= 🗺️ MEMpLEX Map

on - notation

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implicate/named interpretations means of combination means of abstraction

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

The manuscript by Raices et al., provides novel insights into the role and interactions between SPO-11 accessory proteins in C. elegans. The authors propose a model of meiotic DSBs regulation, critical to our understanding of DSB formation and ultimately crossover regulation and accurate chromosome segregation. The work also emphasizes the commonalities and species-specific aspects of DSB regulation.

Strengths:

This study capitalizes on the strengths of the C. elegans system to uncover genetic interactions between a large number of SPO-11 accessory proteins. In combination with physical interactions, the authors synthesize their findings into a model, which will serve as the basis for future work, to determine mechanisms of DSB regulation.

Weaknesses:

The methodology, although standard, lacks quantification. This includes the mass spectrometry data , along with the cytology. The work would also benefit from clarifying the role of the DSB machinery on the X chromosome versus the autosomes.

• We have uploaded the MS data and added a summary table with the number of peptides and coverage.

• We have added statistics to the comparisons of DAPI body counts.

• We have provided additional images of the change in HIM-5 localization

• We have quantified the overlap (or lack thereof) between XND-1 and HIM-17 and the DNA axis

Reviewer #2 (Public Review):

Summary:

Meiotic recombination initiates with the formation of DNA double-strand break (DSB) formation, catalyzed by the conserved topoisomerase-like enzyme Spo11. Spo11 requires accessory factors that are poorly conserved across eukaryotes. Previous genetic studies have identified several proteins required for DSB formation in C. elegans to varying degrees; however, how these proteins interact with each other to recruit the DSB-forming machinery to chromosome axes remains unclear.

In this study, Raices et al. characterized the biochemical and genetic interactions among proteins that are known to promote DSB formation during C. elegans meiosis. The authors examined pairwise interactions using yeast two-hybrid (Y2H) and co-immunoprecipitation and revealed an interaction between a chromatin-associated protein HIM-17 and a transcription factor XND-1. They further confirmed the previously known interaction between DSB-1 and SPO-11 and showed that DSB-1 also interacts with a nematodespecific HIM-5, which is essential for DSB formation on the X chromosome. They also assessed genetic interactions among these proteins, categorizing them into four epistasis groups by comparing phenotypes in double vs. single mutants. Combining these results, the authors proposed a model of how these proteins interact with chromatin loops and are recruited to chromosome axes, offering insights into the process in C. elegans compared to other organisms.

Weaknesses:

This work relies heavily on Y2H, which is notorious for having high rates of false positives and false negatives. Although the interactions between HIM-17 and XND-1 and between DSB-1 and HIM-5 were validated by co-IP, the significance of these interactions was not tested, and cataloging Y2H interactions does not yield much more insight.

We appreciate that the reviewer recognized the value of our IP data, but we beg to differ that we rely too heavily on the Y2H. We also provide genetic analysis on bivalent formation to support the physical interaction data. We do acknowledge that there are caveats with Y2H, however, including that a subset of the interactions can only be examined with proteins in one orientation due to auto-activation. While we acknowledge that it would be nice to have IP data for all of the proteins using CRISPR-tagged, functional alleles, these strains are not all feasible (e.g. no functional rec-1 tag has been made) and are beyond the scope of the current work.

Moreover, most experiments lack rigor, which raises serious concerns about whether the data convincingly supports the conclusions of this paper. For instance, the XND-1 antibody appears to detect a band in the control IP; however, there was no mention of the specificity of this antibody.

We previously showed the specificity of this antibody in its original publication showing lack of staining in the xnd-1 mutant by IF (Wagner et al., 2010). To further address this, however, we have now included a new supplementary figure (Figure S1) demonstrating the specificity of the XND-1 antibody by Western blot. The antibody detects a distinct band in extracts from wild-type (N2) worms, but this band is absent in two independent xnd-1 mutant strains. This confirms that the antibody specifically recognizes XND-1, supporting the validity of the IP results shown in the main figures.

Additionally, epistasis analysis of various genetic mutants is based on the quantification of DAPI bodies in diakinesis oocytes, but the comparisons were made without statistical analyses.

We have added statistical analysis to all datasets where quantification was possible, strengthening the rigor and interpretation of our findings.

For cytological data, a single representative nucleus was shown without quantification and rigorous analysis. The rationale for some experiments is also questionable (e.g. the rescue by dsb-2 mutants by him-5 transgenes in Figure 2), making the interpretation of the data unclear. Overall, while this paper claims to present "the first comprehensive model of DSB regulation in a metazoan", cataloging Y2H and genetic interactions did not yield any new insights into DSB formation without rigorous testing of their significance in vivo. The model proposed in Figure 4 is also highly speculative.

Regarding the cytology, we provide new images and quantification of HIM-17 and XND-1 overlap with the DNA axes. We also added full germ line images showing HIM-5 localization in wild type and dsb-1 mutants, to provide a more complete and representative view of the observed phenotype. To further support our findings, we’ve also included images demonstrating that this phenotype is consistently observed with both in live worm with the the him-5::GFP transgene and in fixed worms with an endogenously tagged version of HIM-5.

Reviewer #3 (Public Review):

During meiosis in sexually reproducing organisms, double-strand breaks are induced by a topoisomerase-related enzyme, Spo11, which is essential for homologous recombination, which in turn is required for accurate chromosome segregation. Additional factors control the number and genome-wide distribution of breaks, but the mechanisms that determine both the frequency and preferred location of meiotic DSBs remain only partially understood in any organism.

The manuscript presents a variety of different analyses that include variable subsets of putative DSB factors. It would be much easier to follow if the analyses had been more systematically applied. It is perplexing that several factors known to be essential for DSB formation (e.g., cohesins, HORMA proteins) are excluded from this analysis, while it includes several others that probably do not directly contribute to DSB formation (XND-1, HIM-17, CEP-1, and PARG-1).

We respectfully disagree with the reviewer’s statement regarding the selection of factors included in our analysis. In this work, our focus was specifically on SPO-11 accessory factors — proteins that directly interact with or regulate SPO-11 activity during doublestrand break formation. Cohesins and chromosome axis proteins (such as the HORMA domain proteins) are essential for establishing the correct chromosome architecture that supports DSB formation, but there is no evidence that they are direct accessory factors of SPO-11. Therefore, they were intentionally excluded from this study to maintain a clear and focused scope on proteins that more directly modulate SPO-11 function.

Conversely, XND-1, HIM-17, CEP-1, and PARG-1 have all been implicated in regulating aspects of SPO-11-mediated DSB formation or its immediate environment. Although their contributions mayinvolve broader chromatin or DNA damage response regulation, prior literature supports their inclusion as relevant modulators of SPO-11 activity, justifying their analysis within the context of this work.

The strongest claims seem to be that "HIM-5 is the determinant of X-chromosome-specific crossovers" and "HIM-5 coordinates the actions of the different accessory factors subgroups." Prior work had already shown that mutations in him-5 preferentially reduce meiotic DSBs on the X chromosome. While it is possible that HIM-5 plays a direct role in DSB induction on the X chromosome, the evidence presented here does not strongly support this conclusion. It is also difficult to reconcile this idea with evidence from prior studies that him-5 mutations predominantly prevent DSB formation on the sex chromosomes, while the protein localizes to autosomes.

HIM-5 is not the only protein that is autosomally enriched but preferentially affects the X chromosome: MES-4 and MRG-1 are both autosomally-enriched but influence silencing of the X chromosome. While HIM-5 appears autosomally-enriched, it does not appear to be autosomal-exclusive. While we would ideally perform ChIP to determine its localization on chromatin, this method for assaying DSB sites is likely insufficient to identify DSB sites which differ in each nucleus and for which there are no known hotspots in the worm.

him-5 mutants confer an ~50% reduction in total number of breaks and a very profound change in break dynamics (seen by RAD-51 foci (Meneely et al., 2012)). Since the autosomes receives sufficient breaks in this context to attain a crossover in >98% of nuclei, this indicates that the autosomes are much less profoundly impacted by loss of DSB functions than is the X chromosome. Indeed, prior data from co-author, Monica Colaiacovo, showed that fewer breaks occur on the X (Gao, 2015) likely resulting from differences in the chromatin composition of the X and autosome resulting from X chromosome silencing.

The conclusion that HIM-5 must be required for breaks on the X comes from the examination of DSB levels and their localization in different mutants that impair but do not completely abrogate breaks. In any situation where HIM-5 protein expression is affected (xnd-1, him-17, and him-5 null alleles), breaks on the X are reduced/ eliminated. By contrast, in dsb-2 mutants, where HIM-5 expression is unaffected, both X and autosomal breaks are impacted equally. As discussed above, in the absence of HIM-5 function, there are ~15 breaks/ nucleus. The Ppie1::him-5 transgene is expressed to lower levels than Phim-5::him-5, but in the best case, the ectopic expression of this protein should give a maximum of ~15 breaks (the total # of breaks is thought to be ~30/nucleus). By these estimates, Ppie-1::him-5; him-17 and him-5 null mutants have the same number of breaks. Yet, in the former case, breaks occur on the X; whereas in the latter they do not. The best explanation for this discrepancy is that HIM-5 is sufficient to recruits the DSB machinery to the X chromosome.

The one experiment that seems to elicit the conclusion that HIM-5 expression is sufficient for breaks on the X chromosome is flawed (see below). The conclusion that HIM-5 "coordinates the activities of the different accessory sub-groups" is not supported by data presented here or elsewhere.

We have reorganized the discussion to more directly address the reviewers’ concerns. We raise the possibility that HIM-5 has an important role in bringing together the SPO-11 and its interacting components (DSB-1/2/3) with the other DSB inducing factors, including those factors that regulating DSB timing (XND-1), coordination with the cell cycle (REC-1), association with the chromosome axis (PARG-1, MRE-11), and coupling to downstream resection and repair (MRE-11, CEP-1).  

This raises a natural question: if HIM-5 has such a central role, why are the phenotypes of HIM-5 so mild? We propose that while the loss of DSBs on the X appears mild, more profound effects are seen in the total number, timing, and placement of the DSBs across the genome- all of which are diminished or altered in the absence of HIM-5. The phenotypes of him-5 loss reminiscent of those observed in Prdm9-/- in mice where breaks are relocated to transcriptional start sites and show significant delay in formation. As with PRDM9, the comparatively subtle phenotypes of HIM-5 loss do not diminish its critical role in promoting proper DSB formation in most mammals.

Like most other studies that have examined DSB formation in C. elegans, this work relies on indirect assays, here limited to the cytological appearance of RAD-51 foci and bivalent chromosomes, as evidence of break formation or lack thereof. Unfortunately, neither of these assays has the power to reveal the genome-wide distribution or number of breaks. These assays have additional caveats, due to the fact that RAD-51 association with recombination intermediates and successful crossover formation both require multiple steps downstream of DSB induction, some of which are likely impaired in some of the mutants analyzed here. This severely limits the conclusions that can be drawn. Given that the goal of the work is to understand the effects of individual factors on DSB induction, direct physical assays for DSBs should be applied; many such assays have been developed and used successfully in other organisms.

We appreciate the reviewer’s thoughtful comments. We agree that RAD-51 foci are an indirect readout of DSB formation and that their dynamics can be influenced by defects in downstream repair processes. However, in C. elegans, the available methods for directly detecting DSBs are limited. Unlike other organisms, C. elegans lacks γH2AX, eliminating the possibility of using γH2AX as a DSB marker. TUNEL assays, while conceptually appealing, have proven unreliable and poorly reproducible in the germline context. Similarly, RPA foci do not consistently correlate with the number of DSBs and are influenced by additional processing steps.

Given these limitations, RAD-51 foci remain the most widely accepted surrogate for monitoring DSB formation in C. elegans. While we fully acknowledge the caveats associated with this approach — particularly the potential effects of downstream repair defects — RAD-51 analysis continues to provide valuable insight into DSB dynamics and regulation, especially when interpreted in combination with other phenotypic assessments.

Throughout the manuscript, the writing conflates the roles played by different factors that affect DSB formation in very different ways. XND-1 and HIM-17 have previously been shown to be transcription factors that promote the expression of many germline genes, including genes encoding proteins that directly promote DSBs. Mutations in either xnd-1 or him-17 result in dysregulation of germline gene expression and pleiotropic defects in meiosis and fertility, including changes in chromatin structure, dysregulation of meiotic progression, and (for xnd-1) progressive loss of germline immortality. It is thus misleading to refer to HIM-17 and XND-1 as DSB "accessory factors" or to lump their activities with those of other proteins that are likely to play more direct roles in DSB induction.

It is clear that we will not reach agreement about the direct vs indirect roles here of chromatin remodelers/transcription factors in break formation. In yeast, there is a precedent for SPP1 and in mouse for Prdm9, both of which could be described as transcription factors as well, as having roles in break formation by creating an open chromatin environment for the break machinery. We envision that these proteins function in the same fashion. The changes in histone acetylation in the xnd-1 mutants supports such a claim.

We do not know what the reviewer is referring to in statement that “XND-1 and HIM-17 have previously been shown to be transcription factors that promote the expression of many germline genes.” While the Carelli et al paper indeed shows a role for HIM-17 in expression of many germline genes, there is only one reference to XND-1 in this manuscript (Figure S3A) which shows that half of XND-1 binding sites overlap with the co-opted germline promoters. There is no transcriptional data at all on xnd-1 mutants, save our studies (referenced herein) that XND-1 regulates him-5 expression.

For example, statements such as the following sentence in the Introduction should be omitted or explained more clearly: "xnd-1 is also unique among the accessory factors in influencing the timing of DSBs; in the absence of xnd-1, there is precocious and rapid accumulation of DSBs as monitored by the accumulation of the HR strand-exchange protein RAD-51.

We are not sure what is confusing here. The distribution of RAD-51 foci is significantly altered in xnd-1 mutants and peak levels of breaks are achieved as nuclei leave the transition zone (Wagner et al., 2010; McClendon et al., 2016). There is no other mutation that causes this type of change in RAD-51 distribution.

"The evidence that HIM-17 promotes the expression of him-5 presented here corroborates data from other publications, notably the recent work of Carelli et al. (2022), but this conclusion should not be presented as novel here.

We have clarified this in the text. We note that this paper showed alterations in him-5 levels by RNA-Seq but they did not validate these results with quantitative RT-PCR. Thus, our studies do provide an important validation of their prior results.

The other factors also fall into several different functional classes, some of which are relatively well understood, based largely on studies in other organisms. The roles of RAD50 and MRE-11 in DSB induction have been investigated in yeast and other organisms as well as in several prior studies in C. elegans. DSB-1, DSB-2, and DSB-3 are homologs of relatively well-studied meiotic proteins in other organisms (Rec114 and Mei4) that directly promote the activity of Spo11, although the mechanism by which they do so is still unclear.

Whilst we agree that we understand some of the functions of the homologs, there are clearly examples in other processes of conserved proteins adopting unique regulatory function. We should not presume evolutionary conservation until proven. Indeed the comparison between the Mer2 proteins becomes particularly relevant here. For example, the RMM complex in plants does not contain PRD3, although this protein is thought to have function in DSB formation and repair (Lambing et al, 2022; Vrielynck et al., 2021; Thangavel et al., 2023). In Sordaria, as well, the Mer2 homolog has distinct functions (Tesse et al., 2017).  

Mutations in PARG-1 (a Poly-ADP ribose glycohydrolase) likely affect the regulation of polyADP-ribose addition and removal at sites of DSBs, which in turn are thought to regulate chromatin structure and recruitment of repair factors; however, there is no convincing evidence that PARG-1 directly affects break formation.

Our prior collaborative studies on PARG-1 showed that is has a non-catalytic function that promote DSBs that is independent of accumulation of PAR (Janisiw et al., 2020; Trivedi et al., 2022)

CEP-1 is a homolog of p53 and is involved in the DNA damage response in the germline, but again is unlikely to directly contribute to DSB induction.

We respectfully disagree with the reviewer’s statement. While CEP-1 is indeed a homolog of p53 and plays a major role in the DNA damage response, prior work from Brent Derry’s lab and from our group (Mateo et al., 2016) demonstrated that specific cep-1 separationof-function alleles affect DSB induction and/or repair pathway choice independently of canonical DNA damage checkpoint activation. In particular, defects in DSB formation observed in certain cep-1 mutants can be rescued by exogenous irradiation, supporting a direct or closely linked role in promoting DSB formation rather than merely responding to damage. Thus, based on these functional data, we considered CEP-1 a relevant factor to include in our analysis. We have now clarified this rationale in the revised manuscript.

HIM-5 and REC-1 do not have apparent homologs in other organisms and play poorly understood roles in promoting DSB induction. A mechanistic understanding of their functions would be of value to the field, but the current work does not shed light on this. A previous paper (Chung et al. G&D 2015) concluded that HIM-5 and REC-1 are paralogs arising from a recent gene duplication, based on genetic evidence for a partially overlapping role in DSB induction, as well as an argument based on the genomic location of these genes in different species; however, these proteins lack any detectable sequence homology and their predicted structures are also dissimilar (both are largely unstructured but REC-1 contains a predicted helical bundle lacking in HIM-5). Moreover, the data presented here do not reveal overlapping sets of genetic or physical interactions for the two genes/proteins. Thus, this earlier conclusion was likely incorrect, and this idea should not be restated uncritically here or used as a basis to interpret phenotypes.

Actually, there is quite good bioinformatic analysis that the rec-1 and him-5 loci evolved from a gene duplication and that each share features of the ancestral protein (Chung et al., 2015). We are sorry if the reviewer casts aspersions on the prior literature and analyses. The homology between these genes with the ancestral protein is near the same degree as dsb-1, dsb-2, or dsb-3 to their ancestral homologs (<17%).

DSB-1 was previously reported to be strictly required for all DSB and CO formation in C. elegans. Here the authors test whether the expression of HIM-5 from the pie-1 promoter can rescue DSB formation in dsb-1 mutants, and claim to see some rescue, based on an increase in the number of nuclei with one apparent bivalent (Figure 2C). This result seems to be the basis for the claim that HIM-5 coordinates the activities of other DSB proteins. However, this assay is not informative, and the conclusion is almost certainly incorrect. Notably, a substantial number of nuclei in the dsb-1 mutant (without Ppie-1::him-5) are reported as displaying a single bivalent (11 DAPI staining bodies) despite prior evidence that DSBs are absent in dsb-1 mutants; this suggests that the way the assay was performed resulted in false positives (bivalents that are not actually bivalents), likely due to inclusion of nuclei in which univalents could not be unambiguously resolved in the microscope. A slightly higher level of nuclei with a single unresolved pair of chromosomes in the dsb-1; Ppie-1::him-5 strain is thus not convincing evidence for rescue of DSBs/CO formation, and no evidence is presented that these putative COs are X-specific. The authors should provide additional experimental evidence - e.g., detection of RAD-51 and/or COSA-1 foci or genetic evidence of recombination - or remove this claim. The evidence that expression of Ppie-1::him-5 may partially rescue DSB abundance in dsb-2 mutants is hard to interpret since it is currently unknown why C. elegans expresses 2 paralogs of Rec114 (DSB-1 and DSB-2), and the age-dependent reduction of DSBs in dsb-2 mutants is not understood.

We have removed this claim in part because we have been unable to create the triple mutants strains to analyze COSA-1 foci.

To the point about 11 vs 12 DAPI bodies: the literature is actually replete with examples of 11 DAPI bodies vs 12 in mutants with no breaks:

Hinman al., 2021: null allele of dsb-3 has an average of 11.6 +/- 0.6 breaks;

Stamper et al, 2013, show just over 60% of dsb-1 nuclei with 12 DAPI bodies and 5-10% with 10 DAPI bodies. (Figure 1);

In addition, we also previously showed (Machovina et al., 2016) that a subset of meiotic nuclei have a single RAD-51 focus and can achieve a crossover. RAD-51 foci in spo-11 were also reported in Colaiacovo et al., 2003.

Several of the factors analyzed here, including XND-1, HIM-17, HIM-5, DSB-1, DSB-2, and DSB-3, have been shown to localize broadly to chromatin in meiotic cells. Coimmunoprecipitation of pairs of these factors, even following benzonase digestion, is not strong evidence to support a direct physical interaction between proteins.

Similarly, the super-resolution analysis of XND-1 and HIM-17 (Figure 1EF) does not reveal whether these proteins physically interact with each other, and does not add to our understanding of these proteins functions, since they are already known to bind to many of the same promoters. Promoters are also likely to be located in chromatin loops away from the chromosome axis, so in this respect, the localization data are also confirmatory rather than novel.

While the binding to promoters would be expected to be on DNA loops, that has not been definitively shown in the worm germ line. The supplemental data of the Carelli paper suggests that there are ~250 binding sites for each protein at these coopted promoters. This could not account for crossover map seen in C. elegans.

The reviewer states correct that we do not reveal that these proteins interact, but we have shown that the two proteins co-IP and have a Y2H interaction. This interaction is supporedt by a recent publication (Blazickova et al., 2025) corroborating this conclusion and identifies XND-1 in HIM-17 co-IPs also in the presence of benzonase. We do now show, however, by immuno-localization that the two proteins appear to be adjacent, but nonoverlapping. As now described in the text, AlphaFold 3 modeling and structural analysis suggests that the two proteins do interact directly and that the tagged 5’ end of HIM-17 used in our studies is likely to be at least 200nm from the putative XND-1 binding interface, a distance that is consistent with our confocal images showing frequent juxtaposition of the two proteins.

The phenotypic analysis of double mutant combinations does not seem informative. A major problem is that these different strains were only assayed for bivalent formation, which (as mentioned above) requires several steps downstream of DSB induction. Additionally, the basis for many of the single mutant phenotypes is not well understood, making it particularly challenging to interpret the effects of double mutants. Further, some of the interactions described as "synergistic" appear to be additive, not synergistic. While additive effects can be used as evidence that two genes work in different pathways, this can also be very misleading, especially when the function of individual proteins is unknown. I find that the classification of genes into "epistastasis groups" based on this analysis does not shed light on their functions and indeed seems in some cases to contradict what is known about their functions. ‘

As described above, each of the proteins analyzed is thought to have a direct role in regulating meiotic DSB formation and single mutant phenotypes are consistent with this interpretation. In almost all-if not all- of these cases, IR induced breaks suppress univalent phenotypes (or uncover a downstream repair defect (e.g. in mre-11)) supporting this conclusion. We have changed the terminology from “epistasis groups” since this is not strict epistasis, but rather, “functional groups”.  

The yeast two-hybrid (Y2H) data are only presented as a single colony. While it is understandable to use a 'representative' colony, it is ideal to include a dilution series for the various interactions, which is how Y2H data are typically shown.

The Y2H data are presented as spots on a plate and are from three to four individual transformants per interaction tested, and are not individual colonies. The experiment was repeated in triplicate from different transformations. We have now made this clearer in the materials and methods section. This approach has been successfully used to examine protein interactions in our prior manuscripts of yeast and human proteins [Gaines et al (2015) Nat. Comms 6:7834; Kondrashova et al (2017) Cancer Discovery 7:984; Garcin et al (2019) PLoS Genetics 15:e1008355; Bonilla et al (2021) eLife 1: e68080) Prakash et al (2022) PNAS 119: e2202727119, etc]

Additional (relatively minor) concerns about these data:

(1) Several interactions reported here seem to be detected in only one direction - e.g., MRE-11-AD/HIM-5-BD, REC-1-AD/XND-1-BD, and XND-1-AD/HIM-17-BD - while no interactions are seen with the reciprocal pairs of fusion proteins. I'm not sure if some of this is due to pasting "positive" colony images into the wrong position in the grid, but this should be addressed.

The asymmetry in the interactions observed is due to the well-known phenomenon in yeast two-hybrid (Y2H) assays where certain plasmids exhibit self-activation when fused in one orientation, making interpretation of reciprocal interactions challenging. In our experiment, some of the plasmids indeed showed self-activation in one direction, which likely accounts for the lack of interaction seen with the reciprocal pairs of fusion proteins. We have clarified this point in the Methods.

(2) DSB-3 was only assayed in pairwise combinations with a subset of other proteins; this should be explained; it is also unclear why the interaction grids are not symmetrical about the diagonal.

We have now completed the analysis by adding the interactions of DSB-3 with the remaining proteins that were missing from the initial set.

(3) I don't understand why the graphic summaries of Y2H data are split among 3 different figures (1, 2, and 3).

We chose to split the graphic summaries of the Y2H data across Figures 1, 2, and 3 because we felt this organization better aligns with the flow of the results presented in each figure. Each set of interactions is shown in the context of the specific experiments and findings discussed in those sections, which we believe helps provide a clearer and more logical presentation of the data.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

Figure 1: B) The IP is difficult to interpret - there is a band of the corresponding size to XND-1 in the control lane calling into question the specificity of the IP/Western.

We added a supplemental figure with the specificity of the antibody showing that there is a background non-specific band.

C) More information about the mass spectrometry should be included. No indication of the number of times a peptide was identified, or the overall coverage of the identified proteins.

Done

This is important as in the results section (line 114) the authors indicate that there was "strong" interaction yet there is no way to assess this.

D) Why wasn't hatching measured in the him-5p::him-5; him-17(ok424) strain?

Great question. I guess we need to do this while back out for review. If anyone has suggestions of what to say here. Clearly we overlooked this point but do have the strain.

E) Quantification of the cytology should be included.

We have now quantified overlap between XND-1 and HIM-17

Figure 2: C) Statistics should be included.

Done

E) Quantification should be included for the cytology. I recommend changing the eals15 to HIM-5.

We included better images showing whole gonads instead of one or two nuclei. We were not sure what the reviewers want us to quantify here since the relocalization of the protein to the cytoplasm is very clear.

I have a general issue with the use of the term epistasis - this is used to order gene function based on different mutant phenotypes, usually with null alleles. While I think the authors have valid points with how they group the different SPO-11 accessory proteins, I do not think they should use the word epistasis, but rather genetic interactions.

We appreciate the reviewers thoughts on this matter and have removed the term epistasis and use functional groups or genetic interactions throughout the text.

Figure 4 and the nature of the X chromosome: First, I think it would help the non-C. elegans reader to include a little more information on the X chromosome with respect to its differences compared to the autosomes. I also think that, if possible, it would be beneficial to include a model of the X in Figure 4.

We have added more about X/autosome differences in the intro and during the discussion of HIM-5 function and have added a figure showing difference in the behavior of the X/autosomes during DSB/crossover formation.

Minor points:

Abstract: Given the findings of Silva and Smolikove on SPO-11 breaks, I recommend removing "early" from line 28 in the Abstract.

Done

Introduction (line 93): I think "biochemical studies" is a stretch here - I recommend "interaction studies".

Done

Results: (lines 160-161): mutations are not required for breaks. Line 172, there is a problem with the sentence.

Corrected

Reviewer #2 (Recommendations For The Authors):

Major comments:

(1) Figure 1B- The signal for XND-1 seems to appear both in the control and him-17::HA IP. Do the authors have tested the specificity of the XND-1 antibody?

We included a supplementary figure demonstrating the specificity of the XND-1 antibody by Western blot. This was also previously published (Wagner et al., 2010)

(2) Figure 1D - can the authors provide an explanation why the him-5p::him-5 transgene that drives a higher expression than pie-1p::him-5 fails to suppress the Him phenotype seen in him-17? What are the HIM-5 levels like in these two strains compared to N2 and him-17 null mutants? Can this information provide explanation for the differential effect of the him-5 transgenes?

We previously reported that him-5p::him-5 drives higher expression than pie-1p::him-5 (McClendon et al, 2016).

The reason that him-5p::him-5 does not rescue, despite higher wild type expression is that HIM-17 directly regulates expression of him-5. Since HIM-17 does not regulate the pie-1 promoter, the pie-1p::him-5 construct can at least partially suppress the him-17 mutation.

We have (hopefully) explained this better in the text.  

(3) Line 102- the subheading "HIM-5 is the essential factor for meiotic breaks in the Xchromosome" may not be appropriate for this section. This is what has previously been known. However, the results in Figure 1 demonstrate that a him-5 transgene can partially rescue the him-17 and ¬xnd-1 phenotype, but not that it is essential for meiotic DSB formation on X chromosomes.

We think some of the concern here is sematic and have changed the phraseology to say that HIM-5 is SUFFICIENT for DSBs on the X… which had not previously been shown.

Vis-à-vis the X chromosome, in all genetic backgrounds examined, the absence of HIM-5 consistently results in a complete lack of DSBs on the X. For instance, in dsb-2 mutants— where HIM-5 is still expressed—DSBs are reduced genome-wide, but the X chromosome occasionally retains breaks. In contrast, even a weak allele of him-17 results specifically in the loss of X chromosome breaks, underscoring a unique requirement for HIM-5 in promoting DSBs on the X. While Figure 1 shows that a him-5 transgene can partially rescue him-17 and xnd-1 phenotypes, the consistent observation that X breaks are absent without HIM-5 supports its classification as sufficient for DSB formation on the X chromosome.

(4) Figure 1E - please consider enlarging the images and showing multiple examples.

Done.

I also suggest that the authors perform a more rigorous analysis to support the conclusion that XND-1 and HIM-17 localize away from the axis by quantifying multiple images and doing line-scan analysis.

Provided. New images are provided in both, the main and supplemental figures, and quantification is included. There is no detectable overlap of the two protein with one another or the DNA axes (see quantification of overlap in Fig. 1).

(5) Line 162 - This is the first mention of DSB-1, DSB-2, and DSB-3 in the paper. DSB-1 and DSB-2 are Rec114 homologs in C. elegans (Tesse et al., 2017), while DSB-3 is a homolog of Mei4 (Hinman et al., 2021). These proteins should be properly introduced in the introduction with appropriate citations.

Done. We appreciate the reviewer pointing out that this was the first reference to these genes.

(6) Line 169 - the rationale for this experiment is unclear. Why did the Y2H interaction between HIM-5 and DSB-1 prompt the authors to test the rescue of dsb-1 or dsb-2 phenotypes by the ectopic expression of him-5? Do the authors have evidence that HIM-5 level is reduced in dsb-1 or dsb-2 mutants?

We have reorganized this section to better explain the motivation for looking at these interactions. We did see a difference in the localization in HIM-5 in the dsb-1 mutant animals and we did have a sense that HIM-5 was critical for breaks on the X. We reasoned that it could have independent functions in promoting breaks that were not yet appreciated so wanted to do this experiment.

(7) Line 172 - "very slightly reduced". This claim requires statistical analysis.

We added statistical analysis, but we also removed this claim.

(8) Figures 2C and 2D - Can the authors provide an explanation why the pie-1p::him-5 transgene fails to suppress the phenotypes in dsb-1, while the him-5p::him-5 trasgene can? Again, the rationale for these experiments is unclear. Because of this, the interpretation is also unclear.

The difference in rescue between the pie-1p::him-5 and him-5p::him-5 transgenes likely reflects differences in expression levels. As previously shown (McClendon et al., 2016), the him-5p::him-5 construct results in significantly higher expression of HIM-5 protein compared to pie-1p::him-5. This elevated expression likely explains its ability to partially rescue the dsb-1 phenotype. In contrast, the lower expression driven by the pie-1 promoter is insufficient to compensate for the absence of dsb-1 function. We have clarified the rationale and interpretation of these experiments in the revised manuscript to better reflect this point.

(9) Lines 184-185 - the data for endogenously tagged HIM-5::3xHA are not shown anywhere in the paper. This must be shown.

We have added this in the supplemental figures.

(10) Figure 2D and 2E - what does the localization of pie-1p::him-5::GFP (eaIs15) and him5p::him-5::GFP (eaIs4) look like in wild-type and dsb-1 mutants? Are the cytoplasmic aggregates caused by increased levels of HIM-5 expression? Can the differential behavior of him-5 transgenes provide explanation for differential rescues?

We now show both live and fixed images of Phim-5::him-5::gfp transgenes, as well as the localization of the endogenously HA-tagged HIM-5 locus (Figure 2 and S3). In all cases, the protein is initially nuclear and then absent from meiotic nuclei with similar timing. The Ppie1::him-5 transgene was very difficult to image due to low expression (even in wild type) so it not shown here. We presume it is the slightly elevated level of expression of the Phim5::him-5::gfp that can explain the differential rescue.

(11) Lines 221-222, where are the results shown? Please refer to Figure S3.

Done

(12) Figure S3 - these need statistical analyses.

Done

(13) Lines 230-231 - what about the rec-1; parg-1; cep-1 triple mutant?

This is an excellent suggestion and not one we have not yet pursued. Given the lack of strong phenotypes in all combination of double mutants, we prioritized other experiments . However, we agree that examining the rec-1; parg-1; cep-1 triple mutant would provide a valuable test of whether these factors act in the same pathway, and we appreciate the reviewer highlighting this potential future direction.

(14) Line 298 - I suggest the authors take a look at the Alphafold prediction of DSB-1/DSB-2/DSB-3 and the comparison to human and budding yeast Rec114/Mei4 complex in Guo et al., 2022 eLife, which could provide insights into the Y2H results.

We thank the reviewer for these comments and have indeed used these interactions and predicted homologies to zero in a region of interaction between these proteins that resembles what is seen in humans and yeast with a dimer of REC114 like proteins wraps stabilizing a central Mei4 helix . This is now shown in Figure 3H, I. Satisfyingly, this modeling predicts that a trimer comprised of 2 DSB-1 proteins with DSB-3 is more stable than a DSB1-DSB-2-DSB-3 trimer. This might explain why DSB-2 is not required in young adults and only becomes essential as DSB-1 levels drop in older animals (Rosu et al., 2013)

(15) Can the authors introduce mutations within the DSB-1 interfaces that disrupt the interaction to either SPO-11 or DSB-2?

We have begun to address this question by introducing targeted mutations within DSB-1. As shown in Figure 3E and 3F, mutations in the C-terminal region of DSB-1—which includes a core of four α-helices—disrupt its interaction with DSB-2 and DSB-3, but not with SPO-11. These findings suggest that the C-terminus mediates interactions specifically with DSB2 and DSB-3

(16) Line 323 - The him-5 phenotypes are too weak to support the idea that it serves as the linchpin for the whole DSB complex. Do the authors have an explanation for why him-5 mutants exhibit X-chromosome-specific DSB defects?

In response to the reviewer, above, and in the text, we have included a more detailed explanation of why we think HIM-5 has a key role in coordinating meiotic break formation. Although, identified for its role on the X, the phenotypes associated with DSB formation in the mutant are really quite pleiotropic and severe.

(17) Line 436 - C. elegans lacks DSB hotspots.

Removed

Minor comments:

(1) Figure 1A - please show the raw data for the yeast two-hybrid.

We show representative yeast colonies in Figure S3.

(2) It looks like the labeling for Figure 1B and 1C are switched.

Fixed.

(3) Figure 1B - what does the red box indicate? Please explain it in the legend.

It indicates the XND-1 band. We added that information in the legend.

(4) Figure 1C - in the legend, it was noted that the results are from GFP pulldowns of HIM17::GFP. However, the method for Figure 1B and the method section noted that HIM-17 was tagged with 3xHA, and the pull-down was performed using anti-HA affinity matrix. Please reconcile this discrepancy.

That’s because they were done in two different sets of experiments. For the IPs we used a HIM-17::HA strain and for the MS, a HIM-17::GFP strain.

(5) Also in Figure 1C - please call Table S2 in the main text when discussing the mass spec results. Also, it is not clear what HIM-17 and GFP indicate in the table. What makes CKU80 different from the other proteins listed under GFP? Please explain more clearly in the legend.

We have move the table to supplemental data where we have included all of the peptide counts and gene coverage. We have included in the revised method rationale for inclusion in this table which explains why CKU-80 differs.

(6) Line 527 - it is unclear what experiment was done for HIM-17. Please revise it to indicate that this is for "HIM-17 immunoprecipitation". Also please indicate the strain used for HIM17 pull-down (AV280?).

(7) Line 113- please be specific about how the HIM-17 IP was performed. Which epitope and strains are used for pull-downs?

This indeed was AV280. This has been added to the text and methods.

(8) Figure 1D- What does ND mean? In the text, it was stated that there was only a minor suppression of hatching rates. The hatching rate for him-5p::him-5; him-17 must have been measured, and the data must be presented.

ND does mean not determined. We have removed the statement about “minor suppression”. We only tested the overall population dynamics in the Phim-5::him-5;him17(ok424) and the DAPI body counts. The failure to suppress the latter suggests there would be no enect on hatching rates, although we did not test this directly. Since we had done this for the Ppie-1::him-5;him-17 strain, we provided this information to further support the claims of genetic rescue by ectopic expression.

(9) Line 151 - please specify that STED was used.

We have removed the STED images, and just show the confocal images with Lightning Processing.

(10) Figure 1E- the authors suggested that HIM-17 and XND-1 mainly localize to autosomes but not the X chromosome. However, there is not enough evidence that the chromosome excluded from HIM-17 staining is indeed an X chromosome.

(11) Figure 1E (Line 154) - what are the active chromatin markers examined? Where are the data?

We have previously shown that the chromosome lacking XND-1 staining is the X (Wagner et al., 2010). The X is heterochromatic and chromatin marks associated with active transcription, including H3K4me3 and HTZ-1 (a variant H2A), preferentially localize to autosomes, effectively anti-marking the X chromosome. As shown in the new Figure 1E, a single chromosome has very little XND-1 and HIM-17 associated proteins. This is the X chromosome.

(12) Line 172 - It should be a comma instead of the period after "In dsb-1 mutants".

Fixed

(13) Figure S3H-K - I suggest the authors indicate the alleles of mre-11 (null vs. iow1) on the graph, similarly to him-5(e1490) to avoid confusion.

Done

(14) Lines 294 and 600 - Guo et al. 2022 is now published in eLife. The authors must cite the published paper, not the preprint.

Fixed

(15) Line 407 - the reference Carelli et al., 2022 is missing.

Added

(16) Line 766 - please remove "is" before nuclear.

Done

Reviewer #3 (Recommendations For The Authors):

Major issues:

In my view, the most interesting mechanistic finding in the paper is the evidence that HIM-5 may not bind to chromatin in the absence of DSB-1. If validated, this would suggest that HIM-5 is likely to be directly involved in a process that promotes break formation, in contrast to factors such as HIM-17 and XND-1. It does not, however, support the idea that HIM-5 is at the top of a hierarchy of DSB factors, as it is interpreted here. More importantly, the data supporting this claim are unconvincing; only a single image of an unfixed gonad from an animal expressing HIM-5::GFP is shown. Immunofluorescence should be performed and the results must be quantified.

We have provided additional images of the HIM-5 relocalization to show that we observed this in both fixed and live worms with two different tagged strains. The exclusion from the nucleus is seen in all scenarios. Whether the protein now accumulates exclusively in the cytoplasm/ is destabilized is challenging to address with the fixed images due to the arbitrariness of defining “background” staining.

More generally, this type of analysis, looking at the interdependence of different factors for their association with chromosomes, is much more informative than the genetic interaction data presented in the paper, which does not seem to provide any mechanistic insights into the functions of the factors analyzed. The paper could potentially be greatly improved through a more extensive, systematic analysis of the interdependence of DSBpromoting factors for their localization to chromosomes.

We have at least added this for XND-1 and HIM-17 and show they are not interdependent for chromosome association. We also provide for the first time data on the localization of HIM-5 in the dsb-1 mutant. Many of the other interactions have already been shown in the literature and/or were not warranted base on the lack of genetic interaction we present here.

Minor issues:

The title is vague and inconclusive. A more concrete title summarizing the major findings would help readers to assess whether the work is of interest.

We have discussed the title extensively with all authors and all would like to keep the current title.

The authors claim that the expression of HIM-5 from a different promoter (Ppie-1::him-5) but not its endogenous promoter (Phim-5::him-5) can partially rescue the DSB defect in him-17 mutants. To support this claim, they should really quantify the germline expression of HIM-5 in wild-type, him-17, him-17; Ppie-1::him-5, and Phim-5::him-5; him-17.

We had previously reported the expression in the N2 background of both transgenes (McClendon et al., 2016)

Panel O appears to be missing from Figure S3.

Fixed

The evidence for chromosome fusions in cep-1; mre-11 mutants shown in S4D is not convincing and the claim should be removed unless stronger evidence can be obtained.

A clearer image has been added

The basis of the following statement is unclear: "Furthermore, rec-1;him-5 double mutants give an age-dependent severe loss of DSBs (like dsb-2 mutants) suggesting that the ancestral function of the protein may have a more profound effect on break formation." The manuscript does not seem to include data regarding age-dependent loss of DSBs and no other publication is cited to support this claim. The interpretation is also perplexing; I think that it may be predicated on the idea that REC-1 and HIM-5 are paralogs, but as stated above, this claim is not well supported and is likely specious.

We have added the reference. This was shown in Chung et al., 2013 – the paper that presented the cloning of the rec-1 locus.

Google Colaboratory

Colab is a hosted Jupyter Notebook service

DOI: 10.1016/j.celrep.2025.116341

Resource: (BioLegend Cat# 317428, RRID:AB_1186122)

Curator: @scibot

SciCrunch record: RRID:AB_1186122

What is this?

This text shows that clearly one of the issues why multicultural education is not being implemented is because teacher's do not have the information and training they need to implement it into their routines. This issue makes it almost impossible for multicultural aspects to be seen in the classroom because the teacher has no knowledge about it as talked about in the text. Higher up departments should be held accountable to put these trainings into place and to implement these concepts into schools for there to be an actual change.

Sí, puede ser importante no hacer sobreúso de términos. No decir que todo es genocidio, o genocida.

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• Tema: 950
• Processo(s): RE 632.115
• Relator: Min. Luís Roberto Barroso
• Título: Responsabilidade civil objetiva do Estado por atos protegidos por imunidade parlamentar.

O Tribunal fixou a seguinte tese: - 1. A imunidade material parlamentar (art. 53, caput, c/c art. 27, § 1º, e art. 29, VIII, CF/1988) configura excludente da responsabilidade civil objetiva do Estado (art. 37, § 6º, CF/1988), afastando qualquer pretensão indenizatória em face do ente público por opiniões, palavras e votos cobertos por essa garantia.

• 2. Nas hipóteses em que a conduta do parlamentar extrapolar os limites da imunidade material, eventual responsabilização recairá de forma pessoal, direta e exclusiva sobre o próprio parlamentar, sob o regime de responsabilidade civil subjetiva.

<u>HEMENÊUTICA CONSTITUCIONAL</u>

MÉTODO TÓPICO-PROBLEMÁTICO - Fonte: A Tópica e a Argumentação Jurídica, por Thomas da Rosa de Bustamante

Resumo: A Tópica Jurídica de Theodor Viehweg

I. Definição, Contexto e Propósito

Marco no Pensamento Jurídico: A obra Tópica e Jurisprudência (1953) rompeu com o positivismo dominante, que via o jurista como um mero aplicador da lei.

Crítica ao Positivismo: Em um contexto pós-guerra, a Tópica surgiu como resposta à incapacidade do positivismo de lidar com juízos de valor de forma racional. Viehweg buscou recuperar a razão prática para controlar a racionalidade das decisões.

II. Os Três Pilares Conceituais

1. A Aporia (O Problema)
   • Definição: Uma "questão estimulante e ineludível" que admite mais de uma resposta.
   • Aporia Fundamental do Direito: "O que é justo aqui e agora".
   • Função: É o ponto de partida do pensamento, estimulando a busca por uma solução.
2. A Tópica (O Método)
   • Definição: Uma técnica de pensamento orientada pelo problema (techne), não uma ciência com verdades absolutas (episteme).
   • Função Principal: É uma "arte de invenção" (ars inveniendi) para buscar e encontrar as premissas do raciocínio.
   • Natureza: Dialética, partindo de opiniões verossímeis e aceitas (endoxa).
3. O Topos (A Ferramenta)
   • Definição: Os topoi (plural) são "pontos de vista utilizáveis e aceitáveis" que servem como "fórmulas de procura" para argumentos.
   • Tipos: A Tópica pode ser de primeiro grau (busca casual de topoi) ou de segundo grau (uso de catálogos de topoi já consolidados).

III. Tópica vs. Pensamento Sistemático

Pensamento Sistemático: Parte de um sistema fechado para deduzir soluções. A prioridade é o sistema.

Pensamento Tópico: Parte do problema concreto (aporia) para buscar, em vários sistemas, a melhor solução. A prioridade é o problema.

IV. Legado e Críticas

Legado:

• Ponto de partida das teorias modernas da argumentação jurídica.
• Revelou o raciocínio jurídico como um processo dialógico e discursivo.
• Mudou o foco da linguagem jurídica para sua dimensão pragmática.

Críticas:

• Não estabelece hierarquia ou regras de preferência entre topoi conflitantes.
• Não deixa claro o papel da lei, que poderia ser vista como apenas mais um topos.
• Partiu de um conceito restrito de "sistema", que o próprio Viehweg reviu posteriormente.

Quem organiza o SNCTI é o regime de colaboração entre entes públicos e privados. Não compete apenas à União organizar o SNCTI

• Informativo 1110
• RE 1017365 / SC - Tema nº 1031
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EDSON FACHIN
• Julgamento: 27/09/2023 (Presencial)
• Ramo do Direito: Constitucional, Administrativo
• Matéria: Ordem Social; Índios; Demarcação de Terras; Posse Tradicional; Direito Originário Territorial/Domínio Público; Terras Indígenas; Procedimento Declaratório de Direito Originário

Demarcação de terras tradicionalmente indígenas: desnecessidade de um marco temporal como parâmetro à declaração do direito originário territorial

Tese fixada - I - A demarcação consiste em procedimento declaratório do direito originário territorial à posse das terras ocupadas tradicionalmente por comunidade indígena;

• II - <u>A posse tradicional indígena é distinta da posse civil</u>, consistindo na ocupação das terras habitadas em caráter permanente pelos indígenas, nas utilizadas para suas atividades produtivas, nas imprescindíveis à preservação dos recursos ambientais necessários a seu bem-estar e nas necessárias a sua reprodução física e cultural, segundo seus usos, costumes e tradições, nos termos do § 1º do artigo 231 do texto constitucional;

• III - A proteção constitucional aos direitos originários sobre as terras que tradicionalmente ocupam independe da existência de um marco temporal em 05 de outubro de 1988 ou da configuração do renitente esbulho, como conflito físico ou controvérsia judicial persistente à data da promulgação da Constituição;

• IV – Existindo ocupação tradicional indígena ou renitente esbulho contemporâneo à promulgação da Constituição Federal, aplica-se o regime indenizatório relativo às benfeitorias úteis e necessárias, previsto no § 6º do art. 231 da CF/88;

• V – Ausente ocupação tradicional indígena ao tempo da promulgação da Constituição Federal ou renitente esbulho na data da promulgação da Constituição, são válidos e eficazes, produzindo todos os seus efeitos, os atos e negócios jurídicos perfeitos e a coisa julgada relativos a justo título ou posse de boa-fé das terras de ocupação tradicional indígena, assistindo ao particular direito à justa e prévia indenização das benfeitorias necessárias e úteis, pela União; e, quando inviável o reassentamento dos particulares, caberá a eles indenização pela União (com direito de regresso em face do ente federativo que titulou a área) correspondente ao valor da terra nua, paga em dinheiro ou em títulos da dívida agrária, se for do interesse do beneficiário, e processada em autos apartados do procedimento de demarcação, com pagamento imediato da parte incontroversa, garantido o direito de retenção até o pagamento do valor incontroverso, permitidos a autocomposição e o regime do § 6º do art. 37 da CF;

• VI – Descabe indenização em casos já pacificados, decorrentes de terras indígenas já reconhecidas e declaradas em procedimento demarcatório, ressalvados os casos judicializados e em andamento;

• VII – É dever da União efetivar o procedimento demarcatório das terras indígenas, sendo admitida a formação de áreas reservadas somente diante da absoluta impossibilidade de concretização da ordem constitucional de demarcação, devendo ser ouvida, em todo caso, a comunidade indígena, buscando-se, se necessário, a autocomposição entre os respectivos entes federativos para a identificação das terras necessárias à formação das áreas reservadas, tendo sempre em vista a busca do interesse público e a paz social, bem como a proporcional compensação às comunidades indígenas (art. 16.4 da Convenção 169 OIT);

• VIII – A instauração de procedimento de redimensionamento de terra indígena não é vedada em caso de descumprimento dos elementos contidos no artigo 231 da Constituição da República, por meio de pedido de revisão do procedimento demarcatório apresentado até o prazo de cinco anos da demarcação anterior, sendo necessário comprovar grave e insanável erro na condução do procedimento administrativo ou na definição dos limites da terra indígena, ressalvadas as ações judiciais em curso e os pedidos de revisão já instaurados até a data de conclusão deste julgamento;

• IX - O laudo antropológico realizado nos termos do Decreto nº 1.775/1996 é um dos elementos fundamentais para a demonstração da tradicionalidade da ocupação de comunidade indígena determinada, de acordo com seus usos, costumes e tradições, na forma do instrumento normativo citado;

• X - As terras de ocupação tradicional indígena são de posse permanente da comunidade, cabendo aos indígenas o usufruto exclusivo das riquezas do solo, dos rios e lagos nelas existentes;

• XI - As terras de ocupação tradicional indígena, na qualidade de terras públicas, são inalienáveis, indisponíveis e os direitos sobre elas imprescritíveis;

• XII – A ocupação tradicional das terras indígenas é compatível com a tutela constitucional do meio ambiente, sendo assegurado o exercício das atividades tradicionais dos povos indígenas;

• XIII – Os povos indígenas possuem capacidade civil e postulatória, sendo partes legítimas nos processos em que discutidos seus interesses, sem prejuízo, nos termos da lei, da legitimidade concorrente da FUNAI e da intervenção do Ministério Público como fiscal da lei.”

Resumo - O reconhecimento do direito às terras tradicionalmente ocupadas pelos indígenas não se sujeita ao marco temporal da promulgação da Constituição Federal (5/10/1988) nem à presença de conflito físico ou controvérsia judicial existentes nessa mesma data.

• Em mudança de posicionamento jurisprudencial, esta Corte concluiu pela inaplicabilidade da teoria do fato indígena e pela prevalência da teoria do indigenato, segundo a qual a posse dos indígenas sobre as terras configura um direito próprio dos povos originários e cuja tradicionalidade da ocupação deve ser considerada conforme os parâmetros expressamente previstos no texto constitucional (CF/1988, art. 231, §§ 1º e 2º).

• Se houver ocupação tradicional indígena ou renitente esbulho contemporâneo à data de promulgação da Constituição Federal de 1988, são assegurados aos não índios o direito à indenização pelas benfeitorias úteis e necessárias (CF/1988, art. 231, § 6º). Porém, na hipótese de inexistir quaisquer dessas situações, consideram-se válidos e eficazes os atos e negócios jurídicos perfeitos e a coisa julgada relativos a justo título ou posse de boa-fé das terras de ocupação tradicional indígena. Neste caso, o particular tem direito a ser previamente indenizado pela União ao valor correspondente às benfeitorias necessárias e úteis, ou, quando inviável o seu reassentamento, ao valor da terra nua (1).

• Com base nesses e em outros entendimentos, o Plenário, por maioria, ao apreciar o Tema 1.031 da repercussão geral, deu provimento ao recurso extraordinário para anular o acórdão recorrido e reformar a sentença de primeiro grau, julgando, por conseguinte, improcedentes os pedidos deduzidos na petição inicial.

• ADI 4269
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EDSON FACHIN
• Julgamento: 18/10/2017
• Publicação: 01/02/2019

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. DIREITO CONSTITUCIONAL E ADMINISTRATIVO. REGULARIZAÇÃO FUNDIÁRIA DAS TERRAS DE DOMÍNIO DA UNIÃO NA AMAZÔNIA LEGAL. IMPUGNAÇÃO AOS ARTIGOS 4º, §2º, 13, 15, INCISO I, §§ 2º, 4º E 5º, DA LEI Nº 11.952/2009. PREJUÍZO PARCIAL DA AÇÃO. ALTERAÇÃO SUBSTANCIAL E REVOGAÇÃO DE DISPOSITIVOS PROMOVIDA POR LEI SUPERVENIENTE. ADEQUADA PROTEÇÃO ÀS TERRAS QUILOMBOLAS E DE OUTRAS COMUNIDADES TRADICIONAIS AMAZÔNICAS. INCONSTITUCIONALIDADE DA INTERPRETAÇÃO QUE CONCEDE ESSAS TERRAS A TERCEIROS. INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO. ARTIGOS 216, INCISO II, DO TEXTO CONSTITUCIONAL E 68 DO ADCT. AUSÊNCIA DE VISTORIA PRÉVIA NA REGULARIZAÇÃO DE IMÓVEIS DE ATÉ QUATRO MÓDULOS FISCAIS. PROTEÇÃO DEFICIENTE AO MEIO AMBIENTE SE DESACOMPANHADA DE MEIOS EFICAZES PARA FISCALIZAÇÃO DOS REQUISITOS DE INGRESSO NO PROGRAMA TERRA LEGAL. INTERPRETAÇÃO CONFORME À CONSTITUIÇÃO. RESPEITO AO ARTIGO 225, CAPUT, DA CONSTITUIÇÃO. - 1. Há prejuízo parcial da ação direta de inconstitucionalidade quando lei superveniente promova alteração substancial ou revogue dispositivo impugnado em demanda de controle concentrado, conforme jurisprudência pacífica desta Corte. No caso, a superveniência da Lei nº 13.465, de 11 de julho de 2017, alterou a redação do artigo 15, inciso I e §2º, bem como revogou expressamente seus §§ 4º e 5º, circunstância que impede o conhecimento da ação, no ponto. - 2. O direito ao meio ambiente equilibrado foi assegurado pela Constituição da República, em seu artigo 225, bem como em diversos compromissos internacionais do Estado Brasileiro. A região amazônica, dada a diversidade biológica, cultural, etnográfica e geológica, mereceu tutela especial do constituinte, tornando-se imperiosa a observância do desenvolvimento sustentável na região, conjugando a proteção à natureza e a sobrevivência humana nas áreas objeto de regularização fundiária.

• 3. Revela-se de importância ímpar a promoção de regularização fundiária nas terras ocupadas de domínio da União na Amazônia Legal, de modo a assegurar a inclusão social das comunidades que ali vivem, por meio da concessão de títulos de propriedade ou concessão de direito real de uso às áreas habitadas, redução da pobreza, acesso aos programas sociais de incentivo à produção sustentável, bem como melhorando as condições de fiscalização ambiental e responsabilização pelas lesões causadas à Floresta Amazônica.

• 4. O artigo 4º, §2º da Lei nº 11.952/2009 vai de encontro à proteção adequada das terras dos remanescentes de comunidades quilombolas e das demais comunidades tradicionais amazônicas, ao permitir interpretação que possibilite a regularização dessas áreas em desfavor do modo de apropriação de território por esses grupos, sendo necessária interpretação conforme aos artigos 216, I da Constituição e 68 do ADCT, para assegurar a relação específica entre comunidade, identidade e terra que caracteriza os povos tradicionais.

• 5. Exige interpretação conforme à Constituição a previsão do artigo 13 da Lei nº 11.952/2009, ao dispensar a vistoria prévia nos imóveis rurais de até quatro módulos fiscais, a fim de que essa medida de desburocratização do procedimento seja somada à utilização de todos os meios eficazes de fiscalização do meio ambiente, como forma de tutela à biodiversidade e inclusão social dos pequenos proprietários que exercem cultura efetiva na área.

• 6. Ação Direta de Inconstitucionalidade conhecida parcialmente e, na parte conhecida, julgada parcialmente procedente.

Legislação LEG-FED ADCT ANO-1988 ART-00068 ATO DAS DISPOSIÇÕES CONSTITUCIONAIS TRANSITÓRIAS

Outras ocorrências Doutrina (1)

• ADI 3239
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CEZAR PELUSO
• Redator(a) do acórdão: Min. ROSA WEBER
• Julgamento: 08/02/2018
• Publicação: 01/02/2019

AÇÃO DIRETA DE INCONSTITUCIONALIDADE. DECRETO Nº 4.887/2003. PROCEDIMENTO PARA IDENTIFICAÇÃO, RECONHECIMENTO, DELIMITAÇÃO, DEMARCAÇÃO E TITULAÇÃO DAS TERRAS OCUPADAS POR REMANESCENTES DAS COMUNIDADES DOS QUILOMBOS. ATO NORMATIVO AUTÔNOMO. ART. 68 DO ADCT. DIREITO FUNDAMENTAL. EFICÁCIA PLENA E IMEDIATA. INVASÃO DA ESFERA RESERVADA A LEI. ART. 84, IV E VI, "A", DA CF. INCONSTITUCIONALIDADE FORMAL. INOCORRÊNCIA. CRITÉRIO DE IDENTIFICAÇÃO. AUTOATRIBUIÇÃO. TERRAS OCUPADAS. DESAPROPRIAÇÃO. ART. 2º, CAPUT E §§ 1º, 2º E 3º, E ART. 13, CAPUT E § 2º, DO DECRETO Nº 4.887/2003. INCONSTITUCIONALIDADE MATERIAL. INOCORRÊNCIA. IMPROCEDÊNCIA DA AÇÃO. - 1. Ato normativo autônomo, a retirar diretamente da Constituição da República o seu fundamento de validade, o Decreto nº 4.887/2003 apresenta densidade normativa suficiente a credenciá-lo ao controle abstrato de constitucionalidade. - 2. Inocorrente a invocada ausência de cotejo analítico na petição inicial entre o ato normativo atacado e os preceitos da Constituição tidos como malferidos, uma vez expressamente indicados e esgrimidas as razões da insurgência. - 3. Não obsta a cognição da ação direta a falta de impugnação de ato jurídico revogado pela norma tida como inconstitucional, supostamente padecente do mesmo vício, que se teria por repristinada. Cabe à Corte, ao delimitar a eficácia da sua decisão, se o caso, excluir dos efeitos da decisão declaratória eventual efeito repristinatório quando constatada incompatibilidade com a ordem constitucional. - 4. O art. 68 do ADCT assegura o direito dos remanescentes das comunidades dos quilombos de ver reconhecida pelo Estado a propriedade sobre as terras que histórica e tradicionalmente ocupam – direito fundamental de grupo étnico-racial minoritário dotado de eficácia plena e aplicação imediata. Nele definidos o titular (remanescentes das comunidades dos quilombos), o objeto (terras por eles ocupadas), o conteúdo (direito de propriedade), a condição (ocupação tradicional), o sujeito passivo (Estado) e a obrigação específica (emissão de títulos), mostra-se apto o art. 68 do ADCT a produzir todos os seus efeitos, independentemente de integração legislativa. - 5. Disponíveis à atuação integradora tão-somente os aspectos do art. 68 do ADCT que dizem com a regulamentação do comportamento do Estado na implementação do comando constitucional, não se identifica, na edição do Decreto 4.887/2003 pelo Poder Executivo, mácula aos postulados da legalidade e da reserva de lei. Improcedência do pedido de declaração de inconstitucionalidade formal por ofensa ao art. 84, IV e VI, da Constituição da República. - 6. O compromisso do Constituinte com a construção de uma sociedade livre, justa e solidária e com a redução das desigualdades sociais (art. 3º, I e III, da CF) conduz, no tocante ao reconhecimento da propriedade das terras ocupadas pelos remanescentes das comunidades dos quilombos, à convergência das dimensões da luta pelo reconhecimento – expressa no fator de determinação da identidade distintiva de grupo étnico-cultural – e da demanda por justiça socioeconômica, de caráter redistributivo – compreendida no fator de medição e demarcação das terras. - 7. Incorporada ao direito interno brasileiro, a Convenção 169 da Organização Internacional do Trabalho – OIT sobre Povos Indígenas e Tribais, consagra a "consciência da própria identidade" como critério para determinar os grupos tradicionais aos quais aplicável, enunciando que Estado algum tem o direito de negar a identidade de um povo que se reconheça como tal. - 8. Constitucionalmente legítima, a adoção da autoatribuição como critério de determinação da identidade quilombola, além de consistir em método autorizado pela antropologia contemporânea, cumpre adequadamente a tarefa de trazer à luz os destinatários do art. 68 do ADCT, em absoluto se prestando a inventar novos destinatários ou ampliar indevidamente o universo daqueles a quem a norma é dirigida. O conceito vertido no art. 68 do ADCT não se aparta do fenômeno objetivo nele referido, a alcançar todas as comunidades historicamente vinculadas ao uso linguístico do vocábulo quilombo. Adequação do emprego do termo “quilombo” realizado pela Administração Pública às balizas linguísticas e hermenêuticas impostas pelo texto-norma do art. 68 do ADCT. Improcedência do pedido de declaração de inconstitucionalidade do art. 2°, § 1°, do Decreto 4.887/2003. - 9. Nos casos Moiwana v. Suriname (2005) e Saramaka v. Suriname (2007), a Corte Interamericana de Direitos Humanos reconheceu o direito de propriedade de comunidades formadas por descendentes de escravos fugitivos sobre as terras tradicionais com as quais mantêm relações territoriais, ressaltando o compromisso dos Estados partes (Pacto de San José da Costa Rica, art. 21) de adotar medidas para garantir o seu pleno exercício. - 10. O comando para que sejam levados em consideração, na medição e demarcação das terras, os critérios de territorialidade indicados pelos remanescentes das comunidades quilombolas, longe de submeter o procedimento demarcatório ao arbítrio dos próprios interessados, positiva o devido processo legal na garantia de que as comunidades tenham voz e sejam ouvidas. Improcedência do pedido de declaração de inconstitucionalidade do art. 2º, §§ 2º e 3º, do Decreto 4.887/2003. - 11. Diverso do que ocorre no tocante às terras tradicionalmente ocupadas pelos índios – art. 231, § 6º – a Constituição não reputa nulos ou extintos os títulos de terceiros eventualmente incidentes sobre as terras ocupadas por remanescentes das comunidades dos quilombos, de modo que a regularização do registro <u>exige o necessário o procedimento expropriatório</u>. A exegese sistemática dos arts. 5º, XXIV, 215 e 216 da Carta Política e art. 68 do ADCT impõe, quando incidente título de propriedade particular legítimo sobre as terras ocupadas por quilombolas, seja o processo de transferência da propriedade mediado por regular procedimento de desapropriação. Improcedência do pedido de declaração de inconstitucionalidade material do art. 13 do Decreto 4.887/2003. Ação direta de inconstitucionalidade julgada improcedente.

• ADI 7008
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. ROBERTO BARROSO
• Julgamento: 22/05/2023
• Publicação: 06/06/2023

Direito constitucional e administrativo. Ação direta de inconstitucionalidade. Lei nº 16.260/2016, do Estado de São Paulo. Concessão de áreas estaduais para exploração de atividades de ecoturismo e extração comercial de madeira e subprodutos florestais. - 1. Ação direta de inconstitucionalidade contra a Lei nº 16.260/2016, do Estado de São Paulo, que autoriza a concessão à iniciativa privada de áreas estaduais para exploração de atividades de ecoturismo e extração comercial de madeira e subprodutos florestais. - 2. O ato normativo veicula autorização legislativa dada ao Poder Executivo estadual para a concessão da exploração de serviços ou do uso, total ou parcial, de áreas em próprios estaduais. Ato normativo de caráter genérico que não afasta a incidência de normas editadas pela União em matéria ambiental ou o dever de consulta prévia às comunidades indígenas e tradicionais eventualmente afetadas. Sendo evidente o sentido da norma, revela-se incabível a interpretação conforme à Constituição para essa finalidade. - 3. O art. 231 da Constituição consagrou o caráter originário do direito dos índios às terras por eles “tradicionalmente ocupadas”, reservando-lhes, com exclusividade, o usufruto das riquezas do solo, dos rios e dos lagos nelas existentes. Além disso, essas terras foram incluídas entre os bens da União (art. 20, XI, da CF/88). Trata-se, portanto, de território pertencente à União e de usufruto exclusivo dos povos indígenas, sendo inconstitucional a sua concessão pelo Estado à iniciativa privada. - 4. Também a proteção às terras ocupadas por comunidades tradicionais e de remanescentes quilombolas é essencial à preservação de sua identidade e seus “modos de criar, fazer e viver” (arts. 215 e 216 da Constituição; art. 68 do ADCT e Convenção nº 169 da OIT). É inconstitucional a concessão dessas áreas, pelo Estado, à iniciativa privada, para exploração florestal madeireira e do ecoturismo, independentemente do <u>status de regularização fundiária</u> e da <u>morosidade</u> do Estado em efetivar seu dever de demarcá-las e protegê-las. - 5. Pedido julgado parcialmente procedente, para conferir interpretação conforme a Constituição à Lei nº 16.260/2016, do Estado de São Paulo, de modo a afastar sua incidência relativamente às terras tradicionalmente ocupadas por comunidades indígenas, remanescentes quilombolas e demais comunidades tradicionais. - 6. Fixação da seguinte tese de julgamento: “1.* É constitucional norma estadual que, sem afastar a aplicação da legislação nacional em matéria ambiental (inclusive relatório de impacto ambiental) e o dever de consulta prévia às comunidades indígenas e tradicionais, quando diretamente atingidas por ocuparem zonas contíguas, autoriza a concessão à iniciativa privada da exploração de serviços ou do uso de bens imóveis do Estado; 2.* A concessão pelo Estado não pode incidir sobre áreas tradicionalmente ocupadas por povos indígenas, remanescentes quilombolas e demais comunidades tradicionais”.

Tese - 1. É constitucional norma estadual que, sem afastar a aplicação da legislação nacional em matéria ambiental (inclusive relatório de impacto ambiental) e o dever de consulta prévia às comunidades indígenas e tradicionais, quando diretamente atingidas por ocuparem <u>zonas contíguas</u>, autoriza a concessão à iniciativa privada da exploração de serviços ou do uso de bens imóveis do Estado;

• 2. A concessão pelo Estado não pode incidir sobre áreas tradicionalmente ocupadas por povos indígenas, remanescentes quilombolas e demais comunidades tradicionais.

Ver Decreto nº 4.887/2003

Art. 17. A titulação prevista neste Decreto será reconhecida e registrada mediante outorga de título <u>coletivo</u> e <u>pró-indiviso</u> às comunidades a que se refere o art. 2o, caput, com obrigatória inserção de cláusula de inalienabilidade, imprescritibilidade e de impenhorabilidade.

• Parágrafo único. As comunidades serão representadas por suas associações legalmente constituídas.

• Informativo 1179
• RE 1326178 / SC - Tema 1.156
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. CRISTIANO ZANIN
• Julgamento: 23/05/2025 (Virtual)
• Ramo do Direito: Constitucional
• Matéria: Precatórios; Débitos da Fazenda Pública; Fracionamento; Créditos Superpreferenciais; Requisição de Pequeno Valor

Créditos de natureza superpreferencial: pagamento da parcela por meio de RPV

Tese fixada - O pagamento de crédito superpreferencial (art. 100, § 2º, da CF/1988) deve ser realizado por meio de precatório, exceto se o valor a ser adimplido encontrar-se dentro do limite estabelecido por lei como pequeno valor.

Resumo - É inconstitucional — por violar o art. 100, §§ 2º e 8º, da Constituição Federal de 1988 — o pagamento parcial de valores de natureza alimentícia pertencente a credores superpreferenciais por meio de requisição de pequeno valor (RPV), se o montante devido ultrapassar o limite legalmente fixado para essa modalidade.

• O texto constitucional estabelece que os créditos chamados de superpreferenciais — de natureza alimentícia e de titularidade de idosos, pessoas com deficiência ou portadores de doenças graves — devem ser pagos por meio de precatório, salvo se o montante exigível estiver dentro do limite definido como de pequeno valor. Isso, porque a expedição de RPV é medida excepcional, condicionada à existência de previsão legal que defina as obrigações passíveis de quitação por essa via (1).

• Conforme jurisprudência desta Corte (2), o fracionamento de precatórios superpreferenciais para possibilitar o pagamento por meio de RPV, além de representar risco de impacto orçamentário significativo, não encontra amparo na Constituição Federal, uma vez que o pagamento dos créditos contra a Fazenda Pública deve ser realizado de forma integral pelo <u>mesmo rito</u> (RPV ou precatório), <u>sem que se mesclem as modalidades</u>.

• Na espécie, o acórdão do Tribunal Regional Federal da 4ª Região manteve decisão que reconheceu a possibilidade de fracionamento do precatório, permitindo o pagamento da parcela superpreferencial (até 180 salários-mínimos) por meio de RPV, reservando-se o excedente para quitação via precatório judicial.

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, ao apreciar o Tema 1.156 da repercussão geral: (i) deu provimento ao recurso extraordinário para reconhecer a violação ao art. 100, §§ 2° e 8° da Constituição Federal de 1988; (ii) determinou que o pagamento dos créditos superpreferenciais seja adimplido por meio de expedição de precatórios; e (iii) fixou a tese anteriormente citada.

(1) CF/1988: “Art. 100. Os pagamentos devidos pelas Fazendas Públicas Federal, Estaduais, Distrital e Municipais, em virtude de sentença judiciária, far-se-ão exclusivamente na ordem cronológica de apresentação dos precatórios e à conta dos créditos respectivos, proibida a designação de casos ou de pessoas nas dotações orçamentárias e nos créditos adicionais abertos para este fim (...) § 2º Os débitos de natureza alimentícia cujos titulares, originários ou por sucessão hereditária, tenham 60 (sessenta) anos de idade, ou sejam portadores de doença grave, ou pessoas com deficiência, assim definidos na forma da lei, serão pagos com preferência sobre todos os demais débitos, até o valor equivalente ao triplo fixado em lei para os fins do disposto no § 3º deste artigo, admitido o fracionamento para essa finalidade, sendo que o restante será pago na ordem cronológica de apresentação do precatório. (...) § 8º É vedada a expedição de precatórios complementares ou suplementares de valor pago, bem como o fracionamento, repartição ou quebra do valor da execução para fins de enquadramento de parcela do total ao que dispõe o § 3º deste artigo.” (2) Precedentes citados: ADI 6.556 MC-Ref; e RE 1.300.190, RE 1.310.690, RE 1.310.475, no RE 1.312.089, RE 1.293.528, RE 1.306.206, RE 1.297.760 e RE 1.304.973 (decisões monocráticas).

Legislação: CF/1988: art. 100, § 2º e 8º.

Precedentes: ADI 6.556 MC-Ref; e RE 1.300.190, RE 1.310.690, RE 1.310.475, no RE 1.312.089, RE 1.293.528, RE 1.306.206, RE 1.297.760 e RE 1.304.973 (decisões monocráticas).

Obs.: Não se admite o pagamento de crédito superpreferencial, que tem limite de até 3 vezes o fixado para RPV, por requisição de pequeno valor. Não há que se falar em pagamento de partes em RPV e de outra parte em precatório. Os pagamentos dos superpreferenciais somente devem ser veiculados por RPV se estritamente dentro do limite delimitado para esta forma excepcional de pagamento. Via de regra, é por meio de precatórios o pagamento de tais verbas.

• Informativo 1081
• ADI 5421 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. GILMAR MENDES
• Julgamento: 16/12/2022 (Virtual)
• Ramo do Direito: Constitucional, Processual Civil
• Matéria: Precatório; Requisição de Pequeno Valor; Valor Limite; Repartição de Competência; Execução contra a Fazenda Pública

RPV e autonomia dos estados e municípios

Resumo - Os estados e municípios podem redefinir o valor limite da Requisição de Pequeno Valor (RPV) visando à adequação de suas respectivas capacidades financeiras e especificidades orçamentárias.

• É inconstitucional — por violar a competência privativa da União para legislar sobre direito processual (CF/1988, art. 21, I), uma vez que as normas que dispõem sobre RPV têm caráter eminentemente processual (2) — legislação estadual que transfere ao credor a responsabilidade pelo encaminhamento da documentação necessária para solicitação do pagamento do RPV diretamente ao órgão público devedor, bem como determina a suspensão do prazo para pagamento.

• Os estados e municípios podem redefinir o valor limite da Requisição de Pequeno Valor (RPV) visando à adequação de suas respectivas capacidades financeiras e especificidades orçamentárias.

• Os entes federados, desde que respeitado o princípio da proporcionalidade, gozam de autonomia para estabelecer o montante correspondente às obrigações de pequeno valor e, dessa forma, afastar a aplicação do sistema de precatórios. Eles só não podem estabelecer valor demasiado <u>além</u> ou <u>aquém</u> do razoável, tendo como parâmetro as suas disponibilidades financeiras (1)

• É inconstitucional — por violar a competência privativa da União para legislar sobre direito processual (CF/1988, art. 21, I), uma vez que as normas que dispõem sobre RPV têm caráter eminentemente processual (2) — legislação estadual que transfere ao credor a responsabilidade pelo encaminhamento da documentação necessária para solicitação do pagamento do RPV diretamente ao órgão público devedor, bem como determina a suspensão do prazo para pagamento.

• Ademais, a lei estadual impugnada não se aplica aos processos judiciais de competência da justiça federal, ainda que no exercício da competência federal delegada, já que para eles prevalece o conteúdo de norma editada pelo Conselho da Justiça Federal (CJF), atualmente a Resolução 458/2017.

• Com base nesses entendimentos, o Plenário, por unanimidade, julgou parcialmente procedente a ação para (i) declarar a inconstitucionalidade do caput e do parágrafo único do art. 6º da Lei 14.757/2015 do Estado do Rio Grande do Sul; e (ii) dar interpretação conforme a Constituição aos incisos do mesmo art. 6º, para limitar sua aplicação aos processos judiciais de competência da justiça estadual, de modo que eles não deverão ser aplicados aos processos julgados no exercício da competência federal delegada, os quais devem ser regidos pela Resolução do CJF (3).

(1) Precedentes citados: ADI 2.868 e ADI 4.332.

(2) Precedentes citados: RE 632.550 AgR; RE 293.231 e ADI 5.534.

(3) Lei 14.757/2015 do Estado do Rio Grande do Sul: “Art. 1º Serão consideradas de pequeno valor, para os fins do disposto no § 3.º do art. 100 da Constituição Federal, as obrigações que o Estado do Rio Grande do Sul, suas Autarquias e Fundações devam quitar em decorrência de decisão judicial transitada em julgado cujo valor, devidamente atualizado, não exceda a 10 (dez) salários mínimos. Art. 2º O crédito de pequeno valor não estará sujeito ao regime de precatórios e deverá ser pago, mediante depósito judicial, no prazo de até 60 (sessenta) dias, contados da data em que for protocolada, perante o órgão competente, a requisição expedida pelo juízo da execução. Parágrafo único. Nas requisições de pequeno valor expedidas por meio eletrônico, o prazo será contado da data de expedição. Art. 3º São vedados o fracionamento, a repartição ou a quebra do valor da execução para que o pagamento se faça, em parte, na forma estabelecida no ‘caput’ do art. 2º desta Lei e, em parte, com a expedição de precatório. Art. 4º Se o valor da execução ultrapassar o montante estabelecido no art. 1º desta Lei, o pagamento far-se-á por meio de precatório, sendo facultada à parte exequente a renúncia ao crédito do valor excedente, para que possa optar pelo pagamento do saldo sem o precatório, na forma prevista no art. 2º desta Lei. Parágrafo único. A opção pelo recebimento do crédito na forma prevista nesta Lei implica a renúncia ao restante dos créditos porventura existentes oriundos do mesmo processo judicial. Art. 5º As requisições de pequeno valor cujo trânsito em julgado da decisão tenha ocorrido antes da entrada em vigor desta Lei observarão o limite de 40 (quarenta) salários mínimos. Art. 6º A requisição de pequeno valor expedida em meio físico será encaminhada diretamente pelo credor, ou seu representante, ao ente devedor responsável pelo pagamento da obrigação, e deverá ser instruída com os seguintes documentos e informações: I - indicação do número do processo judicial em que foi expedida a requisição; II - indicação da natureza da obrigação a que se refere o pagamento; III - comprovante de situação cadastral das partes e dos advogados no Cadastro de Pessoa Física - CPF - ou no Cadastro Nacional de Pessoa Jurídica - CNPJ - do Ministério da Fazenda; IV - cópia da memória completa do cálculo definitivo, ainda que objeto de renúncia ao valor estabelecido nesta Lei; V - indicação do período compreendido para efeito de cálculo do imposto de renda e das contribuições aos sistemas de previdência e saúde; e VI - cópia da manifestação da Procuradoria-Geral do Estado de concordância com o valor do débito. Parágrafo único. A requisição de pequeno valor que não preencher os requisitos do ‘caput’ deste artigo não será recebida pela autoridade competente, ficando suspenso o prazo do seu pagamento até a apresentação pelo credor dos documentos ou informações faltantes. Art. 7º Esta Lei entra em vigor na data de sua publicação. Art. 8º Revoga-se a Lei nº 13.756, de 15 de julho de 2011.”

Legislação: Lei 14.757/2015 do Estado do Rio Grande do Sul

Precedentes: ADI 2.868, ADI 4.332, RE 632.550 AgR; RE 293.231 e ADI 5.534

• Informativo 1066
• RE 1359139 RG / CE
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. PRESIDENTE
• Julgamento: 01/09/2022 (Virtual)
• Ramo do Direito: Processual Civil, Constitucional
• Matéria: Execução contra a Fazenda Pública; Requisição de Pequeno Valor/ Organização Político-Administrativa; Poder Judiciário

RPV: valor previsto no ADCT e fixação de quantia referencial inferior por ente federado

Tese fixada - (I) As unidades federadas podem fixar os limites das respectivas requisições de pequeno valor em patamares inferiores aos previstos no artigo 87 do ADCT, desde que o façam em consonância com sua capacidade econômica. - (II) A aferição da capacidade econômica, para este fim, deve refletir não somente a receita, mas igualmente os graus de endividamento e de litigiosidade do ente federado.

• (III) A ausência de demonstração concreta da desproporcionalidade na fixação do teto das requisições de pequeno valor impõe a deferência do Poder Judiciário ao juízo político-administrativo externado pela legislação local.”

Resumo - Ao editar norma própria, o ente federado, desde que em consonância com sua <u>capacidade econômica</u> e com o princípio da <u>proporcionalidade</u>, pode estabelecer quantia inferior à prevista no art. 87 do ADCT como teto para o pagamento de seus débitos judiciais por meio de Requisição de Pequeno Valor (RPV).

• O patamar provisório fixado no ADCT (1) para o pagamento de RPV não é irredutível, cabendo a cada unidade federativa estipular o valor máximo para essa especial modalidade de pagamento de acordo com sua capacidade econômica, cuja aferição deve considerar, além do quantum das receitas auferidas, os graus de endividamento e de litigiosidade do ente público.

• No tocante à atuação do Poder Judiciário, deve ser adotada uma postura de autocontenção quando não houver demonstração concreta da desproporcionalidade na fixação do valor referencial.

• Com base nesse entendimento, o Plenário, por unanimidade, reconheceu a existência da repercussão geral da questão constitucional suscitada (Tema 1.231 RG) e, no mérito, também por unanimidade, reafirmou a jurisprudência dominante sobre a matéria (2) para dar provimento ao recurso extraordinário, assentando a constitucionalidade da Lei 10.562/2017 do Município de Fortaleza/CE, que fixa como teto para pagamento das RPVs o equivalente ao maior benefício do Regime Geral de Previdência Social. Não se manifestou o ministro André Mendonça.

(1) ADCT: “Art. 87. Para efeito do que dispõem o § 3º do art. 100 da Constituição Federal e o art. 78 deste Ato das Disposições Constitucionais Transitórias serão considerados de pequeno valor, até que se dê a publicação oficial das respectivas leis definidoras pelos entes da Federação, observado o disposto no § 4º do art. 100 da Constituição Federal, os débitos ou obrigações consignados em precatório judiciário, que tenham valor igual ou inferior a: I – quarenta salários-mínimos, perante a Fazenda dos Estados e do Distrito Federal; II – trinta salários-mínimos, perante a Fazenda dos Municípios. Parágrafo único. Se o valor da execução ultrapassar o estabelecido neste artigo, o pagamento far-se-á, sempre, por meio de precatório, sendo facultada à parte exequente a renúncia ao crédito do valor excedente, para que possa optar pelo pagamento do saldo sem o precatório, da forma prevista no § 3º do art. 100.”

(2) Precedentes citados: ADI 2868; ADI 4332; e ADI 5100.

Legislação: ADCT, art. 87 Lei 10.562/2017-Fortaleza/CE

Precedentes: ADI 2868; ADI 4332; e ADI 5100.

• Informativo nº 834
• 26 de novembro de 2024.
• SEGUNDA TURMA
• Compartilhe:
• Processo: AgInt no RMS 66.132-RS, Rel. Ministro Afrânio Vilela, Segunda Turma, por unanimidade, julgado em 12/11/2024, DJe 18/11/2024.

Ramo do Direito DIREITO ADMINISTRATIVO

TemaPaz, Justiça e Instituições Eficazes <br /> Servidor Público. Aposentadoria voluntária com proventos integrais. Regra de transição prevista no art. 3º da EC n. 47/2005. Data do ingresso no serviço público. Regime celetista em Fundação prestadora de serviço público. Não abrangência pela regra de transição.

Destaque - A regra de transição prevista no art. 3º, caput, da EC n. 47/2005, a qual garantiu aposentadoria com proventos integrais a servidor que tenha ingressado no serviço público anteriormente a 16/12/1998, não se aplica à prestação de serviço em fundação pública sob o <u>regime celetista</u> e por meio de <u>contrato administrativo</u>.

Informações do Inteiro Teor - A controvérsia cinge-se a aferir se o período laborado em Fundação prestadora de serviço público, como celetista, antes de ser ocupante de cargo efetivo, deve ser considerado como tempo de efetivo serviço para o fim de aposentadoria com proventos integrais, nos termos do previsto no art. 3º, caput, da Emenda Constitucional n. 47/2005.

• O caput do art. 3º da EC n. 47/2005, expressamente, garante o direito à aposentadoria com proventos integrais ao servidor "da União, dos Estados, do Distrito Federal e dos Municípios, incluídas suas autarquias e fundações" cujo ingresso no serviço público tenha ocorrido anteriormente a 16/12/1998.

• Não se contesta a natureza de serviço público prestado em Fundação estadual (pessoa jurídica de direito privado). O que se discute é a existência de vínculo efetivo, com o fim de integralizar a aposentadoria pelo regime próprio.

• Despicienda a natureza jurídica da Fundação estadual, cujo serviço prestado era inequivocamente de caráter público. O que importa para a solução da controvérsia é a natureza do vínculo empregatício.

• O trabalho junto à Fundação se deu por meio de contrato administrativo, regido pela CLT e com contribuição, portanto, ao Regime Geral da Previdência Social - RGPS. Embora o tempo laborado junto à Fundação seja computado para sua aposentadoria, a contribuição naquele período difere-se daquela como servidor público concursado e não é apta a integralizar a sua aposentadoria voluntária como almejado.

• Assim, conclui-se que a regra prevista no art. 3º da EC n. 47/2005 destina-se aos servidores ocupantes de <u>cargo efetivo</u>. A expressão "ingresso no serviço público" refere-se à investidura em cargo público, nos termos do art. 37 da Constituição Federal, que expressamente prevê, no inciso II, que "a investidura em cargo ou emprego público depende de aprovação prévia em concurso público".

Obs.: O precedente traz à discussão o direito de servidor poder se aposentar com proventos integrais. Mas a EC delimitou que esse direito somente é aplicável para o servidor efetivo que tenha ingressado no serviço público até 16 de dezembro de 1998, inclusive efetivos alocados em fundações e autarquias.

No entanto, o caso concreto julgado versa sobre pessoa que, embora tenha ingressado no serviço público anteriormente a 16/12/1998, não ingressou na qualidade de efetivo, mas sim como servidor regido pela CLT. Logo, descaracteriza-se o direito à aposentadoria por proventos integrais.

• Informativo 1.190
• RE 1469887 / AL - Tema 1.424
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. PRESIDENTE
• Julgamento: 12/09/2025 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: Militar; Concurso Público; Requisitos Básicos para a Investidura; Altura Mínima

Polícia Militar: altura mínima para investidura em cargo da carreira Tese fixada - A exigência de altura mínima para ingresso em cargo do Sistema Único de Segurança Pública pressupõe a existência de lei e da observância dos parâmetros fixados para a carreira do exército (Lei federal nº 12.705/2012, 1,60m para homens e 1,55m para mulheres).

Resumo - É inconstitucional — por violar o princípio da razoabilidade — lei estadual que exige, como requisito para ingresso na Polícia Militar, altura mínima superior à prevista para ingresso nas carreiras do Exército.

• A imposição, pelo legislador estadual, de requisitos mais rigorosos do que os previstos na legislação federal para o Exército, sem qualquer justificativa relacionada às atribuições do cargo, configura afronta aos princípios da razoabilidade e da proporcionalidade (1).
• Na espécie, o Tribunal de Justiça do Estado de Alagoas manteve a eliminação de candidata em concurso público para a Polícia Militar por ela não possuir a altura mínima de 1,65m exigida pela legislação estadual (2).
• Com base nesses entendimentos, o Plenário, por unanimidade, reconheceu a existência de repercussão geral da questão constitucional suscitada (Tema 1.424 da repercussão geral) e, no mérito, por maioria: (i) reafirmou a jurisprudência dominante sobre a matéria (3); (ii) deu provimento ao recurso extraordinário para reformar o acórdão recorrido e determinar o prosseguimento da candidata no concurso público; e, por fim, (iii) fixou a tese anteriormente citada.

(1) Lei nº 12.705/2012: “Art. 2º A matrícula para o ingresso nos cursos de formação de oficiais e sargentos de carreira do Exército depende de aprovação prévia em concurso público, atendidos os seguintes requisitos, dentre outros estabelecidos na legislação vigente: (...) XIII - ter altura mínima de 1,60 m (um metro e sessenta centímetros) ou, se do sexo feminino, a altura mínima de 1,55 m (um metro e cinquenta e cinco centímetros).” (2) Lei nº 5.346/1992 do Estado de Alagoas: “Art. 7º O ingresso na Polícia Militar do Estado de Alagoas é facultado a todos os brasileiros, sem distinção de raça, sexo, cor ou credo religioso, mediante matrícula ou nomeação, após aprovação em concurso público de prova ou provas e títulos, desde que observadas as seguintes condições: (...) III – altura mínima de 1,65m (um metro e sessenta e cinco centímetros), se do sexo masculino, e 1,60m (um metro e sessenta centímetros), se do sexo feminino;” (3) Precedentes citados: ADI 5.044, ARE 1.459.395 AgR, RE 1.465.829 AgR e RE 1.480.201, bem como ARE 1.562.570, ARE 1.511.877 e RE 1.500.883 (decisões monocráticas).

Legislação: Lei nº 12.705/2012: art. 2º, XIII. Lei nº 5.346/1992 do Estado de Alagoas: art. 7º, III.

Precedentes: ADI 5.044, ARE 1.459.395 AgR, RE 1.465.829 AgR e RE 1.480.201, bem como ARE 1.562.570, ARE 1.511.877 e RE 1.500.883 (decisões monocráticas).

• Informativo 1163
• ADI 3516 / CE
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. EDSON FACHIN
• Julgamento: 13/12/2024 (Virtual)
• Ramo do Direito: Administrativo
• Matéria: Servidor Público; Inativos e Pensionistas; Gratificações; Proibição de Vinculação da Receita de Impostos; Atividades de Administração Tributária; Princípio da Eficiência

Impossibilidade de vinculação de receita de imposto a pagamento de Prêmio por Desempenho Fiscal a inativos e pensionistas

Resumo - São inconstitucionais — pois afrontam o art. 167, IV, da CF/1988 — dispositivos de lei estadual que vinculam a receita de impostos ao pagamento de Prêmio por Desempenho Fiscal (PDF) ou de gratificação a inativos e pensionistas.

• A ressalva contida no dispositivo acima citado (1) autoriza a vinculação da receita tributária ao pagamento do PDF apenas aos servidores <u>em atividade</u> na administração tributária. Ela tem respaldo no princípio da eficiência (CF/1988, art. 37, caput), na medida em que visa ao aumento da produtividade dos fiscais, e se fundamenta no incremento da arrecadação, no alcance de metas fixadas em regulamento, bem como na instituição de programas de qualidade e produtividade no serviço público, a ser viabilizado sob a forma de adicional ou prêmio de produtividade (2).

• À luz do caráter contributivo do sistema previdenciário, a concessão de vantagem remuneratória a servidores inativos sem o devido desconto da contribuição previdenciária também é inconstitucional, sob pena de desvirtuamento do equilíbrio atuarial e financeiro.

• Com base nesses e em outros entendimentos, o Plenário, por unanimidade, conheceu em parte da ação e, nessa extensão, a julgou parcialmente procedente para declarar a inconstitucionalidade dos arts. 1º, § 1º; 1º-A e 5º-A, da Lei nº 13.439/2004, com a redação da Lei nº 14.969/2011, ambas do Estado do Ceará (3).

(1) CF/1988: “Art. 167. São vedados: (...) IV - a vinculação de receita de impostos a órgão, fundo ou despesa, ressalvadas a repartição do produto da arrecadação dos impostos a que se referem os arts. 158 e 159, a destinação de recursos para as ações e serviços públicos de saúde, para manutenção e desenvolvimento do ensino e para realização de atividades da administração tributária, como determinado, respectivamente, pelos arts. 198, § 2º, 212 e 37, XXII, e a prestação de garantias às operações de crédito por antecipação de receita, previstas no art. 165, § 8º, bem como o disposto no § 4º deste artigo;”

(2) CF/1988: “Art. 39. (...) § 7º Lei da União, dos Estados, do Distrito Federal e dos Municípios disciplinará a aplicação de recursos orçamentários provenientes da economia com despesas correntes em cada órgão, autarquia e fundação, para aplicação no desenvolvimento de programas de qualidade e produtividade, treinamento e desenvolvimento, modernização, reaparelhamento e racionalização do serviço público, inclusive sob a forma de adicional ou prêmio de produtividade.”

(3) Lei nº 13.439/2004 do Estado do Ceará: “Art. 1º Fica instituído para os servidores públicos ativos, integrantes do Grupo Ocupacional Tributação, Arrecadação e Fiscalização - TAF, o Prêmio por Desempenho Fiscal - PDF, a ser concedido mensalmente, desde que implementadas as condições previstas para a sua concessão, nos valores e limites fixados nesta Lei, com o objetivo de estimular os aumentos de produtividade da Secretaria da Fazenda que impliquem no incremento. (Redação dada pela Lei n.º 14.969, de 01.08.11) I - da arrecadação tributária anual, inclusive multas e juros e outras receitas previstas na legislação tributária; II - de outros indicadores de desempenho referidos nesta Lei ou que venham a ser estabelecidos em regulamento. § 1º. O Prêmio por Desempenho Fiscal (PDF) de que trata o caput será extensivo a pensionistas de servidores fazendários, conforme disposto em regulamento. (...) Art. 1º-A Aos aposentados na data da publicação desta Lei e aos que estejam em processo de aposentadoria instaurados nesta mesma data, bem como aos pensionistas de ex-servidores fazendários é devida gratificação em substituição ao valor percebido no mesmo título, na data de vigência desta Lei, totalmente desvinculado da sistemática de apuração e distribuição prevista na Lei n° 13.439, de 16 de janeiro de 2004, correspondente a 97,34% (noventa e sete vírgula trinta e quatro por cento) do valor da 1ª Classe, referência ‘C’ da Tabela B, do anexo III, da Lei n° 13.778, de 6 de junho de 2006, com a redação dada pela Lei n° 14.350, de 19 de maio de 2009, e alterações posteriores, observando-se, para os pensionistas, a proporcionalidade da pensão, submetida exclusivamente à revisão geral dos servidores, a serem custeados com recursos do PDF, Grupo I, conforme disposição em regulamento. Parágrafo único. No prazo de 90 (noventa) dias, a contar da data da publicação da presente Lei, a Secretaria da Fazenda - SEFAZ, juntamente com a Secretaria de Planejamento e Gestão – SEPLAG, e Procuradoria Geral do Estado-PGE, deverão apresentar os atos normativos e legais necessários à realização dos ajustes dos atos de aposentadoria, concedidas até a data de publicação desta Lei. (redação dada pela Lei n.º 14.969, de 01.08.11) (Revogado pela Lei n.º 17.393, de 26/02/2021) (...) Art. 5º-A O Prêmio por Desempenho Fiscal - PDF, será devido ao servidor efetivo do grupo TAF que venha a se aposentar após a publicação desta Lei, nos seguintes termos: I – aos servidores que implementarem as regras dos arts. 3º ou 6º da Emenda Constitucional nº 41, de 19 de dezembro de 2003, ou do art. 3º da Emenda Constitucional nº 47, de 5 de julho de 2005, o Prêmio por Desempenho Fiscal – PDF, será calculado pela média aritmética simples de valores mensais percebidos, a esse título, pelo servidor fazendário nos 24 (vinte e quatro) meses anteriores ao pedido de aposentadoria; II – para os servidores que implementarem as regras dos arts. 3º ou 6º da Emenda Constitucional nº 41, de 19 de dezembro de 2003, ou do art. 3º da Emenda Constitucional nº 47, de 5 de julho de 2005, cujo período de percepção por ocasião do pedido de aposentadoria seja menor do que 24 (vinte e quatro) meses, será observada a média aritmética do período de percepção, multiplicado pela fração cujo numerador será o número correspondente ao total de meses trabalhado e o denominador será sempre o numeral 24; III – para os que implementarem os requisitos de aposentadoria previstos no art. 40, da Constituição Federal, com a redação dada pela Emenda Constitucional nº 41, de 19 de dezembro de 2003, nos termos da legislação federal. Parágrafo único. Nas hipóteses dos incisos I e II deste artigo, o PDF não poderá ser inferior ao limite mínimo definido no art. 4º-A desta Lei. (Redação dada pela Lei n.º 14.969, de 01.08.11)”

Legislação: CF/1988: art. 37, caput, XXII; art. 39, § 7º; art. 165, §§ 4º e 8º; art. 167, IV; art. 198, § 2º; art. 212. Lei nº 13.439/2004 do Estado do Ceará: art. 1º, § 1º; 1º-A e 5º-A.

• Informativo nº 862
• 16 de setembro de 2025.
• SEGUNDA TURMA
• Processo: REsp 2.026.929-ES, Rel. Ministro Marco Aurélio Bellizze, Segunda Turma, por unanimidade, julgado em 9/9/2025.

Ramo do Direito DIREITO CIVIL

TemaPaz, Justiça e Instituições Eficazes <br /> Protestos. Ausência de comunicação prévia às autoridades competentes. Paralisação de diversas vias de acesso. Dano moral coletivo. Caracterização.

Destaque - A realização de protestos sem comunicação prévia às autoridades e com obstrução de diversas vias públicas de acesso à capital do Estado por lapso temporal considerável configura dano moral coletivo in re ipsa.

Informações do Inteiro Teor - Cinge-se a controvérsia em saber se a realização de protestos <u>sem comunicação prévia às autoridades</u> e com <u>paralisação de diversas vias de acesso à capital do Estado</u> configura dano moral coletivo, justificando a condenação ao pagamento de indenização.

• A configuração do dano moral coletivo requer que a conduta antijurídica afete intoleravelmente os valores e interesses coletivos fundamentais, mediante conduta de grave lesão.

• A jurisprudência desta Corte Superior firmou-se no sentido de que o dano moral coletivo se configura in re ipsa, ou seja, <u>independentemente</u> da comprovação de dor, sofrimento ou abalo psicológico.

• No caso, ficou demonstrado o abuso no exercício do direito de reunião, configurando ofensa intolerável aos interesses coletivos, capaz de ensejar a condenação por dano moral coletivo. Isso porque, a pretexto de defender seus associados, o sindicato olvidou-se de que <u>o exercício da cidadania pressupõe o respeito ao direito dos demais indivíduos</u>, tendo obstruído importantes vias públicas de acesso à capital do Estado por lapso temporal considerável, até mesmo com a interrupção total em uma delas, com o uso de material inflamável e a queima de pneus na via, colocando em risco não só a população em geral, mas os próprios manifestantes.

• ADI 7688 MC-Ref
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. FLÁVIO DINO
• Julgamento: 19/08/2024
• Publicação: 16/10/2024

DIREITO CONSTITUCIONAL. AÇÃO DIRETA DE INCONSTITUCIONALIDADE. ART 166-A, INCISO I E PARÁGRAFOS DA CONSTITUIÇÃO. DISPOSITIVOS QUE TRATAM DAS TRANSFERÊNCIAS ESPECIAIS CONHECIDAS COMO “EMENDAS PIX”. INADEQUAÇÃO DOS MECANISMOS DE TRANSPARÊNCIA E RASTREABILIDADE DAS TRANSFERÊNCIAS ESPECIAIS. RISCO DE GRAVE DANO AO ERÁRIO. CAUTELAR DEFERIDA EM PARTE.

• 1. A transparência requer a ampla divulgação sobre a origem e o destino dos recursos públicos, conforme decidido pelo STF na ADPF 854. Imperativo assegurar o controle institucional e social sobre o orçamento público. A probabilidade do direito está demonstrada mediante dados que apontam para a inexistência dos instrumentos de planejamento, bem como para a inadequação de mecanismos de controle quanto às transferências especiais (“emendas PIX”).

• 2. Há risco de dano ao erário e à ordem constitucional caso a realização das transferências especiais (“emendas PIX”), previstas no art. 166-A da Constituição, continue a ocorrer sem mecanismos que assegurem a transparência e a rastreabilidade dos dados (art. 163-A da Constituição).

• 3. Decisão liminar obriga a existência prévia de planos de trabalho, com o registro em plataforma eletrônica sobre a destinação e aplicação de parcela muito expressiva do Orçamento da União. No mesmo sentido de obediência à Constituição Federal, a decisão liminar dispõe sobre a incidência plena dos controles externo e interno constantes dos artigos 70, 71 e 74 da Carta Magna.

• 4. Tutela liminar deferida não é impeditiva de realização de transferências especiais (“emendas PIX”), desde que observados os trilhos constantes da Constituição Federal.

• 5. Medida cautelar referendada.

• ADPF 854 Ref
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. FLÁVIO DINO
• Julgamento: 04/12/2024
• Publicação: 14/03/2025

DIREITO CONSTITUCIONAL. ARGUIÇÃO DE DESCUMPRIMENTO DE PRECEITO FUNDAMENTAL E AÇÕES DIRETAS DE INCONSTITUCIONALIDADE. TRANSPARÊNCIA E RASTREABILIDADE NO PROCESSO ORÇAMENTÁRIO. ARTS. 163 E SEGUINTES DA CF. SUPERVENIÊNCIA DA LC Nº. 210/2024. INEXISTÊNCIA DE BLOQUEIO GENERALIZADO À EXECUÇÃO DE EMENDAS PARLAMENTARES. MEDIDA CAUTELAR REFERENDADA.

• 1. A execução de recursos oriundos de emendas parlamentares exige o cumprimento dos pressupostos constitucionais da transparência e da rastreabilidade (163-A da CF).
• 2. A LC nº. 210/2024 constitui avanço no cumprimento das determinações do Plenário desta Corte, ao estabelecer regras acerca da proposição e execução de emendas parlamentares. A referida lei complementar deve ser aplicada em consonância com a Constituição, interpretada pelas decisões do Plenário do STF.
• 3. Inexiste bloqueio generalizado à execução de emendas parlamentares, cabendo ao ordenador de despesas competente a análise e deliberação motivada, caso a caso, acerca do cumprimento das determinações desta Corte e da LC nº. 210/2024 para a continuidade da execução das emendas.
• 4. Medida cautelar referendada.

• Informativo 1089
• ADI 6270 / DF
• Órgão julgador: Tribunal Pleno
• Relator(a): Min. LUIZ FUX
• Julgamento: 29/03/2023 (Presencial)
• Ramo do Direito: Administrativo, Constitucional
• Matéria: Serviços Públicos, Transporte Terrestre, Concessão, Permissão e Autorização, Licitação, Causas de Inexigibilidade; Assimetria Regulatória, Princípios da Administração Pública

Dispensa delicitação para a outorga de serviços de transporte coletivo de passageiros desvinculados da exploração de infraestrutura

Resumo - É constitucional dispositivo de lei federal que altera o regime de outorga da prestação regular de serviços de transporte terrestre coletivo de passageiros desvinculados da exploração de obras de infraestrutura, permitindo sua realização mediante mera autorização estatal, <u>sem a necessidade de licitação prévia</u>, desde que cumpridos requisitos específicos.

• A assimetria regulatória estabelece a possibilidade de outorga da titularidade do serviço público estatal de transporte mediante autorização, sem a necessidade de licitação, se atendidos requisitos objetivos estabelecidos pela respectiva agência reguladora, no caso, a Agência Nacional de Transporte Terrestres - ANTT (CF/1988, arts. 21, XII, e; e 174, caput) (2).

• A Constituição Federal elegeu setores que, em razão da sua dinâmica de funcionamento, abrigam atividades cuja oferta pode ser compartilhada entre vários agentes, sem prejuízo dos atributos de continuidade, atualidade e adequação do serviço público. Assim, a dispensa de licitação não significa que faltará rigidez na seleção das transportadoras.

• Nesse contexto, a escolha política de permitir a descentralização operacional possibilita a ampliação da competitividade em benefício do consumidor e gera uma alocação mais eficiente de recursos, aumentando o bem-estar da sociedade.

• Isso porque a maior oferta de prestadores contribui para a universalização dos serviços, atingindo uma maior capilaridade no atendimento de destinos e rotas, de forma a garantir o direito de locomoção, a redução de desigualdades regionais, o desenvolvimento nacional, bem como a integração política e cultural dos povos da América Latina (CF/1988, art. 4º, parágrafo único).

• Com base nesse entendimento, o Plenário, em apreciação conjunta, por maioria, conheceu parcialmente da ADI 6.270/DF e integralmente da ADI 5.549/DF; e, quanto ao mérito, por maioria, as julgou improcedentes. Em obiter dictum, o Tribunal entendeu que o Poder Executivo e a ANTT devem providenciar as formalidades complementares introjetadas no acórdão do Tribunal de Contas da União e na Lei 14.298/2022.

(1) Lei 12.996/2014: “Art. 3ºALei nº10.233, de 5 de junho de 2001, passa a vigorar com as seguintes alterações: “Art. 13 (...) IV -permissão, quando se tratar de: a) prestação regular de serviços de transporte terrestre coletivo interestadual semiurbano de passageiros desvinculados da exploração da infraestrutura; b) prestação regular de serviços de transporte ferroviário de passageiros desvinculados da exploração de infraestrutura; V -autorização, quando se tratar de: (...) e)prestação regular de serviços de transporte terrestre coletivo interestadual e internacional de passageiros desvinculados da exploração da infraestrutura.”

(2) CF/1988: “Art. 21. Compete à União: (...) XII - explorar, diretamente ou mediante autorização, concessão ou permissão: (...) e) os serviços de transporte rodoviário interestadual e internacional de passageiros; (...) Art. 174.Como agente normativo e regulador da atividade econômica, o Estado exercerá, na forma da lei, as funções de fiscalização, incentivo e planejamento, sendo este determinante para o setor público e indicativo para o setor privado. (...)”

Legislação: CF/1988: arts. 4º; 21, XII, e; e 174, caput. Lei 14.298/2022. Lei 12.996/2014: art. 3º. Lei 10.233/2001: art 13, IV, a e b, e V, e.

Observação: Julgamento conjunto: ADI 5.549/DF e ADI 6.270/DF (relator Ministro Luiz Fux)

O CBS começará a ser cobrado somente em 2027

• Tema: 1.196
• Processo(s): RE 1.347.526
• Relator: Min. Cristiano Zanin
• Título: Constitucionalidade da Medida Provisória 739/2016, substituída pela Medida Provisória 767/2017 e convertida na Lei 13.457/2017, as quais alteraram a Lei 8.213/1991, inserindo preceito sobre prazo estimado para a duração do benefício.

O Tribunal fixou a seguinte tese: - Não viola os artigos 62, caput e § 1º, e 246 da Constituição Federal a estipulação de prazo estimado para a duração de benefício de auxílio-doença, conforme estabelecido nos §§ 8º e 9º do art. 60 da Lei 8.213/1991, com redação dada pelas medidas provisórias 739/2016 e 767/2017, esta última convertida na Lei 13.457/2017.

Regra Geral (Art. 14, I): A vedação à participação do autor do anteprojeto, projeto básico ou executivo na licitação é a regra geral. O objetivo é proteger os princípios da isonomia, moralidade e impessoalidade, evitando direcionamento e conflito de interesses.

Exceção Principal - Concessões e Permissões: Esta vedação NÃO se aplica aos processos de licitação para concessões e permissões de serviços públicos.

• Fundamento Legal Expresso: A permissão é garantida pelo Art. 31 da Lei nº 9.074/1995, que autoriza expressamente a participação dos autores dos projetos nesses certames.

• Justificativa: No modelo de concessão, o risco é transferido ao particular, e o foco está na eficiência da prestação do serviço, o que mitiga os riscos de direcionamento do projeto da obra em si.

Exceção Decorrente - Procedimento de Manifestação de Interesse (PMI): A participação do autor dos estudos também é permitida na licitação subsequente.

• Justificativa: A proibição inviabilizaria o próprio instituto do PMI, cujo objetivo é justamente atrair a expertise do setor privado para desenvolver projetos para a Administração. O vencedor da licitação, caso não seja o autor dos estudos, geralmente o ressarcirá.

Aplicação em Estatais (Lei 13.303/2016): Importante notar que a Lei das Estatais (Art. 44, I) segue a regra geral de vedação, aplicando a mesma proibição deste Art. 14.

Art. 75. É dispensável a licitação:

[...]

IV - para contratação que tenha por objeto:

[...]

• f) bens ou serviços produzidos ou prestados no País que envolvam, cumulativamente, alta complexidade tecnológica e defesa nacional;

g) materiais de uso das Forças Armadas, com exceção de materiais de uso pessoal e administrativo, quando houver necessidade de manter a padronização requerida pela estrutura de apoio logístico dos meios navais, aéreos e terrestres, mediante autorização por ato do comandante da força militar;

V - para contratação com vistas ao cumprimento do disposto nos arts. 3º, 3º-A, 4º, 5º e 20 da Lei nº 10.973, de 2 de dezembro de 2004, observados os princípios gerais de contratação constantes da referida Lei;

VI - para contratação que possa acarretar comprometimento da segurança nacional, nos casos estabelecidos pelo Ministro de Estado da Defesa, mediante demanda dos comandos das Forças Armadas ou dos demais ministérios;

XII - para contratação em que houver transferência de tecnologia de produtos estratégicos para o Sistema Único de Saúde (SUS), conforme elencados em ato da direção nacional do SUS, inclusive por ocasião da aquisição desses produtos durante as etapas de absorção tecnológica, e em valores compatíveis com aqueles definidos no instrumento firmado para a transferência de tecnologia;

XVI - para aquisição, por pessoa jurídica de direito público interno, de insumos estratégicos para a saúde produzidos por fundação que, regimental ou estatutariamente, tenha por finalidade apoiar órgão da Administração Pública direta, sua autarquia ou fundação em projetos de ensino, pesquisa, extensão, desenvolvimento institucional, científico e tecnológico e de estímulo à inovação, inclusive na gestão administrativa e financeira necessária à execução desses projetos, ou em parcerias que envolvam transferência de tecnologia de produtos estratégicos para o SUS, nos termos do inciso XII deste caput, e que tenha sido criada para esse fim específico em data anterior à entrada em vigor desta Lei, desde que o preço contratado seja compatível com o praticado no mercado;

Os contratos celebrados pela Administração Pública sempre deverão prever o foro da sede da Administração para dirimir qualquer questão contratual. O foro da sede será o competente inclusive quando o contratado residir no exterior. No entanto, há exceções.

Muito embora o plano de contratações anual seja facultativo, a sua publicação, uma vez realizado, é obrigatória.

cô ấy chờ ở xe bán đồ ăn

I like this as a first step, because most people might just think logically as in "o yes she has to do those things because that's where she's currently at now and it's her fault and she just needs to stick through it" but when in fact you can ask the questions around it that might lead to a bigger picture and therefore help fix the world of the underlying struggles might be?

This paragraph shows how philosophy has sort of a surface and a deeper factor to it. providing the skills to live life in a more logical and clear way on the surface but more deeper teaching how to seek out new ideas and gain knowledge in a disciplined and structured way. It shows that you should be able to gain more knowledge the more you put into obtaining said knowledge which is an important skill to have.

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Prazos de <u>24 horas</u>

• Art. 813, § 1º: Em casos especiais que exijam a realização de audiências fora da sede do juízo ou tribunal, o edital de designação do novo local deve ser afixado com antecedência mínima de 24 (vinte e quatro) horas.
• Art. 888, § 4º: O arrematante que não efetuar o pagamento do preço da arrematação dentro de 24 (vinte e quatro) horas perderá o sinal que deu em benefício da execução.

Prazos de <u>48 horas</u>

• Art. 774, parágrafo único: Caso a notificação postal não seja entregue, o Correio é obrigado a devolvê-la ao tribunal de origem no prazo de 48 horas.
• Art. 802: Após a apresentação de uma exceção de suspeição, o juiz ou tribunal deve designar uma audiência dentro de 48 (quarenta e oito) horas para instruir e julgar a questão.
• Art. 841: Uma vez recebida a reclamação trabalhista, o escrivão ou secretário tem 48 (quarenta e oito) horas para remeter a segunda via da petição ao reclamado, notificando-o para a audiência.
• Art. 851, § 2º: A ata da audiência de julgamento deve ser juntada ao processo pelo presidente ou juiz, devidamente assinada, no prazo improrrogável de 48 horas.
• Art. 880: Na fase de execução, o juiz expedirá um mandado de citação para que o executado pague a dívida em 48 (quarenta e oito) horas ou garanta a execução, sob pena de penhora.
• Art. 880, § 3º: Se o executado for procurado por duas vezes em um espaço de 48 (quarenta e oito) horas e não for encontrado, a citação será feita por edital.
• Art. 886: No processo de execução, após a inquirição de testemunhas, o escrivão ou secretário tem 48 (quarenta e oito) horas para fazer os autos conclusos ao juiz para que profira a decisão.

Dispositivo criado para assegurar ao empregado público o direito à transferência horizontal, para o mesmo quadro de pessoal, para filial localizada na mesma localidade para a qual o convivente/cônjuge servidor/militar foi transferido no interesse da administração pública.

A transferência se faz mediante solicitação do empregado público e às suas custas (não-incidência do art. 470). Esse direito do empregado público não está vinculado à discricionariedade do órgão público.

Com isso, não haverá o direito à transferência acaso cônjuge/convivente servidor peça a transferência. Será incabível se a localidade pretendida pelo empregado público não obtiver filial do órgão em que trabalha.

Súmula nº 74/TST CONFISSÃO (atualizada em decorrência do CPC de 2015) - Res. 208/2016, DEJT divulgado em 22, 25 e 26.04.2016 - I - Aplica-se a confissão à parte que, expressamente intimada com aquela cominação, não comparecer à audiência em prosseguimento, na qual deveria depor. (ex-Súmula nº 74 - RA 69/1978, DJ 26.09.1978) - II - A prova pré-constituída nos autos pode ser levada em conta para confronto com a confissão ficta (arts. 442 e 443, do CPC de 2015 - art. 400, I, do CPC de 1973), não implicando cerceamento de defesa o indeferimento de provas posteriores. (ex-OJ nº 184 da SBDI-I - inserida em 08.11.2000) - III- A vedação à produção de prova posterior pela parte confessa somente a ela se aplica, não afetando o exercício, pelo magistrado, do poder/dever de conduzir o processo.

Súmula nº 122/TST REVELIA. ATESTADO MÉDICO - A reclamada, ausente à audiência em que deveria apresentar defesa, é revel, <u>ainda que presente seu advogado munido de procuração</u>, podendo ser ilidida a revelia mediante a apresentação de atestado médico, que deverá declarar, expressamente, a impossibilidade de locomoção do empregador ou do seu preposto no dia da audiência.

Obs.: Vide que houve superação parcial dessa súmula, visto que a Reforma Trabalhista inseriu o § 5º no art. 844 para descaracterizar a revelia, mesmo que ausente o Reclamado, mas presente à audiência o seu procurador.

Súmula nº 398/TST - AÇÃO RESCISÓRIA. AUSÊNCIA DE DEFESA. INAPLICÁVEIS OS EFEITOS DA REVELIA - Na ação rescisória, o que se ataca é a decisão, ato oficial do Estado, acobertado pelo manto da coisa julgada. Assim, e considerando que a coisa julgada envolve questão de ordem pública, a revelia não produz confissão na ação rescisória.

Tema Repetitivo nº 135/TST

• O indeferimento da prova testemunhal fundamentado na presunção de veracidade decorrente de confissão ficta por desconhecimento dos fatos controvertidos pela parte ou seu preposto, em depoimento pessoal, não configura cerceamento de defesa.

Precedentes: - A jurisprudência do TST consagrou-se no sentido de que o desconhecimento do preposto dos fatos relativo à controvérsia <u>enseja a aplicação da confissão ficta</u> , não caracterizando cerceamento de defesa o indeferimento de oitiva de testemunha." (AgR-RR-86100-22.2007.5.01.0078, 1ª Turma, Relator Ministro Hugo Carlos Scheuermann, DEJT 15/08/2016).

• Com efeito, a jurisprudência desta Corte Superior, se consolidou no sentido de que o desconhecimento dos fatos pelo preposto da reclamada implica a presunção relativa de veracidade das alegações aduzidas na inicial. O Tribunal Superior do Trabalho também firmou o seu entendimento no sentido de que o indeferimento da produção de prova testemunhal, ocasionado pela aplicação da confissão ficta, não gera cerceamento do direito de defesa, tendo em vista que o art. 443, I, do CPC possibilita que o magistrado indefira a oitiva da testemunha sobre fatos provados ou confessados pela parte. Nesse contexto, uma vez comprovados os fatos articulados na exordial, em razão da aplicação da confissão ficta, não há que se falar em cerceamento do direito de defesa, na medida em que a decretação da confissão ficta produz o encerramento da produção de prova, nos termos do artigo 374, II, do CPC. (Ag-AIRR-1001230-16.2022.5.02.0614, 2ª Turma, Relatora Ministra Liana Chaib, DEJT 24/05/2024).

Using positive body language is so important. This is an area I want to grow in, and also ask for feedback as I. might not be aware of how I'm coming across.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public Review):

Summary:

Cai et al have investigated the role of msiCAT-tailed mitochondrial proteins that frequently exist in glioblastoma stem cells. Overexpression of msiCAT-tailed mitochondrial ATP synthase F1 subunit alpha (ATP5) protein increases the mitochondrial membrane potential and blocks mitochondrial permeability transition pore formation/opening. These changes in mitochondrial properties provide resistance to staurosporine (STS)-induced apoptosis in GBM cells. Therefore, msiCAT-tailing can promote cell survival and migration, while genetic and pharmacological inhibition of msiCAT-tailing can prevent the overgrowth of GBM cells.

Strengths:

The CAT-tailing concept has not been explored in cancer settings. Therefore, the present provides new insights for widening the therapeutic avenue. 

Your acknowledgment of our study's pioneering elements is greatly appreciated.

Weaknesses:

Although the paper does have strengths in principle, the weaknesses of the paper are that these strengths are not directly demonstrated. The conclusions of this paper are mostly well-supported by data, but some aspects of image acquisition and data analysis need to be clarified and extended.

We are grateful for your acknowledgment of our study’s innovative approach and its possible influence on cancer therapy. We sincerely appreciate your valuable feedback. In response, this updated manuscript presents substantial new findings that reinforce our central argument. Moreover, we have broadened our data analysis and interpretation, as well as refined our methodological descriptions.

Reviewer #2 (Public Review):

This work explores the connection between glioblastoma, mito-RQC, and msiCAT-tailing. They build upon previous work concluding that ATP5alpha is CAT-tailed and explore how CAT-tailing may affect cell physiology and sensitivity to chemotherapy. The authors conclude that when ATP5alpha is CAT-tailed, it either incorporates into the proton pump or aggregates and that these events dysregulate MPTP opening and mitochondrial membrane potential and that this regulates drug sensitivity. This work includes several intriguing and novel observations connecting cell physiology, RQC, and drug sensitivity. This is also the first time this reviewer has seen an investigation of how a CAT tail may specifically affect the function of a protein. However, some of the conclusions in this work are not well supported. This significantly weakens the work but can be addressed through further experiments or by weakening the text.

We appreciate the recognition of our study's novelty. To address your concerns about our conclusions, we have revised the manuscript. This revision includes new data and corrections of identified issues. Our detailed responses to your specific points are outlined below.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) In Figure 1B, please replace the high-exposure blots of ATP5 and COX with representative results. The current results are difficult to interpret clearly. Additionally, it would be helpful if the author could explain the nature of the two different bands in NEMF and ANKZF1. Did the authors also examine other RQC factors and mitochondrial ETC proteins? I'm also curious to understand why CAT-tailing is specific to C-I30, ATP5, and COX-V, and why the authors did not show the significance of COX-V.

We appreciate your inquiry regarding the data.  Additional attempts were made using new patient-derived samples; however, these results did not improve upon the existing ATP5⍺, (NDUS3)C-I30, and COX4 signals presented in the figure.  This is possibly due to the fact that CAT-tail modified mitochondrial proteins represent only a small fraction of the total proteins in these cells.  It is acknowledged that the small tails visible above the prominent main bands are not particularly distinct. To address this, the revised version includes updated images to better illustrate the differences. We believe the assertion that GBM/GSCs possess CAT-tailed proteins is substantiated by a combination of subsequent experimental findings. The figure (refer to new Fig. 1B) serves primarily as an introduction. It is important to note that the CAT-tailed ATP5⍺ plays a vital role in modulating mitochondrial potential and glioma phenotypes, a function which has been demonstrated through subsequent experiments.

It is acknowledged that the CAT-tail modification is not exclusive to the ATP5⍺protein.  ATP5⍺ was selected as the primary focus of this study due to its prevalence in mitochondria and its specific involvement in cancer development, as noted by Chang YW et al.  Future research will explore the possibility of CAT tails on other mitochondrial ETC proteins. Currently, NDUS3 (C-I30), ATP5⍺, and COX4 serve as examples confirming the existence of these modifications. It remains challenging to detect endogenous CAT-tailing, and bulk proteomics is not yet feasible for this purpose. COX4 is considered significant.  We hypothesize that CAT-tailed COX4 may function similarly to the previously studied C-I30 (Wu Z, et al), potentially causing substantial mitochondrial proteostasis stress.  

Concerning RQC proteins, our blotting analysis of GBM cell lines now includes additional RQC-related factors. The primary, more prominent bands (indicated by arrowheads) are, in our assessment, the intended bands for NEMF and ANKZF1.  Subsequent blotting analyses showed only single bands for both ANKZF1 and NEMF, respectively. The additional, larger molecular weight band of NEMF, which was initially considered for property analysis (phosphorylation, ubiquitination, etc.), was not examined further as it did not appear in subsequent experiments (refer to new Fig. S1C).

References:

Chang YW, et al. Spatial and temporal dynamics of ATP synthase from mitochondria toward the cell surface. Communications biology. 2023;6(1).

Wu Z, et al. MISTERMINATE Mechanistically Links Mitochondrial Dysfunction With Proteostasis Failure. Molecular cell. 2019;75(4).

(2) In addition to Figure 1B, it would be interesting to explore CAT-tailed mETC proteins in cancer tissue samples.

This is an excellent point, and we appreciate the question. We conducted staining for ATP5⍺ and key RQC proteins in both tumor and normal mouse tissues. Notably, ATP5⍺ in GBM exhibited a greater tendency to form clustered punctate patterns compared to normal brain tissue, and not all of it co-localized with the mitochondrial marker TOM20 (refer to new Fig. S3C-E). Crucially, we observed a significant increase in NEMF expression within mouse xenograft tumor tissues, alongside a decrease in ANKZF1 expression (refer to new Fig. S1A, B). These findings align with our observations in human samples.

(3) Please knock down ATP5 in the patient's cells and check whether both the upper band and lower band of ATP5 have disappeared or not.

This control was essential and has been executed now. To validate the antibody's specificity, siRNA knockdown was performed. The simultaneous elimination of both upper and lower bands upon siRNA treatment (refer to new Fig. S2A) confirms they represent genuine signals recognized by the antibody.

(4) In Figure 1C and ID, add long exposure to spot aggregation and oligomer. Figure 1D, please add the blots where control and ATP5 are also shown in NHA and SF (similar to SVG and GSC827).

New data are included in the revised manuscript to address the queries. Specifically, the new Fig 1D now displays the full queue as requested, featuring blots for Control, ATP5α, AT3, and AT20. Our analysis reveals that AT20 aggregates exhibit higher expression and accumulation rates in GSC and SF cells.

Fig. 1C has been updated to include experimental groups treated with cycloheximide and sgNEMF. Our results show that sgNEMF effectively inhibits CAT-tailing in GBM cell lines, whereas cycloheximide has no impact. After consulting with the Reporter's original creator and optimizing expression conditions, we observed no significant aggregates with β-globin-non-stop protein, potentially due to the length of endogenous CAT-tail formation (as noted by Inada, 2020, in Cell Reports). Our analysis focused on the ratio of CAT-tailed (red box blots) and non-CAT-tailed proteins (green box blots). Comparing these ratios revealed that both anisomycin treatment and sgNEMF effectively hinder the CAT-tailing process, while cycloheximide has no effect.

(5) In Figure 1E, please double-check the results with the figure legend. ATP5A aggregated should be shown endogenously. The number of aggregates shown in the bar graph is not represented in micrographs. Please replace the images. For Figure 1E, to confirm the ATP5-specific aggregates, it would be better if the authors would show endogenous immunostaining of C-130 and Cox-IV.

Labels in Fig. 1E were corrected to reflect that the bar graph in Fig. 1F indicates the number of cells with aggregates, not the quantity of aggregates per cell. The presence of endogenous ATP5⍺ is accurately shown. To address the specificity of ATP5⍺, immunostaining for endogenous NUDS3 was conducted. This revealed NUDS3 aggregation in GBM cells (SF and GSC) lacking TOM20, as demonstrated in the new Fig. S3A, B. These findings suggest NUDS3 also undergoes CAT-tailing modification, similar to ATP5⍺.

(6) Figure 3A. Please add representative images in the anisomycin sections. It is difficult to address the difference.

We appreciate your feedback. Upon re-examining the Calcein fluorescence intensity data in Fig. 3A, we believe the images accurately represent the statistical variations presented in Fig. 3B. To address your concerns more effectively, please specify which signals in Fig. 3A you find potentially misleading. We are prepared to revise or substitute those images accordingly.

(7) Figure 3D. If NEMF is overexpressed, is the CAT-tailing of ATP 5 reversed?

Thank you. Your prediction aligns with our findings. We've added data to the revised Fig. S6A, B, which demonstrates that both NEMF overexpression and ANKZF1 knockdown lead to elevated levels of CRC. This increase, however, was not statistically significant in GSC cells. A plausible explanation for this discrepancy is that the MPTP of GSC cells is already closed, thus any additional increase in CAT-tailing activity does not result in further amplification.

(8) Figure 3G. Why on the BN page are AT20 aggregates not the same as shown in Figure 2E?

We appreciate your inquiry regarding the ATP5⍺ blots, specifically those in the original Fig. 3G (left) and 2E (right). Careful observation of the ATP5⍺ band placement in these figures reveals a high degree of similarity. Notably, there are aggregates present at the top, and the diffuse signals extend downwards. Given that this is a gradient polyacrylamide native PAGE, the concentration diminishes towards the top. Consequently, the non-rigid nature of the Blue Native PAGE gel may lead to slight variations in the aggregate signals; however, the overall patterns are very much alike. To mitigate potential misinterpretations, we have rearranged the blot order in the new Fig. 3M.

(9) Figure 4D. The amount of aggregation mediated by AT20 is more compared to AT3. Why are there no such drastic effects observed between AT3 and AT20 in the Tunnel assay?

The previous Figure 4D presents the quantification of cell migration from the experiment depicted in Figure 4C. But this is a good point. TUNEL staining results are directly influenced by mitochondrial membrane potential and the state of mitochondrial permeability transition pores (MPTP), not by the degree of protein aggregation. Our previous experiments showed comparable effects of AT3 and AT20 on mitochondria (Fig. 2E, 3K), which aligns with the expected similar outcomes on TUNEL staining. As for its biological nature, this could be very complicated. We hope to explore it in future studies.

(10) Figure 5C: The role of NEMF and ANKZF1 can be further clarified by conducting Annexin-PI assays using FACS. The inclusion of these additional data points will provide more robust evidence for CAT-tailing's role in cancer cells.

In response to your suggestion, we have incorporated additional data into the revised version.

Using the Annexin-PI kit, we labeled apoptotic cells and detected them using flow cytometry (FACS). Our findings indicate that anisomycin pretreatment, NEMF knockdown (sgNEMF), and ANZKF1 upregulation (oeANKZF1) significantly increase the rate of STS-induced apoptosis compared to the control group (refer to new Fig. S9D-G).

(11) Figure 5F: STS is a known apoptosis inhibitor. Why it is not showing PARP cleavage?

Also, cell death analysis would be more pronounced, if it could be shown at a later time point. What is the STS and Anisomycin at 24h or 48h time-point? Since PARP is cleaved, it would also be better if the authors could include caspase blots.

I guess what you meant to say here is "Staurosporine is a protein kinase inhibitor that can induce apoptosis in multiple mammalian cell lines." Our study observed PARP cleavage even in GSCs, which are typically more resistant to staurosporine-induced apoptosis (C-PARP in Fig. S9B). The ratio of C-PARP to total PARP increased. We selected a 180-minute treatment duration because longer treatments with STS + anisomycin led to a late stage of apoptosis and non-specific protein degradation (e.g., at 24 or 48 hours), making PARP comparisons less meaningful. Following your suggestion, we also examined caspase 3/7 activity in GSC cells treated with DMSO, CHX, and anisomycin. We found that anisomycin treatment also activated caspases (Fig. S9A).

(12) In Figure 5, the addition of an explanation, how CAT-tailing can induce cell death, would add more information such as BAX-BCL2 ratio, and cytochrome-c release from the mitochondria.

Thank you for your suggestion. In this study, we state that specific CAT-tails inhibit GSC cell death/apoptosis rather than inducing it. Therefore, we do not expect that examining BAX-BCL2 and mitochondrial cytochrome c release would offer additional insights.

(13) To confirm the STS resistance, it would be better if the author could do the experiments in the STS-resistant cell line and then perform the Anisomycin experiments.

Thank you. We should emphasize that our data primarily originates from GSC cells. These cells already exhibit STS-resistance when compared to the control cells (Fig. S8A-C).

(14) It would be more advantageous if the author could show ATP5 CATailed status under standard chemotherapy conditions in either cell lines or in vivo conditions.

This is an interesting question. It's worth exploring this question; however, GSC cells exhibit strong resistance to standard chemotherapy treatments like temozolomide (TMZ).

Additionally, we couldn't detect changes in CAT-tailed ATP5⍺ and thus did not include that data.

(15) In vivo (cancer mouse model or cancer fly model) data will add more weight to the story.

We appreciate your intriguing question. An effective approach would be to test the RQC pathway's function using the Drosophila Notch overexpression-induced brain tumor model. However, Khaket et al. have conducted similar studies, stating, "The RNAi of Clbn, VCP, and Listerin (Ltn), homologs of key components of the yeast RQC machinery, all attenuated NSC over-proliferation induced by Notch OE (Figs. 5A and S5A–D, G)." This data supports our theory, and we have incorporated it into the Discussion. While the mouse model more closely resembles the clinical setting, it is not covered by our current IACUC proposal. We intend to verify this hypothesis in a future study.

Reference:

Khaket TP, Rimal S, Wang X, Bhurtel S, Wu YC, Lu B. Ribosome stalling during c-myc translation presents actionable cancer cell vulnerability. PNAS Nexus. 2024 Aug 13;3(8):pgae321.

Reviewer #2 (Recommendations For The Authors):

Figure 1B, C: To demonstrate that Globin, ATP5alpha, and C-130 are CAT-tailed, it is necessary to show that the high mobility band disappears after NEMF deletion or mutagenesis of the NFACT domain of NEMF. This can be done in a cell line. The anisomycin experiment is not convincing because the intensity of the bands drops and because no control is done to show that the effects are not due to translation inhibition (e.g. cycloheximide, which inhibits translation but not CAT tailing). Establishing ATP5alpha as a bonafide RQC substrate and CAT-tailed protein is critical to the relevance of the rest of the paper.

Thank you for suggesting this crucial control experiment.

To confirm the observed signal is indeed a bona fide CAT-tail, it's essential to demonstrate that NEMF is necessary for the CAT-tailing process. We have incorporated data from NEMF knockdown (sgNEMF) and cycloheximide treatment into the revised manuscript. Our findings show that both sgNEMF and anisomycin treatment effectively inhibit the formation of CAT-tailing signals on the reporter protein (Fig. 1C). Similarly, NEMF knockdown in a GSC cell line also effectively eliminated CAT-tails on overexpressed ATP5⍺ (Fig. S2B).

In general, the text should be weakened to reflect that conclusions were largely gleaned from artificial CAT tails made of AT repeats rather than endogenously CAT-tailed ATP5alpha. CAT tails could have other sequences or be made of pure alanine, as has been suggested by some studies.

Thank you for your reminder. We have reviewed the recent studies by Khan et al. and Chang et al., and we found their analysis of CAT tail components to be highly insightful. We concur with your suggestion regarding the design of the CAT tail sequence. We aimed to design a tail that maintained stability and resisted rapid degradation, regardless of its length. In the revised version, we clarify that our conclusions are based on artificial CAT tails, specifically those composed of AT repeat sequences (p. 9). We acknowledge that the presence of other sequence components may lead to different outcomes (p. 19).

Reference:

Khan D, Vinayak AA, Sitron CS, Brandman O. Mechanochemical forces regulate the composition and fate of stalled nascent chains. bioRxiv [Preprint]. 2024 Oct 14:2024.08.02.606406. Chang WD, Yoon MJ, Yeo KH, Choe YJ. Threonine-rich carboxyl-terminal extension drives aggregation of stalled polypeptides. Mol Cell. 2024 Nov 21;84(22):4334-4349.e7. 

Throughout the work (e.g. 3B, C), anisomycin effects should be compared to those with cycloheximide to observe if the effects are specific to a CAT tail inhibitor rather than a translation inhibitor.

We agree that including cycloheximide control experiments is crucial. The revised version now incorporates new data, as depicted in Fig. S5A, B, illustrating alterations in the on/off state of MPTP following cycloheximide treatment. Furthermore, Fig. S6A, B present changes in Calcium Retention Capacity (CRC) under cycloheximide treatment. The consistency of results across these experiments, despite cycloheximide treatment, suggests that anisomycin's role is specifically as a CAT tail inhibitor, rather than a translation inhibitor.

Line 110, it is unclear what "short-tailed ATP5" is. Do you mean ATP5alpha-AT3? If so this needs to be introduced properly. Line 132: should say "may indicate accumulation of CAT-tailed protein" rather than "imply".

We acknowledge your points. We have clarified that the "short-tailed ATP5α" refers to ATP5α-AT3 and incorporated the requested changes into the revised manuscript.

Figure 1C: how big are those potential CAT-tails (need to be verified as mentioned earlier)?

They look gigantic. Include a ladder.

In the revised Fig. 1D, molecular weight markers have been included to denote signal sizes. The aggregates in the previous Fig. 1C, also present in the control plasmid, are likely a result of signal overexposure. The CAT-tailed protein is observed just above the intended band in these blots. These aggregates have been re-presented in the updated figures, and their signal intensities quantified.

Line 170: "indicating that GBM cells have more capability to deal with protein aggregation".

This logic is unclear. Please explain.

We appreciate your question and have thoroughly re-evaluated our conclusion. We offer several potential explanations for the data presented in Fig. 1D: (1) ATP5α-AT20 may demonstrate superior stability. (2) GSC (GBM) cells might lack adequate mechanisms to monitor protein accumulation. (3) GSC (GBM) cells could possess an increased adaptive capacity to the toxicity arising from protein accumulation. This discussion has been incorporated into the revised manuscript (lines 166-169).

Line 177: how do you know the endogenous ATP5alpha forms aggregates due to CAT-tailing? Need to measure in a NEMF hypomorph.

We understand your concern and have addressed it. Revised Fig. 3G, H demonstrates that a reduction in NEMF levels, achieved through sgNEMF in GSC cells, significantly diminishes ATP5α aggregation. This, in conjunction with the Anisomycin treatment data presented in revised Fig. 3E, F, confirms the substantial impact of the CAT-tailing process on this aggregation.

Line 218: really need a cycloheximide or NEMF hypomorph control to show this specific to CAT-tailing.

We have revised the manuscript to include data from sgNEMF and cycloheximide treatments, specifically Fig. 3G, H, and Fig. S5C, D, as detailed in our response above.

Lines 249,266, Figure 5A: The mentioned experiments would benefit from controls including an extension of ATP5alpha that was not alanine and threonine, perhaps a gly-ser linker, as well as an NEMF hypomorph.

We sincerely appreciate your insightful comments. In response, the revised manuscript now incorporates control data for ATP5α featuring a poly-glycine-serine (GS) tail. This data is specifically presented in Figs. S2E-G, S4E, S7A, D, E, and S8F, G. Our experimental findings consistently demonstrate that the overexpression of ATP5α, when modified with GS tails, had no discernible impact on protein aggregation, mitochondrial membrane potential, GSC cell mobility, or any other indicators assessed in our study.

Figure S5A should be part of the main figures and not in the supplement.

This has been moved to the main figure (Fig. 5C).

Bir yığın tuğla ve harcı nasıl kullandığımız, onu bir “ev” yapar; ona dair ne hissettiğimiz, ne düşündüğümüz ve ne söylediğimiz ise, o evi bir “yuva”ya dönüştürür.

Kültür, bir dönemin klasik edebiyat, resim, müzik ve felsefe eserlerinde temsil edilen büyük fikirlerin toplamıdır — yani o dönemin “yüksek kültürü”dür.

APEO * A - architecture -- centralized, distributed (federated vs. non-federated) * P - processing -- (ACID, BASE, CAP theorem) * E - environment -- (pre-prod, prod, sandbox) * O - organization -- (relational, non-relational, hierarchical)

APEO A - architecture P - processing type E - environment O - organization

this is also a weak way to introduce what the topic o your paper is about. It should be more engaged and less bland.

* Rip = destrozar. * yap = ladrido molesto o chillón. * as much = tanto. * Puppy = cachorro. * bark = ladrar.

Goals of the academy

interesting

These two lines stick out to me as references to spiritual redemption and innocence. First, the washing of feet in soda water draws a remarkable parallel to the biblical scene of Jesus washing the feet of his disciples. In addition to demonstrating humility and selfless compassion towards others, washing involves the cleansing of dirt from the skin, which could also, by extension, mean the cleansing of sin or guilt from the soul. However, in "The Waste Land", Mrs. Porter and her daughter "wash their feet in soda water"- a fragment which, I discovered upon further research, originates from a vulgar soldier's WWI song (line 199). The vulgarity of the song, the change to "soda water", and the grotesque scenery of the waste land indicate that this act of foot washing is not indicative of any act of spiritual cleansing or redemption at all: humanity has fallen, but ignores the path towards good.

In the next part of the highlighted text, "et O ces voix d’enfants, chantant dans la coupole!", most nearly translates to "And, O those children’s voices singing in the dome!" (line 202). The excerpt, taken from Paul Verlaine's poem “Parsifal”, emphasizes the good and innocence inherent in the tale's protagonist, which leads to his ability to receive the Holy Grail and heal the wounded king. This further adds to the notion of spiritual redemption and innocence as appearing as parodic versions of themselves, thus expanding upon Eliot's larger theme of the loss of spirituality in TWL.

Para que corra este código es necesario el paquete gtools, porque sino sale error, sugiero agregar la instalación del paquete antes de esto o al inicio.

DOI: 10.1016/j.immuni.2025.09.001

Resource: (BioLegend Cat# 105811, RRID:AB_313220)

Curator: @scibot

SciCrunch record: RRID:AB_313220

What is this?

DOI: 10.1016/j.cell.2025.08.038

Resource: (Thermo Fisher Scientific Cat# PA5-23091, RRID:AB_2540618)

Curator: @scibot

SciCrunch record: RRID:AB_2540618

What is this?

redundant o

acento

Esto parece más intro o justifiación que conclusiones. Entiendo que lo escribes para introducir a los siguientes párrafos que sí se ven como conclusiones pero usaste 9 líneas, prácticamente un párrafo para ello - entonces, si consideras necesario mencionarlo, resúmelo a 3 líneas máximo.

Falta un espacio al inicio. Además creo que puedes borrar este enunciado o moverlo a trabajo futuro. Si se mueve, no conviene usar "Desafortunadamente", esa palabra no es técnica (desafortunadamente para quién? para ti puede ser pero para otros no, i.e., es una palabra que involucra lo personal).

Además de la corrección anterior, hay que agregar una discusión de los resultados, aunque sea cualitativa o visual, no es solo poner las gráficas. Describe lo que ves en los resultados, argumenta algo, y da entrada a la discusión cuantitativa.

Discute o concluye un poco, tanto en esta como en la Sección 13.1.2, sobre porqué estos resultados de entrenamiento y preliminares son buenos para tus resultados principales (Sección 14).

The television set was sold to its consumers as an indicator of good family value and togetherness. It was sold to paint the picture of the good old American family spending time together. Once again it was meant to be an activity where the family agreed to one thing.

(añadir)-> en adentrarse en el funcionamiento de las herramientas,

Eliminar

Twentse Belofte: Voorkomen van voortijdig schoolverlaten in Twente

Twentse Belofte is een initiatief dat zich inzet voor het voorkomen van voortijdig schoolverlaten onder jongeren in de regio Twente. De organisatie wil deze jongeren een positief toekomstperspectief bieden en erkent het belang van het behalen van een diploma voor hun welzijn en gevoel van bijdrage .

Kernmissie en aanpak

• **Voorkomen van voortijdig schoolverlaten: ** Het primaire doel van de Twentse Belofte is om te voorkomen dat jongeren, vooral kwetsbare, voortijdig van school gaan .
• **Regionale samenwerking: ** Dit bereiken ze door een regionale aanpak te ontwikkelen in samenwerking met verschillende partners in de hele keten. Dit bevordert een professioneel netwerk waar overheid, onderwijs en zorginstellingen effectief samenwerken .
• **Ondersteuning op maat: ** Het initiatief ontwikkelt benaderingen die zijn afgestemd op de specifieke situaties waarmee jongeren worden geconfronteerd, variërend van moeilijkheden bij de overgang tussen scholen (V (S) O naar MBO) tot uitdagingen zoals schulden die leiden tot schoolverzuim .

Gedefinieerde situaties en projecten

De Twentse Belofte heeft vijf specifieke situaties geschetst als leidraad voor hun samenwerkingsprojecten en interventies: * Van school naar school: ** Overgangen tussen verschillende onderwijsniveaus aanpakken . * Op school: ** Ondersteuning van leerlingen die momenteel op school zijn ingeschreven . * Van school zonder startkwalificatie: ** Met de nadruk op degenen die de school verlaten zonder een basiskwalificatie . * Specifieke doelgroepen: ** Ondersteuning op maat voor bepaalde kwetsbare groepen . * **Escalatietabellen: ** Verwijst waarschijnlijk naar gestructureerde interventies voor complexe gevallen .

Betrokkenheid en contact

• **Doelgroep: ** De website is bedoeld voor professionals binnen en buiten Twente die werken met kwetsbare jongeren. Het is bedoeld om samenwerking binnen het Twentse Belofte-netwerk te inspireren en te motiveren.
• **Uitnodiging om deel te nemen: ** Gemeenten, zorgorganisaties en kennisinstellingen worden aangemoedigd om bij te dragen aan de opbouw van een sterk netwerk. Professionals kunnen meer informatie vinden of in contact komen met projectleiders via de sectie 'Situaties' .
• **Contactgegevens: ** Voor meer informatie kunnen individuen contact opnemen met de organisatie via e-mail op info@twentsebelofte.nl .

De Twentse Belofte legt de nadruk op een collectieve inspanning om een positieve toekomst voor jongeren te verzekeren door sterke partnerschappen aan te moedigen en gerichte strategieën te ontwikkelen om voortijdig schoolverlaten tegen te gaan.

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

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Reply to the reviewers

We thank the reviewers for their thoughtful comments and overall very supportive feedback.

Reviewer #1 writes: "The study is very thorough and the experiments contain the appropriate controls. (...) The findings of the study can have relevance for human conditions involving disrupted mitochondrial dynamics, caused for example by mutations in mitofusins." Reviewer #2 writes: "The dataset is rich and the time-resolved approach strong." Reviewer #3 writes: "I admire the philosophy of the research, acknowledging an attempt to control for the many possible confounding influences. (...) This is a powerful and thoughtful study that provides a collection of new mechanistic insights into the link between physical and genetic properties of mitochondria in yeast."

We address all points below. We have not yet updated our text and figures since we expect substantial additions from new experiments. But we have included Figure R1 with some additional analyses of existing data at the bottom of the manuscript.

Reviewer1

1.1 Statistical comparisons are missing throughout the manuscript (with the exception of Fig. 2c). Appropriate statistical tests, along with p-values, should be used and reported where different gorups are compared, for example (but not limited to) Fig. 3d and most panels of Fig. 4.

We initially decided not to add too many extra labels to the already very busy plots, given that the magnitude of change mostly speaks for itself. However, we will try to find meaningful statistical tests together with a sensible graphical representation for all of the figures. For one example see Figure R1A.

1.2. I do not agree with the use of Atp6 protein as a direct read-out of mtDNA content. While Atp6 protein levels will decrease with decreasing mtDNA content, the inverse is not necessarily true: decreased Atp6 protein levels do not necessarily indicate decreased mtDNA levels, because they could alternatively or additionally be caused by decreased transcription and/or translation. Therefore, please do not equate Atp6 protein levels to mtDNA levels, and instead rephrase the text referencing the Atp6 experiments in the Results and Discussion sections to measure "mtDNA expression" or "mt-encoded protein" or similar. For example, on p. 14 line 431 should read "mtDNA expression" rather than "decreased synthesis of mtDNA", and line 440 on the same page "mean mtDNA levels" should be "mtDNA expression" or similar.

All three reviewers agree that using Atp6-NG as a direct proxy for mtDNA requires more validation, or at least rephrasing of the text. We agree that this is the most important point to address. We had previously tried using the mtDNA LacO array (Osman et al. 2015) to directly assess the amount of nucleoids per cell. However, the altered mitochondrial morphology of the Fzo1 depleted cells combined with the LacI-GFP which is still in mitochondria even when mtDNA is gone, increases the noise level to a point that we cannot interpret the signal. However, as this manuscript was in the submission process, the Schmoller lab (co-authors #2 and #7) adapted the HI-NESS system to label mtDNA in live yeast cells(Deng et al. 2025). This system promises much better signal to noise and we expect we can address all concerns regarding the actual count of nucleoids per cell. Should this unexpectedly fail for technical reasons, we will try to calibrate the Atp6-levels with DAPI staining at defined time points and will rephrase the text as the reviewer suggests.

1.3. In Fig. 3, the authors use the fluorescence intensity of a mitochondrially-targeted mCardinal as a read-out of mitochondrial mass. Please provide evidence that this is not affected by MMP, either with relevant references or by control experiments (e.g. comparing it to N-acridine orange or other MMP-independent dyes or methods).

Whether or not the import of any mitochondrial protein is dependent on the MMP depends largely on the signal sequence. The preSu9-signaling sequence was previously characterized as largely independent of the MMP compared to other presequences (Martin, Mahlke, and Pfanner 1991), which is why Vowinckel (Vowinckel et al. 2015) and others (Di Bartolomeo et al. 2020; Perić et al. 2016; Ebert et al. 2025) have previously used this as a neutral reference to the strongly MMP-dependent pre-Cox4 signal to estimate MMP. As one control in our own data, we consider that the population-averaged mitochondrial fluorescent signal Figure S3C stays constant in the first few hours, in agreement with the total averaged mitochondrial proteome (Fig R1E). As additional controls, we plan to compare the signal to an MMP independent dye as the reviewer suggests.

1.4. In Fig. 2e-f, the authors use a promoter reporter with Neongreen to answer whether the reduced levels of the nuclear-encoded mitochondrial proteins Mrps5 and Qcr7 are due to decreased expression or to protein degradation, and find no evidence of degradation of the Neongreen reporter protein. However, subcellular localization might affect the availability of the protein to proteases. Although not absolutely required, it would be relevant to know if the Neongreen fusion protein is found in the same subcellular compartment as Mrps5 and Qcr7 at 0h and 9h after Fzo1 depletion.

Here, it seems we need to explain the set-up and interpretation of the data better. The key point we are trying to make with the promoter-Neongreen construct is that the regulation is not mainly at the level of transcription. We are showing that the reduction in the levels of the actual protein (orange bars) is not (mainly) explained by a reduction in expression, since the promoter is similarly active at 0 and at 9 hours (grey bars). If expression from the promoter were strongly reduced, the Neongreen would be diluted with growth and would also decrease, but this is not the case. The fluorophore itself is just floating around in the cytosol and is not subject to the same post-translational regulation as Mrps5 and Qcr7, so there is no reason to expect degradation.

1.5. Fzo1 depletion leads to a very rapid drop in MMP during the first hour of depletion. In the Discussion, can the authors speculate on the possible mechanism of this rapid MMP drop that occurs well before mtDNA or mt-encoded proteins are decreased in level?

This is indeed an interesting point. We think there are likely three reasons causing this initial drop: Firstly, due to the fragmentation the mixing of mitochondrial content is disturbed and smaller fragments may have suboptimal stoichiometry of components (see also (Khan et al. 2024) who look at this in detail including the Fzo1 deletion); secondly, already fairly early, some mitochondrial fragments may not contain any mtDNA and therefore will be unable to synthesize ETC proteins; thirdly, altered morphological features like changes in the surface-to-volume ratios may play a role. Sadly, mechanistically following up on this is not possible with the tools in our hands and therefore outside of the scope of this manuscript. But we are happy to include these speculations in our discussion.

1.6. In Fig. 2a, the mtDNA copy number of Fzo1-depleted cells is ca 1.3-fold of the control cells at the 0h timepoint. Why might this be? Is it an impact of one of the inducers? If so, we might be looking at the combination of two different processes when measuring copy number: one that is an induction caused by the inducer(s), and the other a consequence of Fzo1 depletion itself.

We believe that this 30% increase is within the noise of the experiment rather than an effect of the induction. Since we normalize to t=0 uninduced, the first black data point does not have error bars, emphasizing this difference. None of the protein data suggests that there is an increase in mtDNA encoded proteins (see e.g. 2B, or Atp6 fluorescence data). In the planned HI-NESS experiment, we will see in our single cell data whether there is an actual increase in mtDNA upon TIR induction. Additionally, we will run a qPCR to carefully determine mtDNA levels of untreated wild-type cells, tetracycline treated wild-type cells and tetracycline induced TIR expressing cells to exclude effects of tetracycline as well as the expression of TIR on mtDNA.

Minor comments:

1.7. p. 3, line 71: "ten thousands of dividing cells.." should be "tens of thousands of dividing cells".

Thank you, will correct.

1.8.-p.4, line 116: please be even more clear with what the "depleted" cells and controls are treated with: are depleted cells treated with both inducers, and controls with neither?

We will make this more clear. Depleted cells are treated with both inducers, the control cells are not. However, in Figure 1A and in S1 we do controls to show that inducing TIR per se or adding aTC per se does not change growth rate or mitochondrial morphology. We will make this more clear.

1.9. -p.5, lines 147-148: the authors write "the rate with which the abundance of Cox2 and Var1 proteins decreases was similar to the rate of mtDNA loss" though the actual rate is not shown. Please calculate and show rates for these processes side by side to make comparison possible, or alternatively rephrase the statement.

Indeed this was not phrased well. We will call it dynamics rather than rates.

1.10. -Fig. 2d: changing the y-axis numbering to match those in panels a and b would facilitate comparisons.

Makes sense, we will change this.

1.11. Fig. 2e: it is recommended to label the western blot panels to indicate what protein is being imaged in each (Neongree,, Mrps5, Qcr7).

We will adapt the labelling to make it more clear.

1.12. -p.9, line 262: I suggest referencing Fig. 4e at the end of the first sentence for clarity.

We will modify the sentence as suggested.

1.13. -In the sections related to Fig. 3a and Fig. 5a as well as the connected supplemental data, the authors discuss both the median and the mean of mitochondrial mass and Atp6 protein, respectively. For purposes of clarity, I suggest decreasing the focus on the mean (that is provided only in the supplemental data) and focusing the text mainly on the median. The two show differing trends and it is very good that both are shown, but the clarity of the text can be improved by focusing more on the median where possible.

We will check the phrasing and simplify.

1.14. -p. 14, line 435: the statement that mt mass is maintained over the first 9h of depletion is only true for the mean mt mass, not for the median. Please make this clear or rephrase.

We will check phrasing, make it more clear and also point out the extended proteomics data (see Fig R1), which corresponds to the mean of the populations

1.15.-p.14, line 452: "mitofusions" should be "mitofusins".

Thanks for catching this.

Reviewer 2:

2.1. While inducible TIR is used to reduce background, the manuscript should rigorously exclude auxin/TIR off-targets (growth, mitochondrial phenotypes, gene expression). Please include full matched controls: (plus minus)auxin, (plus minus)TIR, epitope tag alone, and a degron control on an unrelated mitochondrial membrane protein.

We agree that rigorous controls are crucial for the interpretation of the results. However, we think we have already included most of the controls the reviewer is asking for, but we might have not pointed this out clearly enough. For example, in Fig 1A, we could make it more clear by adding more labels in which samples we added aTC, which is only described in the figure legend.

Here is a list of all the controls:

• Each depletion experiment is always matched with an experiment of the same strain without induction. So the genetic background as well as effects such as light exposure, time spent in the microfluidics systems, etc are controlled for.
• Figure S1D shows that the growth rate is wildtype like in a strain containing either the AID tag or the TIR protein AND upon addition of both chemicals. It also shows that the final genetic background (AID-tag and TIR) also grows like wildtype if the inducers are not added. This conclusively shows that neither the tags/constructs nor the chemicals per se affect growth rate
• In Figure S1C we show the mitochondrial morphology of the same controls. We will make sure to label them more consistently to match panel D, and include an actual wildtype and a FLAG-AID-Fzo1 strain without TIR treated with both aTC and 5-Ph-IAA as direct comparison
• In figure 1A we compare the Fzo1 protein levels of a strain with and without TIR. We show that in absence of TIR, adding either aTC or Auxin does not change Fzo1 levels and that the levels are comparable in the strain that is able to deplete Fzo1 directly before addition of 5-Ph-IAA (after 2 h of induction of TIR through addition of tetracycline)
• Additionally, in Figure S2C we show that two hours after adding aTC, the entire proteome does not change significantly apart from a strong induction of TIR. We can also make this more clear in the figure legend.
• Additionally, we will run a qPCR to carefully determine mtDNA levels of untreated wild-type cells, tetracycline treated wild-type cells and tetracycline induced TIR expressing cells to exclude effects of tetracycline as well as the expression of TIR on mtDNA. (also in response to 1.6.) In summary, we think we have controlled sufficiently for all confounding parameters and most importantly showed that addition of either aTC or Auxin as well as the FLAG-AID tag per se does not disturb mitochondria or cell growth. We do not see what a degron control on an unrelated protein will tell us. Depending on the nature of the protein, it may or may not have a phenotype that may or may not be related to morphology changes etc.

2.2. The Mitoloc preSu9 vs Cox4 import ratio is only a proxy of mitochondrial membrane potential (ΔΨm) and itself depends on mitochondrial mass, protein expression, matrix ATP, and import saturation. The authors need to calibrate ΔΨm with orthogonal dyes (TMRE/TMRM) and pharmacologic titrations (FCCP/antimycin/oligomycin) to generate a response curve; show that Mitoloc tracks dye-based ΔΨm across the relevant range and corrects for mass/photobleaching. Report single-cell ΔΨm vs mass residuals.

We completely agree that the MitoLoc system is only a rough proxy for the actual membrane potential. That is why we make no quantitative claims on the absolute value or absolute difference between groups of cells. We also make very clear in Fig 3B what we are actually measuring and can emphasize again in the text that this is only a proxy. We agree that it is a good idea to compare MitoLoc values to TMRE staining as the reviewer suggests, we will do these experiments in depleted and control cells at different timepoints. Please note though that also dye staining has its caveats, especially in dynamic live cell experiments. TMRM for example is not compatible with the acidic pH 5 medium that is typically used for yeast and subjecting cells to washing steps and higher pH may change both morphology of mitochondria and the MMP, especially in cells that are already “stressed”. We prefer not to complete elaborate pharmacological titration experiments because firstly, this was extensively done in the original MitoLoc paper by the Ralser lab ((Vowinckel et al. 2015), cited 120 times); secondly, the value of the MMP is not the most critical claim of the manuscript. See also 3.12. Please note that in Figure S4D we had already plotted MMP vs mitochondrial concentration.

2.3. To use Atp6-mNeon as a proxy for mtDNA is an assumption. Interpreting Atp6 intensity as "functional mtDNA" could be confounded by translation, turnover, or assembly. Please (i) report mtDNA copy number time courses (you have qPCR), nucleoid counts (DAPI/PicoGreen or TFAM/Abf2 tagging), and (ii) assess translation (e.g., 35S-labeling or puromycin proxies) and turnover (proteasome/AAA protease inhibition, mitophagy mutants -some data are alluded to- plus mRNA levels for mtDNA-encoded genes). This will support the "reduced synthesis" versus "increased degradation" conclusion.

We agree with all three reviewers that Atp6 is only a proxy for mtDNA (Jakubke et al. 2021; Roussou et al. 2024) and the correlation should be checked more carefully. We will use the very recently established Hi-NESS system to follow nucleoids/ mtDNA during depletion experiments. See detailed reply to 1.2.

(ii) in Figure 2C we inhibit mitochondrial translation and show that in this case control and depleted cells have the same level of Cox2, at least suggesting that degradation is not the key mechanism controlling the levels of mtDNA encoded proteins. We cannot do proteasome inhibitor assays since the nature of the AID-TIR systems requires an active proteasome. In figure S5C we show that the Atp6 depletion is similar in an atg32 deletion. This does not completely exclude a contribution of mitophagy to the observed phenotype, but does confirm that mitophagy is not the primary reason for cells becoming petite.

2.4. The promoter-NeonGreen reporters argue against transcriptional down-regulation of nuclear OXPHOS. Please add mRNA (RT-qPCR/RNA-seq) for representative genes and a pulse-chase or degradation-pathway dependency (e.g., proteasome/mitophagy/autophagy mutants) to firmly assign active degradation. The authors need to normalize proteomics to mitochondrial mass (e.g., citrate synthase/porin) to separate organelle abundance from protein turnover.

While we are happy to perform qPCR experiments for selected genes, a full RNA-seq experiment seems outside the scope of this study. As explained above, a proteasome inhibitor experiment is not possible in this set-up. Bulk mitophagy/autophagy seems unlikely to be the cause of the decrease of the nuclear-encoded OXPHOS proteins, since most other mitochondrial proteins do not decrease on average on population level in the first hours. This data is now plotted as additional figure (see below) and will be included in the supplementary of the revised manuscript (Fig R1E).

2.5. Using preSu9-mCardinal intensity as "mitochondrial concentration" is sensitive to expression, import competence, and morphology/segmentation. The authors should provide validation that this metric tracks 3D volume across fragmentation states (e.g., correlation with mito-GFP volumetrics; detergent-free CS activity; TOMM20/Por1 immunoblot per cell).

We agree that this is an important point and the co-authors discussed this point quite intensively. In figure S3A and B we show (using confocal data) that there is a very strong correlation between the total fluorescence signal and the 3D volume reconstruction. However, the slope of the correlation is different between tubular and fragmented mitochondria (compare panels A and B) and see figure legend. Since we are dealing with diffraction-limited objects it is likely that the 3D reconstruction is sensitive to morphology, especially if mitochondria are “clumping”. We therefore think that the total fluorescence signal is actually a better estimate of mitochondrial mass per cell than the 3D volume reconstruction (especially for our data obtained with a conventional epifluorescence microscope). The mean of the total mitochondrial fluorescence also better matches the population average mitochondrial proteome (Fig R1E). To consolidate this assumption, we will additionally compare our data to a strain with Tom70-Neongreen and to MMP independent dyes.

Notably, since the morphology is similarly altered in mothers and buds this is of minor impact for our main point – the unequal distribution between mother and buds.

2.6. The unequal mother-daughter distribution is compelling, but causality remains inferred. Test whether modulating inheritance machinery (actin cables/Myo2, Num1, Mmr1) or altering fission (Dnm1 inhibition) modifies segregation defects and rescues mtDNA/Atp6 decline. Complementation with Fzo1 re-expression at defined times would help order the phenotype cascade.

We agree that rescue experiments would be very useful. We have some preliminary data for tether experiments, for example with Num1. The general problem is that the fragmented mitochondria clump together. We have not found a method to restore an equal distribution between mother and daughter cells. We will try to optimize the assay, but are not overly confident it will work. Mmr1 deletion aggravates the Fzo1 phenotype, likely also because the distribution becomes even more heterogeneous, but we have not rigorously analyzed this.

We like the idea of the Fzo1 re-expression and will run such experiments. This will be especially powerful in combination with the new HI-NESS mtDNA reporter. We may be able to track exactly when cells reach the point-of-no return and become petite. This will also help connecting our mathematical model more directly to the data.

2.7. The model is useful but should include parameter sensitivity (segregation variance, synthesis slopes, initial nucleoid number) and prospective validation (e.g., predict rescue upon partial restoration of synthesis or inheritance, then test experimentally).

We will refine our model to include the to-be-measured nucleoids/mtDNA values. We will include a parameter sensitivity analysis with the updated model.

Reviewer 3:

3.1. About the use of Atp6 as a good proxy for mtDNA content. This is assumed from l285 onwards, based on a previous publication. As the link is fairly central to part of the paper's arguments, and the system in this study is being perturbed in several different ways, a stronger argument or demonstration that this link remains intact (and unchanged, as it is used in comparisons) would seem important.

We agree, see 1.2.

3.2. About confounding variables and processes. The study does an admirable job of being transparent and attempting to control for the many different influences involved in the physical-genetic link. But some remain less clearly unpacked, including some I think could be quite important. For example, there is a lot of focus on mito concentration -- but given the phenotypes are changing the sizes of cells, do concentration changes come from volume changes, mito changes, or both? In "ruling out" mitophagy -- a potentially important (and intuitive) influence, the argument is not presented as directly as it could be and it's not completely clear that it can in fact be ruled out in this way. There are a couple of other instances which I've put in the smaller points below.

Thank you for acknowledging our efforts to show transparent and well-controlled experiments! We address each of the specific points below.

3.3. full genus name when it first appears

We will add the full name.

3.4. I may be wrong here, but I thought the petite phenotype more classically arises from mtDNA deletion mutations, not loss? The way this is phrased implies that mtDNA loss is [always] the cause. Whether I'm wrong on that point or not, the petite phenotype should be described and referenced.

We can expand the text and cite additional relevant papers. The term “petite” refers to any strain that is respiratory incompetent and leads to small colonies (not necessarily small cells!) (Seel et al. 2023). This can be mutations or gene loss (fragments) on the mtDNA (these are called cytoplasmic petite), or chemically induced loss of mtDNA (e.g. EtBr), or mutations of nuclear genes required for respiration (these are termed nuclear petite; some nuclear petites show loss of mtDNA in addition to the mutation in the nuclear genome) (Contamine and Picard 2000).

3.5. para starting l59 -- should mention for context that mitochondria in (healthy, wildtype) yeast are generally much more fused than in other organisms

ok.

3.6. Fig 1C -- very odd choice of y-axis range! either start at zero or ensure that the data fill as much vertical space of the plot as possible

True, this was probably some formatting relic. We will adapt the axis to fill the full space. Most of our axes start at 0, but that doesn’t make so much sense here, since we consider the solidity in the control as “baseline”.

3.7. "wild-type like more tubular mitochondria" reads rather awkwardly. "more tubular mitochondria (as in the wild-type)"?

Thank you, sounds better.

3.8. l106 -- imaging artefacts? are mitos fragmenting because of photo stress? -- this is mentioned in l577-8 in the Methods, but the data from the growth rate and MMP comparison isn't given -- an SI figure would be helpful here. It would be reassuring to know that mito morphology wasn't changing in response to phototoxicity too.

In the methods we just briefly point out that we have done all our “due diligence” controls to check that we do not generate phototoxicity, something that we highlight in the cited review. We do not explicitly have a figure for this, but figure S1A shows that the solidity of the mitochondrial network in control cells stays the same over 9 hours, even though these cells are exposed to the same cultivation and imaging regime as the depleted cells. We will also add a picture of control cells after 9 h. In S1B we show that control cells containing TIR but no AID tag treated with both chemicals imaged over 9 hours also show the same solidity (~mitochondrial morphology) as untreated control. Also, the doubling times of cells grown in our imaging system (Fig R1B) are very similar to the shake flask (Fig R1A). All in all, we are very confident that our imaging settings did not impact our reported phenotypes.

3.9. para l146 -- so this suggests mtDNA-encoded proteins have a very rapid turnover, O(hours) -- is this known/reasonable?

Reference (Christiano et al. 2014) suggests that respiratory chain proteins are shorter lived than the average yeast protein. However, based on Figure 2C we think the dynamics mostly speak for a dilution by growth.

3.10. section l189 -- it's hard to reason fully about these statistics of mitochondrial concentration given that the petite phenotype is fundamentally affecting overall cell volume. can we have details on the cell size distribution in parallel with these results? to put it another way -- how does mitochondrial *amount* per cell change?

This is a good point. We report mostly on mitochondrial “concentrations” because we think this is what the cell actually cares about (mitochondrial activity in relationship to cytosolic activity). But we will include additional graphs on mitochondrial amount as well as size distributions (Fig R1C, related to Fig 4F). We can already point out that the size distribution of the population does not change much in the first hours. The “petite” phenotype refers to small colonies on growth medium with limited supply of a fermentable carbon source, not to smaller size of single cells.

3.11. l199 the mean in Fig S3C certainly does change -- it increases, clearly relative both to control and to its initial value. rather than sweeping this under the carpet we should look in more detail to understand it (a consequence of the increased skew of the distribution)?

This relates somewhat to the previous point. The increase in average concentration is not due to an increased amount in the population, but due to the fact that it is the small buds that get a very high amount of the mitochondria which “exaggerates” the asymmetric/heterogenous distribution. This will be clarified by the figures we mention in the point above.

3.12. para line 206 -- this doesn't make it clear whether your MMP signal is integrated over all mitochondria in the cell, or normalised by mitochondrial content? this matters quite a lot for the interpretation if the distributions of mitochondrial content are changing. reading on, this is even more important for para line 222. Reading further on, there is an equation on l612 that gives a definition, but it doesn't really clarify (apologies if I'm misunderstanding).

For each cell, we basically calculate the relative mitochondrial enrichment of the MMP sensitive vs the MMP insensitive pre-sequence.

So, MMP= (total intensity of mitochondrial pre-Cox4 Neongreen/ total intensity of mitochondrial pre-Su9 Cardinal) / (total cytosolic pre-Cox4 Neongreen/ total cytosolic pre-Su9 Cardinal).

We calculate this value for each cell, but we do not have the optical resolution to calculate it for individual mitochondrial fragments.

Both constructs are driven by the same strong promoter, so transcription of the fluorophore should never limit the uptake. Also, in Figure 3D we compare control and depleted cells with similar total mitochondrial concentration, so the difference must be due to a different import of the two fluorophores, see also Fig S4D. The calculated “MMP” value is of course only a crude proxy for the actual membrane potential in millivolts and we do not want to make any claims on absolute values or quantitative differences. But essentially what we are interested in is “mitochondrial health/activity” and we think the system is good at reporting this. See also 2.2.

3.13. l230 -- a point of personal interest -- low mito concentrations are connected to low "function" (MMP) and give extended division times -- this is interestingly exactly the model needed to reproduce observations in HeLa cells (https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002416). That model went on to predict several aspects of downstream cellular behaviour -- it would be very interesting to see how compatible that picture (parameterised using HeLa observations) is with yeast!

Thank you for pointing out your interesting paper, which we will include in our discussion. Another recent preprint about fission yeast (Chacko et al. 2025) also fits into this picture. Since you were kind enough to disclose your identity, we would be happy to discuss this further with you in person if we can maybe follow-up on this.

3.14. l239 "less mitochondria" -- a bit tricky but I'd say "fewer mitochondria" or "less mitochondrial content"

Thanks, we will think about how to best rephrase this, probably less mitochondrial content.

3.15. Section l234 So here (and in Fig 4) the focus is on overall distributions of mitochondrial concentration in different cells (mother-to-be, mother, bud; gen 1, gen >1). But we've just seen that one effect of fzo1 is to broader the distribution of mitochondrial concentration across cells. Can't we look in more depth at the implications of this heterogeneity? For example in Fig 4F (which is cool) we look at the distribution of all fzo1 mothers-to-be, mothers, and buds. But this loses information about the provenance. For example, do mothers-to-be with extremely low mito concentrations just push everything to the bud, while mothers-to-be with high mito concentrations distribute things more evenly? It would seem very easy and very interesting to somehow subset the distribution of mothers-to-be by concentration and see how different subsets behave

This is a good point. When analyzing the data, we pretty much plotted everything against everything and then chose the graphs that we think will best guide the reader through the story-line. We can make additional supplementary plots where we show the starting concentrations/amounts of the mother in relationship to the resulting split ratio at the end of the cycle (Fig R1D).

3.16. l285 -- experimental design -- do we know that Atp6 will continue to be a good proxy for functional mtDNA in the face of the perturbations provided by Fzo1 depletion? Especially if there is impact on the expression of mitoribosomes, the relationship between mtDNA and Atp6 may look rather different in the mutant?

This is actually our top-priority experiment now. We will use the HI-NESS system and possibly DAPI staining to make a more direct link to mtDNA/ nucleoid numbers, see 1.2.

3.17. l290 -- ruled out mitophagy. This message could be much clearer. Comparing Fig S5C and Fig 3A side-by-side is a needlessly difficult task -- put Fig 3A into Fig S5. Then we see that when mitophagy is compromised, the distribution of mitochondrial concentration has a lower median and much lower upper quartile than in the mitophagy-equipped Fzo1 mutant? What is going on here? For a paper motivated by disentangling coupled mechanisms, this should be made clearer!

Thanks for pointing this out. We can of course easily include the control in the corresponding figure. Compromising mitophagy is likely to generally affect mitochondrial health and turnover a little bit, independent of what is going on with Fzo1. The second evidence that speaks against large-scale mitophagy is the proteomics data: On population level the dynamics of the respiratory chain proteins are very different from those of other (nuclear encoded) mitochondrial proteins. We will add additional supplementary figures to make this more clear, see Fig R1E. Most mitochondrial proteins in the proteomics experiment stay constant in the first few hours, consistent with the imaging data showing that the mean mitochondrial content of the population does not change initially. This again highlights that it is the unequal distribution which is the problem and not massive degradation of mitochondria.

3.18. With the Atp6 signal, how do we know that fluorescence from different cells is comparable? Buds will be smaller than mother cells for example, potentially leading to less occlusion of the fluorescent signal by other content in the cytoplasm

This is of course a general problem that anyone faces doing quantitative fluorescence microscopy. From the technical side, we have done the best we could by taking a reasonable amount of z-slices and by choosing fluorophores that are in a range with little cellular background fluorescence (e.g. Neongreen is much better than GFP). From a practical standpoint, we are always comparing to the control, which is subject to the same technical limitations as the depleted cells and the cell sizes are very similar. So, even if we are systematically overestimating the Atp6 concentration in the bud by a few %, the difference to the control would still be qualitatively true. We therefore do not think that any of our conclusions are affected by this.

3.19. l343 -- maintenance of mtDNA -- here the point about l285 (is the Atp6-mtDNA relationship the same in the Fzo1 mutant) is particularly important, as we're directly tying findings about the protein product to implications about the mtDNA

We will carefully address this, see above.

3.20. l367 -- on a first read this description of the model feels like lots of choices have been made without being fully justified. Why a log-normal distribution (when the fit to the data looks rather flawed); why the choice of 5 groups for nucleoid number (why not 3? or 8?); the process used for parameter fitting is very unclear (after reading the methods I think some of these values are read directly from the data, but the shapes of the distributions remain unexplained). l705 -- presumably the ratio was drawn from a log-normal distribution and then the corresponding nucleoid numbers were rounded to integers? the ratio itself wasn't rounded? (also l367) How were the log-normal distributions fitted to experiments (Figs. S7A,B)? Just by eye?

We will update our model based on measured nucleoid counts and then explain more stringently the choices we make/ parameters we select.

3.21. l711 by random selection -- just at random? ("selection" could be confusing) Overall, it feels like the model may be too complicated for what it needs to show. Either (a) the model should show qualitatively that unequal inheritance and reduced production leads to rapid loss -- which a much simpler model, probably just involving a couple of lines of algebra, could show. Or (b) the model should quantitatively reproduce the particular numerical observations from the experiments -- it's not totally clear that it does this (do the cell-cycle-based decay timescales in Fig 7 correspond to the hour-based decay timescales in other plots, for example). At the moment the model is at a (b) level of detail but it's only clear that it's reporting the (a) level of results.

If the HI-NESS and Fzo1 re-addition experiments work as explained above, all parameters will have direct experimental data, and we should get much closer to (a).

3.22. A lot of the discussion repeats the results; depending on editorial preferences some of this text could probably be pared back to focus on the literature connections and context.

We will think about streamlining the discussion once some of the additional material alluded to above has been added.

3.23. Data availability -- it looks like much of the data required to reproduce the results is not going to be made available. Images and proteomic data are promised, but the data associated with mitochondrial concentration and other features are not mentioned. For FAIR purposes all the data (including statistics from analysis of the images) should be published.

We maybe didn’t phrase this clearly. All data will be made available. Where technically feasible, this will be directly accessible in a repository, otherwise by request to the corresponding author.

On our OMERO server, we have deposited many TB of raw images as well as all the intermediate steps such as segmentation masks, and the csv files with all the extracted data for each cell (including background corrections etc). Additionally, we can include csvs with the data grouped in a way that we used to generate all the box blots etc. As of now, the OMERO data is unfortunately only available by requesting a personal guest login from our bioinformatics facility, but we were promised that with the next technical update there will be a public link available. The proteomics data and the model are already fully accessible. The raw western blot images with corresponding ponceau staining will be included with the final publication either as additional supplementary material or in whatever format matches the journal requirements.

3.24 l660 -- can an overview of the EM protocol be given, to avoid having to buy the Mayer 2024 article?

The cited paper is open access. But we can also include more details in our method section.

References:

Chacko, L. A., H. Nakaoka, R. Morris, W. Marshall, and V. Ananthanarayanan. 2025. 'Mitochondrial function regulates cell growth kinetics to actively maintain mitochondrial homeostasis', bioRxiv.

Christiano, R., N. Nagaraj, F. Frohlich, and T. C. Walther. 2014. 'Global proteome turnover analyses of the Yeasts S. cerevisiae and S. pombe', Cell Rep, 9: 1959-65.

Contamine, V., and M. Picard. 2000. 'Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast', Microbiol Mol Biol Rev, 64: 281-315.

Deng, Jingti, Lucy Swift, Mashiat Zaman, Fatemeh Shahhosseini, Abhishek Sharma, Daniela Bureik, Francesco Padovani, Alissa Benedikt, Amit Jaiswal, Craig Brideau, Savraj Grewal, Kurt M. Schmoller, Pina Colarusso, and Timothy E. Shutt. 2025. 'A novel genetic fluorescent reporter to visualize mitochondrial nucleoids', bioRxiv: 2023.10.23.563667.

Di Bartolomeo, F., C. Malina, K. Campbell, M. Mormino, J. Fuchs, E. Vorontsov, C. M. Gustafsson, and J. Nielsen. 2020. 'Absolute yeast mitochondrial proteome quantification reveals trade-off between biosynthesis and energy generation during diauxic shift', Proc Natl Acad Sci U S A, 117: 7524-35.

Ebert, A. C., N. L. Hepowit, T. A. Martinez, H. Vollmer, H. L. Singkhek, K. D. Frazier, S. A. Kantejeva, M. R. Patel, and J. A. MacGurn. 2025. 'Sphingolipid metabolism drives mitochondria remodeling during aging and oxidative stress', bioRxiv.

Jakubke, C., R. Roussou, A. Maiser, C. Schug, F. Thoma, R. Bunk, D. Horl, H. Leonhardt, P. Walter, T. Klecker, and C. Osman. 2021. 'Cristae-dependent quality control of the mitochondrial genome', Sci Adv, 7: eabi8886.

Khan, Abdul Haseeb, Xuefang Gu, Rutvik J. Patel, Prabha Chuphal, Matheus P. Viana, Aidan I. Brown, Brian M. Zid, and Tatsuhisa Tsuboi. 2024. 'Mitochondrial protein heterogeneity stems from the stochastic nature of co-translational protein targeting in cell senescence', Nature Communications, 15: 8274.

Martin, J., K. Mahlke, and N. Pfanner. 1991. 'Role of an energized inner membrane in mitochondrial protein import. Delta psi drives the movement of presequences', J Biol Chem, 266: 18051-7.

Osman, C., T. R. Noriega, V. Okreglak, J. C. Fung, and P. Walter. 2015. 'Integrity of the yeast mitochondrial genome, but not its distribution and inheritance, relies on mitochondrial fission and fusion', Proc Natl Acad Sci U S A, 112: E947-56.

Perić, Matea, Peter Bou Dib, Sven Dennerlein, Marina Musa, Marina Rudan, Anita Lovrić, Andrea Nikolić, Ana Šarić, Sandra Sobočanec, Željka Mačak, Nuno Raimundo, and Anita Kriško. 2016. 'Crosstalk between cellular compartments protects against proteotoxicity and extends lifespan', Scientific Reports, 6: 28751.

Roussou, Rodaria, Dirk Metzler, Francesco Padovani, Felix Thoma, Rebecca Schwarz, Boris Shraiman, Kurt M. Schmoller, and Christof Osman. 2024. 'Real-time assessment of mitochondrial DNA heteroplasmy dynamics at the single-cell level', The EMBO Journal, 43: 5340-59-59.

Seel, A., F. Padovani, M. Mayer, A. Finster, D. Bureik, F. Thoma, C. Osman, T. Klecker, and K. M. Schmoller. 2023. 'Regulation with cell size ensures mitochondrial DNA homeostasis during cell growth', Nat Struct Mol Biol, 30: 1549-60.

Vowinckel, J., J. Hartl, R. Butler, and M. Ralser. 2015. 'MitoLoc: A method for the simultaneous quantification of mitochondrial network morphology and membrane potential in single cells', Mitochondrion, 24: 77-86.

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Referee #3

Evidence, reproducibility and clarity

This article addresses the connection between perturbed mitochondrial structure and genetics in yeast. When mitochondrial fusion is compromised, what is the chain of causality -- the mechanism -- that leads to mtDNA populations becoming depleted? This is a fascinating question, linking physical cell biology to population genetics. I admire the philosophy of the research, acknowledging and attempt to control for the many possible confounding influences. The manuscript describes the context and the research tightly and digestibly; the figures illustrate the results in a clear and natural way.

For transparency, I am Iain Johnston and I am happy for this review to be treated as public domain. To my eyes my most important shortcoming as a review is my relative lack of familiarity with the yeast fzo1 mutant; while I am familiar with analysis of yeast mito morphology and mtDNA segregation, a reviewer familiar with the nuances of this strain and its culture would be a useful complement.

I have a few more general points and a collection of smaller points below that I believe might help make the story more robust.

General points

1. About the use of Atp6 as a good proxy for mtDNA content. This is assumed from l285 onwards, based on a previous publication. As the link is fairly central to part of the paper's arguments, and the system in this study is being perturbed in several different ways, a stronger argument or demonstration that this link remains intact (and unchanged, as it is used in comparisons) would seem important.
2. About confounding variables and processes. The study does an admirable job of being transparent and attempting to control for the many different influences involved in the physical-genetic link. But some remain less clearly unpacked, including some I think could be quite important. For example, there is a lot of focus on mito concentration -- but given the phenotypes are changing the sizes of cells, do concentration changes come from volume changes, mito changes, or both? In "ruling out" mitophagy -- a potentially important (and intuitive) influence, the argument is not presented as directly as it could be and it's not completely clear that it can in fact be ruled out in this way. There are a couple of other instances which I've put in the smaller points below.

Smaller points

l47 full genus name when it first appears

l58 I may be wrong here, but I thought the petite phenotype more classically arises from mtDNA deletion mutations, not loss? The way this is phrased implies that mtDNA loss is [always] the cause. Whether I'm wrong on that point or not, the petite phenotype should be described and referenced.

para starting l59 -- should mention for context that mitochondria in (healthy, wildtype) yeast are generally much more fused than in other organisms

Fig 1C -- very odd choice of y-axis range! either start at zero or ensure that the data fill as much vertical space of the plot as possible

l105 "wild-type like more tubular mitochondria" reads rather awkwardly. "more tubular mitochondria (as in the wild-type)"?

l106 -- imaging artefacts? are mitos fragmenting because of photo stress? -- this is mentioned in l577-8 in the Methods, but the data from the growth rate and MMP comparison isn't given -- an SI figure would be helpful here. It would be reassuring to know that mito morphology wasn't changing in response to phototoxicity too.

para l146 -- so this suggests mtDNA-encoded proteins have a very rapid turnover, O(hours) -- is this known/reasonable?

section l189 -- it's hard to reason fully about these statistics of mitochondrial concentration given that the petite phenotype is fundamentally affecting overall cell volume. can we have details on the cell size distribution in parallel with these results? to put it another way -- how does mitochondrial amount per cell change?

l199 the mean in Fig S3C certainly does change -- it increases, clearly relative both to control and to its initial value. rather than sweeping this under the carpet we should look in more detail to understand it (a consequence of the increased skew of the distribution)?

para line 206 -- this doesn't make it clear whether your MMP signal is integrated over all mitochondria in the cell, or normalised by mitochondrial content? this matters quite a lot for the intepretation if the distributions of mitochondrial content are changing. reading on, this is even more important for para line 222. Reading further on, there is an equation on l612 that gives a definition, but it doesn't really clarify (apologies if I'm misunderstanding).

l230 -- a point of personal interest -- low mito concentrations are connected to low "function" (MMP) and give extended division times -- this is interestingly exactly the model needed to reproduce observations in HeLa cells (https://journals.plos.org/ploscompbiol/article?id=10.1371/journal.pcbi.1002416). That model went on to predict several aspects of downstream cellular behaviour -- it would be very interesting to see how compatible that picture (parameterised using HeLa observations) is with yeast!

l239 "less mitochondria" -- a bit tricky but I'd say "fewer mitochondria" or "less mitochondrial content"

Section l234 So here (and in Fig 4) the focus is on overall distributions of mitochondrial concentration in different cells (mother-to-be, mother, bud; gen 1, gen >1). But we've just seen that one effect of fzo1 is to broader the distribution of mitochondrial concentration across cells. Can't we look in more depth at the implications of this heterogeneity? For example in Fig 4F (which is cool) we look at the distribution of all fzo1 mothers-to-be, mothers, and buds. But this loses information about the provenance. For example, do mothers-to-be with extremely low mito concentrations just push everything to the bud, while mothers-to-be with high mito concentrations distribute things more evenly? It would seem very easy and very interesting to somehow subset the distribution of mothers-to-be by concentration and see how different subsets behave

l285 -- experimental design -- do we know that Atp6 will continue to be a good proxy for functional mtDNA in the face of the perturbations provided by Fzo1 depletion? Especially if there is impact on the expression of mitoribosomes, the relationship between mtDNA and Atp6 may look rather different in the mutant?

l290 -- ruled out mitophagy. This message could be much clearer. Comparing Fig S5C and Fig 3A side-by-side is a needlessly difficult task -- put Fig 3A into Fig S5. Then we see that when mitophagy is compromised, the distribution of mitochondrial concentration has a lower median and much lower upper quartile than in the mitophagy-equipped Fzo1 mutant? What is going on here? For a paper motivated by disentagling coupled mechanisms, this should be made clearer!

With the Atp6 signal, how do we know that fluorescence from different cells is comparable? Buds will be smaller than mother cells for example, potentially leading to less occlusion of the fluorescent signal by other content in the cytoplasm

l336 -- similar to the Jajoo et al. mechanism in fission yeast -- but are you talking about feedback control of the mtDNA or the protein (or mRNA) product?

l343 -- maintenance of mtDNA -- here the point about l285 (is the Atp6-mtDNA relationship the same in the Fzo1 mutant) is particularly important, as we're directly tying findings about the protein product to implications about the mtDNA

l367 -- on a first read this description of the model feels like lots of choices have been made without being fully justified. Why a log-normal distribution (when the fit to the data looks rather flawed); why the choice of 5 groups for nucleoid number (why not 3? or 8?); the process used for parameter fitting is very unclear (after reading the methods I think some of these values are read directly from the data, but the shapes of the distributions remain unexplained). l705 -- presumably the ratio was drawn from a log-normal distribution and then the corresponding nucleoid numbers were rounded to integers? the ratio itself wasn't rounded? (also l367) How were the log-normal distributions fitted to experiments (Figs. S7A,B)? Just by eye? l711 by random selection -- just at random? ("selection" could be confusing) Overall, it feels like the model may be too complicated for what it needs to show. Either (a) the model should show qualitatively that unequal inheritance and reduced production leads to rapid loss -- which a much simpler model, probably just involving a couple of lines of algebra, could show. Or (b) the model should quantitatively reproduce the particular numerical observations from the experiments -- it's not totally clear that it does this (do the cell-cycle-based decay timescales in Fig 7 correspond to the hour-based decay timescales in other plots, for example). At the moment the model is at a (b) level of detail but it's only clear that it's reporting the (a) level of results.

A lot of the discussion repeats the results; depending on editorial preferences some of this text could probably be pared back to focus on the literature connections and context.

Data availability -- it looks like much of the data required to reproduce the results is not going to be made available. Images and proteomic data are promised, but the data associated with mitochondrial concentration and other features are not mentioned. For FAIR purposes all the data (including statistics from analysis of the images) should be published.

l660 -- can an overview of the EM protocol be given, to avoid having to buy the Mayer 2024 article?

Significance

This is a powerful and thoughtful study that provides a collection of new mechanistic insights into the link between physical and genetic properties of mitochondria in yeast. Cell biologists, geneticists, and the mitochondrial field will find this of potentially deep interest. Because of the mode and dynamics of inheritance in budding yeast, findings here may not be directly transferrable to other eukaryotes, but these insights are still of interest for researchers outside of yeast for their insight into how this well-studied system manages its mitochondrial populations.

Es una tecnología que permite el compromiso, la participación o mejora la relación entre las personas y el gobierno al mejorar las comunicaciones ciudadanas y la decisión pública.

civic tech

Esta reflexión sobre lo abstracto o ambiguo que puede ser el lenguaje que usamos para denominar las cosas, y el impacto que puede tener nombrarlas en mayor detalle y siendo más descriptivos me resulta interesante porque en términos de la accesibilidad al conocimiento, la terminología es una de las barreras que puede limitarnos la comprensión e interacción con el mismo.

La tecnología pensada para comunidades debe práctica y no solo teórica, y para lograrlo se pueden usar dos estrategias: la envolvente, que ofrece una herramienta integral como Grafoscopio, o la incrustada, que mejora las herramientas que la gente ya utiliza, como se muestra con Cardumem. La idea es encontrar que entre estas dos formas se alinee para que la tecnología llegue a las necesidades reales de una comunidad y no solo el entorno académico u operativo de la programación.

En algunos lugares la gente habla de los desarrolladores y los que programan, pero aquí, en Colombia, la tecnología se hace pensando en algo más aterrizado que es parte de la cultura colombiana como los pueblos indígenas o campesinos, es así que los sistemas se vuelven como un medio o herramienta que no está tan suelta sino que puede ser parte de las personas.

debate sobre el “Not Invented Here syndrome” invita a reflexionar sobre cuándo reinventar herramientas en lugar de usar las ya existentes surge al decidir entre desarrollar repositorios propios o usar software de código abierto ya probado

En algunos pueblos o comunidades), la gente crea ideas para resolver problemas del día a día: cómo cuidar su idioma y otros para vivir mejor juntos etc., En otros lugares (como universidades grandes), prefieren hacer ideas muy generales que no siempre sirven de inmediato. Normalmente dicen que un trabajo es importante si está en un libro o en internet y lo leen muchos científicos, pero es más funcional revisar que es más efectivo en el impacto de ayuda a las personas en este caso proteger su cultura como su lengua y tradiciones, esto permite que el lenguaje tenga la interpretación correcta y que no la misma palabra se entienda distinto.

Parece una contradicción.

• pero se refiere al conocimiento que no tiene en cuenta o no armoniza el propósito de Dios, lo que lo hace frustrante y cansador por entender que nunca se sabe todo

• y también puede relacionarse con adquirir mucho conocimiento solo pone de manifiesto lo poco que sabemos y lo breve de nuestra vida, y éso también es frustrante e irritante

No sé refiere a una labor especial o un trabajo, sino se refiere a lo que las cosas que la humanidad realiza por toda su vida, cómo la necesidad de acumular, alimento, ropa, vivienda, y nosotros sabemos que estás cosas son necesarias, y confiamos en Jehová pero muchos hacen de estás cosas y , el acumular dinero lo más importante en su vida.

Atalaya del 77. La repetición de los ciclos puede afectar de tal manera al hombre que su sentido de la vista y su sentido del oído no se satisfagan, sino que deseen algo nuevo o novedoso. Y sin embargo realmente no hay nada nuevo en los ciclos naturales ni en los acontecimientos del vivir cotidiano común.

Viento, forma parte de un ciclo y de las leyes naturales, que fortalece nuestra fe cuando meditamos en ello. La ciencia No sabe mucho sobre el viento, pero muchas personas conocen en su poder, para navegar, generar energía o incluso protegerse. Nosotros tampoco sabemos cómo funciona, pero lo que si sabemos es que Jehová, es el creador y todo lo que ha hecho, declara sobre su sabiduría, poder amor y justicia.

portanto, nem apenas o conjunto

5.º

5.º

4.º

5.º

Author response:

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

Reviewer #1 (Public review): 

Summary: 

In this manuscript entitled "Molecular dynamics of the matrisome across sea anemone life history", Bergheim and colleagues report the prediction, using an established sequence analysis pipeline, of the "matrisome" - that is, the compendium of genes encoding constituents of the extracellular matrix - of the starlet sea anemone Nematostella vectensis. Re-analysis of an existing scRNA-Seq dataset allowed the authors to identify the cell types expressing matrisome components and different developmental stages. Last, the authors apply time-resolved proteomics to provide experimental evidence of the presence of the extracellular matrix proteins at three different stages of the life cycle of the sea anemone (larva, primary polyp, adult) and show that different subsets of matrisome components are present in the ECM at different life stages with, for example, basement membrane components accompanying the transition from larva to primary polyp and elastic fiber components and matricellular proteins accompanying the transition from primary polyp to the adult stage. 

Strengths: 

The ECM is a structure that has evolved to support the emergence of multicellularity and different transitions that have accompanied the complexification of multicellular organisms. Understanding the molecular makeup of structures that are conserved throughout evolution is thus of paramount importance. 

The in-silico predicted matrisome of the sea anemone has the potential to become an essential resource for the scientific community to support big data annotation efforts and understand better the evolution of the matrisome and of ECM proteins, an important endeavor to better understand structure/function relationships. This study is also an excellent example of how integrating datasets generated using different -omic modalities can shed light on various aspects of ECM metabolism, from identifying the cell types of origins of matrisome components using scRNA-Seq to studying ECM dynamics using proteomics. 

We greatly appreciate the positive feedback regarding the design of our study and the evolutionary significance of our findings.

Weaknesses: 

My concerns pertain to the three following areas of the manuscript: 

(1) In-silico definition of the anemone matrisome using sequence analysis: 

a) While a similar computational pipeline has been applied to predict the matrisome of several model organisms, the authors fail to provide a comprehensive definition of the anemone matrisome: In the text, the authors state the anemone matrisome is composed of "551 proteins, constituting approximately 3% of its proteome (see page 6, line 14), but Figure 1 lists 829 entries as part of the "curated" matrisome, Supplementary Table S1 lists the same 829 entries and the authors state that "Here, we identified 829 ECM proteins that comprise the matrisome of the sea anemone Nematostella vectensis" (see page 17, line 10). Is the sea anemone matrisome composed of 551 or 829 genes? If we refer to the text, the additional 278 entries should not be considered as part of the matrisome, but what is confusing is that some are listed as glycoproteins and the "new_manual_annotation" proposed by the authors and that refer to the protein domains found in these additional proteins suggest that in fact, some could or should be classified as matrisome proteins. For example, shouldn't the two lectins encoded by NV2.3951 and NV2.3157 be classified as matrisome-affiliated proteins? Based on what has been done for other model organisms, receptors have typically been excluded from the "matrisome" but included as part of the "adhesome" for consistency with previously published matrisome; the reviewer is left wondering whether the components classified as "Other" / "Receptor" should not be excluded from the matrisome and moved to a separate "adhesome" list. 

In addition to receptors, the authors identify nearly 70 glycoproteins classified as "Other". Here, does other mean "non-matrisome" or "another matrisome division" that is not core or associated? If the latter, could the authors try to propose a unifying term for these proteins? Unfortunately, since the authors do not provide the reasons for excluding these entries from the bona fide matrisome (list of excluding domains present, localization data), the reader is left wondering how to treat these entries. 

Overall, the study would gain in strength if the authors could be more definitive and, if needed, even propose novel additional matrisome annotations to include the components for now listed as "Other" (as was done, for example, for the Drosophila or C. elegans matrisomes). 

The reviewer is correct to point out the confusing terminology used throughout our manuscript, where both the total of 829 proteins constituting the curated list of ECM domain proteins and the actual matrisome (excluding "others") were referred to as "matrisomes". In general, we followed the example set by Naba & Hynes in their 2012 paper (Mol Cell Proteomics. 2012 Apr;11(4):M111.014647. doi: 10.1074/mcp.M111.014647), where they define the "matrisome" as encompassing all components of the extracellular matrix ("core matrisome") and those associated with it ("matrisome-associated" proteins). This corresponds to our group of 551 proteins, comprising both core matrisome and matrisomeassociated proteins. The Naba & Hynes paper also contains the inclusive and exclusive domain lists for the matrisome that we applied for our dataset. In the revised manuscript, we have now labelled the group of 829 proteins as "curated ECM domain proteins/genes", which includes all proteins positively selected for containing a bona fide ECM domain. After excluding non-matrisomal proteins such as receptors, we arrive at the 551 proteins that constitute the "Nematostella matrisome". We have maintained this terminology throughout the revised manuscript and have revised Figures 1B and 4B accordingly.

Regarding the category of "other" proteins, which by definition are not part of the matrisome although containing ECM domains, we have taken the reviewer's advice and classified these in more detail. We categorized all receptors as "adhesome" (202 proteins).  The remaining group of “other” secreted ECM domain proteins were then further subcategorized. Those exhibiting significant matches in the ToxProt database were subclassified as "putative venoms" (15 proteins). This group also includes the two lectins (NV2.3951 and NV2.3157), which had been originally shifted to the “other” category due to their classification as venoms. We categorized as “adhesive proteins” (28 proteins) factors such as coadhesins that due to their domain architecture resemble bioadhesive proteins described in proteomic studies of other invertebrate species, such as corals or sponges (see also https://doi.org/10.1016/j.jprot.2022.104506). Further sub-categories are stress/injury response proteins (9 proteins) and ion channels (6 proteins). The remaining 17 proteins were categorized as “uncharacterized ECM domain proteins”. These include highly diverse proteins possessing either single ECM domains or novel domain combinations. We decided to retain those in our dataset as candidates for future functional characterization.

b) It is surprising that the authors are not providing the full currently accepted protein names to the entries listed in Supplementary Table S1 and have used instead "new_manual_annotation" that resembles formal protein names. This liberty is misleading. In fact, the "new_manual_annotation" seems biased toward describing the reason the proteins were positively screened for through sequence analysis, but many are misleading because there is, in fact, more known about them, including evidence that they are not ECM proteins. The authors should at least provide the current protein names in addition to their "new_manual_annotations". 

c) To truly serve as a resource, the Table should provide links to each gene entry in the Stowers Institute for Medical Research genome database used and some sort of versioning (this could be added to columns A, B, or D). Such enhancements would facilitate the assessment of the rigor of the list beyond the manual QC of just a few entries. 

d) Since UniProt is the reference protein knowledge database, providing the UniProt IDs associated with the predicted matrisome entries would also be helpful, giving easy access to information on protein domains, protein structures, orthology information, etc. 

e) In conclusion, at present, the study only provides a preliminary draft that should be more rigorously curated and enriched with more comprehensive and authoritative annotations if the authors aspire the list to become the reference anemone matrisome and serve the community. 

Table S1 has been updated to include links to the respective Stowers Institute IDs (first two columns), as well as SwissProt IDs and current descriptions from both the Stowers Institute (SI) and Swissprot.

In our manual annotations, we prioritized these over automated ones due to the considerable effort invested in examining each sequence individually. The cnidaria-specific minicollagens and NOWA proteins might serve as an example. According to the SI descriptions, the minicollagens are annotated as “keratin-associated protein, predicted or hypothetical protein, collagen-like protein and pericardin”. We classified these as minicollagens on the basis of overall domain architecture and of signature domains and sequence motifs, such as minicollagen cysteine-rich domains (CRDs) and polyproline stretches (doi: 10.1016/j.tig.2008.07.001). NOWA is a CTLD/CRD-containing protein that is part of nematocyst tubules (doi:10.1016/j.isci.2023.106291). The first two NOWA isoforms, according to Si descriptions, were annotated as aggrecan and brevican core proteins, which is very misleading. We therefore feel that our manual annotations better serve the cnidarian research community in classifying these proteins.

Automated annotations of ECM proteins often rely on similarities between individual domains, neglecting overall domain composition. For example, Swissprot descriptions annotate 31 TSP1 domain-containing proteins in our list as "Hemicentin-1", but closer inspection reveals that only one sequence (NV2.24790) qualifies as Hemicentin-1 due to its characteristic vWFA, Ig-like, TSP1, G2 nidogen, and EGF-like domain architecture. Regarding novel protein annotations, NV2.650 might serve as an example. While SI descriptions annotate this protein as "epidermal growth factor" based on the presence of several EGF-like domains, our analysis reveals two integrin alpha N-terminal domains that classify this sequence as integrin-related. We have therefore assigned a description (Secreted integrin-N-related protein) that references this defining domain and avoids misclassification within the EGF family.

In cases where the automated annotation (including those in Genbank) matched our own findings, we adopted the existing description, as seen with netrin-1 (NV2.7734). We acknowledge that our manual annotations are not flawless and will be refined by future research. Nonetheless, we offer them as an approximation to a more accurate definition of the identified protein list.

(2) Proteomic analysis of the composition of the mesoglea during the sea anemone life cycle: 

a) The product of 287 of the 829 genes proposed to encode matrisome components was detected by proteomics. What about the other ~550 matrisome genes? When and where are they expressed? The wording employed by the authors (see line 11, page 13) implies that only these 287 components are "validated" matrisome components. Is that to say that the other ~550 predicted genes do not encode components of the ECM? This should be discussed. 

Obviously, our wording was not sufficiently accurate here. In the revised Fig. 1B we indicated that 210 of the 551 matrisome (core and associated) proteins were confirmed by mass spectrometry. In total, 287 proteins were identified by mass spectrometry, meaning that 77 of those are non-matrisomal proteins belonging to the “adhesome” (47) and “other” (30) groups. The fact that the remaining 542 proteins of the matrisome predicted by our in silico analysis could not be identified has two major reasons: (1) Our study was focussed on the molecular dynamics of the mesoglea. Therefore, only mesogleas were isolated for the mass spectrometry analysis and nematocysts were mostly excluded by extensive washing steps. As nematocysts contribute significantly to the predicted matrisome, this group of proteins is underrepresented in the mass spectrometry analysis. (2) A significant fraction of the predicted ECM proteins constitutes soluble factors and transmembrane receptors. These might not be necessarily part of the mesoglea isolates. In addition, the isolation and solubilization method we applied might have technical limitations. Although we used harsh conditions for solubilizing the mesoglea samples (90°C and high DTT concentrations), we cannot exclude that we missed proteins which resisted solubilization and thus trypsinization. We confirmed that all genes predicted by the in silico analysis have transcriptomic profiles as demonstrated in supplementary table S4. We have clarified these points in the revised results part (p.6) and also revised the statement in line 16, page 13.

b) Can the authors comment on how they have treated zero TMT values or proteins for which a TMT ratio could not be calculated because unique to one life stage, for example? 

We did not include these proteins in the analysis of the respective statistical comparison. This involved only very few proteins (about 10).  

c) Could the authors provide a plot showing the distribution of protein abundances for each matrisome category in the main figure 4? In mammals, the bulk of the ECM is composed of collagens, followed by fibrillar ECM glycoproteins, the other matrisome components being more minor. Is a similar distribution observed in the sea anemone mesoglea? 

We have included such a plot showing protein abundances across life stages and protein categories (Fig. 4A). Collagens and basement membrane proteoglycans (perlecan) are the most abundant protein categories in the core matrisome while secreted factors dominate in the matrisome-associated group.

d) Prior proteomic studies on the ECM of vertebrate organisms have shown the importance of allowing certain post-translational modifications during database search to ensure maximizing peptide-to-spectrum matching. Such PTMs include the hydroxylation of lysines and prolines that are collagen-specific PTMs. Multiple reports have shown that omitting these PTMs while analyzing LC-MS/MS data would lead to underestimating the abundance of collagens and the misidentification of certain collagens. The authors may want to reanalyze their dataset and include these PTMs as part of their search criteria to ensure capturing all collagen-derived peptides. 

Thank you for this suggestion. We have re-analyzed our dataset including lysine and proline hydroxylation as PTM. While we obtained in total 70 more proteins using this approach, this additional group did not contain any large collagen or minicollagen we had not detected before. We only obtained two additional collagen-like proteins with very short triple helical domains (V2t013973001.1, NV2t024002001.1), one being a fragment. We don’t feel this justifies implementing a re-analysis of the proteome in our study.

e) The authors should ensure that reviewers are provided with access to the private PRIDE repository so the data deposited can also be evaluated. They should also ensure that sufficient meta-data is provided using the SRDF format to allow the re-use of their LCMS/MS datasets. 

We apologize for not providing the reviewer access in our initial submission and have asked the editorial office to forward the PRIDE repository link to all reviewers immediately after receiving the reviews. We did upload a metadata.csv file with the proteomics dataset. This file contains an annotation of all TMT labels to the samples and conditions and replicates used in the manuscript. It contains similar information as an SRDF format file. In addition, the search output files on protein and psm level have been provided. So, from our point of view, we provided all necessary information to reproduce the analysis.

(3) Supplementary tables: 

The supplementary tables are very difficult to navigate. They would become more accessible to readers and non-specialists if they were accompanied by brief legends or "README" tabs and if the headers were more detailed (see, for example, Table S2, what does "ctrl.ratio_Larvae_rep2" exactly refer to? Or Table S6 whose column headers using extensive abbreviations are quite obscure). Similarly, what do columns K to BX in Supplementary Table S1 correspond to? Without more substantial explanations, readers have no way of assessing these data points. 

We have revised the tables and removed any redundant data columns. We also included detailed explanations of the used abbreviations, both in the headers and in a separate README file. Some of the information was apparently lost during the conversion to pdf files. We will therefore upload the original .xls files when submitting the revised manuscript.

Reviewer #2 (Public review): 

This work set out to identify all extracellular matrix proteins and associated factors present within the starlet sea anemone Nematostella vectensis at different life stages. Combining existing genomic and transcriptomic datasets, alongside new mass spectometry data, the authors provide a comprehensive description of the Nematostella matrisome. In addition, immunohistochemistry and electron microscopy were used to image whole mount and decellularized mesoglea from all life stages. This served to validate the de-cellularization methods used for proteomic analyses, but also resulted in a very nice description of mesoglea structure at different life stages. A previously published developmental cell type atlas was used to identify the cell type specificity of the matrisome, indicating that the core matrisome is predominantly expressed in the gastrodermis, as well as cnidocytes. The analyses performed were rigorous and the results were clear, supporting the conclusions made by the authors. 

Thank you. We greatly appreciate the positive assessment of our study.

Reviewer #3 (Public review): 

Summary: 

This manuscript by Bergheim et al investigates the molecular and developmental dynamics of the matrisome, a set of gene products that comprise the extracellular matrix, in the sea anemone Nematostella vectensis using transcriptomic and proteomic approaches. Previous work has examined the matrisome of the hydra, a medusozoan, but this is the first study to characterize the matrisome in an anthozoan. The major finding of this work is a description of the components of the matrisome in Nematostella, which turns out to be more complex than that previously observed in hydra. The authors also describe the remodeling of the extracellular matrix that occurs in the transition from larva to primary polyp, and from primary polyp to adult. The authors interpret these data to support previously proposed (Steinmetz et al. 2017) homology between the cnidarian endoderm with the bilaterian mesoderm. 

Strengths: 

The data described in this work are robust, combining both transcriptome and proteomic interrogation of key stages in the life history of Nematostella, and are of value to the community. 

Thank you for your positive assessment of our dataset. 

Weaknesses: 

The authors offer numerous evolutionary interpretations of their results that I believe are unfounded. The main problem with extending these results, together with previous results from hydra, into an evolutionary synthesis that aims to reconstruct the matrisome of the ancestral cnidarian is that we are considering data from only two species. I agree with the authors' depiction of hydra as "derived" relative to other medusozoans and see it as potentially misleading to consider the hydra matrisome as an exemplar for the medusozoan matrisome. Given the organismal and morphological diversity of the phylum, a more thorough comparative study that compares matrisome components across a selection of anthozoan and medusozoan species using formal comparative methods to examine hypotheses is required. 

Specifically, I question the author's interpretation of the evolutionary events depicted in this statement: 

"The observation that in Hydra both germ layers contribute to the synthesis of core matrisome proteins (Epp et al. 1986; Zhang et al. 2007) might be related to a secondary loss of the anthozoan-specific mesenteries, which represent extensions of the mesoglea into the body cavity sandwiched by two endodermal layers." 

Anthozoans and medusozoans are evolutionary sisters. Therefore, the secondary loss of "anthozoan-like mesenteries" in hydrozoans is at least as likely as the gain of this character state in anthozoans. By extension, there is no reason to prefer the hypothesis that the state observed in Nematostella, where gastroderm is responsible for the synthesis of the core matrisome components, is the ancestral state of the phylum. Moreover, the fossil evidence provided in support of this hypothesis (Ou et al. 2022) is not relevant here because the material described in that work is of a crown group anthozoan, which diversified well after the origin of Anthozoa. The phylogenetic structure of Cnidaria has been extensively studied using phylogenomic approaches and is generally well supported (Kayal et al. 2018; DeBiasse et al. 2024). Based on these analyses, anthozoans are not on a "basal" branch, as the authors suggest. The structure of cnidarian phylogeny bifurcates with Anthozoa forming one clade and Medusozoa forming the other. From the data reported by Bergheim and coworkers, it is not possible to infer the evolutionary events that gave rise to the different matrisome states observed in Nematostella (an anthozoan) and hydra (a medusozoan). Furthermore, I take the observation in Fig 5 that anthozoan matrisomes generally exhibit a higher complexity than other cnidarian species to be more supportive of a lineage-specific expansion of matrisome components in the Anthozoa, rather than those components being representative of an ancestral state for Cnidaria. Whatever the implication, I take strong issue with the statement that "the acquisition of complex life cycles in medusozoa, that are distinguished by the pelagic medusa stage, led to a secondary reduction in the matrisome repertoire." There is no causal link in any of the data or analyses reported by Bergheim and co-workers to support this statement and, as stated above, while we are dealing with limited data, insufficient to address this question, it seems more likely to me that the matrisome expanded in anthozoans, contrasting with the authors' conclusions. While the discussion raises many interesting evolutionary hypotheses related to the origin of the cnidarian matrisome, which is of vital interest if we are to understand the origin of the bilaterian matrisome, a more thorough comparative analysis, inclusive of a much greater cnidarian species diversity, is required if we are to evaluate these hypotheses. 

DeBiasse MB, Buckenmeyer A, Macrander J, Babonis LS, Bentlage B, Cartwright P, Prada C, Reitzel AM, Stampar SN, Collins A, et al. 2024. A Cnidarian Phylogenomic Tree Fitted With Hundreds of 18S Leaves. Bulletin of the Society of Systematic Biologists [Internet] 3. Available from: https://ssbbulletin.org/index.php/bssb/article/view/9267

Epp L, Smid I, Tardent P. 1986. Synthesis of the mesoglea by ectoderm and endoderm in reassembled hydra. J Morphol [Internet] 189:271-279. Available from: https://pubmed.ncbi.nlm.nih.gov/29954165/ 

Kayal E, Bentlage B, Sabrina Pankey M, Ohdera AH, Medina M, Plachetzki DC, Collins AG, Ryan JF. 2018. Phylogenomics provides a robust topology of the major cnidarian lineages and insights on the origins of key organismal traits. BMC Evol Biol [Internet] 18:1-18. Available from: https://bmcecolevol.biomedcentral.com/articles/10.1186/s12862-018-1142-0

Ou Q, Shu D, Zhang Z, Han J, Van Iten H, Cheng M, Sun J, Yao X, Wang R, Mayer G. 2022. Dawn of complex animal food webs: A new predatory anthozoan (Cnidaria) from Cambrian. The Innovation 3:100195 

Steinmetz PRH, Aman A, Kraus JEM, Technau U. 2017. Gut-like ectodermal tissue in a sea anemone challenges germ layer homology. Nature Ecology & Evolution 2017 1:10 [Internet] 1:1535-1542. Available from: https://www.nature.com/articles/s41559-017-0285-5

Zhang X, Boot-Handford RP, Huxley-Jones J, Forse LN, Mould AP, Robertson DL, Li L, Athiyal M, Sarras MP. 2007. The collagens of hydra provide insight into the evolution of metazoan extracellular matrices. J Biol Chem [Internet] 282:6792-6802. Available from: https://pubmed.ncbi.nlm.nih.gov/17204477/ 

We agree with the reviewer that only the analysis of several additional anthozoan and medusozoan representatives will yield a valid basis for a reconstruction of the ancestral cnidarian matrisome and allow statements about ancestral or novel features within the phylum. We have therefore revised our statements in the discussion part of the manuscript by implementing the cited literature and also findings from medusozoan genome analysis (e.g. Gold et al., 2018) demonstrating that changes in gene content are as common in the anthozoans as in medusozoans, which questioned the previously stated “basal” state of Nematostella or of anthozoans in general.

Reviewer #1 (Recommendations for the authors): 

(1) In Figure 2A, an "o" is missing in the labeling of the "developing cnidcytes" population. 

Thank you, we have corrected the typo.

(2) It would be helpful to have the different life stages indicated as headers of the heat maps presented in Figure 4. 

We have included symbolic representations for the different life stages on top of the heat maps in addition to the respective labels at the bottom.

Reviewer #2 (Recommendations for the authors): 

Important changes: 

(1) Figure 2B The x-axis tissue names should be changed to something more easily readable/understandable - some are clear, but others are not. Perhaps abbreviations could be expanded in the legend. 

We have expanded the legend in Fig. 2B to render it more easily readable. We have also rotated the maps in A to have them aligned with the ones in Fig.3B.

(2) Figure 3B This figure would be improved by the inclusion of cluster names, to understand better the mapping. 

We have added relevant cluster names to Fig. 3B and as stated above aligned the orientation of the maps in Fig. 2B and Fig. 3B.

(3) Figure 3C As with 2B, I find the y-axis cnidocyte cell state names to be unclear at times. Perhaps abbreviations could be expanded in the legend. 

All abbreviations were expanded in Fig.3C axis labels.

(4) Many of the supplementary tables are not well exported or easily readable as is (gene names are truncated, headers truncated, etc), which means that they may not be easily usable by researchers in the field interested in following up on this work in other contexts. Indeed, to be more usable, please consider sharing these supplementary data as .csv files, for example, instead of as .pdfs. 

We are sorry for this inconvenience, which was obviously caused by the conversion to pdf files. We will upload the original csv files when submitting the revised manuscript.

Smaller nitpicky comments: 

(5) Page 2 line 4 & page 3 line 7: Please consider a term other than "pre-bilaterian". The drawing/ordering of a phylogeny of extant species is not meaningful in terms of more or less ancestral. e.g. if the tips are flipped in the drawing of the tree, can we say that bilaterians are pre-cnidarians? What does that mean? 

We have used that term on the basis that cnidarians existed before the appearance of bilaterians according to the fossil record and molecular phylogenies (McFadden et al., 2021; Adoutte et al., 2000;Cavalier-Smith et al., 1996; Collins, 1998; Kim et al., 1999; Medina et al., 2001; Wainright et al., 1993). To acknowledge remaining uncertainties in the timing of origin of animals, we will use the term “early-diverging metazoans” instead, which is widely accepted in the cnidarian community. 

(6) Page 3 line 9 I was confused by the use of "gastrula-shaped body" to describe cnidarians, which are on the whole very morphologically diverse and don't all resemble gastrulae (that can also be quite diverse). 

This term is sometimes used to refer to the diploblastic cnidarian body plan (outer ectoderm, inner endoderm) with a mouth that corresponds to the blastopore. To avoid misunderstandings, we changed it in the revised manuscript to “Cnidarians, the sister group to bilaterians, are characterized by a simple body plan with a central body cavity and a mouth opening surrounded by tentacles.”

Reviewer #3 (Recommendations for the authors): 

(1) In general, I felt there was a lot of discussion about protein structure and diversity that is difficult to follow without a figure. I think some of the information in Supplementary Figures S5, S9, and S11 should be in the main figures. 

Following the reviewer’s suggestion, we have integrated Fig. S5 (collagens) into the main Fig. 2 and Fig. S9 (polydoms) into Fig. 4. As metalloproteases are not extensively discussed in the manuscript (and also due to the large size of the figure) we have kept Fig. S11 as a supplementary figure.

(2) Page 3, Line 7: The use of the term "pre-bilaterian" is inappropriate. Cnidarians and bilaterians are evolutionary sisters. Therefore, each lineage derives from the same split and is the same age. The cnidarian lineage is not older than the bilaterian lineage. 

Following a similar request by reviewer 2 we have replaced this term by “early diverging metazoans”.

(3) Page 5, Line 10. How were in silico matrisomes from early-branching metazoan species predicted? 

We applied the same bioinformatic pipeline as for the Nematostella matrisome. We clarified this in the respective methods part.

(4) Page 16, Line 8: This should be Thus. 

Obviously, the wording of this sentence was ambiguous. We changed it to ”In contrast, the adult mesoglea is significantly enriched in elastic fiber components, such as fibrillins and fibulin. This compositional shift likely adds to the visco-elastic properties (Gosline 1971a, b) of the growing body column (Fig. 4B,D, supplementary table S7).”

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Author response:

General Statements

In this paper we demonstrate that the lipid packing of the plasma membrane has a huge impact on the stability of caveolae. By using interdisciplinary techniques, we show that the widely used dynamin inhibitor Dyngo-4a adsorbs and inserts to lipid bilayers leading to a decreased lipid packing and hence reduced caveolae dynamics and internalization even in cells lacking dynamin. We have added experiments that validates that Dyngo-4a treatment does not result in fragmentation or disassembly of the caveolae.  A FRAP assay of cytosolic caveolae has been employed to address questions concerning scission. Moreover, as suggested by the reviewers, we have also included new simulation data that show and expand on the fact that Dyngo-4a positions in the lipid leaflet similar to cholesterol and preferentially associates with cholesterol clusters, affecting the spatial distribution of cholesterol in the membrane. We believe that these added data have greatly improved the paper and strengthened our conclusions that the lipid packing is a critical determinant in the balance between internalization and stable plasma membrane association of membrane vesicles.

As requested, we have expanded the introduction to provide more detailed information about previous findings in the field. Changes and addition to the text has been highlighted in red for easier tracking.

Point-by-point description of the revisions

Reviewer #1 (Evidence, reproducibility and clarity):

The authors use Dyngo-4a, a known Dynami inhibitor to test its influence on caveolar assembly and surface mobility. They investigate, whether it incorporates into membranes with Quartz-Crystal Microbalance, they investigate how it is organized in membranes using simulations. Finally, they use lipid-packing sensitive dyes to investigate lipid packing in the presence of Dyngo-4a, membrane stiffness using AFM and membrane undulation using fluorescence microscopy. They also use a measure they call "caveola duration time" to claim that something happens to caveolae after Dyngo-4a addition and using this parameter, they do indeed see an increase in it in response to Dyngo-4a, which is reduced back to the baseline after addition of cholesterol.

Overall, the authors claim: 1) Dyngo-4a inserts into the membrane and this 2) results in "a dramatic dynamin-independent inhibition of caveola scission". 3) Dyngo-4a was inserted and positioned at the level of cholesterol in the bilayer and 4) Dyngo-4a-treatment resulted in decreased lipid packing in the outer leaflet of the plasma membrane 5) but Dyngo-4a did not affect caveola morphology, caveolae-associated proteins, or the overall membrane stiffness 6) acute addition of cholesterol counteracts the block in caveola scission caused by Dyngo-4a.

Overall, in this reviewers opinion, claims 1, 3, 4, 5 are well-supported by the presented data from electron and live cell microscopy, QCM-D and AFM.

However, there is no convincing assay for caveolar endocytosis presented besides the "caveola duration" which although unclearly described seems to be the time it takes in imaging until a caveolae is not picked up by the tracking software anymore in TIRF microscopy.

Since the main claim of the paper is a mechanism of caveolar endocytosis being blocked by Dyngo-4a, a true caveolar internalization assays is required to make this claim. This means either the intracellular detection of not surface connected caveolar cargo or the quantification of caveolar movement from TIRF into epifluorescence detection in the fluorescence microscope. Otherwise, the authors could remove the claim and just claim that caveolar mobility is influenced.

We thank the reviewer for the nice constructive comments, and we very much appreciate the positive critique. We have now included a FRAP experiment of endocytic Cav1-GFP supporting the effect on internalization. In addition, we are currently preforming CTxB HRP experiments to quantify the number of caveolae at PM using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.

Reviewer #1 (Significance):

A number of small molecule inhibitors for the GTPase dynamics exist, that are commonly used tools in the investigation of endocytosis. This goes as far that the use of some of these inhibitors alone is considered in some publications as sufficient to declare a process to be dynamin-dependent. However, this is not correct, as there are considerable off-target effects, including the inhibition of caveolar internalization by a dynamin-independent mechanism. This is important, as for example the influence of dynamin small molecule inhibitors on chemotherapy resistance is currently investigated (see for example Tremblay et al., Nature Communications, 2020).

The investigation of the true effect of small molecules discovered as and used as specific inhibitors and their offside effects is extremely important and this reviewer applauds the effort. It is important that inhibitors are not used alone, but other means of targeting a mechanism are exploited as well in functional studies. The audience here thus is besides membrane biophysicists interested in the immediate effect of the small molecule Dyngo-4a also cell biologists and everyone using dynamic inhibitors to investigate cellular function.

Reviewer #2 (Evidence, reproducibility and clarity):

This manuscript uses the small molecule dynamin inhibitors dynasore and dyngo to show that in dynamin triple knockout cells that these inhibitors impact lipid packing and organization in the plasma membrane. Data showing that dyngo affects caveolin dynamics using tirf microscopy is also shown and is interpreted to reflect inhibition of caveolae scission from the membrane.

This data showing that dyngo and dynasore target membrane order is quite compelling and argues that the effects of these inhibitors is not dynamin specific and that inhibition of endocytosis by these small molecule inhibitors is dynamin-independent. The in vitro and in vivo data they provide is convincing.

Similarly, the data showing that dynasore and dyngo affect caveolin dynamics and clathrin endocytosis (transferrin) is quite convincing and argues that altered lipid packing is impacting membrane dynamics at the plasma membrane.

What is less convincing is the conclusion that dyngo is preventing caveolae scission from the membrane. Study of caveolae endocytosis is based on a TIRF assay that has inherent limitations:

- Caveolae are defined as bright cav1-positive spots in diffraction limited TIRF and their disappearance presumed to be endocytic events. Cav1 spots are presumed to be caveolae but the authors do not consider that they may be flat non-caveolar oligomers. The diffraction limited TIRF approach interprets the large structures as caveolae but evidence to that effect is lacking.

This is a valid comment and to address this we have now included data showing colocalization of cavin1 and EHD2 to the Cav1-GFP spots. We can however not determine if they are flat or invaginated. We do have extensive experience imaging caveolae using TIRF microscopy and carefully chose cells that display low expression of fluorescently labelled caveolin to avoid non-caveolar structures.

- The analysis (and the diagram presented in figure 4) considers that caveolae can either diffuse laterally in the membrane or internalize and does not consider that caveolae can flatten and possibly fragment in the membrane. Is it not possible that loss of Cav1 spots is a fragmentation event and not necessarily a scission event?

This is a good question, yet, fragmentation and disassembly would result in shorter track durations and this is not what is observed in data. We have now also included data showing that cavin1 is persistently associated with the Cav1 spots identified as caveolae during Dyngo-4a treatment indicating that these are caveolae. Furthermore, IF stainings showing colocalization of Cav1GFP with cavin1 or EHD2 after Dyngo-4a treatment have also been added. We have now also expanded on the different interpretations of the data in the results section.

- The analysis is based on overexpression of Cav1-GFP that may alter the stoichiometry between Cav1 and cavin1 such that while caveolae may be expressed, larger non-caveolar structures may accumulate.

Yes, this is correct, we have specifically imaged cell expressing low levels of Cav1-GFP to avoid accumulated non-caveolar structures that can be spotted in cells with high expression.

- Cav1 has been shown to be internalized via the CLIC pathway (Chaudary et al, 2014) and if dyngo is impacting clathrin then maybe it is also impacting CLIC endocytosis and thereby Cav1 endocytosis via this pathway?

Dyngo-4a has been shown to not affect CLIC endocytosis (McCluskey et al., 2013) and in our data we do not see internalization following Dyngo-4a treatment.

- The longer Cav1 TIRF track time and shorter displacement with dyngo is consistent with inhibition of caveolae scission. However, as the authors discuss, could not reduced membrane undulations due to dyngo's impact on membrane order be responsible for the longer tracks? Alternatively, perhaps the altered lipid packing is corralling Cav1 movement and reducing non-caveolar Cav1 endocytosis, resulting in shorter tracks of longer duration? The proposed interaction of dyngo with cholesterol could prevent scission but also stabilize large (flat?) Cav1 oligomers in the membrane, perhaps reducing Cav1 oligomer fragmentation.

We completely agree that membrane undulations contribute to instability of the TIRF-field and therefore disruption of cav1-GFP tracks as we discuss in the results section and have been described in previous work (Larsson et al., 2023). Yet, we have also shown that internalization of caveolae results in shorter tracks (Hubert et al., 2020; Larsson et al., 2023; Mohan et al., 2015). Furthermore, the tracked Cav1-GFP spots are persistently positive for cavin1 both with and without Dyngo-4a treatment showing that the majority do not disassemble become internalized by other pathways. Additionally, the added IF stainings after 30 min Dyngo-4a treatment also show that the Cav1-GFP spots remain positive for cavin1 and EHD2 just as ctrl-treated cells.

My point here is not to discredit the data but only to suggest that the TIRF approach used is an indirect measure of caveolae scission from the membrane that requires substantiation using other approaches.

We appreciate these comments and have tried to address these by adding new data and discussions on the interpretation of the tracking data in the results section.

Dyngo is certainly generally affecting lipid packing via cholesterol and thereby affecting Cav1 dynamics in the plasma membrane. The claim of caveolae scission should be qualified and alternative possibilities considered and discussed. If the authors persist in arguing that dyngo is affecting caveolae scission then the effect should be substantiated by accumulation of caveolae by quantitative EM and high spatial and temporal resolution imaging of Cav1 and cavin1 to define the endocytic events. As the latter represents a new, and potentially very challenging, line of experimentation, I would suggest that it is beyond the scope of the current study. As indicated above the additional experiments are not necessary and qualification of the claims would be sufficient.

We have now included a FRAP experiment of endocytic Cav1-GFP supporting the effect on internalization. We are also currently preforming CTxB HRP experiments to quantify the number of caveolae at the PM using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.

Other points

Figure 1C - Cav1 positive spots cannot be interpreted to be caveolae from diffraction limited confocal images. Same comment applies to Fig 4G - caveola? duration.

We completely agree with this and that the claims should be qualified. We have added IF stainings showing that the Cav1-GFP structures are also positive for cavin1. We have now clarified that we cannot distinguish between flat or different curved states of caveolae using this methodology. We have also changed the labelling of Fig. 4G.

Figure 4C - it is not clear why this EM data is not quantified - for both the number of caveolae and clathrin coated pits - as this would help clarify the interpretation of the effect reported.

We are currently preforming CTxB HRP experiments to quantify the number of caveolae using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long.

Figure 4D - the AFM experiments should perhaps be repeated as the non-significant effect of dyngo on the Young's modulus may be a result of insufficient n values.

We would like to clarify that to ensure the robustness of our AFM measurements, we performed the experiments with sufficient biological and technical replicates. Specifically, each data point shown in Figure 4D represents a Young’s modulus value averaged from approximately sixty force-distance curves per cell. For each condition, we collected force-distance maps on eight to nine individual cells, obtained from two separate petri dishes per day. We repeated this process on two independent days. In total, we analysed thirty-one cells for the DMSO control and thirty-three cells for the Dyngo-4a treatment. We performed the “student’s t-test with Welch’s correction” to access the statistical significance between the two conditions, as described in the main text. We believe that the sample size and statistical approach are sufficient to support the conclusions presented. Furthermore, we also analysed cell stiffness by calculating the slope of the linear portion of the force-distance curves. This analysis also did not reveal any statistically significant differences between the conditions (data not shown), further supporting our conclusion that Dyngo-4a treatment does not significantly alter the Young’s modulus under our experimental setup (or conditions).

Reviewer #2 (Significance):

This data showing that dyngo and dynasore target membrane order is quite compelling and argues that the effects of these inhibitors is not dynamin specific and that inhibition of endocytosis by these small molecule inhibitors is dynamin-independent. The in vitro and in vivo data they provide is convincing.

Similarly, the data showing that dynasore and dyngo affect caveolin dynamics and clathrin endocytosis (transferrin) is quite convincing and argues that altered lipid packing is impacting membrane dynamics at the plasma membrane.

What is less convincing is the conclusion is that dyngo is preventing caveolae scission from the membrane.

Reviewer #3 (Evidence, reproducibility and clarity):

Larsson et al present experimental and computational data on the role of Dyngo4a (a compound that was developed to inhibit dynamin) on the dynamics of caveolae. The manuscript mostly documents effects of Dyngo on caveolae, with one experiment to suggest a mechanism for this result. This one rather unconvincing result forms the focus of the manuscript contributing to a disconnect between the data and the presentation. Additionally, there are concerns with data interpretation. The writing could also benefit from revision to address grammar mistakes, strengthen referencing, and increase precision. Overall, the manuscript requires substantial revisions before being considered for publication. The central claim, in particular, needs stronger evidence to support the proposed mechanism.

We thank the reviewer for the thorough review and for experimental suggestions that we believe has strengthened our data further.

Significant issues (in approximate order of importance):

(1) The data supporting the central mechanistic explanation appears limited. There is no evidence that Dyngo remains in one leaflet

The simulations show that the energy barrier for moving in between bilayers is very high. Furthermore, simulations of C-Laurdan has shown that it does not readily flip in between membrane leaflets (Barucha-Kraszewska et al., 2013) supporting that it reports on the outer lipid leaflet when added to cells. We have however now changed this and state that Dyngo-4a decreased the lipid order in the plasma membrane.

- the GP of the PM is very low compared to previous measurements,

The absolute GP-values will vary between setups depending on what filters are used so they are not comparable between laboratories. What is of importance is that we found a significant change in the relative GP-values in cells treated with Dyngo-4a and control cells. It is this change that we report. We have not performed any GP-measurements on this cell type earlier so it is unclear what previous measurements reviewer #3 are referring to.

- effects on other membranes are not explored,

The order of the intracellular membranes is as expected lower than that of the plasma membrane. Differentiating different intracellular membranes of interest like endocytotic vesicles from other intracellular membranes would be very difficult but, more importantly, our study is focused on what is happening in the plasma membrane where caveolae reside and would be of minor interest for plasma membrane dynamics.

- dynamin-directed effects of Dyngo are not considered,

In the discussion section we discuss the difficulties with disentangling dynamin-direct and indirect effects.

(2) The QCM-D measurements and claims require explanation as several aspects remains unclear. In Fig S2, the 'softness' (what does this mean?) changes by 4-fold with DMSO alone (what does this mean?), then fractionally more with Dyngo. Then fractionally more again when Dyngo is removed (why?). Then it remains somewhat higher when both Dyngo and DMSO are removed, which is somehow interpreted as Dyngo remaining in the bilayer, but not DMSO.

We understand the confusion of the reviewer and hope our explanations provide clarity. QCM-D measurements are based on an oscillating quartz crystal sensor. Specifically, alterations in oscillation frequency (ΔF) and the rate of energy dissipation from the sensor surface (ΔD) are what is measured. Allowing the measurement of: 1) materials adsorbing to the sensor surface, 2) changes in the viscoelastic properties of a solution in contact with the sensor surface, 3) changes in the material adsorbed to the sensor surface upone exposure to different solutions. The ratio of ΔD/-ΔF reports the mechanical softness or rigidity of an adsorbed material, in this case the SLB.

A “buffer shift” is the term used when there is not an adsorption to the sensor surface, but rather an effect from altering the solution above the sensor surface. One reason is because different solutions can have different densities (e.g., a DMSO-buffer mixture vs buffer alone), which impacts the oscillations of the sensor. It was observed that the DMSO-buffer mixture alone gave a large buffer shift in comparison to the adsorption of the Dyngo-4a into the SLB, thereby muddling the data interpretation. Thus, in Fig. S2 the system was first equilibrated with the DMSO-buffer mixture prior to addition of the Dyngo-4a solution to allow for clearer visualization of the two events. In QCMD to assess if something has made a permeant change to the system you change back to the solutions used before the addition, thus first we washed with a DMSO-Buffer mixture followed by buffer alone. Control experiments were carried out in which no Dyngo-4a was added (also shown in Fig. S2). The control shows the same “buffer shift” from the DMSO-buffer mixture occurs in both systems and that upon returning to a buffer only condition there is no permanent change to the system caused from exposure to the DMSO. In contrast, once the system that received Dyngo-4a is changes back to a buffer only system we see that mass has been added to the system (ΔF) with little change to the dissipation (ΔD), thereby resulting in a lower ratio of ΔD/-ΔF, which is to say that the SLB after the adsorption of Dyngo-4a was more rigid that the SLB without Dyngo-4a.

These interpretations are difficult to grasp, as the authors seem to be implying simple amphiphilic partitioning into the membrane, which should all be removable by efficient washing.

Amphiphilic partitioning is not fully reversible by “efficient washing” it depends on partitioning coefficients.

I do not doubt that this compound interacts with membranes, but the quantifications appear ambiguous. A bilayer with 16 mol% (or worse, 30% if all in one leaflet) Dyngo is very unlikely (to remain a bilayer). Even if such a bilayer was conceivable, the authors are claiming an ADDITION of Dyngo that would INCREASE the area of one leaflet by 30%, which needs explanation as it appears unlikely.

We understand that in our attempt provide numbers in the results section for the amount of binding observed in QCM-D, this can easily be interpreted as this is what is observed to insert into the PM. However, as discussed in the discussion, we also see aggregations of Dyngo-4a that associate with the membrane in the simulations which likely could contribute to the binding observed in QCM-D prior to washing. The precise amount of membrane inserted Dyngo-4a is difficult to measure as we discuss in the text. In order to make this clearer, we have now moved all these details to the discussion section where we elaborate on this. Furthermore, since Dyngo-4a, like cholesterol, is intercalating in between the head groups of the lipids the area would not increase in direct proportion to the mol%.

Also, there are no replicates shown, so unclear how reproducible these effects are?

For clarity, only single experiments are shown. However, multiple experiments were performed and the range in measured values for 3 technical repeats can be observed in the standard deviations found in the main text (e.g., 6 ± 2 mol%).

(3) The simulations are insufficiently described and difficult to interpret. How big are these systems? Why do the figures show the aqueous system with lateral boundaries?

There are no explicit boundaries used in the simulations, periodic boundary conditions are applied in all three dimensions. The lateral boundaries observed in the figures correspond to the simulation box edges and are a visual artifact of 2D projections with QuickSurf representation. No artificial wall or constraints were introduced laterally. Additional technical details, including the system size and periodic boundary conditions have now been added to the methods section.

It seems quite important that multiple Dyngo molecules aggregate rather than partition into membranes - is this likely to occur in experiment?

Yes, this is important and with the additional simulation experiments suggested by Reviewer #3 it has been clarified that they contribute a great deal to the change in lipid packing of lipid bilayers containing cholesterol.  However, it is hard to test aggregation is the cellular system, but we believe that this happens and contribute to the effect on membranes. We have now emphasized the effect of the aggregates in the text.

PMF simulations are strongly suggesting that Dyngo does not spontaneously cross membranes, which is inconsistent with its drug-like amphiphilicity (cLogP~2.5 is optimally suited for membrane permeation) and known effects on intracellular proteins. This suggests an artefact in these PMFs.

As stated in the submitted version of the manuscript, logP was used to validate the topology and the observed value was in a very good agreement with cLogP. Moreover, this validation complemented the standard procedure of CHARMM-GUI ligand modelling, that provided a reasonable penalty score (around 20) for the Dyngo-4a topology. POPC and cholesterol molecules are standard in the force field and validated by numerous studies. The parameters used for the membrane simulations and AWH in particular are very common for this type of studies. Thus, we do not see what may cause any artifacts in the free energy profile construction. In fact, amphiphilicity of the molecule may be one of the key reasons that Dyngo-4a molecule remains at the aqueous interface of the membrane and does not cross the membrane spontaneously. Also, we believe that the energy barrier of 40-60 kJ/mol is not prohibitively high and Dyngo-4a molecules may still overcome the barrier eventually, though we expect majority to reside in the upper leaflet.

The authors should experimentally measure the permeation of Dyngo through bilayers (or lack thereof), to more robustly support their finding that Dyngo does not cross membranes spontaneously.

We thank the reviewer for the suggestion, however this if very technically challenging and would require establishment of precise systems which is beyond the scope of this manuscript.

(4) Why not measure effect of Dyngo on lipid packing directly and more broadly in model membranes?

With the added modelling experiments supporting the previous simulations and the calculated GP values from the C-Laurdan experiments on cellular plasma membrane, we do not find it necessary to include more model membranes experiments than the already existing ones on lipid monolayers and supported lipid bilayers.

(5) Statistics should not be done on individual cells (n>26), but rather on independent experiment (N=3?)

We have performed the statistics on live cell particle tracking according to previous literature on similar systems (Boucrot et al., 2011; Larsson et al., 2023; Shvets et al., 2015; Stoeber et al., 2012).

(6) Fig 1G is important but rather unclear. Firstly, these kymographs are an odd way to show that the caveolae are not moving. More importantly, caveolae in normal cells have been shown to be quite stable and immobile (eg doi: 10.1074/jbc.M117.791400), yet here they are claimed to be very mobile.

Although this might be an odd and unconventional way to depict dynamic processes, we believe that this is a very illustrative way to show track stability over time in bulk rather than just a kymograph over a few structures in a cell. Furthermore, we are not claiming that caveolae are very mobile but rather the opposite very stable in agreement with previous work (Boucrot et al., 2011; Larsson et al., 2023; Mohan et al., 2015). We have now edited the text to make this even clearer.

Also, if Dyngo prevents caveolae scission, there should be more of them at the membrane - why no quantification like Fig 1C to show accumulation of caveolae upon Dyngo treatment? Or directly counting caveolae via EM, as in Fig 4C?

We are currently preforming CTxB HRP experiments using EM but due to reasons out of our control we have not managed to finish these on time, they will be included in the manuscript once they are ready in hopefully not too long. However, Dynasore has previously been shown, by EM, to increase the number of caveolae at the PM (Moren et al., 2012; Sinha et al., 2011).

(7) The writing can be made more precise and referencing could be strengthened.

The introduction was written in a short format, and we have now extended this and made it more precise.

Some examples:

(a) 'scissoned' is not a word in English,

Thanks, we have now changed this.

(b) what is meant by "Cav1 assembly is driven by high chol content"? There are many types of caveolin assemblies.

We agree that this can be made more precise and have now clarified this in the introduction.

(c) "This generates a unique membrane domain with distinct lipid packing and a very high curvature." Unclear what 'this' refers to and there is no reference here, so what is the evidence for either of these claims? Caveolin-8S oligomers are not curved. Perhaps 'this' is caveolae, but they are relatively large and also not very highly curved and I am unaware of measurements of lipid packing therein.

Caveolae are around 50 nm which in biology is a very high curvature of a membrane. It has been extensively proven that caveolae have a distinct lipid composition highly enriched in cholesterol and sphingolipids, which thereby also will generate a unique lipid packing as compared to the surrounding membrane. Yet, the reviewer is correct that lipid packing has not been measured in a caveola for obvious technical challenges. Thus, we have now changed the text to “special lipid composition”.

The sentence following that one again makes a specific, but unreferenced, claim.

(d) intro claims that lipid packing is critical for fission, but it is unclear quite what is meant by this claim. The references do not help, as they are often about the basic biophysics of lipids, rather than how packing affects fission.

We have now edited the text.  

(e) intro strongly implies that caveolae remain membrane attached because of stalled scission. How strong is the evidence for this? The fact that EHD2 is at the neck is not definitive,

We used the term stalled scission to describe that all omega shaped membrane invaginations do not scission in the same automatic way as clathrin coated vesicles. We have now changed this in the text. Caveolae are shown to be released (undergo scission) and be detected as internal caveolae if the protein EHD2 is removed. Hence this must be interpreted as if EHD2 stalls scission. The evidence includes data compiled over the last 12 years from others and us which include for example: 1) Caveolae with EHD2 have a longer duration time (Larsson et al., 2023; Mohan et al., 2015; Moren et al., 2012; Stoeber et al., 2012), Knock down of EHD2 results in more internalized caveolae as measured by CTxB HRP using EM (Moren et al., 2012) and shorter duration time at the PM (Hubert et al., 2020; Larsson et al., 2023; Mohan et al., 2015; Stoeber et al., 2012). 2) EHD2 overexpression results in less internalized caveolae as measured by CTxB HRP using EM (Stoeber et al., 2012). Furthermore, 3) overexpression or acute addition of purified EHD2 via microinjection counteracts lipid induced scission of caveolae and hence result in caveolae stabilization at the PM (Hubert et al., 2020). It is very hard to see that the release and internalization of caveolae could result from anything else than that these have undergone scission. EHD2 has been found around the rim of caveolae (Matthaeus et al., 2022) and overexpression of EHD2 oligomerizing mutants have been shown to expand the caveola neck (Hoernke et al., 2017; Larsson et al., 2023).

(f) unclear what is meant by 'lipid packing frustration' and how Dyngo supposedly induces it.

Lipid packing frustration refers to what is usually referred to as lipid packing defect, but since lipid membranes are describe as a fluid system it should not have defects whereby, we believe that lipid packing frustration is more accurate. However, we have now changed the text and use “decreased lipid packing” or “decreased lipid order” more thoroughly to describe the effect on the plasma membrane.

(8) IF of Cav1 is insufficient to claim puncta as caveolae. Co-stained puncta of caveolin with cavin are much stronger evidence. Same issue for Cav1-GFP puncta.

We agree and have now provided IF showing cavin1 and EHD2 colocalization to Cav1GFP in non and Dyngo-4a-treated cells.

(9) Fig 3E claims that "preferred position of Dyngo-4a was closer to the head groups" but the minimum looks to be in similar place as Fig 3B without cholesterol. Response:

We appreciate the reviewer’s observation. The PMF minima in the POPC and POPC:Chol membranes are indeed close in absolute position (~1.1–1.2 nm from the bilayer center). However, as clarified in the revised text, the presence of cholesterol leads to a slight shift of Dyngo-4a closer to the headgroup region and broadens the positional distribution. This is also evident from the added density profiles (Fig. S3A) and is now described more precisely in the manuscript.

Critically, these results do not support the notion that Dyngo affects lipid packing sufficiently, which is not measured in the simulations (though could be).

We thank the reviewer for the excellent suggestion. In response, we have now included a detailed analysis of Dyngo-4a’s effect on lipid packing in the simulations. As described in the revised manuscript, we measured deuterium order parameters, area per lipid (APL), and lipid–Dyngo–cholesterol spatial distributions (Figs. 3-H, S3C-E). The results demonstrate that Dyngo-4a decreases lipid order in POPC:Chol membranes. Both single molecules and clusters reduce the order parameter by up to 0.04 units, particularly in the upper leaflet, where Dyngo-4a reside.The reduction is most pronounced in the midchain region of the sn1 tail and around the double bond of the sn2 tail. These effects were accompanied by increased APL in POPC:Chol membranes and by colocalization of Dyngo-4a near cholesterol-rich regions. Together, these data confirm that Dyngo-4a perturbs membrane organization and lipid packing in a composition-dependent manner. We believe these additions directly address the concern and demonstrate that the simulations indeed support the conclusion that Dyngo-4a modulates lipid packing.

Finally, the simulation data do not show "that Dyngo-4a is competing with cholesterol"; it is unclear what 'competition' means in this context, but regardless, the data only shows that Dyngo sits at a similar location as cholesterol.

We agree with the reviewer that “competition” was an imprecise term. We have rephrased the relevant sections to clarify that Dyngo-4a and cholesterol localize to overlapping regions and exhibit spatial coordination. As now stated in the manuscript, cholesterol appears to partially displace Dyngo-4a from its preferred depth seen in pure POPC, broadens its membrane distribution, and alters lipid packing. According to the order parameters there is an interplay between chol and Dyngo-4a and the heatmaps show that the distribution of chol in the membrane gets less uniform in the presence of Dyngo-4a. These interactions suggest that Dyngo-4a perturbs cholesterol-rich domains.

As new analysis routines were added to the study, we have now also added the details on those to the Methods section of the text.

(10) AFM measures the stiffness of the cell (as correctly explained in Results section) not "overall stiffness of the PM" as stated in the Discussion.

We thank the reviewer for pointing this out, we have now altered this in the discussion section.

(11) Fig2A: what was the starting lipid surface pressure? How does Dyngo insertion depend on initial lipid packing?

The starting pressure lipid pressure was 20 mN m<sup>-1</sup which we now have incorporated in the figure legend. We performed several such experiments with a starting pressure ranging from 20-23 mN m^-1 showing consistent results which we described in the materials and methods section. Given that we also performed QCMD analysis and simulations on bilayers showing that Dyngo-4a adsorbed and inserted respectively, we have not performed a titration of starting pressures resulting in a MIP of Dygo-4a.

(12) Fig 4B is a strange approach to measure membrane motion. Why not RMSD or some other displacement based method? As its shown, it implies that the area of the cell changes.

The method that we used to quantify the area of the cell which is attached (or close to) the glass and thereby is visible in TIRF microscopy. This is area indeed changes over time which has been frequently observed and used to describe and quantify the mobility, lamellipodia and filopodia formation among other things. We agree that RMSD can also be used to analyze the data before and after treatments and we have now included RMSD­­­­ analysis in the manuscript.

Reviewer #3 (Significance):

The title, abstract, and introduction of the manuscript are largely framed around lipid packing, but most of the data investigate other unexpected effects of treating cells with Dyngo4a. The only measurement for lipid packing (or any other membrane properties) is Fig 4E-F. Therefore, this paper is effectively an investigation of an artefact of a common reagent, which itself could be a valuable contribution. However, the mechanism to explain its effect requires stronger evidence, and its broad biological significance needs further exploration.

Overall, the impact of documenting the effects of Dyngo4a on membranes appears modest but may be valuable to the membrane trafficking community.

Barucha-Kraszewska, J., S. Kraszewski, and C. Ramseyer. 2013. Will C-Laurdan dethrone Laurdan in fluorescent solvent relaxation techniques for lipid membrane studies? Langmuir. 29:1174-1182.

Boucrot, E., M.T. Howes, T. Kirchhausen, and R.G. Parton. 2011. Redistribution of caveolae during mitosis. J Cell Sci. 124:1965-1972.

Hoernke, M., J. Mohan, E. Larsson, J. Blomberg, D. Kahra, S. Westenhoff, C. Schwieger, and R. Lundmark. 2017. EHD2 restrains dynamics of caveolae by an ATP-dependent, membrane-bound, open conformation. Proc Natl Acad Sci U S A. 114:E4360-E4369.

Hubert, M., E. Larsson, N.V.G. Vegesna, M. Ahnlund, A.I. Johansson, L.W. Moodie, and R. Lundmark. 2020. Lipid accumulation controls the balance between surface connection and scission of caveolae. Elife. 9.

Larsson, E., B. Moren, K.A. McMahon, R.G. Parton, and R. Lundmark. 2023. Dynamin2 functions as an accessory protein to reduce the rate of caveola internalization. J Cell Biol. 222.

Matthaeus, C., K.A. Sochacki, A.M. Dickey, D. Puchkov, V. Haucke, M. Lehmann, and J.W. Taraska. 2022. The molecular organization of differentially curved caveolae indicates bendable structural units at the plasma membrane. Nat Commun. 13:7234.

McCluskey, A., J.A. Daniel, G. Hadzic, N. Chau, E.L. Clayton, A. Mariana, A. Whiting, N.N. Gorgani, J. Lloyd, A. Quan, L. Moshkanbaryans, S. Krishnan, S. Perera, M. Chircop, L. von Kleist, A.B. McGeachie, M.T. Howes, R.G. Parton, M. Campbell, J.A. Sakoff, X. Wang, J.Y. Sun, M.J. Robertson, F.M. Deane, T.H. Nguyen, F.A. Meunier, M.A. Cousin, and P.J. Robinson. 2013. Building a better dynasore: the dyngo compounds potently inhibit dynamin and endocytosis. Traffic. 14:1272-1289.

Mohan, J., B. Moren, E. Larsson, M.R. Holst, and R. Lundmark. 2015. Cavin3 interacts with cavin1 and caveolin1 to increase surface dynamics of caveolae. J Cell Sci. 128:979-991.

Moren, B., C. Shah, M.T. Howes, N.L. Schieber, H.T. McMahon, R.G. Parton, O. Daumke, and R. Lundmark. 2012. EHD2 regulates caveolar dynamics via ATP-driven targeting and oligomerization. Mol Biol Cell. 23:1316-1329.

Shvets, E., V. Bitsikas, G. Howard, C.G. Hansen, and B.J. Nichols. 2015. Dynamic caveolae exclude bulk membrane proteins and are required for sorting of excess glycosphingolipids. Nat Commun. 6:6867.

Sinha, B., D. Koster, R. Ruez, P. Gonnord, M. Bastiani, D. Abankwa, R.V. Stan, G. Butler-Browne, B. Vedie, L. Johannes, N. Morone, R.G. Parton, G. Raposo, P. Sens, C. Lamaze, and P. Nassoy. 2011. Cells respond to mechanical stress by rapid disassembly of caveolae. Cell. 144:402-413.

Stoeber, M., I.K. Stoeck, C. Hanni, C.K. Bleck, G. Balistreri, and A. Helenius. 2012. Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin. EMBO J. 31:2350-2364.

Creo que no tiene caso dividir el texto con esta subsección si solo hay una subsección. Puede quedar todo el texto (4 párrafos o 3 si quitas el anterior) de la sección 10 sin divisiones.

Por otro lado, no son aplicaciones tan recientes, la más nueva es de hace 7 años, échale un ojo a estas referencias: https://www.scielo.org.mx/scielo.php?pid=S1405-55462018000401573&script=sci_arttext https://www.cell.com/heliyon/fulltext/S2405-8440(24)16140-3 https://www.sciencedirect.com/science/article/pii/S0017931023008438?casa_token=wbiNgqg10poAAAAA:DrtwAK4QYSucDxmgD9UILQje9Jz_0sTD3WMmK3_ycliMbSg94LD3cQi_FQ0T34AplbJWmNNF0cs

o busca bioheat equation en scholar google

Creo que aquí solo está refraseada info que ya habías dado antes, por lo que sugiero quitar este párrafo y añadir más literatura nueva

Modificar este enunciado. Sigue sin quedarme claro a qué te refieres con "aprende el operador general" y cambiar "muchas" por "múltiples" o "varias"

Mismos comentarios que para el ejemplo anterior. Además, hay que señalar qué condiciones iniciales representan cada color. Esto puede ser mediante una leyenda o en el pie de figura. Porqué la f(x) es con dashed lines y la u(x9) con continuas?

El pie de figura está escrito como si fueran instrucciones. Modifícalo a "Se construye" o "Construcción de". "Se especifica", etc.

Me confundí un poco con este enunciado, hay que tener cuidado al escribir con negaciones o dobles negaciones. Según yo, se está afirmando que si el problema original NO está bien planteado, entonces hay pocas razones ...........? pero si SI está bien planteado, entonces no hay pocas razones?

Creo que aquí es "o" en lugar de "y"

sustituir por "Dentro del banco de métodos para encontrar soluciones particulares de una EDP lineal, uno de los más comunes .........." o aunque sea modificar lo de "métodos que pueden intentarse"

Acento

"Reconocer las dificultades" no es un lenguaje muy técnico se oye como algo personal, cómo decir: reconozco que está difícil, reconozco que me equivoqué, reconozco que eres bueno.

Sugiero sustituirlo por "Resaltar las limitaciones en la implementación .........." o "Definir los límites en la implementación ........." o "Enmarcar las ventajas/desventajas al implementar ............"

Creo que "aprende" no es el verbo indicado o quisiste decir "aprende de un"

marco cartilaginoso, pericondrio y piel delgada

o: i think this is a darker statement said, but is true.

O: Antigone hasn't showed much care in her personal life aside her brother and uncle; how does her character view aspects such as marriage that could've been parts of her life?

O: the chorus perspective seems to be changing. In the first part of the play they centered around Kreon and added on to his ideologies, however they are showing more sympathy for antigone.

O: This is the most Kreon has spoken against the gods

O: diffrent ways of saying her eternal resting place

O: I this this whole monologue is so good, the language is interesting

O: I agree with Kendyls observation

O: put "law" in italics maybe to seem more important or something to pay attention to more

O: writing this passage in this format makes it seem more important and caught my attention more than others

O: throughout the entirety of the play Antigone has had distinctly values on law and family, which differ drastically from her sister. This time it feels different. She sees death almost as a reward for doing something honorable, while characters like the guard see it as punishment.

O: in relation to our in class discussion, both sisters seem to be looking out for one another in this argument. Though generally ismene is more caring for antigone than the other way around

LMFAOOO ANTIGONE IDGAF QUEEN

O: Antigone’s line “then can we get this over with” sounds casual, but it’s portraying a display of courage and defiance against Kreon. The back-and-forth between them shows their conflicting personalities and beliefs really well here despite it being simple; Kreon’s cold rigidity vs. Antigone’s bold refusal to cower. It’s like a power struggle condensed into three lines, which is so friggin tuff.

O. There seems to be a lot of mention of each character's self philosophy or edicts. This may allude to their actions later in the book as foreshadowing. But it also could be word-building or character development done by the author.

O: This is Antigone recognizing the differences between her and Ismene. Antigone is more rash and hot-headed while Ismene is more logical and technical. This is a contrast between the two sisters. Could this also have been between the two brothers?

O: Antigone sees her own death, not as a punishment, but as her religious duty for her brothers. She wants the gods over the state laws and Kreon. This has come up multiple times where she mentions death and it's almost like she's actively wanting it. This also will (probably) come up again when she dies(Dr. Callaghan said she dies it's not a spoiler) and also a reason why she seeks death.

O: This shows hoe Kreon is feared by the public and how he is driven by power

O: The contrast between Antigone and Ismene is quite strong because Antigone feels as though it is her duty to serve her brother even though it is unlawful, while Ismene continues repeating the idea that she is powerless/that she's a woman and is unable to help like her sister is planning to

O: Wait why's Antigone serving cunt and diva here.. Okay but seriously this line stood out to me because it gives the audience an impression of Antigone's personality and how that may affect her fate later in the play

O: Sophokles writing styles seems to have characters repeat their third-to-last line as their last line (chorus demonstrates this also). This puts emphasis on the meaning and importance of that particular line.

O: she makes death seem less of a punishment and more of a place of reunion. This shows her relationship with death and contrasts with Ismene in where she clings to life.

O: Antigone shows loyalty to her family and goes against the edict

O. if u look you'll see the repetition of “every question put to him.” This creates a pattern that emphasizes the subject’s capacity. he seems all-knowing, reliable, and consistent. But the repetition also sets up an expectation that is immediately broken by the line that follows: “but one.”

o: this is a sitution between Antigone and Ismene, Ismene is telling Antigone not to go and what he wants is the imposible.

O: at the end of page 16 and 18 theres repetition in each sentence.

O: Creon uses very harsh words to showcase how badly he wants to punish Polyneikes. It also shows the big contrast with how Polyneikes and Eteokles are treated.

El presente artículo tiene como finalidad estructurar una visión general sobre el streaming de videojuegos como agente generador de empleo y nuevas carreras profesionales en el Ecuador. En la metodología se planteó un diseño no experimental de alcance descriptivo y correlacional, de enfoque mixto, empleando el método Delphi en el componente cualitativo, mientras que se ejecutaron encuestas para solventar el componente cuantitativo. En este sentido, se pudo observar una mayor participación de la generación Z y millennials en este tipo de actividades, lo que a su vez refleja el interés por formar parte del streaming de videojuegos, mientras que otros que ya son usuarios han percibido un aporte o beneficio económico por jugar o crear contenido. Además, se resalta al género masculino como el principal participante en estas plataformas. Se concluyó que hay relación entre las personas que dedican horas a los videojuegos y el deseo de obtener réditos económicos a través de este modelo de negocio. De igual forma se observa una tendencia de los gamers en profesionalizarse en carreras de marketing digital y producción audiovisual y multimedia, lo cual sirve para que las instituciones de educación superior ajusten y fortalezcan los programas académicos relacionados, así como enfocar de manera más efectiva sus estrategias de marketing hacia este público objetivo. [ABSTRACT FROM AUTHOR]

• Informativo nº 847
• 15 de abril de 2025.
• RECURSOS REPETITIVOS
• Processo:

REsp 2.129.162-MG, Rel. Ministro Paulo Sérgio Domingues, Primeira Seção, por unanimidade, julgado em 9/4/2025. (Tema 1298).

REsp 2.131.059-MG, Rel. Ministro Paulo Sérgio Domingues, Primeira Seção, por unanimidade, julgado em 9/4/2025 (Tema 1298).

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes

Ação de desapropriação por utilidade pública ou de constituição de servidão administrativa. Desistência. Honorários sucumbenciais. Limites percentuais do art. 27, § 1º, do DL n. 3.365/41. Incidência. Base de cálculo dos honorários. Valor atualizado da causa. Arbitramento por apreciação equitativa (Art. 85, § 8º, do CPC). Cabimento apenas quando o valor da causa é muito baixo. Tema 1298.

Destaque - Aplicam-se os percentuais do art. 27, § 1º, do DL n. 3.365/1941 no arbitramento de honorários sucumbenciais devidos pelo autor em caso de <u>desistência</u> de ação de desapropriação por utilidade pública ou de <u>constituição de servidão administrativa</u>, os quais terão como base de cálculo o valor atualizado da causa. Esses percentuais não se aplicam somente se o valor da causa for muito baixo, caso em que os honorários serão arbitrados por apreciação equitativa do juiz, na forma do art. 85, § 8º, do CPC.

Informações do Inteiro Teor - Cinge-se a controvérsia em definir se os limites percentuais previstos no art. 27, § 1º, do Decreto-Lei n. 3.365/1941 devem ser observados no arbitramento de honorários sucumbenciais em caso de desistência de ação de desapropriação por utilidade pública ou de constituição de servidão administrativa.

• A previsão do art. 27, § 1º, do DL n. 3.365/1941 veio para estabelecer normas especiais para os honorários advocatícios em ações expropriatórias seja quanto à base de cálculo de tal verba, seja quanto aos percentuais que devem incidir sobre a base arbitrada. Embora amalgamadas em um único preceito (texto), subsiste relativa independência entre as normas jurídicas contidas no dispositivo legal, de modo que alterações circunstanciais na base de cálculo não devem afastar, obrigatoriamente, a incidência da lex specialis relativa aos percentuais estabelecidos para o arbitramento dos honorários advocatícios.

• Assim, em havendo desistência da ação de desapropriação ou de constituição de servidão administrativa, é evidente que cai por terra a possibilidade de arbitramento dos honorários sucumbenciais tomando por base de cálculo a diferença entre o preço ofertado pelo expropriante e a indenização fixada na sentença, tal como previsto em norma especial inserida no texto do art. 27, § 1º, do DL n. 3.365/1941, uma vez que a sentença, nessa excepcional circunstância, não estabelecerá indenização alguma.

• Nesse cenário ocasional, embora não haja condenação, o princípio da causalidade impõe que o ente (não mais) expropriante seja declarado sucumbente de modo que os honorários correrão a sua conta, porque deu causa ao ajuizamento da demanda e dela desistiu (art. 90 do Código de Processo Civil).

• À falta de condenação ou de proveito econômico efetivo, já foi dito que não há suporte jurídico para o estabelecimento da base de cálculo dos honorários nos moldes do art. 27, § 1º, do DL 3.365/1941, de modo que essa base será fixada de acordo com norma jurídica supletiva prevista no art. 85, § 2º, do CPC, tomando-se em conta, então, o valor atribuído à causa.

• O socorro à norma supletiva do CPC faz-se porque não existe suporte jurídico para a aplicação da norma especial do DL 3.365/1941 apenas no que toca à base de cálculo dos honorários sucumbenciais. Ora, a desistência da ação não implica desaparecimento do suporte jurídico de aplicação dessa lex specialis, de modo que não há razão jurídica para se recorrer, quanto aos percentuais, a outras normas jurídicas que pudessem ser aplicadas de forma supletiva ou subsidiária.

• Dessarte, mesmo em caso de desistência da ação expropriatória, os percentuais a serem observados devem ser os estabelecidos no art. 27, § 1º, do DL3.365/1941.

• Ressalte-se, contudo, que haverá casos em que o valor da causa, mesmo que atualizado, corresponderá a valor ínfimo a implicar honorários irrisórios caso aquele valor seja mantido como base para a incidência das alíquotas do art. 27, § 1º, do DL n. 3.365/1941.

• Nessa excepcional hipótese, portanto, afasta-se completamente a aplicação do art. 27, § 1º, do DL n. 3.365/1941 para a fixação dos honorários sucumbenciais - seja quanto à base de cálculo estabelecida no preceito, seja quanto aos percentuais ali estabelecidos -, uma vez que a verba honorária será arbitrada pelo juiz por apreciação equitativa, com fundamento no art. 85, § 8º, do CPC, a fim de impedir que a verba honorária seja fixada em patamar incompatível com a dignidade do trabalho advocatício.

• Dessa forma, deve ser fixada a seguinte tese jurídica de eficácia vinculante: Aplicam-se os percentuais do art. 27, § 1º, do DL 3.365/41 no arbitramento de honorários sucumbenciais devidos pelo autor em caso de desistência de ação de desapropriação por utilidade pública ou desconstituição de servidão administrativa, os quais terão como base de cálculo o valor atualizado da causa. Esses percentuais não se aplicam somente se o valor da causa for muito baixo, caso em que os honorários serão arbitrados por apreciação equitativa do juiz, na forma do art. 85, § 8º, do CPC.

• Informativo nº 724
• 14 de fevereiro de 2022.
• PRIMEIRA SEÇÃO
• Compartilhe:
• Processo: CC 174.764-MA, Rel. Min. Mauro Campbell Marques, Primeira Seção, por unanimidade, julgado em 09/02/2022.

Ramo do Direito DIREITO ADMINISTRATIVO, DIREITO CONSTITUCIONAL, DIREITO PROCESSUAL CIVIL

TemaPaz, Justiça e Instituições Eficazes

Conflito negativo de competência. Juízos estadual e federal. Ação de improbidade administrativa ajuizada por ente municipal. Prestação de contas de verbas federais. Mitigação das súmulas 208/STJ e 209/STJ. Competência cível da Justiça Federal absoluta em razão da pessoa. Art. 109, I, da CF. Ausência de ente federal em qualquer dos polos da relação processual. Competência da Justiça Estadual.

Destaque - Nas ações de improbidade administrativa, a competência da Justiça Federal é definida em razão da presença das pessoas jurídicas de direito público previstas no art. 109, I, da Constituição Federal na relação processual, e não em razão da natureza da verba federal sujeita à fiscalização da Tribunal de Contas da União.

Informações do Inteiro Teor - No caso, o ente municipal ajuizou ação de improbidade administrativa, em razão de irregularidades na prestação de contas de verbas federais decorrentes de convênio.

• A competência para processar e julgar ações de ressarcimento ao erário e de improbidade administrativa, relacionadas à eventuais irregularidades na utilização ou prestação de contas de repasses de verbas federais aos demais entes federativos, estava sendo dirimida por esta Corte Superior sob o enfoque das Súmulas 208/STJ ("Compete à Justiça Federal processar e julgar prefeito municipal por desvio de verba sujeita à prestação de contas perante órgão federal") e 209/STJ ("Compete à Justiça Estadual processar e julgar prefeito por desvio de verba transferida e incorporada ao patrimônio municipal").

• O art. 109, I, da Constituição Federal prevê, de maneira geral, a competência cível da Justiça Federal, delimitada objetivamente em razão da <u>efetiva</u> presença da União, entidade autárquica ou empresa pública federal, na condição de autoras, rés, assistentes ou oponentes na relação processual. Estabelece, portanto, competência absoluta em razão da pessoa (ratione personae), configurada pela presença dos entes elencados no dispositivo constitucional na relação processual, independentemente da natureza da relação jurídica litigiosa.

• Por outro lado, o art. 109, VI, da Constituição Federal dispõe sobre a competência penal da Justiça Federal, especificamente para os crimes praticados em detrimento de bens, serviços ou interesse da União, entidades autárquicas ou empresas públicas. Assim, para reconhecer a competência, em regra, bastaria o <u>simples interesse</u> da União, inexistindo a necessidade da efetiva presença em qualquer dos polos da demanda.

• Nesse contexto, a aplicação dos referidos enunciados sumulares, em processos de natureza cível, tem sido mitigada no âmbito deste Tribunal Superior. A Segunda Turma afirmou a necessidade de uma distinção (distinguishing) na aplicação das Súmulas 208 e 209 do STJ, no âmbito cível, pois tais enunciados provêm da Terceira Seção deste Superior Tribunal, e versam hipóteses de fixação da competência em matéria penal, em que basta o interesse da União ou de suas autarquias para deslocar a competência para a Justiça Federal, nos termos do inciso IV do art. 109 da CF. Logo adiante concluiu que a competência da Justiça Federal, em matéria cível, é aquela prevista no art. 109, I, da Constituição Federal, que tem por base critério objetivo, sendo fixada tão só em razão dos figurantes da relação processual, prescindindo da análise da matéria discutida na lide (REsp 1.325.491/BA, Rel. Ministro Og Fernandes, Segunda Turma, julgado em 05/06/2014, DJe 25/06/2014).

• Assim, nas ações de ressarcimento ao erário e improbidade administrativa ajuizadas em face de eventuais irregularidades praticadas na utilização ou prestação de contas de valores decorrentes de convênio federal, <u>o simples fato das verbas estarem sujeitas à prestação de contas perante o Tribunal de Contas da União, por si só, não justifica a competência da Justiça Federal</u>.

• O Supremo Tribunal Federal já afirmou que o fato dos valores envolvidos transferidos pela União para os demais entes federativos estarem eventualmente sujeitos à fiscalização do Tribunal de Contas da União não é capaz de alterar a competência, pois a competência cível da Justiça Federal exige o efetivo cumprimento da regra prevista no art. 109, I, da Constituição Federal.

• Igualmente, a mera transferência e incorporação ao patrimônio municipal de verba desviada, no âmbito civil, não pode impor de maneira absoluta a competência da Justiça Estadual. Se houver manifestação de interesse jurídico por ente federal que justifique a presença no processo, (v.g. União ou Ministério Público Federal) regularmente reconhecido pelo Juízo Federal nos termos da Súmula 150/STJ, a competência para processar e julgar a ação civil de improbidade administrativa será da Justiça Federal.

• Em síntese, é possível afirmar que a competência cível da Justiça Federal é definida em razão da presença das pessoas jurídicas de direito público previstas no art. 109, I, da CF na relação processual, seja como autora, ré, assistente ou oponente e não em razão da natureza da verba federal sujeita à fiscalização da Corte de Contas da União.

• No caso, não figura em nenhum dos pólos da relação processual ente federal indicado no art. 109, I, da Constituição Federal, o que afasta a competência da Justiça Federal para processar e julgar a referida ação. Ademais, não existe nenhuma manifestação de interesse em integrar o processo por parte de ente federal e o Juízo Federal consignou que o interesse que prevalece restringe-se à órbita do Município autor, o que atrai a competência da Justiça Estadual para processar e julgar a demanda.

Obs.: Ou seja, para fins de fixação de competência, não basta a mera indicação da origem do recurso (se estadual ou se federal). Para haver competência da Justiça Federal, no âmbito cível, deve haver efetiva presença da União; não basta mero interesse da União como ocorre na seara penal. Importante destacar que as súmulas 208 e 209, com indicado, provêm da seção especializada em Direito Criminal.

cada innovación técnica busca ahorrar costos de trabajo humano y aumentar la productividad, esto provoca desempleo, inseguridad laboral. en vez de usarse como herramientas, se ocupa mas en la sustitución en el trabajo humano.

valor de uso y valor de cambio. el valor como objetivo de satisfacer necesidades, solo lo que se domina es que tenga precio y se pueda vender

significa literalmente "en el estado en que" y se refiere al estado o condición existente de un asunto o situación en un momento determinado

I can understand this comparison because it take times to adjust to things even in writing.

Repeating these lines emphasizes the importance of the Nile and has almost a praise like tone to it.

Keeps repeating this idea that the Nile is praised and worthy of these grand festivals, etc.

contribute your own thinking

OJO, MIRAR

RELEVANTE

OJO

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Reply to the reviewers

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

The manuscript by Xu et al. investigated split gene drive systems by targeting multiple female essential genes involved in fertility and viability in Drosophila. The authors evaluate the suppression efficiency through individual corsses and cage trials. Resistance allele formation and fitness costs are explored by examining the sterility and fertility of each line. Overall, the experimental design is sound and methods are feasible. The work is comprehensive, and conclusions are well supported by the data. This work offers informative insights that could guide the design of suppression gene drive systems in other invasive disease vectors or agricultural pests.

However, several points requiring clarification or improvement:

1 Methodological clarity: Some experimental details are indufficiently described, for example, regarding the setup of genetic crosses involving different Cas9 derivatives. In line 197-198, "the mated females, together with females that were mated with Cas9 only males", it is unclear whether the latter group refers to gRNA-females.

-We thank the reviewer for pointing out this ambiguity. The latter group refers to Cas9 females crossed to Cas9 males. We have clarified this both in the methods (line 207) and results (line 505-509).

2.Regarding the inheritance rates, you included the reverse orientation of CG4415-Cas9, as I understood, it means this component is in reverse orientation with fluorescent marker. Since it is standard to design adjacent components in opposite direction to avoid transcriptional interference, the rationale for including this comparison should be better justified.

• In our construct, ‘CG4415 (reverse orientation)’ indicates that Cas9 was oriented in the same direction as the fluorescent marker, while the other Cas9 constructs (nanos-Cas9 and CG4415-Cas9) places them in opposite directions. “reverse” just indicates a change from a “standard” in another study. Our previous publication showed that Cas9 orientation relative to the marker had little apparent effect on drive performance at the yellow-G locus. In this study, we compared both orientations in a fertility gene and again observed similar results, suggesting that orientation relative to the marker does not substantially affect drive efficiency in our system. We have clarified this in the figure legend text.

Embryo resistance is inferred from the percentage of sterile drive females derived from drive mothers. How many female individuals were analysed per line and why deep sequencing was not employed to directly detect resistance alleles.

-Embryo resistance can mean slightly different things for different applications. The most important is probably the fraction of females that have little to no fertility due to embryo resistance. Some of these may not have complete embryo resistance alleles, but instead, have mosaicism, with a sufficient level of resistance to still cause sterility. It is unclear exactly what proportion of resistance to wild-type may cause this, and thus, proportions from pooled sequencing, which could include both complete and all levels of mosaicism, may not be sufficient to measure this parameter. Another relevant parameter that we did not measure is the fraction of males rendered unable to do drive conversion (this value should be closer to the complete resistance rate, but probably still lower because of the multiple gRNAs). Even in this case, deep sequencing would not allow us to determine exactly what is happening in males, making individual sequencing a preferred approached. It is very nice, of course, for characterizing which resistance alleles are present overall, but in this study, we wanted to put a bit more emphasis on the effect of resistance, rather than its sequence characterizing.

We analyzed 30 females per line for lines targeting nox, oct, dec and stl, 9 females for ndl and 276 individuals for line tra-v2 (Data Set S4). We believe such individual analyses sufficiently detected embryo resistance causing sterility within reasonable error. Note that we did also randomly genotype several sterile females and found mutations at target sites that disrupted gene functions.

In response to this comment, we have added some text to justify our measurement of resistance alleles and include some of this discussion:

“Note also that this defines embryo resistance as sufficient to induce sterility, but these may be mosaic rather than complete resistance. Further, note that the multiplex gRNA design in males may allow for continued drive conversion with a complete (as opposed to mosaic) embryo resistance allele, if some sites remain wild-type.”

Masculinisation phenotypes were observed upon disruption of tra gene. How strong intersexes were distinguished from males? What molecular markers were used to determine genetic sex. This information should be clearly provided.

-We observed two types of strong masculinisation phenotypes (Figure S2), one with bigger body size than wildtype males, and the other was identical to wildtype males. The homozygosity of the drive allele could be assessed by the brightness of red fluorescence in the eyes. However, we also randomly genotyped these masculinized females (as part of a batch that included males) to confirm their sex using primers for the Y-linked gene PP1Y2. A specific band was detected in wild-type males but not in masculinized females, confirming their genetic sex. This information has been added to the manuscript (lines 477-480).

It would be more appropriate to use "hatchability"rather than "fertility" when referring to egg-to-larva viability.

-Thank you for the suggestion. We used egg-to-adult survival rates as a proxy for the fertility of their parents because they usually laid similar number of eggs. However, it still technically incorrect language. We have fixed this in line 582 and elsewhere in the section.

In cage trials, a complete gene drive is mimicked by introducing Cas9 to the background population, but this differs from actual complete gene drive, due to potential effects from separate insertion sites (different chromosome or loci). These difference could impact the system's performance and should be discussed.

-We appreciate this point and have added discussion on the limitations of mimicking a complete gene drive using split components (line 766-779).

7.Given the large amount of data presented, it would improve readability and interpretation if each result section concluded with a concise summary highlighting the key findings and implications.

-Thank you for the suggestion. We have added brief summaries at the end of each results section to highlight the key findings and their significance.

Reviewer #1 (Significance (Required)):

The authors evaluate suppression efficiency through individual courses and cage trials. Resistance allele formation and fitness costs are explored by examining the sterility and fertility of each line. Overall, the experimental design is sound and methods are feasible. The work is comprehensive, and conclusions are well supported by the data. This work offers informative insights that could guide the design of suppression gene drive systems in other invasive disease vectors or agricultural pests.

-We appreciate the reviewer’s positive assessment of our work.

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

Paper summary

The manuscript by Xu. et al presents an insightful and valuable contribution to the field of gene drive research. The manuscript by Xu et al. presents an insightful and valuable contribution to the field of gene drive research. The strategy of targeting and disrupting female fertility genes using selfish homing genetic elements was first proposed by Burt in 2003. However, for this approach to be effective, the phenotypic constraints associated with gene disruption have meant that the pool of suitable target genes remains relatively small - notwithstanding the significant expansion in accessible targets enabled by CRISPR-based genome editing nucleases. Population suppression gene drives are well developed as proof-of-principle systems, with some now in the late stages of development as genetic control strains. However, advancing the pipeline will require a broader set of validated target genes - both to ensure effectiveness across diverse species and to build redundancy into control strategies, reducing reliance on any single genetic target.

In their paper, the authors conduct a systematic review of nine female fertility genes in Drosophila melanogaster to assess their potential as targets for homing-based suppression gene drives. The authors first conduct a thorough bioinformatic review to select candidate target genes before empirically testing candidates through microinjection and subsequent in vivo analyses of drive efficiency, population dynamics, and fitness costs relating to fecundity and fertility. After finalising their results, the authors identify two promising candidate target genes - oct and stl - which both demonstrate high gene conversion rates and, regarding the latter, can successfully suppress a cage population at a high release frequency. However, the manuscript suffers from a lack of in-depth discussion of a key limitation in its experimental design - namely, that the authors utilise a split-drive design to assess population dynamics and fitness effects when such a drive will not reflect release scenarios in the field. The review below highlights some major strengths and weaknesses of the paper, with suggestions for improvement.

Key strengths

The study's most significant strength is in its systematic selection and empirical testing of nine distinct genes as targets for homing-based gene drive, hence providing a valuable resource that substantially expands the pool of potential targets beyond the more commonly studied target genes (e.g. nudel, doublesex, among others). The identification of suitable target genes presents a significant bottleneck in the development of gene drives and the work presented here provides a foundational dataset for future research. The authors bolster the utility of their results by assessing the conservation of candidate genes across a range of pest species, suggesting the potential for broader application.

A key finding in the paper is the successful suppression of a cage population using a stl-targeting gene drive (albeit at a high release frequency). This provides a critical proof-of-principal result demonstrating that stl is a viable target for a suppression drive. While in the paper suppression was not possible at lower release frequencies, together, the results provide evidence for complex population dynamics and threshold effects that may govern the success or failure of a gene drive release strategy - hence moving the conversation from a technical perspective ("can it work") to how a gene drive may be implemented. Moreover, the authors also employ a multiplexed gRNA strategy for all their gene drive designs and in particular their population suppressive gene drive targeting stl. This provides further proof-of-principal evidence for multiplexed gRNAs in order to combat the evolution of functional resistance following gene drive deployment.

Finally, a further strength of this paper is in the clever dissection of fitness effects resulting from maternal Cas9 deposition. The authors design and perform a robust set of crosses to elucidate the parental source of fitness effects (i.e. maternally, paternally, or biparentally derived Cas9), finding (as they and others have before) that embryonic fitness was significantly reduced when Cas9 was inherited from a maternal source. As discussed, the authors conclude that maternal deposition is particularly pronounced in the context of split drives as opposed to complete drives, with the implication being that a complete drive might succeed where a split-drive has failed; thus providing a key directive for future study.

Concerns

The manuscript's central weakness lies in its interpretation of the results from the cage experiments - namely that a split-drive system was used to "mimic the release of a complete drive". In the study, mosquitoes carrying the drive element (i.e. the gRNA) were introduced into a population homozygous for the Cas9 element over several generations. This design is likely not representative of a real-world scenario and, as the authors state, likely exaggerates fitness costs. This is because the females carrying Cas9 will maternally deposit Cas9 protein into her eggs, with activity spanning several generations. When mated with a drive-carrying male the gRNA will immediately co-exist with maternally deposited Cas9, leading to early somatic cleavage and significant fitness costs (reflected in the author's own fertility crosses). This is fundamentally different to how a complete drive would function in a real-world release, where complete-drive males would mate with wild-type females not carrying Cas9. Their offspring would carry the drive element but would not be exposed to maternally deposited cas9, thus deleterious maternal effects would only begin to appear in the subsequent generation from females carrying the drive. Fitness costs measured from split-drive designs are therefore likely substantially overestimated compared to what would occur during the initial but critical release phase of a complete drive. This flaw weakens the paper's ability to predict the failure or success of the screened targets in a complete drive design, thus weakening the interpretation of the results from the cage trials. As a suggestion for improvement, the authors should explicitly and more prominently discuss the limitations of their split-drive model compared to complete drive models, both in the Results and Discussion. It is also recommended to include a schematic for both strategies that contrasts the experimental setup design (i.e. release of the drive into a Cas9 homozygous background) with a complete-drive release, clearly illustrating differences in maternal deposition pathways. This will not only contextualise the results and support the author's conclusion that observed fitness costs are likely an overestimate but will further strengthen the arguments that the candidate target genes found in this study may still be viable in a complete-drive system.

-We sincerely appreciate the thoughtful review and the valuable comments and suggestions provided, which have helped improve both the clarity and readability of this study. We have revised several parts in the discussion of the manuscript and hope that these changes adequately address the concerns raised. We have also made Figure S5 to illustrate the differences between two release strategies (biparental-Cas9 split drive in our study and complete drive in real release).

Please note that this type of fitness cost may have partially undermined our cage study (the fitness effect is notable, but still small compared to total fitness costs), but this is also among the first studies to propose and investigate this phenomenon in the first place (it is also noted in another preprint from our lab but to our knowledge not proposed elsewhere). Thus, part of the impact of our manuscript is showing that this is important, which may inform future cage studies in our lab and elsewhere.

A second weakness in the manuscript relates to its limited explanation and discussion of key concepts. For example, the manuscript reports a stark difference in outcome of the two stl-targeting drives, where a high initial release in cage 1 led to population elimination versus a failure of the drive to spread in cage 2. The authors attribute this to vague "allele effects" and stochastic factors such as larval competition; however the results appear reminiscent of the Allee effect, which is a well-characterised phenomenon describing the correlation of population size (or density) and individual fitness (or per capita population growth rate). Using their results as an example, is it plausible that the high-frequency initial release in cage 1 imposed enough genetic load to quickly drive the population density below the Allee threshold thus quickly leading to population eradication. In cage 2, the low-frequency at initial release was insufficient to cross the Allee threshold. Omitting mention of this ecological principal greatly weakens the Discussion, and further presents a missed opportunity to discuss one of the more crucial strengths of the paper - that is, in providing a deeper insight into the practical requirements for successful field implementation.

-While we do indeed mention this Allee effect (the “allele effect” noted above is a misspelling that we have corrected), we were hesitant to give it much discussion, considering that the specific Allee effect in our cages is likely of a very different nature than one would find in nature (we explain that it is likely due to bacterial growth that occurs when fewer larvae are present). However, it is perhaps still a good excuse to cover it in the discussion, while still noting that the specific Allee effect in our cage may not be representative. We have added the following text: “Nonetheless, the successful result in the cage with high release study may point to a potential field strategy for a drive that is less efficient (perhaps even one found to be less efficient in initial field tests compared to laboratory tests). If the initial release frequency of the drive is sufficiently high and widespread, then short-term high genetic load may substantially reduce the population, perhaps enough for Allee effects to become important. At this point, even if average genetic load is slowly declining without additional drive releases, persistent moderate genetic load coupled with the Allee effect may be sufficient to ensure population elimination.”

In a similar vein, the authors provide only a superficial mechanistic discussion into the fitness costs associated with drives targeting key candidate genes. The paper would benefit from a deeper discussion regarding the specific molecular functions of top-performing genes (stl, oct, nox) and how unintended Cas9 activity could disrupt their activity, integrating known molecular functions with observed fitness costs. For instance, oct encodes a G-protein coupled receptor essential for ovulation and oviduct muscle relaxation, thus disruption to the oct gene would directly impair egg-laying which would account for the observed phenotypic effects. A deeper discussion linking unintended Cas9 activity to the specific, sensitive functions of target genes would elevate the paper from a descriptive screen to a more insightful mechanistic study.

-We appreciate the reviewer’s comment. We have added a discussion to further explain fitness cost caused by unintended Cas9 activity disrupting target gene functions. However, keep in mind that the exact timing of Cas9 cleavage and the exact timing of these gene’s essential functions is still somewhat uncertain, which may limit insights from this line of analysis compared to a situation where ideal, high quality data is available for both of these. Here is the new material in the discussion:

“The functions of the top-performing genes suggests a mechanistic basis for the observed fitness costs. Aside from germline cells, nanos has expression in other ovary cells as well. CG4415 lacks this expression, but our Cas9 construct with this promoter may have a different expression pattern that the native gene, as evidenced by its support for good drive conversion in females. stl is essential for ovarian follicle development, and its disruption likely in non-germline ovary cells could compromise egg chamber development and fertility. oct encodes the octopamine β2 receptor, a G-protein coupled receptor critical for ovulation and fertilization, so if it were similarly lost, egg-laying would be directly impaired. nox, which encodes NADPH oxidase, contributes to calcium flux and smooth muscle contraction during ovulation, so its disruption may prevent egg laying. tra is needed in the whole body for sexual development, but may also play an important role in ovary function. Thus, unintended Cas9 activity at these non-germline ovary cells can directly interfere with sensitive reproductive functions, potentially explaining the fertility costs observed in drive carriers. This issue could potentially be overcome if promoters were available that were truly restricted to germline cells rather than other reproductive cells, though it remains unclear if such promoters both exist and would retain their expression pattern at a non-native locus.”

It is curious that the authors chose two genes on the X chromosome as targets. In insects (such as Drosophila here) that have heterogametic sex chromosomes, homing is not possible in the heterogametic sex as there is no chromosome to home to - so there will be no homing in males. On top of that, there is usually some fitness effect in carrier (heterozygous) females, so in a population these are nearly always bad targets for drives - unless there is some other compelling reason to choose that target?

-Our rationale for testing X-linked targets is twofold. First, these genes are likely to play important roles in sex-specific functions and may have a different expression pattern (which is why specifically Dec was included), potentially reducing fitness costs. Although homing cannot occur in males, if drive conversion at these sites in females is very high and fitness costs are minimal, the resulting genetic load could still be sufficient to suppress populations (thus, such candidates could be superior even in diploids if they happen to have a lower fitness costs). Second, X-linked targets may have broader relevance for suppression drives in haplodiploid pests (e.g., fire ants), which has the same population dynamics as an X-linked target in a diploid populations. Our results therefore could have provided useful insights for such scenarios (such as for fire ants: Liu et al., bioRxiv 2025) if drive performance was sufficient for followup testing.

Minor comments

• Enhanced clarity in the Figures and data presentation would greatly improve readability. For example, Figure 5 is critical yet difficult to interpret; consider changing x-axis labels from icons to explicit text (e.g. "biparental Cas9", "maternal cas9", "paternal Cas9"). Similarly, Figure 4 is difficult to read and the y-axis label "population size" is ambiguous; consider adding shapes or dashes (rather than relying solely on colour) and clarifying the y-axis (e.g. no. adults collected) in the legend.

-We appreciate the reviewer’s comment and have revised Figure 4 as suggested. Regarding Figure 5, we attempted to replace the icons with text labels; however, this was not possible because there is very little horizontal space and two generations to specify. Instead, we have revised the figure legend to provide a clearer explanation, which can hopefully improve clarity..

• Expand on or include a schematic to show the differences in construction between the tra-v1 and tra-v2 constructs to better contextualise the discrepancies in results (e.g. inheritance rates of 61%-66% for tra-v1 and 81%-83% for tra-v2 between the two.

-We have expanded Figure 2 to compare the constructs of tra-v1 and tra-v2. The further explanation of these two constructs was added into the result section: ‘When targeting tra, we originally tested the 4-gRNA construct tra-v1. However, the drive inheritance rate was relatively low (61%-66%), and sequencing revealed that only the middle two gRNAs were active (Table S3). Lack of cleavage at the outmost sites is particularly detrimental to achieving high drive conversion. Therefore, a second construct tra-v2 was tested that retained the two active gRNAs and included two new gRNAs. It showed substantially improved drive inheritance (81%-83%). ’

• Minor typos e.g.:

o Line 87: "form" to "from"

o Line 484: "expended" to "expanded

o Line 560: "foor" to "for"

o Line 732: "conversed" to "conserved

-We have revised these typos.

• Clarify the split drive system: the authors introduce split drive for the first time in Line 118. They should at least give a clear definition and explanation of split drive and complete drive in the introduction.

-We have included an introduction of split drive and complete drive in the introduction (line 47-53).

• Line 237-238., The fitness evaluation lacks a clear description of controls. How were non-drive flies generated and validated as controls?

-Drive heterozygotes were crossed with Cas9 homozygotes to generate the flies used for fitness evaluation. From the same cross, non-drive progeny were obtained and used as controls, ensuring they shared a comparable genetic background and rearing conditions with the drive-carrying individuals. We have now clarified in the manuscript results that “these served as the controls because they had the same environment and parents as the drive flies”.

• Line 409-412.,line 423.,The high inheritance rates of stl and oct drives are impressive; however, variation in results across Cas9 promoters should be explained further in the discussion.

-In the discussion section (lines 751-765), we included a dedicated paragraph addressing the variation observed between the nanos and CG4415 promoters. We have now expanded it to briefly note some differences:

“Our previous works showed that both nanos and CG4415 have high drive conversion rates8, but nanos failed to suppress target populations in a homing drive targeting the female fertility gene yellow-G due to its fitness cost in drive females27. CG4415 had much lower maternal deposition, which allowed the elimination of cage populations by targeting yellow-G8. Here, we tested both promoters with drives targeting oct and stl, with both showing slightly higher drive efficiency than the drive targeting yellow-G in small-scale crosses. CG4415 has slightly worse though still good performance in females, likely due to male-biased expression compared to nanos.”

• Line 414: The CG4415 promoter yielded reduced drive conversion rates in females, yet is still referred to as a promising promoter. This conclusion seems optimistic and should be clarified/more justified.

-Based on our previous study cited in this context, CG4415 shows relatively lower germline conversion rates compared to nanos, although still remaining at a high level. Importantly, CG4415 also exhibits reduced maternal deposition relative to nanos, which could help mitigate fitness costs associated with maternal deposition—an important consideration for suppression systems. Taken together, while its conversion efficiency is lower (but only slightly), the potential benefits of reduced maternal deposition and perhaps even fitness costs provide a rationale for regarding CG4415 as a promising promoter. We state this when first introducing the promoter in the “Drive efficiency assessment” results subsection.

• Specify the number of flies released, sex ratio, and cage size per generation (Line 466). This is essential for reproducibility.

-We appreciate the reviewer’s comment and have revised the text to clarify our release approach, which differed from that used in other studies (which tend to have substantial fitness differences between lines in the first generation that can complicate analysis and change results). Rather than directly releasing drive males or females into cages, we first crossed drive males with non-drive females and then mixed them with non-drive females mated to non-drive males. The offspring (including males and females) from these crosses were recorded as the G0 generation, and their ratios were recorded as release frequency. We have specified the release ratio adult numbers in the following paragraph and supplementary file.

Reviewer #2 (Significance (Required)):

Overall the manuscript presents a valuable and timely resource for gene drive research, in particular for its systematic appraisal of potential target genes for population suppression drives and its rigorous assessment of the impact of maternal Cas9 deposition. The value in the generation and empirical testing of a novel multiplexed stl-targeting gene drive that led to population eradication in a cage trial should not be understated. While several key aspects of the discussion of the manuscript should be strengthened, the study presents a meaningful contribution to the field, extending previous work and and outlines important considerations for the design and implementation of effective gene drive systems.

-We thank the reviewer for their encouraging and constructive comments. We are pleased that the systematic evaluation of target genes, the analysis of maternal Cas9 deposition, and the multiplexed stl-targeting drive were recognized as valuable contributions. We have strengthened the discussion as suggested, and we believe these revisions further enhance the manuscript as an aid for the design and implementation of future gene drive systems.

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

In this study, Xu and colleagues explored how CRISPR-based homing gene drives could be used to suppress insect populations by targeting female fertility genes in Drosophila melanogaster. They engineered split gene drives with multiplexed guide RNAs to target nine candidate genes, seeking to prevent functional resistance and achieve high drive conversion with minimal fitness costs.

Here my comments about this work:

Abstract: While the stated aim of the study on line 16 is to "maintain high drive conversion efficiency with low fitness costs in female drive carriers," the conclusion in lines 29-31 shifts focus toward the broader challenges and future optimization of gene drive systems. This conclusion does not clearly highlight the specific results of the study or how they relate directly to the original objective. It would be more effective to emphasize the actual findings, such as which target genes performed best and under what conditions, and how these findings support or contradict the stated goals. The study primarily aimed to assess the efficiency of specific female fertility genes and to evaluate strategies for minimizing the formation of functional resistance alleles, rather than proposing a protocol for optimization. Therefore, better alignment is needed between the study's aim, experimental design, and concluding statements. Clarifying this alignment would also help refine the paper's focus and more accurately communicate its contribution, including whether it is exploratory, comparative, or methodologically driven.

-We have revised the abstract to clarify the alignment as suggested by the reviewer. We note that this discrepancy is due to the initial aim of our study being different than some of the important lessons learned along the way regarding fitness effects from Cas9 deposition in split drives. Still, we agree that it would be better to be more consistent in our wording and conclusions.

Introduction: One of the key design elements in this study is the use of multiplexed gRNAs. It is reasonable to assume that this strategy may influence fitness costs, potentially in more than one way. Given that assessing fitness cost is a major focus of the study, it would be helpful to include a brief discussion of previous research examining how multiplexed gRNAs may impact fitness in gene drive systems. A short review of relevant studies, if available, would provide important context for interpreting the results and could help clarify whether any observed fitness costs might be attributed, at least in part, to the multiplexing strategy itself. This addition could be appropriately placed around line 102, where gRNA design is discussed.

-We have added an explanation in the Discussion to mention this. However, it has not been conclusively shown that multiplexed gRNAs have any effect on fitness. Indeed, there have been some multiplexed constructs that seem to have no fitness effect, and some that have high fitness costs. This doesn’t rule out the potential for multiplexed gRNAs to influence fitness itself, but it means that the mechanism may be complex. The new text reads:

“Another potential though unconfirmed source of fitness cost arises from increased cleavage events associated with multiplexed gRNAs, where the greater number of gRNAs can enhance the overall cut rate compared to single-gRNA designs.”

Line 42: Cas12a also showed efficacy using gene drives in yeast and Drosophila.

-We now mention Cas12a at the beginning of the introduction.

Line 133: The paragraph begins by stating that homologs of the target genes were identified and aligned. To improve clarity, especially for readers who are new to gene drive research, it would be helpful to begin the paragraph with a brief introductory sentence explaining the purpose of this step. For example, you could state the importance of identifying and aligning homologs to assess the conservation of target sites across species, which is critical for evaluating the broader applicability of gene drive strategies. This context would guide the reader and clarify the relevance of the analysis.

-We have added the explanation as suggested.

Lines 144-145: You mention that "the exception was tra, for which two constructs containing different gRNA sets were generated." For clarity, it would be helpful to provide a brief explanation of why two different gRNA sets were used for tra, and whether this differs from the approach taken with the other target genes. It's currently unclear whether all other genes were targeted using a single, standardized set of gRNAs, and this should be explicitly stated here for consistency, even though it is mentioned later in the plasmid construction section. Additionally, I suggest combining the sections on gRNA target design and plasmid construction. Since these components are closely related and sequential in the experimental workflow, presenting them together would improve the logical flow and help readers follow the methodology more smoothly.

-We have combined both the gRNA target design and plasmid construction sections. We also discuss the two tra constructs early in the results section (see response to reviewer 2).

Line 210: The analysis of the cage experiments was based on models from previous studies that used a simplified assumption of a single gRNA at the target site. While I understand this approach has precedent, it raises important questions about potential limitations. Specifically, could simplifying the analysis to one gRNA affect the conclusions of this study, given that the experimental design involves multiplexed gRNAs with four distinct target sites? The implications of using this simplified model should be clearly addressed, as the dynamics of drive efficiency, resistance formation, and fitness effects may differ when multiple gRNAs are employed. Additionally, while I am not a statistician, it is worth asking whether more sophisticated modeling approaches could be applied to account for all four gRNAs, rather than reducing the system to a single-gRNA framework. A discussion of the modeling choices and their potential consequences would strengthen the interpretation of the results.

-We have clarified this. While we have modeled multiple gRNAs with high fidelity in SLiM, the maximum likelihood method is not very amenable to such treatment. It may cause our fitness estimate to be a small overestimate, but give the low fitness inferences, would certainly not have a large enough effect to fundamentally change any conclusion (and should be of a consistent level across all cages). We now discuss this in the methods section.

Lines 297-300: Your results show that the expression of all target genes was higher in females, except for oct, which had higher expression in males. Additionally, oct expression decreased in adults. Given that oct is functionally important for ovulation and fertilization, processes that are primarily required in adult females, this pattern is somewhat unexpected. Could there be a possible explanation for the lower expression of oct, particularly in females and especially in adults, where its function would presumably be most critical? A brief discussion or hypothesis addressing this discrepancy would help clarify the biological relevance and interpretation of the expression data.

-Based on transcriptome data from FlyBase, derived from Graveley et al. (2011), Oct is indeed expressed slightly higher in adult males than in adult females. This difference may be attributed to the fact that the female flies used in the study were virgins; Oct expression could be upregulated post-mating to mediate ovulation. Additionally, Oct is expressed not only in reproductive tissues but also in other organs such as the nervous system, where sex-specific differences in cell type composition or neural activity may contribute to the observed expression bias. However, high expression does not necessarily correlate with essential expression. Though Oct could have multiple functions, it’s still possible that the only apparent phenotype upon knockout is female sterility. We have added the following text: “This male-biased expression may result from the use of virgin females in the dataset, as oct is likely upregulated after mating. Moreover, oct is also expressed in non-reproductive tissues such as the nervous system, which may contribute to sex-specific differences in expression38. While oct may have multiple functions, it is possible that it is only essential for female fertility.”

Lines 346-347: What is the distance between the gRNA target sites within each gene? Are all of the gRNAs confirmed to be active? It would be valuable to include a table summarizing the distance between target sites for each gene, the activity levels of the individual gRNAs, and the corresponding homing rates. This would help determine whether there is a correlation between gRNA spacing and drive efficiency. For example, Lopez del Amo et al. (Nature Communications, 2020) demonstrated that even a 20-nucleotide mismatch at each homology arm can significantly reduce drive conversion. Including such a comparative analysis in your study could provide important insights into how gRNA arrangement influences overall drive performance and would be incredibly helpful for future multiplexing designs.

-We have showed previously that close spacing of gRNAs should help maintain high drive conversion efficiency, and this is alluded to indirectly in the introduction (we now mention it more directly). In our study, gRNAs were positioned in close proximity without overlap, with the general distance between the outermost cut sites within each gene being We have added a summary table (Table S3) presenting the sequencing results, which also showed gRNA activity levels. Notably, most but not all gRNAs were active, at least for embryo resistance (low to moderate activity may still be present in the germline). Coupled with varying activity levels for those that were active, this likely contributed to reduced drive conversion due to mismatches at the homology arms. This observation supports the notion that drive performance could be optimized by selecting and arranging more active gRNAs. Consistent with this, our second construct targeting tra (tra-v2) exhibited a higher inheritance rate than the original construct, suggesting that gRNA arrangement and activity critically influence drive efficiency. Testing the activity of every single gRNA requires the construction of multiple gRNA lines, since in vitro or ex vivo tests will not be accurate as in vivo transformation test. However, in our study, as long as drive conversion rates were reasonably high, further optimization was not needed. Therefore, the multiplexing gRNA design can not only maximize drive conversion, but also reduce labor filtering an increased number of 1-gRNA designs with lower performance.

Line 434: I was not able to find any sequencing data. This is important to evaluate gRNA activities and establish correlations with drive efficiency.

-We have added a summary of the sequencing results in Table S3, though these are for embryo resistance alleles. Note that while high gRNA activity is correlated with high drive inheritance, these are not directly related. For suppression drives, germline resistance rates are usually of low importance compared to drive inheritance, so we did not assess these in detail (and pessimistically assumed complete germline resistance in our cage models).

Line 482: Did the authors test Cas9-only individuals (without the drive) against a wild-type population? This would help determine whether Cas9 alone has any unintended fitness effects. Additionally, is Cas9 expression stable over time and across generations? It would be helpful to include any observations or thoughts on the long-term stability and potential fitness impact of Cas9 in the absence of the drive element.

-We did not perform a direct comparison of Cas9-only individuals and wild-type flies in this study. However, previous studies (Champer et al., Nature Communications, 2020 - Langmuller et al., eLife 2022), which we now cite in the discussion, found no significant fitness difference between very similar Cas9-expressing lines and wild type in the absence of a drive element, indicating no significant fitness impact from Cas9 alone (though we cannot exclude a small effect, it certainly could not come close to explaining our results). In our experiments, Cas9 expression was generally stable across generations, as indicated by consistent drive inheritance and fertility test results obtained from independent batches. Separate from this study, we did observe rare instability in one nanos-Cas9 line, which had remained stable for over five years but recently became inactive (low population maintenance size may have caused stochastic removal of the functional allele). It is something to watch out for, but probably not on the timescale of a single study.

Discussion: I would appreciate a more direct and clearly stated conclusion that summarizes the key findings of the study. While the discussion addresses the main outcomes in depth, presenting a concise concluding paragraph, either at the end of the discussion or as a standalone conclusion section, would provide a stronger and more definitive closing statement. This would help reinforce what the study ultimately achieved and ensure the main takeaways are clearly communicated to the reader.

-We have revised and expanded the last paragraph of the discussion section to make our findings more direct and clear.

Overall, I believe this is an important study that offers valuable insights for advancing the design of CRISPR-based gene drives. The findings contribute to the development of more efficient and practical gene drive prototypes, bringing the field closer to real-world applications.

Reviewer #3 (Significance (Required)):

In this study, Xu and colleagues explored how CRISPR-based homing gene drives could be used to suppress insect populations by targeting female fertility genes in Drosophila melanogaster. They engineered split gene drives with multiplexed guide RNAs to target nine candidate genes, seeking to prevent functional resistance and achieve high drive conversion with minimal fitness costs. Among the targets, the stall (stl) and octopamine β2 receptor (oct) genes performed better, showing the highest inheritance rates in lab crosses. When tested in population cages, the stl drive was able to completely eliminate a fly population, but only when released at a high enough frequency, while other cages failed. These failures were traced and explained by fitness cost in drive-carrying females, caused largely by maternally deposited Cas9, which led to embryo resistance and reduced fertility. Through additional fertility assays and modeling, the team confirmed that the origin and timing of Cas9 expression, particularly from mothers, significantly impacted drive success. Surprisingly, even when Cas9 was driven by promoters with supposedly low somatic activity, such as nanos, fitness still persisted. The study revealed that while gene drives can be powerful, their effectiveness relies on finely balanced factors like promoter choice, drive architecture, and gene function. Overall, the research offers valuable lessons for designing robust, next-generation gene drives aimed at ecological pest control.

-We sincerely appreciate the reviewer’s positive and thoughtful comments. We agree that the points raised highlight the importance of our findings and hope that our revisions have further improved both the clarity and overall content of the manuscript.

Henriques, S. O., Rzayeva, N., Pinfield, S., & Waltman, L. (2023, October 13). Preprint review services: Disrupting the scholarly communication landscape?. https://doi.org/10.31235/osf.io/8c6xm

Decreto nº 9.830/2019

Responsabilização na hipótese de dolo ou erro grosseiro

Art. 12. O agente público somente poderá ser responsabilizado por suas decisões ou opiniões técnicas se agir ou se omitir com dolo, direto ou eventual, ou cometer erro grosseiro, no desempenho de suas funções.

• § 1º Considera-se erro grosseiro aquele manifesto, evidente e inescusável praticado com culpa grave, caracterizado por ação ou omissão com elevado grau de negligência, imprudência ou imperícia.

• § 2º Não será configurado dolo ou erro grosseiro do agente público se não restar comprovada, nos autos do processo de responsabilização, situação ou circunstância fática capaz de caracterizar o dolo ou o erro grosseiro.

• § 3º O mero nexo de causalidade entre a conduta e o resultado danoso não implica responsabilização, exceto se comprovado o dolo ou o erro grosseiro do agente público.

• § 4º A complexidade da matéria e das atribuições exercidas pelo agente público serão consideradas em eventual responsabilização do agente público.

• § 5º O montante do dano ao erário, ainda que expressivo, não poderá, por si só, ser elemento para caracterizar o erro grosseiro ou o dolo.

• § 6º A responsabilização pela opinião técnica não se estende de forma automática ao decisor que a adotou como fundamento de decidir e somente se configurará se estiverem presentes elementos suficientes para o decisor aferir o dolo ou o erro grosseiro da opinião técnica ou se houver conluio entre os agentes.

• § 7º No exercício do poder hierárquico, só responderá por culpa in vigilando aquele cuja omissão caracterizar erro grosseiro ou dolo.

• § 8º O disposto neste artigo não exime o agente público de atuar de forma diligente e eficiente no cumprimento dos seus deveres constitucionais e legais.

Acórdão 1525/2025 Primeira Câmara (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Omissão no dever de prestar contas. Débito. Multa.

• A omissão no dever de prestar contas constitui grave inobservância do dever de cuidado no trato com a coisa pública, revelando a existência de culpa grave, uma vez que se distancia do que seria esperado de um administrador médio, o que caracteriza erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb), incluído pela Lei 13.655/2018, legitimando a condenação em débito do responsável e a aplicação da sanção pecuniária prevista no art. 57 da Lei 8.443/1992.

Acórdão 755/2025 Plenário (Tomada de Contas Especial, Relator Ministro Jhonatan de Jesus)

Responsabilidade. Culpa. Erro grosseiro. Conduta. Avaliação. Sanção.

• Para fins do exercício do poder sancionatório do TCU, o erro grosseiro a que alude o art. 28 do Decreto-lei 4.657/1942 (Lindb) fica configurado quando a conduta do agente público se distancia acentuadamente daquela que seria esperada do administrador médio, parâmetro que retrata o dever de cuidado objetivo esperado de um gestor comum, capaz e prudente.

Acórdão 1089/2025 - Plenário (Tomada de Contas Especial, Relator Ministro Benjamin Zymler)

Responsabilidade. Culpa. Parecerista. Fundamentação. Parecer jurídico.

• Os pareceres jurídicos desprovidos de fundamentação adequada, favoráveis a contratações manifestamente ilegais ou que deixem de considerar jurisprudência pacificada do TCU podem ensejar a responsabilização do seu autor, se o ato concorrer para eventual irregularidade praticada pela autoridade que nele se embasou.

Acórdão 2467/2025 - Segunda Câmara (Tomada de Contas Especial, Relator Ministro-Substituto Marcos Bemquerer)

Responsabilidade. Culpa. Erro grosseiro. Convênio. Execução física. Plano de trabalho.

• Para fins do exercício do poder sancionatório do TCU, considera-se erro grosseiro (art. 28 do Decreto-lei 4.657/1942 - Lindb) a execução do objeto conveniado em desacordo com o plano de trabalho aprovado pelo concedente, sem qualquer justificativa.

Acórdão 5284/2025 - Segunda Câmara (Recurso de Reconsideração, Relator Ministro Augusto Nardes)

Responsabilidade. Débito. Culpa. Dolo. Lei de Introdução às Normas do Direito Brasileiro. Erro grosseiro.

• A regra prevista no art. 28 do Decreto-lei 4.657/1942 (Lindb), que estabelece que o agente público só responderá pessoalmente por suas decisões ou opiniões técnicas em caso de dolo ou erro grosseiro, <u>não se aplica à responsabilidade financeira por dano ao erário</u>. O dever de indenizar prejuízos aos cofres públicos permanece sujeito à comprovação de dolo ou culpa, sem qualquer gradação, tendo em vista o tratamento constitucional dado à matéria (art. 37, § 6º, da Constituição Federal).

• 1. O eventual acolhimento do pedido na ação de usucapião acarreta perda patrimonial imediata, ou seja, perda da propriedade do imóvel, gerando enorme prejuízo para os credores da massa falida. Assim, deve-se reconhecer a competência do juízo universal da falência para apreciar demandas dessa natureza.
• 2. "A arrecadação é ato de apreensão judicial executiva que visa à guarda e conservação dos bens do falido para futura alienação, em benefício dos credores. Sendo assim, nada mais coerente que todas as questões relacionadas aos bens arrecadados sejam decididas pelo juízo falimentar." (CC 84.752/RN, Rel. Ministra NANCY ANDRIGHI, SEGUNDA SEÇÃO, julgado em 27/06/2007, DJ 01/08/2007, p. 433)
• 3. Conflito de competência não conhecido em relação aos Juízos da 16ª e 17ª Varas Cíveis de Brasília/DF e, quanto ao incidente suscitado em face do Juízo da 11ª Vara Cível de Brasília/DF e do Tribunal de Justiça do Distrito Federal e dos Territórios, conflito conhecido para declarar a competência do Juízo da 11ª Vara Cível de Goiânia/GO. (CC n. 114.842/GO, relator Ministro Luis Felipe Salomão, Segunda Seção, julgado em 25/2/2015, DJe de 3/3/2015.)

1. Nos termos da jurisprudência consolidada pela Segunda Seção do STJ, no julgamento do CC 114.842/GO, em 25/02/2015, eventual acolhimento do pedido na ação de usucapião acarreta perda patrimonial imediata, ou seja, perda da propriedade do imóvel, gerando enorme prejuízo para os credores da massa falida, devendo ser reconhecida a competência do juízo universal da falência para apreciar demandas dessa natureza. (AgInt no REsp n. 2.004.910/CE, relatora Ministra Nancy Andrighi, Terceira Turma, julgado em 28/11/2022, DJe de 30/11/2022.)

Yes! Cf De Gaulle in SSA

I often find that my gut intuition leads me the right way, so if i don't have a lot of confidence in my answer, I'll spend more time checking it.

with their son in law

No me identifico personalmente con esto, pero tengo un hermano que se fue a tener hijos y siguió viviendo en casa de mis padres con su conyuge. Les llevó un tiempo conseguir su propio lugar, pero al final lo consiguieron. Dicho esto, entiendo que esto sea cierto en cierto modo.

Transformar o cambiar algo mudando alguna de sus características. Usado también como pronominal.

Expresar, generalmente con vehemencia, su queja o disconformidad

unidad singular e irrepetible que forma parte de un sistema más grande, ya sea biológico, social o estadístico.

conjunto de uno o mas hijos y uno o dos padres.

O: This line shows the actor being real with the audience. He doesn't try to act like a star but admits his own struggles as a normal actor. It makes him easier to relate to and makes the scene more personal and honest.

O & Q: Oates consistently uses first person language and makes an effort to make this writing very personal. I think this is to make a more impactful argument because it shows more emotion, and therefore could mean more to the reader. But is this just a normal practice for this form of writing? Or is it specific to this, setting it aside from other writings?