I'm also curious about the potential backlash that Ball's performance may have caused during this time, and wonder how these challenges to traditional depictions of femininity would be viewed in the modern day and vice-versa. I feel that many gender-related portrayals that we don't give a second thought to today would lead to public backlash during the time period in which I Love Lucy was on air.

I find it interesting that Shaw's commentary on women's roles and values were incorporated into the many roles that she chose to play. However, I also wonder whether these characters were based on stereotypical ideas about what place a wealthy French woman, Italian immigrant girl, and so on, would have in their respective societies.

It is always amazing to me to hear about television shows that were able to bend gender roles and expectations this early in the history of television and broadcasted media. It also reminds me that these now-archaic mediums were once one the most culturally progressive centers in American society.

It is not typical to acknowledge the role of vaudeville as a contributing factor to the modern sitcom. Obviously, stylistically, this had a huge impact. I wonder what other types of impacts that this type of performance actually had on the medium.

This is akin to the argument that radio was a live music killer. In reality, radio (and other forms of consuming music) actually enhance and help to grow the medium of live music. It shows that all new mediums actually help promote the content that they are housing.

I wonder if this aspect of Ball's performance caused public backlash. I cannot imagine that pushing these types of cultural boundaries happened without some sort of response from the more conservative members of society.

Throughout history, so many of the most legendary entertainment performances have been ones that challenge traditional ideas about gender and sexual identities. These are always the ideas that engage people the most.

knowing what I know about inner city children and public school systems this statement hit home because i was and know that most teachers spend more time disciplining the students than being able to teach.

More children and behavioral health needs than professionals?

according to CHAPTER 50-06 DEPARTMENT OF HEALTH AND HUMAN SERVICES states the obligation and definition of Human service professional encountering behavioral health.

The inability to cope as stated would create the question of what is needed from the students standpoint to "cope"

this shows a form of alarm to the reader that the professional sees a increase in anxiety issues etc,

Author of this Article shows to be Michael Standaert. An credited Journalist.

Perhaps true, but in the case of presidents, I think it’s more of a function of people’s pocketbooks.

This speaks directly to Daniel Conman’s book, ”Thinking, Fast and Slow.”

These may also just illustrate how people act in social situations, where they have to comply with or behave according to various norms, also in conjunction with their own feelings about saying or doing something overly offensive to someone. For example, a guy may not like Chinese, but doesn’t want the social stigma of publically denying one a meal.

I was having a beer with a friend one day and talking politics, when she told me that she considered herself objective and a critical thinker. Then in the next sentence or two, she told me she always votes a straight party ticket. My comment was, “then, you are not a critical thinker.” I think I was correct, and she indeed had second thoughts about what she had said.

This may be true, but it may also be a reach. It might be that someone is simply biased against something new and innovative, if for example, they are a traditionalist. I remember, for example, in high school that my sister-in-law named their daughter’s honey Lee and Melody very different names for a generation That was traditionally named after Judeo-Christian Saints or other or other traditional Anglo-Saxon names. I have some white friends who named their son “Ranger.” Let’s face it, that’s just a weird name to have show up on a staffing chart.

My question is, if they are that blasé about it, then is what they feel really an attitude or simply a comment about something they still don’t really care one way or the other about. For example, they may exercise to maintain their health without either liking or disliking it. True, maybe not a common situation but certainly possible.

Fascinating; and yet, more insightful than surprising. I say this because it brings to mind the adage, “Actions speak louder than words.” People often comment on their feelings about this or that, but then you watch what they do and you have to say, “Hmmmm. Really?”

I have a problem with this. In my mind, this illustrates role modeling in children as when when they learn right and wrong from copying their parents behavior. The way they describe it here has nothing to do with emotion, other than perhaps wanting her father‘s approval. But they don’t say that. Instead, they imply that the child had an emotional reaction one way or the other, that is an affective reaction from playing with the black child, but the way they describe it that’s not what happened. It obviously was pleasurable playing with her or she wouldn’t of continued doing it

This is so difficult to tell sometimes. I sometimes delete messages but wonder if they are valid? I got one from AT&T that I thought was slightly weird but it WAS real. It shows how effective and informed these scammers can be.

I definitely understand the impact of this and how it can be difficult, but I also wonder because of how much online shopping happening that is getting tracked, is there a point in consistently rejecting cookies? Websites make it difficult sometimes.

This is unfortunately something I am so guilty of, and I am rethinking that I said earlier that privacy and security online was a strength of mine. I might have behaved uninformed.

I do wonder how posts people made before they were an adult can impact their future. If employers see something a young teenager posted, how often does it hurt someone's chances years later?

Where would be the line between hacking to help someone in trouble and invading someone's privacy.

To me, I think that ethics plays a massive role in what can and can't be posted online. It serves as a sort of self-moderation that people could hold themselves accountable to while calling out "unethical" acts or posts. The case of Justine Sacco is a good example of what can be considered an "unethical" post.

This shows how Earthjustice's get information from its knowledge about law as opposed to scientific research. They call themselves as a watchdog organization, which means their objective is suing. Although their impact is serious in legal and policy debates, it should be looked against scientific facts on CCS.

The article does not have a actual author but the author is Earthjustice. Which is an nonprofit environmental organization that offers a valid analysis. That advocates to clean energy and reducing pollution. They create credibility by having a long history of engaging in environmental law and policy, making it trustworthy. However it does not mean they are a main source on carbon capture. They fight problems against industries that harm the environment.

Harmonic Cosmic Ecology (HCE) 7.1 is here, introducing refined models and groundbreaking research paths! We're diving deeper into the fascinating realm of the Informational Gravity Hypothesis (IGH), quantum gravity, neutron star resonances, and the harmonic patterns that link cosmic and biological scales.

Highlights include:

Clarified and improved mathematical formalism for IGH, removing earlier speculative ULDM associations to strengthen theoretical rigor.

Enhanced empirical support via quantum simulations with NV centers, bridging quantum physics with gravitational research.

New testable predictions leveraging LIGO gravitational-wave data, particularly focusing on quasinormal modes (QNMs) in black hole mergers.

Join the discussion: We're continually refining this evolving framework, so your insights and feedback are invaluable. Let’s collaborate and explore how quantum information shapes our cosmic tapestry!

Next Steps: Expect further empirical validations, interdisciplinary integration, and detailed simulations to support these exciting hypotheses.

Thank you to everyone who’s been contributing to and supporting the growth of the Harmonic Cosmic Ecology project. Onward to new scientific horizons!

ColorDarwin

Taxon: Zebra

ColorDarwin

Taxon: Zebra

Twitter makes money off of advertisements which play based on being on the site. This means more people spending more time on twitter directly translates to more money for the company. Features that lead to controversy or a lot of people talking about a topic (whether positive or negative) lead to more money for the company. This could include things like retweets and the later quote-tweet.

Hola

RevisionColorDarwin

I plan to use this fact about Bryan's speech in my essay to express how despite the fact that this era had many bad effects, it also brought people together and served as a historical preventative measure for future monopolies and as encouragement for better workers rights.

I'm struggling to understand why the wealthy individuals were still at risk in their current positions. What caused the need for investors?

ModeloBiocolores

RevisionColorDarwin

This article represents the news value of prominence as it has to do with celebrities disagreeing, and bringing in other people who are famous.

This metaphor stated by Wallen shows that he feels like SNL is not a place of purity or a place he wanted to be, kind of bashing SNL for their sense of humor.

Saying it was visible suggests that Morgan Wallen didn't seem to care what was perceived of this decision to leave early.

The comparison to Prince shows that sometimes celebrities do not interact with the cast, but it isn't common.

This metaphor is leaning into how Wallen does not appear to be friendly or outgoing towards others.

The phrase "spike in the norm" is a metaphor suggesting that it was out of the ordinary.

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

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Reply to the reviewers

__Reviewer #1 __

(Evidence, reproducibility and clarity (Required)):

The manuscript identified a novel role of Intraflagellar Transport Protein 20 (IFT20) in the function and homeostasis of lymphatic endothelial junctions. The authors showed that IFT20 regulates VE-cadherin localization at adherens junctions in lymphatic endothelial cells. The authors performed impressive in vivo work that shows the requirement for IFT20 for the homeostasis of intercellular junctions, lymphangiogenesis, and drainage function of lymphatic vessels. In contrast, the cell biology part of the paper was underwhelming and will need significant revisions to support the proposed model. In the result section, several conclusions have to be toned down to match the actual results. The study employs in vivo mouse models, immunofluorescence, biochemical assays, and loss-of-function experiments to support their conclusions.

Major comments - The authors present disrupted localization of VE-cadherin. Is this a mislocalization and/or protein stability issue in IFT20 KD cells? A western blot can help assess protein levels, and a phase-chase endocytosis assay of VE-cadherin can strengthen evidence. The authors did not confirm the permeability phenotype seen in vivo.

We thank the reviewer for this helpful suggestion.

Planned 1: Western blot to assess total VE-cadherin protein levels in IFT20 WT and KD cells.

Planned 2: Immunofluorescence staining for cell-surface VE-cadherin using permeabilized and non-permeabilized IFT20 WT and KD cells during VEGF-C stimulation and washout.

Together, these two experiments will assess VE-cadherin stability and more directly test the hypothesis that VE-cadherin does not recycle effectively back to the cell surface in the absence of IFT20.

• While the authors focused on IFT20 and rab5, we do not have a clear idea about the vesicular dynamics as well as the status of early, late, and recycling endosomes in IFT20 KD cells. Is IFT 20 localized to non-rab5+ endosomes, and if yes, what are the species? A more general endosomal profiling would help strengthen the authors' message. For example, in Fig. 4-5, the authors will have to stain for other early endosomal markers as well as late, and recycling endosomal markers in control and IFT20 KD cells.

Thank you for this helpful suggestion.

Planned 3: Immunofluorescence staining for EEA1 (early endosome), RAB7 (late endosome), RAB4 (fast recycling), RAB11 (recycling endosome) along with IFT20 to determine its localization pattern.

This experiment will determine the localization of IFT20 relative to various endosomal compartments.

• In fig. 6C, a majority of VE-cadherin is not associated with Rab5. Staining with additional endosomal markers might help identify other endosomal species colocalizing with VE-cadherin. It will be critical to add to Fig. 6c the intensity profiles depicting colocalizations. The authors can also live image a fluorescently (f)-tagged VE-cadherin (maybe with another f-tagged rab5) and assess their association dynamics in IFT20 KD cells (similar to fig6C).

Thank you for this helpful suggestion.

Planned 4: Immunofluorescence staining for EEA1 (early endosome), RAB7 (late endosome), RAB4 (fast recycling), RAB11 (recycling endosome) along with VE-cadherin in IFT20 WT and KD cells to determine its localization pattern.

Planned 5: Additional colocalization analysis such as adding intensity profiles and possibly proximity ligation assay.

Beyond the scope of this manuscript 1: While we agree that imaging the dynamics of FP-tagged VE-cadherin in live cells would provide more detail about its localization, we feel that this is beyond the scope of the current manuscript.

These experiments will determine the localization of VE-cadherin across various endosomal compartments and strengthen the current colocalization data.

• Primary cilia do seem to regulate vascular plexus in the mouse retina as well as endothelial permeability through mediating subcellular localization of junction proteins. The authors do not clearly exclude the ciliary function of IFT20 in mediating lymphatic endothelial cell-cell junctions. A rescue experiment can help settle this question by targeting IFT20 exclusively to cilia (or not) and assessing, for example, VE-cadherin localization. The following is optional: It is also unclear whether the described regulation is specific to IFT20 or can be phenocopied by the ablation of another IFT subunit and/or cilia ablation through the depletion of a non-IFT cilia assembly regulator.

Thank you for this helpful suggestion. We propose an alternative strategy.

Planned 6: To determine the role of ciliary vs. nonciliary functions, we will knockdown IFT74, an IFT protein in the same IFT complex B as IFT20 that is required for cilia assembly and function but is not known to participate in vesicular trafficking. We will assess VE-cadherin localization in IFT74 WT and KD cells by immunofluorescence.

Beyond the scope of the manuscript 2: We have not optimized reagents for targeting IFT20 to the cilium (e.g. ciliary targeting sequence) and believe that assessing the effects of a protein from the same IFT complex (IFT74) without known nonciliary functions will alleviate the reviewer’s concern.

• Figs. 7A and B do not seem very convincing. The control vs. IFT20 KD western blot levels look mostly similar between the two conditions. The result section does not translate the actual data in Fig. 7A and B. Additionally, there are no statistical comparisons between control and KD conditions in the graphs. Except for a potential pVEGFR-3 increase at 30 min VEGF-C in IFT20 KD cells, but after washout the level is similar to control. This figure does not support well the model presented in fig. 8. The conclusion in lines 456-459 has to be toned down.

Thank you for this helpful suggestion.

Planned 7: We will remove these western blot data with the exception of pVEGFR-3 and add phospho-tyrosine immunofluorescence. We will use immunofluorescence to quantify phosphorylated tyrosine levels and repeat western blots for pVEGFR-3 at different concentrations and time points of VEGF-C stimulation in IFT20 WT and KD cells. We will remove the other western blot data and revise the text accordingly. We will also attempt to pull down total VEGFR-3 and then blot for pVEGFR-3 to improve sensitivity of this assay.

These experiments will focus our analysis on the activation of VEGFR-3.

• The authors were not able to stain for pVEGFR3. It would still be helpful to see a colocalization between total VEGFR3, IFT20, and VE-cadherin in control cells and IFT20 KD cells (VEGFR3 and VE-cadherin).

Thank you for this helpful suggestion.

Planned 8: We will perform immunofluorescence for VEGFR-3, IFT20, and VECAD and assess their localization.

Minor comments - The control used in Figures 1 and 2 does not seem ideal. The proper control would be IFT20fl/fl cre neg. Is there a reason why the authors excluded a lox allele in control? Also, the authors have to provide the mice age used in these figures and when the Cre kicks in in the result section.

Thank you for this helpful suggestion.

Planned 9: We will clarify the use of control genotypes, and add mouse ages and Cre details to results/methods. This is a constitutive LYVE-1 Cre.

• Please describe the overall mouse phenotype(s) of the LYVE1 CRE-IFT20 flox.

Thank you for pointing out this oversight.

Planned 10: We will include a description of the overall phenotypes of LYVE1 Cre IFT20 KOs in the text. One notable phenotype is abdominal ascites.

• Line 109:'By expression, the authors probably mean immunostained.

Thank you for pointing out this oversight.

Planned 11: We will change to “immunostaining for”.

• Many graphs exhibit undefined Y-axis labels and units. Please clarify these as well as the way they were quantified. Include such information in figure legends and/or in the materials and methods section. The figures in question are fig1C, E and F, fig2E, fig3E, fig4b and D, fig6b and D, fig7B and C.

Thank you for this helpful suggestion.

Planned 12: We will clarify the quantification strategies and units in the text and figure legends and make sure the axes are clearly labeled.

• Line 295:'homeostasis"-the authors probably mean in a serum-rich condition.

Thank you for this helpful suggestion.

Planned 13: That is indeed what we meant. We will merge this sentence and the next sentence to be clearer.

• Fig4C specifically the lower two images on the right side: the images do not seem to represent the corresponding graphs.

Thank you for this helpful suggestion.

Planned 14: We will double check these images and adjust if necessary.

• Please add the statistical tests used to evaluate significance in all figure legends.

Thank you for pointing out this oversight.

Planned 15: We will be sure statistical tests are named in all figure legends.

Reviewer #1 (Significance (Required)):

This study provides novel insights into IFT20's role in VE-cadherin trafficking and endothelial junction stability, with its strongest aspect being the in vivo data in Figures 1 and 2, demonstrating lymphatic defects upon IFT20 loss. This represents a conceptual advance by extending IFT protein function beyond cilia (if one of the major comments is addressed) to vascular integrity. However, mechanistic depth is lacking, and ciliary role was not tested-additional rescue and colocalization experiments are needed to confirm the model. The study will interest vascular and lymphatic biologists, as well as cell biologists studying intracellular trafficking and cilia.

Expertise: cilia and mouse genetics

__Reviewer #2 __

(Evidence, reproducibility and clarity (Required)):

Paulson et al. use an in vivo model of IFT20 deletion (Lyve1-Cre) and primary lymphatic endothelial cell (LEC) cultures to investigate the role of IFT20 in controlling LEC-LEC junction dynamics. The key findings/suggestions include: i) Authors show alterations in the VE-cadherin (or ZO-1) staining at the LEC junctions upon IFT20 deletion or silencing. ii) They also show evidence of the IFT20 localization to RAB5 endosomes and alteration of RAB5 endosome dynamics upon IFT20 silencing.

In the current manuscript, some of the key data are not convincing. Further experimentation and analysis (also of the existing data) are needed to solidify the authors' statements as detailed below. I expect that the suggested experiments can be executed in 3-to-6 months and require, at least, antibodies, which have not been used in the current manuscript.

Major comments

1. The data information, presented in the figure legends, is difficult to understand. The authors should always indicate how many biological replicates and independent experiments the data is derived from. This holds also for the representative images. Now, it seems that some of the quantified data are derived from only 1 experiment (see, for example, rows 423-425: "Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition"). The quantifications should be based on data from at least three independent experiments.

Often data points represent the field of views from a single sample, thus, biasing the statistical testing. The data points should represent biological replicates or independent experiments to allow the reader to make conclusions, about whether the findings are statistically significant and can be repeated.

Thank you for this helpful critique.

Planned 16: We will be sure to indicate biological and technical replicates and ensure that quantifications are representative of at least three independent experiments. We will also ensure that quantifications are statistically robust.

The Lyve1-Cre is not specific for lymphatic vasculature (for example https://www.jax.org/strain/012601# and Lee LK et al. 2020, Cell Reports), as also stated by the authors (row 112). However, this is not shown in the data and complicates the interpretation of the data. Here, authors can stain the IFT20 with their existing mouse IFT20-specific antibody to show the loss in the lymphatic and/or blood vasculature. If IFT20 is lost in both vasculature types, it is not possible to say "lymphatic specific" (for example, row 143) and draw conclusions that the observed phenotypes would be primary to IFT20 loss in the lymphatic vasculature.

Thank you for this helpful suggestion.

Planned 17: We will assess IFT20 KO in blood vasculature and tone down lymphatic-specific language in the text.

The authors write (rows 164-168) "Lymphatic vessels in the IFT20 KO or VE-cadherin KO embryonic dorsal skin exhibited increased and variable lumen size and excessive branching, suggesting that impaired lymphatic organization and function contributed to the fluid homeostasis defect. Here, immunofluorescence staining for LYVE-1 in the ear skin revealed similar patterning defects in adult IFT20 KO lymphatic vessels (Figure 2A), that have also been described in VE-cadherin KO mice (Hägerling et al., 2018)." However, based on Figure 2A, it is not obvious that there would be excessive lymphatic vessel branching, impaired organization or similarities to VE-cadherin deleted lymphatic vessels. To justify their statement, the authors should provide quantification of the branching (at least 3 mice/genotype).

Thank you for this helpful critique.

Planned 18: Based on the suggestion from Reviewer 3, we will remove these morphological and skin drainage data.

IFT20 deletion or silencing causes alterations in the cell junction pattern/VE-cadherin intensity. The authors' interpretation that IFT20 deletion/silencing would cause discontinuous or "button-like" junctions is not supported by the provided images (Figures 1E, 3F, 6A, 6C). Rather, it seems that the levels of VE-cadherin in vivo are decreased, whereas the "continuity" of the junction is not altered. In cell culture, IFT20 silencing seems to cause wider and, to some extent, overlapping VE-cadherin junctions and not "discontinuous". These junctions may represent a more immature state. The authors should change the nomenclature accordingly or provide additional data. Using the existing cell culture experiment images, it would be more appropriate to analyze the width of the VE-cadherin junctions, instead of the "granularity".

Thank you for this helpful suggestion.

To assess VE-cadherin levels in vitro, we will perform western blots as described in Planned 1 above.

Planned 19: We will measure widths of junctions from IFT20 KD and WT images and adjust the language in the text.

Paulson et al. show images of IFT20 and RAB5 double-stained samples. The co-localization seems to happen mostly at the weakly IFT20 positive puncta (Figure 3A-B). Authors should show the disappearance of the signal in the siIFT20 treated samples (in comparison to siControl samples) to highlight the specificity of the weak signal.

Thank you for this helpful suggestion.

Planned 20: We will add data showing the IFT20 KD more clearly at high magnification.

1. The Authors analyze the co-localization of VE-cadherin and RAB5 as co-localization area (Figure 6C-D). The images show that the co-localization is stated to happen at LEC periphery/junctions. LEC periphery is notoriously thin and microscope Z-resolution does not allow distinction of truly co-localizing or "on top of each other" signal. Based on row 607 co-localization would be expected to happen at least in EEA1+ vesicles, which are located perinuclearly (not at the junctions) in LECs (Korhonen et al. 2022, JCI). Authors could use EEA1, RAB5, and VE-cadherin triple staining for the quantification.

Thank you for this helpful suggestion.

Please see Planned 3 and Planned 4 above where we propose experiments to address this concern.

In the current experiments, authors cannot conclude whether the VE-cadherin signal is at the cell junction (non-internalized), in endosomes (internalized during the experiment), or newly produced VE-cadherin on its way to the plasma membrane. To allow conclusions about the internalized VE-cadherin, and its localization in RAB5 vesicles, authors should conduct, for example, a classical endocytosis assay: incubation of live cells with non-blocking anti-VE-cadherin antibody, followed by acid wash to remove the non-internalized antibody, fixation and staining for RAB5. Also, shorter VEGF-C treatment would allow conclusions about the VE-cadherin dynamics.

Thank you for this helpful suggestion.

In Planned 2 above, we will perform immunofluorescence staining for cell-surface VE-cadherin using permeabilized and non-permeabilized IFT20 WT and KD cells during VEGF-C stimulation at various timepoints and washout to address this concern.

siRNAs can have off-target effects and, thus, the use of at least two independent methods/oligos for silencing is needed. Paulson et al. use a pool of 4 oligos for silencing. They should rather test the efficacy of the single oligos and then use the two best oligos (1/sample) to show and quantify the same phenotype. This is needed at least for the key experiments shown in Figures 4C-D, Figure 6A-B (see also comment #3), Figure 7A-B

Thank you for this helpful suggestion. We chose these reagents based on pooled siRNAs at low concentration minimizing off-target effects while still achieving strong KD vs. single siRNAs at higher concentration. Please see this technical note for further information about minimizing off-target effects by the use of pooled siRNAs vs. single siRNAs: https://horizondiscovery.com/-/media/Files/Horizon/resources/Application-notes/off-target-tech-review-technote.pdf?sc_lang=en

1. “SMARTpool siRNA reagents pool four highly functional SMARTselection designed siRNAs targeting the same gene. Studies show that strong on-target gene knockdown can be achieved with minimal off-target effects if a pool consisting of highly functional multiple siRNA is subsituted for individual duplexes. This finding is in contrast to speculation that mixtrues of siRNAs can compound off-target effects. … [Their data show that] while individual duplexes delivered at 100 nM can induce varying numbers of off-targeted genes, transfection of the corresponding SMARTpool siRNA (100 nM total concentration) induces only a fraction of the total off-target profile.”
2. “Our scientists have identified a unique combination of [chemical] modifications that eliminate as much as 80% of off-target effects.”
3. “The ON-TARGETplus product line is comprised of four individual siRNAs, and SMARTpool reagents which are chemically modified and rationally designed to minimize off-target effects.”

   OPTIONAL: Paulson et al stated in the first article (2021, Front. Cell Dev. Biol.) that IFT20 deletion/silencing causes lymphatic endothelial phenotypes due to its role in primary cilia, whereas here the authors conclude that IFT20 controls VE-cadherin dynamics at the RAB5 vesicles. However, the current experiments cannot dissect the role of IFT20 in these two distinct locations. For this, authors could delete/silence another gene required for primary cilia or RAB5 endosomes and then analyze, which IFT20 phenotypes are recapitulated.

Thank you for this helpful suggestion. Please see Planned 6 above where we propose to determine the role of ciliary vs. nonciliary IFT functions by knocking down IFT74, an IFT protein in the same IFT complex B as IFT20 that is required for cilia assembly and function but is not known to participate in vesicular trafficking. We will assess VE-cadherin localization in IFT74 WT and KD cells by immunofluorescence.

The data shown in Figure 2 B-E (Lymphatic drainage) is not necessary for the current manuscript ("IFT20 regulates VE-cadherin traffic in LECs") and can be removed. As the authors state in the manuscript, the drainage phenotype may be due to lymphatic vessel valve defects (rows 584-585) rather than primary for LEC-LEC junction defects. The data does not justify the abstract sentence "and lymph transport is impaired by intracellular sequestration of VE-cadherin" (row 42).

Thank you for this helpful suggestion. Please see Planned 18 above, where we propose to remove these data.

Minor comments

1. For some of the images, the signal should be enhanced to allow visual inspection also in the paper version (Figures 5A-B and 6C, magenta).

Thank you for this helpful suggestion.

Planned 21: We will enhance the signal in the indicated figures.

Authors show representative Western Blots and quantification of several biological replicates/sample types to investigate signaling responses upon VEGF-C treatment of control and siIFT20 cells. The authors state that the P-levels of VEGFR3, ERK, VE-cadherin, and AKT have different dynamics in control and IFT20-silenced cells. To justify this conclusion, authors should test the statistical significance between the siControl and siIFT20 samples at each time point. The current quantification (Figure 7B) shows that there is, at least, a trend of increased p-VEGFR3, p-VE-cadherin, p-ERK, and p-AKT in IFT20 silenced cells. However, the representative Western Blot image does not display a clear difference (Figure 7A). Authors should include the original western blots, used for quantification, as supplements.

Thank you for this helpful suggestion. Please see Planned 7 above where we propose to remove these data with the exception of pVEGFR-3 and add corresponding immunofluorescence data. We will ensure blots are included as supplemental figures.

The authors use western blot quantification to show that the altered LEC junctions affect VEGFR3 signaling. They further hypothesize that the increased VEGFR3 signaling may be a consequence of VEGFR3 localization in endosomes. The authors did not detect any signal using the phospho-specific VEGFR3 antibody (rows 441-442). To analyze the location of VEGFR3 upon VEGF-C treatment in siControl and siIFT20 LECs, the authors should use anti-VEGFR3 (total) antibodies that have been shown to detect VEGFR3 in similar assays.

Thank you for this helpful suggestion.

Please see Planned 8 above where we will perform immunofluorescence for VEGFR-3, IFT20, and VECAD and assess their localization.

The normality of the data should be tested before the selection of the statistical test. If this has been done, please, indicate it in the materials and methods or re-run the statistical analysis, if some of the data is not normally distributed.

Thank you for this helpful suggestion.

Planned 22: We will double check the statistics and normality for all quantifications.

The authors should use arrows, arrowheads, etc. to highlight examples of relevant features in the images. For example, in Figure 3C, the increased stress fiber formation is not obvious to the reader.

Thank you for this helpful suggestion.

Planned 23: We will add arrows etc. where appropriate.

Reviewer #2 (Significance (Required)):

Lymphatics are essential for fluid, leukocyte, and lipid trafficking to lymph nodes and/or systemic circulation. Recent findings have promoted lymphatics as a potential target to control the level of adaptive immunity in inflammation-associated diseases, including tumorigenesis (for example Song et al 2020, Nature). Early work on lymphatic endothelium in vivo, highlighted the dynamics of lymphatic endothelial junction, which, reversibly, can alter between continuous and discontinuous ("button-like") states (Baluk 2007, Am. Jour. Pathol.; Yao 2012, Am J. Pathol.). These changes may have an effect on fluid drainage capacity, lymphatic vessel growth, and prevention of pathogen dissemination to the systemic circulation. Recently, lymphatic junctions have been shown to present hubs of VEGFR3 signaling, VEGFR3 and VE-cadherin dynamics, and leukocyte transmigration (Sung et al. 2022, Nat. Cardiovasc. Res.; Hagerling et al. 2018, EMBO J.; Liaqat et al. 2024, EMBO J.). Thus, the manuscript by Paulson et al. investigates a topical subject.

The authors suggest a role for IFT20 in the control of VE-cadherin dynamics. Based on my expertise in lymphatic endothelial biology, I envision that the manuscript can potentially increase knowledge on the regulators of the lymphatic endothelial junctions, which might have physiological, and in the long term, translational significance. However, in the current manuscript, the exact mechanisms of how IFT20 controls lymphatic endothelial junctions are left open. In addition to the lymphatic research field, the study is, potentially of interest to researchers working on blood vasculature or, even, epithelium, i.e. tissues where junctional dynamics play a major role in health and disease.

Furter controls, analysis, and experimentation are needed to warrant the authors' statements. In their future work, the authors should also consider means to rigorously dissect the IFT20 functions in primary cilia and endosomes.

__Reviewer #3 __

(Evidence, reproducibility and clarity (Required)):

In this manuscript, the group of Fink and coworkers investigates mechanistic aspects of the intraflagellar transport protein 20 (IFT20) function in lymphatic endothelial cells (LECs). In a previous study, this group had demonstrated the presence of primary cilia on LECs and shown that loss of IFT20 during development resulted in edema, lymphatic vessel dilation and altered branching. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute lymphangiogenesis after corneal suture. In this manuscript, Paulson et al. recapitulate the suture-induced hyper-lymphangiogenesis after lymphatic-specific IFT20 KO using a LYVE1-Cre delete strain and demonstrate a reduced, more discontinuous VE-Cadherin (VECad) staining in newly formed lymphatic vessels (LVs). Prompted by distended and hyperbranching dermal vessels, the performed functional tracer injection experiments and demonstrate increased lymphatic backflow and leakage into the interstitium. To gain further mechanistic insights the authors turned to reductionist cell culture models, starting with a mouse LEC line, in which IFT20 had been deleted using CRISPR/Cas9 resulting in loss of primary cilia, increased stress fibre formation and impaired junctional integrity. More importantly, similar effects were detected in human dermal (HD)LECs after IFT20 KD. Further IFT20 KD HDLECs showed accumulation of RAB5+ vesicles indicating defective endosome maturation. Indistinguishable formation of RAB5+ endosomes after VEGF-C stimulation in HDLECs and IFT20KD HDLECs indicated that endocytosis and formation of early endosomes occur independent if IFT20. Through starvation, stimulation and wash-out experiments the authors provide colocalization data suggesting that after VEGF-C stimulation IFT20 is recruited to endosomes where it contributes to VECad recycling. Finally, the authors addressed if the increase in RAB5+ endosomes following VEGF-C stimulation resulted in prolonged retention of signaling-active VEGFR-3 in endosomes. Western blotting for phosphorylation of VEGFR-3 and its downstream signaling components after activation of starved HDLECs or IFT20KD HDLECs and subsequent factor wash out provided evidence towards this model.

Subsequently open question and potential suggestions for improvement are listed: The authors describe a slight leakiness of the LYVE1-Cre deleter strain to result in massive hemangiogenesis (line112). How extensive is the resulting deletion in blood endothelial cells? What are the consequences for VECad distribution in BEC junctions i.e. for blood vessels and vascular permeability? Are the defects described specific for LECs or are the manifestation of generic defects in LECs?

Thank you for these helpful suggestions.

Please see Planned 17 above where we will assess IFT20 KO in blood vasculature and tone down lymphatic-specific language in the text.

Fig. 1 E, what is the distribution of LYVE1 in IFT20 KO LECs at higher magnification, is LYVE1 excluded from the VECad expression domain?

Thank you for this helpful suggestion.

Planned 24: We will review our corneal confocal data to address this question.

Fig.1 F, what does VECad-positive LV (%) area (line 154 - 155) refer to, given that all LECs are VECad+ but the junctional distribution of the protein is distinctly different?

Thank you for pointing out our need to clarify. This quantification measures the overlap of VE-cadherin with LYVE-1 as a way to measure the area covered by adherens junctions between lymphatic endothelial cells. Where junctions are punctate, they have smaller area vs. long continuous junctions.

Planned 25: We will update the text to clarify this measurement.

In the discussion, the authors speculate that the development of valves could be potentially impaired in IFT20 LEC KO mice. Ear skin would be an excellent tissue to stain the valves and analyse their structure in collecting LVs. Of particular interest in this context are Int a9, VECad, FOXC2 and PROX1 expression. The later two are required for valve formation and upregulated in valve forming areas in response to oscillatory shear stress (Sabine A et al. (2012) Dev Cell 22 (2):430-445. doi:10.1016/j.devcel.2011.12.020).

Thank you for this helpful suggestion. Based on the suggestion from Reviewer 2, we will remove the ear lymph drainage data and focus on the cell biology in this manuscript. Our current experiments focus more on lymphatic valve formation in this context and these data can be moved to a separate manuscript.

Planned 26: We will revise the text to remove speculation about valve development in this model and address this in a later manuscript.

Does IFT20 KO and loss of the primary cilium impair OSS sensing and result in a failure to express sufficient levels of PROX1 for valve formation (Fig. 7 C).

Thank you for this helpful comment. We will address the role of cilia in OSS sensing and valve formation in a forthcoming manuscript.

A larger area view including pre-collectors and collectors would be informative and reveal changes in the overall structure of the lymphatic vessel bed in absence of IFT20.

Based on the suggestion from Reviewer 2, we will remove these data.

Fig. 2 A, (line 187 - 190) please indicate the age of analysed animals.

Planned 27: We will add the ages of mice used.

With respect to Fig.1, LVs in the area are mainly capillaries, what is the distribution of VECad? Are the LVs comprised of oak-leave shaped LECs, higher magn. pictures would be required.

Thank you for this helpful suggestion.

Planned 28: We will include higher magnification images of capillaries.

Fig. 2 (C - E) Line 201 - 203 the description of retrograde flow using a clock terminology is unusual and not clear to the reader. Is this meant relative to the point of injection with 12 being at the top or relative to the injection axis (i.e. forward / backward direction)? It would seem that indication of the angle in combination with a sketch of the analysis would help the reader to interpret these data.

Thank you for this helpful critique. We will remove these data based on this suggestion and that of Reviewer 2.

The application of cell culture models is appropriate, however, the value of the mLEC model is questionable given that VECad is not detectable in these cells and PROX1 and VEGFR-3 staining are not shown. Therefore, the HDLEC model bears significantly more relevance. In Fig. 3D, were mLECS mitotically arrested during the 24hrs transwell migration, to exclude division and crowding effects during the observation time?

Thank you for this helpful critique.

Planned 29: We will clarify the methods for this experiment in the text.

Fig. 6 It is commendable that the authors report their lack of success to directly visualize VEGFR-3 endocytosis by IF and attempt a WB analysis instead. However given the spread of the results normalization to ß-actin as a loading control appears inappropriate. Phosphorylated forms of VEGFR-3 and VECad should be normalized to the expression of the total protein as measured with a non-phospospecific antibody, exactly the way done here for ERK1/2 and AKT. Generally, IP-WB experiments provide superior data in this type of setting.

Thank you for this helpful suggestion. Based on suggestions from the other reviewers, we will remove these WB data with the exception of pVEGFR-3 and add corresponding immunofluorescence. We will include additional time points and include blots used for quantifications as supplements.

Line 597 - 599: "VEGFR-3 signaling is required for the establishment of VE-cadherin button junctions as lymphatic collecting vessels mature but is not required for their maintenance (Jannaway et al., 2023)." Collecting LVs are characterized by zipper junctions, but not button junctions. Therefore, this sentence needs clarification.

Thank you for this helpful suggestion.

Planned 30: We will clarify this text.

Reviewer #3 (Significance (Required)):

The role of IFT20 in formation of the primary cilium and endocytic vesicle transport warrants its investigation in lymphatic endothelial cells. Therefore, this study addresses relevant questions and provides important first insights into the cell biological function of IFT20 in this cell type. IFT20 has so far not been implicated in endocytosis and recycling of VECad and VEGFR-3 and the model suggested by the authors is compelling and adds to the mechanistic understanding of previous studies on the role of VECad in LECs. In particular, it could be of relevance for the enigmatic formation of button junction in lymphatic capillaries and the mechano-response of LECS underlying valve formation. At this point, the picture obtained from the endocytosis assays is more conclusive compared to the analysis of the impact of IFT20 loss on button junction formation. Clearly the study is of interest for a general cell biological audience as well as vascular biologists.

• *

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Referee #3

Evidence, reproducibility and clarity

In this manuscript, the group of Fink and coworkers investigates mechanistic aspects of the intraflagellar transport protein 20 (IFT20) function in lymphatic endothelial cells (LECs). In a previous study, this group had demonstrated the presence of primary cilia on LECs and shown that loss of IFT20 during development resulted in edema, lymphatic vessel dilation and altered branching. Lymphatic-specific deletion of IFT20 cell-autonomously exacerbated acute lymphangiogenesis after corneal suture. In this manuscript, Paulson et al. recapitulate the suture-induced hyper-lymphangiogenesis after lymphatic-specific IFT20 KO using a LYVE1-Cre delete strain and demonstrate a reduced, more discontinuous VE-Cadherin (VECad) staining in newly formed lymphatic vessels (LVs). Prompted by distended and hyperbranching dermal vessels, the performed functional tracer injection experiments and demonstrate increased lymphatic backflow and leakage into the interstitium. To gain further mechanistic insights the authors turned to reductionist cell culture models, starting with a mouse LEC line, in which IFT20 had been deleted using CRISPR/Cas9 resulting in loss of primary cilia, increased stress fibre formation and impaired junctional integrity. More importantly, similar effects were detected in human dermal (HD)LECs after IFT20 KD. Further IFT20 KD HDLECs showed accumulation of RAB5+ vesicles indicating defective endosome maturation. Indistinguishable formation of RAB5+ endosomes after VEGF-C stimulation in HDLECs and IFT20KD HDLECs indicated that endocytosis and formation of early endosomes occur independent if IFT20. Through starvation, stimulation and wash-out experiments the authors provide colocalization data suggesting that after VEGF-C stimulation IFT20 is recruited to endosomes where it contributes to VECad recycling. Finally, the authors addressed if the increase in RAB5+ endosomes following VEGF-C stimulation resulted in prolonged retention of signaling-active VEGFR-3 in endosomes. Western blotting for phosphorylation of VEGFR-3 and its downstream signaling components after activation of starved HDLECs or IFT20KD HDLECs and subsequent factor wash out provided evidence towards this model.

Subsequently open question and potential suggestions for improvement are listed: The authors describe a slight leakiness of the LYVE1-Cre deleter strain to result in massive hemangiogenesis (line112). How extensive is the resulting deletion in blood endothelial cells? What are the consequences for VECad distribution in BEC junctions i.e. for blood vessels and vascular permeability? Are the defects described specific for LECs or are the manifestation of generic defects in LECs? Fig. 1 E, what is the distribution of LYVE1 in IFT20 KO LECs at higher magnification, is LYVE1 excluded from the VECad expression domain? Fig.1 F, what does VECad-positive LV (%) area (line 154 - 155) refer to, given that all LECs are VECad+ but the junctional distribution of the protein is distinctly different?

In the discussion, the authors speculate that the development of valves could be potentially impaired in IFT20 LEC KO mice. Ear skin would be an excellent tissue to stain the valves and analyse their structure in collecting LVs. Of particular interest in this context are Int a9, VECad, FOXC2 and PROX1 expression. The later two are required for valve formation and upregulated in valve forming areas in response to oscillatory shear stress (Sabine A et al. (2012) Dev Cell 22 (2):430-445. doi:10.1016/j.devcel.2011.12.020). Does IFT20 KO and loss of the primary cilium impair OSS sensing and result in a failure to express sufficient levels of PROX1 for valve formation (Fig. 7 C). A larger area view including pre-collectors and collectors would be informative and reveal changes in the overall structure of the lymphatic vessel bed in absence of IFT20. Fig. 2 A, (line 187 - 190) please indicate the age of analysed animals. With respect to Fig.1, LVs in the area are mainly capillaries, what is the distribution of VECad? Are the LVs comprised of oak-leave shaped LECs, higher magn. pictures would be required. Fig. 2 (C - E) Line 201 - 203 the description of retrograde flow using a clock terminology is unusual and not clear to the reader. Is this meant relative to the point of injection with 12 being at the top or relative to the injection axis (i.e. forward / backward direction)? It would seem that indication of the angle in combination with a sketch of the analysis would help the reader to interpret these data.

The application of cell culture models is appropriate, however, the value of the mLEC model is questionable given that VECad is not detectable in these cells and PROX1 and VEGFR-3 staining are not shown. Therefore, the HDLEC model bears significantly more relevance. In Fig. 3D, were mLECS mitotically arrested during the 24hrs transwell migration, to exclude division and crowding effects during the observation time?

Fig. 6 It is commendable that the authors report their lack of success to directly visualize VEGFR-3 endocytosis by IF and attempt a WB analysis instead. However given the spread of the results normalization to ß-actin as a loading control appears inappropriate. Phosphorylated forms of VEGFR-3 and VECad should be normalized to the expression of the total protein as measured with a non-phospospecific antibody, exactly the way done here for ERK1/2 and AKT. Generally, IP-WB experiments provide superior data in this type of setting. Line 597 - 599: "VEGFR-3 signaling is required for the establishment of VE-cadherin button junctions as lymphatic collecting vessels mature but is not required for their maintenance (Jannaway et al., 2023)." Collecting LVs are characterized by zipper junctions, but not button junctions. Therefore, this sentence needs clarification.

Significance

The role of IFT20 in formation of the primary cilium and endocytic vesicle transport warrants its investigation in lymphatic endothelial cells. Therefore, this study addresses relevant questions and provides important first insights into the cell biological function of IFT20 in this cell type. IFT20 has so far not been implicated in endocytosis and recycling of VECad and VEGFR-3 and the model suggested by the authors is compelling and adds to the mechanistic understanding of previous studies on the role of VECad in LECs. In particular, it could be of relevance for the enigmatic formation of button junction in lymphatic capillaries and the mechano-response of LECS underlying valve formation. At this point, the picture obtained from the endocytosis assays is more conclusive compared to the analysis of the impact of IFT20 loss on button junction formation. Clearly the study is of interest for a general cell biological audience as well as vascular biologists.

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---

Referee #2

Evidence, reproducibility and clarity

Paulson et al. use an in vivo model of IFT20 deletion (Lyve1-Cre) and primary lymphatic endothelial cell (LEC) cultures to investigate the role of IFT20 in controlling LEC-LEC junction dynamics. The key findings/suggestions include: i) Authors show alterations in the VE-cadherin (or ZO-1) staining at the LEC junctions upon IFT20 deletion or silencing. ii) They also show evidence of the IFT20 localization to RAB5 endosomes and alteration of RAB5 endosome dynamics upon IFT20 silencing.

In the current manuscript, some of the key data are not convincing. Further experimentation and analysis (also of the existing data) are needed to solidify the authors' statements as detailed below. I expect that the suggested experiments can be executed in 3-to-6 months and require, at least, antibodies, which have not been used in the current manuscript.

Major comments

1. The data information, presented in the figure legends, is difficult to understand. The authors should always indicate how many biological replicates and independent experiments the data is derived from. This holds also for the representative images. Now, it seems that some of the quantified data are derived from only 1 experiment (see, for example, rows 423-425: "Graphs show one representative biological replicate of two, each comprising two technical replicates with 100+ cells per condition"). The quantifications should be based on data from at least three independent experiments.

Often data points represent the field of views from a single sample, thus, biasing the statistical testing. The data points should represent biological replicates or independent experiments to allow the reader to make conclusions, about whether the findings are statistically significant and can be repeated. 2. The Lyve1-Cre is not specific for lymphatic vasculature (for example https://www.jax.org/strain/012601# and Lee LK et al. 2020, Cell Reports), as also stated by the authors (row 112). However, this is not shown in the data and complicates the interpretation of the data. Here, authors can stain the IFT20 with their existing mouse IFT20-specific antibody to show the loss in the lymphatic and/or blood vasculature. If IFT20 is lost in both vasculature types, it is not possible to say "lymphatic specific" (for example, row 143) and draw conclusions that the observed phenotypes would be primary to IFT20 loss in the lymphatic vasculature. 3. The authors write (rows 164-168) "Lymphatic vessels in the IFT20 KO or VE-cadherin KO embryonic dorsal skin exhibited increased and variable lumen size and excessive branching, suggesting that impaired lymphatic organization and function contributed to the fluid homeostasis defect. Here, immunofluorescence staining for LYVE-1 in the ear skin revealed similar patterning defects in adult IFT20 KO lymphatic vessels (Figure 2A), that have also been described in VE-cadherin KO mice (Hägerling et al., 2018)." However, based on Figure 2A, it is not obvious that there would be excessive lymphatic vessel branching, impaired organization or similarities to VE-cadherin deleted lymphatic vessels. To justify their statement, the authors should provide quantification of the branching (at least 3 mice/genotype). 4. IFT20 deletion or silencing causes alterations in the cell junction pattern/VE-cadherin intensity. The authors' interpretation that IFT20 deletion/silencing would cause discontinuous or "button-like" junctions is not supported by the provided images (Figures 1E, 3F, 6A, 6C). Rather, it seems that the levels of VE-cadherin in vivo are decreased, whereas the "continuity" of the junction is not altered. In cell culture, IFT20 silencing seems to cause wider and, to some extent, overlapping VE-cadherin junctions and not "discontinuous". These junctions may represent a more immature state. The authors should change the nomenclature accordingly or provide additional data. Using the existing cell culture experiment images, it would be more appropriate to analyze the width of the VE-cadherin junctions, instead of the "granularity". 5. Paulson et al. show images of IFT20 and RAB5 double-stained samples. The co-localization seems to happen mostly at the weakly IFT20 positive puncta (Figure 3A-B). Authors should show the disappearance of the signal in the siIFT20 treated samples (in comparison to siControl samples) to highlight the specificity of the weak signal. 6. The Authors analyze the co-localization of VE-cadherin and RAB5 as co-localization area (Figure 6C-D). The images show that the co-localization is stated to happen at LEC periphery/junctions. LEC periphery is notoriously thin and microscope Z-resolution does not allow distinction of truly co-localizing or "on top of each other" signal. Based on row 607 co-localization would be expected to happen at least in EEA1+ vesicles, which are located perinuclearly (not at the junctions) in LECs (Korhonen et al. 2022, JCI). Authors could use EEA1, RAB5, and VE-cadherin triple staining for the quantification.

In the current experiments, authors cannot conclude whether the VE-cadherin signal is at the cell junction (non-internalized), in endosomes (internalized during the experiment), or newly produced VE-cadherin on its way to the plasma membrane. To allow conclusions about the internalized VE-cadherin, and its localization in RAB5 vesicles, authors should conduct, for example, a classical endocytosis assay: incubation of live cells with non-blocking anti-VE-cadherin antibody, followed by acid wash to remove the non-internalized antibody, fixation and staining for RAB5. Also, shorter VEGF-C treatment would allow conclusions about the VE-cadherin dynamics. 7. siRNAs can have off-target effects and, thus, the use of at least two independent methods/oligos for silencing is needed. Paulson et al. use a pool of 4 oligos for silencing. They should rather test the efficacy of the single oligos and then use the two best oligos (1/sample) to show and quantify the same phenotype. This is needed at least for the key experiments shown in Figures 4C-D, Figure 6A-B (see also comment #3), Figure 7A-B 8. OPTIONAL: Paulson et al stated in the first article (2021, Front. Cell Dev. Biol.) that IFT20 deletion/silencing causes lymphatic endothelial phenotypes due to its role in primary cilia, whereas here the authors conclude that IFT20 controls VE-cadherin dynamics at the RAB5 vesicles. However, the current experiments cannot dissect the role of IFT20 in these two distinct locations. For this, authors could delete/silence another gene required for primary cilia or RAB5 endosomes and then analyze, which IFT20 phenotypes are recapitulated. 9. The data shown in Figure 2 B-E (Lymphatic drainage) is not necessary for the current manuscript ("IFT20 regulates VE-cadherin traffic in LECs") and can be removed. As the authors state in the manuscript, the drainage phenotype may be due to lymphatic vessel valve defects (rows 584-585) rather than primary for LEC-LEC junction defects. The data does not justify the abstract sentence "and lymph transport is impaired by intracellular sequestration of VE-cadherin" (row 42).

Minor comments

1. For some of the images, the signal should be enhanced to allow visual inspection also in the paper version (Figures 5A-B and 6C, magenta).
2. Authors show representative Western Blots and quantification of several biological replicates/sample types to investigate signaling responses upon VEGF-C treatment of control and siIFT20 cells. The authors state that the P-levels of VEGFR3, ERK, VE-cadherin, and AKT have different dynamics in control and IFT20-silenced cells. To justify this conclusion, authors should test the statistical significance between the siControl and siIFT20 samples at each time point.

The current quantification (Figure 7B) shows that there is, at least, a trend of increased p-VEGFR3, p-VE-cadherin, p-ERK, and p-AKT in IFT20 silenced cells. However, the representative Western Blot image does not display a clear difference (Figure 7A). Authors should include the original western blots, used for quantification, as supplements. 12. The authors use western blot quantification to show that the altered LEC junctions affect VEGFR3 signaling. They further hypothesize that the increased VEGFR3 signaling may be a consequence of VEGFR3 localization in endosomes. The authors did not detect any signal using the phospho-specific VEGFR3 antibody (rows 441-442). To analyze the location of VEGFR3 upon VEGF-C treatment in siControl and siIFT20 LECs, the authors should use anti-VEGFR3 (total) antibodies that have been shown to detect VEGFR3 in similar assays. 13. The normality of the data should be tested before the selection of the statistical test. If this has been done, please, indicate it in the materials and methods or re-run the statistical analysis, if some of the data is not normally distributed. 14. The authors should use arrows, arrowheads, etc. to highlight examples of relevant features in the images. For example, in Figure 3C, the increased stress fiber formation is not obvious to the reader.

Significance

Lymphatics are essential for fluid, leukocyte, and lipid trafficking to lymph nodes and/or systemic circulation. Recent findings have promoted lymphatics as a potential target to control the level of adaptive immunity in inflammation-associated diseases, including tumorigenesis (for example Song et al 2020, Nature). Early work on lymphatic endothelium in vivo, highlighted the dynamics of lymphatic endothelial junction, which, reversibly, can alter between continuous and discontinuous ("button-like") states (Baluk 2007, Am. Jour. Pathol.; Yao 2012, Am J. Pathol.). These changes may have an effect on fluid drainage capacity, lymphatic vessel growth, and prevention of pathogen dissemination to the systemic circulation. Recently, lymphatic junctions have been shown to present hubs of VEGFR3 signaling, VEGFR3 and VE-cadherin dynamics, and leukocyte transmigration (Sung et al. 2022, Nat. Cardiovasc. Res.; Hagerling et al. 2018, EMBO J.; Liaqat et al. 2024, EMBO J.). Thus, the manuscript by Paulson et al. investigates a topical subject.

The authors suggest a role for IFT20 in the control of VE-cadherin dynamics. Based on my expertise in lymphatic endothelial biology, I envision that the manuscript can potentially increase knowledge on the regulators of the lymphatic endothelial junctions, which might have physiological, and in the long term, translational significance. However, in the current manuscript, the exact mechanisms of how IFT20 controls lymphatic endothelial junctions are left open. In addition to the lymphatic research field, the study is, potentially of interest to researchers working on blood vasculature or, even, epithelium, i.e. tissues where junctional dynamics play a major role in health and disease.

Furter controls, analysis, and experimentation are needed to warrant the authors' statements. In their future work, the authors should also consider means to rigorously dissect the IFT20 functions in primary cilia and endosomes.

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Referee #1

Evidence, reproducibility and clarity

The manuscript identified a novel role of Intraflagellar Transport Protein 20 (IFT20) in the function and homeostasis of lymphatic endothelial junctions. The authors showed that IFT20 regulates VE-cadherin localization at adherens junctions in lymphatic endothelial cells. The authors performed impressive in vivo work that shows the requirement for IFT20 for the homeostasis of intercellular junctions, lymphangiogenesis, and drainage function of lymphatic vessels. In contrast, the cell biology part of the paper was underwhelming and will need significant revisions to support the proposed model. In the result section, several conclusions have to be toned down to match the actual results. The study employs in vivo mouse models, immunofluorescence, biochemical assays, and loss-of-function experiments to support their conclusions.

Major comments

• The authors present disrupted localization of VE-cadherin. Is this a mislocalization and/or protein stability issue in IFT20 KD cells? A western blot can help assess protein levels, and a phase-chase endocytosis assay of VE-cadherin can strengthen evidence. The authors did not confirm the permeability phenotype seen in vivo.
• While the authors focused on IFT20 and rab5, we do not have a clear idea about the vesicular dynamics as well as the status of early, late, and recycling endosomes in IFT20 KD cells. Is IFT 20 localized to non-rab5+ endosomes, and if yes, what are the species? A more general endosomal profiling would help strengthen the authors' message. For example, in Fig. 4-5, the authors will have to stain for other early endosomal markers as well as late, and recycling endosomal markers in control and IFT20 KD cells.
• In fig. 6C, a majority of VE-cadherin is not associated with Rab5. Staining with additional endosomal markers might help identify other endosomal species colocalizing with VE-cadherin. It will be critical to add to Fig. 6c the intensity profiles depicting colocalizations. The authors can also live image a fluorescently (f)-tagged VE-cadherin (maybe with another f-tagged rab5) and assess their association dynamics in IFT20 KD cells (similar to fig6C).
• Primary cilia do seem to regulate vascular plexus in the mouse retina as well as endothelial permeability through mediating subcellular localization of junction proteins. The authors do not clearly exclude the ciliary function of IFT20 in mediating lymphatic endothelial cell-cell junctions. A rescue experiment can help settle this question by targeting IFT20 exclusively to cilia (or not) and assessing, for example, VE-cadherin localization. The following is optional: It is also unclear whether the described regulation is specific to IFT20 or can be phenocopied by the ablation of another IFT subunit and/or cilia ablation through the depletion of a non-IFT cilia assembly regulator.
• Figs. 7A and B do not seem very convincing. The control vs. IFT20 KD western blot levels look mostly similar between the two conditions. The result section does not translate the actual data in Fig. 7A and B. Additionally, there are no statistical comparisons between control and KD conditions in the graphs. Except for a potential pVEGFR-3 increase at 30 min VEGF-C in IFT20 KD cells, but after washout the level is similar to control. This figure does not support well the model presented in fig. 8. The conclusion in lines 456-459 has to be toned down.
• The authors were not able to stain for pVEGFR3. It would still be helpful to see a colocalization between total VEGFR3, IFT20, and VE-cadherin in control cells and IFT20 KD cells (VEGFR3 and VE-cadherin).

Minor comments

• The control used in Figures 1 and 2 does not seem ideal. The proper control would be IFT20fl/fl cre neg. Is there a reason why the authors excluded a lox allele in control? Also, the authors have to provide the mice age used in these figures and when the Cre kicks in in the result section.
• Please describe the overall mouse phenotype(s) of the LYVE1 CRE-IFT20 flox.
• Line 109:'By expression, the authors probably mean immunostained.
• Many graphs exhibit undefined Y-axis labels and units. Please clarify these as well as the way they were quantified. Include such information in figure legends and/or in the materials and methods section. The figures in question are fig1C, E and F, fig2E, fig3E, fig4b and D, fig6b and D, fig7B and C.
• Line 295:'homeostasis"-the authors probably mean in a serum-rich condition.
• Fig4C specifically the lower two images on the right side: the images do not seem to represent the corresponding graphs.
• Please add the statistical tests used to evaluate significance in all figure legends.

Significance

This study provides novel insights into IFT20's role in VE-cadherin trafficking and endothelial junction stability, with its strongest aspect being the in vivo data in Figures 1 and 2, demonstrating lymphatic defects upon IFT20 loss. This represents a conceptual advance by extending IFT protein function beyond cilia (if one of the major comments is addressed) to vascular integrity. However, mechanistic depth is lacking, and ciliary role was not tested-additional rescue and colocalization experiments are needed to confirm the model. The study will interest vascular and lymphatic biologists, as well as cell biologists studying intracellular trafficking and cilia.

Expertise: cilia and mouse genetics

What sense exactly? This seems really shallow to me ... If only some axis of x is getting heavily changed, would that be considered appropriate?

This sounds like correctional officers' commit crimes that can be described/ compared to, that of predation. To prey on an entrapped group of prey. Why exactly do we have such high statistics? How come the jails and prisons don't have a clause that only permits same sex correctional officers to inmates in order to minimize the claims? I know it wouldn't eliminate the abuse altogether, but it would certainly minimize it! Shouldn't the officer then be charged the same as a person who is charged for pedophilia, rape, things of that nature? Not just relieved of their jobs?

If you like the annotated conversation here, you can also join in on the margins of the paper itself.

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I purley just think that this fact is very interesting. I find it to be interesting because today you would not think that showing a pregnant woman on tv would be something that needed to be addressed. It is fascinating that was viewed controversial at one time. One of the many milestones and history markers this show had produced for the mass population.

I wonder how many television shows are like this. It takes hundreds of people to put together a show, but we rarely hear about any other than the producer and director. There are so many immense talents that we unfortunately do not hear about.

This is an interesting glimpse of the politics involved with unions, which has been historically very common in the entertainment industry. It is unfortunate, because it gets in the way of creative people making their art. On the other hand, they are totally necessary in our society and have brought working people many rights.

This is a very interesting concept, and it makes me think about the "secret" creators behind modern television shows. Throughout history, there have always been quiet hitmakers, who choose to sit in the background rather than make themselves known. After all, it makes more sense for a studio to put a celebrity's name on a show than an unrecognized name.

This is not something that's very common nowadays. I feel like actors who become producers usually do so on different films, rather than opting to act in their own projects.

All of these things in tandem is very wild! It is almost impossible to visualize a hit this large in today's media. Now, there are not even shows that come close to being this unifying within culture.

The stress it puts on students!

Agreed!

Elementary students should not have homework especially the younger grades!!

Teachers need to start think: 'is this really helping the students or am I just giving homework just to give it?'

This is interesting and something I have not thought about before! Most of the time when parents want to 'help' they do the assignment for the student.

I remember first watching "I Love Lucy" in my freshman year of high school and I have come across it at least 3 separate times in my academic career since then. I think it is interesting how much you can learn not only from the show but also from how the show was made. This show is so versatile with all the different lessons and things it could show its audience. It makes sense why it is not only so widely known but also enjoyed.

Ah I see .. so a showrunner doesn't have to just be what was listed, but rather can be anyone .... I wonder if even an influencer that promotes a certain tv show or film could be accredited to be a "showrunner" ... is it more than just making the show garner attention or is it including behind the scenes stuff too? do people who run behind the scenes but aren't talked about still get that showrunner role/credit?

I beg to differ- I feel as though if "I Love Lucy" was a contemporary series, I believe that the success of the show would be attributed to the actors rather than those behind the scenes. I feel like enjoying those behind the scenes and their hard work is not common these days and people tend to care about WHO is on the screen rather than the one who is presenting what is on the screen as a form of their own artwork and passion.

The development of the "hyphenate" writer-producer (described as one that creates and produces television content) is an under appreciated but important influence of I Love Lucy that is highlighted in this sentence. This was controversial at the time of the episode because television production was still developing and industry specialists were usually limited to certain jobs and not very versatile.

I feel that the rise of writer-producers was a positive for film and television, as consistency and quality became more sought after by the public. Although many people were and are still responsible for bringing these scripts to life, problems can arise if writers and producers are fighting to tell the story in the way they see fit. When one person is in charge and finds a successful formula, it becomes much easier to remain consistent and develop storylines in ways that both make sense and appeal to the audience.

It's interesting to consider how the landscape of Hollywood has changed over the years, especially when comparing the modern the day to the 1950s. While it is not uncommon today for people to take on multiple roles at once, such as writer-producer, actor-producer, and so on, these hyphenate positions used to be rare.

hello!

Everything is highlighted footnotes, and citations that are used throughout the paper. If you click on them they will bring you to the link where they either cited, or paraphrased others findings. All this links will be mostly to google scholar accounts, showing the validity of this journal.

Could there be any relationship to flagella of unicellular eukaryotes?

It seems possible some degree of oxygen respiration could have been acquired on the branch leading to FECA. I think much is still unknown about this.

As this is mainly a news report, this state was a new report in this location for California residents

This is a 4-6th grade reading

https://mvcc.libguides.com/kidlit/lexile https://www.booksource.com/reading-level-chart

Cite in MLA 9th edition but could be put in APA also with the help from these people "Thomas Fuller reported from Napa, and Richard Pérez-Peña and Jonah Engel Bromwich from New York. Reporting was contributed by Carol Pogash from Santa Rosa, California; Stacey Solie from Petaluma, Calif.; Julie Turkewitz from Las Vegas; and Scott Bransford from Fairfield, California." many interviews and person experience .

The reporters added new 360 videos to see how it would've been being there with a sorts of videos about fire and firefighters.

New York Times has a high Factual rating of around 1.4. which is high with high credibility but can be very bias.

https://www.allsides.com/news-source/new-york-times-opinion-media-bias https://mediabiasfactcheck.com/new-york-times/

This title kinda foreshadows the impact of the fire and how it affected residents who faced the challenge of rebuilding their lives. This occurrence demonstrates the importance of being adequately prepared for emergencies.

This is the website with full photos and everything. for more info

https://www.nytimes.com/2017/10/10/us/california-fires.html link.gale.com/apps/doc/A508978861/OVIC?u=midlandstc&sid=summon&xid=baebbc3d.

Some in-text citations I did. This is important because on the website they interviewed many and they all didn't take the warning seriously. When they did it was too late.

If you click on any of the author's names, it will send you to a page of other articles they have published and them. All New York Times reporters for over 10+ years.

I assume this is referring to services like Auth0 where people out-source authentication instead of keeping it directly part of your own code base

To me, I feel like the use of religion as a guideline morally is fine, but I disagree more with the idea of Divine Command Theory. Different religious groups will interpret scripture differently, which means much of it should be left to be interpreted based on reasoning. An example of difference that comes to mind is Catholics and Baptists. Both follow the Christian faith, but seem to differ in their interpretation of the Bible and what they value more in the religion.

Egoism is interesting because it implies in the goal that if everyone does what will maximize their own net good, overall the goodness in the world outweighs the evil. But every culture, person, and place has it's own relative perspective on what goodness is and what the goodness that one ought to do includes. This makes the entire theory relative because it is subjective.

Another aspect of effective entertainment is things that make audience members feel like they are "in" on something. It's a way to engage them and create a sense of belonging among viewers. This is a great strategy to foster this feeling.

I think some of the most effective comedy is simply situations that people relate to. It is the formula most often applied to sitcoms. Shows like Seinfield and Modern Family come to mind.

This model is definitely common nowadays. Social media and the internet has allowed creators to create large-scale merchandise brands. When you walk into a convenience store, you'll be greeted by countless influencer-endorsed candy bars and energy drinks. It is cool to see that this model was established long ago.

I think that even when the popular culture is conservative, people are still longing for ideas that challenge their worldview. The key is to do it in a way that doesn't outright scare or offend them. I Love Lucy and it's depiction of feminism is a perfect example.

This reminds me of a phenomenon that is very common in today's media: parasocial relationships. It seems that I Love Lucy was harnessing this power in the same way that social media influencers do. When viewers think that they have a real connection with the creators of media, they are simply more likely to tune in.

In past geography classes taken in high school, I was never prompted to use spatial thinking. It was more about memorizing facts. I can see how learning about geography with a mind open to spatial concepts can be much more efficient and purposeful than simply trying to memorize facts. For what it's worth, I agree that geography is about a lot more than knowing what is done where. It's about learning how communities function and the cool little tidbits that no one would think about but are actually incredibly relevant to bettering the world today.

It is important to note that what we eat can be just as based on societal norms of the time as it can be what is healthy or satisfying to us individually. From what we eat to how much we eat, those who are around us eating habits influence on ours, and ours on them. I found a good article on ScienceDirect explaining how certain societal factors can affect a person's eating choices. It states "We are more likely to follow an eating norm if it is perceived to be relevant based on social comparison."(Social Influences On Eating) people that are seeking to be approved by others are more likely to identify and follow social eating norms, whether they are fully conscience of what they are doing or not. That can help explain why a certain region is more prone to eat more of something that is just as available to it as a region where the people do not eat as much of it.

Social Influences On Eating.Current Opinion in Behavioral Sciences.https://www.sciencedirect.com/science/article/pii/S235215461500131X#bbib0350. Accessed 31 march 2025.

When you see the background of people and inventors that originally thought of what are now popular items, you can see more of why they created what they did and how their everyday lives compelled them to create something to fix an issue or fill a gap, just like the Kellogg brothers were hoping to create a breakfast food that would satisfy their religious requirements and their nutritional needs.

This website uses pictures of people smiling to show that their costumers are happy and unbothered.

The publisher of this website is TIAA they are a qualified financial planning company founded by Adrew Carnegie.

This is used as a source to provide extra information about where they are based in. This is a corporate source written in discloser format.

The purpose of this article is to advertise the good thing the company has and what they are efficient in. Essentially they want to sell theyre qualifications and work

This uses words that target the audience of people who do not have very much growth in money.

This implies the audiance of financial advisors showing that the team TIAA has put together is well qualified.

Neel Mukherjee helped create this homepage for TIAA. He completed a PhD at Pembroke College and Cambridge and went on to take a master's in creative writing at the University of East Anglia.

It was cool how the October Revolution gave mostly unknown musicians a chance to perform, instead of relying on big names. This made the event feel more raw and spontaneous, letting fresh voices experiment and take risks without pressure. The focus on new compositions and breaking traditional jazz rules added to that feeling of unpredictability. It really showed how powerful and exciting music can be when artists are free to explore.

This article uses a picture of a arrow moving up towards a percent sign sitting on money showing that this article is a guide to maximizing your capital counts.

This article is published my Invictus they have many credible blogs written my credible authors.

Stress testing and capital planning is highlighted in this sentence to give a direct source to finding more information.

This article is to explain that investing is not being nonconservative but being wise with money.

These few sentances show that this article may be for someone more advanced in this field of financial planning, Making it more suitable for a particular audience.

This does not directly target an audiance but it does have a theme of many banks across America making it easy to identify whos doing the regulatory "safe harbor"

The author of this article is Adam Mustafa. Adam Mustafa he has overseen the design and implementation of fully customized capital testing, capital management and financial planning systems. He has also advised high growth banks.

adjusted formatting

Digital infrastructure on taxes varies based on advanced or developing countries.

Will read this source after

Domestic Revenue mobilization refers to increasing government revenues.

This highlights the importance of strategic competitiveness in business. When legal protections are weak, success depends on superior execution in areas like pricing, distribution, and sales. Entrepreneurs must continuously innovate and adapt to stay ahead of rivals.

The author of this article is Patti Casaleggio. Patti is a executive assistant at Invictus currently but was a retirement financial planner before.

Se hace enfasis, junto con el parrafo anterior, que en general, las muertes fetales, primero se van a a dar mayormente en embarazos a termino y que la presencia de factores de riesgos, no aumentan mucho el riesgo, el unico que si, es una muerte fetal previa

Buena tabla, enfatizar sobre el puro riesgo de estar embarazada sin ninguna comorbilidad ya tiene riesgo

Se hace una anotación sobre si el SARVS CoV 2 puede ser factor de riesgo o no sobre la muerte fetal, teniendo 2 casos, donde en uno si aumenta y en otro no

This article, rather than having a works cited towards the bottom, it has hyperlinks to the webpages of other sources. They use this technique a few more times in this article.

This sentence shows that this problem isn't just affecting the 'Big Guys', but the smaller businesses as well. With this having been stated it shows that this article is meant for a general audience of people who are interested, and affected.

This is the problem that Microsoft, with its AI agents are trying to solve for the better of network defenders and cybersecurity.

This video is a great addition to this article to because it shows a business founders point of view with internal conflicts. That everyone can have an argument and a falling out.

The author of this article is Jennifer Conrad. She is the Senior writer/reporter for Inc. She has also written article for many other big companies, e.g. Wired, Vogue, The Wire China. She also has a masters degree in Chinese studies and economics.

More articles from this writer: https://www.inc.com/author/jennifer-conrad

Very nice results! I have a question about the confocal microscopy images in Figure 2c. I noticed there are a few cells visible in the DAPI channel (particularly in the top row) that show minimal or no discernible APC or GFP signal. Are these representative of the small percentage (<3%) of CAR-expressing cells that weren't successfully stained by the multimers, or are they potentially CAR-negative cells included in the sample? I'm curious about how these unstained cells relate to the high specificity rates you reported.

based on the size or height of the patient?

A waveform image would be very helpful

"additions" is similar to adding new pieces to a structure or puzzle, emphasizing growth or reconstruction which is another metaphor.

Not physically under, but worked for

potentially a taylor swift reference

This is a metaphor since he isn't physically "shaking" up the longhorns staff. Shaking in this title means switching, or moving around staff members of the new team he is a part of.

Picture again, to demonstrate the placement in the circuit

picture would be helpful.

This article has a few graphics displayed throughout the article. While they are not used to serve a statistical purpose, but are there to give the reader a simple image to look at and help the reader imagine the situation. To help give the mental vision of the situation.

The authors are Frank Cespedes and Georg Krentzel.

Frank Cespedes is the Senior Lecturer of Business Administration in the Entrepreneurial Management Unit. https://www.linkedin.com/in/frankcespedes/

Georg Kretzel is a Senior partner with Globalpraxis.

These two questions are extremely relevant, especially as AI and social engineering grows more prevalent in social media platforms. AI is being used to spread disinformation or misinformation to large crowds of people. For example, during the 2024 election. Donald Trump used AI generated images to exaggerate the celebrity support he had and natural disasters in the US. His use of AI to convince people of false information was extremely dangerous because individuals did not see the difference between real life and the social media posts they were viewing. While Social Media can be great to connect with a large network of people. For example, I am able to keep in contact with friends from middle school who now live in different countries, it is also exteremly scary because individuals can use the widespread tech to share manipulated or fake content.

https://www.nytimes.com/2025/03/29/climate/lee-zeldin-epa.html

https://www.nytimes.com/2025/03/27/climate/global-sea-ice-record-low.html

https://theconversation.com/climate-change-isnt-fair-but-tony-junipers-new-book-explains-how-a-green-transition-could-be-just-250671?utm_source=dlvr.it&utm_medium=mastodon

@Janrae.Valencia this has been edited now

VERY personal

something that I empathize with a lot nature threatening you with the existence of god

poetic and nature

meter + sibilance

Nature

in poetry we personify nature a lot, this also builds off the other one I highlighted about nature threatening you with the existence of god

nature

Tone

goes with the rambling style of prose that he keeps using

personal

Poetic

"Grief is" metaphor

Tone

all of the #death stuff, mentioned in the blurb

poetic

I just really like this passage

Personal

Tone combines nature with fucking...I think...

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I thank the Referees for their...

Referee #1

1. The authors should provide more information when...

Responses + The typical domed appearance of a hydrocephalus-harboring skull is apparent as early as P4, as shown in a new side-by-side comparison of pups at that age (Fig. 1A). + Though this is not stated in the MS 2. Figure 6: Why has only...

Response: We expanded the comparison

Minor comments:

1. The text contains several...

Response: We added...

Referee #2

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Referee #2

Evidence, reproducibility and clarity

Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB. The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

Specific comments/concerns are listed below.

1. In Figure 3, quantifications of the fluorescence at HLBs for mCherry-RBP1 and MXC-mScarlet should be provided.
2. In Figure 5C, both 5' and 3' transcripts are observed in G214 cells. However, their accumulation in the cytoplasm is not visible. How do the authors explain this result? What happens in S14 cells?
3. In Figure 6, the authors observed RD histone 3' transcripts only in replicating cells (EdU positive) while they detected 5' transcripts in both replicating and non-replicating cells. They argue that the appearance of 3' transcripts is due to the release from transcriptional pausing. To further support particular states in the transcriptional arrest, data by immunofluorescence using specific antibodies recognizing either RNA pol II ser5P or ser2P would determine whether the presence of 3' transcripts is associated with the accumulation at HLB of RNA pol II ser2P (elongating polymerase). Moreover, is there a correlation between P-MXC and RNA pol II ser2P?
4. In Figure 7 panels C and D, the 5' transcripts should be shown. Although RD histone 3' transcripts accumulate in CyE+ embryonic cells, unfortunately, their presence at HLBs (pointed by arrows) is not visible in the image of panel E. To firm up conclusions quantifications of the 3' and 5' transcripts should be provided for CycE+ and CycEnull cells. In Hur et al., 2020, the authors looked at RD histone transcripts in WT embryo and CycE+/-/Cdk2+/- mutant. They found that the amount of H3 transcripts using a probe corresponding to the coding sequence is not changed in the mutant as compared to the WT. In contrast, they found that there is an increase of transcripts that are not correctly processed using probes downstream the stem-loop region. This seems inconsistent with the results presented here where a decrease of 3' transcripts is observed. This needs an explanation/discussion. Are such incorrectly processed transcripts observed in CycEnull mutant?
5. The authors suggest that active Cyclin E/Cdk2 triggers the release of RNA pol II promoter-proximal pausing and therefore induces transcriptional elongation at RD histone genes when cells enter S phase. To further support this hypothesis, determining whether there is an enrichment of the elongation factor p-TEFb at HLB when Cyclin E/Cdk2 is active would help.
6. Instead of using cycling E mutants, to determine whether it is the phosphorylation of MXC which directly impacts the elongation of RD histone genes, it would be interesting to generate phospho-null or phospho-mimetic mutant of MXC.
7. In Suzuki et al., 2022, the authors described 3' RNA pol II pausing at RD histone genes. Although this study used human cells, it would be interesting to discuss that in addition to a promoter-proximal pausing that regulates transcription elongation, a 3' pausing could also regulate the transcription termination and 3' processing.
8. In the discussion, the authors should point out some limitations of their studies linked to the method and could propose for the future that a more precise and molecular view of the pausing mechanism could be carried out using sequencing methods such as ChIP-seq of various isoforms of the RNA pol II (total, ser2P, ser5P) and elongation regulators (p-TEFb.....) and PRO-seq.

Minor points:

1. In Figure 1, for panels B and D as well as for panels C and E, to falicitate comparison of the localization of the different proteins, it would help to show the same developmental stages and the same image scales.
2. In Figures 3 and 7 (C-F), the developmental stages should be indicated on the images, as it is done in the other figures.
3. In the legend of Figure 7, it is indicated (D) and (E) instead of (C) and (D) in the sentence: "Endocycling midgut cells in (D) contain cytoplasmic histone mRNA which is absent in (E) (boxed regions)."

Significance

Kemp et al. aimed to explore the transcriptional cell cycle regulation of replication-dependent (RD) histone genes at histone locus body (HLB) in Drosophila. They evaluate the accumulation of RNA pol II and RD histone transcripts at HLB during the cell cycle using live and fixed imaging of Drosophila tissues at different stages of development. They find that RNA pol II is enriched at HLB, not only during S phase when RD histone genes are transcribed but throughout the cell cycle. Outside of S phase, they detect short but not full-length RD histone transcripts suggesting a mechanism of RNA pol II pausing. Full length RD transcripts are only produced upon cyclin E/Cdk2 activation when cells enter S phase, arguing that Cyclin E/Cdk2 can activate transcription elongation. They propose that the elongation release triggered by Cyclin E/Cdk2 is the critical step linking RD histone gene expression and cell cycle progression rather than the recruitment of RNA pol II to HLB.

The data are interesting and robust, however, additional experiments could reinforce the findings and the proposed model.

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Referee #1

Evidence, reproducibility and clarity

Summary:

Kemp et al. seek to define the molecular interactions that limit replication-dependent histone gene transcription to S-phase of the cell cycle. They use the Drosophila model system and leverage live-imaging tools, such as tagged proteins and Jabba trap, and RNA FISH in several tissues to determine that RNA Pol II is enriched at the locus throughout the cell cycle and is paused outside of S-phase. Therefore, they conclude that it is not Pol II recruitment to the locus that couples histone transcription to S-phase, but release of Pol II pausing.

Major comments:

The data presented are clean and well-presented. The claims are supported by the data without exaggeration. It would be helpful to provide -omics support for this entirely image-based analysis (e.g. PRO- or GRO-seq data from synchronized, sorted Drosophila cells may already exist- OPTIONAL).

A major requirement is that the authors make clear in Introduction and Discussion that the observation of Pol Ii pausing at RD histone genes is not novel. This requires, at minimum, a discussion of Liu (2024) and Suzuki (2022). This allows readers to focus on the advance novel to this work, which is specifically the cell cycle coupling of Pol II pausing.

As the authors are claiming different dynamics between Spt6 and RPB1 in Figure 1, they should provide similarly-staged embryos for comparison. For example, the authors should show RPB1 in early/mid S of NC 14, as this is when they see Spt6 variability. In theory, this should be relatively easy as these are stills from the live videos.

Minor comments:

The use of Spt6 live imaging early on was slightly confusing. The authors should consider moving this data later in the results or providing more written justification for why they investigated Spt6 (further than "to further explore the regulation of RNA pol II dynamics... p6). Similarly, Spt6 is included in the model figure, which might be a stretch given the only Spt6 data involves the timing of Spt6 colocalization with Mxc during the cell cycle.

Misleading language/missed citations:

p3: "600 kB array" is misleading. The whole locus is ~ 600 kB.

p3: Mxc may remain at the locus throughout the cell cycle, so the whole HLB does not disassemble (Terzo, 2015).

p4: H1-specific factors include cramped (Gibert and Karch, 2011; Bodner et al. 2024 bioRxiv)

p4: Hodkinson, 2023 is not the correct reference. The correct reference is Hodkinson, 2024, Genetics.

p5: The Drosophila HLB is detectable at NC 10 (White, 2011; Terzo, 2015) not White, 2007

p5: A typo: "imagining"

p7: The section title "RNA pol II is necessary for HLB assembly" is incorrect, as Figure 3 shows that pol II is NOT necessary for Mxc recruitment, but for HLB growth. Mxc, however, is necessary for pol II recruitment.

p9: The authors should clarify what "HisC" means as this is the first usage.

Figures/experiments:

Fig 2: The authors should show the gating in Figure I that led to the three categories in Figure J. The legend/colors in Figure J are not necessary.

An "easy" experiment would be to use the FUCCI cell lines and 5'/3' RNA FISH in combination (assuming fluorophores allow) - OPTIONAL

Discussion:

p13: The reference to the work of Gugliemlmi, 2013 should first come up in the Introduction, as it provides rationale.

p13: "without engaging in transcription" is misleading, as pol II is transcribing, but paused.

p15: It makes sense for pol II to pause at histone genes in G1, as they are preparing for the rapid burst of histone transcription needed in S phase. But what might be the functional rationale for pol II pausing in G2, if the HLB disassembles in M?

Methods:

It should be made clear how embryos were staged for live imaging, as it is likely by timing after cell cycle events. What is this timing? It would be best if this detail is not just mentioned in the methods, but also in the main text. This is especially important for readers not familiar with Drosophila embryogenesis. Please cite/acknowledge DGRC for Fly-FUCCI line (if appropriate)

Significance

This study provides convincing evidence that pol II is enriched at the histone locus and paused outside of S-phase. What limits the significance is that several prior studies concluded that Pol II is paused at the histone locus:

Lu et al. bioRxiv 2024, "Integrator-mediated clustering of poised RNA polymerase II synchronizes histone transcription"

Suzuki et al. Nat Comm 2022, "The 3' Pol II pausing at replication-dependent histone genes is regulated by Mediator through Cajal bodies' association with histone locus bodies"

Neither of these studies is discussed or even cited in the manuscript, which is disappointing. Therefore, the advance is limited to the cell-cycle coupling of pausing. This is still important, as a major knowledge gap as outlined by the authors is that it is not clear how histone transcription is coupled to S-phase and they rule out Pol II pausing as a possible mechanism, and point toward Pol II pausing release.

Moreover, there is also evidence (from these authors) that Mxc phosphorylation is not always coupled to histone transcription in Drosophila ovaries. This work is also not discussed or cited:

Potter-Birriel et al. J Cell Sci 2021, "A region of SLBP outside the mRNA-processing domain is essential for deposition of histone mRNA into the Drosophila egg"

The current research may be of interest to the broad cell cycle field, but it may also be useful as a model for those conducting basic, foundational research who seek to describe how Pol II is released from pausing. The histone locus may be of interest as a novel, facile model for pausing.

Reviewer expertise: Drosophila, chromatin, gene expression

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Reviewer #1

__Evidence, reproducibility and clarity __

This is a well-written manuscript that describes a thorough study of the functionality of individual residues of a central component of the ESX-3 type VII secretion system of Mycobacterium smegmatis, EccD3, in the essential role of this protein transport system in iron acquisition. Using the powerful and unbiased approach of deep mutational scanning (DMS), the authors assessed the impact of different mutations on a large number of residues of this component. This carefully executed research highlights the importance of hydrophobic residues at the center the ubiquitin-like domain, specific residues of the linker domain that connects this domain with the transmembrane domains and specific residues that connect EccD3 with the MycP3 component.

Major comments

Since the LOF effects in the iron-sufficient and iron-deficient condition differ less than expected, the differences of the DMS results between these two conditions should be better presented, explained and discussed: 1. The authors discuss: "Of the 270 LOF mutations seen in the iron-deficient condition, 37 (13.7%) were tolerant in the iron sufficient condition, and 39 (14.44%) had strong LOF effects but weak LOF effects in the iron sufficient condition." Do the authors mean that 39 (14.44%) had strong LOF effects in the iron-deficient condition, but weak LOF effects in the iron-sufficient condition. In turn, does this mean that the remaining mutants (71.9%) had similar LOF effects in the two conditions?

We thank this reviewer for their comment and for highlighting a lack of clarity. We have updated the main text to more effectively communicate our point - that 270 mutants had LOF effects in the iron-deficient media. 37 of these 270 mutants were tolerant in the iron-sufficient media. 39 of these 270 mutants had strong LOF effects in iron-deficient media, but were weak LOF in iron-sufficient media. The remaining 124/270 mutants had weak LOF effects in both conditions. The larger point is that removing iron leads to stronger selection - tolerant mutants become LOF, weak LOF become strong LOF. Removing iron pushes mutants at the bounds over the limit.

__ The diagonal shape of the scatter plot in Fig. 2C, which shows the correlation of the Enrich2 scores of all mutants in the two conditions, indicates that the growth of most mutants is affected similarly in these conditions, but in Fig. 2D lower graph, which shows only the Enrich2 scores of missense mutants, there are clear differences between the two conditions. How can this be explained?__

We apologize for any confusion created by this presentation of our data. We hoped to highlight that while effects are largely similar across conditions, there are some differences. As communicated in our first response, 270 out of our ~2700 missense mutations had LOF effects in the iron-deficient condition. 37 of these 270 mutants were tolerant in the iron-sufficient media. 39 of these 270 mutants had strong LOF effects in iron-deficient media, but were weak LOF in iron-sufficient media. The remaining 124 mutations had weak LOF effects in both conditions.

While Figure 2C shows this difference, it is hard to see by nature of using a scatter plot. We have added contours to highlight how our data is distributed. Our density plots in Figure 2D are meant to try to highlight these differences, where the top plot is showing the effects of all missense mutations. Negatively scored mutations represent LOF effects, mutations with scores around 0 are considered tolerant, and the extremely rare scores with positive scores have GOF effects. Our bottom plot specifically zooms into the negatively scored mutations, to show the 270 LOF mutants we discussed. Specifically, we were hoping to highlight the 39 mutations that have strong LOF effects in iron-deficient media (so the purple line scores are more negative), but weak LOF effects in iron-sufficient media (the green line scores are less negative).

__ Regarding the authors' explanation for the observed LOF effects in the permissive condition, "This speaks to the sensitivity of next-generation sequencing compared to the strong differences observed between conditions in phenotypic growth curves." But this sensitivity does not explain the observed large LOF effects but no growth difference in the permissive condition, unless the analysis is less quantitative than expected? Could it be that there is local iron depletion in this mixed culture, causing selection pressure even in the iron-sufficient condition? Moreover, the severity of the growth defect at the time of sampling, i.e., after 24 hours of growth, is unclear. Indeed, the growth curve in Fig. 1 shows that the growth of the double mutant in iron-deficient conditions is significantly impaired at that timepoint. In the growth curve in Fig. 2B (and also slightly in Fig. 2F), however, the growth defect is less pronounced: the double mutant has a similar OD600 as the WT strain, although the error bar is larger. Is this variability between replicates also seen in the DMS analysis? In general, no statistics are shown for the DMS analysis and there is no information on the significance of the observed LOF effects. In addition, the legend should explain how many replicates the DMS data are based on.__

We thank this reviewer for their comment and for highlighting a point of confusion. In addition to increased sensitivity in next generation sequencing compared to our growth curve experiments, our data analysis and variant scoring was performed by comparing growth rates of our mutant strains to our wild type strain. So, any effect on viability or growth rates seen by expression mutant variants will be more notable in our DMS scoring, as they are relative to wild type. In contrast, our growth curves are plotted as the raw OD600 values of each strain. We believe this difference underlies the difference seen in our heatmaps and growth rates.

It is also a relevant and important point that our libraries are grown as mixed cultures, where there is competition over the limited iron in their growth media, as we highlight in our discussion.

While the double mutant does show a stark growth defect at 24 hours in Figure 1 compared to the WT and complement, it grows just as well as those strains in Figure 2B. The growth defect becomes notable after 24 hours. Within this experiment, we observed variability in growth at the 24hr timepoint for the negative control strain, but also selection when compared to the positive control and library growth at later time points. We analyzed our DMS data in accordance with typical methods used in the field (see: https://doi.org/10.1186/s13059-017-1272-5). We include statistics for the DMS analysis as supplemental Figure 1. We apologize for any confusion regarding the figure caption, however in our manuscript we do point out that our library growth in Figure 2B was repeated in triplicate in the figure caption, and the samples collected during that experiment were the ones used to generate the DMS data.

Minor comments

1. Line and page numbering should be added to the manuscript to facilitate the reviewing process.

We have updated our manuscript to include line and page numbering.

__ "Knockout of the entire ESX-3 operon leads to inhibited M. smegmatis growth in a low-iron environment. When individual components of the ESX-3 system are deleted, growth is only available under impaired if the additional siderophore exochelin formyltransferase fxbA is also knocked out20." First, a reference should be added to the first sentence. Second, Siegrist et al. did not exactly show this. They showed that the fxbA/eccC3 double mutant grows slower that the fxbA single mutant. To my knowledge there is no publication showing that single esx-3 component mutants grow as WT in iron-deficient conditions. Do the authors have data demonstrating this? If true, it is surprising that mutating EccD3 has a milder phenotype compared the complete region deletion, as it is a crucial ESX-3 component.__

We apologize for any confusion. We had the relevant reference two lines prior, and have since added it to that sentence as well.

The reviewer is correct that Siegrest et al did not show the effects of just ESX-3 component single deletions. However, Siegrest et al. 2009 demonstrated that deleting the entire ESX-3 operon results in growth similar to the wild type strain in low-iron media. In contrast, the fxbA single knockout exhibits a notable growth defect, and the fxbA/ESX-3 double knockout has an even more severe growth defect. Following the logic that a double knockout is needed to observe a growth defect in low-iron media, Siegrest et al. 2014 demonstrated this also extends to single ESX-3 component knockouts, such as the fxbA/eccD3 double knockout strain. To ensure clarity and accuracy, I will edit the sentence to say "When individual components of the ESX-3 system are deleted, growth is significantly impaired when the additional siderophore exochelin formyltransferase fxbA is also knocked out."

__ Reference to Table 1, should be a reference to Table S1.__

We have updated our manuscript to correct this reference.

__ "Our heatmaps surprisingly reveal residues where substitutions are deleterious specifically in the iron-sufficient condition" Refer here to Fig. S2.__

We have updated our manuscript to include this reference.

__ "In the iron-deficient condition, 6/551 (1.08%) missense mutations have a weak LOF effect, and 0 have strong effects." More clearly explain this refers to the residues of the transmembrane region.__

We have updated our manuscript to provide more clarity.

__ "The MycP transmembrane helix has been hypothesized to be required for ESX complex specificity, targeting MycP to associate with the correct ESX homologue." I miss a reference here. And I thought that the transmembrane domain of MycP was required for complex stability not for specificity?__

We thank the reviewer for pointing out our missing citation, and asking us to clarify our point. I believe the literature suggests that both the protease and transmembrane domains of MycP are required for both complex stability and specificity. van Winden et al. 2016 https://doi.org/10.1128/mbio.01471-16 show that MycP5 needs to be present for secretion. The protease activity can be abolished and the ESX-5 complex can still secrete and be pulled down, as seen by BN-PAGE. van Winden et al. 2019 https://doi.org/10.1074/jbc.RA118.007090 show that truncated mutants missing either the protease domain or the transmembrane domain cannot rescue ESX-5 secretion or complex stability in a MycP knockout strain. More relevant, they attempted to rescue MycP1 and MycP5 mutants by creating chimeric proteins that either had the MycP1 protease domain and MycP5 transmembrane domain, or the MycP5 protease domain and MycP1 transmembrane domain. If the protease and transmembrane domains were required for complex stability and NOT specificity, we would see MycP5 rescue ESX-1 secretion in the MycP1 mutant strains and vice versa. We would also see the chimera proteins rescue both ESX-1 and ESX-5 secretion and complex stability. Instead, we see that neither chimera rescued ESX-1 nor ESX-5 secretion or complex stability, implying that both MycP domains are necessary.

We will amend our paper text to reference MycP's role in complex stability instead of specificity, and soften the language: "The MycP transmembrane helix has been shown to be required for ESX complex stability, as MycP knockouts and truncated mutants abolish ESX secretion and pulldowns of the entire complex."

__ "....role in ESX function relating to EccB3 and EccC3. In the transmembrane, ..... we" Insert "region" after "transmembrane"__

We have updated our manuscript to include this update.

Significance

The study provides insight into individual residues of a central component of the ESX-3 type VII secretion system for functionality, which is useful for those studying the functioning of mycobacterial type VII secretion systems. Moreover, because this system is essential for the growth of the important pathogen M. tuberculosis, this knowledge can be used to design new anti-tuberculosis compounds that block the ESX-3 system. Although the results mainly confirm previous observations (highlighting specific residues important for the stability of ubiquitin and residues of other parts of EccD important for protein-protein interactions within the ESX-3/ESX-5 membrane complex), to my knowledge this is the first time DMS has been applied to mycobacteria. This study is therefore of interest to mycobacteriologists.

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Reviewer #2

__Evidence, reproducibility and clarity __

This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system. 1. The authors engineered an M. smegmatis knockout strain with deletions of fxbA and eccD3. Deletion of fxbA renders the exocholin iron uptake system non-functional, forcing the bacteria to rely on siderophore-mediated iron uptake under iron-limiting conditions. This process, in turn, depends on ESX-3 secretion activity, as PPE4, a known ESX-3 substrate, has been previously implicated in iron utilization in M. tuberculosis (Tufariello et al., 2016). This experimental setup links EccD3 function to a growth phenotype under iron-limiting conditions, as mutations impairing ESX-3 secretion disrupt iron utilization and mycobacterial growth. 2. By complementing the knockout strain with a library of EccD3 mutant variants, the authors systematically identify residues essential for protein-protein interactions within the ESX-3 core complex. Structural analysis corroborates the functional relevance of these residues, specifically those mediating interactions between EccD3 and other ESX-3 components, or those disrupting the hydrophobic core of the EccD3 ubiquitin-like (Ubl) domain. 3. Structural comparisons with the MycP5-bound ESX-5 complex allow the authors to predict residues within EccD3 that may interact with MycP3 during ESX-3 core complex assembly. Furthermore, comparisons with the ESX-5 hexamer suggest residues that may stabilize or drive oligomerization of the ESX-3 dimer into its putative hexameric state. These insights are significant and provide testable hypotheses for future studies. 4. The methodology is limited to ESX-3. The authors exploit the essentiality of ESX-3 for siderophore-dependent growth under iron-limiting conditions. However, this functional readout cannot be directly transferred to other ESX systems (ESX-1, ESX-2, ESX-4, ESX-5), which have distinct substrates, biological roles, and regulatory mechanisms.

Significance

This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

Thank you for your thoughtful and supportive feedback. We appreciate your time and effort in reviewing our study.

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Reviewer #3

__Evidence, reproducibility and clarity __

The manuscript by Trinidad et al. provides a deep mutational scanning (DMS) analysis to investigate the functional roles of residues from the EccD3 subunit of the Type VII ESX-3 secretion apparatus from M. smegmatis. A previously published structure of ESX-3 from M. smegmatis by the Rosenberg group (Oren Rosenberg is also an author of this paper) is used as basis for structural interpretation of the DMS data presented in this contribution. A shortcoming of the previous structure, despite being very rich in terms of structural details, was in the lack of hexameric pore formation, which has been established more recently by structures of the related ESX-5 system.

Technically, DMS is state-of-the art and a powerful approach to systematically scan residues of potential functional interest. Therefore, the data presented here, provide a remarkable repository for further interpretation in this contribution and by other future investigations. The experimental data have been deposited in Github enabling access by others in the future.

Overall, the paper would benefit from an improved overall organisation. I found in part hard to extract some of the main points from the way the data are presented. In essence, two separate screens were performed, the first one focusing on the EccD3 Ubl domain and adjacent linker regions and a second one on the EccD3 TM region. I think the paper could be better structured accordingly. Tables of residues with strong effects in iron-deficient and iron-sufficient media, together with their structural annotation, would facilitate extracting main messages from this manuscript. Without going too much in detail, there is also scope for improvement of most of the structural figures. More consistency in terms of color coding with the previous paper by Powileit et al. (2019) would also help navigation.

A potential weakness of the paper is in the limited scope of interpretation of the data in the context of the dimeric ESX-3 assembly, which is actually acknowledged by the authors. Computational AI-based methods should allow generating a complete pore model of ESX-3, which would allow interpretation of some of the data in a more functional relevant context. This would enhance the validity of the current interpretations presented.

We acknowledge the lack of a hexameric ESX-3 structure, and would love to base our analysis on such a structure. Unfortunately, experimentally purifying and determining such a structure is beyond the scope of this manuscript. While AI-based methods are certainly exciting and helpful to make sense of mutational data, they are not able to computationally predict such large structures. The AlphaFold3 server website is commonly used for these purposes and allows predictions of up to 5000 tokens (or amino acids). An ESX-3 hexamer would be composed of 6x EccB proteins (519 AA each), 6x EccC proteins (1326 AA each), 12x EccD proteins (476 AA each), and 6x EccE proteins (310 AA each). Together, this complex would be made up of 18,642 amino acids.

We tried using alphafold to predict an ESX-5 dimer complex, as well as reproduce the ESX-3 dimer complex, and were unable to produce these structures. Each ESX protomer is assembled correctly, as each protein within the complex makes appropriate contacts with each other. We see the EccD-dimers still form the membrane vestibule within each ESX complex. The issue is the ESX dimer complex has not assembled correctly: the EccC transmembrane helix 1 of a protomer should interact with the EccB transmembrane helix of the neighboring protomer; and, the N-terminus of EccB in one protomer should interact with the loop between the EccD transmembrane helices 10 and 11 in the neighboring protomer. Instead, Alphafold creates contacts along the EccD proteins from both complexes. We have included a "top-down" view of the ESX-5 dimer, where the periplasmic domains of EccB have been cleaved off for clarity.

A side view:

Here we have the ESX-3 dimer structure published by Poweleit et al. side-by-side with the ESX-3 dimer predicted by alphafold, visualized in Pmyol. The alphafold structure largely has each proteins' domains and folds properly predicted, including even the EccD3 dimer found in each ESX protomer. However, the protomers are not assembled into a dimer properly as compared to the purified ESX-3 dimer from PDB: 6umm. We included a "front" and "side view", as well as a "top down" view where the cytoplasmic domains have been hidden for visual clarity.

The use of full names and acronyms needs to be more consistent. As an example, the terms "ubiquitin-like" and ubiquitin-like (Ubl) and UBl are used in parallel throughout the manuscript. The percentages given in various places of the paper could be reduced to integers, as they generally relate to relatively small data sets. Please express numbers with a precision, reasonable matching expected statistical significance.

We apologize for the lack of consistency in how we referred to the ubiquitin-like domain. I originally wrote "ubiquitin-like (Ubl)" once per section (intro, results, discussion). I have edited these all to just "Ubl" after the introduction, except for figure and section titles. We have also reduced our percentages to integers.

Some of the DMS experiments have been repeated three-fold, which should be a minimal number to allow extracting statistical significance, other experiments have only been repeated two-fold. Could this be clarified, please?

We apologize for this oversight, and thank the reviewer for pointing this out. All experiments were done in triplicate, the exception being the site-directed mutant growth curves, which were performed in duplicate. We have repeated this experiment in triplicate in response to this point. As we repeated this experiment, mutant R134A dropped out due to technical reasons, and so we did not include it in the updated growth curves.

Specific comments on text and figures:

Figure 1: The EM densities shown considerably deviate from those that were shown in the original publication by Poweleit et al (2019). If there is an aim is to reinterpret the data this needs to be described in sufficient technical detail. There may be a case for this, in light of recent advances in computational AI-vased structural biology.

We acknowledge this may be confusing and we apologize for that, as the EM density I have shown in this manuscript uses the same map we used to create the one seen in the original publication Poweleit et al 2019. There are existing crystal structures of EccB1 and the ATPase domains of EccC1 that we used to create homology models of EccB3 and EccC3 using the structure-prediction software RaptorX for the 2019 publication. These homology models were then combined with a low resolution EM density to create the model seen in the 2019 eLife paper. I did not include those homology models in this manuscript, as I did not believe those predictions were relevant to this study. I wanted to include the highest resolution and thus most accurate depiction of our ESX-3 structure.

Introduction, statement "We made comparisons to a prior DMS on ubiquitin to increase signal-to-noise in our interpretation of the Ubl domain mutagenesis data." Could this be further explained please? I could not find anything in addition in the Methods section and elsewhere.

__ __We apologize for the confusion!

EccD3 Ubl domain and ubiquitin DMS dataset comparisons

To compare the DMS data of EccD3 Ubl with that of ubiquitin, we first identified homologous residues in each structure. This was achieved by aligning the EccD3 Ubl domain with ubiquitin (PDB: 1ubq) using PyMOL and assessing the positional correspondence of side chains (e.g., ubiquitin residue I3 aligned with EccD3 residue V12). Next, we referenced missense mutation datasets to calculate the average DMS score for each residue position in both proteins. We then generated a scatter plot to compare the average missense scores for ubiquitin and EccD3 Ubl using ggplot2. Data points were color-coded according to the functional roles assigned to ubiquitin, with residues forming the hydrophobic patch and core highlighted, while all other residues were represented in grey.

Description of "vestibule" as a core feature of the ESX-3 structure. As mentioned above, this is very much a result of the presented dimeric arrangement. In the context of a complete pore model, these features may change or even disappear.

While we would certainly welcome an ESX-3 hexamer model to definitively determine whether this feature persists, such a model is not currently available. However, the highly homologous ESX-5 complex retains these EccD vestibules, and there is no reason to believe these features would change or disappear. Therefore, based on our interpretation of the ESX-3 dimer and ESX-5 hexamer we believe that the EccD membrane vestibule is not just an artifact of the ESX-3 dimer complex.

It is possible that the reviewer misunderstood what we were referring to as the vestibule. We updated the language in the text to improve clarity. However the vestibule is not a consequence of ESX-3 complex dimer formation. It is an inherent feature of the ESX monomer complexes, where two EccD proteins dimerize to form said vestibule. Furthermore, there is no evidence to suggest that this feature would be lost in a hexameric state.

Structurally, the ESX-3 dimer consists of two ESX-3 monomer complexes, each containing one EccB, one EccC, one EccE, and two EccD proteins. Therefore, each ESX-3 monomer inherently includes an EccD dimer. The presence of the EccD dimer is not exclusive to the ESX-3 dimer but is a fundamental component of each ESX-3 complex. Similarly, the ESX-5 hexamer retains the EccD dimer within each ESX-5 complex, further supporting the idea that this structural feature is conserved.

Figure 2, panel B: Isn't right that "positive" and "negative" need to exchanged? Perhaps, there is something I misunderstood.

We apologize for the confusion, and appreciate the reviewer pointing out this inconsistency. We have updated the manuscript to correct this.

Figure 2, panel F: it is hard to extract the assignments from the overlaid curves.

We apologize for a lack of clarity in how this growth curve was presented. We have included labels at the end point to show where each sample is.

Figure 3, caption "from low (red) to white (tolerant)": for the sake of consistency, please either put the color in parentheses, or functional description. Does this statement relate to panel A or B? "All other residues are colored white". I can't see this.

We apologize for the inconsistency, and have updated this label. We hope we have clarified the fact that the entire structure is white except for the residues we colored red.

Results text "In contrast to ubiquitin, all hydrophobic core residues in the EccD3 Ubl domain are equally intolerant to charged residue swaps. Unsurprisingly, residues important for ubiquitin's specific degradation interactions are not sensitive to substitutions in the EccD3 Ubl domain." Does this mean that proper folding of Ubl is less critical for ESX_3 function? Please elaborate on this further.

We apologize for any confusion. Our data shows that residues which side chains extend into the hydrophobic core of the Ubl domain are intolerant to swaps to charge residues. We hypothesize these missense mutations disrupt this hydrophobic core, and lead to destabilization of this domain. These intolerant missense mutations each have negative Enrich2 scores, implying a loss of ESX-3 function, and that proper folding of the Ubl is critical for ESX-3 function. We have updated our text to clarify this point:

Unsurprisingly, residues important for ubiquitin function's specific interactions are not sensitive to substitutions in the EccD3 Ubl domain. There is no simple discernable preference within the Ubl domain to any side that maintains protein-protein interactions, implying that the scores are dominated by stability effects and that the Ubl domain must maintain a stable β-grasp fold for ESX-3 function.

Figure 4, panel C: the surface does not provide residue-specific information, hence this panel is not very informative.

We agree with the reviewer that Figure 4 panel C was not very informative, and so we have removed it from Figure 4 for the sake of brevity.

Results text "T148 extends out from transmembrane helix 1 into a hydrophobic pocket between transmembrane helices 1, 2, and 3." Could this please be illustrated in one of the structural presentations?

We have updated figure 5 to include a snapshot of this residue and the hydrophobic pocket it extends into, as panel E.

Results text, last paragraph, Figure 5C-D: interpretation of the experimental ESX-3 data based on ESX-5 models is problematic, without showing proof of conservation of relevant sequence/structural features. As mentioned above, I would encourage the authors to establish a hexameric ESX-3 model and interpret the data starting from there. Extrapolation of the interpretation of data to other ESX systems, including ESX-5, would expand the scope by generalization, which however would open another chapter. The ESX-5 structure does not explain e.g. why W227 when mutated is less sensitive to iron depletion as opposed to iron being present.

We do not believe we can use AI to predict a hexameric ESX-3 model. We will update our supplement to include a figure showing proof of conservation between the EccD3 and EccD5 sequences. We can superpose the ESX-3 dimer structure onto the ESX-5 hexamer structure, and see that this dimeric complex overlays quite well on top of an ESX-5 subcomplex. We can imagine this hexamer as a trimer of dimers, where three copies of this dimeric complex interact to form the hexamer. The superposition is not perfect and there are slight rearrangements to different helices to allow for hexamer formation, but these do not imply we cannot compare these two homologous structures.

We have included a new structure snapshot in Figure 5, where panel D is the ESX-3 dimer (PDB: 6umm) shown as a side and top-down view. This allows for a comparison with panel C, the snapshot of the ESX-5 complex (PDB: 7np7) where in two protomers the EccB, EccC, and EccD proteins are colored the same way as ESX-3, and the other ESX-5 protomers are colored white. Note that in this hexamer, EccE is missing. We see the EccD membrane vestibule is conserved in both structures.

Significance

Strength and Limitations: already assessed under "Evidence, reproducibility and clarity".

There is scope for further interpretation using experimental structural and modeling data. There is also scope for applying complementary assays for selected mutants, most likely within a lower throughput format.

Advance: The contribution demonstrates well the power of DMS for systematic screening, in the context of Type VII secretion. The main advance is in the raw data generated and deposited.

Audience: microbiology with a specific interest in secretion, structural biology

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #3

Evidence, reproducibility and clarity

The manuscript by Trinidad et al. provides a deep mutational scanning (DMS) analysis to investigate the functional roles of residues from the EccD3 subunit of the Type VII ESX-3 secretion apparatus from M. smegmatis. A previously published structure of ESX-3 from M. smegmatis by the Rosenberg group (Oren Rosenberg is also an author of this paper) is used as basis for structural interpretation of the DMS data presented in this contribution. A shortcoming of the previous structure, despite being very rich in terms of structural details, was in the lack of hexameric pore formation, which has been established more recently by structures of the related ESX-5 system.

Technically, DMS is state-of-the art and a powerful approach to systematically scan residues of potential functional interest. Therefore, the data presented here, provide a remarkable repository for further interpretation in this contribution and by other future investigations. The experimental data have been deposited in Github enabling access by others in the future.

Overall, the paper would benefit from an improved overall organisation. I found in part hard to extract some of the main points from the way the data are presented. In essence, two separate screens were performed, the first one focusing on the EccD3 Ubl domain and adjacent linker regions and a second one on the EccD3 TM region. I think the paper could be better structured accordingly. Tables of residues with strong effects in iron-deficient and iron-sufficient media, together with their structural annotation, would facilitate extracting main messages from this manuscript. Without going too much in detail, there is also scope for improvement of most of the structural figures. More consistency in terms of color coding with the previous paper by Powileit et al. (2019) would also help navigation.

A potential weakness of the paper is in the limited scope of interpretation of the data in the context of the dimeric ESX-3 assembly, which is actually acknowledged by the authors. Computational AI-based methods should allow generating a complete pore model of ESX-3, which would allow interpretation of some of the data in a more functional relevant context. This would enhance the validity of the current interpretations presented.

The use of full names and acronyms needs to be more consistent. As an example, the terms "ubiquitin-like" and ubiquitin-like (Ubl) and UBl are used in parallel throughout the manuscript. The percentages given in various places of the paper could be reduced to integers, as they generally relate to relatively small data sets. Please express numbers with a precision, reasonable matching expected statistical significance.

Some of the DMS experiments have been repeated three-fold, which should be a minimal number to allow extracting statistical significance, other experiments have only been repeated two-fold. Could this be clarified, please?

Specific comments on text and figures:

Figure 1: The EM densities shown considerably deviate from those that were shown in the original publication by Poweleit et al (2019). If there is an aim is to reinterpret the data this needs to be described in sufficient technical detail. There may be a case for this, in light of recent advances in computational AI-vased structural biology.

Introduction, statement "We made comparisons to a prior DMS on ubiquitin to increase signal-to-noise in our interpretation of the Ubl domain mutagenesis data." Could this be further explained please? I could not find anything in addition in the Methods section and elsewhere.

Description of "vestibule" as a core feature of the ESX-3 structure. As mentioned above, this is very much a result of the presented dimeric arrangement. In the context of a complete pore model, these features may change or even disappear.

Figure 2, panel B: Isn't right that "positive" and "negative" need to exchanged? Perhaps, there is something I misunderstood.

Figure 2, panel F: it is hard to extract the assignments from the overlaid curves.

Figure 3, caption "from low (red) to white (tolerant)": for the sake of consistency, please either put the color in parentheses, or functional description. Does this statement relate to panel A or B? "All other residues are colored white". I can't see this.

Results text "In contrast to ubiquitin, all hydrophobic core residues in the EccD3 Ubl domain are equally intolerant to charged residue swaps. Unsurprisingly, residues important for ubiquitin's specific degradation interactions are not sensitive to substitutions in the EccD3 Ubl domain." Does this mean that proper folding of Ubl is less critical for ESX_3 function? Please elaborate on this further.

Figure 4, panel C: the surface does not provide residue-specific information, hence this panel is not very informative.

Results text "T148 extends out from transmembrane helix 1 into a hydrophobic pocket between transmembrane helices 1, 2, and 3." Could this please be illustrated in one of the structural presentations?

Results text, last paragraph, Figure 5C-D: interpretation of the experimental ESX-3 data based on ESX-5 models is problematic, without showing proof of conservation of relevant sequence/structural features. As mentioned above, I would encourage the authors to establish a hexameric ESX-3 model and interpret the data starting from there. Extrapolation of the interpretation of data to other ESX systems, including ESX-5, would expand the scope by generalization, which however would open another chapter. The ESX-5 structure does not explain e.g. why W227 when mutated is less sensitive to iron depletion as opposed to iron being present.

Referee cross-commenting

I especially second the comments of referee #1, major comments, point 3 (statistical significance of the data). Addressing this point is crucial for the paper. Referee #2, significance section "The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system." I was considering making the same point but did not at the end. Of course, ultimately, it would be great if all components of ESX-3 could be analyzed they way it was done for the EccD3 component. However, I am afraid such exercise could become quite open ended. Already by now, there is some compromise on the depth of mechanistic interpretation in light of a large data set generated.

Significance

Strength and Limitations: already assessed under "Evidence, reproducibility and clarity".

There is scope for further interpretation using experimental structural and modeling data. There is also scope for applying complementary assays for selected mutants, most likely within a lower throughput format.

Advance: The contribution demonstrates well the power of DMS for systematic screening, in the context of Type VII secretion. The main advance is in the raw data generated and deposited.

Audience: microbiology with a specific interest in secretion, structural biology

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #2

Evidence, reproducibility and clarity

This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

1. The authors engineered an M. smegmatis knockout strain with deletions of fxbA and eccD3. Deletion of fxbA renders the exocholin iron uptake system non-functional, forcing the bacteria to rely on siderophore-mediated iron uptake under iron-limiting conditions. This process, in turn, depends on ESX-3 secretion activity, as PPE4, a known ESX-3 substrate, has been previously implicated in iron utilization in M. tuberculosis (Tufariello et al., 2016). This experimental setup links EccD3 function to a growth phenotype under iron-limiting conditions, as mutations impairing ESX-3 secretion disrupt iron utilization and mycobacterial growth.
2. By complementing the knockout strain with a library of EccD3 mutant variants, the authors systematically identify residues essential for protein-protein interactions within the ESX-3 core complex. Structural analysis corroborates the functional relevance of these residues, specifically those mediating interactions between EccD3 and other ESX-3 components, or those disrupting the hydrophobic core of the EccD3 ubiquitin-like (Ubl) domain.
3. Structural comparisons with the MycP5-bound ESX-5 complex allow the authors to predict residues within EccD3 that may interact with MycP3 during ESX-3 core complex assembly. Furthermore, comparisons with the ESX-5 hexamer suggest residues that may stabilize or drive oligomerization of the ESX-3 dimer into its putative hexameric state. These insights are significant and provide testable hypotheses for future studies.
4. The methodology is limited to ESX-3. The authors exploit the essentiality of ESX-3 for siderophore-dependent growth under iron-limiting conditions. However, this functional readout cannot be directly transferred to other ESX systems (ESX-1, ESX-2, ESX-4, ESX-5), which have distinct substrates, biological roles, and regulatory mechanisms.

Significance

This work provides valuable insights into EccD3 function, a core component of the ESX-3 secretion system. The strength of this study lies in the development of a robust functional assay for the systematic mapping of functionally relevant amino acids in EccD3. The approach could potentially be expanded to analyze other ESX-3 components but remains limited to the ESX-3 secretion system.

Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

Learn more at Review Commons

---

Referee #1

Evidence, reproducibility and clarity

This is a well-written manuscript that describes a thorough study of the functionality of individual residues of a central component of the ESX-3 type VII secretion system of Mycobacterium smegmatis, EccD3, in the essential role of this protein transport system in iron acquisition. Using the powerful and unbiased approach of deep mutational scanning (DMS), the authors assessed the impact of different mutations on a large number of residues of this component. This carefully executed research highlights the importance of hydrophobic residues at the center the ubiquitin-like domain, specific residues of the linker domain that connects this domain with the transmembrane domains and specific residues that connect EccD3 with the MycP3 component.

Major comments

Since the LOF effects in the iron-sufficient and iron-deficient condition differ less than expected, the differences of the DMS results between these two conditions should be better presented, explained and discussed:

1. The authors discuss: "Of the 270 LOF mutations seen in the iron-deficient condition, 37 (13.7%) were tolerant in the iron sufficient condition, and 39 (14.44%) had strong LOF effects but weak LOF effects in the iron sufficient condition." Do the authors mean that 39 (14.44%) had strong LOF effects in the iron-deficient condition, but weak LOF effects in the iron-sufficient condition. In turn, does this mean that the remaining mutants (71.9%) had similar LOF effects in the two conditions?
2. The diagonal shape of the scatter plot in Fig. 2C, which shows the correlation of the Enrich2 scores of all mutants in the two conditions, indicates that the growth of most mutants is affected similarly in these conditions, but in Fig. 2D lower graph, which shows only the Enrich2 scores of missense mutants, there are clear differences between the two conditions. How can this be explained?
3. Regarding the authors' explanation for the observed LOF effects in the permissive condition, "This speaks to the sensitivity of next-generation sequencing compared to the strong differences observed between conditions in phenotypic growth curves." But this sensitivity does not explain the observed large LOF effects but no growth difference in the permissive condition, unless the analysis is less quantitative than expected? Could it be that there is local iron depletion in this mixed culture, causing selection pressure even in the iron-sufficient condition? Moreover, the severity of the growth defect at the time of sampling, i.e., after 24 hours of growth, is unclear. Indeed, the growth curve in Fig. 1 shows that the growth of the double mutant in iron-deficient conditions is significantly impaired at that timepoint. In the growth curve in Fig. 2B (and also slightly in Fig. 2F), however, the growth defect is less pronounced: the double mutant has a similar OD600 as the WT strain, although the error bar is larger. Is this variability between replicates also seen in the DMS analysis? In general, no statistics are shown for the DMS analysis and there is no information on the significance of the observed LOF effects. In addition, the legend should explain how many replicates the DMS data are based on.

Minor comments

1. Line and page numbering should be added to the manuscript to facilitate the reviewing process.
2. "Knockout of the entire ESX-3 operon leads to inhibited M. smegmatis growth in a low-iron environment. When individual components of the ESX-3 system are deleted, growth is only available under impaired if the additional siderophore exochelin formyltransferase fxbA is also knocked out20." First, a reference should be added to the first sentence. Second, Siegrist et al. did not exactly show this. They showed that the fxbA/eccC3 double mutant grows slower that the fxbA single mutant. To my knowledge there is no publication showing that single esx-3 component mutants grow as WT in iron-deficient conditions. Do the authors have data demonstrating this? If true, it is surprising that mutating EccD3 has a milder phenotype compared the complete region deletion, as it is a crucial ESX-3 component.
3. Reference to Table 1, should be a reference to Table S1.
4. "Our heatmaps surprisingly reveal residues where substitutions are deleterious specifically in the iron-sufficient condition" Refer here to Fig. S2.
5. "In the iron-deficient condition, 6/551 (1.08%) missense mutations have a weak LOF effect, and 0 have strong effects." More clearly explain this refers to the residues of the transmembrane region.
6. "The MycP transmembrane helix has been hypothesized to be required for ESX complex specificity, targeting MycP to associate with the correct ESX homologue." I miss a reference here. And I thought that the transmembrane domain of MycP was required for complex stability not for specificity?
7. "....role in ESX function relating to EccB3 and EccC3. In the transmembrane, ..... we" Insert "region" after "transmembrane"

Significance

The study provides insight into individual residues of a central component of the ESX-3 type VII secretion system for functionality, which is useful for those studying the functioning of mycobacterial type VII secretion systems. Moreover, because this system is essential for the growth of the important pathogen M. tuberculosis, this knowledge can be used to design new anti-tuberculosis compounds that block the ESX-3 system. Although the results mainly confirm previous observations (highlighting specific residues important for the stability of ubiquitin and residues of other parts of EccD important for protein-protein interactions within the ESX-3/ESX-5 membrane complex), to my knowledge this is the first time DMS has been applied to mycobacteria. This study is therefore of interest to mycobacteriologists.

BIG 5

Taxonomic biases of the training data likely also play an important role here. The data sources aren't equal in how they've sampled the protein universe. This is especially apparent when comparing the structure and sequence databases. For example, certain taxa (e.g., humans) are overrepresented in the PDB, while others dominate UniRef.

It's not hard to imagine how the distribution differences in the t-SNE might reflect this, especially given the strong overlap of sequence-based methods with the UniRef samples. Do you know if the same is true for the structure-based methods? If you visualized where, say, PDB proteins are, would there be strong overlap?

Any ideas on how to disentangle approach from the taxonomic makeup of training data?

The author of this article is Adam Mustafa he is a banker and financial planner who gives advice to people who dont understand. He has no rewards for his work

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How can we make sure that using this technology can be used for ethical use and what are the regulations to use it

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This brings you to another IAEA article written by Joanne Liou. I think it's interesting that the IAEA published this article given that their entire brand is international collaboration regarding nuclear and atomic energy.