the overall increase of functional hematopoietic progenitors is fully demonstrated, the assays proposed in the manuscript do not clarify whether this is due to a specific effect at the endothelial level or to an increased proliferation rate of the generated hematopoietic progenitors. Similar conclusions can be applied to the functional validation of IGFBP2 in vitro.

The overall conclusions are sometimes vague and not always supported by the data. For instance, the authors state that the CRISPR activation strategy resulted in transcriptional remodeling and a steer in cell identity, but they do not specify which cell types are involved and at what level of the EHT process this is happening. In the discussion, the authors also claim that they provided evidence to support that RUNX1T1 could regulate IGFBP2 expression. However, this is exclusively based on the enrichment of RUNX1T1 gRNA in cells expressing higher levels of IGFBP2 and it does not demonstrate any direct or indirect association of the two factors.

We thank the reviewer for the positive comments about the importance of our work and have now addressed the points raised as weaknesses by performing additional analysis and experiments, adding a new schematic of the mechanism, and rewording our claims.

We have clarified the different effects mediated by the activation and the IGFBP2 addition in a summary section at the end of the results and added Figure 6, showing this in visual form. We have also clearly stated the limitations related to the correlation between RUNX1T1 and IGFBP2 in the discussion and toned down our claims regarding this throughout the entire paper. We have also reworded the text to clarify the specific cell types identified in the sequencing data that we refer to.

Reviewer #2 (Public Review):

To enable robust production of hematopoietic progenitors in-vitro, Petazzi et al examined the role of transcription factors in the arterial hemogenic endothelium. They use IGFBP2 as a candidate gene to increase the directed differentiation of iPSCs into hematopoietic progenitors. They have established a novel induced-CRISPR mediated activation strategy to drive the expression of multiple endogenous transcription factors and show enhanced production of hematopoietic progenitors through expansion of the arterial endothelial cells. Further, upregulation of IGFBP2 in the arterial cells facilitates the metabolic switch from glycolysis to oxidative phosphorylation, inducing hematopoietic differentiation. While the overall study and resources generated are good, assertions in the manuscript are not entirely supported by the experimental data and some claims need further experimental validation.

We thank the reviewer for the positive comments, and we have provided new data and analysis to make sure that all our assertations are clearly supported and also reworded those where limitations were identified by the reviewers.

Recommendations for the authors:

Reviewing Editor (Recommendations For The Authors):

The assessment could change from "incomplete" to "solid" if the authors: i) improve data analysis (for both scRNAseq and functional assays) by providing additional information that could strengthen their conclusions, as suggested in the specific comments by both reviewers; ii) either provide new functional evidence supporting their mechanistic conclusion or alternatively tone down the claims that are not fully supported by data and acknowledge the limitations raised by reviewers in the discussion; (iii) the issue of paracrine signaling to expand only hematopoietic progenitors needs to be addressed.

We have now improved the data analysis and provided additional functional tests to strengthen our conclusions and toned down those that were identified by the reviewers as not supported enough and included a discussion on these limitations. We have also reworded the section about the paracrine signaling throughout the paper.

Reviewer #1 (Recommendations For The Authors):

Figure 1 contains exclusively published data. It might be more appropriate to use it as a supplementary figure or as part of a more exhaustive figure (maybe combining Figures 1 and 2 together?).

Figure 1 contained novel bioinformatic analyses that represent the base of our research and it has a different content and focus to figure 2, which is already a large figure. We therefore believe it is better to keep it as a separate figure, containing a new panel now too. 

It seems there is an issue with Figure S3 labelling:

• In line 112, Figure S2A-B does not display genomic PCR and sequencing results;

• In line 123, Figure S3D-E does not show viability and proliferation data;

• In line 127, Figure S3G does not show mCherry expression in response to DOX;

We apologies for the confusion with the numbers, we have now correctly labelled the figures.

It would be more informative to include gates and frequency on flow cytometry plots in Figure S3, to be able to evaluate the extent of the reduction in mCherry expression.

We have now included the gating and frequency of mCherry-expressing cells in Supplementary Figure 3D.

It is not clear from the text and figures whether the SB treatment was maintained throughout the hematopoietic differentiation protocol (line 122):

• If so, it would be important to confirm that HDAC treatment does not affect EHT cultures

• If not, can the authors provide some evidence that transgene silencing is not occurring during hematopoietic differentiation?

We have clarified that we decided to treat the cells with SB exclusively in maintenance condihons because HDACs have been shown to be essenhal for the EHT (lines 138-142). We have now also included addihonal data showing the high expression of the mCherry tag reporhng the iSAM expression on day 8 (Supplementary Figure 4F).

Can the authors provide a simple diagram summarizing the experimental strategy for each differentiation experiment in the respective supplementary figure? For instance, at what stage of the protocol was DOX added in Figure 3? Or at what stage IGFBP2 was added in Figure 5? It would be a very useful addition to the interpretation of the results.

We have now included three schemahcs for all the experiments in the manuscript in supplementary figure 4 A-C.

In Figure 3, the authors should provide more detailed information about the data filtering of the scRNAseq experiment, and more specifically:

• How many cells were included in the analysis for each library after QC and filtering?

• How "cells in which the gRNAs expression was detected" were selected? Do they include only cells showing expression of gRNAs for all 9 TF?

This informahon is now included in the method sechon lines 773-781; the detailed code is available on the GitHub link provided in the same sechon. We have filtered the cells expressing one gRNA for the non-targehng gRNA (iSAM_NT) control and more than one for the iSAM_AGM sample. 

In Figure 3A, it is not clear whether the expression of the 9 factors is consistently detected in all cells or just a subset of them, and the heatmap in Figure 3A does not provide this information. It would be more accurate to provide expression on a per-cell basis, for instance, as a violin plot displaying single dots representing each cell. 

We have now included this violin plot in Supplementary Figure 4G as requested. However, this visualisation is difficult to interpret because some of the target genes’ expression seems variable in both experimental and control conditions. We had envisaged that this could have been the case and so this is why we had included the three different controls.  For this reason we chose to show the normalised expression which takes all the different variables into account (Figure 3A). 

In Figure 3B-C, it seems that clusters EHT1 and EHT2 do not express endothelial markers anymore. Are these fully differentiated hematopoietic cells rather than cells undergoing EHT? In general, it would be quite important to provide evidence of expressed marker genes characterizing each cluster (eg. heatmap summarizing top DEG in the supplementary figure?). 

We have now provided a spreadsheet containing the clusters’ markers that we used in

Supplementary Table 1) a heatmap in Figure 3E. Furthermor,e we have now edited Figure 3C to include Pan Endothelial markers (PECAM1 and CDH5). These data show that the EHT1 and EHT2 cluster both express endothelial markers but are progressively downregulated as expected during endothelial to hematopoietic transition. We have also included and discussed this in the manuscript lines 192-195 and a schematic for the mechanism in Figure 6.

In Figure 3E, displaying the proportion of clusters within each sample/library would be a more accurate way of comparing the cell types present in each library (removing potential bias introduced by loading different numbers of cells in each sample).

We have now included the requested data in Supplementary Figure 4I and it confirms again the expansion of arterial cells in the activated cells.    

In Figure 3G, by plating 20,000 total CD34+, the assay does not account for potential differences in sample composition. It is then hard to discriminate between the increased number of progenitors in the input or an enhanced ability of HE to undergo EHT. This is an important aspect to consider to precisely identify at what level the activation of the 9 factors is acting. A proper quantification of flow cytometry data summarizing the % of progenitors, arterial cells, etc. would be useful to interpret these results.

Lines 204-205 reworded. We are very much aware of the fact that the CD34+ cell population consists of a range of cells across the EHT process and this is precisely why we carried out this single cell sequencing analyses.  We purposely tested the effect of the observed changes in composition by colony assays

In Figure 3G, it seems that NT cells w/o DOX have very little CFU potential (if any). Can the authors provide an explanation for this?

We think that the limited CFU potential is due to the extensive genetic manipulation and selection that the cells underwent for the derivation of all the iSAM lines but this did not impede us from observing an effect of gene activation on CFU numbers. This is one of the primary reasons that we then validated our overall findings using the parental iPSC line in control condition and with the addition of IGFBP2. We show that the parental iPSC line gives rise to hematopoietic progenitor, both immunophenotypically (Figure 4D) and functionally, at expected levels (Figure 4B left column).

Figure 4A shows an upregulation of IGFBP2 in arterial cells as a result of TF activation. However, from the data presented here, it is not possible to evaluate whether this is specific to the arterial cluster, or it is a common effect shared by all cell types regardless of their identity. 

Data has now been included in Supplementary Figure 4H, which shows that all the cells show an increase in IGFBP2, but arterial cells show the highest increase. We have now edited the text to reflect this, in lines 228-230.

In Figure 5A-B only a minority of arterial cells express RUNX1 in response to IGFBP2 treatment. Is this sufficient to explain the very significant increase in the generation of functional hematopoietic progenitors described in Figure 4? Quantification and statistical analysis of RUNX1 upregulation would strengthen this conclusion.

We have now provided the statistical analysis showing significant upregulation of RUNX1 upon IGFBP2 addition. The p values are now provided in the figure 5 legend.

In Figure 5 the authors conclude that IGFBP2 remodels the metabolic profile of endothelial cells. However, it is not clear which cell types and clusters were included in the analysis of Figure 5C-G. Is the switch from Glycolysis to Oxidative Phosphorylation specific to endothelial cells? Or it is a more general effect on the entire culture, including hematopoietic cells? 

We based this conclusion on the fact that the single-cell RNAseq allows to verify that the metabolic differences are obtained in the endothelial cells. Given that we sorted the adherent cells, the majority of these are endothelial cells as shown in Figure 5A. The Seahorse pipeline includes a number of washing steps resulting in the analyses being performed on the adherent compartment which we know consists primarily of endothelial cells. We cannot exclude some contamination from non-endothelial cells but we highlight to this reviewer that the initial observation of the metabolic changes was identified in endothelial cells in the single cell sequencing data. Taken together, we believe that this implies that metabolic changes are specific to this population. We have clarified this in the line 317.

In the discussion, the authors conclude that they "provide evidence to support the hypothesis that RUNX1T1 could regulate IGFBP2 expression". To further support this conclusion, the authors could provide a correlation analysis of the expression of the two genes in the cell type of interest. 

Following the observation of the IGFBP2 high expression across clusters, we have now reworded this sentence in lines 382-385  We have tried to perform the correlation analysis but we believe this not to be appropriate due to the detection level of the gRNA, we have now included this as a limitation point in the discussion lines 416-427, and also toned down the conclusion we did draw about RUNX1T1 throughout the whole manuscript.

As mentioned by the authors, IGFBP2 binds IGF1 and IGF2 modulating their function. Both IGF1 (http://dx.doi.org/10.1016/j.ymeth.2015.10.001) and IGF2 (doi:10.1038/nbt.3702) have been used in iPSC differentiation into definitive hematopoietic cells. It would be relevant to discuss/reference this in the discussion.

We have now included the suggested reference in the section where we discuss the role of IGFBP2 in binding IGF1 and IGF2.

Reviewer #2 (Recommendations For The Authors):

(1) Figure 1 compares the transcriptome of human AGM and in-vitro derived hemogenic endothelial cells (HECs). It is not clear why only the genes downregulated in the latter were chosen. Are there any significantly upregulated genes, knockdown/knockout which could also serve a similar purpose? Single-cell transcriptome database analysis is very preliminary. A detailed panel with differences in cluster properties of HECs between the two systems should be provided. A heatmap of all differentially expressed genes between the two samples must be generated, along with a logical explanation for choosing the given set of genes. 

We have now included another panel in figure 1 to better clarify the logic behind the strategy used to identify our target genes (Figure 1A).

(2) Figure 2 - a panel describing the workflow of gRNA design and targeting for the 9 candidate genes, along with lentiviral packaging and transduction would make it easier to follow. 

We have now included three schematics for all the experiments in the manuscript in supplementary figure 4 A-C. 

(3) Figure 3- to assess the effect of arterial cell expansion on the emergence of hematopoietic progenitors, CD34+ Dll4+ cells should be sorted for OP9 co-culture assay.

Using only CD34+ cells does not answer the question raised. Also, the CFU assay performed does not fully support the claim of enhanced hematopoietic differentiation since only CFU-E and CFU-GM colonies are increased in Dox-treated samples, with no effect on other colony types. OP9 co-culture assay with these cells would be required to strengthen this claim. 

We wanted to clarify that the effect on the methylcellulose coming from the activated cells was not limited to CFU-E, as the reviewer reported; instead, it also affected CFU-GM and CFU-M. 

We have now performed additional experiments where we sorted the CD34+ compartment into DLL4- and DLL4+ in Supplementary Figure 5D-E, which we discussed in lines 250-258. 

(4) In Figure 3F, there appears to be a lot of variation in the DLL4% fold change values for

DOX treated iSAM_AGM sample, which weakens the claim of increased arterial expansion.

Can the authors explain the probable reason? It is suggested that the two other controls (iSAM_+DOX and iSAM_-DOX) should be included in this analysis. It is imperative to also show % populations rather than just fold change to gain confidence.

We agree that there is a lot of variability. That is because differentiation happens in 3D in embryoid bodies, which contain many different cell types that differentiate in different proportions across independent experiments. We have now included the raw data in Supplementary Figure 4 D, with additional statistical analysis to show the expansion of arterial cells including also the suggested additional controls.

(5) How does activation of these target genes cause increased arterialization? Is the emergence of non-HE populations suppressed? Or is it specific to the HE? The data on this should be clarified and also discussed. ANTO/Lesley text

We have provided additional data clarifying the connection between increased arterialisation and hemogenic potential. We showed that the activation induces increased arterialisation and that IGFBP2 acts by supporting the acquisition of hemogenic potential. We have discussed this in lines 326-348 and provided a new figure to explain this in detail (figure 6)

(6) Considering that IGFBP2 was chosen from the activated target gene(s) cluster, can the authors explain why the reduced CFU-M phenomenon observed in Figure 3G does not appear in the MethoCult assay for IGFBP2 treated cells (Figure 4B)?

The difference could be explained by the fact that in Figure 3G, the cells underwent activation of multiple genes, while in Figure 4B, they were only exposed to IGFBP2. Our results show that IGFBP2 could at least partially explain the phenotype that we see with the activation, but we believe that during the activation experiments, there might be other signals available that might not be induced by IGFBP2 alone. We have also added a summary section and a figure to clarify the different mechanisms of action of the gene activation and IGFBP2.

(7) Figure 4- while the experiments conducted support the role of IGFBP2 in increasing hematopoietic output, there is no experimental evidence to prove its function through paracrine signalling in HECs. The authors need to provide some evidence of how IGFBP2 supplementation specifically expands only the hematopoietic progenitors. Experimental strategies involving specifically targeting IGFBP2 in hemogenic/arterial endothelial cells are required to prove its cell type specific function. Additionally, assessing the in vivo functional potential of the hematopoietic cells generated in the presence of IGFBP2, by bone-marrow transplantation of CD34+ CD43+ cells, is essential. 

The role of IGFBP2 in the context of HSC production and expansion was not the topic of our research, and we have not claimed that IGFBP2  affects the long-term repopulating capacity of HSPCs. Therefore, we believe that the requested experiments are not required to support the specific claims that we do make. We have now provided more experiments and bioinformatic analysis that support the role of IGFBP2 in inducing the progression of EHT from arterial cells to hemogenic endothelium, and to avoid misunderstandings, we have toned down our claims by editing the text regarding its paracrine effect s. 

(8) Figure 4C-D -It is recommended to plot % populations along with fold change value. As this is a key finding, it is important to perform flow cytometry for additional hematopoietic markers- CD144, CD235a and CD41a to demonstrate whether this strategy can also expand erythroid-megakaryocyte progenitors. Telma

Figure 4C already shows the percentage values; we have now added the percentage for Figure 4D in SF5C. We have also performed additional analysis as requested and added the data obtained to Supplementary Figure 5D.

(9) In Figure 5, analysis showing the frequency of cells constituting different clusters, between untreated and IGFBP2-treated samples in the single-cell transcriptome analysis is essential. Additional experiments are required to validate the function of IGFBP2 through modulation of metabolic activity. Inhibition of oxidative phosphorylation in the IGFBP2treated cells should reduce the hematopoietic output. Authors should consider doing these experiments to provide a stronger mechanistic insight into IGFBP2-mediated regulation of hematopoietic emergence.

We have now included the requested cluster composition in Supplementary Figure 5F. We decided not to include further tests on the metabolic profile of IGFBP2 as we already discussed in other papers that showed, using selective inhibitors, that the EHT coincides with a glycol to OxPhos switch. 

(10) It is very striking to see that IGFBP2 supplementation changes the transcriptional profile of developing hematopoietic cells by increasing transcription of OXPHOS-related genes with concomitant reduction of glycolytic signatures, particularly at Day 13. However, the mitochondrial ATP rate measurements do not seem convincing. The bioenergetic profiles show that when mitochondrial inhibitors are added, both groups exhibit decreased OCR values and, on the other hand, higher ECAR. This indicates that both groups have the capability to utilize OXPHOS or glycolysis and may only differ in their basal respiration rates.

Differences in proliferation rate can cause basal respiration to change. There is no information on how the bioenergetic profile was normalized (cell no./protein amount). Given that IGFBP2 has been shown to increase proliferation, it is very likely that the cells treated with IGFBP2 proliferated faster and therefore have higher OCR. The data needs to be normalized appropriately to negate this possibility.

We have previously tested whether IGFBP2 causes an increase in proliferation by analysing the cell cycle of cells treated with it, as we initially thought this could be a mechanism of action. We have now provided the quantification of the cell cycle in the cells treated with IGFBP2, showing no effect was observed in cell cycle Supplementary Figure 4E. Following this analysis, we decided to plate the same number of cells and test their density under the microscope before running the experiment; each experiment was done in triplicate for each condition. We have now added this info to the method sections lines 806-813.  We did not comment on the basal difference, which we agree might be due to several factors, but we only compared the difference in response to the inhibitors, which isn’t affected by the basal level but exclusively by their D values. We have also included the formulas used to calculate the ATP production rate.

Overall, it appears that IGFBP2 does not seem to primarily cause metabolic changes, but simply accelerates the metabolic dependency on OXPHOS. Hence, the term 'metabolic remodelling' must be avoided unless IGFBP2 depletion/loss of function analysis is shown.

We thank the reviewer for suggesting how to interpret the data about the dependency on OXPHOS. We have now changed the conclusions and claims about the effect of IGFBP2. We have also included a cell cycle analysis of the hematopoietic cells derived upon IGFBP2 addition to show that they don’t show differences in proliferation that could cause the increase in colony formation we observed. Regarding the assay, we have plated the same number of cells for each group to make sure we were comparing the same number of cells, which we also assessed in the microscope before the test, and we eliminated the suspension cells during the washes that preceded the measurement. The review is correct in indicating that there is a basal difference in the value of OCR and ECAR where the IGFBP2 is lower at the start and not higher, which would not conceal higher proliferation. Finally, the ATP production rate is calculated on the variation of OCR and ECAR upon the addition of inhibitors, which normalizes for the basal differences.

Temps Forts de la Vidéo "Peut on être adultes avec nos ados"

Voici les temps forts de la vidéo avec une description des sujets abordés :

• Introduction (0:00-3:00): Présentation de Cynthia Fleury, philosophe et psychanalyste, et du thème de la conférence : "Peut-on être adulte avec nos adolescents ?"
• Statistiques sur la santé mentale des adolescents (3:00-7:00): Présentation des chiffres de Santé France sur la dégradation de la santé mentale des adolescents, notamment depuis la Covid-19. Discussion sur la conscientisation accrue de la santé mentale et les obstacles à la consultation d'un professionnel.
• Clinique de l'adolescence (7:00-9:00): Exploration des transformations physiologiques et psychiques de l'adolescence, incluant la puberté, le développement cognitif et la construction de l'identité.
• Le concept de "care" (9:00-14:00): Analyse du "care" comme élément central de l'individuation et de la construction d'un sujet en relation avec le monde. Discussion des travaux de Winnicott, Gilligan et Tronto sur l'éthique du "care".
• Définition d'un adulte (14:00-23:00): Réflexion sur la définition d'un adulte selon le Larousse et proposition d'une dialectique pour le développement d'un sujet basée sur l'imagination vraie, le "pretium doloris" (prix de la douleur) et la "vis comica" (force comique).
• L'adolescence comme expérience d'un "corps mutant" (23:00-28:00): Discussion du texte de Jean-Pierre Benoît, "L'adolescence, un excès de corps," et exploration des défis posés par les transformations corporelles et la découverte de la sexualité.
• L'adolescence comme découverte de la vie comme "maladie chronique" (28:00-35:00): Analogie entre l'expérience de la maladie chronique et l'adolescence, toutes deux impliquant des ruptures biographiques, des atteintes à l'image de soi et des remises en question des projets de vie.
• Déconnexion des adolescents et conduites à risque (35:00-38:00): Analyse de la déconnexion croissante des adolescents par rapport à la réalité du monde adulte et des conduites à risque comme moyen de se réapproprier son corps et son existence.
• L'impact du Covid-19 (38:00-42:00): Discussion sur l'impact profond du confinement et de la pandémie sur la santé mentale des adolescents et des adultes, et sur la perte de chances pour les plus jeunes.
• L'importance du lien (42:00-44:00): Recommandations pour maintenir le lien avec les adolescents, en utilisant la verbalisation, le non-verbal et le partage d'expériences communes.
• Conclusion (44:00-46:00): Dernière question sur les activités offertes aux MNA pour vivre une vie d'adolescent et discussion sur la nécessité d'inclure le risque dans le processus de soin.

● Adolescence: Ce tag est essentiel car la vidéo explore de nombreux aspects de l'adolescence, tels que les transformations physiques et psychiques, la construction de l'identité, les conduites à risque et la relation aux adultes. ● Éducation: La vidéo aborde la question de l'éducation des adolescents, notamment le rôle des parents et la nécessité d'une autorité bienveillante. ● Santé Mentale: Les statistiques sur la santé mentale des adolescents et l'impact du Covid-19 occupent une part importante de la vidéo, justifiant ce tag. ● Philosophie: La vidéo s'appuie sur des concepts philosophiques pour analyser l'adolescence et la relation adulte-adolescent, notamment les travaux de Kant, Nietzsche et Ricker. ● Psychanalyse: Les théories psychanalytiques, en particulier celles de Winnicott, Anna Freud et Ronald Laing, sont utilisées pour comprendre le développement de l'adolescent. Concepts Clés: ● "Care": Ce concept central est analysé en profondeur, notamment à travers les travaux de Winnicott, Gilligan et Tronto. ● Individuation: La vidéo explore le processus d'individuation de l'adolescent, en lien avec le concept de "care". ● Rupture Biographique: Ce concept est utilisé pour illustrer les transformations profondes que traverse l'adolescent, en lien avec l'expérience de la maladie chronique. ● Corps Mutant: La vidéo s'intéresse à l'importance du corps dans l'expérience adolescente et aux défis posés par ses transformations. ● Conduites à Risque: Les conduites à risque, telles que la scarification et les tentatives de suicide, sont abordées dans la vidéo comme des manifestations de la quête d'identité et de la confrontation au réel. Autres Tags Pertinents: ● Parents ● Enfance ● Développement Personnel ● Psychologie ● Sociologie ● Communication ● Relation Adulte-Enfant ● Autorité ● Bienveillance ● Écrans ● Réseaux Sociaux ● Pandémie ● Confinement

reply to u/Pawps4895 at https://old.reddit.com/r/typewriters/comments/1h1dcil/help_ink_ribbon_not_moving/

That ghosting effect you're seeing may be down to your typing technique. Computer keyboard typing technique is different than typewriter technique. If you're pressing hard and/or bottoming the keys out, you may not be getting your fingers out of the way and causing the key to double strike while you're lifting your finger up.

Instead, type as if they keys are hot lava. Strike and release them as quickly as possible and that ghosting should clear up. For more on technique, try: https://hypothes.is/users/chrisaldrich?q=tag%3A%22typing+technique%22

If that isn't the issue, is that ghosting happening on all the keys or just a few? Cleaning things out certainly couldn't hurt: https://boffosocko.com/2024/08/09/on-colloquial-advice-for-degreasing-cleaning-and-oiling-manual-typewriters/

Reviewer #2 (Public review):

In this study, Kavaklıoğlu et al. investigated and presented evidence for a role for domesticated transposon protein L1TD1 in enabling its ancestral relative, L1 ORF1p, to retrotranspose in HAP1 human tumor cells. The authors provided insight into the molecular function of L1TD1 and shed some clarifying light on previous studies that showed somewhat contradictory outcomes surrounding L1TD1 expression. Here, L1TD1 expression was correlated with L1 activation in a hypomethylation dependent manner, due to DNMT1 deletion in HAP1 cell line. The authors then identified L1TD1 associated RNAs using RIP-Seq, which display a disconnect between transcript and protein abundance (via Tandem Mass Tag multiplex mass spectrometry analysis). The one exception was for L1TD1 itself, is consistent with a model in which the RNA transcripts associated with L1TD1 are not directly regulated at the translation level. Instead, the authors found L1TD1 protein associated with L1-RNPs and this interaction is associated with increased L1 retrotransposition, at least in the contexts of HAP1 cells. Overall, these results support a model in which L1TD1 is restrained by DNA methylation, but in the absence of this repressive mark, L1TD1 is expression, and collaborates with L1 ORF1p (either directly or through interaction with L1 RNA, which remains unclear based on current results), leads to enhances L1 retrotransposition. These results establish feasibility of this relationship existing in vivo in either development or disease, or both.

Comments on revised version:

In general, the authors did an acceptable job addressing the major concerns throughout the manuscript. This revision is much clearer and has improved in terms of logical progression.

Reviewer #1 (Public review):

Summary<br /> Roseman et al. use a new inhibitor of the maintenance DNA methyltransferase DNMT1 to probe the role of methylation on binding of the CTCF protein, which is known to be involved chromatin loop formation. As previous reported, and as expected based on our knowledge that CTCF binding is methylation-sensitive, the authors find that loss of methylation leads to additional CTCF binding sites and increased loop formation. By comparing novel loops with the binding of the pre-mRNA splicing factor SON, which localizes to the nuclear speckle compartment, they propose that these reactivated loops localize to near speckles. This behavior is dependent on CTCF whereas degradation of two speckle proteins does not affect CTCF binding or loop formation. The authors propose a model in which DNA methylation controls the association of genome regions with speckles via CTCF-mediated insulation.

Strengths<br /> The strengths of the study are 1) the use of a new, specific DNMT1 inhibitor and 2) the observation that genes whose expression is sensitive to DNMT1 inhibition and dependent on CTCF (cluster 2) show higher association with SON than genes which are sensitive to DNMT1 inhibition but are CTCF insensitive, is in line with the authors' general model.

Weaknesses<br /> There are a number of significant weaknesses that as a whole undermine many of the key conclusions, including the overall mechanistic model of a direct regulatory role of DNA methylation on CTCF-mediated speckle association of chromatin loops.

(1) The authors frequently make quasi-quantitative statements but do not actually provide the quantitative data, which they actually all have in hand. To give a few examples: "reactivated CTCF sites were largely methylated (p. 4/5), "many CTCF binding motifs enriched..." (p.5), "a large subset of reactivated peaks..."(p.5), "increase in strength upon DNMT1 inhibition" (p.5); "a greater total number....." (p.7). These statements are all made based on actual numbers and the authors should mention the numbers in the text to give an impression of the extent of these changes (see below) and to clarify what the qualitative terms like "largely", "many", "large", and "increase" mean. This is an issue throughout the manuscript and not limited to the above examples.<br /> Related to this issue, many of the comparisons which the authors interpret to show differences in behavior seem quite minor. For example, visual inspection suggests that the difference in loop strength shown in figure 1E is something like from 0 to 0.1 for K562 cells and a little less for KCT116 cells. What is a positive control here to give a sense of whether these minor changes are relevant. Another example is on p. 7, where the authors claim that CTCF partners of reactivated peaks tend to engage in a "greater number" of looping partners, but inspection of Figure 2A shows a very minor difference from maybe 7 to 7.5 partners. While a Mann-Whitney test may call this difference significant and give a significant P value, likely due to high sample number, it is questionable that this is a biologically relevant difference.

(2) The data to support the central claim of localization of reactivated loops to speckles is not overly convincing. The overlap with SON Cut&Tag (figure 2F) is partial at best and although it is better with the publicly available TSA-seq data, the latter is less sensitive than Cut&Tag and more difficult to interpret. It would be helpful to validate these data with FISH experiments to directly demonstrate and measure the association of loops with speckles (see below).

(3) It is not clear that the authors have indeed disrupted speckles from cells by degrading SON and SRRM2. Speckles contain a large number of proteins and considering their phase separated nature stronger evidence for their complete removal is needed. Note that the data published in ref 58 suffers from the same caveat.

(4) The authors ascribe a direct regulatory role to DNA methylation in controlling the association of some CTCF-mediated loops to speckles (p. 20). However, an active regulatory role of speckle association has not been demonstrated and the observed data are equally explainable by a more parsimonious model in which DNA methylation regulates gene expression via looping and that the association with speckles is merely an indirect bystander effect of the activated genes because we know that active genes are generally associated with speckles. The proposed mechanism of a regulatory role of DNA methylation in controlling speckle association is not convincingly demonstrated by the data. As a consequence, the title of the paper is also misleading.

(5) As a minor point, the authors imply on p. 15 that ablation of speckles leads to misregulation of genes by altering transcription. This is not shown as the authors only measure RNA abundance, which may be affected by depletion of constitutive splicing factors, but not transcription. The authors would need to show direct effects on transcription.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

In the work: "Endosomal sorting protein SNX4 limits synaptic vesicle docking and release" Josse Poppinga and collaborators addressed the synaptic function of Sortin-Nexin 4 (SNX4). Employing a newly developed in vitro KO model, with live imaging experiments, electrophysiological recordings, and ultrastructural analysis, the authors evaluate modifications in synaptic morphology and function upon loss of SNX4. The data demonstrate increased neurotransmitter release and alteration in synapse ultrastructure with a higher number of docked vesicles and shorter AZ. The evaluation of the presynaptic function of SNX4 is of relevance and tackles an open and yet unresolved question in the field of presynaptic physiology.

Strengths:

The sequential characterization of the cellular model is nicely conducted and the different techniques employed are appropriate for the morpho-functional analysis of the synaptic phenotype and the derived conclusions on SNX4 function at presynaptic site. The authors succeeded in presenting a novel in vitro model that resulted in chronical deletion of SNX4 in neurons. A convincing sequence of experimental techniques is applied to the model to unravel the role of SNX4, whose functions in neuronal cells and at synapses are largely unknown. The understanding of the role of endosomal sorting at the presynaptic site is relevant and of high interest in the field of synaptic physiology and in the pathophysiology of the many described synaptopathies that broadly result in loss of synaptic fidelity and quality control at release sites.

We thank the reviewer for their positive evaluation of our manuscript.

Weaknesses:

The flow of the data presentation is mostly descriptive with several consistent morphological and functional modifications upon SNX loss. The paper would benefit from a wider characterization that would allow us to address the physiological roles of SNX4 at the synaptic site and speculate on the underlying molecular mechanisms. In addition, due to the described role of SNX4 in autophagy and the high interest in the regulation of synaptic autophagy in the field of synaptic physiology, an initial evaluation of the autophagy phenotype in the neuronal SNX4KO model is important, and not to be only restricted to the discussion section.

We thank the reviewer for their suggestions and agree that broader characterization would help us speculate on the underlying mechanism. To address this, we have conducted additional independent experiments investigating the role of SNX4 in neuronal autophagy, as suggested by this reviewer. These experiments are now included in the main figures and are no longer limited to the discussion section. Please see the detailed responses to this reviewer's recommendations below.

Reviewer #2 (Public Review):

Summary:

SNX4 is thought to mediate recycling from endosomes back to the plasma membrane in cells. In this study, the authors demonstrate the increases in the amounts of transmitter release and the number of docked vesicles by combining genetics, electrophysiology, and EM. They failed to find evidence for its role in synaptic vesicle cycling and endocytosis, which may be intuitively closer to the endosome function.

Strengths:

The electrophysiological data and EM data are in principle, convincing, though there are several issues in the study.

We thank the reviewer for their positive evaluation of our manuscript.

Weaknesses:

It is unclear why the increase in the amounts of transmitter release and docked vesicles happened in the SNX4 KO mice. In other words, it is unclear how the endosomal sorting proteins in the end regulate or are connected to presynaptic, particularly the active zone function.

We thank the reviewer for their suggestions and agree that further characterization would help to understand how endosomal sorting proteins regulate presynaptic neurotransmission. We have now added extra data on electrophysiological recordings clarifying SNX4’s role in the synapse. Please see the detailed responses to this reviewer's recommendations below.

Reviewer #3 (Public Review):

Summary:

The study aims to determine whether the endosomal protein SNX4 performs a role in neurotransmitter release and synaptic vesicle recycling. The authors exploited a newly generated conditional knockout mouse to allow them to interrogate the SNX4 function. A series of basic parameters were assessed, with an observed impact on neurotransmitter release and active zone morphology. The work is interesting, however as things currently stand, the work is descriptive with little mechanistic insight. There are a number of places where the data appear to be a little preliminary, and some of the conclusions require further validation.

Strengths:

The strengths of the work are the state-of-the-art methods to monitor presynaptic function.

We thank the reviewers for their positive evaluation of our manuscript.

Weaknesses:

The weaknesses are the fact that the work is largely descriptive, with no mechanistic insight into the role of SNX4. Further weaknesses are the absence of controls in some experiments and the design of specific experiments.

We thank the reviewer for their suggestions and agree that addition of extra control groups and experiments would strengthen interpretation of the observed phenotype. To address this, we have now performed experiments to investigate the miniature excitatory postsynaptic currents and added extra control groups such as overexpression of SNX4 on control background. In addition, we assessed SNX4-mediated neuronal autophagy as a potential molecular mechanism by which SNX4 affects synaptic output. Please see the detailed responses to this reviewers’ recommendations below.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The characterization of the neurite outgrowth presented in Figure 1 is a necessary starting point for the characterization of the model and the interpretation of the following data. Being the analysis conducted at 21 DIV, a significant portion of the neurite tree is out of the analyzed field. Adding sholl analysis will better indicate the complexity of the that appears to be influenced by SNX4 loss in the representative images shown in Figure 1f.

We fully agree and have now performed a Sholl analysis of dendrite branches to investigate dendritic complexity. (Figure 1(i), page 2-3, line 86-88). SNX4 depletion does not affect dendrite length or dendrite branching.

(2) Analogously, the characterization of synapse number is of relevance for the interpretation of the data. For a better flow of the data, Figure 4 might be presented as Figure 2 (without the repetition of panel h in Figure 1). An explanation of how VAMP2 puncta are processed is necessary in the method section. A double labelling with a postsynaptic marker would allow trafficking organelles to be distinguished from mature synaptic contacts. Indeed, the analysis of VAMP2 intensity along neurite in mature 21DIV neurons should reveal peaks in the intensity profile that represent synaptic contacts. For unexplained reasons, the profile is rather flat in the two experimental groups. Focusing on axonal branches will surely result in a peaked profile for VAMP2 labelling.

We fully agree that the characterization of synapses is relevant for the interpretation of the data. We have now added a section in our Material and Methods how the VAMP2 puncta are processed (p14 line 517-520). Instead of labeling mature synapses using double labeling of VAMP2 and PSD95, we analyzed the number of active synapses in live neurons using SypHy (Fig. 3g). The reviewer is correct that the VAMP2 data presented in Fig 1I and Fig 4 is part of the same dataset and we have clarified this in the figure legend. In Fig 1I only the total number of VAMP2 puncta is plotted as a marker for synapse number, while in Fig 4 we assess VAMP2 as potential SNX4 sorting cargo (Ma et al., 2017). Because of these different aims, we prefer to keep the figures separate. The analysis of VAMP2 intensity along the distance of the soma is a Sholl analysis (Fig. 4d), represents the average VAMP2 intensity over distance from the soma of 35-41 neurons per group. In contrast to a line scan of a single neurite, this average profile lacks the peaks of individual synapses.

(3) Miniature excitatory postsynaptic currents recordings would strengthen the synaptic characterization and complement the electrophysiological recordings shown in Figure 2. Analyzing frequency and amplitude parameters would complement the data on the number of synaptic connections defined by the pre and postsynaptic colocalization puncta as suggested above and may support the data shown in Figure 3 g that suggests a decreased number of active synapses in SNX4-KO cells.

We fully agree that the characterization of miniature excitatory postsynaptic currents would strengthen the synaptic characterization and complement the other electrophysiological data. Therefore, we have now added additional experiments showing the mEPSCs (Fig. 2k-m, page 4) in SNX4 cKO neurons versus control. This data shows that the amplitude and frequency of spontaneous miniature EPSCs (mEPSCs) were not affected upon SNX4 depletion, consistent with a normal first evoked EPSC and RRP estimate. Furthermore, these data suggest that it is unlikely that the observed increase in neurotransmission is due to post-synaptic effects.

(4) Recordings on the first evoked response shown in Figure 2 b and quantified in Figures c and d suggest that SNX4 overexpression per se exerts some effect on the Amplitude and the Charge of the first evoked response. This is also evident in the supplementary Figure 2 with lower frequency trains. An additional experimental group, namely control+SNX4 is needed for the correct interpretation of the observed phenotype. The possibility that SNX4 per se exerts an effect on evoked transmission could be discussed in terms of putative mechanisms and interactions.

We thank the reviewer for their suggestion and agree that an additional experimental group (control + SNX4) would strengthen interpretation of the observed phenotype. We have now added a new experimental condition with overexpression of SNX4 on a control background (Supplementary Fig. 3, page 20). This data shows that the amplitude and charge of the first evoked response were not affected in control + SNX4 neurons compared to control, and no differences were detected in the response to the 40 Hz stimulation train (Supplementary Fig. 3a-e).  Together, these data suggest that SNX4 overexpression in itself does not affect the neurotransmission protocols studied in SNX4 cKO experiments.

(5) To correctly interpret the SyPhy experiments and exclude an effect of SNX silencing on SV recycling, it is suggested to repeat the experiments shown in Figure 3 in the absence and in the presence of bafilomycin. Indeed, the quantifications shown in Figure 3 d and f do not represent "release fraction" as stated (lines 139/140) but they rather refer to an average difference between release fraction and recovered fraction. With the use of bafilomycin, the comparison of the deltaFmax/deltaFNH4Cl with and without bafilomycin would enable the release fraction to be correctly evaluated and compared.

We appreciate the reviewer’s suggestion and agree on the importance of considering the impact of SV recycling when evaluating the released fraction. We agree that the presence of bafilomycin is critical to isolate the released component during stimulation. We have now rephrased this conclusion. To assess synaptic recycling in these assays, bafilomycin in not critically required and we show by multiple independent experiments, including SypHy and FM64 dye assays, that SV recycling is either not affected or the effect is too small to be detected by these methods.

(6) In the ultrastructural analysis, additional quantifications are needed to exclude the accumulation of endosome-like structures. It is not clear if, in the evaluation of total SV number (Figure 5e), the authors counted all vesicles or vesicles < 50nm. This has to be explained and additional quantification of # of SV < 50nm and # SV > 50nm is informative, taking into account the endosomal nature of SNX4. Indeed, although the average size of SV is not changed (fig. 5 d), the density of "bigger vesicle" may result from endosomal-like structure accumulation. An additional suggested quantification is on vesicle # SV > 80nm as previously reported in the cited references dealing with endosomal proteins and presynaptic morphology.

We fully agree that the characterization of vesicle size is important and that it was not clearly stated which vesicles were included in the total number of SV (Fig. 5e). We have now added this to the figure description. We have also added a histogram that contains the vesicle numbers of different bin sizes for SNX4 cKO synapses and control synapses (Supplementary Fig. 4, page 21) including # SVs > 80nm. (Whilst it seems that there are more “bigger” vesicles in the KO, further analysis revealed that this is mostly driven by one experiment and this effect is not consistent.)

(7) Due to the high scientific interest in presynaptic autophagy for SV recycling and degradation, and the paucity of experimental work assessing the proteins involved, an initial evaluation of the neuronal autophagy process (by western blot analysis and immunocytochemistry) for the characterization of the model will better support the paragraph in the discussion (lines 314-322) and contribute to future work in the field. Although very rare, autophagosomes quantification at presynaptic sites can also be performed from the already acquired images. A double membrane structure with the material inside is evident in the representative control image presented!

We appreciate the reviewer’s suggestion and agree that presynaptic autophagy is an interesting potential mechanism that would elaborate our current working model. To address the reviewers’ suggestion, we added multiple independent experiments to investigate basal autophagy markers such as ATG5 using western blot analysis, characterization of p62 levels using immunohistochemistry and performed additional morphometric analysis on the electron microscopy data (Supplementary Fig. 5). In SNX4 cKO neurons, there was no significant difference in P62 puncta numbers or P62 somatic intensity under basal conditions or after blocking autophagic P62 degradation by bafilomycin treatment, suggesting that autophagic flux remains normal. Also, no changes in total ATG5 protein levels were observed and ultrastructural analysis revealed no differences in the total number of autophagosomes. Collectively, these data indicate that SNX4 depletion does not impact the basal autophagic flux, ATG5 protein levels, or the number of autophagosomes.

Minor points:

(1) Dorrbaun et al. 2018 is missing from the reference list. In the legend to figure 1 there is an incorrect reference to Figure 6, rather than Figure 4.

We have now adjusted the figure legend and added the reference (page 16, line 604).

(2) Information on the construct employed for the rescue is missing. Is it a fluorescent tag construct? Representative images of the three autaptic neurons (control, KO, KO+SNX4) would nicely complement data presentation in Figure 2. 

We have now elaborated on this in material and methods section (p12, line 418-421). Unfortunately, we did not obtain pictures of autaptic neurons used for electrophysiology experiments.

Reviewer #2 (Recommendations For The Authors):

(1) Figure 2d and f are somewhat inconsistent. Total charges for the 1st EPSCs differ almost 2-fold in the same condition.

We appreciate the reviewer’s concern. The average EPSCs charge of the first evoked was 89, 122 and 57 pC for control, KO and rescued neurons respectfully. The average charge of the first pulse of 40Hz train was 41,58 and 32 pC for control, KO and rescued neurons respectfully, which is roughly 50% of the naïve response of the same cells. These trains were recorded after 2 or 3 other stimulation paradigms, which can have affected the total charge released in the 40Hz train. That said, the proportional difference between groups is high comparable, with a 37% increased average charge released in SNX4 cKO compared to control in the naïve response and 41% increased response in the first response of the 40 Hz train, and rescued cells show a 53% reduction in average released charge compared to control in the naïve response compared to a 44% reduction in the first response of the 40 Hz train. Although the absolute values differ between these readouts, we conclude that the biological comparison between groups is consistent.

(2) Figure 2h. This type of analysis has a drawback. See Neher (2015) for the problems associated with this analysis.

We fully agree with the reviewer’s comment. As noted in our discussion (page 9 line 285), while this analysis has its limitations, it can still provide an indication of the ready releasable pool.   

(3) The EPSC phenotype may be due to postsynaptic effects. This should be excluded by additional experiments (mEPSC analysis) or further clarification.

We fully agree that the characterization of miniature excitatory postsynaptic currents recording would strengthen the synaptic characterization and complement the electrophysiological recordings. Therefore, we have now added additional experiments showing the mEPSCs (Fig. 2k-m) in SNX4 cKO neurons versus control. This data shows that the amplitude and frequency of spontaneous miniature EPSCs (mEPSCs) were not affected upon SNX4 depletion, suggesting that it is unlikely that the observed increase in neurotransmission is due to post-synaptic effects.

(4) The increased number of docked vesicles observed in EM and the increased slope (vesicle recruitment, Figure 2h) are not consistent with each other. Maybe the definition of docked vesicles is unclear in this version of the manuscript.

As noted in our material & methods (page 15, line 547-548), SVs were defined as docked if there was no distance visible between the SV membrane and the active zone membrane. We have added the pixel size for clarification. Indeed, we do not observe an increase in release probability or first evoked response, which would correspond with an increased docked pool. However, we think that the increase in docked vesicles might contribute to an enhanced SV recruitment (see discussion).

(5) Figure 3: Vesicle cycling was monitored in only a limited condition. It is known that there are multiple pathways of vesicle cycling. Ideally, these pathways should be dissected. At least, the authors mention the possibility that they have missed some "positive" conditions.

We fully agree with the reviewer’s comment that vesicle recycling is complex with several parallel pathways involved. While we did not study individual endocytosis pathways, we used different assays covering various recycling pathways. The SypHy assay (Fig. 3c & f) combined with the 100 AP stimulation paradigm at room temperature predominantly addresses clathrin-mediated endocytosis. Additionally, the FM-64 dye assay at 37 degrees Celsius covers ultrafast endocytosis pathways as well as bulk endocytosis routes. Since neither assay showed major effects, we decided not to pursue further experiments focusing on different endocytosis pathways.

Reviewer #3 (Recommendations For The Authors):

Major points:

(1) Since all of the work here is culture-focussed, the in vivo phenotype is not as relevant, however the in vitro properties are. The incomplete Cre-dependent removal of SNX4 is concerning (especially axonal SNX4 levels identified via immunofluorescence), however, the main concern is that there was no profiling of the other molecular changes within these cultures. This is important, since there may be considerable alterations in the expression of a number of presynaptic proteins which may explain the observed phenotypes. Ideally, these cultures could have been profiled in an unbiased manner via mass spectrometry to identify potential changes in the presynaptic proteome, or at the very least the levels of key fusion molecules would have been assessed via Western blotting.

We thank the reviewer for their suggestion and agree that mass spectrometry would strengthen the interpretation of the observed phenotype. However, due to contractual constraints, we are unable to pursue a mass spectrometry follow-up experiment. We agree that characterizing key fusion molecules is of potential interest. Therefore, based on literature, we selected a likely candidate, VAMP2, which did not show any alterations in expression levels when knocking out SNX4. Given the previously described role of SNX4 in the degradation pathway, one would expect increased degradation of key fusion molecules if they are recycled by SNX4. Other literature indicates that reduced levels of key fusion molecules, such as synaptotagmin or SNAP-25 (Broadie et al., 1994; Washbourne et al., 2001) , do not mimic our phenotype.

(2) The experiments reported in Figure 2, in particular those in 2c and 2d, suggest that overexpression of SNX4 has a dominant-negative effect on neurotransmitter release. This is strongly supported by the supplementary data during a stimulus train (particularly the start point of the 5 Hz train in Supplementary Figure 2). Therefore, the perceived rescue of EPSC charge in Figure 2f, 2g may be a result of SNX4 inhibiting neurotransmitter release. A determination of the impact of SNX4 overexpression (and level of overexpression) in WT neurons is essential to show that this is a bonefide rescue, rather than a direct inhibition by SNX4 overexpression.

We thank the reviewer for their suggestion and agree that an additional experimental group (control + SNX4) would strengthen interpretation of the observed phenotype. We have now added a new experiment with an extra experimental condition with overexpression of SNX4 on a control background (Supplementary Fig. 3 page 21). This data shows that the amplitude and charge of the first evoked response were not affected in control + SNX4 neurons compared to control, and no differences were detected in the response to the 40 Hz stimulation train (Supplementary Fig. 3a-e).  Together, these data suggest that SNX4 overexpression in itself does not affect the neurotransmission protocols studied in SNX4 cKO experiments.

(3) The experiments in Figure 3 clearly reveal a lack of effect of SNX4 depletion on synaptic vesicle endocytosis. However, the assumption that synaptic vesicle recycling is unaffected is a little premature. The fact that the second evoked SypHy peak is significantly larger than the first (Figures 3c-e) suggests that more vesicles may be recycling in KO neurons. Furthermore, the FM dye experiments do not aid interpretation, since there may be insufficient time (10 min) for new vesicles to be generated from endosomal intermediates experiments. Therefore, to confirm an absence of effect on recycling, the authors could either 1) perform the same experiment as 3c, but with 4 stimulation trains (to drive the system harder to reveal any phenotype) or 2) repeat the FM dye experiment but increase the time between loading and unloading to 30 min.

We fully agree with the reviewers' comment that vesicle recycling is an important component to consider and is complex with several parallel pathways involved. We conducted multiple independent experiments covering the most significant recycling pathways. The SypHy assay (Fig. 3c & f) combined with the 100 AP stimulation paradigm at room temperature predominantly addresses clathrin-mediated endocytosis. Additionally, the FM-64 dye assay at 37 degrees Celsius covers ultrafast endocytosis pathways as well as bulk endocytosis routes. To further challenge the system and reveal recycling phenotypes, we included a second 100 AP stimulation in our SypHy assay. While only the increase of the second SypHy peak is significant, the absolute numbers do not differ much from the first peak (0,17 for control and 0,21 for KO second peak and 0,19 for control and 0,22 for KO first peak, Supplementary table1). We nevertheless do not see any effects on recycling after the second peak (mean decay time is 27 for control and 26 for KO Supplementary Table 1). A single 100 AP 40 Hz train depletes all the synchronous release (not shown) and most of the evoked charge (see Fig 2f), hence two of these trains with one minute recovery is already a very demanding protocol. Although increasing the time between loading and unloading to 30 minutes might uncover other recycling components, it has been shown that ultrafast endocytosis occurs within 30 seconds (Watanabe et al., 2013), suggesting that 10 minutes should provide enough time for synaptic vesicle recycling. This is also evident from the fact that we can significantly destain synapses loaded with FM dye by electrical stimulation (Fig 3j), indicating that synaptic vesicle recycling took place. Since neither assay showed major effects, we concluded that under these circumstances, synaptic recycling is not significantly affected. However, we cannot exclude the possibility that recycling deficits in SNX4 cKO neurons could be detected in other paradigms,

(4) There is no obvious effect on VAMP2 levels or location in SNX4 KO neurons (Figure 4). However, when one considers that SNX4 is proposed to have a role in VAMP2 trafficking, it is surprising that an experiment examining the live trafficking of VAMP2-SypHy was not performed. This would have revealed activity-dependent alterations that would have been missed by simply measuring VAMP2 expression and localization, and potentially provided a molecular explanation for the enhanced neurotransmitter release during a stimulus train.

We appreciate the reviewer’s suggestion and agree that it could be a valuable experiment However, overexpressing a VAMP2-pHluorin construct might obscure potential phenotypes related to VAMP2 trafficking. SNX4 is expected to be involved in VAMP2 recycling, even with activity-dependent changes. Mis-sorted VAMP2 would accumulate in acidic vesicles, which could be masked by the VAMP2-pHluorin construct. Similarly, mis-sorting of other SNX4 cargo, such as the transferrin receptor, has been identified through lysosomal degradation, as shown by Western blot analysis of expression levels of the endogenous protein. We did not detect any differences in endogenous levels of VAMP2 within 21 days of SNX4 deletion (Fig 4), indicating that SNX4-dependent endosome sorting is not essential for VAMP2 recycling.

(5) The morphological data in Figure 5 report a series of small changes in docked vesicles and active zone length. In many cases, significance is obtained due to synapses being used as the experimental n, and thus inflating the statistical power. When one considers that no significant effect was observed on evoked release (apart from during a stimulus train), it suggests that the number of docked vesicles does not alter release probability in this system (which the authors point out). Instead, they suggest that an increased supply of vesicles is responsible, via increased recruitment to RRP/releasable pool (but not via increased recycling). If this is the case, it should have been reflected as an increase in the evoked SypHy response in Fig 2c,d (which is borderline significant). What may help is to determine the morphological landscape immediately after a stimulus strain, since this is the only condition where enhanced release is observed, and thus provide a morphological correlate to the physiological data.

We fully agree with the reviewer’s suggestion that an ultrastructural characterization immediately after a stimulus train would be informative. Unfortunately, contract constraints prevent us from performing this experiment. For our ultrastructural morphological data, we treated synapses as individual experimental n since it is not possible to determine whether synapses in a micronetwork on one sapphire originate from the same neuron. We used 18 independent sapphires from 3 independent pups to ensure the technical and biological replication of our data and measuring independent neurons. We fully agree with the reviewers comment to be careful with ‘inflating the statistical power’ due to potential nesting effects when using synapses as experimental n. To mitigate the potential nesting effect of analyzing multiple synapses per neuron, the intracluster correlation (ICC) is calculated per variable and per nesting effect. If ICC was close to 0.1, indicating that a considerable portion of the total variance can be attributed to e.g. synapse or sapphire, multilevel analysis was performed to accommodate nested data (Aarts et al., 2014).

Minor points

(1) When a new mouse model is generated, it is usually accompanied by a thorough characterization of its properties. However, in this case, there was no information provided about the conditional SNX4 knockout mouse. This is surprising and at a minimum, the following should be provided a) the background strain, b) method of generation, c) the number of animals used to establish the colony, d) breeding strategy, e) backcrossing strategy, f) genotyping protocol.

We apologize that a thorough characterization of our novel mouse model was lacking and therefore added this to our material & methods section (page 11, line 377-391).

(2) There is a noticeable difference between WT and KO neurons during train stimulation in Figure 2f, however, this appears to be due to the fact that there is a far higher EPSC charge to begin with in KO neurons. Why is there such a disparity when there is no difference in response to single pulses (Figures 2b-d) or presynaptic plasticity (Figure 2e)?

We understand the reviewer’s concern. We excluded an outlier (3x SD) in the KO dataset that drove the initial far higher EPSC charge in the graph (was already excluded for the statistics, Supplementary table 1). The average charge of the first pulse of 40Hz train is 41 pC and for KO neurons 58 pC, which did not differ significantly.  These trains of Fig. 2f were recorded after 2 or 3 other stimulation paradigms, which can have affected the total charge released in the 40Hz train. That said, the proportional difference between groups is high comparable between Fig 2b-d and 2f, with a 37% increased average charge released in SNX4 cKO compared to control in the naïve response (Fig. 2d) and 41% increased response in the first response of the 40 Hz train (Fig. 2f), and rescued cells show a 53% reduction in average released charge compared to control in the naïve response compared to a 44% reduction in the first response of the 40 Hz train. Although the absolute values differ between these readouts, we conclude that the biological comparison between groups is consistent.

(3) Line 343-344 - "(Supplementary Figure 1a)" should be "(Figure 1a)".

We thank the reviewer for this comment and adjusted this in the manuscript.

Welcome back to stage two of this advanced demo lesson and again have included full instructions attached to this lesson.

And this stage of the demo will be another one where you're entering lots of commands because you're going to automate the build of the WordPress application instance.

So again, I would recommend opening the instructions for this demo lesson and copy and pasting the commands rather than typing them out by hand.

Now at this point in the advanced demo series, you're going to have a leftover instance that you used to manually install WordPress in the previous stage.

It should be called WordPress - Manual.

So I'm going to want you to go ahead and right click on that and select terminate instance and confirm that process to remove this instance from your AWS account.

We're going to be setting up exactly the same single instance deployment of WordPress, so both the database and the application on the same instance.

But instead of manually building this, we're going to be using a launch template.

So from the EC2 console, just go ahead and click on launch templates under instances.

The first step is to create a launch template for our WordPress application.

So go ahead and click on create launch template.

Now launch templates are actually a new version of launch configurations that were previously used with auto scaling groups.

Launch templates allow you to either launch instances manually using the template or they can be part of auto scaling groups.

But what a launch template allows you to do is to specify all of the configuration in advance to launch an instance and that template can be used to launch one or many instances.

So we're going to create a launch template which will automate the installation of WordPress, MariaDB and perform all of the configuration.

And a launch template can actually have many different versions, which is a feature we'll use throughout this demo series as we evolve the design.

So the first step is to name this template and we're going to call it WordPress.

Under template version description, go ahead and enter single server DB and app.

And then check this box which says provide guidance to help me set up a template that I can use with EC2 auto scaling.

We're not immediately going to set it up as part of an auto scaling group, but it will help us highlight any options which are required if we want to use it with an auto scaling group.

Now launch templates can actually be created from scratch or they can be based on a previous template version.

If we expand source template, you're able to specify a template which this template is based on.

But in this case, we're creating one from scratch so we won't set any of those options.

Now just scroll down.

So the next thing we're going to define in this launch template is the AMI that we're going to use.

So go ahead and click on Quickstart.

And once this has changed, we're going to use the same AMI we've been using previously.

So I want you to go ahead and click on Amazon Linux, specifically Amazon Linux 2023.

It should be the SSD volume type.

It should be listed as free tier eligible and just make sure that you've got 64 bit x86 selected.

And then scroll down further still and in the instance type drop down, we're looking for the T series of instances.

And then you need to select the one that's free tier eligible.

In most cases, this will be T2.micro, but select whichever is free tier eligible.

We want to keep this advanced demo as much as possible within the free tier.

Scroll down again and for key pair, just make sure that it says don't include in the launch template.

Move down further still to network settings.

Then make sure select existing security group is selected.

And then in the security groups drop down, click in that and make sure that you select the A4L.

VPC - SG WordPress.

So this is the security group which will automatically be associated with any instances launched using this launch template.

So select A4L.

VPC - SG WordPress and there will be some randomness after this.

That's fine.

Just make sure you select the SG WordPress group and then we can scroll down further still.

Now we can leave storage volumes as default.

We won't set any resource tags.

We won't do any configuration of network interfaces, but I will want you to expand advanced details.

There are a few things that we need to set within advanced details.

The first is an IAM instance profile.

So click in this drop down and then make sure that you pick A4L.

VPC - SG WordPress instance profile.

Again, there will be some randomness.

That's fine.

What this is doing is creating the configuration which will attach an instance role to this EC2 instance.

And this instance role is going to provide all the permissions required to interact with the parameter store and the elastic file system and anything else that this instance requires.

And this was pre-created on your behalf using the cloud formation template.

Next, scroll down further still and look for credit specification.

Remember, this is the same option that you set when launching an instance manually.

Now, as before, it's always best to set this to unlimited.

But if you are using a brand new AWS account, then it's possible that AWS won't allow you to use this option.

So you should probably go ahead and pick standard.

It won't make that much of a difference.

I'm going to pick unlimited, but I do suggest if you are using a fairly new account, you go ahead and select standard.

So that's the configuration for the instance, the base level configuration.

What I want you to do now though is to scroll all the way down to the bottom and there's a user data box.

This user data allows us to specify bootstrapping information to automatically configure our EC2 instances.

So into this user data box, I want you to paste the entire code snippet within stage 2B of this stages instructions.

And again, they're attached to this lesson.

The top line should be hash bang forward slash bin forward slash bash and then a space hyphen XE.

And then if you scroll all the way down to the bottom, the last line should be RM space forward slash TMP forward slash DB dot setup.

And now we can see we've pasted this entire user data.

Once you've done that, go ahead and click on create launch template.

Now that user data that you just pasted in is essentially all of the commands that you ran in the previous stage of the demo.

Only instead of pasting them one by one, you've defined them within the user data.

So this simply automates the process end to end.

So to test this, go ahead and click on launch templates towards the top of the screen.

It should show that you have a single launch template.

It's called WordPress.

The default version is one and the latest version is one.

And as we move throughout this demo series, the latest version and the default version will change.

So just keep an eye on those as we go.

For now, though, I want you to click in the checkbox next to this launch template, click on actions and then launch instance from template.

So this is going to launch an EC2 instance using this launch template.

We're asked to choose a launch template and a version and define the number of instances and we can leave all of these as the defaults.

If we just scroll down, you'll see how it's pre-populating all of these values with the configuration from the launch template.

And that's what we want.

Under key pair name, just select to proceed without a key pair not recommended.

And that's the default value.

Scroll down further still.

Even the networking configuration is partially pre-populated.

The only thing we need to do is specify a subnet that this instance will be launched into.

And when we configure auto scaling groups to use this launch template, the auto scaling group will configure the subnets on our behalf.

Because we're launching an instance directly from the launch template, we have to specify this subnet.

So click in the subnet dropdown and then look for SN-PUB-A.

Because we're going to deploy this WordPress instance into the public subnet in Availability Zone A.

So select that.

Scroll down.

Look for the resource tag section and click on add tag.

We're going to add a tag to the instance launched by this template.

So into key, just type name and then for value, use WordPress-LT.

And this will just tell us that this is an instance launched using the launch template.

Once you've entered those, just scroll all the way down to the bottom and click launch instance.

And this will launch an EC2 instance using this template.

And this will automate everything that we had to do in the previous stage manually.

So this saves us significant time and it enables us to use automation in later stages of this demo series.

So now go ahead and click on the instance ID in this success box and this will take you to the EC2 console.

Just give this instance a couple of minutes to finish its build process.

Even though we're automating the process, it does still take some time to perform the installation and the configuration of all of those different components.

So go ahead and just copy the public IP version for address of this instance into your clipboard.

And then after you've waited a few minutes, open that in a new tab.

If you get an error or it opens with a blank page, then you just need to give it a few minutes longer.

But when it's finished, it should show the same WordPress installation screen.

Once it does load the installation screen, we're going to follow the same process.

So site title is Categorum, username is Admin.

Enter the same password and then enter the fake test at test.com email address.

Then click on install WordPress.

Then click on login.

Enter admin again.

Enter the password.

Click on login.

It looks as though our automated WordPress build has worked because the dashboard has loaded.

Click on posts.

Delete the default post.

Click on add new.

For the title, the best animals again, click on the plus, select gallery, click on upload.

And again, pick a selection of animal pictures and click on open.

Remember, this is a new EC2 instance.

So the one we previously terminated will have also deleted the data on that previous instance.

Once these images have uploaded, click on publish and then publish again to upload the images to the EC2 instance and store the data within the database.

So remember two components, the data stored in the database and the images or media stored locally on the EC2 instance.

Click on view post to make sure that this loads correctly.

It does.

So that means the automatic build has worked okay.

Everything's functioning as we expect.

This has been an automatic build of a functional WordPress application.

Now, the only thing that's changed from the previous stage of this advanced demo series is we've automated the build of this instance.

It still has much the same limitations as the previous stage.

So while we can improve the build time and we can use launch templates to support further automation, the database and application are still on the same instance.

So neither can scale without the other.

The database of the application is still located on that instance, meaning scale in or out operations risk this data.

The WordPress content store is also stored locally on the instance.

So again, any scale in or out operations risk the media that's stored locally as well as the database.

Customers still connect directly to the instance, which means we can't perform health checks or automatically heal any failed instances.

For this, we need a load balancer which we'll be looking at in later stages of this demo series.

And of course, the IP address of the instance is still hard coded into the database.

So this is something else we need to resolve as we move through the demo series.

With that being said, though, that is everything that you needed to do in stage two of this demo series.

So in this stage, you've automated the build of the WordPress instance using a launch template.

Now, in stage three, you're going to migrate the data from the local database on EC2 into RDS.

And this will move the data out of the lifecycle of the EC2 instance.

And this makes it easier to scale.

So in stage three, you're going to perform that migration and then update the launch template to take account of that configuration change.

So go ahead and complete this stage of the demo lesson.

And when you're ready, I'll look forward to you joining me in the next.

Still using race as a tag here. Not a super fan but if carefully explained how this is being used as a measure for systemic and structural racism and show how it correlates with income and thus likely other unmeasured determinants.

Why not also have Model 1 + %AGA if part of the point is that doing this does tag mortality - I would like to see that here

This will probably tag a lot of other things

in the start tag

يتم تحديد السمات دائمًا في علامة البداية

Author response:

Public reviews:

Reviewer #1:

Epigenetic regulation complex (PRC2) is essential for neural crest specification, and its misregulation has been shown to cause severe craniofacial defects. This study shows that Eed, a core PRC2 component, is critical for craniofacial osteoblast differentiation and mesenchymal proliferation after neural crest induction. Using mouse genetics and single-cell RNA sequencing, the researcher found that conditional knockout of Eed leads to significant craniofacial hypoplasia, impaired osteogenesis, and reduced proliferation of mesenchymal cells in post-migratory neural crest populations.

Overall, the study is superficial and descriptive. No in-depth mechanism was analyzed and the phenotype analysis is not comprehensive.

We thank the reviewer for sharing their expertise and for taking the time to provide a helpful suggestion to improve our study. We are gratified that the striking phenotypes we report from Eed loss in post-migratory neural crest craniofacial tissues were appreciated. The breadth and depth of our phenotyping techniques, including skeletal staining, micro-CT, echocardiogram, immunofluorescence, histology, and unbiased single-cell gene expression analysis, provide comprehensive data in support our conclusion that PRC2 is required for craniofacial osteoblast differentiation. We hypothesize that epigenetic regulation of chromatin accessibility downstream of PRC2 activity is the molecular mechanism that underlies these phenotypes. To test this hypothesis in our revision, we are using CUT&Tag to profile H3K27me3 epigenetic modifications genome-wide and at the loci encoding the differentially expressed genes revealed by our single-cell transcriptomics in developing craniofacial structures. We anticipate that these experiments will reveal an epigenetic mechanism underlying the phenotypes we report from Eed loss in post-migratory neural crest craniofacial tissues.

Reviewer #2:

Summary:The role of PRC2 in post-neural crest induction was not well understood. This work developed an elegant mouse genetic system to conditionally deplete EED upon SOX10 activation. Substantial developmental defects were identified for craniofacial and bone development. The authors also performed extensive single-cell RNA sequencing to analyze differentiation gene expression changes upon conditional EED disruption.

Strengths:

(1) Elegant genetic system to ablate EED post neural crest induction.

(2) Single-cell RNA-seq analysis is extremely suitable for studying the cell type-specific gene expression changes in developmental systems.

We thank the reviewer for their generous and helpful comments on our study. We are pleased that our mouse genetic and single-cell RNA sequencing approaches were appropriate in pairing the craniofacial phenotypes we report with distinct gene expression changes in post-migratory neural crest tissues upon Eed deletion.

Weaknesses:

(1) Although this study is well designed and contains state-of-the-art single-cell RNA-seq analysis, it lacks the mechanistic depth in the EED/PRC2-mediated epigenetic repression. This is largely because no epigenomic data was shown.

Thank you for this suggestion. As described in response to Reviewer #1, we will include H2K27me3 CUT&Tag data in craniofacial tissue harvested from E12.5 and E16.5 Sox10-Cretg+ Eedfl/fl and Sox10-Cretg+ Eedfl/wt  embryos in our revision. Our analyses will including genome-wide and targeted metaplot visualizations across genotypes and developmental timepoints and assess how H3K27me3 occupancy relates to gene expression changes in our single-cell RNA sequencing data.

(2) The mouse model of conditional loss of EZH2 in neural crest has been previously reported, as the authors pointed out in the discussion. What is novel in this study to disrupt EED? Perhaps a more detailed comparison of the two mouse models would be beneficial.

We acknowledge the study the reviewer has indicated (Schwarz et al. Development 2014). This elegant investigation uses Wnt1-Cre to delete Ezh2 and found a similar phenotype to ours in the form of catastrophic craniofacial hypoplasia. We sought to add depth to the study of PRC2’s vital role in neural crest development by ablating Eed, which has a unique function in the PRC2 complex by binding to H3K27me3 and allosterically activating Ezh2. In this sense, we sought to test if phenotypes arising from deletion of Eed, the PRC2 “reader”, differ from phenotypes arising from deletion of Ezh2, the PRC2 “writer”, in neural crest derived tissues. Due to limitations associated with the Wnt1-Cre transgene (Lewis et al. Developmental Biology 2013), we used the Sox10-Cre allele which targets the migratory neural crest and is completely recombined by E10.5, instead of Wnt1-Cre which targets pre-migratory neural crest cells. A more detailed comparison of these mouse models will be included in the Discussion section of our revised manuscript, and we thank the reviewer for this thoughtful suggestion.

(3) The presentation of the single-cell RNA-seq data may need improvement. The complexity of the many cell types blurs the importance of which cell types are affected the most by EED disruption.

We agree with the reviewer’s critique of the scRNA-seq data presentation. Because Sox10+ cells were not sorted (via FACS, for example) from craniofacial tissues before single-cell RNA sequencing, we identified a breath of cell types in UMAP space unrelated to epigenetic disruption of neural crest derived tissues. We will include subcluster visualization plots in the figures of our revised manuscript to highlight specific changes in clusters, such as osteoblasts and mesenchymal stem cells, that arise from Eed loss in post-migratory neural crest craniofacial tissues.

(4) While it's easy to identify PRC2/EED target genes using published epigenomic data, it would be nice to tease out the direct versus indirect effects in the gene expression changes (e.g Figure 4e).

We agree with the reviewer that our single-cell RNA sequencing data do not provide insight into direct versus indirect changes in gene expression downstream of PRC2. We hope that the aforementioned CUT&Tag experiment will provide the necessary mechanistic insight into H3K27me3 occupancy and direct effects on gene expression resulting from PRC2 inactivation in our mouse model.

مثل ال P

Author response:

Reviewer #1 (Public review):

Summary:

This work investigated the role of CXXC-finger protein 1 (CXXC1) in regulatory T cells. CXXC1-bound genomic regions largely overlap with Foxp3-bound regions and regions with H3K4me3 histone modifications in Treg cells. CXXC1 and Foxp3 interact with each other, as shown by co-immunoprecipitation. Mice with Treg-specific CXXC1 knockout (KO) succumb to lymphoproliferative diseases between 3 to 4 weeks of age, similar to Foxp3 KO mice. Although the immune suppression function of CXXC1 KO Treg is comparable to WT Treg in an in vitro assay, these KO Tregs failed to suppress autoimmune diseases such as EAE and colitis in Treg transfer models in vivo. This is partly due to the diminished survival of the KO Tregs after transfer. CXXC1 KO Tregs do not have an altered DNA methylation pattern; instead, they display weakened H3K4me3 modifications within the broad H3K4me3 domains, which contain a set of Treg signature genes. These results suggest that CXXC1 and Foxp3 collaborate to regulate Treg homeostasis and function by promoting Treg signature gene expression through maintaining H3K4me3 modification.

Strengths:

Epigenetic regulation of Treg cells has been a constantly evolving area of research. The current study revealed CXXC1 as a previously unidentified epigenetic regulator of Tregs. The strong phenotype of the knockout mouse supports the critical role CXXC1 plays in Treg cells. Mechanistically, the link between CXXC1 and the maintenance of broad H3K4me3 domains is also a novel finding.

Weaknesses:

(1) It is not clear why the authors chose to compare H3K4me3 and H3K27me3 enriched genomic regions. There are other histone modifications associated with transcription activation or repression. Please provide justification.

Thank you for highlighting this important point. We prioritized H3K4me3 and H3K27me3 because they are well-established markers of transcriptional activation and repression, respectively. These modifications provide a robust framework for investigating the dynamic interplay of chromatin states in Treg cells, particularly in regulating the balance between activation and suppression of key genes. While histone acetylation, such as H3K27ac, is linked to enhancer activity and transcriptional elongation, our focus was on promoter-level regulation, where H3K4me3 and H3K27me3 are most relevant. Although other histone modifications could provide additional insights, we chose to focus on these two to maintain clarity and feasibility in our analysis. We are happy to further elaborate on this rationale in the manuscript if necessary.

(2) It is not clear what separates Clusters 1 and 3 in Figure 1C. It seems they share the same features.

We apologize for not clarifying these clusters clearly. Cluster 1 and 3 are both H3K4me3 only group, with H3K4me3 enrichment and gene expression levels being higher in Cluster 1. At first, we divided the promoters into four categories because we wanted to try to classify them into four categories: H3K4me3 only, H3K27me3 only, H3K4me3-H3K27me3 co-occupied, and None. However, in actual classification, we could not distinguish H3K4me3-H3K27me3 co-occupied group. Instead, we had two categories of H3K4me3 only, with cluster 1 having a higher enrichment level for H3K4me3 and gene expression levels.

(3) The claim, "These observations support the hypothesis that FOXP3 primarily functions as an activator by promoting H3K4me3 deposition in Treg cells." (line 344), seems to be a bit of an overstatement. Foxp3 certainly can promote transcription in ways other than promoting H3K3me3 deposition, and it also can repress gene transcription without affecting H3K27me3 deposition. Therefore, it is not justified to claim that promoting H3K4me3 deposition is Foxp3's primary function.

We appreciate the reviewer’s thoughtful observation regarding our claim about FOXP3’s role in promoting H3K4me3 deposition. We acknowledge that FOXP3 is a multifunctional transcription factor with diverse mechanisms of action, including transcriptional activation independent of H3K4me3 deposition and transcriptional repression that does not necessarily involve H3K27me3 deposition.

Our intention was not to imply that promoting H3K4me3 deposition is the exclusive or predominant function of FOXP3 but rather to highlight that this mechanism contributes significantly to its role in regulating Treg cell function. We agree that our wording may have overstated this point, and we will revise the text to provide a more nuanced interpretation. Specifically, we will clarify that our observations suggest FOXP3 can facilitate transcriptional activation, in part, by promoting H3K4me3 deposition, but this does not preclude its other regulatory mechanisms.

(4) For the in vitro suppression assay in Figure S4C, and the Treg transfer EAE and colitis experiments in Figure 4, the Tregs should be isolated from Cxxc1 fl/fl x Foxp3 cre/wt female heterozygous mice instead of Cxxc1 fl/fl x Foxp3 cre/cre (or cre/Y) mice. Tregs from the homozygous KO mice are already activated by the lymphoproliferative environment and could have vastly different gene expression patterns and homeostatic features compared to resting Tregs. Therefore, it's not a fair comparison between these activated KO Tregs and resting WT Tregs.

Thank you for this insightful comment and for pointing out the potential confounding effects associated with using Treg cells from homozygous Foxp3Cre/Cre (or Cre/Y) Cxxc1fl/fl mice. We agree that using Treg cells from _Foxp3_Cre/+ _Cxxc1_fl/fl (referred to as “het-KO”) and their littermate _Foxp3_Cre/+ _Cxxc1_fl/+ (referred to as “het-WT”) female mice would provide a more balanced comparison, as these Treg cells are less likely to be influenced by the activated lymphoproliferative environment present in homozygous KO mice.

To address this concern, we will perform additional experiments using Treg cells isolated from _Foxp3_Cre/+ _Cxxc1_fl/fl (“het-KO”) and their littermate _Foxp3_Cre/+ _Cxxc1_fl/+ (“het-WT”) female mice. We will update the manuscript with these new data to provide a more accurate assessment of the impact of CXXC1 deficiency on Treg cell function.

(5) The manuscript didn't provide a potential mechanism for how CXXC1 strengthens broad H3K4me3-modified genomic regions. The authors should perform Foxp3 ChIP-seq or Cut-n-Taq with WT and Cxxc1 cKO Tregs to determine whether CXXC1 deletion changes Foxp3's binding pattern in Treg cells.

Thank you for your insightful comments and valuable suggestions. We greatly appreciate your recommendation to explore the potential mechanism by which CXXC1 enhances broad H3K4me3-modified genomic regions.

In response, we plan to conduct CUT&Tag experiments for Foxp3 in both WT and Cxxc1 cKO Treg cells.

Reviewer #2 (Public review):

FOXP3 has been known to form diverse complexes with different transcription factors and enzymes responsible for epigenetic modifications, but how extracellular signals timely regulate FOXP3 complex dynamics remains to be fully understood. Histone H3K4 tri-methylation (H3K4me3) and CXXC finger protein 1 (CXXC1), which is required to regulate H3K4me3, also remain to be fully investigated in Treg cells. Here, Meng et al. performed a comprehensive analysis of H3K4me3 CUT&Tag assay on Treg cells and a comparison of the dataset with the FOXP3 ChIP-seq dataset revealed that FOXP3 could facilitate the regulation of target genes by promoting H3K4me3 deposition.

Moreover, CXXC1-FOXP3 interaction is required for this regulation. They found that specific knockdown of Cxxc1 in Treg leads to spontaneous severe multi-organ inflammation in mice and that Cxxc1-deficient Treg exhibits enhanced activation and impaired suppression activity. In addition, they have also found that CXXC1 shares several binding sites with FOXP3 especially on Treg signature gene loci, which are necessary for maintaining homeostasis and identity of Treg cells.

The findings of the current study are pretty intriguing, and it would be great if the authors could fully address the following comments to support these interesting findings.

Major points:

(1) There is insufficient evidence in the first part of the Results to support the conclusion that "FOXP3 functions as an activator by promoting H3K4Me3 deposition in Treg cells". The authors should compare the results for H3K4Me3 in FOXP3-negative conventional T cells to demonstrate that at these promoter loci, FOXP3 promotes H3K4Me3 deposition.

We appreciate the reviewer’s critical observation regarding our claim about FOXP3’s role in promoting H3K4me3 deposition. We acknowledge that FOXP3 is a multifunctional transcription factor with diverse mechanisms of action, including transcriptional activation independent of H3K4me3 deposition and transcriptional repression that does not necessarily involve H3K27me3 deposition.

Our intention was not to imply that promoting H3K4me3 deposition is the exclusive or predominant function of FOXP3 but rather to highlight that this mechanism contributes significantly to its role in regulating Treg cell function. We agree that our wording may have overstated this point, and we will revise the text to provide a more nuanced interpretation. Specifically, we will clarify that our observations suggest FOXP3 can facilitate transcriptional activation, in part, by promoting H3K4me3 deposition, but this does not preclude its other regulatory mechanisms.

We will compare H3K4me3 levels at the promoter loci of interest between FOXP3-negative conventional T cells and FOXP3-positive regulatory T cells. This comparison will help elucidate whether FOXP3 directly promotes H3K4me3 deposition at these loci.

(2) In Figure 3 F&G, the activation status and IFNγ production should be analyzed in Treg cells and Tconv cells separately rather than in total CD4+ T cells. Moreover, are there changes in autoantibodies and IgG and IgE levels in the serum of cKO mice?

We appreciate the reviewer’s constructive feedback on the analyses presented in Figures 3F and 3G and the additional suggestion to investigate autoantibodies and serum immunoglobulin levels.

Regarding Figures 3F and 3G, we agree that separating Treg cells and Tconv cells for analysis of activation status and IFN-γ production would provide a more precise understanding of the cellular dynamics in Cxxc1 cKO mice.

To address this, we will reanalyze the data to examine Treg and Tconv cells independently and include these results in the revised manuscript.

As for the changes in autoantibodies and serum IgG and IgE levels, we acknowledge that these parameters are important indicators of systemic immune dysregulation.

We will now measure serum autoantibodies and immunoglobulin levels in Cxxc1 cKO mice and WT controls.

(3) Why did Cxxc1-deficient Treg cells not show impaired suppression than WT Treg during in vitro suppression assay, despite the reduced expression of Treg cell suppression assay -associated markers at the transcriptional level demonstrated in both scRNA-seq and bulk RNA-seq?

Thank you for your thoughtful question. We appreciate your interest in understanding the apparent discrepancy between the reduced expression of Treg-associated suppression markers at the transcriptional level and the lack of impaired suppression observed in the in vitro suppression assay.

There are several potential explanations for this observation:

(1) Functional Redundancy: Treg cell suppression is a complex, multi-faceted process involving various effector mechanisms such as cytokine production (e.g., IL-10, TGF-β), cell-cell contact, and metabolic regulation. Thus, even though the transcriptional signature of suppression-associated genes is altered, compensatory mechanisms may still allow Cxxc1-deficient Treg cells to retain functional suppression capacity under these specific in vitro conditions.

(2) In Vitro Assay Limitations: The in vitro suppression assay is a simplified model of Treg function that may not capture all the complexities of Treg-mediated suppression in vivo. While we observed altered gene expression in Cxxc1-deficient Treg cells, this might not directly translate to a functional defect under the specific conditions of the assay. In vivo, additional factors such as cytokine milieu, cell-cell interactions, and tissue-specific environments may be required for full suppression, which could be missing in the in vitro assay.

(4) Is there a disease in which Cxxc1 is expressed at low levels or absent in Treg cells? Is the same immunodeficiency phenotype present in patients as in mice?

Thank you for your insightful question regarding the role of CXXC1 in Treg cells and its potential link to human disease. To our knowledge, no specific human disease has been identified where CXXC1 is expressed at low levels or absent specifically in Treg cells. There is currently no direct evidence of an immunodeficiency phenotype in human patients that parallels the one observed in Cxxc1-deficient mice.

Reviewer #3 (Public review):

In the report entitled "CXXC-finger protein 1 associates with FOXP3 to stabilize homeostasis and suppressive functions of regulatory T cells", the authors demonstrated that Cxxc1-deletion in Treg cells leads to the development of severe inflammatory disease with impaired suppressive function. Mechanistically, CXXC1 interacts with Foxp3 and regulates the expression of key Treg signature genes by modulating H3K4me3 deposition. Their findings are interesting and significant. However, there are several concerns regarding their analysis and conclusions.

Major concerns:

(1) Despite cKO mice showing an increase in Treg cells in the lymph nodes and Cxxc1-deficient Treg cells having normal suppressive function, the majority of cKO mice died within a month. What causes cKO mice to die from severe inflammation?

Considering the results of Figures 4 and 5, a decrease in Treg cell population due to their reduced proliferative capacity may be one of the causes. It would be informative to analyze the population of tissue Treg cells.

We thank the reviewer for this insightful comment and acknowledge the importance of understanding the causes of severe inflammation and early mortality in cKO mice. Based on our data and previous studies, we propose the following explanations:

(1) Reduced Treg Proliferative Capacity: As shown in Figure 5I, the decreased proportion of FOXP3+Ki67+ Treg cells in cKO mice likely reflects impaired proliferative capacity, which may limit the expansion of functional Treg cells in response to inflammatory cues, particularly in peripheral tissues where active suppression is required.

(2) Altered Treg Function and Activation: Cxxc1-deficient Treg cells exhibit increased expression of activation markers (Il2ra, Cd69) and pro-inflammatory genes (Ifng, Tbx21). This suggests a functional dysregulation that may impair their ability to suppress inflammation effectively, despite their presence in lymphoid organs.

(3) Tissue Treg Populations: Although our study focuses on lymph node-resident Treg cells, tissue-resident Treg cells play a crucial role in maintaining local immune homeostasis. It is plausible that Cxxc1 deficiency compromises the accumulation or functionality of tissue Treg cells, contributing to uncontrolled inflammation in non-lymphoid organs. Unfortunately, we currently lack data on tissue Treg populations, which limits our ability to directly address this hypothesis.

Regarding the suggestion to analyze tissue Treg populations, we agree that this would be an important next step in understanding the cause of the severe inflammation and early mortality in Cxxc1-deficient mice.

We plan to perform detailed analyses of Treg cell populations in various tissues, including the gut, lung, and liver, to determine if there are specific defects in tissue-resident Treg cells that could contribute to the observed phenotype.

(2) In Figure 5B, scRNA-seq analysis indicated that Mki67+ Treg subset are comparable between WT and Cxxc1-deficient Treg cells. On the other hand, FACS analysis demonstrated that Cxxc1-deficient Treg shows less Ki-67 expression compared to WT in Figure 5I. The authors should explain this discrepancy.

Thank you for pointing out the apparent discrepancy between the scRNA-seq and FACS analyses regarding Ki-67 expression in Cxxc1-deficient Treg cells.

In Figure 5B, the scRNA-seq analysis identified the Mki67+ Treg subset as comparable between WT and Cxxc1-deficient Treg cells. This finding reflects the overall proportion of cells expressing Mki67 transcripts within the Treg population. In contrast, the FACS analysis in Figure 5I specifically measures Ki-67 protein levels, revealing reduced expression in Cxxc1-deficient Treg cells compared to WT.

To address this discrepancy more comprehensively, we will further analyze the scRNA-seq data to directly compare Mki67 mRNA expression levels between WT and Cxxc1-deficient Treg cells.

In addition, the authors concluded on line 441 that CXXC1 plays a crucial role in maintaining Treg cell stability. However, there appears to be no data on Treg stability. Which data represent the Treg stability?

We appreciate the reviewer’s observation and recognize that our wording may have been overly conclusive. Our data primarily highlight the impact of Cxxc1 deficiency on Treg cell homeostasis and transcriptional regulation, rather than providing direct evidence for Treg cell stability. Specifically, the downregulation of Treg-specific suppressive genes (Nt5e, Il10, Pdcd1) and the upregulation of pro-inflammatory markers (Gzmb, Ifng, Tbx21) indicate a shift in functional states. While these findings may suggest an indirect disruption in the maintenance of suppressive phenotypes, they do not constitute a direct measure of Treg cell stability.

To address the reviewer’s concern, we will revise our conclusion to more accurately state that our data support a role for CXXC1 in maintaining Treg cell homeostasis and functional balance, without overextending claims about Treg cell stability. Thank you for bringing this to our attention, as it will help us improve the clarity and precision of our manuscript.

(3) The authors found that Cxxc1-deficient Treg cells exhibit weaker H3K4me3 signals compared to WT in Figure 7. This result suggests that Cxxc1 regulates H3K4me3 modification via H3K4 methyltransferases in Treg cells. The authors should clarify which H3K4 methyltransferases contribute to the modulation of H3K4me3 deposition by Cxxc1 in Treg cells.

Thank you for pointing out the need to clarify the role of H3K4 methyltransferases in the modulation of H3K4me3 deposition by CXXC1 in Treg cells.

In our study, we found that Cxxc1-deficient Treg cells exhibit reduced H3K4me3 levels, as shown in Figure 7. CXXC1 has been previously reported to function as a non-catalytic component of the Set1/COMPASS complex, which contains H3K4 methyltransferases such as SETD1A and SETD1B. These methyltransferases are the primary enzymes responsible for H3K4 trimethylation.

References:

(1) Lee J.H., Skalnik D.G. CpG-binding protein (CXXC finger protein 1) is a component of the mammalian Set1 histone H3-Lys4 methyltransferase complex, the analogue of the yeast Set1/COMPASS complex. J. Biol. Chem. 2005; 280:41725–41731.

(2). J. P. Thomson, P. J. Skene, J. Selfridge, T. Clouaire, J. Guy, S. Webb, A. R. W. Kerr, A. Deaton, R. Andrews, K. D. James, D. J. Turner, R. Illingworth, A. Bird, CpG islands influence chromatin structure via the CpG-binding protein Cfp1. Nature 464, 1082–1086 (2010).

(3) Shilatifard, A. 2012. The COMPASS family of histone H3K4 methylases: mechanisms of regulation in development and disease pathogenesis. Annu. Rev. Biochem. 81:65–95.

(4) Brown D.A., Di Cerbo V., Feldmann A., Ahn J., Ito S., Blackledge N.P., Nakayama M., McClellan M., Dimitrova E., Turberfield A.H. et al. The SET1 complex selects actively transcribed target genes via multivalent interaction with CpG Island chromatin. Cell Rep. 2017; 20:2313–2327.

Furthermore, it would be important to investigate whether Cxxc1-deletion alters Foxp3 binding to target genes.

Thank you for this important suggestion regarding the impact of Cxxc1 deletion on FOXP3 binding to target genes. We agree that understanding whether Cxxc1 deficiency affects FOXP3’s ability to bind to its target genes would provide valuable insight into the regulatory role of CXXC1 in Treg cell function.

To address this, we plan to perform CUT&Tag experiments to assess FOXP3 binding profiles in Cxxc1-deficient versus wild-type Treg cells. These experiments will allow us to determine if Cxxc1 loss disrupts FOXP3’s occupancy at key regulatory sites, which may contribute to the observed functional impairments in Treg cells.

(4) In Figure 7, the authors concluded that CXXC1 promotes Treg cell homeostasis and function by preserving the H3K4me3 modification since Cxxc1-deficient Treg cells show lower H3K4me3 densities at the key Treg signature genes. Are these Cxxc1-deficient Treg cells derived from mosaic mice? If Cxxc1-deficient Treg cells are derived from cKO mice, the gene expression and H3K4me3 modification status are inconsistent because scRNA-seq analysis indicated that expression of these Treg signature genes was increased in Cxxc1-deficient Treg cells compared to WT (Figure 5F and G).

Thank you for the insightful comment. To clarify, the Cxxc1-deficient Treg cells analyzed for H3K4me3 modification in Figure 7 were indeed derived from Cxxc1 conditional knockout (cKO) mice, not mosaic mice.

The scRNA-seq analysis presented in Figures 5F and G revealed an upregulation of Treg signature genes in Cxxc1-deficient Treg cells. This finding suggests that the loss of Cxxc1 drives these cells toward a pro-inflammatory, activated state, underscoring the pivotal role of CXXC1 in maintaining Treg cell homeostasis and suppressive function.

Regarding the apparent discrepancy between the reduced H3K4me3 levels and the increased expression of these genes, it is important to note that H3K4me3 primarily functions as an epigenetic mark that facilitates chromatin accessibility and transcriptional regulation, acting as an upstream modulator of gene expression. However, gene expression levels are also influenced by downstream compensatory mechanisms and complex inflammatory environments. In this context, the reduction in H3K4me3 likely reflects the direct role of CXXC1 in epigenetic regulation, whereas the upregulation of gene expression in Cxxc1-deficient Treg cells may result as a side effect of the inflammatory environment.

To further substantiate our findings, we performed RNA-seq analysis on Treg cells from Foxp3_Cre/+ _Cxxc1_fl/fl (“het-KO”) and their littermate _Foxp3_Cre/+ _Cxxc1_fl/+ (“het-WT”) female mice, as presented in Figure S6C. This analysis revealed a notable reduction in the expression of key Treg signature genes, including _Icos, Ctla4, Tnfrsf18, and Nt5e, in het-KO Treg cells. Importantly, the observed changes in gene expression were consistent with the altered H3K4me3 modification status, further supporting the epigenetic regulatory role of CXXC1. These results further emphasize the critical role of CXXC1 promotes Treg cell homeostasis and function by preserving the H3K4me3 modification.

Reviewer #2 (Public review):

FOXP3 has been known to form diverse complexes with different transcription factors and enzymes responsible for epigenetic modifications, but how extracellular signals timely regulate FOXP3 complex dynamics remains to be fully understood. Histone H3K4 tri-methylation (H3K4me3) and CXXC finger protein 1 (CXXC1), which is required to regulate H3K4me3, also remain to be fully investigated in Treg cells. Here, Meng et al. performed a comprehensive analysis of H3K4me3 CUT&Tag assay on Treg cells and a comparison of the dataset with the FOXP3 ChIP-seq dataset revealed that FOXP3 could facilitate the regulation of target genes by promoting H3K4me3 deposition.

Moreover, CXXC1-FOXP3 interaction is required for this regulation. They found that specific knockdown of Cxxc1 in Treg leads to spontaneous severe multi-organ inflammation in mice and that Cxxc1-deficient Treg exhibits enhanced activation and impaired suppression activity. In addition, they have also found that CXXC1 shares several binding sites with FOXP3 especially on Treg signature gene loci, which are necessary for maintaining homeostasis and identity of Treg cells.

The findings of the current study are pretty intriguing, and it would be great if the authors could fully address the following comments to support these interesting findings.

Major points:

(1) There is insufficient evidence in the first part of the Results to support the conclusion that "FOXP3 functions as an activator by promoting H3K4Me3 deposition in Treg cells". The authors should compare the results for H3K4Me3 in FOXP3-negative conventional T cells to demonstrate that at these promoter loci, FOXP3 promotes H3K4Me3 deposition.

(2) In Figure 3 F&G, the activation status and IFNγ production should be analyzed in Treg cells and Tconv cells separately rather than in total CD4+ T cells. Moreover, are there changes in autoantibodies and IgG and IgE levels in the serum of cKO mice?

(3) Why did Cxxc1-deficient Treg cells not show impaired suppression than WT Treg during in vitro suppression assay, despite the reduced expression of Treg cell suppression assay -associated markers at the transcriptional level demonstrated in both scRNA-seq and bulk RNA-seq?

(4) Is there a disease in which Cxxc1 is expressed at low levels or absent in Treg cells? Is the same immunodeficiency phenotype present in patients as in mice?

Author response:

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

eLife Assessment

This important study addresses how 3' splice site choice is modulated by the conserved spliceosome-associated protein Fyv6. The authors provide compelling evidence Fyv6 functions to enable selection of 3' splice sites distal to a branch point and in doing so antagonizes more proximal, suboptimal 3' splice sites. The study would be improved through a more nuanced discussion of alternative possibilities and models, for instance in discussing the phenotypic impact of Fyv6 deletion.

We thank the editors and reviewers for their supportive comments and assessment of this manuscript. We have improved the discussion at several points as suggested by the reviewers to include discussion of alternative possibilities.

Public Reviews:

Reviewer #1 (Public Review):

Summary:

A key challenge at the second chemical step of splicing is the identification of the 3' splice site of an intron. This requires recruitment of factors dedicated to the second chemical step of splicing and exclusion of factors dedicated to the first chemical step of splicing. Through the highest resolution cyroEM structure of the spliceosome to-date, the authors show the binding site for Fyv6, a factor dedicated to the second chemical step of splicing, is mutually exclusive with the binding site for a distinct factor dedicated to the first chemical step of splicing, highlighting that splicing factors bind to the spliceosome at a specific stage not only by recognizing features specific to that stage but also by competing with factors that bind at other stages. The authors further reveal that Fyv6 functions at the second chemical step to promote selection of 3' splice sites distal to a branch point and thereby discriminate against proximal, suboptimal 3' splice site. Lastly, the authors show by cyroEM that Fyv6 physically interacts with the RNA helicase Prp22 and by genetics Fyv6 functionally interacts with this factor, implicating Fyv6 in 3'SS proofreading and mRNA release from the spliceosome. The evidence for this study is robust, with the inclusion of genomics, reporter assays, genetics, and cyroEM. Further, the data overall justify the conclusions, which will be of broad interest.

Strengths:

(1) The resolution of the cryoEM structure of Fyv6-bound spliceosomes at the second chemical step of splicing is exceptional (2.3 Angstroms at the catalytic core; 3.0-3.7 Angstroms at the periphery), providing the best view of this spliceosomal intermediate in particular and the core of the spliceosome in general.

(2) The authors observe by cryoEM three distinct states of this spliceosome, each distinguished from the next by progressive loss of protein factors and/or RNA residues. The authors appropriately refrain from overinterpreting these states as reflecting distinct states in the splicing cycle, as too many cyroEM studies are prone to do, and instead interpret these observations to suggest interdependencies of binding. For example, when Fyv6, Slu7, and Prp18 are not observed, neither are the first and second residues of the intron, which otherwise interact, suggesting an interdependence between 3' splice site docking on the 5' splice site and binding of these second step factors to the spliceosome.

(3) Conclusions are supported from multiple angles.

(4) The interaction between Fyv6 and Syf1, revealed by the cyroEM structure, was shown to account for the temperature-sensitive phenotypes of a fyv6 deletion, through a truncation analysis.

(5) Splicing changes were observed in vivo both by indirect copper reporter assays and directly by RT-PCR.

(6) Changes observed by RNA-seq are validated by RT-PCR.

(7) The authors go beyond simply observing a general shift to proximal 3'SS usage in the fyv6 deletion by RNA-seq by experimentally varying branch point to 3' splice site distance experimentally in a reporter and demonstrating in a controlled system that Fyv6 promotes distal 3' splice sites.

(8) The importance of the Fyv6-Syf1 interaction for 3'SS recognition is demonstrated by truncations of both Fyv6 and of Syf1.

(9) In general, the study was executed thoroughly and presented clearly.

We thank the reviewer for their recognition of the strengths of our multi-faceted approach that led to highly supported conclusions.

Weaknesses:

(1) Despite the authors restraint in interpreting the three states of the spliceosome observed by cyroEM as sequential intermediates along the splicing pathway, it would be helpful to the general reader to explicitly acknowledge the alternative possibility that the difference states simply reflect decomposition from one intermediate during isolation of the complex (i.e., the loss of protein is an in vitro artifact, if an informative one).

We thank the reviewer for noticing our restraint in interpreting these structures, and we agree that the scenario described by the reviewer is a possibility. We have now explicitly mentioned this in the Discussion on lines 755-757.

(2) The authors acknowledge that for prp8 suppressors of the fyv6 deletion, suppression may be indirect, as originally proposed by the Query and Konarska labs - that is, that defects in the second step conformation of the spliceosome can be indirectly suppressed by compensating, destabilizing mutations in the first step spliceosome. Whereas some of the other suppressors of the fyv6 deletion can be interpreted as impacting directly the second step spliceosome (e.g., because the gene product is only present in the second step conformation), it seems that many more suppressors beyond prp8 mutants, especially those corresponding to bulky substitutions, which would more likely destabilize than stabilize, could similarly act indirectly by destabilization of first step conformation. The authors should acknowledge this where appropriate (e.g., for factors like Prp8 that are present in both first and second step conformations).

We agree that this is also a possibility and have now included this on lines 480-486.

Reviewer #2 (Public Review):

In this manuscript, Senn, Lipinski, and colleagues report on the structure and function of the conserved spliceosomal protein Fyv6. Pre-mRNA splicing is a critical gene expression step that occurs in two steps, branching and exon ligation. Fyv6 had been recently identified by the Hoskins' lab as a factor that aids exon ligation (Lipinski et al., 2023), yet the mechanistic basis for Fyv6 function was less clear. Here, the authors combine yeast genetics, transcriptomics, biochemical assays, and structural biology to reveal the function of Fyv6. Specifically, they describe that Fyv6 promotes the usage of distal 3'SSs by stabilizing a network of interactions that include the RNA helicase PRP22 and the spliceosome subunit SYF1. They discuss a generalizible mechanism for splice site proofreading by spliceosomsal RNA helicases that could be modulated by other, regulatory splicing factors.

This is a very high quality study, which expertly combines various approaches to provide new insights into the regulation of 3'SS choice, docking, and undocking. The cryo-EM data is also of excellent quality, which substantially extends on previous yeast P complex structures. This is also supported by the authors use of the latest data analysis tools (Relion-5, AlphaFold2 multimer predictions, Modelangelo). The authors re-evaluate published EM densities of yeast spliceosome complexes (B*, C,C*,P) for the presence or absence of Fyv6, substantiate Fyv6 as a 2nd step specific factor, confirm it as the homolog of the human protein FAM192A, and provide a model for how Fyv6 may fit into the splicing pathway. The biochemical experiments on probing the splicing effects of BP to 3'SS distances after Fyv6 KO, genetic experiments to probe Fyv6 and Syf1 domains, and the suppressor screening add substantially to the study and are well executed. The manuscript is clearly written and we particularly appreciated the nuanced discussions, for example for an alternative model by which Prp22 influences 3'SS undocking. The research findings will be of great interest to the pre-mRNA splicing community.

We thank the reviewer for their positive comments on our manuscript.

We have only few comments to improve an already strong manuscript.

Comments:

(1) Can the authors comment on how they justify K+ ion positions in their models (e.g. the K+ ion bridging G-1 and G+1 nucleotides)? How do they discriminate e.g. in the 'G-1 and G+1' case K+ from water?

The assignment of K+ at this position is justified by both longer coordination distances and relatively high cryo-EM density compared to structured water molecules in the same vicinity. We have added a panel to figure3-figure supplement 4C to show the density for the G-1/G+1 bridging K+ ion and to show the adjacent density for putative water molecules which coordinate the ion. The K+ ion density is larger and has stronger signal than the adjacent water molecules. The coordination distances are also longer than would be expected for a Mg2+. For these reasons and because K+ was present in the purification buffer, we modelled the density as K+.

(2) The authors comment on Yju2 and Fyv6 assignments in all yeast structures except for the ILS. Can the authors comment on if they have also looked into the assignment of Yju2 in the yeast ILS structure in the same manner? While it is possible that Fyv6 could dissociate and Yju2 reassociate at the P to ILS transition, this would merit a closer look given that in the yeast P complex Yju2 had been misassigned previously.

We thank the reviewer for pointing out this very interesting topic! We have used ModelAngelo to analyze the S. cerevisiae ILS structure for support of density assignment as Yju2 (and not Fyv6). This analysis supports the assignment as Yju2 in this structure and we have no evidence to doubt its presence in those particular purified spliceosomes. We have updated Figure 4- figure supplement 1B accordingly.

That being said, we do think that this issue should be studied more carefully in the future. The S. cerevisiae ILS structure (5Y88) was determined by purifying spliceosome complexes with a TAP-tag on Yju2. So the conclusion that Yju2 is part of the ILS spliceosome involves some circular logic: Yju2 is part of ILS spliceosome complexes because it is present in ILS complexes purified with Yju2. We also note that Yju2 was absent in ILS complexes recently determined from metazoans by the Plaschka group.  We have added some additional nuance to the Discussion to raise this important mechanistic point at lines 711-718.

(3) For accessibility to a general reader, figures 1c, d, e, 2a, b, would benefit from additional headings or labels, to immediately convey what is being displayed. It is also not clear to us if Fig 1e might fit better in the supplement and be instead replaced by Supplementary Figure 1a (wt) , b (delta upf1), and a new c (delta fyv6) and new d (delta upf1, delta fyv6). This may allow the reader to better follow the rationale of the authors' use of the Fyv6/Upf1 double deletion.

We thank the reviewer for the suggestion and have updated Figures 1 C-E to include additional information in the headings and labels. We have not changed the labels in Figures 2A, B but have added additional clarifying language to the legend.

In terms of rearranging the figures, we thank the reviewer for the suggestion but have decided that the figures are best left in their current ordering.

(4) The authors carefully interpret the various suppressor mutants, yet to a general reader the authors may wish to focus this section on only the most critical mutants for a better flow of the text.

We thank the reviewer for this suggestion. While this section of the manuscript does contain (to quote Reviewer #3) “extensive new information regarding functional interactions”, it was a bit long. We have reduced this section of the manuscript by ~200 words for a more focused presentation for general readers.

Reviewer #3 (Public Review):

In this manuscript the authors expand their initial identification of Fyv6 as a protein involved in the second step of pre-mRNA splicing to investigate the transcriptome-wide impact of Fyv6 on splicing and gain a deeper understanding of the mechanism of Fyv6 action.

They first use deep sequencing of transcripts in cells depleted of Fyv6 together with Upf1 (to limit loss of mis-spliced transcripts) to identify broad changes in the transcriptome due to loss of Fyv6. This includes both changes in overall gene expression, that are not deeply discussed, as well as alterations in choice of 3' splice sites - which is the focus of the rest of the manuscript

They next provide the highest resolution structure of the post-catalytic spliceosome to date; providing unparalleled insight into details of the active site and peripheral components that haven't been well characterized previously.

Using this structure they identify functionally critical interactions of Fyv6 with Syf1 but not Prp22, Prp8 and Slu7. Finally, a suppressor screen additionally provides extensive new information regarding functional interactions between these second step factors.

Overall this manuscript reports new and essential information regarding molecular interactions within the spliceosome that determine the use of the 3' splice site. It would be helpful, especially to the non-expert, to summarize these in a table, figure or schematic in the discussion.

We thank the reviewer for the positive comments and suggestions. We did include a summary figure in panel 7H. However, it was a bit buried. To highlight the summary figure more clearly, we have moved panel 7H to its own figure (Fig. 8).

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) The resolution of some panels is poor, nearly illegible (e.g., Supp Fig 1A, B).

The resolution of panels in supplemental figure 1 has been increased. However, this may be an artifact of the PDF conversion process. We will pay attention to this during the publication process.

(2) Panel S6B: 6HYU is a structure of DHX8, not DDX8

We have corrected DDX8 to DHX8 in Supplemental Fig. S6D and associated figure legend.

(3) The result that Syf1 truncations can suppress the Fyv6 deletion is impressive. The subsequent discussion seems muddled. A discussion of Fyv6 binding at the first step, instead of Yju2, doesn't seem relevant here (though worthy of consideration in the discussion), given that the starting mutation is the Fyv6 deletion. Further, conjuring rebinding of Yju2 based on the data in the paper seems unnecessarily speculative (assumes that biochemical state III is on pathway), unless I am unaware of some other evidence for such rebinding. Instead, a simpler explanation would seem to be that in the absence of Fyv6, Syf1 inappropriately binds Yju2 instead at the second step and that deletion of the common Fyv6/Yju2 binding site on Syf1 suppresses this defect. In this case, the ts phenotype of the Fyv6 deletion would result from inappropriate binding of Yju2, and the splicing defect would be due to loss of Fyv6 activity. Alternatively, especially considering the work of the labs of Query and Konarska, the authors should consider the possibility that i) the Fyv6 deletion destabilizes the second step conformation, shifting an equilibrium to the first step conformation, and that ii) the Syf1 truncation destabilizes binding of Yju2, thereby restoring the equilibrium. In this case the ts phenotype of the Fyv6 deletion is due to a disturbed equilibrium and the splicing defect is due to the failure of Fyv6 to function at the second step.

We believe the reviewer is specifically referencing the final paragraph of this Results section (the paragraph that comes just before the section “Mutations in many different splicing factors…”). In retrospect, we agree that our discussion was convoluted. In particular, we emphasized rebinding of Yju2 based on its presence in the cryo-EM structure of the yeast ILS complex. However, given some uncertainties about whether or not Yju2 is a bona fide ILS component (as discussed above). We don’t think it is appropriate to over-emphasize rebinding of Yju2 and have decided to incorporate the elegant mechanisms proposed by the reviewer. This paragraph has now been edited accordingly (lines 386-395).

(4) The authors imply they have performed biochemical studies, which I think is misleading. Of course, RT-PCR and primer extension assays for example are performed in vitro, but these are an analysis of RNA events that occurred in vivo. In my view a higher threshold should be used for defining "biochemistry". To me "biochemistry" would imply that the authors have, for example, investigated 3' splice site usage in splicing extracts of the fyv6 deletion or engaged in an analysis of the Syf1-Fyv6 interaction involving the expression of the interacting domains in bacteria followed by a binding analysis in the test tube.

We disagree with the reviewer on this point. Biochemistry is defined as the “branch of sciences concerned with the chemical substances, reactions, and physico chemical processes which occur within living organisms; biological or physical chemistry.” (Oxford English Dictionary). Biochemical studies are not defined by whether or not they take place in vitro, in vivo, or even in silico. Indeed, much of the history of biochemistry (especially in studies of metabolism, for example) involved experiments occurring in vivo that reported on the molecular properties and mechanisms of biological processes. We think many of our experiments fall into this category including our structure/function analysis of splicing factors and the use of the ACT1-CUP1 reporter substrate.

(5) The monovalents are shown; inositol phosphate is shown; is the binding of Prp22 to RNA shown?

We have added a panel to Figure 3-figure supplement 4D showing density for the 3' exon within Prp22.

(6) The authors invoke undocking of the 3'SS in the P complex. Where is the 3'SS in the ILS? The author's model predicts: undocked.

In all ILS structures to date, the 3′ SS is undocked, in agreement with this prediction. We have now noted this observation in line 760.

(7) Would be helpful to show fyv6 deletion in Fig 1b.

We have included growth data for an additional fyv6 deletion strain (in a cup1Δ background) in Figure 1b. The results are quite similar to the upf1_Δ_ background except with slightly worse growth at 23°C.

Reviewer #2 (Recommendations For The Authors):

Minor comments

(1) Fig.3b is the arrow indicating the right rotation?

This typo has been fixed.

(2) Fig.4b, panel H is annotated, which should read 'F'.

This typo has been fixed.

(3) Line 178: "Finally, we analyzed the sequence features of the alternative 3ʹ SS activated by loss of Fyv6." We would suggest 'used after' instead of 'activated by'.

We have replaced ‘activated by’ with ‘with increased use after’.

(4) In Line 544, the authors speculate on a Slu7 requirement for 3'SS docking and on 3'SS docking maintenance. In the results section (Line 265) they however only mention the latter possibility. These statements should be consistent.

We thank the reviewer for pointing this out. We have added a reference to docking maintenance to the results section at line 325.

(5) Line 476: "Unexpectedly, Prp22 I1133R was actually deleterious when Fyv6 was present for this reporter." We suggest removing "actually".

We have removed ‘actually’.

(6) The authors describe the observed changes in splicing events in absolute numbers (e.g. in Fig 1c). To better assess for the reader whether these numbers reflect large or small effects of Fyv6 in defining mRNA isoforms, it would be more useful to state these as percent changes of total events or to provide a reference number for how many introns are spliced in S.c. See for example the statements in Lines 132 and 145.

We have added a percentage at line 138 that indicates ~20% of introns in yeast showed splicing changes.

Reviewer #3 (Recommendations For The Authors):

Do the authors have a proposed explanation for the observed DGE in non-intron containing genes in the Fyv6 depleted cells?

The simplest explanation is that this is an indirect effect due to splicing changes occurring in other genes (such as transcription factors, ribosomal protein genes, etc..). It is possible that this can be further dissected in the future using shorter-term knockdown of Fyv6 using Anchors Away or AID-tagging. However, that is beyond the scope of the current manuscript, and we do not wish to comment on these non-intron containing genes further at present.

Figure 2A - What is going on with the events that show no FAnS value under one condition (i.e. are up against the X or Y axis)? These are of interest as most on the Y- axis are blue.

The events along one of the axes denote alternative splice sites that are only detected under one condition (either when Fyv6 is present or when it is absent). At this stage, we do not wish to interpret these events further since most have a relatively low number of reads overall.

Charles, talk about a freaking amazing academic writer! I’m so jealous of your expansive vocabulary! Especially in your false dreams writing, your highly descriptive and emotional sentences really shone through – you’ve definitely got an ear for music! For this and the other writings, each one of your sentences contained so much meaning and clearly related to the purpose of the writing – especially with your concluding ideas!

Most of the comments I made were very minor ones - usually relating to grammar or sentence structure - because I thought you clearly stated your thoughts. One big suggestion is to try and implement shorter sentences to add a sort of “attitude to your writing.” You are fantastic at formally stating your ideas, yet sometimes I find that the most influential writings use a conversational tone here and there. I also noticed that many of your sentences end with a comma then -ing word; therefore, maybe look to change this up somehow by making shorter sentences, placing the dependent clause at the beginning of the sentence, or using different punctuation to help with structure variation. Also, I understand you didn’t have enough time to do this yet, but definitely implement some media to help your project’s visual appeal.

I like the linear structure that you went with. I think (other than possibly switching “The Duality of Vulnerability Between ‘u’ and ‘i’” and “How Kendrick Lamar Transformed Cultural Trauma Into To Pimp a Butterfly”) the order you have is great. One thing that I suggest would be to incorporate more of the tag feature at the bottom of your pages. For example, tag the bibliography page at the bottom of each page where you use cites and the lyrics page after you cite one of the song’s lyrics.

In the end, I really enjoyed your project. Of course I knew who Kendrick Lamar was, but I had no clue of all the details about his young life. I enjoyed your unique approach to this writing: analyzing two songs instead of one. The relationship that you discussed between these songs (especially in “The Duality of Vulnerability Between ‘u’ and ‘i’”) is very informative and overall intriguing – I love it when music artists tell a story with multiple songs (just as you did in false dreams)! For me, analyzing one song was hard enough but two…that’s just impressive. And the best part: you did it beautifully!

I think it would be awesome if every time in your project you talked bout the lyrics, you would hyperlink it back to here and tag this page at the bottom of your writing. It would help give your project more connections and give this lyrics page more purpose!

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

Manuscript number: RC-2024-02640

Corresponding author(s): Purusharth I, Rajyaguru; Stephan Vagner

1. General Statements

This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

In the manuscript titled, "RGG motif-containing Scd6/LSM14A proteins regulate the translation of specific mRNAs in response to hydroxyurea-induced genotoxic stress" we elucidate a conserved role of an RNA-binding protein with low-complexity sequences (RGG-motifs) in genotoxic stress response. This work uncovers HU-stress mediated translation regulation of SRS2, Ligase IV and RTEL1 transcripts by Scd6 (yeast)/LSM14 (human). It further identifies RNP condensates and arginine methylation as sites and means of this regulation.

We heartily thank all three reviewers for their overall encouraging comments about the significance of this manuscript. Specifically, we appreciate their view that the manuscript provides new functional insights into the role of RGG-motif-containing RNA-binding protein in genotoxic stress response. They further agree that such knowledge will impact and interest the general audience of RNA biology and stress biology.

We have carefully noted all the comments raised by three reviewers. We have addressed almost all the comments, including several by performing new experiments. The new results and their analysis have helped us improve the manuscript, allowing us to provide a stronger mechanistic and functional insight underlying the findings presented in this work. We thank the reviewers for their insightful comments. Below, we provide a point-by-point response to each of the comments.

2. Description of the planned revisions

Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

Reviewer 3

Major Comment 4: Page 7, top: '...indicating that Scd6 regulated the expression of SRS2 in a HU-dependent manner.' In my opinion, the results so far suggest that Scd6 and SRS2 are somehow functionally connected during HU-treatment. To substantiate the statement of the authors, they should provide a Western blot showing that the levels of SRS2 change upon Scd6 KO or OE during HU-treatment. This will also substantiate the results shown in Figs 2G-H.

Response: We thank the reviewer for this comment. Detecting Srs2 protein has been technically challenging. The SRS2 construct used in this study is untagged. Unfortunately, the commercial SRS2 antibody has been discontinued. We requested several groups who have used SRS2 antibody in their past studies but they have either closed down their labs or are unable to find an aliquot to share. We have tried tagging SRS2 with 6xHis/1XFLAG/3xFLAG tags at N and C-terminal, but unfortunately, the protein was undetectable in the Western blot analysis using either of the tag-specific antibodies. We have also tried western blot analysis using SRS2-GFP strain, but the protein does not get detected by anti-GFP antibody, probably because of very low expression.

Since we will not be able to provide western blots for Srs2 protein levels due to technical challenges, we shall provide western blots for RTEL1 (human homolog of Srs2) protein levels upon Lsm14A knockdown in the presence and absence of HU. This will validate the polysome data we have of RTEL1 regulation by LSM14A, and would, by extension, substantiate the SRS2 polysome data.

Major Comment 5: Figs 3: How are the localization of Scd6 protein and SRS2 mRNA to granules, and the levels of Srs2 protein, in cells exposed to HU after deletion of Hmt1? This would substantiate a role of Hmt1 in vivo.

Response: We will provide the data for Scd6 protein localization and SRS2 mRNA localization in granule enriched fraction upon HU treatment in Δhmt1 background. This experiment is ongoing.

3. Description of the revisions that have already been incorporated in the transferred manuscript

Reviewer 1

Major Comment 1: Fig. 1 F/G: were the delta RGG and LSM variants expressed at an equivalent level to the WT protein in these experiments?

Response: We thank the reviewer for this comment. We have quantified the total fluorescence intensity of GFP from the existing microscopy images for WT and domain deletion mutants for both Scd6 and Sbp1 (Now Figure 3A and 3D). This result (added as a new figure panel Fig 3C and 3F) indicates that the levels of Scd6∆RGG mutant is more whereas Scd6∆Lsm protein levels are comparable than WT. Similarly, Sbp1∆RGG mutant expression is comparable to WT in the given experimental conditions.

Major Comment 2: Fig. 3G: The 6 data points for the delta LSM variant are literally spread evenly up and down the graph, making these data appear highly questionable as to whether one can draw a definitive conclusion from them.

Response: We agree with the reviewer that the data points are varied. To address the scatter in data, we have performed additional experiments and added those to the existing results. Even though there is a spread in the points, except for one data point, all others show an increase in methylation of LSM domain deletion mutant compared to WT, which is statistically significant. The old blot and graph (Old Figure 3F and 3G) have now been replaced with new ones (Figure 5F and 5G) which look more convincing. The result and conclusion derived from it remain unchanged.

Minor Comments

Comment 1: Abstract: the acronym NHEJ likely will need to be defined for the general reader.

Response: The acronym has been expanded in the abstract and explained in the introduction.

Comment 2: Introduction, first paragraph: change gene expression to 'transcription' in the phrase 'Even if the contribution of gene expression to GSR..' as I assume this is what is meant here. Gene expression consists of synthesis, processing, translation and decay.

Response: The required change has been made.

Comment 3: Pg. 3 Introduction: Since they are liquid-liquid phase condensates and ribonucleoproteins (RNPs) refer to any protein-RNA interaction, I think that referring to PBs and SGs as mRNPs is a bit misleading (especially the 'major mRNPs').

Response: The statement has been rewritten.

Comment 4: Introduction: are PBs truly 'sites' of mRNA decay as stated? There are papers in the literature that would argue otherwise.

Response: The statement has been modified with more citations.

Comment 5: Pg. 3, three lines from bottom. Change LSM14 to LSM14A

Response: The addition has been done.

Comment 6: Pg. 4 top - What is an 'LCS' - containing protein? The acronym has not been defined

Response: The acronym has been defined now. We have also defined acronyms wherever they were missing.

Comment 7: Fig. S1 - there are a lot of important data in this figure that demonstrate the coordinated movement of Scd6 and Sbp1 to granules. They should be moved into the main body of the manuscript in my opinion. Likewise, a whole section of the Results is dedicated to Fig. S2 - thus I would suggest moving these data into the main body of the manuscript to assist the reader.

Response: We thank the reviewer for pointing this out. Figure S1 has now been added to the main body of the manuscript as Figure 2. Figure S2 has now been added to Figure 1 and new Figure 3. This rearrangement has improved the flow of the manuscript.

Comment 8: Fig. 1F should be flipped in the figure with panel G since G is discussed in the results section before F

Response: Figure 1F and 1G are now Figure 3A and 3D and in the same order as mentioned in the text.

Comment 9: Be sure to define all acronyms for the reader.

Response: All acronyms in the manuscript have been defined wherever applicable.

Comment 11: Fig. 3H/I: It might be optimal to calculate and compare Kd's for the methylated and unmethylated variants. Also, the labels at the top of 3H do not line up with the wells of the EMSA gel.

Response: We have calculated the Kd’s for the EMSA, and it has been added to the results section. We have also aligned the labels at the top of the EMSA gel (now Figure 5I) to match with the wells.

Reviewer 2

Major Comment 1: Fig. 2A, B. While there seems to be an effect on the lag phase, it could be revealing if the authors pls. calculate the doubling times for the strains and treatments (taking through the exponential growth phase). Furthermore, it would be good if the authors can show the rescue of phenotypes for deletion strains (ie. reintroduction of respective gene on ARS-CEN based plasmids or (if not available) with the OE plasmids.

Response: We thank the reviewer for this remark. We have calculated the doubling times for the strains in the tested conditions and added in the text. We have analyzed the effect of complementing the deletion strains with the respective genes on the CEN plasmid. We observe that Δscd6 shows tolerance to HU stress as previously seen, which gets rescued almost completely upon complementation with WT SCD6. This result has been included in the manuscript as a new figure panel (Figure S1A) . Δsbp1 also shows marginal tolerance to HU stress, but complementation with WT SBP1 only slightly rescues the phenotype, which is not statistically significant (Figure S1B). This result highlights a more important role of Scd6 as compared to Sbp1 in genotoxic stress response.

Major Comment 2 (part 1): Fig. 3H. The authors tested the 5'UTR of SRS2 for interaction with recombinant Scd6. Firstly, it is unclear why the authors have chosen the 5'UTR for investigation? Can the authors explain.

Response: We thank the reviewer for this important comment. During experimentation and analysis, we assayed Scd6 binding to two different fragments of SRS2 mRNA: 5’ and 3’UTR of same lengths (200 bases). We used the UTR fragments because there are numerous reports indicating the role of UTRs in the regulation by RNA binding proteins (https://doi.org/10.1093/bfgp/els056, https://doi.org/10.1126/science.aad9868, https://doi.org/10.1093/jxb/erae073). RNA EMSAs with purified Scd6 and in vitro transcribed UTR RNA fragments revealed a significantly better binding of Scd6 with the 5’ UTR fragment of SRS2 mRNA compared to the 3’ UTR. Therefore, we proceeded with the 5’ UTR fragment for further analysis. We have now added this as a supplementary figure panel and explanation in the manuscript text (Figure S2B).

Major Comment 2 (part 2): Secondly, the affinities are relatively low (µM), and the gel shift assay lacks a negative control. The authors should test an unrelated RNA fragment of approximately the same size to control for specificity (negative control). It is unclear whether the protein could interact with any RNA fragment through a charged RNA backbone.

Response: Our in vivo data suggests that the binding of Scd6 with SRS2 mRNA is condition and RNA-specific and is regulated by methylation (now Figure 5C, S2A and 5E). As the reviewer mentioned, Scd6, in principle, could bind to any RNA molecule given the affinity of an RNA-binding protein (with positively charged amino acids such as arginine) to RNA molecule. Nevertheless, the significant difference in the binding of Scd6 to the 5’UTR and 3’UTR fragments itself acts as a relative control for EMSA. The aim of the in vitro experiment (EMSA) was to establish the difference, if any, in the binding affinities of unmethylated vs methylated Scd6, like the in vivo data, where we observe significantly increased binding to SRS2 mRNA upon decreased Scd6 methylation.

Major Comment 2 (part 3): Thirdly, it would be good if the authors could show a Coomassie gel for the recombinant protein used in those assays.

Response: The Coomassie gel which was provided as part the supplementary data (now Figure S2C), have now been added as another gel image to the main figure (Figure 5H), next to the EMSA, for better clarity.

Major Comment 3: Methods and Materials: The Materials and Methods section lacks important information and requires further details to evaluate the study (see below 11 – 17)

Response: The comment has been duly noted.

Minor Comments

Results:

Comment 4: The numbering of Figure S1, S2 is confused in the first part of the results section. The authors should check numbering. In general, numbering should follow in the order of the text - pls. check.

Response: Based on the comment#7 by Reviewer 1, Figure S1 and S2 have now been added to the main figure, and the changes in the text have been made accordingly.

Comment 5: Pg. 5. CHX treatment leads to a decrease in Scd6-GFP and SBP-1 GFP granules. Essentially, CHX blocks translation elongation so the result indicates that puncta depend on active translation. The authors may want to add this liaising point towards the claim that mRNAs could be present in those puncta. How this results integrates with data shown in Fig. S5B*.

*

Response: We thank the reviewer for this comment. Since granules are dynamic structures that depend on active translation, CHX treatment leads to the dissociation of Scd6 and Sbp1 granules. This indicate that most of the mRNAs present in these granules could be recycled for translation in polysomes. This strategy has been used in multiple research articles for similar deductions (10.1091/mbc.E08-05-0499, https://doi.org/10.1083/jcb.151.6.1257, https://doi.org/10.1093/nar/gku582). We have now modified the text in the manuscript to accommodate this point. It has been previously reported that core components of stress granules, once formed are stable and resistant to RNase, EDTA and NaCl treatment ex vivo (https://doi.org/10.1016/j.cell.2015.12.038), even when these structures have RNA. Figure S5B (now S3C) indicates that the granule enriched fraction derived from untreated and treated cells indeed behaves like stress granule cores and not protein aggregates allowing us to proceed with downstream experiments.

Comment 6: Fig. 2H. It would be helpful to the reader, if the authors could mark the respective fraction in the polysomes taken for analysis of relative enrichments. How was this relative enrichment was calculated needs further description.

Response: The modification has been made (now Figure 4G) and added to the methods and materials.

Comment 7: Fig. S5B. 1% SDS treatment cause absence for Scd6 signal from the pellet fraction. Based on this result, I am not clear how based on this result they can claim for presence of higher order mRNA-protein complexes? Why does it exclude the possibility for Scd6 aggregates accumulating in the pellet? The authors need to explain/ modify this statement. Related to earlier findings that showed dependency of puncta upon CHX treatment, one wonders how this result matches to this earlier observation (ie.EDTA should dissassemble ribosomes)? Can the authors explain?

Response: The very stable β-zipper interactions present in prion like domains, which leads to aggregation, is resistant to 1-2% SDS treatment (https://doi.org/10.1016/j.cell.2015.12.038). Hence, we think that solubilization upon 1% SDS treatment indicates that these are not aggregates. EDTA and NaCl are capable of disrupting interactions, which are stabilized mainly by electrostatic forces. Our observations (now Figure S3C) indicate that Scd6 could be part of the more stable mRNP condensate core structure and are therefore resistant to these treatments. Such observations have been previously reported, for example, stress granules in yeast are not affected by EDTA and NaCl treatments (https://doi.org/10.1016/j.cell.2015.12.038).

Comment 8 (part 1): Fig. 5E, F. For the RNA-seq, the authors compared polysomes with free RNAs (up to 80S) and found enrichment of LIG4 and RTEL1. However, the polysomal profiling mainly shows a slight shift of those mRNAs in higher polysomes; while there is no difference compared to free fractions. How can this be explained?

Response: We observed a shift from lower polysome fractions (11-12-13) (not from free fractions) to higher polysome fractions (14-15) indicating an increased number of ribosomes translating the RTEL1 mRNA.

Comment 8 (part 2): On the line, the authors should indicate clearly what fractions were pooled for RNA seq analysis. It is also not clear how the authors quantified percentage of RNA in individual fractions (have they spiked-in an RNA?) - this needs to be stated in the M&M section.

Response: We have now added the requested information in the Materials and Methods section. Fractions 13 to 17 were pooled for RNAseq analysis. The % of RNA in each fraction was calculated as described in Panda AC et al. Bio Protoc . 2017 Feb 5;7(3):e2126. doi: 10.21769/BioProtoc.2126

Comment 9: At the end, if may be beneficial to the reader if the authors could provide a simple scheme depicting the model develop during this study.

Response: We thank the reviewer for this comment. We have included a model derived from our study as a new figure (Figure 8).

Comment 10: Supplemental Data set (.xls) The adjusted p-values are clustered and >0.05. Can the authors check and describe how those were calculated. How does it match with Volcano plots.

Response: The adjusted p-values are indeed >0.05. The p-values (and not the adjusted p-values) are plotted in the Volcano plot (now Fig. 7E)

Materials and Methods:

Comment 11: A list of primers should be given with specification of their use.

Response: The list has been added in the supplementary files (Table S3)

Comment 12: The plasmids constructed for (over)expression of proteins/ production of recombinant proteins should be added. If published, references should be added accordingly.

Response: The list has been added in the supplementary files (Table S4)

Comment 13: RIP: the media for growing yeast cells should be added. Check also other section if defined.

Response: The information has been added wherever required.

Comment 14: RT-qPCR is not sufficiently described. RT kit needs specification, PCR reaction cycles should be given.

Response: The information has been added

Comment 15: Quantification of mRNA levels in polysomes is unclear. How was the distribution of mRNA profiles determined? Have the authors added some RNA spikes to fractions?

See above.

Response: The % of RNA in each fraction was calculated as described in Panda AC et al. Bio Protoc . 2017 Feb 5;7(3):e2126. doi: 10.21769/BioProtoc.2126. Details have now been added in the Mat and Meth section.

Comment 16: The calculation for the enrichments in IPs is not described conclusively and should be added.

Response: The calculation has now been elaborated and added to the methods and materials section.

Comment 17: Polysomes fractionation (mammalian). It is indicated that the resultant supernatant was adjusted to 5M NaCl and 1 M MgCl2. This seems to be very high - is this a typo? OR why such high concentrations have been chosen?

Response: The sentence has been removed. There is no need for such adjustment.

Review 3

Major Comment 2: Fig 2A-F: The effects of Scd6 and Sbp1 deletion upon HU-treatment are very small. A more convincing effect is observed upon over-expression of both SRS2 and SCD6. What is the effect of over-expression of SCD6 and SBP1 alone (i.e. without SRS2 over-expression)?

Response: We thank the reviewer for this comment. The effects are indeed small but consistent and reproducible with two different kinds of assays (growth curve and plating assay, now Figure 4A-C). Overexpression of Scd6 or Sbp1 alone when expressed from a CEN/2u plasmid does not have any phenotype in the presence of HU (Figure S1A and S1B). Although, it has been previously reported that galactose-inducible Scd6 causes a severe growth defect (https://doi.org/10.1093/nar/gkw762), we performed spot assays with galactose inducible Scd6 and Sbp1 on control and HU plates, but did not see any difference in the extent of growth upon HU treatment. This data has now been presented as Figure S1C.

Major Comment 3: Fig 2E: Why is there an opposite effect of deletion of Scd6 and Sbp1in the SRS2 over-expression background?

Response: We thank the reviewer for this comment; however, we respectfully disagree with the idea that overexpression of SRS2 yields opposite phenotypes in SCD6 and SBP1 deletion backgrounds. Figure 2E (now Figure 4E) gives the impression that SRS2 overexpression in SBP1 deletion grows significantly more for two reasons. There was an increased spotting of Dsbp1 cells overexpressing SRS2 (row#6) as compared to Dscd6 cells overexpressing SRS2 (row#4), which is evident in the plate without HU (left panel). Additionally, there is also reduced spotting of wild-type cells overexpressing SRS2 (row#2) as compared to Dscd6 cells overexpressing SRS2 (row#4). We have now replaced these panels with another image with better loadings. Quantitation of five experiments (Figure S1F) indicates that Dsbp1 grows slightly better in both EV and SRS2 over-expression background, but the increase is not statistically significant. We interpret this data to suggest that SRS2 is not a direct target of Sbp1. Another protein perhaps performs the specific role of Sbp1 in assisting Scd6 in genotoxic stress response in Dsbp1 background.

Major Comment 6: Fig 3C: Is the increased interaction of SRS2 mRNA with Scd6 due to increased levels of SRS2 mRNA upon HU treatment? See also comment below.

Response: Based on RT-qPCR of total RNA, SRS2 mRNA levels do not seem to increase, which has now been added as a Supplementary figure (Figure S3D, left panel). Moreover, quantification of SRS2 mRNA from the FISH data also does not support an increase in mRNA levels (Figure 6D, left panel).

Major Comment 7: Fig 4A: There seems to be an enrichment of SRS2 mRNA both in the granule-enriched pellet and in the supernatant upon HU treatment in the Scd6-GFP context, suggesting increased SRS2 mRNA levels altogether. The enrichment in granules upon HU is difficult to see, as one should measure the distribution of the mRNA in the pellet relative to the supernatant. Can the authors represent the ratio pellet/supernatant normalized to a control transcript? A similar calculation can be done for the protein normalized to a control protein.

Response: As mentioned earlier, RT-qPCR data with SRS2 mRNA levels in total lysate has been added to supplementary data (Figure S3D, left panel). Based on RT-qPCR of total RNA, SRS2 mRNA levels do not seem to increase.

The quantification of SRS2 mRNA and Scd6 protein enrichment is done such that the supernatant and pellet fractions are separately normalized to their respective controls (Scd6GFP, untreated sample) and therefore do not represent the mRNA distribution but relative mRNA enrichment. However, as per the recommendation by the reviewer, the data has been replotted as a ratio of supernatant and pellet with the addition of two more data points and has been added in the main figure (Figure 6E). The data concludes increased enrichment of SRS2 mRNA in granules upon HU treatment. The previous data has been included in the supplementary data as Supplementary figure (Figure S3D, right panel).

Major Comment 8: Fig 4B: Increased juxtaposition of SRS2 mRNA and Scd6 granules upon HU treatment does not really mean increased colocalization. Granules are likely significantly apart such that increased interactions between the two partners are not explained by increased juxtaposition. Please, comment, tune-down and provide examples where increased granule juxtaposition is associated with increased interaction.

Response: We believe that the usage of term ‘juxtaposition’ is leading to misinterpretation of the data. Therefore, we have replaced it with ‘percentage area overlap’ analysis to demonstrate that the SRS2 mRNA foci indeed overlap/localize with Scd6GFP foci up to an average of 43.5% in HU stress. This analysis has been added as an additional panel (Figure 6C), indicating that the SRS2 mRNA interacts with Scd6 in the granules. Even though the granules do not overlap/localize completely, the observed area of granule overlap (43.5%) is functionally effective as it leads to the physical interaction of Scd6 and SRS2 (Figure 6E & 5C) and, consequently, repression (Figure 4H). The FISH data, granule enrichment, and RNA immunoprecipitation data demonstrate Scd6 protein and SRS2 mRNA interaction in granules.

Major Comment 9: Fig 4D: These results are in direct contradiction with those shown in Fig 1C.

Response: We thank the reviewer for this comment. Figure 1C (now Figure 1B and 1C) demonstrates that Scd6 localization to puncta, when expressed from a CEN plasmid, significantly increases upon HU stress. The same trend is visible in Figure 4D (now Figure 6D) where Scd6 is expressed from a 2μ plasmid; however, it is not significant. The data in 1C and 4D (now 1C and 6D respectively) are rather inconsistent with each other than being contradictory. Nevertheless, we understand this reviewer’s concern and address it below.

The initial localization experiments were performed using Scd6 expressed from CEN plasmid or genomically tagged Scd6. Since both these versions of Scd6 are not detectable using western blotting, we used Scd6 expressed from 2μ plasmid. Localization to condensates by liquid-liquid phase separation is a concentration-driven phenomenon. Therefore, when Scd6 is expressed from a 2μ plasmid amounting to increased protein levels, its localization to puncta increases even in the absence of stress, which is visible in the quantitation provided in the figure (Figure 6D) as compared to Figure 1C. We have now analyzed the percentage granular localization (granule intensity) of Scd6 (2µ), which significantly increases upon HU stress (Figure S3A). Thus although number of Scd6 granules does not increase upon HU stress when expressed from a 2µ plasmid, there is significant increase in localization of Scd6 to granule upon HU stress (Figure S3A).

Major comment 10: Fig 5E: Can the authors provide a GO analysis of the up- and down- regulated transcripts?

Response: We have now provided a GO analysis (Table S2). However, due to the low number of regulated genes, only a few GO terms with weak scores appeared in the analysis.

Minor comments:

Comment 11: Figures S1 and S2 seem to be swapped. Please make sure that Figures and panels are arranged in the order they are mentioned in the main text.

Response: We thank the reviewer for pointing it out. Based on the comment#7 by Reviewer 1, Figure S1 and S2 have now been added to the main figure, and the changes in the text have been made accordingly. We have ensured that the order of figures matches the text.

Comment 12: Page 5, sentence: 'our results argue for the role of Scd6 and Sbp1 in HU-mediated stress response'. I do not agree, as no functional assays showing that these proteins affect HU-mediated stress response have been provided at this point of the story. Please, delete.

Response: We have removed the sentence from the existing paragraph.

Comment 13: Page 6: The authors state 'Since Dscd6 and Dsbp1 showed tolerance to chronic HU exposure...'. Where is this shown?

Response: The growth curve in Figure 2A and 2B (now Figure 4A and 4B) and the plating assay in Figure 2C (now Figure 4C) was done with hydroxyurea in the media/plate. Hence, we state that deletion of either SCD6 or SBP1 shows tolerance to chronic (or continuous) HU stress.

Comment 14: Fig 2F: The rescue by SCD6 OE is not complete, as mentioned in the main text.

Response: We have now included the quantification of the spot assay in 2F (now Figure 4F) to show that the rescue by SCD6 overexpression is complete (Fig S1G).

Comment 15: Figure 2G-H: Please, indicate in the figure what the authors consider 'translated' and 'untranslated’ fractions.

Response: The fractions have now been labelled to indicate the missing information in Figure 2G (now Figure 4G).

4. Description of analyses that authors prefer not to carry out

Review 1

Minor Comment 10: Pg. 8/Fig. S3D/4A: It would be interesting to complete the story and determine the functional relationship of Scd6 to the DNL4 mRNA

Response: It is indeed an interesting observation and is currently being pursued as part of another story. We believe it is beyond the scope of the current manuscript.

Review 3

Major Comment 1: Page 5 and Fig S2E-F: The CLHX experiment to conclude that mRNA is present in Scd6 and Sbp1 puncta is rather indirect. The fact that RNase treatment of a granule-enriched pellet has no effect (Fig S5B) does not help. The authors should perform RNase treatment of intact cells and see that the puncta disappear.

Response: We thank the reviewer for this comment. Cycloheximide treatment is a well-accepted assay to detect the presence of mRNA in granules. Since granules are dynamic structures, and these depend on active translation, CHX treatment leads to the dissociation of Scd6 and Sbp1 granules. This indicates that granule assembly depends on the availability of mRNA derived from translating ribosomes. The observation that Scd6 puncta are sensitive to cycloheximide but not to RNase A treatment is not surprising. It indeed is consistent with the properties of some of the condensates reported in the literature. For example, stress granule cores that are sensitive to cycloheximide, like Scd6 puncta, are resistant to RNase treatment in lysate, indicating that once formed, these structures are quite stable (https://doi.org/10.1016/j.cell.2015.12.038). It is interpreted to suggest that the RNAs in these condensates are protected by the RNA-binding proteins. Also, subsequently, in the study, we do RNA immunoprecipitation and granule enrichment experiments and show specific RNA enrichment with Scd6 (Figure 5C, 6A).

I noticed that you have a works cited page on each of your writings and that is very important. But I wonder if it would help to also have your entire bibliography page connected to the notes as an indirect connections (you can do this by editing the page and adding a tag)

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annotation meta: may need new tag: - Is it worth the investment? - Is it just a passing fad?

Reviewer #2 (Public review):

Summary:

The authors tried to determine how PA28g functions in oral squamous cell carcinoma (OSCC) cells. They hypothesized it may act through metabolic reprogramming in the mitochondria.

Strengths:

They found that the genes of PA28g and C1QBP are in an overlapping interaction network after an analysis of a genome database. They also found that the two proteins interact in coimmunoprecipitation and pull-down assays using the lysate from OSCC cells with or without expression of the exogenous genes. They used truncated C1QBP proteins to map the interaction site to the N-terminal 167 residues of C1QBP protein. They observed the levels of the two proteins are positively correlated in the cells. They provided evidence for the colocalization of the two proteins in the mitochondria and the effect on mitochondrial form and function in vitro and in vivo OSCC models, and the correlation of the protein expression with the prognosis of cancer patients.

Weaknesses:

Many data sets are shown in figures that cannot be understood without more descriptions either in the text or the legend, e.g., Fig. 1A. Similarly, many abbreviations are not defined.

The revision addressed these issues.

Some of the pull-down and coimmunoprecipitation data do not support the conclusion about the PA28g-C1QBP interaction. For example, in Appendix Fig. 1B the Flag-C1QBP was detected in the Myc beads pull-down when the protein was expressed in the 293T cells without the Myc-PA28g, suggesting that the pull-down was not due to the interaction of the C1QBP and PA28g proteins. In Appendix Fig. 1C, assume the SFB stands for a biotin tag, then the SFB-PA28g should be detected in the cells expressing this protein after pull-down by streptavidin; however, it was not. The Western blot data in Fig. 1E and many other figures must be quantified before any conclusions about the levels of proteins can be drawn.

The revision addressed these problems.

The immunoprecipitation method is flawed as it is described. The antigen (PA28g or C1QBP) should bind to the respective antibody that in turn should binds to Protein G beads. The resulting immunocomplex should end up in the pellet fraction after centrifugation, and analyzed further by Western blot for coprecipitates. However, the method in the Appendix states that the supernatant was used for the Western blot.

The revision corrected this method.

To conclude that PA28g stabilizes C1QBP through their physical interaction in the cells, one must show whether a protease inhibitor can substitute PA28q and prevent C1QBP degradation, and also show whether a mutation that disrupt the PA28g-C1QBP interaction can reduce the stability of C1QBP. In Fig. 1F, all cells expressed Myc-PA28g. Therefore, the conclusion that PA28g prevented C1QBP degradation cannot be reached. Instead, since more Myc-PA28g was detected in the cells expressing Flag-C1QBP compared to the cells not expressing this protein, a conclusion would be that the C1QBP stabilized the PA28g. Fig. 1G is a quantification of a Western blot data that should be shown.

The binding site for PA28g in C1QBP was mapped to the N-terminal 167 residues using truncated proteins. One caveat would be that some truncated proteins did not fold correctly in the absence of the sequence that was removed. Thus, the C-terminal region of the C1QBP with residues 168-283 may still bind to the PA29g in the context of full-length protein. In Fig. 1I, more Flag-C1QBP 1-167 was pull-down by Myc-PA28g than the full-length protein or the Flag-C1QBP 1-213. Why?

The interaction site in PA28g for C1QBP was not mapped, which prevents further analysis of the interaction. Also, if the interaction domain can be determined, structural modeling of the complex would be feasible using AlphaFold2 or other programs. Then, it is possible to test point mutations that may disrupt the interaction and if so, the functional effect.

The revision added AlphaFold models for the protein interaction. However, the models were not analyzed and potential mutations that would disrupt the interact were not predicted, made and tested. The revision did not addressed the request for the protease inhibitor.

Décryptage du porno mainstream et exploration du porno alternatif : L'industrie, les normes et l'impact sur la perception de la sexualité

I. Datagueule #85 : "Datagaule et clitodonnées : le plaisir à la chaîne"

A. L'industrie du porno en ligne : Une domination par les "tubes"

Présentation des données clés de l'industrie du porno en ligne: Trafic, téléchargements, évolution depuis l'arrivée de l'internet haut débit.

Focus sur Pornhub, un des géants du secteur, illustrant l'ampleur du phénomène et la rapidité de consommation.

Ascension de la société MGeek, qui a racheté des studios historiques du X fragilisés par la crise de 2008.

Fonctionnement des "tubes" qui offrent un accès gratuit aux vidéos, impactant les revenus des studios.

B. Le porno mainstream : Des normes et des dérives

Le porno mainstream, majoritairement produit pour un public masculin hétérosexuel et blanc, impose ses normes.

Illustration de ces normes à travers la popularité du tag "lesbien" et la stigmatisation des scènes gays pour les acteurs.

L'émergence du "pro-am" (productions professionnelles d'amateurs) et ses conditions de tournage précaires et parfois dangereuses.

Problèmes liés aux contrats, au consentement et à la difficulté de faire retirer des contenus des plateformes.

Conditions de travail des acteurs masculins : Salaires faibles, recours à des médicaments pour la performance sexuelle et risques associés.

C. Addictivité et tabou : Des idées reçues à déconstruire

L'argument de l'addictivité du porno, souvent utilisé pour la censure, est démenti scientifiquement.

L'Organisation Mondiale de la Santé a rejeté l'ajout du visionnage de pornographie dans sa liste des troubles addictifs.

Le porno, érigé en tabou, échappe aux questionnements légitimes qui entourent les autres productions culturelles.

II. Interview de Camille Emmanuel, journaliste et auteur de "Sex Power"

A. Le regard masculin dominant dans le porno mainstream

L'industrie du porno traditionnellement dominée par une vision masculine, centrée sur le plaisir masculin et la pénétration.

Le porno mainstream reproduit les schémas traditionnels de la sexualité, ignorant le plaisir féminin et la diversité des pratiques.

Le discours dominant sur la sexualité féminine est déconstruit par des études scientifiques sur le clitoris et l'orgasme féminin.

B. L'émergence du porno alternatif : Un contre-pouvoir nécessaire

Le mouvement du porno alternatif initié par des femmes dans les années 80, pour proposer une vision différente de la sexualité.

Ce mouvement, encore niche, met en avant la diversité des pratiques, des corps et des sexualités.

Le porno alternatif se distingue par ses modes de production éthiques, respectueux du consentement et du droit du travail.

C. L'impact du porno sur la perception de la sexualité

Le porno mainstream véhicule une vision normée et limitée de la sexualité, pouvant influencer négativement la perception du public.

Le porno alternatif, en proposant une vision plus diverse et inclusive, permet de questionner les normes et de s'ouvrir à d'autres possibilités.

L'importance de se questionner sur sa propre consommation de porno et de réfléchir à l'imaginaire pornographique proposé aux générations futures.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

It is evident that studying leukocyte extravasation in vitro is a challenge. One needs to include physiological flow, culture cells and isolate primary immune cells. Timing is of utmost Importance and a reproducible setup essential. Extra challenges are met when extravasation kinetics in different vascular beds is required, e.g., across the blood-brain barrier. In this study, the authors describe a reliable and reproducible method to analyze leukocyte TEM under physiological flow conditions, including this analysis. That the software can also detect reverse TEM is a plus.

Strengths:

It is quite a challenge to get this assay reproducible and stable, in particular as there is flow included. Also for the analysis, there is currently no clear software analysis program, and many labs have their own methods. This paper gives the opportunity to unify the data and results obtained with this assay under label-free conditions. This should eventually lead to more solid and reproducible results.

Also, the comparison between manual and software analysis is appreciated.

We thank the Reviewer for their positive evaluation of our manuscript and highlighting the value of obtaining more reproducible and unbiases results, as well as detection of forward and reverse transmigration with UFMTrack.

Weaknesses:

The authors stress that it can be done in BBB models, but I would argue that it is much more broadly applicable. This is not necessarily a weakness of the study but more an opportunity to strengthen the method. So I would encourage the authors to rewrite some parts and make it more broadly applicable.

We thank the Reviewer for this suggestion. In the revised version of our manuscript, we have now emphasized the broader applicability of UFMTrack to analyze the interaction of immune cells with 2dimensional endothelial monolayers in various contexts in the abstract, introduction, and discussion sections.

Reviewer #2 (Public Review):

Summary:

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications.

Strengths:

Algorithm is almost as accurate as manual tracking and importantly saves time for researchers.

We thank the Reviewer for this positive evaluation of our work.

Weaknesses:

Applicability can be questioned because the device used is 2D and physiological biology is in 3D. Comparisons to other automated tools was not performed by the authors.

We thank the Reviewer for pointing our attention to these weaknesses in our manuscript.

We have clarified in the revised manuscript that using 2D endothelial monolayer models in parallel laminar flow chambers is still a state-of-the-art methodology for studying the multi-step extravasation process of immune cells across endothelial monolayers under physiological flow by in vitro live cell imaging. These models provide excellent optical quality that is not yet achieved in 3D models. We have extended the introduction to emphasize the limitations of existing tools that motivated us to establish UFMTrack. We have furthermore extended the discussion section to highlight the features unique to our UFMTrack framework.

Reviewer #3 (Public Review):

Summary:

The authors aimed to establish a faster and more efficient method of tracking steps of T-cell extravasation across the blood brain barrier. The authors developed a framework to visualize, recognize and track the movement of different immune cells across primary human and mouse brain microvascular endothelial cells without the need for fluorescence-based imaging. The authors succinctly describe the basic requirements for tracking in the introduction followed by an in-depth account of the execution.

We thank the Reviewer for their positive evaluation of our manuscript and highlighting the value of label-free analysis of the multistep immune cell extravasation cascade with UFMTrack.

Weaknesses and Strengths:

Materials & methods and results:

(1) The methods section also lacks details of the microfluidic device that the authors talk about in the paper. Under physiological sheer stress, the T-cells detach from the pMBMEC monolayer, and are hence unable to be detected; however, this observation requires an explanation pertaining to the reason of occurrence and potential solutions to circumvent it to ensure physiologically relevant experimental parameters.

We thank the Reviewer for pointing out this oversight. We have used a custom-made microfluidic device that has been published and described in detail before. This information has now been included in the Methods Section under Point 7, and the two references describing the flow chamber in depth are mentioned below and have been included in the manuscript.  

Coisne Caroline, Ruth Lyck and Britta Engelhardt. 2013. Live cell imaging techniques to study T cell trafficking across the blood-brain barrier in vitro and in vivo. Fluids and Barriers of the CNS 10:7 doi:10.1186/20458118-10-7; 21 January 2013

Lyck R, Hideaki Nishihara, Sidar Aydin, Sasha Soldati and Britta Engelhardt. 2022. Modeling brain vasculature immune interactions in vitro. Angogenesis, 2nd edition. Editors PatriciaD’Amore and Diane Bielenberg Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a041185

T cell detachment is a physiologically relevant parameter besides T cell arrest, polarization, crawling, probing, and transmigration during the interaction with an endothelial monolayer. T cell detachment means that post-arrest, the T cell cannot engage adhesion molecules required for subsequent polarization and, eventually, transmigration. 

(2) The author describes a method for debris exclusion using UFMTrack that eliminates objects of <30 pixels in size from analysis based on a mean pixel size of 400 for T lymphocytes. However, this mean pixel size appears to stem from in-vitro activated CD8 T cells, which rapidly grow and proliferate upon stimulation. In line with this, activated lymphocytes exhibit increased cytoplasmic area, making them appear less dense or “brighter” by phase microscopy compared to naïve lymphocytes, which are relatively compact and subsequently appear dimmer. Given this, it is not clear whether UFMTrack is sufficiently trained to identify naïve human lymphocytes in circulating blood, nor smaller, murine lymphocytes. Analysis of each lymphocyte subtype in terms of pixel size and intensity would be beneficial to strengthen the claim that UFMTrack can identify each of these populations. Additionally, demonstrating that UFMTrack can correctly characterize the behavior of naïve versus activated lymphocytes isolated from murine and human sources would strengthen the claim that UFMTrack can be broadly applied to study lymphocyte dynamics in diverse models without additional training

We thank the Reviewer for the suggestion to more precisely evaluate the range of cell sizes that can be analyzed by our framework. We have included a visualization of crawling cell sizes successfully analyzed by the UFMTrack in Supplementary Figure 7. It demonstrates that the human peripheral blood mononuclear cells, that are almost twice as small as the activated mouse CD4 T cells used in these assays, can be successfully segmented, tracked, and analyzed with the UFMTrack framework. Thus, our UFMTrack framework is suitable for a broad application to differentially sized immune cells during their interaction with the endothelial cell monolayer under flow. 

(3) Average precision was compared to the analysis of UFMTrack but it is unclear how average precision was calculated. This information should have been included in the methods section

We thank the Reviewer for pointing our attention to the missing information. We have added a subsection, “Performance Analysis”, to the Materials and Methods section, where we describe the statistical methods and the performance metrics used to evaluate the UFMTrack framework.

(4) CD4 and CD8 T cells exhibit distinct biology and interaction kinetics driven in part by their MHC molecule affinity and distinct receptor expression profiles. Thus, it is unclear why two distinct mechanisms of endothelial cell activation are needed to see differences between the populations.

We thank the Reviewer for pointing out that different cytokine stimulations of endothelial cells were used in the assays used here to test our UFMTrack to analyze CD4 and CD8 T cell interactions with the endothelial monolayer. While the Reviewer is correct that CD4 and CD8 T cells use different mechanism to cross the pMBMEC monolayer as show by us (doi: 10.1002/eji.201546251.) and others and that recognition of cognate antigen on MHC class I on pMBMECs will arrest CD8 T cells and lead to CD8 T-cell mediated apoptosis ( doi: 10.1038/s41467-023-38703-2.) the focus of the present study was not on comparing CD4 and CD8 T cell interactions with the pMBMEC monolayer but rather to test suitability of UFMTrack to study the different multi-step transmigration of these T cell subsets across the endothelial monolayer. 

(5) The BMECs are barrier tissues but were cultured on µdishes in this study. To study the transmigration of T-cells across the endothelium, the model would have been more relevant on a semi-permeable membrane instead of a closed surface.

We understand the critique of the Reviewer, but laminar flow chambers with endothelial monolayers still provide a state-of-the-art and established methodology to study immune cell migration across endothelial monolayers by in vitro live cell imaging including endothelial cells forming the blood-brain barrier.  

(6) Methods are provided for the isolation and expansion of human effector and memory CD4+ T cells. However, there is no mention of specific CD4+ T cell populations used for analysis with UFMTrack, nor a clear breakdown of tracking efficiency for each subpopulation. Further, there is no similar method for the isolation of CD8+ T cell compartments. A clear breakdown of the performance efficiency of UFMTrack with each cell population investigated in this study would provide greater insight into the software’s performance with regard to tracking the behavior and movement of distinct immune populations.

We thank the Reviewer for this comment. Since a fair performance evaluation requires collecting reliable and consistent manual annotations, in this work we have performed such analysis only for the mouse CD8 T-cell population migrating on the pMBMEC monolayer. We have chosen this as a reference since it is a different cell population than the one the segmentation model was trained on. This provides an insight into how high performance is expected when other immune cell types are studied than the ones used for model development.

(7) The results section is quite extensive and discusses details of establishment of the framework while highlighting both the pros and cons of the different aspects of the process, for example the limitation of the two models, 2D and 2D+T were highlighted well. However, the results section includes details which may be more fitting in the methods section.

We thank the Reviewer for highlighting the extensive work carried out in the development of our UFMTrack framework. We decided to include in the results section only the description of key elements and design decisions taken when developing the framework, such as the need to include a time series of images for successful segmentation of the transmigrated cells. At the same time, the majority of implementational details can be found in the Supplementary Material.

(8) A few statements in the results section lacked literary support, which was not provided in the discussion either, such as support for increased variance of T-cell instantaneous speed on stimulated vs non-stimulated pMBMECs. Another example is the enhancement of cytokine stimulation directed T-cell movement on the pMBMECs that the authors observed but failed to relay the physiological relevance of it. The authors don’t provide enough references for developments in the field prior to their work which form the basis and need for this technology.

We thank the Reviewer for this comment and for asking for literature references. However, we cannot provide such references as these are original observations we made by employing the UFMTrack framework.  This shows that UFMTrack observes T-cell behaviors that have previously been overlooked. Their physiological relevance will have to be explored in separate studies. We have extended the introduction section to include the details on the existing methods developed in the field, as well as their weaknesses that motivated the development of the UFMTrack framework.

(9) The rationale for use of OT-1 and 2D2-derived murine lymphocytes is unclear here. The OT-1 model has been generated to study antigen-specific CD8+ T cell responses, while the 2D2 model has been generated to recapitulate CD4 T cell-specific myelin oligodendrocyte glycoprotein (MOG) responses.

To establish and test the UFMTrack framework, we have made use of the specific T-cell subsets and endothelial cell models we generally use within our research context. Especially for animal work, this is according to the 3R rules requesting to reduce animal experimentation.  

Figures and text:

(1) There are certain discrepancies and misarrangement of figures and text. For example, discussion of the effect of sheer flow on T cell attachment as part of the introduction in figure 1 and then mentioning it in the text again in the results section as part of figure 4 is repetitive.

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the label of Figure 4 to emphasize that this effect is correctly captured by the UFMTrack.

(2) Section IV, subsection 1 of the results section, refers to ‘data acquisition section above’ in line 279, however the said section is part of materials and methods which is provided towards the end of the manuscript.

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the text to reflect the correct chapter order.

(3) There are figures in the manuscript that have not been referenced in the results section, for example, figure 3A and B. Figure 1 hasn’t been addressed until subsection 7 of materials and methods

We thank the Reviewer for pointing our attention to this misarrangement. We have adjusted the text to refer to all figure panels and the clarification of the cell multiplicity estimation in the supplementary information section. References to Figure 1 were added in the introduction section to illustrate the in vitro under flow imaging setup as well as the typical T cell behaviors in such experiments.

(4) A lack of significance but an observed trend of increased variance of T cell instantaneous speed is reported in line 296-298; however, the graph (figure 4G) shows a significant change in instantaneous speed between non-stimulated and TNFα-stimulated systems. This is misleading to the readers.

We thank the Reviewer for pointing our attention to this discrepancy. We have expanded the text to indicate a low statistical significance for the TNF and no significance but just a trend for the IL1-beta conditions.

(5) The authors talk about three beginner experimentors testing the manual T cell tracking process but figure 5 only showcases data from two experimentors without stating the reason for excluding experimentor 1.

We thank the Reviewer for pointing our attention to this ambiguity. While both the migration analysis and the manual cell tracking were performed by all three beginner experimenters, the cell tracking data for the first one was unfortunately lost due to a hardware failure.

Discussion:

(1) While the discussion captures the major takeaways from the paper, it lacks relevant supporting references to relate the observation to physiological conditions and applicability.

This study is not about the physiological relevance of the microfluidic devices and immune cells used but rather about advancing methodology to analyze dynamic immune cell behavior on endothelial monolayers under physiological flow. Therefore, the discussion does not extend to comparing the physiological relevance of the specific in vitro models employed in this study.   

(2) The discussion lacks connection to the results since the figures were not referenced while discussing an observed trend

We thank the Reviewer for pointing our attention to this misarrangement. We have included the references to the relevant figures as well as supporting references.

(3) The authors briefly looked into mouse and human BMECs and their individual interaction with Tcells, but don’t discuss the differences between the two, if any, that challenged their framework.

We thank the Reviewer for pointing our attention to this weakness. We have added to the discussion section clarifications on the challenges of analyzing the T cell interactions with the HBMEC and the BMDM interactions with the pMBMEC monolayer.

(4) Even though though the imaging tool relies on difference in appearance for detection, the authors talk about lack of feasibility in detecting transmigration of BMDMs due to their significantly different appearance. The statement lacks a problem solving approach to discuss how and why this was the case.

We thank the Reviewer for pointing our attention to this weakness and apologize for the misleading explanation of the problem of analyzing the BMDM sample. Since the transmigrated part of the macrophages differs in appearance from a transmigrated part of a T cell, its detection by a Deep Neural Network trained on the T cell data is worse than that for the T cells. At the same time, the detection performance before the transmigration is sufficient for the BMDM migration analysis. The potential approaches to alleviate this are added to the discussion section.

Relevance to the field:

Utilizing the framework provided by the authors, the application can be adapted and/or utilized for visualizing a range of different cell types, provided they are different in appearance. However, this would require extensive changes to the script and won’t be adaptable in its current form.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

The authors should announce in the abstract that the software analysis Track is downloadable and free to use for all researchers. They may consider providing some sort of helpdesk, although I realize that that may run into too much time.

As said above, they stress that it can be done in BBB models, but I would argue that it is much more broadly applicable.

We thank the Reviewer for these suggestions. We have emphasized the broader applicability of UFMTrack in the abstract and pointed out the public availability of the code and data.

Can they add an experiment that shows that it also works for neutrophils for example? I understand that on paper yes it should work, but the neutrophils are of course faster etc.

This is an excellent suggestion, but we tested UFMTrack within the current framework of ongoing research, which does not include the investigation of neutrophil transmigration across endothelial monolayers.  

Also, the combination of different leukocytes in one TEM assay would really be a step forward. If the software can detect different-sized leukocytes, then this should be possible.

We thank the Reviewer for this suggestion. We have added Supplementary Figure 7, demonstrating the range of cell sizes that were successfully analyzed by the UFMTrack framework throughout our manuscript. We also added a statement to the discussion that according to this data, “simply by discriminating cells by size, it is possible to extend UFMTrack to study the interaction of several types of immune cells migrating on top of a cellular monolayer under flow.”

Extra challenges: can the method also discriminate between paracellular and transcellular migration modes? In particular for T-cells this is known to happen.

We thank the Reviewer for this suggestion. We have added this to the potential applications of UFMTrack in the discussion section. While this differentiation is not feasible relying solely on the phasecontrast imaging data, UFMTrack can simplify this analysis by providing automatically the predictions of the transmigration locations, for analysis of the fluorescent data of the junctional labels.

Reviewer #2 (Recommendations For The Authors):

This paper develops an under-flow migration tracker to evaluate all the steps of the extravasation cascade of immune cells across the BBB. The algorithm is useful and has important applications. There are several points that need to be addressed, particularly about the claims made by the authors.

Please see the comments below for more details:

• Lines 88-92: Add a citation for the characteristics of the BBB as a barrier

We have added two references accordingly.  

• Lines 94-95: Can the authors indicate what models were used for these studies and how those compare to their in vitro model? In addition, can the authors say whether T cells were manually tracked in this study to translate results to the clinic and whether the results were successful when translated to the clinic? This may enhance the argument that automatic trackers are needed if the translation was not 100% successful

This introductory paragraph summarizes in vivo and in vitro observations from several laboratories. Although these studies include manual tracking of T cells, they do not necessarily distinguish all sequential steps of the multi-step T cell transmigration cascade. Thus, automated tracking may provide additional insights, allowing for increased translation of findings to the clinic.  

• Lines 96-98: Citing the work of Roger Kamm and Noo Li Jeon would be helpful here as they pioneered these BBB microfluidic models and have protocol papers on how to build them and how to use them for cancer cell extravasation studies. Roger Kamm has also worked on several extravasation studies with neutrophils, monocytes, and PBMCs from 3D vasculatures in microfluidic devices, under flow using pressurized fluid or recirculating pumps. Mentioning those would be helpful as they are directly related to what the authors are presenting in their paper.

We thank the Reviewer for this comment, and we consider the work of Roger Kamm and Noo Li Jeon as very valuable for the field. However, these authors have focused on developing functional 3D microfluidic devices, including, e.g., all cells of the neurovascular unit which is not the focus of this present study that solely employed parallel flow chamber devices and endothelial monolayers.  

• Lines 110-116: Can the authors comment on the use of ImageJ or similar automatic tracking tools and how these compare to the under-flow migration tracker developed in this paper? Several groups use ImageJ to track cellular migration successfully and in an automatic manner with short intervals between each frame. One paper that comes to mind is Chen et al: DOI: 10.1073/pnas.1715932115 where neutrophil migration in 3D was assessed with ImageJ in microfluidic devices of the vasculature. If the authors can highlight differences between their tool and what is currently available and used for automatic tracking (e.g. ImageJ), this would help in understanding the advantages of the migration tracker developed in this paper.

• Lines 118-121: Add citations for the current state of the art for T cell extravasation tracking

We thank the Reviewer for these suggestions. We have extended the introduction to add more details on the available tools for tracking migrating immune cells and their limitations, as well as the discussion section to emphasize the features unique to the developed UFMTrack framework.

• Figure 1: The device used by the authors is considered to be a 2D microfluidic device with a monolayer of mouse brain endothelial cells. I would recommend the authors to carefully revise the claims made in the paper to mention that this is a 2D device as opposed to a 3D device, in order to not mislead readers who may be expecting these analyses to be performed in 3D vasculatures.

We thank the Reviewer for this suggestion. We have included in the summary the mention of the 2dimensional nature of the employed BBB model.

• Figure 1: The T cells used in this study are not fluorescently-labeled but the authors mention that this is an issue from current state-of-the-art tools. I would recommend that the authors remove this point as being an issue because it is not addressed in their paper. The T cells are also not labeled in this study so this limitation of other systems is not addressed in this paper.

We apologize to the Reviewer as we do not understand this question. There will be many experimental conditions not allowing to study fluorescently tagged T cells. Therefore, UFMTrack is tailored to follow and analyze T cells and other immune cells during their interaction with endothelial monolayers independent of a fluorescence tag.  

• Figure 1: Was the shear stress controlled manually with a syringe? Or with the use of a pressure controller? I would clarify this aspect and discuss human errors that can be introduced from manually controlling the pressure applied to the monolayer.

We thank the Reviewer for pointing our attention to this ambiguity. We have added a mention of the automated syringe pump used to control the shear stress in the text where the values of shear stress applied to the sample are first mentioned.

• Figure 1: Does T cell attachment occur within the first 5 minutes? Can the authors comment on how they chose this timeline and the percentage of T cells that are washed off at the second step at 1.5 dynes/cm^2? Is 30 seconds enough to ensure all the non-adhered T cells are washed off with 1.5 dyns/cm^2?

Superfusion of the T cells over the endothelial monolayer is performed under 0.5 dynes/cm2 to allow the T cells to settle on the endothelial cell monolayer under flow. After increasing to physiological, flow non adherent T cells detach within 30 seconds, as described by the Reviewer. We have included in the Methods Section Point 7 the references describing in depth the design of the flow chamber device and methods used here.  

• Line 154: How many images were used in the training vs. testing dataset for T cell migrations?

We thank the Reviewer for pointing our attention to this missing information. We have added the sizes of the training and validation datasets. Specifically, the 226MPix of available imaging data was split into 154Mpix training and 37 MPix validation sets. The gap in between was introduced to avoid a correlation between validation and training set that would compromise the performance evaluation.

• Are the supplementary videos at real speed or accelerated?

We thank the Reviewer for pointing our attention to this missing information. The videos are sped up by a factor of 96. We have added this information to the Supplementary video descriptions.  

• Lines 208 216: Can the authors comment on how their initial adhesion timeframe of 30sec before starting the recording at 5.5min affects the number of T cells with rapid displacement? 30 seconds may not be enough to ensure T cells have adhered to the endothelium

Please see our comment above. The methodology used in the present assays has been set up and validated in numerous publications. We have included in the Methods Section under Point 7 the references describing in depth the design of the flow chamber device and the methods used here.  

• Lines 275-277: Was the number of testing images 18? Can the authors comment on how this compares to training dataset size and whether these numbers are enough to achieve robust results?

We apologize for this ambiguity in our manuscript. The framework was evaluated on 18 imaging datasets, each corresponding to 32 minutes of recording, not 18 images. We have added this clarification to the “CD4+ T cell analysis” subsection. The total size of these datasets is 18 datasets * 191 timeframe/dataset * 9.9MPix/frame = 34MPix

• Figure 4B: Can the authors add statistics here? Individual datapoints on the error bars would be helpful too. 

We thank the Reviewer for pointing our attention to this weakness. The data corresponds to the statistical errors as evaluated based on all cells in the 18 datasets. We have added the total number of cells in each of the endothelium stimulation conditions to the text.

• Figure 4C-J: Can the authors put individual datapoints here as well and explain whether they considered each T cell to be one datapoint or each endothelium (averaging all T cells) to be one datapoint? 

We thank the Reviewer for this suggestion. However, adding about one thousand points corresponding to each cell would be impractical. We thus present the distributions of the evaluated from the data metrics as a histogram on the violin plot instead of the swarm plot.

• Figure 4: Did the authors wash the monolayers before introducing T cells? Soluble unbound cytokines may still be present and there are two different questions that would be studied here: “Is the inflamed endothelium affecting T cell migration?” (if washing was performed) or “Is T cell and microenvironmental inflammation affecting T cell migration?” (if no washing was performed)

The endothelial monolayers are “washed” by starting the flow in the flow chamber device and this is before superfusing the T cells over the endothelial monolayer. We agree that our flow chamber device combined with UFMTrack will allow to address all these questions.

• Figure 4I: Are all the T cells decelerating? (negative AM speed)

We thank the Reviewer for this question. The cells are moving along the flow, which, in our experiments, is from left to right. The vector of speed is thus pointing against the x-axis, and thus the AM speed is negative.

• Lines 302 306: Please explain how this compares to ImageJ or similar trackers that can achieve similar outputs. 

We thank the Reviewer for this question. We have added a statement in the “T-cell tracking” section emphasizing that standard trackers are incapable of correctly capturing large displacements.

• Lines 306-309: It is not lower for TNF stimulation though. How do the authors address this? TNF is also a pro-inflammatory cytokine.

We have previously shown that stimulation of pMBMECs with IL-1 and TNF-a induces different cell surface levels of ICAM-1 and VCAM-1, which will influence T cell behavior on the pMBMEC monolayer.  

• Lines 313-315: Could this be because the monolayer was not washed and soluble cytokines affected T cell response directly?

Please see our answer to lines 306-309.  

• Lines 319: Please cite Roger Kamm and Noo Li Jeon’s papers on BBB models with human BMECs, pericytes and astrocytes in 3D microfluidic devices.

We thank the Reviewer again for pointing out these studies. As mentioned above, as our present study does not explore 3D models of the BBB, we think it does not fit into the framework of our study to elaborate on 3D models of the BBB. In addition, this would require the inclusion of a discussion of the work of others like, e.g., Peter Searson and others.  

• Figure 5: Several statistics are missing from parts of the figure. Please add those.

We apologize – but we do not understand which statistical analysis the Reviewer is missing from this Figure.  

• Can the authors comment on the number of T cells perfused over the monolayer and if this ratio of T cells to endothelial cells makes physiological sense? Too many T cells may result in endothelium inflammation and increased diapedesis.

The number of T cells used to suprerfuse over the endothelial monolayer is tested to avoid aggregation of T cells in suspension and thus artificial interactions with the endothelial monolayer. T cell behavior on the pMBMEC monolayer remains the same over the dilution of factor 10.  

• Lines 381 383: How does this compare to analyses that look at the cross-section of the endothelium? It is difficult to assess transmigration looking at the top view of the endothelium. Perhaps, cross-section assessments will identify differences in manual vs. automatic tracking.

There is, to the best of our knowledge, no microscopic device that would allow for in vitro live cell imaging of a live endothelial monolayer – this is in the presence of tissue culture medium – from the side at a resolution that would allow to define transmigration. Our current study rather shows the UFMTrack can distinguish cells moving above or below the endothelial monolayer.  

• Figure 5J: This is probably the most important argument of the paper. If the authors can show statistical differences in their graph, this would greatly help convince readers that this tool is necessary and actually computationally efficient compared to manual work by researchers.

We thank the Reviewer for this suggestion. However, comparing a single data point for automated measurement with four manual experimenter analysts is not a statistically sound comparison. We believe that Figure 5K is clearly showing the factor 5 difference in analysis speed as compared to manual analysis. More importantly, though, the automated analysis is taking the machine time, lifting the need for the experimenter to invest even 1/5th of the original analysis time.

• Figure 6: Did the authors use autologous immune cells and endothelial cells? This is particularly relevant with the use of human-derived T cells (line 436) on the BMEC monolayer. Can the authors comment on non-self reactivity by the T cells encountering BMEC from another human subject?

Autologous T cell interaction with BMECs would only be possible when using hiPSC-derived EECM-BMECs and the T cells from the same individual. All other experimental frameworks will not include autologous interactions. This is the experimental framework used by most authors studying immune cell interactions with commercially available donors. We have not studied alloreactive interactions in our assays and thus cannot further comment.  

• Figure 6M,N,O: How does this compare to ImageJ for tracking of fluorescent cells? I recommend the authors to try that, at least for this section, as this may enhance their argument for their tool vs. standard tools like ImageJ if success rates are higher for their tool.

We thank the Reviewer for this suggestion. We included a note on the analysis of the fluorescent datasets using the  TrackMate plugin for imageJ performed previously in our lab in the “Human T cells on immobilized recombinant BBB adhesion molecules” subsection.

• Figure 6: Please put individual datapoints on the bar or violin plots where they are missing.

We thank the Reviewer for this suggestion. However, adding about one thousand points corresponding to each cell would be impractical. We thus present the distributions of the evaluated from the data metrics as a histogram on the violin plot instead of the swarm plot.

• Lines 467-471: This argument is important and should be mentioned earlier in the introduction.

Another point that can be mentioned is the application of this platform to imaging modalities in vivo (mouse or human) given that there is no fluorescent staining in these cases. This review may be relevant: https://doi.org/10.1002/jcb.10454

We thank the Reviewer for this suggestion. We have clarified in the introduction that UFMTrack does not require fluorescent labels of the imaged migrating cells and relies solely on the phase contrast imaging data.

• Discussion: Please address a few more potential applications to this study. One can be cancer and immune infiltration.

We thank the Reviewer for this suggestion. We have elaborated on additional potential applications to the discussion section.

Reviewer #3 (Recommendations For The Authors):

(1) Line 327-328: The authors talk about ‘As we have previously shown…pMBMEC monolayers differs between CD4+ and CD8+ cells…’. Where was this shown? If it was in a previously published article, please provide a reference.

We have added these missing references.  

(2) Line 353: Please provide clear location on where to find the associated information instead of stating ‘see below’.

We thank the Reviewer for pointing our attention to this ambiguity. We have corrected the phrase to “see next paragraph”

(3) Line 439: Please correct the acronym to BMECs

We thank the Reviewer for pointing our attention to this typo. We have corrected it.

Author response:

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

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript by Choi and co-authors presents "P3 editing", which leverages dual-component guide RNAs (gRNA) to induce protein-protein proximity. They explore three strategies for leveraging prime-editing gRNA (pegRNA) as a dimerization module to create a molecular proximity sensor that drives genome editing, splitting a pegRNA into two parts (sgRNA and petRNA), inserting self-splicing ribozymes within pegRNA, and dividing pegRNA at the crRNA junction. Among these, splitting at the crRNA junction proved the most promising, achieving significant editing efficiency. They further demonstrated the ability to control genome editing via protein-protein interactions and small molecule inducers by designing RNA-based systems that form active gRNA complexes. This approach was also adaptable to other genome editing methods like base editing and ADAR-based RNA editing.

Strengths:

The study demonstrates significant advancements in leveraging guide RNA (gRNA) as a dimerization module for genome editing, showcasing its high specificity and versatility. By investigating three distinct strategies-splitting pegRNA into sgRNA and petRNA, inserting self-splicing ribozymes within the pegRNA, and dividing the pegRNA at the repeat junction-the researchers present a comprehensive approach to achieving molecular proximity and reconstituting function. Among these methods, splitting the pegRNA at the repeat junction emerged as the most promising, achieving editing efficiencies up to 76% of the control, highlighting its potential for further development in CRISPR-Cas9 systems. Additionally, the study extends genome editing control by linking protein-protein interactions to RNA-mediated editing, using specific protein-RNA interaction pairs to regulate editing through engineered protein proximity. This innovative approach expands the toolkit for precision genome editing, demonstrating the feasibility of controlling genome editing with enhanced specificity and efficiency.

Weaknesses:

The initial experiments with splitting the pegRNA into sgRNA and petRNA showed low editing efficiency, less than 2%. Similarly, inserting self-splicing ribozymes within pegRNA was inefficient, achieving under 2% editing efficiency in all constructs tested, possibly hindered by the prime editing enzyme. The editing efficiency of the crRNA and petracrRNA split at the repeat junction varied, with the most promising configurations only reaching 76% of the control efficiency. The RNA-RNA duplex formation's inefficiency might be due to the lack of additional protein binding, leading to potential degradation outside the Cas9-gRNA complex. Extending the approach to control genome editing via protein-protein interactions introduced complexity, with a significant trade-off between efficiency and specificity, necessitating further optimization. The strategy combining RADARS and P3 editing to control genome editing with specific RNA expression events exhibited high background levels of non-specific editing, indicating the need for improved specificity and reduced leaky expression. Moreover, P3 editing efficiencies are exclusively quantified after transfecting DNA into HEK cells, a strategy that has resulted in past reproducibility concerns for other technologies. Overall, the various methods and combinations require further optimization to enhance efficiency and specificity, especially when integrating multiple synthetic modules.

Thank you for this accurate summary and assessment of the strengths and weaknesses of the P3 editing as it stands. Looking ahead, we agree that further optimizations will be important, as will characterizing the performance of P3 editing in additional cellular contexts. The revised Discussion (see below) now makes these points more clearly.

Reviewer #2 (Public Review):

Choi et al. describe a new approach for enabling input-specific CRISPR-based genome editing in cultured cells. While CRISPR-Cas9 is a broadly applied system across all of biology, one limitation is the difficulty in inducing genome editing based on cellular events. A prior study, from the same group, developed ENGRAM - which relies on activity-dependent transcription of a prime editing guide RNA, which records a specific cellular event as a given edit in a target DNA "tape". However, this approach is limited to the detection of induced transcription and does not enable the detection of broader molecular events including protein-protein interactions or exposure to small molecules. As an alternative, this study envisioned engineering the reconstitution of a split prime editing guide RNA (pegRNA) in a protein-protein interaction (PPI)-dependent manner. This would enable location- and content-specific genome editing in a controlled setting.

The authors explored three different design possibilities for engineering a PPI-dependent split pegRNA. First, they tried splitting pegRNA into a functional sgRNA and corresponding prime editing transRNA, incorporating reverse-complementary dimerization sequences on each guide half. This approach, however, resulted in low editing efficiency across 7 different designs with various complementary annealing template lengths (<2% efficiency). They also tried inserting a self-splicing ribozyme within the pegRNA, which produces a functional pegRNA post-transcriptionally. The incorporation of a split-ribozyme, dependent on a PPI, could have been used to reconstitute the split pegRNA in an event-controlled manner. However again, only modest levels of editing were observed with the self-splicing ribozyme design (<2%). Finally, they tried splitting the pegRNA at the repeat:anti-repeat junction that was used to join the original dual-guide system comprised of a crRNA and tracrRNA, into a single-guide RNA. They incorporated the prime editing features into the tracrRNA half, to create petracrRNA. Dimerization was initially induced by different complementary RNA annealing sequences. Using this design, they were able to induce an editing efficiency of ~28% (compared to 37% efficiency using a positive control epegRNA guide).

Having identified a suitable split pegRNA system, they next sought to induce the reconstitution of the two halves in a PPI-dependent manner. They replaced the complementary RNA annealing sequences with two different RNA aptamers (MS2 and BoxB). MS2 detects the MCP protein, while BoxB detects the LambdaN protein. Close proximity between MCP and LambdaN would thus bring together the two split pegRNA halves, creating a functional pegRNA that would enable prime editing at a specific target site. They demonstrated that they could induce MCP-BoxB proximity by fusing them to different dimerizing protein partners: 1) constitutive epitope-nanobody/antibody pairs such as scFv/GCN4 or NbALFA/ALFA-Tag; 2) split-GFP; or 3) chemically-induced protein pairs such as FKBP/FRB or ABI/PYL. For all of these approaches, they could achieve between ~20-60% normalized editing efficiency (relative to positive control editing levels with epegRNA). Additional mutation of the linkers between the RNA and aptamers could increase editing efficiency but also increase non-specific background editing even in the absence of an induced PPI.

Additional applications of this overall strategy included incorporating the design with different DNA base editors, with the most promising examples shown with the base editors CBE4max and ABE8. It should be noted that these specific examples used a non-physiological LambdaN-MCP direct fusion protein as the "bait" that induced reconstitution of the two halves of the guideRNA, rather than relying on a true induced PPI. They also demonstrated that the recently reported RADARS strategy could be incorporated into their system. In this example, they used an ADAR-guide-RNA to drive the expression of a LambdaN-PCP fusion protein in the presence of a specific target RNA molecule, IL6. This induced LambdaN-PCP protein could then reconstitute the split peg-RNAs to drive prime editing. To enable this last application, they replaced the MS2 aptamer in their pegRNA with the PP7 aptamer that binds the PCP protein (this was to avoid crosstalk with RADARS, which also uses MS2/MCP interaction). Using this strategy, they observed a normalized editing efficiency of around 12% (but observed non-specific editing of around 8% in the absence of the target RNA).

Strengths:

The strengths of this paper include an interesting concept for engineering guide RNAs to enable activity-dependent genome editing in living cells in the future, based on discreet protein-protein interactions (either constitutively, spatially, or chemically induced). Important groundwork is laid down to engineer and improve these guide RNAs in the future (especially the work describing altering the linkers in Supplementary Figure 3 - which provides a path forward).

Weaknesses:

In its current state, the editing efficiency appears too low to be applied in physiological settings. Much of the latter work in the paper relies on a LambdaN-MCP direction fusion protein, rather than two interacting protein pairs. Further characterizations in the future, especially varying the transfection amounts/durations/etc of the various components of the system, would be beneficial to improve the system. It will also be important to demonstrate editing at additional sites; to characterize how long the PPI must be active to enable efficient prime editing; and how reversible the reconstitution of the split pegRNA is.

Thank you for this assessment of the strengths and weaknesses of the P3 editing as it stands. Looking ahead, we agree that further optimizations will be important, including along the lines suggested by the reviewer, as will further characterization of the system with respect to dependencies, reversibility, etc. The revised Discussion (see below) now makes these points more clearly.

Recommendations for the authors:

Reviewing Editor comments:

It would be helpful to better describe the nature of improvements (on-targeting and/or off-targeting) that would be needed to effectively use this approach in vitro and in vivo applications.

We agree, and have accordingly revised the last paragraph of our discussion to better describe what improvements are needed for in vitro and in vivo applications:

“In our view, there are four outstanding challenges for P3 editing to be broadly useful: evaluating additional cellular contexts, the method’s efficiency and specificity, understanding the limit of detectable protein-protein interactions, and the development of sensors compatible with multiplex P3 editing within the same cell. First, we have thus far only conducted P3 editing in HEK293T cells, and obviously needs to be tested in additional cell types. Second, both the efficiency and specificity of the P3 editing need to be improved before it can be used as a selective editing tool in model systems. We have explored how modifying the crRNA and petracrRNA pair sequences can tune the efficiency-vs-specificity tradeoff, but alternative avenues to improvement (e.g., better docking of RNA-aptamers such as MS2, BoxB, or PP7 by testing more linker sequences that place crRNA and petracrRNA for duplex formation) may be more fruitful in terms of achieving high efficiency and specificity at once (e.g., >50% editing in the setting of a specific protein-protein interaction, and <1% editing without it). Second, it is not clear whether weak and transient interactions among proteins can be used to trigger P3 editing. Assuming the genome editing complex formation is reversible, improving P3 editing efficiency may be able to capture different strengths of protein-protein interactions, although some interactions may be too transient to promote functional guide RNA formation. Finally, the current P3 editing design uses a pair of RNA aptamers and their corresponding protein binders, limiting the multiplex detection of protein-protein pairs. More orthogonal protein-RNA pairs need to be identified (e.g., using a massively parallel platform (Buenrostro et al., 2014) and/or computational prediction (Baek et al., 2023)) to allow for large numbers of P3 sensors for different protein-protein interactions to be deployed within the same cell. Overcoming these four challenges is necessary for P3 editing to be broadly useful for gating genome editing on physiological levels of specific protein-protein interactions in a multiplex fashion.”

Reviewer #2 (Recommendations For The Authors):

It does not appear that all plasmids necessary to reproduce the results of this paper have been deposited to addgene, but only a small subset. The authors might include that these plasmids are available upon request, if not uploaded to a public repository.

We have added a statement that additional plasmids are available upon request. Our Data Availability Statement reads (with the added sentence underlined):

“Raw sequencing data have been uploaded to Sequencing Read Archive (SRA) with the associated BioProject ID PRJNA1004865. The following plasmids have been deposited to Addgene: pU6-crRNA-MS2, pU6-BoxB-petracrRNA, pCMV-LambdaN-MCP, pCMV-LambdaN-NbALFA,  and pCMV-ALFA-MCP (Addgene ID 207624 - 207628). The rest of the plasmids used in this study are available upon request.”

It could be useful to include somewhere why, specifically, editing the guide RNAs as opposed to the Cas9 itself is advantageous. Light-inducible split Cas9s have been engineered, and I imagine other PPI-inducible split Cas9s have also been engineered. A specific mention of the advantages of using engineered split pegRNAs could put the significance of this work in a better context.

Thanks for raising this, and we agree. We have revised the first paragraph of the Results section to highlight why we think splitting the guide RNAs as opposed to Cas9 might be advantageous:

“In the split architecture, the “dimerization module” is a key sensor component. Although strategies that split the protein component of the genome editing complex have been described (e.g., split-Cas9 (Yu et al., 2020)), we reasoned that having the guide RNA serve as the dimerization module rather than the protein, i.e. by splitting it into two parts, and making the restoration of its function dependent on a molecular proximity event, would afford even more control. For example, if multiple split gRNAs were present within the same cell, they could be independently controlled, whereas a split Cas9 would only allow a single control point.  In our initial experiments, we focused on splitting the pegRNA used in prime editing.”

Welcome back.

This is part two of this lesson.

We're going to continue immediately from the end of part one.

So let's get started.

Okay, so the instance is now in an available state.

Let's just close down this informational dialogue at the top.

And let's just minimize this menu on the left.

Let's maximize the amount of screen space that we have for this specific purpose.

So I just want us to go inside this database instance and explore together the information that we have available.

So I talked in the theory lesson how every RDS instance is given an endpoint name and an endpoint port.

So this is the information that we'll use to connect to this RDS instance.

Networking wise, this instance has been provisioned in US-EAST-1A.

It's in the Animals for Life VPC and it's used our A4L subnet group that we created at the start of this demo.

And that means that it's currently utilizing all three database subnets in the Animals for Life VPC.

But it's chosen because we only deployed one instance to use US-EAST-1A.

Now this is the VPC security group that we're going to need to configure.

So right click on this and open it in a new tab and move to that tab.

This is the security group which controls access to this RDS instance.

So let's expand this at the bottom.

So currently it has my IP address being the only source allowed to connect into this RDS instance.

So the only inbound rule on the security group protecting this RDS instance is allowing my IP address.

So we're going to click on Edit and then click on Add Rule.

And we're going to add a rule which allows our other instances to connect to this RDS instance.

So first in the type drop down click and then type mySQL to get the same option as the line above and then click to select.

Next go ahead and type instance into the source box and then select the migrate to RDS-instance security group.

Now this is the security group that's used by any instances deployed by our one click deployment.

And this allows those instances to connect to our RDS instance and that's what we want.

So go ahead and select that and then click on Save Rules.

And this means now that our WordPress instance can communicate with RDS.

So now let's move back to the RDS tab and then make sure we're inside the A4L WordPress database instance.

So that's the connectivity and the security tab.

We also have the monitoring tab and it's here where you can see various CloudWatch provided metrics about the database instance.

You also have logs and events related to this instance.

So if we go and have a look at recent events we can see all of the events such as when the database instance was created, when its first backup was created.

And you can explore those because they might be different in your environment.

You can click on the Configuration tab and see the current configuration of the RDS instance.

The Maintenance and Backups tab is where you can configure the maintenance and backup processes and then of course you can tag the RDS instance.

Now in other lessons in this section of the course and depending on what course you're taking I will be talking about many of these options, what you can modify and which actions you can perform on RDS instances.

But for now we're just going to move on with this demo.

So the next step is that we need to migrate our existing data into this RDS instance.

So what we're going to do is to click on the Connectivity and Security tab and we're going to leave this open.

We're going to need this endpoint name and port very shortly.

You should still have a tab open to the EC2 console.

If you don't you can reach that by going on Services and then opening EC2 in a new tab.

But I want you to select the A4L-WordPress instance and then right click and connect to it using Instance Connect.

So go ahead and do that.

Once you've done that we're going to start referring to the lesson commands document.

So make sure you've got that open.

We're going to use this command to take a backup of the existing MariaDB database.

So we need to replace a placeholder.

What we need to do is delete this and replace it with the private IP address of the MariaDB EC2 instance.

So go back to the EC2 console, select the DB-WordPress instance and copy the private IP version 4 address into your clipboard.

And then let's move back to the WordPress instance and paste that in.

Go ahead and press Enter and it will prompt you for the password.

And the password is the same Animals for Life strong password that we've been using everywhere.

Copy that into your clipboard.

So this is the password for the A4L WordPress user on the MariaDB EC2 instance.

So paste that in and press Enter and then LS-LA to confirm that we now have this A4L WordPress.SQL database backup file.

And we do, so that's good.

So as we did in the previous demo lesson, we're going to take this backup file and we're going to import it into the new destination database, which is going to be the RDS instance.

To do that, we'll use this command, but we're going to need to replace the placeholder hostname with the CNAME of the RDS instance.

So go ahead and delete this placeholder, then go back to the RDS console and I'll want you to copy the endpoint name into your clipboard.

So select it, right click and then copy.

We won't need the port number because this is the standard MySQL port and if you don't specify it, most applications will assume this default.

So just make sure that you have the endpoint DNS name or endpoint CNAME in your clipboard.

And then back on the WordPress EC2 instance, go ahead and paste this database name into this command and press Enter.

And again, you'll be asked for the password and that's the same Animals for Life strong password.

So copy that into your clipboard, paste that in and press Enter.

And that's imported this A4LWordPress.SQL file into the RDS instance.

So now we need to follow the same process and change WordPress so that it points at the RDS instance.

And we do that by moving to where the WordPress configuration file is.

So cd space forward slash var forward slash ww w forward slash html and press Enter.

And then shudu.

So we have admin privileges, nano, which is a text editor and then wp-config.php and press Enter.

Then we need to scroll down and we're looking for where it says DB host and currently it has a host name here.

Now if you go back to the EC2 console and you look at the A4L-DB-WordPress instance, you'll see that its private IP version for DNS name is what's listed inside this configuration item.

So it's currently pointing at this dedicated database instance.

What we need to do is replace that and we're going to replace it with the RDS database DNS name or the CNAME of this RDS instance.

So copy that into your clipboard and then go ahead and delete this private DNS name for the MariaDB EC2 instance and then paste in the RDS endpoint name, also known as the RDS CNAME.

Once you've done that, control O and Enter to save and control X to exit.

And now our WordPress instance is pointing at the RDS instance for its database.

Now we can verify that by checking WordPress, move back to instances, select the WordPress instance, copy the public IP version for addressing to your clipboard.

Don't use this open address link.

Open that in a new tab.

Go ahead and just click on the best cats ever to verify the functionality and it does look as though it's working.

And to verify that, if we go back to the EC2 console, select the A4L-DB-WordPress instance and right click and then stop that instance.

Now the original database that was providing database services to WordPress is going to move into a stopped state.

And if our WordPress blog continues functioning, we know that it's using the RDS instance.

So let's keep refreshing and wait for this to change into a stopped state.

There we go.

It's stopped.

And if we go back to our WordPress page and refresh, it still loads.

And so we know that it's now using RDS for its database services.

So at this point, that's everything that I wanted you to do in this demo lesson.

You've stepped through the process of provisioning an RDS instance.

So you've created a subnet group, provisioned the instance itself, explored the functionality of the instance, including how to provide access to it by selecting a security group.

And then editing that security group to allow access.

You've performed a database migration and you've explored how the RDS instance is presented in the console.

So that's everything that you need to do within this demo lesson.

And don't worry, we're going to be exploring much more of the advanced functionality of RDS as we move through this section of the course.

For now, though, I want us to clear up the infrastructure that we've created as part of this demo lesson.

Now, because we've provisioned RDS manually outside of CloudFormation, unfortunately, there is a little bit more manual work involved in the cleanup.

So I want you to go to the RDS console, move to databases, select this database, click on actions, and then select delete.

Now it will prompt you to create a final snapshot and we're not going to do that.

We're not going to retain automated backups and so you'll need to acknowledge that upon instance deletion, automated backups including any system snapshots and pointing time recoveries will no longer be available.

And don't worry, I'll be talking about backups and recovery in another lesson in this section of the course.

For now, just acknowledge that and then type delete me into this box and confirm the deletion.

Now this deletion is going to take a few minutes.

It's not an immediate process.

It will start in a deleting state and we need to wait for this process to be completed before we continue the cleanup.

So go ahead and pause this video and wait for this instance to fully delete before continuing.

Now that the instance has been deleted, it vanishes from this list.

Next, we need to delete the subnet group that we created earlier.

So click on subnet groups, select the subnet group and then delete it.

You'll need to confirm that deletion.

Once done, it too should vanish from that list.

Next, go to the tab you've got open to the VPC console, scroll down and select security groups.

Now look through this list and locate the security group that you created as part of provisioning the RDS instance.

It should be called a4LVPC-RDS-SG.

Select that, click on actions and then delete security group and you'll need to confirm that process as well.

Once that's deleted, the final step is to go to the cloud formation console and then you'll need to delete the cloud formation stack that was created using the one-click deployment at the start of the demo.

It should be called migrate to RDS.

Select it, click on delete and confirm that deletion.

And once deleted, the account will be returned into the same state as it was at the start of the demo lesson.

So all of the infrastructure that we've used will be removed from the account and the account will be in the same state as at the start of the demo.

Now I hope you've enjoyed this demo and that we're repeating the same WordPress installation and then the creation of the blog post over and over again.

But I want you to get used to different parts of this process over and over again.

You need to know why not to use a database on EC2.

You need to know why not to perform a lot of these processes manually.

From this point onward in the course, we're going to be using RDS to evolve our WordPress design into something that is truly elastic.

And so all of these processes, the things I'm having you repeat are really useful to aid in your understanding of all of these different components.

So from this point onward, we're going to be automating the creation of RDS and focusing on the specific pieces of functionality that you need to understand.

But at this point, that's everything that you need to do in this demo.

So go ahead, complete the video and when you're ready, I look forward to you joining me in the next.

Author response:

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

Reviewer #4

We sincerely appreciate the time and effort you have taken to review our manuscript. We followed your recommendations to polish the text and make it easier to understand.

Regarding terms and terminology, we changed “non-breeding” everywhere in the text to “over- wintering.”

Regarding the title, as it was suggested by reviewer #1 as his recommendation, we tried to find a compromise and make the changes you suggested but left part of the suggestion from reviewer #1. So, now it’s “Foxtrot migration and dynamic over-wintering range of an arctic raptor”

Thank you for highlighting the importance of snow cover and changes in snow cover as a possible factor of over-wintering movements. We appreciate your feedback and have explored several approaches to address this issue. Specifically, we examined how both snow cover extent and changes in snow cover influenced movement distance. However, we found no effect of either factor on movement distance.

Our data show that birds leave their sites in October and move southwest, even though snow cover is minimal at that time. They also leave their sites in November and in subsequent months, regardless of the snow cover levels. Thus, we observed no pattern of birds leaving sites when snow cover reaches a specific threshold (e.g., 75-80%). Similarly, we found no evidence of birds staying in areas with a certain snow cover extent (e.g., 30%), nor did they leave sites when snow cover increased by a specific amount (e.g., by 10 or 20%).

It is possible that more experienced birds anticipate that October plots will become inaccessible later in the winter and, therefore, leave early without waiting for significant snow accumulation. Alternatively, other factors, such as brief heavy snowfalls, may trigger movement, even if these do not lead to sustained increases in snow cover. Multiple factors, possibly acting asynchronously, could also play a role. This complexity adds an interesting dimension to the study of ecological patterns. However, in this study, we chose to focus on describing the migration pattern itself and its impact on aspects like over-winter range determination and population dynamics. While we have prioritized this approach, we remain committed to further analyzing the data to uncover additional details about this behavior.

In response to your suggestion, we have expanded the Methods sections to clarify that we tested the effects of snow cover and changes in snow cover on distance (Lines 241-246); the Results section (Lines 348-349). We have also included the relevant plots in the Supplementary Materials. In the Discussion, we noted that this approach did not reveal any significant dependence and acknowledged that this issue requires further investigation (Lines 422-459).

---------

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

Reviewer #2:

We sincerely appreciate the time and effort you have taken to review our manuscript. 

First of all, we apologize for publishing the preprint without incorporating certain adjustments outlined in our earlier response, particularly in the Methods section. This was due to an oversight regarding the different versions of the manuscript. We have corrected this mistake. Our response to the feedback on this section (Methods), with line numbers of the changes made, is immediately below this response. In addition, we have included the units of measurement (mean and standard deviation) in both the results and figure captions for clarity.

To focus on the main point regarding wintering strategies, we acknowledge that in the previous versions, this aspect was inadequately addressed and caused some confusion. In the revised edition, both the Introduction and the Discussion have been thoroughly reworked.

As you suggested, we have removed the long introductory paragraph and all references to foxtrot migrations from the Introduction. As a result, the Introduction is now short and to the point. In the second paragraph, we explain why we propose the wintering strategies outlined (L74-81).

In the Discussion, we've added a substantial new section at the beginning that discusses different wintering strategies. We have also updated Figure 4 accordingly. Previously, we erroneously suggested that Montagu's harrier and other African-Palaearctic migrants might adopt wintering strategies similar to those we describe. Upon further investigation, however, we found that almost all African-Palaearctic migrants exhibit an itinerant wintering strategy. Conversely, the strategy we describe is primarily observed in mid-latitude wintering species.

We have shown that, unlike itinerancy, the birds in our study don't pause for 1-2 months at multiple non-breeding sites, but instead migrate significant distances, up to 1000 km, throughout the winter. Furthermore, unlike itinerancy, the sites they reach are consistently snow-free throughout the year. Following the logic of publications on Montagu's harriers (Schlaich et al. 2023), our birds do not wait for favorable conditions at the next site, as is typical of itinerancy. Moreover, this behavior is influenced by external factors such as snow cover dynamics and occurs primarily in mid-latitudes. Researchers studying a species similar to our subject, the Common buzzard, observed a similar pattern and termed it "prolonged autumn migration" rather than itinerancy. Although their transmitters stopped working in mid-winter, precluding a full observation of the annual cycle, they captured the essence of continued migration at a slower pace, distinct from itinerancy. We've detailed all of these findings in a new section.

In addition, we acknowledge the mischaracterization of the implications of our research as ‘Conservation implications’ and have corrected this to ‘Mapping ranges and assessing population trends’, as you suggested.

Finally, we've rewritten the Conclusion, removing overly grandiose statements and simply summarizing the main findings.

We appreciate your time and effort in reviewing our manuscript. With your invaluable input, it has become clearer, more concise, and easier to understand.

Dataset: unclear what is the frequency of GPS transmissions. Furthermore, information on relative tag mass for the tracked individuals should be reported.

We have included this information in our manuscript (L 115-122). We also refer to the study in which this dataset was first used and described in detail (L 123).

Data pre-processing: more details are needed here. What data have been removed if the bird died? The entire track of the individual? Only the data classified in the last section of the track? The section also reports on an 'iterative procedure' for annotating tracks, which is only vaguely described. A piecewise regression is mentioned, but no details are provided, not even on what is the dependent variable (I assume it should be latitude?).

Regarding the deaths, we only removed the data when the bird was already dead. We estimated the date of death and excluded tracking data corresponding to the period after the bird's death. We have corrected the text to make this clear (L 130-131).

Regarding the piecewise regression. We have added a detailed description on lines 136-148.

Data analysis: several potential issues here:

(1) Unclear why sex was not included in all mixed models. I think it should be included.

Our dataset contains 35 females and eight males (L116). This ratio does not allow us to include sex in all models and adequately assess the influence of this factor. At the same time, because adult females disperse farther than males in some raptor species, we conducted a separate analysis of the dependence of migration distance on sex (Table S8) and found no evidence for this in our species. We have written about that in the Methods (L177-181) and after in the Results (L277-278).

(2) Unclear what is the rationale of describing habitat use during migration; is it only to show that it is a largely unsuitable habitat for the species? But is a formal analysis required then? Wouldn't be enough to simply describe this?

Habitat use and snow cover determine the two main phases (quick and slow) of the pattern we describe. We believe that habitat analysis is appropriate in this case, and a simple description would be uninformative and not support our conclusions.

(3) Analysis of snow cover: such a 'what if' analysis is fine but it seems to be a rather indirect assessment of the effect of snow cover on movement patterns. Can a more direct test be envisaged relating e.g. daily movement patterns to concomitant snow cover? This should be rather straightforward. The effectiveness of this method rests on among-year differences in snow cover and timing of snowfall. A further possibility would be to demonstrate habitat selection within the entire non-breeding home range of an individual in relation snow cover. Such an analysis would imply associating presenceabsence of snow to every location within the non-breeding range and testing whether the proportion of locations with snow is lower than the proportion of snow of random locations within the entire nonbreeding home range (95% KDE) for every individual (e.g. by setting a 1/10 ratio presence to random locations).

The proposed analysis will provide an opportunity to assess whether the Rough-legged buzzard selects areas with the lowest snow cover, but will not provide an opportunity to follow the dynamics and will therefore give a misleading overall picture. This is especially true in the spring months. In March-April, Rough-legged buzzards move northeast and are in an area that is not the most open to snow. At this time, areas to the southwest are more open to snow (this can be seen in Figure 3b). If we perform the proposed analysis, the control points for this period would be both to the north (where there is more snow) and to the south (where there is less snow) from the real locations, and the result would be that there is no difference in snow cover. 

A step-selection analysis could be used, as we did in our previous work (Curk et al 2020 Sci Rep) with the same Rough-legged buzzards (but during migration, not winter). But this would only give us a qualitative idea, not a quantitative one - that Rough-legged Buzzards move from snow (in the fall) and follow snowmelt progression (in the spring). 

At the same time, our analysis gives a complete picture of snow cover dynamics in different parts of the non-breeding range. This allows us to see that if Rough-legged buzzards remained at their fall migration endpoint without moving southwest, they would encounter 14.4% more snow cover (99.5% vs. 85.1%). Although this difference may seem small (14.4%), it holds significance for rodent-hunting birds, distinguishing between complete and patchy snow cover.

Simultaneously, if Rough-legged buzzards immediately flew to the southwest and stayed there throughout winter, they would experience 25.7% less snow cover (57.3% vs. 31.6%). Despite a greater difference than in the first case, it doesn't compel them to adopt this strategy, as it represents the difference between various degrees of landscape openness from snow cover.

kalo di notal "pointer to node"

ok intinya ini pake tag node biar bisa memberi instruksi ke preprocessor membuat struct baru node dengan Address yang beda-beda.

pada umumnya itu typedef Node (cara aksesnya)

cuman karna ini pake tag, instead of mengubah, kita membuat baru dengan address yang bisa beda beda

ini typedef struct node* Address (perhatikan bahwa struct di tulis juga)

ini tag

tag dan type nya dibedakan (node dan Node) untuk mengatasi strictnya bahasa C dimana tipe tidak bisa di declare setelah operasi lain. dibuatlah jadi tag, im not to sure so ask again ke GPT

Author response:

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

We are grateful to the reviewers for their positive assessment of the revised version of the article.

Please find below our answers to the last, minor comments of the reviewers.

We thank the reviewer for this important comment. In our live imaging experiments, we actually tracked the dorsal and ventral borders of the omp:yfp positive clusters in control and sly mutant embryos. These measurements showed that the omp:yfp positive clusters are more elongated along the DV axis in mutants as compared with control siblings, as seen on fixed samples (data not shown), suggesting that this difference in tissue shape is not due to fixation.

Reviewer #4 (Public review):

Summary:

In this elegant study XX and colleagues use a combination of fixed tissue analyses and live imaging to characterise the role of Laminin in olfactory placode development and neuronal pathfinding in the zebrafish embryo. They describe Laminin dynamics in the developing olfactory placode and adjacent brain structures and identify potential roles for Laminin in facilitating neuronal pathfinding from the olfactory placode to the brain. To test whether Laminin is required for olfactory placode neuronal pathfinding they analyse olfactory system development in a well-established laminin-gamma-1 mutant, in which the laminin-rich basement membrane is disrupted. They show that while the OP still coalesces in the absence of Laminin, Laminin is required to contain OP cells during forebrain flexure during development and maintain separation of the OP and adjacent brain region. They further demonstrate that Laminin is required for growth of OP neurons from the OP-brain interface towards the olfactory bulb. The authors also present data describing that while the Laminin mutant has partial defects in neural crest cell migration towards the developing OP, these NCC defects are unlikely to be the cause of the neuronal pathfinding defects upon loss of Laminin. Altogether the study is extremely well carried out, with careful analysis of high-quality data. Their findings are likely to be of interest to those working on olfactory system development, or with an interest in extracellular matrix in organ morphogenesis, cell migration, and axonal pathfinding.

Strengths:

The authors describe for the first time Laminin dynamics during the early development of the olfactory placode and olfactory axon extension. They use an appropriate model to perturb the system (lamc1 zebrafish mutant), and demonstrate novel requirements for Laminin in pathfinding of OP neurons towards the olfactory bulb.

The study utilises careful and impressive live imaging to draw most of its conclusions, really drawing upon the strengths of the zebrafish model to investigate the role of laminin in OP pathfinding. This imaging is combined with deep learning methodology to characterise and describe phenotypes in their Laminin-perturbed models, along with detailed quantifications of cell behaviours, together providing a relatively complete picture of the impact of loss of Laminin on OP development.

Weaknesses:

Some of the statistical tests are performed on experiments where n=2 for each condition (for example the measurements in Figure S2) - in places the data is non-significant, but clear trends are observed, and one wonders whether some experiments are under-powered.

We initially planned the electron microscopy experiments in order to analyse 3 embryos per genotype per stage. However, because of technical issues we could not perform the measurements in all the cases, explaining why we have n = 2 in some of the graphs. The trends were quite clear, so we chose to keep these data in the article. We believe they nicely complement the immunostaining data assessing basement membrane integrity in control and mutant embryos.

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

Public Reviews: 

Reviewer #1 (Public Review): 

Summary: 

The authors describe the dynamic distribution of laminin in the olfactory system and forebrain. Using immunohistochemistry and transgenic lines, they found that the olfactory system and adjacent brain tissues are enveloped by BMs from the earliest stages of olfactory system assembly. They also found that laminin deposits follow the axonal trajectory of axons. They performed a functional analysis of the sly mutant to analyse the function of laminin γ1 in the development of the zebrafish olfactory system. Their study revealed that laminin enables the shape and position of placodes to be maintained late in the face of major morphogenetic movements in the brain, and its absence promotes the local entry of sensory axons into the brain and their navigation towards the olfactory bulb. 

Strengths: 

- They showed that in the sly mutants, no BM staining of laminin and Nidogen could be detected around the OP and the brain. The authors then elegantly used electron microscopy to analyse the ultrastructure of the border between the OP and the brain in control and sly mutant conditions. 

- To analyse the role of laminin γ1-dependent BMs in OP coalescence, the authors used the cluster size of Tg(neurog1:GFP)+ OP cells at 22 hpf as a marker. They found that the mediolateral dimension increased specifically in the mutants. However, proliferation did not seem to be affected, although apoptosis appeared to increase slightly at a later stage. This increase could therefore be due to a dispersal of cells in the OP. To test this hypothesis, the authors then analysed the cell trajectories and extracted 3D mean square displacements (MSD), a measure of the volume explored by a cell in a given period of time. Their conclusion indicates that although brain cell movements are increased in the absence of BM during coalescence phases, overall OP cell movements occur within normal parameters and allow OPs to condense into compact neuronal clusters in sly mutants. The authors also analysed the dimensions of the clusters composed of OMP+ neurons. Their results show an increase in cluster size along the dorso-ventral axis. These results were to be expected since, compared with BM, early neurog1+ neurons should compact along the medio-lateral axis, and those that are OMP+ essentially along the dorso-ventral axis. In addition to the DV elongation of OP tissue, the authors show the existence of isolated and ectopic (misplaced) YFP+ cells in sly mutants. 

- To understand the origin of these phenotypes, the authors analysed the dynamic behaviour of brain cells and OPs during forebrain flexion. The authors then quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, and proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected. 

- They then analysed the dynamic behaviour of the axon using live imaging. Thus, olfactory axon migration is drastically impaired in sly mutants, demonstrating that Laminin γ1dependent BMs are essential for the growth and navigation of axons from the OP to the olfactory bulb. 

- The authors therefore performed a quantitative analysis of the loss of function of Laminin γ1. They propose that the BM of the OP prevents its deformation in response to mechanical forces generated by morphogenetic movements of the neighbouring brain. 

Weaknesses: 

- The authors did not analyse neurog1 + axonal migration at the level of the single cell and instead made a global analysis. An analysis at the cell level would strengthen their hypotheses.  

- Rescue experiments by locally inducing Laminin expression would have strengthened the paper. 

- The paper lacks clarity between the two neuronal populations described (early EONs and late OSNs).  

- The authors quantitatively measured brain versus OPs in the sly mutant and found that the OP-brain boundary was poorly defined in the sly mutant compared with the control. Once again, the methods (cell tracks, brain size, proliferation/apoptosis, and the shape of the brain/OP boundary) are elegant but the results were expected. 

- A missing point in the paper is the effect of Laminin γ1 on the migration of cranial NCCs that interact with OP cells. The authors could have analysed the dynamic distribution of neural crest cells in the sly mutant. 

We thank the reviewer for the overall positive assessment of our work, and we carefully responded to all her/his insightful comments below. Live imaging experiments to (1) visualise exit and entry point formation with only a few axons labelled, (2) characterise the behaviour of single neurog1:GFP-positive neurons/axons during OP coalescence and to (3) analyse the migration of cranial NCC are now included in the revised manuscript to address the reviewer’s questions, and reinforce our initial conclusions.

Reviewer #2 (Public Review): 

Summary: 

This manuscript addresses the role of the extracellular matrix in olfactory development. Despite the importance of these extracellular structures, the specific roles and activities of matrix molecules are still poorly understood. Here, the authors combine live imaging and genetics to examine the role of laminin gamma 1 in multiple steps of olfactory development. The work comprises a descriptive but carefully executed, quantitative assessment of the olfactory phenotypes resulting from loss of laminin gamma. Overall, this is a constructive advance in our understanding of extracellular matrix contributions to olfactory development, with a well-written Discussion with relevance to many other systems. 

Strengths: 

The strengths of the manuscript are in the approaches: the authors have combined live imaging, careful quantitative analyses, and molecular genetics. The work presented takes advantage of many zebrafish tools including mutants and transgenics to directly visualize the laminin extracellular matrix in living embryos during the developmental process. 

Weaknesses: 

The weaknesses are primarily in the presentation of some of the imaging data. In certain cases, it was not straightforward to evaluate the authors' interpretations and conclusions based on the single confocal sections included in the manuscript. For example, it was difficult to assess the authors' interpretation of when and how laminin openings arise around the olfactory placode and brain during olfactory axon guidance. 

We thank the reviewer for the overall positive assessment of our work, and we carefully responded to all her/his insightful comments below. To address these comments, live imaging data to visualise exit and entry point formation with a sparse labelling of axons, and z-stacks showing how exit and entry points are organised in 3D, have been added to the revised manuscript.

Reviewer #3 (Public Review): 

This is a beautifully presented paper combining live imaging and analysis of mutant phenotypes to elucidate the role of laminin γ1-dependent basement membranes in the development of the zebrafish olfactory placode. The work is clearly illustrated and carefully quantified throughout. There are some very interesting observations based on the analysis of wild-type, laminin γ1, and foxd3 mutant embryos. The authors demonstrate the importance of a Laminin γ1-dependent basement membrane in olfactory placode morphogenesis, and in establishing and maintaining both boundaries and neuronal connections between the brain and the olfactory system. There are some very interesting observations, including the identification of different mechanisms for axons to cross basement membranes, either by taking advantage of incompletely formed membranes at early stages, or by actively perforating the membrane at later ones. 

This is a valuable and important study but remains quite descriptive. In some cases, hypotheses for mechanisms are stated but are not tested further. For example, the authors propose that olfactory axons must actively disrupt a basement membrane to enter the brain and suggest alternative putative mechanisms for this, but these are not tested experimentally. In addition, the authors propose that the basement membrane of the olfactory placode acts to resist mechanical forces generated by the morphogenetic movement of the developing brain, and thus to prevent passive deformation of the placode, but this is not tested anywhere, for example by preventing or altering the brain movements in the laminin γ1 mutant. 

We thank the reviewer for the overall positive assessment of our work and for suggesting interesting experiments to attempt in the future, and we carefully responded to all her/his constructive comments below.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

In general, it would be easier to draw conclusions and compare data if the authors used similar stages throughout the article. 

Throughout the article we tried to focus on a series of stages that cover both the coalescence of the OP (up to 24 hpf) and later stages of olfactory system development spanning the brain flexure process (28, 32, 36 hpf). However, for technical reasons it was not always possible to stick to these precise stages in some of our experiments. Also, in Fig. 1E-J, we picked in the movies some images illustrating specific cell or axonal behaviours, and thus the corresponding stages could not match exactly the stage series used in Fig. 1A-D and elsewhere in the article. Nevertheless, this stage heterogeneity does not affect our main conclusions.

It would be useful to schematise the olfactory placode and the brain in an insert to clearly visualise the system in each figure. 

We hope that the schematic which was initially presented in Fig. 1K already helps the reader to understand how the system is organised. Although we have not added more schematic views to represent the system in each figure (we think this would make the figures overcrowded), we have added additional legends to point to the OP and the brain in the pictures in order to clarify the localisation of each tissue.

In the Summary, the authors refer to the integrity of the basement membrane. I don't think there is any attempt to affect basement membrane integrity in the article. It would be important to do so to look at the effect on CNS-PNS separation and axonal elongation. 

In the Summary, we use the term « integrity of the basement membrane » to mention that we have analysed this integrity in the sly mutant. Given the results of our immunostainings against three main components of the basement membrane (Laminin, Collagen IV and Nidogen), as well as our EM observations, we see the sly mutant as a condition in which the integrity of the basement membrane is strongly affected.

Rescue experiments by locally inducing Laminin expression would have strengthened the paper. 

We have attempted to rescue the sly mutant phenotypes by introducing the mutation in the transgenic TgBAC(lamC1:lamC1-sfGFP) background, in which Laminin γ1 tagged with sfGFP is expressed under the control of its own regulatory sequences (Yamaguchi et al., 2022). To do so, we crossed sly+/-;Tg(omp:yfp) fish with sly+/-; Tg(lamC1:LamC1-sfGFP) fish. Surprisingly, while a rescue of the global embryo morphology was observed, no clear rescue of the olfactory system defects could be detected at 36 hpf. This could be due to the fact that the expression level of LamC1-sfGFP obtained with one copy of the transgene is not sufficient to rescue the olfactory system phenotypes, or that the sfGFP tag specifically affects the function of the Laminin 𝛾1 chain during the development of the olfactory system, making it unable to rescue the defects. Given the results of our first attemps, we decided not to continue in this direction.

(1) Developing OP & brain are surrounded by laminin-containing BM (already described by Torrez-Pas & Whitlock in 2014). 

"we first noticed the appearance of a continuous Laminin-rich BM surrounding the brain from 14-18 hpf, while around the OP, only discrete Laminin spots were detected at this stage (Fig. 1A, A'). " 

Around 8ss for Torrez-Pas & Whitlock (before 14 hpf). Can you modify the text, or show an 8ss stage embryo? As far as I know, the authors do not show images at 14hpf. Please correct this sentence or show a 14 hpf picture. 

The reviewer is right, we do not show any 14 hpf stage in the images and thus have removed this stage in the text and replaced it by 17 hpf.

In Figure 1A, the labelling of laminin 111 does not appear to be homogeneous along the brain.

Is this true? 

At this stage the brain’s BM revealed by the Laminin immunostaining appears fairly continuous (while the OP’s one is clearly dotty and less defined), but indeed very tiny/local interruptions of the signal can been seen along the structure as detected by the reviewer. We thus modified the text to mention these tiny interruptions.

How is the Laminin antibody used by the authors specific to laminin 111?  

We thank the reviewer for raising this important point. The immunogen used to produce this rabbit polyclonal antibody is the Laminin protein isolated from the basement membrane of a mouse Engelbreth Holm-Swarm sarcoma (EHS). It is thus likely to recognise several Laminin isoforms and not only Laminin 111. We thus replaced Laminin 111 by Laminin when mentioning this antibody in the text and Figures.

Please schematise in Figure 1K the stages you have tested and shown here in the article i.e. stages 18 - 22 - 28 -36 hpf using immunohistochemistry and 17-26-27-29-33 and 38 hpf using transgenics for laminin 111 and LamC1 respectively.  

As suggested by the reviewer, we changed the stages in the schematics for stages we have presented in Figure 1 (analysed either with immunostaining or in live imaging experiments). We chose to represent 17 - 22 - 26 - 33 hpf (and thus adapted some of the schematics for them to match these stages).  

Please specify in the Figure 1 legend for panels A to D whether this is a 3D projection or a zsection.

We indicated in the Figure 1 legend that all these images are single z-sections (as well as for panels E-J).

Furthermore, the schematisation in Fig. 1K does not reflect what the authors show: at 22 hpf laminin 111 labelling appears to be present only near the brain, and no labelling lateral to the olfactory placode and anteriorly and posteriorly. Thus, the schematisation in Figure 1K needs to be modified to reflect what the authors show.

We agree with the reviewer that the Laminin staining at this stage is observed around the medial region of the OP, but not more laterally. We modified the schematic view accordingly in Figure 1K. Anterior and posterior sides of the OP are not represented in this schematic because we chose to represent a frontal view rather than a dorsal view.

The authors suggest that" the laminin-rich BM of OP assembles between 18 and 22 hpf, during the late phase of OP coalescence". However, their data indicate that this BM assembles around 28hpf (Figure 1C). Can they clarify this point?

What we meant with this sentence is that we cleary see two distinct BMs from 22 hpf. However, as noticed by the reviewer, the OP’s BM is only present around the medial/basal regions of the OP and does not surround the whole OP tissue at this stage. We modified the text to clarify this point (in particular by mentioning that the OP’s BM starts to assemble between 18 and 22 hpf), and replaced the image shown in Figure 1B, B’ with a more representative picture (the previous z-section was taken in very dorsal regions of the OP).

It would be useful to disrupt these cells that have a cytoplasmic expression of Laminin-sfGFP, to analyse their contribution to BM and OP coalescence.

Indeed it will be interesting in the future to test specifically the role of the cells expressing cytoplasmic Laminin-sfGFP around and within the OP, as proposed by the reviewer. Laser ablation of these cells could be attempted, but due to their very superficial localisation, close to the skin, we believe these ablations (with the protocol/set-up we currently use in the lab) would impair the skin integrity, preventing us to conclude. We consider that the optimisation of this experiment is out of the scope of the present work.

Tg(-2.0ompb:gapYFP)rw032 marks ciliated olfactory sensory neurons (OSNs) (Sato et al., 2005). The authors should mention this. 

Please see our detailed response to the next point below.

Points to be clarified: 

-Tg(-2.0ompb:gapYFP)rw032 marks ciliated olfactory sensory neurons (OSNs) (Sato et al., 2005). The authors should mention this here. Moreover, the authors refer to "OP neurons" throughout the article. In the development of the olfactory organ, two types of neurons have been described in the literature: early EONs (12hpf-26hpf) and later OSNs. Each could have a specific role in the establishment and maintenance of the BM described by the authors. The authors need to clarify this point as, in Figure 1 for example, they use a marker for Tg(neurog1:GFP) EONs and a marker for ciliated OSNs without distinction. The distinction between EONs and OSNs comes a little late in the text and should be placed higher up. 

As mentioned by the reviewer, according to the initial view of neurogenesis in the OP, OP neurons are born in two waves. A transient population of unipolar, dendrite-less pioneer neurons would differentiate first, in the ventro-medial region of the OP and elongate their axons dorsally out of the placode, along the brain wall. These pioneer axons would then be used as a scaffold by later born OSNs located in the dorso-lateral rosette to outgrow their axons towards the olfactory bulb (Whitlock and Westerfield, 1998). 

Another study further characterised OP neurogenesis and showed that the first neurons to differentiate in the OP (the early olfactory neurons or EONs) express the Tg(neurog1:GFP) transgene (Madelaine et al., 2011). As mentioned by the authors in the discussion of this article, neurog1:GFP+ neurons appear much more numerous than the previously described pioneer neurons, and may thus include pioneers but also other neuronal subtypes.

We would like here to share additional, unpublished observations from our lab that further suggest that the situation is more complex than the pioneer/OSN and EON/OSN nomenclatures. First, in many of our live imaging experiments, we can clearly visualise some neurog1:GFP+ unipolar neurons, initially located in a medial position in the OP, which intercalate and contribute to the dorsolateral rosette (where OSNs are proposed to be located) at the end of OP coalescence, from 22-24 hpf. Second, in fixed tissues, we observed that most neurog1:GFP+ neurons located in the rosette at 32 hpf co-express the Tg(omp:meRFP) transgene (Sato et al., 2005). These observations suggest that at least a subpopulation of neurog1:GFP+ neurons could incorporate in the dorsolateral rosette and become ciliated OSNs during development. We can share these results with the reviewer upon request. Further studies are thus needed to clarify and describe the neuronal subpopulations and lineage relationships in the OP, but this detailed investigation is out of the scope and focus of the present study. 

An additional complication comes from the fact that, as shown and acknowledged by the authors in Miyasaka et al., 2005, the Tg(omp:meYFP) line (6kb promoter) labels ciliated OSNs in the rosette but also some unipolar, ventral neurons (around 10 neurons at 1 dpf, Miyasaka et al. 2005, Figure 3A, white arrowheads). This was also observed using the 2 kb promoter Tg(omp:meYFP) line (see for instance Miyasaka et al., 2007) and in our study, we can indeed detect these ventro-medial neurons labelled in the Tg(omp:meYFP) line (2 kb promoter), see for instance Figure 1C’, D’ or Movie 6. It is unclear whether these unipolar omp:meYFPpositive cells are pioneer neurons or EONs expressing the omp:meYFP transgene, or OSN progenitors that would be located basally/ventrally in the OP at these stages.

For all these reasons, we decided to present in the text the current view of neurogenesis in the OP but instead of attributing a definitive identity to the neurons we visualise with the transgenic lines, we prefer to mention them in the manuscript (and in the rest of the response to the reviewers) as neurons expressing neurog1:GFP or omp:meYFP transgenes (or cells/axons/neurons expressing RFP in the Tg(cldnb:Gal4; UAS:RFP) background).

What we also changed in the text to be more clear on this point:

- we moved higher up in the text, as suggested by reviewer 1, the description of the current model of neurogenesis in the OP,

- we mentioned that neurog1:GFP+ neurons are more numerous than the initially described pioneer neurons, as discussed in Madelaine et al., 2011,

- we wrote more clearly that the Tg(omp:meYFP) line labels ciliated OSNs but also a subset of unipolar, ventral neurons (Miyasaka et al., 2005), and pointed to these ventral neurons in Figure 1C’, D’,

- in the initial presentation of the current view of OP neurogenesis we renamed neurog1:GFP+ into EONs to be coherent with Madelaine et al., 2011.

- To visualise pioneer axons, the authors should use an EONS marker such as neurog1 because, to my knowledge, OMP only marks OSN axons and not pioneer axons.  

To visualise neurog1:GFP+ axons during OP coalescence, we performed live imaging upon injection of the neurog1:GFP plasmid (Blader et al., 2003) in the Tg(cldnb:Gal4; UAS:RFP) background (n = 4 mutants and n = 4 controls from 2 independent experiments). We observed some GFP+ placodal neurons exhibiting retrograde axon extension in both controls and sly mutants. In such experiments it is very difficult to quantify and compare the number of neurons/axons showing specific behaviours between different experimental conditions/genetic background. Indeed, due to the cytoplasmic localisation of GFP, the axons can only be seen in neurons expressing high levels of GFP, and due to the injection the number of such neurons varies a lot in between embryos, even in a given condition. Nevertheless, our qualitative observations reinforce the idea that the basement membrane is not absolutely required for mediolateral movements and retrograde axon extension of neurog1:GFP+ neurons in the OP. We added examples of images extracted from these new live imaging experiments in the revised Fig. S5A, B.

- The authors should analyse the presence of laminin in the OP and forebrain in conjunction with neural crest cell dynamics (using a Sox10 transgenic line for example) to refine their entry and exit point hypotheses. 

As described in the answer to the next point, we performed new experiments in which we visualised NCC migration in the Tg(neurog1:GFP) background, which allowed us to analyse the localisation of NCC at the forebrain/OP boundary, in ventral and dorsal positions, both in sly mutant embryos and control siblings.

- A dynamic analysis of the distribution of neural crest cells in the sly mutant over time and during OP coalescence would be important. 

The dynamics of zebrafish cranial NCC migration in the vicinity of the OP has been previously analysed using sox10 reporter lines (Harden et al., 2012, Torres-Paz and Whitlock, 2014, Bryan et al., 2020). To address the point raised by the reviewer, we performed live imaging from 16 to 32 hpf on sly mutants and control siblings carrying the Tg(neurog1:GFP) and Tg(UAS:RFP) transgenes and injected with a sox10(7.2):KalTA4 plasmid (Almeida et al., 2015). This allows the mosaic labelling of cells that express or have expressed sox10 during their development which, in the head region at these stages, represents mostly NCC and their derivatives. 3 independent experiments were carried out (n = 4 mutant embryos in which 8 placodes could be analysed; n = 6 control siblings in which 10 placodes could be analysed). A new movie (Movie 9) has been added to the revised article to show representative examples of control and mutant embryos.

From these new data, we could make the following observations:

- As expected from previous studies (Harden et al., 2012, Torres-Paz and Whitlock, 2014, Bryan et al., 2020), in control embryos a lot of NCC had already migrated to reach the vicinity of the OP when the movies begin at 16 hpf, and were then seen invading mainly the interface between the eye and the OP (10/10 placodes). Surprisingly, in sly mutants, a lot of motile NCC had also reached the OP region at 16 hpf in all the analysed placodes (8/8), and populated the eye/OP interface in 7/8 placodes (10/10 in controls). Counting NCC or tracking individual NCC during the whole duration of the movies was unfortunately too difficult to achieve in these movies, because of the low level of mosaicism (a high number of cells were labelled) and of the high speed of NCC movements (as compared with the 10 min delta t we chose for the movies). 

- in some of the control placodes we could detect a few NCC that populated the forebrain/OP interface, either ventrally, close to the exit point of the axons (4/10 placodes), or more dorsally (8/10 placodes). By contrast, in sly mutants, NCC were observed in the dorsal region of the brain/OP boundary in only 2/8 placodes, and in the ventral brain/OP frontier in only 2/8 placodes as well. Interestingly, in these 2 last samples, NCC that had initially populated the ventral region of the brain/OP interface were then expelled from the boundary at later stages.

We reported these observations in a new Table that is presented in revised Fig. S6B. In addition, instances of NCC migrating at the eye/OP or forebain/OP interfaces are indicated with arrowheads on Movie 9. Previous Figure S6 was splitted into two parts presenting NCC defects in sly mutants (revised Figure S6) and in foxd3 mutants (revised Figure S7).

Altogether, these new data suggest that the first postero-anterior phase of NCC migration towards the OP, as well as their migration in between the eye and OP tissues, is not fully perturbed in sly mutants. The subset of NCC that populate the OP/forebrain seem to be more specifically affected, as these NCC show defects in their migration to the interface or the maintenance of their position at the interface. Since the crestin marker labels mostly NCC at the OP/forebrain interface at 32 hpf (revised Fig. S6A), this could explain why the crestin ISH signal is almost lost in sly mutants at this stage.

(2) Laminin distribution suggests a role in olfactory axon development 

"Laminin 111 immunostaining revealed local disruptions in the membrane enveloping the OP and brain, precisely where YFP+ axons exit the OP (exit point) and enter the brain (entry point) (Fig. 1C-D')." Can the authors quantify this situation? It would be important to analyse this behaviour on the scale of a neuron and thus axonal migration to strengthen the hypotheses. 

As suggested by the reviewer, to better visualise individual axons at the exit and entry point, we used mosaic red labelling of OP axons. To achieve this sparse labelling, we took advantage of the mosaic expression of a red fluorescent membrane protein observed in the Tg(cldnb:Gal4; UAS:lyn-TagRFP) background. The unpublished Tg(UAS:lyn-TagRFP) line was kindly provided by Marion Rosello and Shahad Albadri from the lab of Filippo Del Bene. We crossed the Tg(cldnb:Gal4; UAS:lyn-TagRFP) line with the TgBAC(lamC1:lamC1-sfGFP) reporter and performed live imaging on 2 embryos/4 placodes, in a frontal view. A new movie (Movie 3 in the revised article) shows examples of exit and entry point formation in this context.This allowed us to visualise the formation of the exit and entry points in more samples (6 embryos and 12 placodes in total when we pool the two strategies for labelling OP axons) and through the visualisation of a small number of axons, and reinforce our initial conclusions. 

(3) The integrity of BMs around the brain and the OP is affected in the sly mutant 

Why do the authors analyse the distribution of collagen IV and Nidogen and not proteoglycans and heparan sulphate? 

We attempted to label more ECM components such as proteoglycans and heparan sulfate, but whole-mount immunostainings did not work in our hands.

A dynamic analysis of the distribution of neural crest cells in the sly mutant over time and during OP coalescence would be important. 

See our detailed response to this point above.  

(4) Role of Laminin γ1-dependent BMs in OP coalescence 

The authors use the size of the Tg(neurog1:GFP)+ OP cell cluster at 22 hpf as a marker.  The authors should count the number of cells in the OP at the indicated time using a nuclear dye to check that in the sly mutant the number of cells is the same over time. Two time points as analysed in Figure S2 may not be sufficient to quantify proliferation which at these stages should be almost zero according to Whitlock & Westerfield and Madelaine et al.

Counting the neurog1:GFP+ cell numbers in our existing data was unfortunately impossible, due to the poor quality of the DAPI staining. We are nevertheless confident that the number of cells within neurog1:GFP+ clusters is fairly similar between controls and sly mutants at 22 hpf, since the OP dimensions are the same for AP and DV dimensions, and only slightly different for the ML dimension. In addition, we analysed proliferation and apoptosis within the neurog1:GFP+ cluster at 16 and 21 hpf and observed no difference between controls and mutants.

(5) Role of Laminin γ1-dependent BMs during the forebrain flexure 

In Figure 4F at 32hpf, the presence of 77% ectopic OMP+ cells medially should result in an increase in dimensions along the M-L? This is not the case in the article. The authors should clarify this point. 

As we explained in the Material and Methods, ectopic fluorescent cells (cells that are physically separated from the main cluster) were not taken into account for the measurement of the OP dimensions. This is now also also mentioned in the legends of the Figures (4 and S3) showing the quantifications of OP dimensions.

Cell distribution also seems to be affected within the OMP+ cluster at 36hpf, with fewer cells laterally and more medially. The authors should analyse the distribution of OMP+ cells in the clusters. in sly mutants and controls to understand whether the modification corresponds to the absence of BM function. 

On the pictures shown in Figure 4F,G, we agree that omp:meYFP+ cells appear to be more medially distributed in the mutant, however this is not the case in other sections or samples, and is rather specific to the z-section chosen for the Figure. We found that the ML dimension is unchanged in mutants as compared with controls, except for the 28 hpf stage where it is smaller, but this appears to be a transient phenomenon, since no change is detected at earlier or later stages (Figure 4A-D and Figure S3A-L). The difference we observe at 28 hpf is now mentioned in the revised manuscript.

The conclusions of Figures 4 and S3 would rather be that laminin allows OMP+ cells to be oriented along the medio-lateral axis whereas it would control their position along the dorsoventral axis. The authors should modify the text. It would be useful to map the distribution of OMP+ cells along the dorsoventral and mediolateral axes. The same applies to Neurog1+ cells. An analysis of skin cell movements, for example, would be useful to determine whether the effects are specific.  

We are confident that the measurements of OP dimensions in AP, DV and ML are sufficient to describe the OP shape defects observed in the sly mutants. Analysing cell distribution along the 3 axes as well as skin cell movements will be interesting to perform in the future but we consider these quantifications as being out of the scope of the present work.

(6) Laminin γ1-dependent BMs are required to define a robust boundary between the OP and the brain 

The authors must weigh this conclusion "Laminin γ1-dependent BMs serve to establish a straight boundary between the brain and OP, preventing local mixing and late convergence of the two OPs towards each other during flexion movement." Indeed, they don't really show any local mixing between the brain and OP cells. They would need to quantify in their images (Figure 5A-A' and Figure S4 A-A') the percentage of cells co-labelled by HuC and Tg(cldnb:GFP). 

We agree with the reviewer and thus replaced « reveal » by « suggest » in the conclusion of this section. 

(7) Role of Laminin γ1-dependent BMs in olfactory axon development 

An analysis of the retrograde extension movement in the axons of OMP+ ectopic neurons in the sly1 mutant condition would be useful to validate that the loss of laminin function does not play a role in this event. 

Indeed, even though we can visualise instances of retrograde extension occurring normally in sly mutants, we can not rule out that this process is affected in a subset of OP neurons, for instance in ectopic cells, which often show no axon or a misoriented axon. We added a sentence to mention this in the revised manuscript.

Minor comments and typos: 

Please check and mention the D-V/L-M or A-P/L-M orientation of the images in all figures. 

This has been checked.

Legend Figure 1: "distalmost" is missing a space "distal most". 

We checked and this word can be written without a space.

Figure 1 panel C: check the orientation (I am not sure that Dorsal is up). 

We double-checked and confirm that dorsal is up in this panel.

Movie 1 Legend: "aroung "the OP should be around the OP. 

Thanks to the reviewer for noticing the typo, we corrected it.

Reviewer #2 (Recommendations For The Authors):

The comments below are relatively minor and mostly raise questions regarding images and their presentation in the manuscript. 

• Figure 1, visualization of exit and entry points: It is a bit difficult to visualize the axon exit and entry points in these images, and in particular, to understand how the exit and entry points in C and D correspond to what is seen in F, F', H, and H'. There appears to be one resolvable break in the staining in C and D, whereas there are two distinct breaks in F-H'. Are these single optical sections? Is it possible to visualize these via 3-dimensional rendering? 

All the images presented in Figure 1 are single z-sections, which is now indicated in the Figure legend. As noticed by the reviewer, Laminin immunostainings on fixed embryos at 28 and 36 hpf suggested that the exit and entry points are facing each other, as shown in Figure 1C-D’. However, in our live imaging experiments we always observed that the exit point is slightly more ventral than the entry point (of about 10 to 20 µm). This discrepancy could be due to the fixation that precedes the immunostaining procedure, which could modify slightly the size and shape of cells/tissues. We added a sentence on this point in the text. In addition, we added new movies of the LamC1-sfGFP reporter with sparse red axonal labelling (Movie 3, see response to reviewer 1), as well as z-stacks presenting the organisation of exit and entry points in 3D (Movie 4), which should help to better illustrate the mechanisms of exit and entry point formation.

• Movie 2, p. 6, "small interruptions of the BM were already present near the axon tips, along the ventro-medial wall of the OP." This is a bit difficult to assess since the movie seems to show at least one other small interruption in the BM in addition to the exit point, in particular, one slightly dorsal to the exit point. Was this seen in other samples, or in different optical sections? 

Indeed the exit and entry points often appear as regions with several, small BM interruptions, rather than single holes in the BM. We now show in revised Movie 4 the two z-stacks (the merge and the single channel for green fluorescence) corresponding to the last time points of the movies showing exit and entry point formation in Movie 2, where several BM interruptions can be seen for both the exit and entry points. We had already mentioned this observation in the legend of Movie 2, and we added a sentence on this point in the main text of the revised manuscript. This is also represented for both exit and entry points in the new schematics in revised Fig. 1K and its legend. 

• Movie 2, p. 6, "The opening of the entry point through the brain BM was concomitant with the arrival of the RFP+ axons, suggesting that the axons degrade or displace BM components to enter the brain." Similar to the questions regarding the exit point, it was a bit difficult to evaluate this statement. There appears to be a broader region of BM discontinuity more dorsal to the arrowhead in Movie 2. A single-channel movie of just the laminin fluorescence might help to convey the extent of the discontinuity. As with above, was this seen in other samples, or in different optical sections?  

See our response to the previous comment.

• Figure 1H, I, "the distal tip of the RFP+ axons migrated in close proximity with the brain's BM." This is again a bit difficult to see, and quite different than what is seen in Figure 4A, in which the axons do not seem close to the BM in this section. Is it possible to visualize this via 3-dimensional rendering? 

In fixed embryos or in live imaging experiments, we observed that, once entered in the brain, the distal tips (the growth cones) of the axons are located close to the BM of the brain. However, this is not the case of the axon shafts which, as development proceeds, are located further away from the BM. This can clearly be seen at 36 hpf in Figure 1D’ and Figure 4A, as spotted by the reviewer. We modified the text to clarify this point.

• Figure 2J, J', p. 7, the gap between the OP and brain cells of sly mutants "was most often devoid of electron-dense material." It is difficult to see this loss of electron-dense material in 2J'. The thickness of the space is quantified well and is clearly smaller, but the change in electron-dense material is more difficult to see.  

We looked at Figure 2 again and it seems clear to us that there is electron-dense material between the plasma membranes in controls, which is practically not seen (rare spots) in the mutants. We added a sentence mentioning that we rarely see electron-dense spots in sly mutants.

• Figure 5E-F': There are concerns about evaluating the shape of a tissue based on nuclear position. Is there a way to co-stain for cell boundaries (maybe actin?), and then quantify distortion of the dlx+ cell population using the cell boundaries, rather than nuclear staining? 

We agree with the reviewer that it is not ideal to evaluate the shape of the OP/brain boundary based on a nuclear staining. As explained in the text, we could not use the Tg(eltC:GFP) or Tg(cldnb:Gal4; UAS:RFP) reporter lines for this analysis, due to ectopic or mosaic expression. However we are confident that the segmentation of the Dlx3b immunostaining reflects the organisation of the cells at the OP/brain tissue boundary: in other data sets in which we performed Dlx3b staining with membrane labelling independently of the present study and in the wild type context, we clearly see that cell membranes are juxtaposed to the Dlx3b nuclear staining (in other words, the cytoplasm volume of OP cells is very small). 

• Figure S5E: It would be helpful to see representative images for each of the categories (Proper axon bundle; Ventral projections; Medial projections) or a schematic to understand how the phenotypes were assessed. 

To address this point we added a schematic view to illustrate the phenotypes assessed in each column of the table in revised Figure S5G.

• Figure 6, p. 12, "Laminin gamma 1-dependent BMs are essential for growth and navigation of the axons...": What fraction of the tracked axons managed to exit the OP? Given the quantitative analyses in Figure 6, one might interpret this to mean that laminin gamma 1 is not essential for axon growth (speed and persistence are largely unchanged), but rather, primarily for navigation. 

As noticed by the reviewer, the speed and persistence of axonal growth cones are largely unchanged in the sly mutants (except for the reduced persistence in the 200-400 min window, and an increased speed in the 800-1000 min window), showing that the growth cones are still motile. However, as shown by the tracks, they tend to wander around within the OP, close to the cell bodies, which results in the end in a perturbed growth of the axons. The navigation issues are rather revealed by the analysis of fixed Tg(omp:meYFP) embryos presented in the table of Figure S5G. We modified the text to separate more clearly the conclusions of the two types of experiments (fixed, transgenic embryos versus live, mosaically labelled embryos).

Reviewer #3 (Recommendations For The Authors):

Testing the hypotheses mentioned in the public review will be interesting experiments for a follow-up study, but are not essential revisions for this manuscript. 

I have only a few minor suggestions for revisions: 

P8 subheading 'Role of Laminin γ1-dependent BMs in OP coalescence' - since no major role was demonstrated here, this heading should be reworded.  

We agree with the reviewer and replaced the previous title by « OP coalescence still occurs in the sly mutant ».

P11, line 3 - the authors conclude that the forebrain is smaller 'due to' the inward convergence of the OPs. I do not think it is possible to assign causation to this when the mutant disrupts Laminin γ1 systemically - it is equally possible that the OPs move inward due to a failure of the brain to form in the normal shape. Thus, the wording should be changed here. (In the Discussion on p15, the authors mention the 'apparent distortion' of the brain, and say that it is 'possibly due' to the inward migration of the placodes', but again this could be toned down.) 

We agree with the reviewer’s comment and changed the wording of our conclusions in the Results section.

P11 and Fig. S5 - The table and text seem to be saying opposite things here. The text on p11 (3rd paragraph) indicates that the normal exit point is ventral and that this is disrupted in the mutant, with axons exiting dorsally. However, in the table, at each time point there is a higher % of axons exiting ventrally in the mutant. Please clarify. The table does not provide a % value for axons exiting dorsally - it might help to add a column to show this value. 

We are grateful to the reviewer for pointing this out, and we apologize for the lack of clarity in the first version of the manuscript. We have modified the text and Figure S5 in order to clarify the different points raised by the reviewer in this comment. The Table in Fig. S5G does not represent the % of axons showing defects, but the % of embryos showing the phenotypes. In addition, an embryo is counted in the ventral or medial projection category if it shows at least one ventral or medial projection (even if its shows a proper bundle). This is now clearly indicated in the title of the columns in the table itself and in the legend. The embryos in which the axons exit dorsally in sly mutants are actually those counted in the left column of the Table (they exit dorsally and form a bundle), as shown by the new schematics added below the table. We also added this information in the title of the left column, and mention in the legend the pictures in which this dorsal exit can be observed in the article (Figures 4B and S3E’). Having more sly mutant embryos with axons exiting dorsally is thus compatible with more embryos showing at least one ventral projection.

Fig. S6, shows the lack of neural crest cells between the olfactory placode and the brain in both laminin γ1 mutants (without a basement membrane) and foxd3 mutants (which retain the membrane). Comparison of the two mutants here is a neat experiment and the result is striking, demonstrating that it is the basement membrane, and not the neural crest, that is required for correct morphology of the olfactory placode. I think this figure should be presented as a main figure, rather than supplementary.  

Our new live imaging characterisation of NCC migration in sly mutants and control siblings (Movie 9) revealed that at 32 hpf, in the vicinity of the OP, NCC (or their derivatives) are much more numerous than the subset of NCC showing crestin expression by in situ hybridisation (compare the end of our control movie – 32 hfp, with crestin ISH shown in Figure S6A for instance). 

Thus, the extent of the NCC migration defects should be analysed in more detail in the foxd3 mutant in the future (using live imaging or other NCC markers), and for this reason we chose to keep this dataset in the supplementary Figures.

One of the first topics covered in the Discussion section is the potential role of Collagen. I was surprised to see the description on P15 'the dramatic disorganization of the Collagen IV pattern observed by immunofluorescence in the sly mutant', as I hadn't picked this up from the Results section of the paper. I went back to the relevant figure (Fig. 2) and description on p7, which does not give the same impression: 'in sly mutants, Collagen IV immunoreactivity was not totally abolished'. This suggested to me that there was only minor (not dramatic) disorganisation of the Collagen IV. This needs clarification.  

The linear, BM-like Collagen IV staining was lost in sly mutants, but not the fibrous staining which remained in the form of discrete patches surrounding the OP. We modified the text in the Results section as well as in the Figure 2 legend to clarify our observations made on embryos immunostained for Collagen IV.

Typos etc 

P5 - '(ii) above of the neuronal rosette' - delete the word 'of'. 

P5 two lines below this - ensheathed. 

P10 - '3 distinct AP levels' (delete s from distincts). 

P10 - distortion (not distorsion) . 

P12 - 'From 14 hpf, they' should read 'From 14 hpf, neural crest cells'. 

P15, line 1 - 'is a consequence of' rather than 'is consecutive of'? 

P22 'When the data were not normal,' should read 'When the data were not normally distributed,'. 

We thank the reviewer for noticing these typos and have corrected them.

General 

Please number lines in future manuscripts for ease of reference. 

This has been done.

Ralph Waldo Emerson, Stephen E. Whicher (1959). “The Early Lectures of Ralph Waldo Emerson”, p.327, Harvard University Press

via https://www.azquotes.com/author/4490-Ralph_Waldo_Emerson/tag/hate

Most of the second half of John Smith account talks about trade relations and exploratory missions between various indigenous tribes in the area. (Smith, John “A True Relation of Such Occurrences) Because of this, there wasn't too much to compare to the movie. For one, the movie is short and I can understand why a lot of the mundane time that is spent setting up a colony wasn't included. ("Goldberg, Eric, and Mike Gabriel. 1995. Pocahontas") The movie also deviates so far from this, and other scholarly sources, that there isn't much to compare. Overall, this source is heavily biased with a fair bit of exaggeration, that modern day historians know not to be true. At the same time, half of what John Smith talks about is a gateway into what life was like in the early days of Jamestown. It talks about disease, conditions of the settlement, various indigenous tribes (which the rest of the world knew relatively little about), the land and new sources of food which was an important source at the time it was published. (Smith, John “A True Relation of Such Occurrences) While Disney's Pocahontas isn't that close to this primary source, nor is it to other sources detailing this part of history, it does a good job in respecting Native American culture. There weren't a lot of films depicting Native Americans in a positive or appropriate light before this movie was made. While there is still shockingly low number of films that tell Native American stories, Pocahontas I think was a small catalyst for future indigenous movies due to its commercial success.

Bibliography: “A True Relation of Such Occurrences and Accidents of Note as Hath Hapned in Virginia Since the First Planting of That Colony, Which Is Now Resident in the South Part Thereof, till the Last Returne from Thence. Written by Captaine Smith One of the Said Collony, to a Worshipfull Friend of His in England.,” April 13, 2005. http://web.archive.org/web/20050413081941/http://etext.lib.virginia.edu/etcbin/toccer-new2?id=J1007.xml&images=images/modeng&data=/texts/english/modeng/parsed&tag=public&part=all.

Goldberg, Eric, and Mike Gabriel. 1995. Pocahontas. United States: Buena Vista Pictures.

reply to @ayjay @annie at https://micro.blog/ayjay/48571448

@ayjay I've long presumed you were digitally commonplacing by means of your websites, so thanks for laying out some of your specific current thoughts on the process.

​@Annie I've been collecting examples of bloggers who are using their personal websites as commonplaces, zettelkasten, and "thought spaces" at https://indieweb.org/commonplace_book. If you're interested, you'll also find examples and details to explore in my own digital commonplace. "Thought spaces" is an interesting entry point.

Doctorow goes through more of his process in which he's saving both his "box of delights" and the refashioning of them into longer pieces in "20 years a blogger". In his case it's (now) all done on Pluralistic and syndicated out from there, in much the same way @dave has done for years.

No tag

Was the cytoplasmic background also brighter here (not just the localized signal)? I'm just naively wondering if the achilles tag is causing some stress that is increasing background autofluorescence in the cell (given the onset of uniform cytoplasmic fluorescence in Fig 1).

Reviewer #1 (Public review):

Summary:

The authors describe a method to probe both the proteins associated with genomic elements in cells, as well as 3D contacts between sites in chromatin. The approach is interesting and promising, and it is great to see a proximity labeling method like this that can make both proteins and 3D contacts. It utilizes DNA oligomers, which will likely make it a widely adopted method. However, the manuscript over-interprets its successes, which are likely due to the limited appropriate controls, and of any validation experiments. I think the study requires better proteomic controls, and some validation experiments of the "new" proteins and 3D contacts described. In addition, toning down the claims made in the paper would assist those looking to implement one of the various available proximity labeling methods and would make this manuscript more reliable to non-experts.

Strengths:

(1) The mapping of 3D contacts for 20 kb regions using proximity labeling is beautiful.

(2) The use of in situ hybridization will probably improve background and specificity.

(3) The use of fixed cells should prove enabling and is a strong alternative to similar, living cell methods.

Weaknesses:

(1) A major drawback to the experimental approach of this study is the "multiplexed comparisons". Using the mtDNA as a comparator is not a great comparison - there is no reason to think the telomeres/centrosomes would look like mtDNA as a whole. The mito proteome is much less complex. It is going to provide a large number of false positives. The centromere/telomere comparison is ok, if one is interested in what's different between those two repetitive elements. But the more realistic use case of this method would be "what is at a specific genomic element"? A purely nuclear-localized control would be needed for that. Or a genomic element that has nothing interesting at it (I do not know of one). You can see this in the label-free work: non-specific, nuclear GO terms are enriched likely due to the random plus non-random labeling in the nucleus. What would a Telo vs general nucleus GSEA look like? (GSEA should be used for quantitative data, no GO). That would provide some specificity. Figures 2G and S4A are encouraging, but a) these proteins are largely sequestered in their respective locations, and b) no validation by an orthogonal method like ChIP or Cut and Run/Tag is used.

You can also see this in the enormous number of "enriched" proteins in the supplemental volcano plots. The hypothesis-supporting ones are labeled, but do the authors really believe all of those proteins are specific to the loci being looked at? Maybe compared to mitochondria, but it's hard to believe there are not a lot of false positives in those blue clouds. I believe the authors are more seeing mito vs nucleus + Telo than the stated comparison. For example, if you have no labeling in the nucleus in the control (Figures 1C and 2C) you cannot separate background labeling from specific labeling. Same with mito vs. nuc+Telo. It is not the proper control to say what is specifically at the Telo.

I would like to see a Telo vs nuclear control and a Centromere vs nuc control. One could then subtract the background from both experiments, then contrast Telo vs Cent for a proper, rigorous comparison. However, I realize that is a lot of work, so rewriting the manuscript to better and more accurately reflect what was accomplished here, and its limitations, would suffice.

(2) A second major drawback is the lack of validation experiments. References to literature are helpful but do not make up for the lack of validation of a new method claiming new protein-DNA or DNA-DNA interactions. At least a handful of newly described proximal proteins need to be validated by an orthogonal method, like ChIP qPCR, other genomic methods, or gel shifts if they are likely to directly bind DNA. It is ok to have false positives in a challenging assay like this. But it needs to be well and clearly estimated and communicated.

(3) The mapping of 3D contacts for 20 kb regions is beautiful. Some added discussion on this method's benefits over HiC-variants would be welcomed.

(4) The study claims this method circumvents the need for transfectable cells. However, the authors go on to describe how they needed tons of cells, now in solution, to get it to work. The intro should be more in line with what was actually accomplished.

(5) Comments like "Compared to other repetitive elements in the human genome...." appear to circumvent the fact that this method is still (apparently) largely limited to repetitive elements. Other than Glopro, which did analyze non-repetitive promoter elements, most comparable methods looked at telomeres. So, this isn't quite the advancement you are implying. Plus, the overlap with telomeric proteins and other studies should be addressed. However, that will be challenging due to the controls used here, discussed above.

Author response:

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors describe a method to probe both the proteins associated with genomic elements in cells, as well as 3D contacts between sites in chromatin. The approach is interesting and promising, and it is great to see a proximity labeling method like this that can make both proteins and 3D contacts. It utilizes DNA oligomers, which will likely make it a widely adopted method. However, the manuscript over-interprets its successes, which are likely due to the limited appropriate controls, and of any validation experiments. I think the study requires better proteomic controls, and some validation experiments of the "new" proteins and 3D contacts described. In addition, toning down the claims made in the paper would assist those looking to implement one of the various available proximity labeling methods and would make this manuscript more reliable to non-experts.

Strengths:

(1) The mapping of 3D contacts for 20 kb regions using proximity labeling is beautiful.

(2) The use of in situ hybridization will probably improve background and specificity.

(3) The use of fixed cells should prove enabling and is a strong alternative to similar, living cell methods.

Weaknesses:

(1) A major drawback to the experimental approach of this study is the "multiplexed comparisons". Using the mtDNA as a comparator is not a great comparison - there is no reason to think the telomeres/centrosomes would look like mtDNA as a whole. The mito proteome is much less complex. It is going to provide a large number of false positives. The centromere/telomere comparison is ok, if one is interested in what's different between those two repetitive elements. But the more realistic use case of this method would be "what is at a specific genomic element"? A purely nuclear-localized control would be needed for that. Or a genomic element that has nothing interesting at it (I do not know of one). You can see this in the label-free work: non-specific, nuclear GO terms are enriched likely due to the random plus non-random labeling in the nucleus. What would a Telo vs general nucleus GSEA look like? (GSEA should be used for quantitative data, no GO). That would provide some specificity. Figures 2G and S4A are encouraging, but a) these proteins are largely sequestered in their respective locations, and b) no validation by an orthogonal method like ChIP or Cut and Run/Tag is used.

You can also see this in the enormous number of "enriched" proteins in the supplemental volcano plots. The hypothesis-supporting ones are labeled, but do the authors really believe all of those proteins are specific to the loci being looked at? Maybe compared to mitochondria, but it's hard to believe there are not a lot of false positives in those blue clouds. I believe the authors are more seeing mito vs nucleus + Telo than the stated comparison. For example, if you have no labeling in the nucleus in the control (Figures 1C and 2C) you cannot separate background labeling from specific labeling. Same with mito vs. nuc+Telo. It is not the proper control to say what is specifically at the Telo.

I would like to see a Telo vs nuclear control and a Centromere vs nuc control. One could then subtract the background from both experiments, then contrast Telo vs Cent for a proper, rigorous comparison. However, I realize that is a lot of work, so rewriting the manuscript to better and more accurately reflect what was accomplished here, and its limitations, would suffice.

(2) A second major drawback is the lack of validation experiments. References to literature are helpful but do not make up for the lack of validation of a new method claiming new protein-DNA or DNA-DNA interactions. At least a handful of newly described proximal proteins need to be validated by an orthogonal method, like ChIP qPCR, other genomic methods, or gel shifts if they are likely to directly bind DNA. It is ok to have false positives in a challenging assay like this. But it needs to be well and clearly estimated and communicated.

(3) The mapping of 3D contacts for 20 kb regions is beautiful. Some added discussion on this method's benefits over HiC-variants would be welcomed.

(4) The study claims this method circumvents the need for transfectable cells. However, the authors go on to describe how they needed tons of cells, now in solution, to get it to work. The intro should be more in line with what was actually accomplished.

(5) Comments like "Compared to other repetitive elements in the human genome...." appear to circumvent the fact that this method is still (apparently) largely limited to repetitive elements. Other than Glopro, which did analyze non-repetitive promoter elements, most comparable methods looked at telomeres. So, this isn't quite the advancement you are implying. Plus, the overlap with telomeric proteins and other studies should be addressed. However, that will be challenging due to the controls used here, discussed above.

We thank the Reviewer for their careful reading of manuscript and constructive suggestions. We plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Reviewer #2 (Public review):

Summary

Liu and MacGann et al. introduce the method DNA O-MAP that uses oligo-based ISH probes to recruit horseradish peroxidase for targeted proximity biotinylation at specific DNA loci. The method's specificity was tested by profiling the proteomic composition at repetitive DNA loci such as telomeres and pericentromeric alpha satellite repeats. In addition, the authors provide proof-of-principle for the capture and mapping of contact frequencies between individual DNA loop anchors.

Strengths

Identifying locus-specific proteomes still represents a major technical challenge and remains an outstanding issue (1). Theoretically, this method could benefit from the specificity of ISH probes and be applied to identify proteomes at non-repetitive DNA loci. This method also requires significantly fewer cells than other ISH- or dCas9-based locus-enrichment methods. Another potential advantage to be tested is the lack of cell line engineering that allows its application to primary cell lines or tissue.

Weaknesses

The authors indicate that DNA O-MAP is superior to other methods for identifying locus-specific proteomes. Still, no proof exists that this method could uncover proteomes at non-repetitive DNA loci. Also, there is very little validation of novel factors to confirm the superiority of the technique regarding specificity.

The authors first tested their method's specificity at repetitive telomeric regions, and like other approaches, expected low-abundant telomere-specific proteins were absent (for example, all subunits of the telomerase holoenzyme complex). Detecting known proteins while identifying noncanonical and unexpected protein factors with high confidence could indicate that DNA O-MAP does not fully capture biologically crucial proteins due to insufficient enrichment of locus-specific factors. The newly identified proteins in Figure 1E might still be relevant, but independent validation is missing entirely. In my opinion, the current data cannot be interpreted as successfully describing local protein composition.

Finally, the authors could have discussed the limitations of DNA O-MAP and made a fair comparison to other existing methods (2-5). Unlike targeted proximity biotinylation methods, DNA O-MAP requires paraformaldehyde crosslinking, which has several disadvantages. For instance, transient protein-protein interactions may not be efficiently retained on crosslinked chromatin. Similarly, some proteins may not be crosslinked by formaldehyde and thus will be lost during preparation (6).

(1) Gauchier M, van Mierlo G, Vermeulen M, Dejardin J. Purification and enrichment of specific chromatin loci. Nat Methods. 2020;17(4):380-9.

(2) Dejardin J, Kingston RE. Purification of proteins associated with specific genomic Loci. Cell. 2009;136(1):175-86.

(3) Liu X, Zhang Y, Chen Y, Li M, Zhou F, Li K, et al. In Situ Capture of Chromatin Interactions by Biotinylated dCas9. Cell. 2017;170(5):1028-43 e19.

(4) Villasenor R, Pfaendler R, Ambrosi C, Butz S, Giuliani S, Bryan E, et al. ChromID identifies the protein interactome at chromatin marks. Nat Biotechnol. 2020;38(6):728-36.

(5) Santos-Barriopedro I, van Mierlo G, Vermeulen M. Off-the-shelf proximity biotinylation for interaction proteomics. Nat Commun. 2021;12(1):5015.

(6) Schmiedeberg L, Skene P, Deaton A, Bird A. A temporal threshold for formaldehyde crosslinking and fixation. PLoS One. 2009;4(2):e4636.

We thank the Reviewer for their constructive feedback on our work. As noted above, we plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Reviewer #3 (Public review):

Significance of the Findings:

The study by Liu et al. presents a novel method, DNA-O-MAP, which combines locus-specific hybridisation with proximity biotinylation to isolate specific genomic regions and their associated proteins. The potential significance of this approach lies in its purported ability to target genomic loci with heightened specificity by enabling extensive washing prior to the biotinylation reaction, theoretically improving the signal-to-noise ratio when compared with other methods such as dCas9-based techniques. Should the method prove successful, it could represent a notable advancement in the field of chromatin biology, particularly in establishing the proteomes of individual chromatin regions - an extremely challenging objective that has not yet been comprehensively addressed by existing methodologies.

Strength of the Evidence:

The evidence presented by the authors is somewhat mixed, and the robustness of the findings appears to be preliminary at this stage. While certain data indicate that DNA-O-MAP may function effectively for repetitive DNA regions, a number of the claims made in the manuscript are either unsupported or require further substantiation. There are significant concerns about the resolution of the method, with substantial biotinylation signals extending well beyond the intended target regions (megabases around the target), suggesting a lack of specificity and poor resolution, particularly for smaller loci. Furthermore, comparisons with previous techniques are unfounded since the authors have not provided direct comparisons with the same mass spectrometry (MS) equipment and protocols. Additionally, although the authors assert an advantage in multiplexing, this claim appears overstated, as previous methods could achieve similar outcomes through TMT multiplexing. Therefore, while the method has potential, the evidence requires more rigorous support, comprehensive benchmarking, and further experimental validation to demonstrate the claimed improvements in specificity and practical applicability.

We thank the Reviewer for providing detailed critiques of our manuscript. As noted above, we plan to substantially revise the framing and presentation of manuscript to address the concerns raised by all three reviewers.

Reviewer #3 (Public review):

Summary:

The study explores the cellular and circuit features that distinguish dentate gyrus semilunar granule cells and granule cells activated during contextual memory formation. The authors tag memory and enriched environment-activated dentate granule cells and semilunar granule cells and show their reactivation in an appropriate context a week later. They perform patch clamp recordings from activated and surrounding neurons to understand cellular driving the selective activation of semilunar granule cells and granule cells. Authors perform dual patch clamp recordings from various pairs of labeled semilunar granule cells, labeled granule cells, unlabeled granule cells, and unlabeled semilunar granule cells. The sustained firing of semilunar granule cells explained their preferential activation. In addition, activated neurons received correlated inputs.

Strengths:

The authors confirmed engram cell properties of activated semilunar granule cells and granule cells in two different paradigms, validated using an enriched environment paradigm.

The authors carefully separate semilunar granule cells from granule cells, using electrophysiology and morphology. Cell filling to confirm morphology further strengthens confidence.

The dual patch recordings, which are technically challenging, are carefully performed, and the presence of synaptic activity is confirmed.

Finally, the correlation analysis of EPSCs on labeled neurons is rigorous.

Weaknesses:

(1) Engram cells are (i) activated by a learning experience, (ii) physically or chemically modified by the learning experience, and (iii) reactivated by subsequent presentation of the stimuli present at the learning experience (or some portion thereof), resulting in memory retrieval. The authors show that exposure to Barnes Maze and the enriched environment-activated semilunar granule cells and granule cells preferentially in the superior blade of the dentate gyrus, and a significant fraction were reactivated on re-exposure. However, physical or chemical modification by experience was not tested. Experience modifies engram cells, and a common modification is the Hebbian, i.e., potentiation of excitatory synapses. The authors recorded EPSCs from labeled and unlabeled GCs and SGCs. Was there a difference in the amplitude or frequency of EPSCs recorded from labeled and unlabeled cells?

(2) The authors studied five sequential sections, each 250 μm apart across the septotemporal axis, which were immunostained for c-Fos and analyzed for quantification. Is this an adequate sample? Also, it would help to report the dorso-ventral gradient since more engram cells are in the dorsal hippocampus. Slices shown in the figures appear to be from the dorsal hippocampus.

(3) The authors investigated the role of surround inhibition in establishing memory engram SGCS and GCs. Surprisingly, they found no evidence of lateral inhibition in the slice preparation. Interneurons, e.g., PV interneurons, have large axonal arbors that may be cut during slicing. Similarly, the authors point out that some excitatory connections may be lost in slices. This is a limitation of slice electrophysiology.

Reviewer #1 (Public review):

In this manuscript, Sun et al report the development of a POST-IT (Pup-On-target for Small molecule Target Identification Technology) approach for drug target identification. Generally, this new technology applies a non-diffusive proximity tagging system by utilizing an engineered fusion of proteasomal accessory factor A (PafA) and HaloTag to transfer prokaryotic ubiquitin-like protein (Pup) to proximal proteins upon directly binding to the small molecule. After the pupylated targets are captured, they are able to be detected by mass spectrometry. Significant optimization (Lys-Arg and other mutations) was conducted to eliminate the interference of self-pupylation, polypupylation, and depupylation, POST-IT was successfully applied for the target identification of 2 well-known drugs: dasatinib and hydroxychloroquine, which yielded SEPHS2 and VPS37C as their new potential targets, respectively. Furthermore, POST-IT was also applied in live zebrafish embryos, highlighting its potential for broad biological research and drug development.

This work was well designed and the experiments were logically conducted. The solid results support POST-IT as a promising technology for new drug target identification.

Weakness and limitations:

(1) The technology requires a halo-tagged derivation of the active compound, and the linked position will have a huge impact on the potential "target hits" of the molecules. Given the fact that most of the active molecules lack of structure-activity relationship information, it is very challenging to identify the optimal position of the halo tag linkage.

(2) Although POST-IT works in zebrafish embryos, there is still a long way to go for the broad application of the technology in other animal models.

(3) The authors identified SEPHS2 as a new potential target of dasatinib and further validated the direct binding of dasatinib with this protein. However, considering the super strong activity of dasatinib against c-Src (sub nanomolar IC50 value), it is hard to conclude the contribution of SEPHS2 binding (micromolar potency) to its antitumor activity.

Technical Feedback (8/20)

HTML Structure (3/5)

• ✓ Good heading variety (h1-h4)
• ✗ Structural issues:
• <h3> nested inside <h3>
• Invalid custom tags (<p1>, <p2>)
• ✓ Links properly embedded
• ⚠ Image implementation:
• Alt attributes present
• Some invalid URLs (\ddd)
• Inconsistent sources

CSS Implementation (4/10)

• ✗ Missing IDs completely
• ✗ No class usage
• ✗ Style issues:
• Invalid selectors (p1, p2)
• Redundant background declarations
• Overuse of inline styles
• ⚠ Limited styling scope:
• Basic font sizing
• Simple background images
• Needs more detailed styling

Code Quality (1/5)

• ✗ Organization problems:
• Excessive <br> tags
• Missing closing tags
• Poor indentation
• ✗ Invalid elements:
• Custom paragraph tags
• Broken image sources
• Improper tag nesting

Priority Fixes:

1. Fix HTML structure:
2. Replace <p1>, <p2> with <p>
3. Correct heading hierarchy
4. Add proper closing tags

5. Fix image URLs

6. Improve CSS:

7. Add 5+ unique IDs
8. Create reusable classes
9. Remove redundant styles

10. Replace inline styling

11. Clean up code:

12. Replace <br> with margins/padding
13. Improve indentation
14. Fix tag nesting

Your content structure shows promise, but needs technical cleanup. Focus first on using valid HTML elements and building a proper CSS structure with IDs and classes.

Technical Feedback (18/20)

HTML Structure (5/5)

• ✓ Excellent heading hierarchy (h1-h3)
• ✓ Well-organized paragraphs
• ✓ Links properly implemented:
• Correct <a> tag usage
• Good use of target="_blank"
• ✓ Images:
• Properly embedded
• Well-styled

CSS Implementation (9/10)

• ✓ Strong ID usage:
• #intro
• #ind
• #iow
• #pel
• ✓ Effective class reuse:
• .div1 across sections
• .div2 across sections
• ✓ Comprehensive tag styling:
• Headers
• Paragraphs
• Images
• ⚠ Minor optimization needed:
• Redundant properties in .div1 and .div2
• Could combine common styles

Code Quality (4/5)

• ✓ Clean, readable structure
• ✓ Proper indentation
• ✓ Good element nesting
• ✗ Missing </head> closing tag

Suggestions for Perfect Score:

1. Optimize CSS:
2. Create shared class for common div styles
3. Combine repeated properties
4. Add missing </head> tag

Excellent work overall! Your code is clean and well-structured. Making these minor improvements would make it perfect. Your use of classes and IDs is particularly strong, showing good understanding of CSS organization.

Technical Feedback (8/20)

HTML Structure (2/5)

• ✗ Critical structure issues:
• <div> inside <h1> (invalid)
• Multiple <body> tags
• Missing closing tags for <a> and <ol>
• ✗ Invalid <frame> usage (should be <iframe>)
• ⚠ Heading issues:
• Uses <h1> and <h3>
• Improper nesting
• ⚠ Custom paragraph ID used instead of <p> tag
• ✓ Images have alt attributes

CSS Implementation (5/10)

• ⚠ Problematic ID usage:
• #heading
• #heading2
• #p (should be tag selector)
• Split styles that should be unified
• ✗ Class issues:
• Invalid .container: syntax
• Missing second reusable class
• ✗ Style problems:
• Redundant h1/h3 styles
• Inline styles in <body>
• Misplaced colons
• Missing semicolons

Code Quality (1/5)

• ✗ Major structural issues:
• Multiple body tags
• Broken closing tags
• Improper element nesting
• ✗ Redundant styling
• ✗ Invalid HTML/CSS syntax
• ✗ Disorganized code structure

Critical Fixes:

1. Fix HTML structure:
2. Remove extra <body> tag
3. Fix tag nesting
4. Close all tags properly
5. Correct element usage:
6. Replace <frame> with <iframe>
7. Use proper <p> tags
8. Fix CSS:
9. Remove .container: syntax error
10. Consolidate heading styles
11. Move inline styles to CSS file
12. Clean up redundant code
13. Fix all closing tags

Hi Collin, I really enjoyed your projects concept; however, your project needs significant technical improvements to function properly. Focus first on fixing the HTML structure and closing tags, then work on organizing your CSS more efficiently.

Technical Feedback (19/20)

HTML Structure (5/5)

• ✓ Excellent heading hierarchy (h1-h5)
• ✓ Proper paragraph structure
• ✓ Media well-implemented (YouTube, Giphy iframes)
• ✓ Images correctly formatted
• ⚠ Minor note: Redundant <a> tag after Giphy embed

CSS Implementation (10/10)

• ✓ Strong ID usage:
• #containerOne
• #leftnote
• #rightnote
• #color
• #define
• #bold
• #big
• ✓ Effective class reuse:
• .notes
• .small
• ✓ Comprehensive tag styling:
• Tables (table, th, td)
• Headings
• Paragraphs

Code Quality (4/5)

• ✓ Clean, readable structure
• ✓ Good separation of concerns
• ✓ Proper indentation
• ⚠ Some inline styles could move to CSS file

Suggestions for Perfect Score:

1. Remove redundant <a> tag after Giphy
2. Move inline styles to CSS file
3. Review table styling for consistency

Outstanding work on HTML structure and CSS implementation. Your code is clean, semantic, and well-organized. Minor tweaks to styling organization, but overall nice job.

Technical Feedback (8/20)

HTML Structure (3/5)

• ✓ Main heading (<h1>) used correctly
• ✗ Custom tags used instead of standard <p> tags (<p1>, <p2>, <p4>)
• ⚠ Links present but have syntax errors (missing >)
• ✓ Image (elio2.jpeg) implemented correctly
• ✗ Needs more section headings

CSS Implementation (4/10)

• ⚠ IDs implemented:
• #containerone
• #containertwo
• #floatingpup
• Need 2 more to meet requirements
• ⚠ Classes:
• .container used well
• Need another reusable class (3+ instances)
• ✗ Style issues:
• Invalid syntax (opacity: .7.5)
• Missing semicolons
• Broken custom tag selectors

Code Quality (1/5)

• ✗ Invalid HTML elements (<div1>, <p1>, <p2>, <p4>)
• ✗ Improper CSS formatting
• ✗ Missing closing brackets
• ✗ Invalid font-family declarations
• ✗ Misused quotes in div1 styling

Priority Fixes:

1. Replace custom tags (<p1>, <p2>, <p4>) with standard <p> tags
2. Add 2 more unique IDs
3. Create another reusable class
4. Fix link syntax
5. Clean up CSS syntax (semicolons, opacity values)

Enjoyed this project overall! Address the technical issues listed here and this will be a solid submission.

Technical Feedback (14/20)

HTML Structure (4/5)

• ✓ Good use of heading hierarchy (h1-h6)
• ✓ Paragraphs properly tagged
• ✗ Two empty <p> tags need content
• ✗ Missing navigation links
• ⚠ Images work but need better organization

CSS Implementation (7/10)

• ✓ Multiple IDs used effectively
• ✓ Good reuse of .container class
• ✗ #rain1 ID exists in CSS but not in HTML
• ✗ .container class missing style definitions
• ✗ Layout issues due to undefined container styles

Code Quality (3/5)

• ✗ HTML syntax errors:
• Missing > in <link> tag
• Unclosed final <div>
• ✗ Unused CSS selectors (p2, h2, #rain1)
• ✗ Inconsistent margin spacing
• ✗ Div structure needs optimization

Key Improvements:

1. Add navigation links
2. Define .container styles
3. Fix HTML syntax errors
4. Clean up unused CSS
5. Improve div organization

Overall I really enjoyed your concept and visuals that reinforced the theme of the page. Focus on cleaning up the technical details and completing the missing style definitions to take this to the next level.

Existing facilities that can filter carbon dioxide out of the air only have the capacity to capture 0.01 million metric tons of CO2 globally today, costing companies like Microsoft as much as $600 per ton of CO2. That’s very little capacity with a very high price tag.

Calma, Justine. “Trying to Reverse Climate Change Won’t Save Us, Scientists Warn.” Msn.Com, 1729, https://www.msn.com/en-us/news/technology/trying-to-reverse-climate-change-won-t-save-us-scientists-warn/ar-AA1sN6OC?ocid=msedgntp&cvid=20987699b6484dd5c9aad7c390f9e4cd&ei=4.

Reviewer #3 (Public review):

Summary:

This work investigated the immune response in the murine retina after focal laser lesions. These lesions are made with close to 2 orders of magnitude lower laser power than the more prevalent choroidal neovascularization model of laser ablation. Histology and OCT together show that the laser insult is localized to the photoreceptors and spares the inner retina, the vasculature, and the pigment epithelium. As early as 1-day after injury, a loss of cell bodies in the outer nuclear layer is observed. This is accompanied by strong microglial proliferation at the site of injury in the outer retina where microglia do not typically reside. The injury did not seem to result in the extravasation of neutrophils from the capillary network constituting one of the main findings of the paper. The demonstrated paradigm of studying the immune response and potentially retinal remodeling in the future in vivo is valuable and would appeal to a broad audience in visual neuroscience. However, there are some issues with the conclusions drawn from the data and analysis that can be addressed to further bolster the manuscript.

Strengths:

Adaptive optics imaging of the murine retina is cutting edge and enables non-destructive visualization of fluorescently labeled cells in the milieu of retinal injury. As may be obvious, this in vivo approach is beneficial for studying fast and dynamic immune processes on a local time scale - minutes and hours, and also for the longer days-to-months follow-up of retinal remodeling as demonstrated in the article. In certain cases, the in vivo findings are corroborated with histology.

The analysis is sound and accompanied by stunning video and static imagery. A few different sets of mouse models are used, (a) two different mouse lines, each with a fluorescent tag for neutrophils and microglia, (b) two different models of inflammation - endotoxin-induced uveitis (EAU) and laser ablation are used to study differences in the immune interaction.

One of the major advances in this article is the development of the laser ablation model for 'mild' retinal damage as an alternative to the more severe neovascularization models. While not directly shown in the article, this model would potentially allow for controlling the size, depth, and severity of the laser injury opening interesting avenues for future study.

Weaknesses:

(1) It is unclear based on the current data/study to what extent the mild laser damage phenotype is generalizable to disease phenotypes. The outer nuclear cell loss of 28% and a complete recovery in 2 months would seem quite mild, thus the generalizability in terms of immune-mediated response in the face of retinal remodeling is not certain, specifically whether the key finding regarding the lack of neutrophil recruitment will be maintained with a stronger laser ablation.

(2) Mice numbers and associated statistics are insufficient to draw strong conclusions in the paper on the activity of neutrophils, some examples are below :

a) 2 catchup mice and 2 positive control EAU mice are used to draw inferences about immune-mediated activity in response to injury. If the goal was to show 'feasibility' of imaging these mouse models for the purposes of tracking specific cell type behavior, the case is sufficiently made and already published by the authors earlier. It is possible that a larger sample size would alter the conclusion.

b) There are only 2 examples of extravasated neutrophils in the entire article, shown in the positive control EAU model. With the rare extravasation events of these cells and their high-speed motility, the chance of observing their exit from the vasculature is likely low overall, therefore the general conclusions made about their recruitment or lack thereof are not justified by these limited examples shown.

c) In Figure 3, the 3-day time point post laser injury shows an 18% reduction in the density of ONL nuclei (p-value of 0.17 compared to baseline). In the case of neutrophils, it is noted that "Control locations (n = 2 mice, 4 z-stacks) had 15 {plus minus} 8 neutrophils per sq.mm of retina whereas lesioned locations (n = 2 mice, 4 z-stacks) had 23 {plus minus} 5 neutrophils per sq.mm of retina (Figure 10b). The difference between control and lesioned groups was not statistically significant (p = 0.19)." These data both come from histology. While the p-values - 0.17 and 0.19 - are similar, in the first case a reduction in ONL cell density is concluded while in the latter, no difference in neutrophil density is inferred in the lesioned case compared to control. Why is there a difference in the interpretation where the same statistical test and methodology are used in both cases? Besides this statistical nuance, is there an alternate possibility that there is an increased, albeit statistically insignificant, concentration of circulating neutrophils in the lesioned model? The increase is nearly 50% (15 {plus minus} 8 vs. 23 {plus minus} 5 neutrophils per sq.mm) and the reader may wonder if a larger animal number might skew the statistic towards significance.

(2) The conclusions on the relative activity of neutrophils and microglia come from separate animals. The reader may wonder why simultaneous imaging of microglia and neutrophils is not shown in either the EAU mice or the fluorescently labeled catchup mice where the non-labeled cell type could possibly be imaged with phase-contrast as has been shown by the authors previously. One might suspect that the microglia dynamics are not substantially altered in these mice compared to the CX3CR1-GFP mice subjected to laser lesions, but for future applicability of this paradigm of in vivo imaging assessment of the laser damage model, including documenting the repeatability of the laser damage model and the immune cell behavior, acquiring these data in the same animals would be critical.

(3) Along the same lines as above, the phase contrast ONL images at time points from 3-day to 2-month post laser injury are not shown and the absence of this data is not addressed. This missing data pertains only to the in vivo imaging mice model but are conducted in histology that adequately conveys the time-course of cell loss in the ONL. It is suggested that the reason be elaborated for the exclusion of this data and the simultaneous imaging of microglia and neutrophils mentioned above. Also, it would be valuable to further qualify and check the claims in the Discussion that "ex vivo analysis confirms in vivo findings" and "Microglial/neutrophil discrimination using label-free phase contrast"

Diaspora: the jews living outside Israel (https://www.merriam-webster.com/dictionary/diaspora)

Interesting musical roots for Reggae... Wonder if this is still present?

Mystical roots.

(Note, I give this the fiction tag because I might want to look into this mystical religion for fiction writing as inspiration)

Logical that marijuana (a drug) is correlated with the mystical concept of communicating with diseased spirits for marijuana makes you hallucinate (or perhaps it's demonic in nature?)

It is important to create a shit tag (literally), to keep track of songs to delete.