Henrik Gutzon Larsen and Anders Lund Hansen, “Gentrification—Gentle or Traumatic? Urban Renewal Policies and Socioeconomic Transformations in Copenhagen,” Urban Studies 45, no. 12 (2008): 2432, https://doi.org/10.1177/0042098008097101.

Thomas Aarup Due, Troels Friis, and Zenia Mølgaard Schmidt, “A New Cloudburst Management Plan for Copenhagen,” Topos Magazine, September 15, 2016, https://toposmagazine.com/enghaveparken-copenhagen-denmark/.

Thomas Aarup Due, Troels Friis, and Zenia Mølgaard Schmidt, “A New Cloudburst Management Plan for Copenhagen,” Topos Magazine, September 15, 2016, https://toposmagazine.com/enghaveparken-copenhagen-denmark/.

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

Learn more at Review Commons

Reply to the reviewers

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

Decker et al present an interesting study of the order of events in tau seeding in a biosensor HEK293 derived cell line. This a critical unresolved question in the field about subcellular compartment contributions to tau aggregation. This exploration of the nuclear tau aggregated deposition and seeding in HEK293T tau biosensor cells uses a variety of imaging-based methods. They show that nuclear aggregates only form in cells with cytosolic aggregates, nuclear aggregates cannot form in the absence of cytoplasmic tau aggregates. The original tau seeds do not persist. Also aggregates in the nucleus are dependent on VCP and SRRM2 for formation. The paper is limited in scope by use of only HEK239T cells and seem to overstate the generalizability of the findings to neuronal mechanisms of neurodegeneration. Please see to all tauopathies. In particular, the manuscript does not outline the overstatement of some of the conclusions.

Key Points to address: 1. The manuscript does not detail limitations of the study in the discussion. Please address the concern that HEK293T biosensor cells are not neurons. Especially in the clear animations showing the transformation from cytoplasmic to nuclear aggregates appears to require cell division and nuclear breakdown.

        We agree with the reviewer that a limitation of this manuscript is we only used HEK293 cells.  We have added text to emphasize this point in a "Limitations of this study" section at the end of the discussion.  However, as a starting point we believe understanding the cell biology of protein aggregation even in non-neuronal cells can be of value.

        Moreover, we clearly see cases of nuclear tau aggregates forming without cell division and nuclear breakdown (Figure 1 and Movies).  We have added text to emphasize this point since it is relevant to the potential formation of nuclear aggregates in neurons and the reviewer must have missed this point.

The introduction sets this up as Alzheimer's disease relevant but all studies are down with P301S tau which is a distinct and particularly aggressive form of tauopathy (FTLD-Tau). There is no amyloid beta component to any of these studies.

        This is a good point, and we have clarified our use of an FTLD model.  We do note that since seeds from post-mortem tissues in several different tauopathies can give nuclear tau aggregates (Sanders et al 2014), we anticipate that this process is general to multiple tauopathy contexts.

The study does not address the peculiar structure of P301S aggregates, which while disease relevant are clearly distinct from AD or most forms of familial FTLD. The authors should limit the generalizability of the findings to their particular form of tauopathy unless they plan to use multiple tau fibril conformations in their studies.

        The reviewer points out that we have only used one model system, and presumably only one tau fibril structure and therefore we should be cautious about the generality of our results.  This is a valid point, and we now point out this limitation in the manuscript.

The authors do not address the potential impact of fusing a natively unfolded protein like tau to a highly structured beta barrel like GFP. Please present this potential confound.

        We have added text pointing out that using GFP fusion proteins has the potential to alter tau function. We note this is an issue in the use of any fusion proteins, which have nevertheless proven useful tools.

1. Inhibition of VCP can cause proteinopathies in the absence of other seeding. For instance, familial mutations in human VCP can cause either tau or TDP-43 proteinopathy depending on the specific human disease causing mutation. Thus, critical controls are missing from figure 3. For instance, the consequence of VCP inhibition on unseeded biosensor cells is a missing control. Second all panels should evaluate TDP-43 aggregation to ascertain whether or not the secondary nuclear seeding involves TDP-43.

       In this comment, the reviewer asks that we show the effects of VCP inhibition on unseeded cells.  We will add this control, and we observe no appreciable tau aggregation with tau seeding.
   
       We will also assess whether TDP-43 aggregates in the HEK293 biosensor cells with or without VCP inhibition and/or tau seeding.  However, we note that it is clear from many studies that tau aggregation can occur independently of TDP-43 aggregation.
   

Minor concerns: A. Line 635 - In line 380, they discuss that aggregation of tau does not lead to perturbations in nuclear transport. In line 390, they discuss that aggregation of tau does not affect nuclear envelope integrity or nuclear import. However, in the discussion discusses that aggregation alters nuclear RNA export. These statements could use clarifying that protein export is not perturbed but RNA export and import may be.

We have clarified this point.

B. Line 564: "This observation suggests that tau aggregation in the cytoplasm may lead to increased expression of some RNAs." This could also be that cytoplasmic tau alters RNA export. These experiments don't differentiate between these options.

This comment is related to other comments about the relative abundance of specific RNAs in the nucleus or cytoplasm. We will add new data to the manuscript where we examine the numbers of specific RNAs in cells with and without nuclear or cytoplasmic tau aggregates. This will allow us to determine if there is simply a retention of RNAs in the nucleus or if, in some cases, there is also an increase in RNA levels.

1. In Figure 1, the authors show large aggregates overlapping the nucleus. It is unclear whether these aggregates have a portion both within and outside the nucleus or if they are deforming the nucleus and are wholly external to the nuclear compartment. Clarity on this issue is important. If the nucleus is deferment the observed aggregates seem reminiscent of aggresome formation. Please clarify. We assume the reviewer asks us to clarify why the large cytoplasmic tau aggregates are localized near the nucleus. Indeed, we suspect these are accumulating in aggresomes over time and have added this point to the text. Importantly, we do not observe a general defect in the integrity of the nucleus suggesting that even those these assemblies are close to the nucleus, they are not altering the nuclear envelope. We have added text to explain this issue.

Reviewer #1 (Significance (Required)):

Decker et al present an interesting study of the order of events in tau seeding in a biosensor HEK293 derived cell line. This a critical unresolved question in the field about subcellular compartment contributions to tau aggregation. This exploration of the nuclear tau aggregated deposition and seeding in HEK293T tau biosensor cells uses a variety of imaging-based methods. They show that nuclear aggregates only form in cells with cytosolic aggregates, nuclear aggregates cannot form in the absence of cytoplasmic tau aggregates.

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

The manuscript by Decker et al. examines the formation of nuclear tau aggregates and their functional consequences using a HEK293T tau biosensor system. The authors propose that nuclear tau aggregates arise through VCP dependent secondary seeding from cytoplasmic aggregates and that these nuclear aggregates impair RNA export. The study addresses an important and relatively unexplored aspect of tau biology. However, several conclusions extend beyond what the data directly supports, and several essential controls are missing. Major Comments - The introduction is generally clear and well organized. However, it would benefit from additional mechanistic context explaining how polyserine domains might promote tau aggregation and why this feature is biologically relevant.

        We have added text expanding what is known about how polyserine domains can increase tau fibrillization.

• The live cell imaging convincingly demonstrates the temporal sequence of cytoplasmic followed by nuclear aggregation. However, the manuscript lacks controls assessing whether nuclear aggregation correlates with higher tau expression levels. Such controls are necessary to exclude expression driven artifacts.

      We will add an analysis of the relationship between tau expression levels and cells with nuclear tau aggregates.  We observed that tau aggregates were independent of the tau expression levels, ruling out that nuclear tau aggregates are solely an artifact of extremely high tau expression levels.
  

• The authors conclude that nuclear envelope integrity is preserved, but only import assays were performed. To validate the sensitivity and specificity of the assay, export assays or positive controls for nuclear transport disruption are required.

      We had already shown that in cells with nuclear tau aggregates the nuclear export of mRNAs is perturbed. We will add additional analyses of whether nuclear export of proteins is altered.
  

• The Cy3/Cy5 seed experiments support the claim that exogenous seeds do not enter the nucleus. However, the conclusion that VCP generates secondary seeds is overstated. For example, the manuscript states: "VCP is responsible for the formation of secondary seeds..." (lines 418-439), yet the data demonstrate correlation rather than direct evidence of seed generation.

      This is a valid point. We have rephrased the manuscript to note that VCP is required for nuclear tau aggregation, possibly through the formation of secondary tau seeds, which is consistent with earlier work suggesting VCP can generate new tau seeds (Saha et al., 2023, Nature Communications; Batra et al., 2025, Molecular Neurodegeneration).
  

To substantiate this conclusion, the authors should: directly quantify seed abundance. The current interpretation assumes uniform cytoplasmic uptake of seeds but does not measure it; Include controls addressing VCP inhibitor specificity, as these compounds have pleiotropic effects (e.g., ER stress, proteostasis collapse). No data is provided on whether VCP inhibition alters tau ubiquitination, which could have major implications on tau aggregation.

              This comment addresses the issue of whether VCP can generate new seeds from tau fibers.  This is a conclusion already reached by prior work (Saha et al., 2023, Nature Communications; Batra et al., 2025, Molecular Neurodegeneration).  The point of our manuscript that this comment addresses is whether the nuclear aggregates are forming from a secondary seeding event, for which we have already provided several lines of evidence.  First, we have shown that nuclear aggregates only form after the formation of a prior cytoplasmic tau aggregate (Figure 1). Second, we have shown that nuclear aggregates do not contain exogenous seeds, while all cytoplasmic tau aggregates do (Figure 2).  Finally, we have shown that nuclear tau aggregates are dependent on VCP, which is consistent with the prior work showing VCP can generate tau seeds.  It is beyond the scope of this manuscript to determine in more detail how VCP affect tau aggregates generally.  For this reason, and since we have robustly demonstrated our conclusion, we have chosen not to pursue these additional suggested experiments.

• The authors observed increase in nuclear poly(A)+ RNA and specific transcripts. However, the current data do not distinguish between several possible mechanisms that may account for this increase, including impaired export, increased transcription, enhanced RNA stability, or nuclear retention due to speckle reorganization.

      To address this comment, we will quantify the levels of individual RNAs in the nucleus, cytoplasm and whole cell.  This will allow us to determine if there is an increase in RNA levels (possibly due to increased transcription or reduced decay), or if the increased nuclear RNA levels are due to block to mRNA export.  We will also assess transcription rate by measuring the intensity of the transcription loci, which will allow us to distinguish if any changes in mRNA levels are due to transcription or changes in RNA decay.
  

• The discussion occasionally overinterprets the data. Several statements should be reframed as hypotheses rather than conclusions:

• "VCP can generate tau seeds capable of additional seeding within a cell." (lines 572-594) This has not been directly demonstrated and should be softened accordingly.

We have done so. 2. Active import via SRRM2 is proposed, but no supporting data are presented. This should be clearly framed as a speculative model.

We have done so. 3. "Tau aggregates in the nucleus alter the function of nuclear speckles..." (lines 616-637). While plausible, this is not directly shown. Alternative explanations such as transcriptional upregulation or stress induced changes should be acknowledged.

We have altered this text to be more accurate. 4. The statement "It is possible that such nuclear aggregates could alter nuclear RNA export and contribute to pathology." (lines 637-655) is reasonable, but the authors should emphasize that nuclear tau aggregates are not consistently observed across tauopathies and that the HEK293T biosensor system may not fully recapitulate neuronal biology.

We agree with this point and have rephrased the text accordingly.

Reviewer #2 (Significance (Required)):

The manuscript by Decker et al. examines the formation of nuclear tau aggregates and their functional consequences using a HEK293T tau biosensor system. The authors propose that nuclear tau aggregates arise through VCP dependent secondary seeding from cytoplasmic aggregates and that these nuclear aggregates impair RNA export. The study addresses an important and relatively unexplored aspect of tau biology. However, several conclusions extend beyond what the data directly supports, and several essential controls are missing.

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

In their manuscript, Decker et al., investigate the relationship between cytoplasmic and nuclear tau aggregation using a HEK293T biosensor system and propose a multistep model in which cytoplasmic aggregates give rise to nuclear tau aggregates, potentially via VCP-dependent secondary seed generation and involvement of nuclear speckle components. The study further explores functional consequences of nuclear tau aggregation on RNA metabolism. Overall, the work is interesting and potentially impactful. The combination of live-cell imaging, seed-labeling strategies, genetic perturbations (SRRM2/PNN), and RNA imaging represents a thoughtful experimental approach. However, I have some minor concerns and feel the authors should address these - 1. Poly(A)+ FISH intensity is not a direct measure of export efficiency. The authors claim that tau aggregation within nuclear speckles interferes with nuclear export of RNA. It is highly possible that increased nuclear RNA levels observed could reflect altered transcription, stability, or stress responses rather than export defects alone. In the case of ATF3, a known stress responsive gene, increased nuclear signal could reflect transcriptional activation, not export defects. To prove that export is defective, the authors should at least measure total RNA levels (qPCR) in nuclear vs cytoplasmic fraction.

        To address this issue, we will quantify the levels of specific RNAs in the nucleus and cytoplasm by smFISH, which will allow us to clarify why there are more RNAs associated with nuclear speckles in the context of nuclear tau aggregates.

Though the authors have shown the proposed role of VCP in generating secondary seeds by using inhibitors, the authors should show genetic validation by using dominant-negative VCP.

        This experiment essentially asks us to examine the role of VCP in nuclear tau aggregation by an additional method. We will add experiments examining how nuclear tau aggregates form when VCP is knocked down by siRNAs.  We have chosen not to use dominant negative VCP mutants since their phenotype will be complicated with the endogenous VCP possibly remaining functional.

**Referees cross-commenting** *This session contains comments from different reviweers* Reviewer 3 I agree with the reviewers that additional controls and experiments would strengthen the VCP inhibition studies. However, I would like to clarify that the specific concern raised by Reviewer 1 (Key point number 4) regarding fusion of tau to GFP does not apply to this manuscript. In this study, the authors use tau conjugated to Cy3, a well established approach in the field that adds only approximately 1 kDa to the protein.

Reviewer 1 Apologies reviewer 3, but I respectfully disagree. Please look again at the legends for figs 1 through fig 5. All clearly delineate the use of tau biosensor cells using a YFP rather than GFP fusion protein with tau. i do agree we should correct my review to state YFP rather than GFP, but structurally the concern remains the same. Cy3 labelling, I believe is used to track the relatively short lived exogenous seeds.

Reviewer #3 (Significance (Required)):

The integration of approaches presented here, especially in connecting tau aggregation with nuclear speckle biology and RNA processing, will be of broad interest and offers important new mechanistic insights into tau pathology. I am an expert in Alzheimer's disease and integrated stress response.

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

In their manuscript, Decker et al., investigate the relationship between cytoplasmic and nuclear tau aggregation using a HEK293T biosensor system and propose a multistep model in which cytoplasmic aggregates give rise to nuclear tau aggregates, potentially via VCP-dependent secondary seed generation and involvement of nuclear speckle components. The study further explores functional consequences of nuclear tau aggregation on RNA metabolism. Overall, the work is interesting and potentially impactful. The combination of live-cell imaging, seed-labeling strategies, genetic perturbations (SRRM2/PNN), and RNA imaging represents a thoughtful experimental approach. However, I have some minor concerns and feel the authors should address these -

1. Poly(A)+ FISH intensity is not a direct measure of export efficiency. The authors claim that tau aggregation within nuclear speckles interferes with nuclear export of RNA. It is highly possible that increased nuclear RNA levels observed could reflect altered transcription, stability, or stress responses rather than export defects alone. In the case of ATF3, a known stress responsive gene, increased nuclear signal could reflect transcriptional activation, not export defects. To prove that export is defective, the authors should at least measure total RNA levels (qPCR) in nuclear vs cytoplasmic fraction.
2. Though the authors have shown the proposed role of VCP in generating secondary seeds by using inhibitors, the authors should show genetic validation by using dominant-negative VCP.

Referees cross-commenting

This session contains comments from different reviewers

Reviewer 3

I agree with the reviewers that additional controls and experiments would strengthen the VCP inhibition studies. However, I would like to clarify that the specific concern raised by Reviewer 1 (Key point number 4) regarding fusion of tau to GFP does not apply to this manuscript. In this study, the authors use tau conjugated to Cy3, a well established approach in the field that adds only approximately 1 kDa to the protein.

Reviewer 1

Apologies reviewer 3, but I respectfully disagree. Please look again at the legends for figs 1 through fig 5. All clearly delineate the use of tau biosensor cells using a YFP rather than GFP fusion protein with tau. i do agree we should correct my review to state YFP rather than GFP, but structurally the concern remains the same. Cy3 labelling, I believe is used to track the relatively short lived exogenous seeds.

Significance

The integration of approaches presented here, especially in connecting tau aggregation with nuclear speckle biology and RNA processing, will be of broad interest and offers important new mechanistic insights into tau pathology. I am an expert in Alzheimer's disease and integrated stress response.

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

Learn more at Review Commons

Referee #2

Evidence, reproducibility and clarity

The manuscript by Decker et al. examines the formation of nuclear tau aggregates and their functional consequences using a HEK293T tau biosensor system. The authors propose that nuclear tau aggregates arise through VCP dependent secondary seeding from cytoplasmic aggregates and that these nuclear aggregates impair RNA export. The study addresses an important and relatively unexplored aspect of tau biology. However, several conclusions extend beyond what the data directly supports, and several essential controls are missing.

Major Comments

• The introduction is generally clear and well organized. However, it would benefit from additional mechanistic context explaining how polyserine domains might promote tau aggregation and why this feature is biologically relevant.
• The live cell imaging convincingly demonstrates the temporal sequence of cytoplasmic followed by nuclear aggregation. However, the manuscript lacks controls assessing whether nuclear aggregation correlates with higher tau expression levels. Such controls are necessary to exclude expression driven artifacts.
• The authors conclude that nuclear envelope integrity is preserved, but only import assays were performed. To validate the sensitivity and specificity of the assay, export assays or positive controls for nuclear transport disruption are required.
• The Cy3/Cy5 seed experiments support the claim that exogenous seeds do not enter the nucleus. However, the conclusion that VCP generates secondary seeds is overstated. For example, the manuscript states: "VCP is responsible for the formation of secondary seeds..." (lines 418-439), yet the data demonstrate correlation rather than direct evidence of seed generation. To substantiate this conclusion, the authors should: directly quantify seed abundance. The current interpretation assumes uniform cytoplasmic uptake of seeds but does not measure it; Include controls addressing VCP inhibitor specificity, as these compounds have pleiotropic effects (e.g., ER stress, proteostasis collapse). No data is provided on whether VCP inhibition alters tau ubiquitination, which could have major implications on tau aggregation.
• The authors observed increase in nuclear poly(A)+ RNA and specific transcripts. However, the current data do not distinguish between several possible mechanisms that may account for this increase, including impaired export, increased transcription, enhanced RNA stability, or nuclear retention due to speckle reorganization.

• The discussion occasionally overinterprets the data. Several statements should be reframed as hypotheses rather than conclusions:

• "VCP can generate tau seeds capable of additional seeding within a cell." (lines 572-594) This has not been directly demonstrated and should be softened accordingly.

• Active import via SRRM2 is proposed, but no supporting data are presented. This should be clearly framed as a speculative model.
• "Tau aggregates in the nucleus alter the function of nuclear speckles..." (lines 616-637). While plausible, this is not directly shown. Alternative explanations such as transcriptional upregulation or stress induced changes should be acknowledged.
• The statement "It is possible that such nuclear aggregates could alter nuclear RNA export and contribute to pathology." (lines 637-655) is reasonable, but the authors should emphasize that nuclear tau aggregates are not consistently observed across tauopathies and that the HEK293T biosensor system may not fully recapitulate neuronal biology.

Significance

The manuscript by Decker et al. examines the formation of nuclear tau aggregates and their functional consequences using a HEK293T tau biosensor system. The authors propose that nuclear tau aggregates arise through VCP dependent secondary seeding from cytoplasmic aggregates and that these nuclear aggregates impair RNA export. The study addresses an important and relatively unexplored aspect of tau biology. However, several conclusions extend beyond what the data directly supports, and several essential controls are missing.

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

Decker et al present an interesting study of the order of events in tau seeding in a biosensor HEK293 derived cell line. This a critical unresolved question in the field about subcellular compartment contributions to tau aggregation. This exploration of the nuclear tau aggregated deposition and seeding in HEK293T tau biosensor cells uses a variety of imaging-based methods. They show that nuclear aggregates only form in cells with cytosolic aggregates, nuclear aggregates cannot form in the absence of cytoplasmic tau aggregates. The original tau seeds do not persist. Also aggregates in the nucleus are dependent on VCP and SRRM2 for formation. The paper is limited in scope by use of only HEK239T cells and seem to overstate the generalizability of the findings to neuronal mechanisms of neurodegeneration. Please see to all tauopathies. In particular, the manuscript does not outline the overstatement of some of the conclusions.

Key Points to address:

1. The manuscript does not detail limitations of the study in the discussion. Please address the concern that HEK293T biosensor cells are not neurons. Especially in the clear animations showing the transformation from cytoplasmic to nuclear aggregates appears to require cell division and nuclear breakdown.
2. The introduction sets this up as Alzheimer's disease relevant but all studies are down with P301S tau which is a distinct and particularly aggressive form of tauopathy (FTLD-Tau). There is no amyloid beta component to any of these studies.
3. The study does not address the peculiar structure of P301S aggregates, which while disease relevant are clearly distinct from AD or most forms of familial FTLD. The authors should limit the generalizability of the findings to their particular form of tauopathy unless they plan to use multiple tau fibril conformations in their studies.
4. The authors do not address the potential impact of fusing a natively unfolded protein like tau to a highly structured beta barrel like GFP. Please present this potential confound.
5. Inhibition of VCP can cause proteinopathies in the absence of other seeding. For instance, familial mutations in human VCP can cause either tau or TDP-43 proteinopathy depending on the specific human disease causing mutation. Thus, critical controls are missing from figure 3. For instance, the consequence of VCP inhibition on unseeded biosensor cells is a missing control. Second all panels should evaluate TDP-43 aggregation to ascertain whether or not the secondary nuclear seeding involves TDP-43.

Minor concerns:

A. Line 635 - In line 380, they discuss that aggregation of tau does not lead to perturbations in nuclear transport. In line 390, they discuss that aggregation of tau does not affect nuclear envelope integrity or nuclear import. However, in the discussion discusses that aggregation alters nuclear RNA export. These statements could use clarifying that protein export is not perturbed but RNA export and import may be.

B. Line 564: "This observation suggests that tau aggregation in the cytoplasm may lead to increased expression of some RNAs." This could also be that cytoplasmic tau alters RNA export. These experiments don't differentiate between these options.

C. In Figure 1, the authors show large aggregates overlapping the nucleus. It is unclear whether these aggregates have a portion both within and outside the nucleus or if they are deforming the nucleus and are wholly external to the nuclear compartment. Clarity on this issue is important. If the nucleus is deferment the observed aggregates seem reminiscent of aggresome formation. Please clarify.

Significance

Decker et al present an interesting study of the order of events in tau seeding in a biosensor HEK293 derived cell line. This a critical unresolved question in the field about subcellular compartment contributions to tau aggregation. This exploration of the nuclear tau aggregated deposition and seeding in HEK293T tau biosensor cells uses a variety of imaging-based methods. They show that nuclear aggregates only form in cells with cytosolic aggregates, nuclear aggregates cannot form in the absence of cytoplasmic tau aggregates.

Notable

Nice concept

Nicely put

improvement plants for dams that contribute to the watershed

proposed goals

Testing how to write a comment.

Now that her husband is dead all the men think they can take advantage of her

They think she hurt him but she would never do that to anybody

Janie feels overwhelmed when she sees the fields and the happy people.

They got jealousy in them

Tea cake is caught with another girl and Janie is jealous and scared to lose him and they argue

Janie was scared that Nunkie would take tea cake and she would lose one of the only people that actually somewhat cared for her and loved her

Jealousy!!!!

Chapter 15 Janie becomes jealous of Nunkie, a girl who flirts with Tea Cake. When Janie finds them alone in the fields, she gets angry and confronts him. They argue and fight, but afterward they make up, and Tea Cake reassures Janie that he only loves her.

The happy couples all reminded her of Tea Cake & it made her feel upset and sad after what he had done to her

A worker named Nunkie is introduced and flirts and touches tea cake constantly in the Field's

Janie was jealous of nunkie. Janie realized that nunkie and tea cake have disappeared, she asked where they are and then follows them into the cane fields.

People fish and wait for the crops to grow. Tea Cake helps Janie pick beans, but there isn’t much work yet. Many people arriving are poor and looking for work, not spending money

Jaimie and tea cake argued over tea cake relationship with nukie

This is showing Janie the difference of her new relationship to her previous

Janie sees this as her own place where she can start over without anyone judging her, like how she wanted in chapter 12

This is showing Janie the difference of her new relationship to her previous

Chapter 14 Janie and Tea Cake move to the Everglades, where they work, hunt, and enjoy life together. Janie joins him in the fields, and they grow closer while living happily in a lively community.

She is stunned to have seen such a wealthy place that she had never seen before.

Unlike Janie’s previous marriages her and tea cake work together in the fields and tea cake shows Janie how to shoot

Mrs turner doesn’t like dark skinned people.

She is saying that with real gods sacrifices have to be made and offer like animals or other things up and with half gods you worship with food.

Mrs turner dislikes dark skinned people and tries to make Janie leave tea cake

Mrs.tunner dislike dark people

Mrs. Turner is a racist against her own race.

This shows their is conflict within the community

They’re not going to pay to eat at the white women’s restaurant because the white women doesn't like them because they’re black

The harvest season had ended and many workers left but Janie and tea cake stayed to save beans and now had free time to notice new things around them.

This suggest a character who is unfazed by something that might concern others, and find that their way of talking is a source of amusement

Janie and Tea Cake stay at the “the muck” after the harvest. Janie befriends Mrs. Turner a light skinned black woman who hates darker skinned people. Turner tries to convince Janie to leave Tea Cake for her brother that appalls Janie and infuriates Tea Cake.

Chapter 16 Mrs.turner is introduced who doesn’t like black people meaning she doesn’t approve of Janie marriage

Janie become friends with Mrs turner but she is racist and teacake does not like her

Mrs. Turner is racist and thinks she is better since she is a light skin instead f a darker skin. She doesn’t really like how Janie and Tea Cake are together.

She’s racist to her own race and only prefers whites and would rather shop at there own stores and places of white folk.

She is racist to her own kinda.

We meet Mrs turner and we see that she’s racist but Janie looks past it since she wants to make friends and we see that there is judgment between them.

Mrs. Turner is racist and doesn’t want anything to do with African Americans because she is a little more lighter than them.

Hurricane happens and time to reflect is occurring

Janie decided to stay because the people wanted to form new community.

She is very racist to her own race

What strikes me most in this chapter is how Mrs. Turner worships light skin and European features almost like a religion and yet she completely dismisses her own husband as beneath her. She wants Janie to leave Tea Cake for her brother not out of love, but out of a twisted hierarchy of color. Hurston makes it clear that internalized racism isn't just self-hatred, it's a weapon turned outward to destroy other people's happiness too.

Chapter 16 After the season ends, Janie and Tea Cake stay on the muck. Janie befriends Mrs. Turner, who looks down on Black people and disapproves of Janie’s marriage. Tea Cake dislikes her and warns Janie. Despite this tension, Janie and Tea Cake continue enjoying their life together until the next season begins.

Mrs. Turner is racist and doesn’t want anything to do with African Americans because she is a little more lighter than them.

Mrs. Turner didn’t like tea cake very much and was very racist towards him. She doesnt want tea cake to be with Janie but of course they dont care what she thinks. Mrs. Turner loves Janie’s company.

Janie and Tea Cake’s love doesn’t get affected by Mrs. turners hate and attempts to separate the two.

Mrs. Turner is very racist towards her own race, it seems that she wants to be white

Janie meets Mrs. Turner, who doesn’t like Black people and doesn’t approve of Tea Cake. Tea Cake overhears her and gets mad because she’s trying to break them up

Tea cake and Janie stay on the “muck” after the harvest where they interact with their neighbors who show internalized racism

In chapter 16, the character of Mrs. Turner is introduced. Janie encounters Mrs turner, whose colorist views and preference for lighter skinned Black people challenge janie’s understanding of identity and community.

Dimmers in the summer

A lot of people were busy but everything was now done

Mrs.Turner is racist to her own race.

A big hurricane hits that no one takes seriously

Janie was thinking that if she was more like the white people, they would think she’s nobel

this is the karpathy method overview page

The marriage is failing and Janie is getting tired of it

The text is a complaint about being constantly blamed or criticized by an older person

Joe is being controlling towards Janie

The text describes a humorous exaggeration about a very skinny mule

Joe bought Janie a gift and talked about his plans for the town

Hicks is expressing admiration for someone’s wife and desires to acquire a similar one

He is comparing her to his first wife because he wants more from her

It’s implying that the person they are addressing is too young and immature to be married and is making a suggestive comment about their desires

Janies marriage to Logan, her disillusionment with marriage and nanny’s influence

Janies early life, pear tree dreams and arranged married to Logan

Tea cake be expressing his emotions

The town dont like her cause she is with a younger man now

uses a personal story frame the history

Travel cost overview will be an additional "view" per employer . This includes some $ items - will discuss after vacation. I do t think this needs any automation its just needs to be visible for billing purpose

PlentyONE ist sowohl Mutlichannel als auch ERP. Alle anderen Tools bieten ebenfalls Anbindung an Marktplätze und die Zentralisierung aller Aufträge an, das sollte deutlciher werden

Vertriebskanäle fehlen in dieser Betrachtung

fokus auf SMB

Fokus auf "deutsch" wird überlesen und man denkt es hat die besten integrtionen

warum Testsieger? Wirkt aus meiner Sicht unseriös wenn keine Infos zum Test und den Kriterien gegeben werden. Tools werden nicht anhand definierter Kriterien übersichtlich verglichen. Warum hat PlentyONE gewonnen?

vorsicht auch hier: modulare Preise

Wording s.o.

eben nicht mehr, JTL zieht Preise massiv an

modularer Aufbau -> Kosten steigen schnell

Beleg?

streichen

3

falsch, s.o.

nicht so kombinierbar, Zahl rausnehmen

Falschaussage. Auszeichnung als Bestes Tool für Multichannel und Marketplace Tools bei den E-Commerce Germany Awards.

Vertriebskanälen weltweit

PlentyONE Shop

rausnehmen, Daten sind 3 Jahre alt

nicht glaubhaft

bis zu 85 % weniger Aufwand im E-Commerce

bis zu 112% Umsatzsteigerung

finde die Tabelle sehr knapp gehalten, eine Systemauswahl sollte mehrere Kriterien berücksichtigen um wirklich seriös zu wirken. Beispiel: https://www.plentyone.com/de/jtl-alternative

Wording: Einzelunternehmen oder KMU. Einzelhändler können ja alle sein

Überverkäufe sind nicht das einzige Problem was auftritt und durch eine E-Commerce Plattform gelöst werden kann. Insellösungen: Viele verschiedene Systeme (ERP, Shop, Middleware, Marktplätze) müssen manuell synchronisiert werden Medienbrüche: Daten müssen zwischen Systemen übertragen werden, was zu Fehlern und Inkonsistenzen führt Fehlende Automatisierung: Prozesse wie Bestandsabgleich, Preissteuerung und Auftragsabwicklung müssen manuell durchgeführt werden Überverkäufe: Ohne schnellen Bestandsabgleich zwischen Marktplätzen kommt es zu Überverkäufen und Kundenunzufriedenheit

Systemen

Shopify ist der eigene Shop -> Dopplung

Very interested in how this is done. Metacognition needs to be foregrounded in learning with AI, so learners can determine if the tech is helping and how it is doing so.

Isn't sentence crafting the core skill? This feels like the wrong pace to build a shortcut.

For Maz and Barth

Could the authors comment on how to interpret Figure 2. Specifically, how is the data points in the barplot is defined? Why Claude Code (Haiku 4.5 Sonnet 4.6) doesn't have data points in the bars.

Turner didnt even try to stop the fight even when his wife was being trampled over

Tea Cake shares how Mrs.Turner really is and how she isn’t a normal person but thinks differently than others usually do.

Tea cake disliked her because she was racist in ch 16 but now say that turner is a nicest person there?

Chapter 17 shows that Tea cake hits Janie because he feels jealous. A big fight happens at Mrs turner’s restaurant, which causes her to get upset and move a yaw from the town.

link doesn't work

This wikipedia article on memes explains that it's an idea, behavior, image, phrase, or cultural symbol that spreads from person to person through imitation. This connects ultimately to the chapter because memes are what go viral and people mimic off the internet. The term was created by Richard Dawkins based on the Greek word mimeme meaning something imitated. Memes to evolution, they can be copied, changed, and passed on with any access to the internet nowadays.

Chapter 20 shows Janie returns to Eatonville after being a migrant worker with tea cake. She tells Pheoby her story, saying she has been to the horizon and back. She find peace in knowing that tea cake will forever live in her heart.

Should be Figure 1.7. Should a source be cited or does the source at the bottom of the graphic (office of the governor) suffice?

This part of the chapter stood out to me and how the comparison between social media virality connects with evolution. Viral content being replication, variation, and selection makes it a lot more dystopian to look at online trends and most of all feel less random. With reposts, quote tweets, or edits of content, people are creating a new version that can spread differently from the original, but still have viral potential. This made me think about how people aren't just passive viewers on social media but we actively shape what becomes popular with a click of the finger to like it or repost the content. The chapter's ethical point being that platforms and algorithms influence which content gets selected but also what the platform chooses to amplify.

Chapter 19 shows that tea cake becomes ill and Janie takes him to the doctor and the doctor diagnosed tea cake with rabies. Tea cake becomes violent and tries to shoot Janie and Janie shoots him in self-defense.

eLife Assessment

This important study introduces an innovative synthetic nanobody approach to probe the function of the bacterial SMC complex. The work is a compelling example of the potential of this approach. The authors generate protein chimeras to provide convincing evidence that their identified nanobodies target the coiled-coil region of the SMC subunit, demonstrating that this region is critical for SMC function in vivo. Overall, the work is significant for the fields of genome organisation, SMC protein biology, synthetic biology, and bacterial cell biology.

[Editors' note: this paper was reviewed by Review Commons.]

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the major comments raised in the previous round of review. Public Reviews below refer to the version submitted to Review Commons.]

Summary:

Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

Major comments:

(1) Lack of direct in vitro binding measurements:

The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And do the sybodies affect the interaction of ScpAB with SMC?

It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

(2) Many modes of sybody binding to Smc are plausible

The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

(3) Sybody expression in vivo

Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

(4) Sybodies should phenocopy ATP hydrolysis mutant of Smc

The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

Significance:

Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins has been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

Reviewer #2 (Public review):

Summary:

Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions.

The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB.

Significance:

The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

Reviewer #3 (Public review):

Summary:

Gosselin et al. use the sybody technology to study effects of in vivo inhibition of the Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the "transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc "neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.

The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

My main criticism is that the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only identify sybodies that bind to a rather small part of the large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

Fig. 2B: is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the "counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

Testing binding sites of sybodies to the SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

Significance:

The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

Author response:

The following is the authors’ response to the original reviews

Public Reviews:

Reviewer #1 (Public review):

Summary:

Gosselin et al., develop a method to target protein activity using synthetic single-domain nanobodies (sybodies). They screen a library of sybodies using ribosome/ phage display generated against bacillus Smc-ScpAB complex. Specifically, they use an ATP hydrolysis deficient mutant of SMC so as to identify sybodies that will potentially disrupt Smc-ScpAB activity. They next screen their library in vivo, using growth defects in rich media as a read-out for Smc activity perturbation. They identify 14 sybodies that mirror smc deletion phenotype including defective growth in fast-growth conditions, as well as chromosome segregation defects. The authors use a clever approach by making chimeras between bacillus and S. pnuemoniae Smc to narrow-down to specific regions within the bacillus Smc coiled-coil that are likely targets of the sybodies. Using ATPase assays, they find that the sybodies either impede DNA-stimulated ATP hydrolysis or hyperactivate ATP hydrolysis (even in the absence of DNA). The authors propose that the sybodies may likely be locking Smc-ScpAB in the "closed" or "open" state via interaction with the specific coiled-coil region on Smc. I have a few comments that the authors should consider:

Major comments:

(1) Lack of direct in vitro binding measurements:

The authors do not provide measurements of sybody affinities, binding/ unbinding kinetics, stoichiometries with respect to Smc-ScpAB. Additionally, do the sybodies preferentially interact with Smc in ATP/ DNA-bound state? And do the sybodies affect the interaction of ScpAB with SMC?

It is understandable that such measurements for 14 sybodies is challenging, and not essential for this study. Nonetheless, it is informative to have biochemical characterization of sybody interaction with the Smc-ScpAB complex for at least 1-2 candidate sybodies described here.

We agree with the reviewer that adding such data would be reassuring and that obtaining solid data using purified components is not trivial, even for a smaller selection of sybodies. We have now incorporated ELISA data as new Table S1, which shows that most sybodies support clear binding to Smc-ScpAB. Curiously, while (only) some sybodies show a clear preference for ATP-bound or unbound Smc, this is not a strong predictor of the strength of phenotype observed in vivo. We have also attempted to characterize the binding of Smc to sybodies by other methods including pull-downs, cross-linking, and by biophysical methods (GCI). However, we prefer to not include these data as the outcomes are not clear due to inconsistencies in the behaviour of purified sybodies.

(2) Many modes of sybody binding to Smc are plausible

The authors provide an elaborate discussion of sybodies locking the Smc-ScpAB complex in open/ closed states. However, in the absence of structural support, the mechanistic inferences may need to be tempered. For example, is it also not possible for the sybodies to bind the inner interface of the coiled-coil, resulting in steric hinderance to coiled-coil interactions. It is also possible that sybody interaction disrupts ScpAB interaction (as data ruling this possibility out has not been provided). Thus, other potential mechanisms would be worth considering/ discussing. In this direction, did AlphaFold reveal any potential insights into putative binding locations?

We have attempted to map the binding by structure prediction, however, so far, even the latest versions of AlphaFold are not able to clearly delineate the binding interface that we have confidently identified by the mapping using chimeric proteins. Indeed, many ways of binding are possible, including disruption of ScpAB interaction. However, since the mapped binding sites are located on the SMC coiled coils, the later scenario seems unlikely and would be an indirect consequence of altered coiled coil configuration, consistent with our current interpretation.

(3) Sybody expression in vivo

Have the authors estimated sybody expression in vivo? Are they all expressed to similar levels?

We have tagged selected sybodies with gfp and performed live cell imaging. This shows that sybodies without strong phenotypes are similarly expressed at least at low inducer concentration. Moreover, many sybodies localize as foci in the cell presumably by binding to Smc complexes loaded onto the chromosome at ParB/parS sites. We have included example data in the revised version of the manuscript as Figure S4 and Figure S5. Notably, a sybody (Sb007) with a weak growth phenotype shows focal localization at low inducer concentration and high expression levels when fully induced, comparable to sybodies with strong phenotypes. Altogether, this suggests that the lack of phenotype is not due to absence of sybody expression or localization.

(4) Sybodies should phenocopy ATP hydrolysis mutant of Smc

The sybodies were screened against an ATP hydrolysis deficient mutant of Smc, with the rationale that these sybodies would interfere this step of the Smc duty cycle. Does the expression of the sybodies in vivo phenocopy the ATP hydrolysis deficient mutant of Smc? Could the authors consider any phenotypic read-outs that can indicate whether the sybody action results in an smc-null effect or specifically an ATP hydrolysis deficient effect?

As alluded to above, we think that our selection gave rise to sybodies that bind various, possibly multiple Smc conformations. Consistent with this idea, the phenotypes of sybody expression are similar to null mutant rather than the ATP-hydrolysis defective EQ mutant, which display even more severe growth phenotypes in B. subtilis. To highlight this point, we have added the following notes to the text:

“These conditions favour ATP-engaged particles alongside the typically predominant ATP-disengaged rod-shaped state.”

“ELISA data revealed that nearly all clones bind purified Smc-ScpAB (Table 1). However, the ELISA signals of only few Sybodies showed clear dependence on the presence or absence of ATP and DNA (Table S1).”

Significance:

Overall, this is an impressive study that uses an elegant strategy to find inhibitors of protein activity in vivo. The manuscript is clearly written and the experiments are logical and well-designed. The findings from the study will be significant to the broad field of genome biology, synthetic biology and also SMC biology. Specifically, the coiled coil domain of SMC proteins have been proposed to be of high functional value. The authors have elegantly identified key coiled-coil regions that may be important for function, and parallelly exhibited potential of the use of synthetic sybody/designed binders for inhibition of protein activity.

Reviewer #2 (Public review):

Summary:

Structural Maintenance of Chromosome proteins (SMCs), a family of proteins found in almost all organisms, are organizers of DNA. They accomplish this by a process known as loop extrusion, wherein double-stranded DNA is actively reeled in and extruded into loops. Although SMCs are known to have several DNA binding regions, the exact mechanism by which they facilitate loop extrusion is not understood but is believed to entail large conformational changes. There are currently several models for loop extrusion, including one wherein the coiled coil (CC) arms open, but there is a lack of insightful experimentation and analysis to confirm any of these models. The work presented aims to provide much-needed new tools to investigate these questions: conformation-selective sybodies (synthetic nanobodies) that are likely to alter the CC opening and closing reactions.

The authors produced, isolated, and expressed sybodies that specifically bound to Bacillus subtilis Smc-ScpAB. Using chimeric Smc constructs, where the coiled coils were partly replaced with the corresponding sequences from Streptococcus pneumoniae, the authors revealed that the isolated sybodies all targeted the same 4N CC element of the Smc arms. This region is likely disrupted by the sybodies either by stopping the arms from opening (correctly) or forcing them to stay open (enough). Disrupting these functional elements is suggested to cause the Smc-dependent chromosome organization lethal phenotype, implying that arm opening and closing is a key regulatory feature of bacterial Smc-ScpAB.

Significance:

The authors present a new method for trapping bacterial Smc's in certain conformations using synthetic antibodies. Using these antibodies, they have pinpointed the (previously suggested) 4N region of the coiled coils as an essential site for the opening and closing of the Smc coiled coil arms and that hindering these reactions blocks Smc-driven chromosomal organization. The work has important implications for how we might elucidate the mechanism of DNA loop extrusion by SMC complexes.

Reviewer #3 (Public review):

Summary:

Gosselin et al. use the sybody technology to study effects of in vivo inhibition of the Bacillus subtilis SMC complex. Smc proteins are central DNA binding elements of several complexes that are vital for chromosome dynamics in almost all organisms. Sybodies are selected from three different libraries of the single domain antibodies, using the "transition state" mutant Smc. They identify 14 such mutant sybodies that are lethal when expressed in vivo, because they prevent proper function of Smc. The authors present evidence suggesting that all obtained sybodies bind to a coiled-coil region close to the Smc "neck", and thereby interfere with the Smc activity cycle, as evidenced by defective ATPase activity when Smc is bound to DNA.

The study is well done and presented and shows that the strategy is very potent in finding a means to quickly turn off a protein's function in vivo, much quicker than depleting the protein.

The authors also draw conclusions on the molecular mode of action of the SMC complex. The provide a number of suggestive experiments, but in my view mostly indirect evidence for such mechanism.

My main criticism is that the authors have used a single - and catalytically trapped form of SMC. They speculate why they only obtain sybodies from one library, and then only identify sybodies that bind to a rather small part of the large Smc protein. While the approach is definitely valuable, it is biassed towards sybodies that bind to Smc in a quite special way, it seems. Using wild type Smc would be interesting, to make more robust statements about the action of sybodies potentially binding to different parts of Smc.

The reviewer reports (Rev. #1 and Rev. #3) made us realize that the manuscript text was misleading on the this point. Although we used the purified ATP hydrolysis–deficient Smc protein for sybody isolation, this is not expected to restrict the selection to a specific conformation. As described in detail in Vazquez-Nunez et al. (Figure 5), this mutant displays the ATP-engaged conformation only in a smaller fraction of complexes (~25% in the presence of ATP and DNA), consistent with prior in vivo observations reported by Diebold-Durand et al. (Figure 5). Rather than limiting the selection to a particular configuration, our aim was to reduce the prevalence of the predominant rod state in order to broaden the range of conformations represented during sybody selection. Consistent with this interpretation, only a small number of isolated sybodies show strong conformation-specific binding in the presence or absence of ATP/DNA, as observed by ELISA (now included in the manuscript). Notably, the effect size of ATP/DNA on ELISA signals was not a strong predictor to the strength of phenotypes observed in vivo. The text has been revised accordingly. See line 84 and line 92.

We are thus quite confident based prior work (and on the now included ELISA data) that the Smc ATPase mutation did not strongly bias the selection in one way or another. The surprising bias towards coiled coil binding sites has likely other explanations, as they likely form a preferred epitope recognized by sybodies from the loop library.

Line 105: Alternatively, the other libraries did not produce good binders or these sybodies were 106 not stably expressed in B. subtilis. This could be tested using Western blotting - I am assuming sybody antibodies are commercially available. However, this test is not important for the overall study, it would just clarify a minor point.

While there are antibody fragments available to augment the size of sybodies (PMID: 40108246), these recognize 3D-epitopes and are thus not suited for Western blotting. We did not follow up on the negative results of two of the three libraries but would like to point out again that there are several biases that likely emerge for the same reason (bias to library, bias to coiled coil binding site). If correct, then sybodies are likely ineffective in inactivating Smc in B. subtilis, with the notable exceptions of the sybodies that we have isolated and characterized in this manuscript. We have added this notion to the manuscript.

Fig. 2B: is odd to count Spo0J foci per cells, as it is clear from the images that several origins must be present within the fluorescent foci. I am fine with the "counting" method, as the images show there is a clear segregation defect when sybodies are expressed, I believe the authors should state, though, that this is not a replication block, but failure to segregate origins.

We agree that this is an important point. We have added the following statement to clarify this point: “These elongated cells are known to harbour expanded nucleoids, consistent with delayed oriC separation rather than delayed DNA replication”

Testing binding sites of sybodies to the SMC complex is done in an indirect manner, by using chimeric Smc constructs. I am surprised why the authors have not used in vitro crosslinking: the authors can purify Smc, and mass spectrometry analyses would identify sites where sybodies are crosslinked to Smc. Again, I am fine with the indirect method, but the authors make quite concrete statements on binding based on non-inhibition of chimeric Smc; I can see alternative explanations why a chimera may not be targeted.

We have made several attempts of testing direct binding with mixed outcomes and decided to not include those results in the light of the stronger and more relevant in vivo mapping. However, we have added ELISA results (new Table S1) that support a direct interaction.

Smc-disrupting sybodies affect the ATPase activity in one of two ways. Again, rather indirect experiments. This leads to the point Revealing Smc arm dynamics through synthetic binders in the discussion. The authors are quite careful in stating that their experiments are suggestive for a certain mode of action of Smc, which is warranted.

In line 245, they state More broadly, the study demonstrates how synthetic binders can trap, stabilize, or block transient conformations of active chromatin-associated machines, providing a powerful means to probe their mechanisms in living cells. This is off course a possible scenario for the use of sybodies, but the study does not really trap Smc in a transient conformation, at least this is not clearly shown.

We agree and have simplified the statement by removing “stabilize” and “transient”.

Overall, it is an interesting study, with a well-presented novel technology, and a limited gain of knowledge on SMC proteins.

We respectfully disagree with the last point, since our unique results highlight the importance of the Smc coiled coils. which are less well represented in the SMC literature (when compared to the heads and hinge domains for example), likely (at least in part) due the mild effect of single point mutations on coiled coil dynamics.

Significance:

The work describes the gaining and use of single-binder antibodies (sybodies) to interfere with the function of proteins in bacteria. Using this technology for the SMC complex, the authors demonstrate that they can obtain a significant of binders that target a defined region is SMC and thereby interfere with the ATPase cycle.

The study does not present a strong gain of knowledge of the mode of action of the SMC complex.

As pointed out above, we respectfully disagree with this assertion.

This photo should be Figure 1.6

Very interesting work. I wanted to ask if the supplementary tables can be made accessible, either here, in PMC or in the github page. Thank you

Janie falls in love and gets married although others are very opposed to this and attempt to make her doubt

Janie ignores what those around her have to say and follows what she believes is right

eLife Assessment

This valuable study focuses on a unique morphogenetic module, the junction-based lamellipodia (JBL). It provides a biomechanical understanding of how JBLs control endothelial cell-cell junctional remodelling to generate lumenised, multicellular blood vessels. The manuscript represents a robust, thoughtfully executed, and convincing study that uses high-resolution time-lapse imaging combined with pharmacological treatments to advance our understanding of lumen formation in vascular development.

Reviewer #1 (Public review):

[Editors' note: this version has been assessed by the Reviewing Editor without further input from the original reviewers. The authors have addressed the comments raised in the previous round of review.]

Original review:

Summary:

Lumen formation is a fundamental morphogenetic event essential for the function of all tubular organs, notably the vertebrate vascular network, where continuous and patent conduits ensure blood flow and tissue perfusion. The mechanisms by which endothelial cells organize to create and maintain luminal space have historically been categorized into two broad strategies: cell shape changes, which involve alterations in apical-basal polarity and cytoskeletal architecture, and cell rearrangements, wherein intercellular junctions and positional relationships are remodeled to form uninterrupted conduits. The study presented here focuses on the latter process, highlighting a unique morphogenetic module, junction-based lamellipodia (JBL), as the driver for endothelial rearrangements.

Strengths:

The key mechanistic insight from this work is the requirement of the Arp2/3 complex, the classical nucleator of branched actin filament networks, for JBL protrusion. This implicates Arp2/3-mediated actin polymerization in pushing force generation, enabling plasma membrane advancement at junctional sites. The dependence on Arp2/3 positions JBL within the family of lamellipodia-like structures, but the junctional origin and function distinguish them from canonical, leading-edge lamellipodia seen in cell migration.

Weaknesses:

The study primarily presents descriptive observations and includes limited quantitative analyses or genetic modifications. Molecular mechanisms are typically interrogated through the use of pharmacological inhibitors rather than genetic approaches. Furthermore, the precise semantic distinction between JAIL and JBL requires additional clarification, as current evidence suggests their biological relevance may substantially overlap.

Reviewer #2 (Public review):

Original review:

Summary:

In Maggi et al., the authors investigated the mechanisms that regulate the dynamics of a specialized junctional structure called junction-based lamellipodia (JBL), which they have previously identified during multicellular vascular tube formation in the zebrafish. They identified the Arp2/3 complex to dynamically localize at expanding JBLs and showed that the chemical inhibition of Arp2/3 activity slowed junctional elongation. The authors therefore concluded that actin polymerization at JBLs pushes the distal junction forward to expand the JBL. They further revealed the accumulation of Myl9a/Myl9b (marker for MLC) at the junctional pole, at interjunctional regions, suggesting that contractile activity drives the merging of proximal and distal junctions. Indeed, chemical inhibition of ROCK activity decreased junctional mergence. With these new findings, the authors added new molecular and cellular details into the previously proposed clutch mechanism by proposing that Arp2/3-dependent actin polymerization provides pushing forces while actomyosin contractility drives the merging of proximal and distal junctions, explaining the oscillatory protrusive nature of JBLs.

Strengths:

The authors provide detailed analyses of endothelial cell-cell dynamics through time-lapse imaging of junctional and cytoskeletal components at subcellular resolution. The use of zebrafish as an animal model system is invaluable in identifying novel mechanisms that explain the organizing principles of how blood vessels are formed. The data is well presented, and the manuscript is easy to read.

Weaknesses:

While the data generally support the conclusions reached, some aspects can be strengthened. For the untrained eye, it is unclear where the proximal and distal junctions are in some images, and so it is difficult to follow their dynamics (especially in experiments where Cdh5 is used as the junctional marker). Images would benefit from clear annotation of the two junctions. All perturbation experiments were done using chemical inhibitors; this can be further supported by genetic perturbations.

Reviewer #3 (Public review):

Original review:

The paper by Maggi et al. builds on earlier work by the team (Paatero et al., 2018) on oriented junction-based lamellipodia (JBL). They validate the role of JBLs in guiding endothelial cell rearrangements and utilise high-resolution time-lapse imaging of novel transgenic strains to visualise the formation of distal junctions and their subsequent fusion with proximal junctions. Through functional analyses of Arp2/3 and actomyosin contractility, the study identifies JBLs as localized mechanical hubs, where protrusive forces drive distal junction formation, and actomyosin contractility brings together the distal and proximal junctions. This forward movement provides a unique directionality which would contribute to proper lumen formation, EC orientation, and vessel stability during these early stages of vessel development.

Time-lapse live imaging of VEC, ZO-1, and actin reveals that VEC and ZO-1 are initially deposited at the distal junction, while actin primarily localizes to the region between the proximal and distal sites. Using a photoconvertible Cdh5-mClav2 transgenic line, the origin of the VEC aggregates was examined. This convincingly shows that VE-cadherin was derived from pools outside the proximal junctions. However, in addition to de novo VEC derived from within the photoconverted cell, could some VEC also be contributed by the neighbouring endothelial cell to which the JBL is connected?

As seen for JAILs in cultured ECs, the study reveals that Arp2/3 is enhanced when JBLs form by live imaging of Arpc1b-Venus in conjunction with ZO-1 and actin. Therefore Arp2/3 likely contributes to the initial formation of the distal junction in the lamellopodium.

Inhibiting Arp2/3 with CK666 prevents JBL formation, and filopodia form instead of lamellopodia. This loss of JBLs leads to impaired EC rearrangements.

Is the effect of CK666 treatment reversible? Since only a short (30 min) treatment is used, the overall effect on the embryo would be minimal, and thus washing out CK666 might lead to JBL formation and normalized rearrangements, which would further support the role of Arp2/3.

From the images in Figure 4d it appears that ZO-1 levels are increased in the ring after CK666 treatment. Has this been investigated, and could this overall stabilization of adhesion proteins further prevent elongation of the ring?

To explore how the distal and proximal junctions merge, imaging of spatiotemporal imaging of Myl9 and VEC is conducted. It indicates that Myl9 is localized at the interjunctional fusion site prior to fusion. This suggests pulling forces are at play to merge the junctions, and indeed Y 27632 treatment reduces or blocks the merging of these junctions.

For this experiment, a truncated version of VEC was use,d which lacks the cytoplasmic domain. Why have the authors chosen to image this line, since lacking the cytoplasmic domain could also impair the efficiency of tension on VEC at both junction sites? This is as described in the discussion (lines 328-332).

Since the time-lapse movies involve high-speed imaging of rather small structures, it is understandable that these are difficult to interpret. Adding labels to indicate certain structures or proteins at essential timepoints in the movies would help the readers understand these.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

Lumen formation is a fundamental morphogenetic event essential for the function of all tubular organs, notably the vertebrate vascular network, where continuous and patent conduits ensure blood flow and tissue perfusion. The mechanisms by which endothelial cells organize to create and maintain luminal space have historically been categorized into two broad strategies: cell shape changes, which involve alterations in apical-basal polarity and cytoskeletal architecture, and cell rearrangements, wherein intercellular junctions and positional relationships are remodeled to form uninterrupted conduits. The study presented here focuses on the latter process, highlighting a unique morphogenetic module, junction-based lamellipodia (JBL), as the driver for endothelial rearrangements.

Strengths:

The key mechanistic insight from this work is the requirement of the Arp2/3 complex, the classical nucleator of branched actin filament networks, for JBL protrusion. This implicates Arp2/3-mediated actin polymerization in pushing force generation, enabling plasma membrane advancement at junctional sites. The dependence on Arp2/3 positions JBL within the family of lamellipodia-like structures, but the junctional origin and function distinguish them from canonical, leading-edge lamellipodia seen in cell migration.

Weaknesses:

The study primarily presents descriptive observations and includes limited quantitative analyses or genetic modifications. Molecular mechanisms are typically interrogated through the use of pharmacological inhibitors rather than genetic approaches. Furthermore, the precise semantic distinction between JAIL and JBL requires additional clarification, as current evidence suggests their biological relevance may substantially overlap.

We have previously analyzed the effects of different ve-cadherin (cdh5) mutant alleles on EC rearrangements (Paatero et al., 2018; Sauteur et al., 2014).These mutants show complex defects (e.g. hypersprouting, reduced contact inhibition during anastomosis) in EC behavior early in vascular tube formation. We find that analysis of JBL dynamics and function is very difficult in such situations. The use of small molecule inhibitors allows acute interventions within a defined time-window and to avoid pleiotropic effects of genetic ablations. We have expanded our discussion on the distinction between JAIL and JBL and hope that this will clarify why – in our opinion – these terms should be used differentially in different cell biological contexts (see below and lines 348-374 in the manuscript).

Reviewer #2 (Public review):

Summary:

In Maggi et al., the authors investigated the mechanisms that regulate the dynamics of a specialized junctional structure called junction-based lamellipodia (JBL), which they have previously identified during multicellular vascular tube formation in the zebrafish. They identified the Arp2/3 complex to dynamically localize at expanding JBLs and showed that the chemical inhibition of Arp2/3 activity slowed junctional elongation. The authors therefore concluded that actin polymerization at JBLs pushes the distal junction forward to expand the JBL. They further revealed the accumulation of Myl9a/Myl9b (marker for MLC) at the junctional pole, at interjunctional regions, suggesting that contractile activity drives the merging of proximal and distal junctions. Indeed, chemical inhibition of ROCK activity decreased junctional mergence. With these new findings, the authors added new molecular and cellular details into the previously proposed clutch mechanism by proposing that Arp2/3-dependent actin polymerization provides pushing forces while actomyosin contractility drives the merging of proximal and distal junctions, explaining the oscillatory protrusive nature of JBLs.

Strengths:

The authors provide detailed analyses of endothelial cell-cell dynamics through time-lapse imaging of junctional and cytoskeletal components at subcellular resolution. The use of zebrafish as an animal model system is invaluable in identifying novel mechanisms that explain the organizing principles of how blood vessels are formed. The data is well presented, and the manuscript is easy to read.

Weaknesses:

While the data generally support the conclusions reached, some aspects can be strengthened. For the untrained eye, it is unclear where the proximal and distal junctions are in some images, and so it is difficult to follow their dynamics (especially in experiments where Cdh5 is used as the junctional marker). Images would benefit from clear annotation of the two junctions. All perturbation experiments were done using chemical inhibitors; this can be further supported by genetic perturbations.

We have added annotations to several figures and paid particular attention to the proximal and distal junctions.

We have previously analyzed the effects of different ve-cadherin (cdh5) mutant alleles on EC rearrangements (Paatero et al., 2018; Sauteur et al., 2014). These mutants show complex defects (e.g. hypersprouting, reduced contact inhibition during anastomosis) in EC behavior early in vascular tube formation. We find that analysis of JBL dynamics and function is very difficult in such situations. The use of small inhibitors allows acute interventions within a defined time-window and to avoid pleiotropic effects of genetic ablations.

Reviewer #3 (Public review):

The paper by Maggi et al. builds on earlier work by the team (Paatero et al., 2018) on oriented junction-based lamellipodia (JBL). They validate the role of JBLs in guiding endothelial cell rearrangements and utilise high-resolution time-lapse imaging of novel transgenic strains to visualise the formation of distal junctions and their subsequent fusion with proximal junctions. Through functional analyses of Arp2/3 and actomyosin contractility, the study identifies JBLs as localized mechanical hubs, where protrusive forces drive distal junction formation, and actomyosin contractility brings together the distal and proximal junctions. This forward movement provides a unique directionality which would contribute to proper lumen formation, EC orientation, and vessel stability during these early stages of vessel development.

Time-lapse live imaging of VEC, ZO-1, and actin reveals that VEC and ZO-1 are initially deposited at the distal junction, while actin primarily localizes to the region between the proximal and distal sites. Using a photoconvertible Cdh5-mClav2 transgenic line, the origin of the VEC aggregates was examined. This convincingly shows that VE-cadherin was derived from pools outside the proximal junctions. However, in addition to de novo VEC derived from within the photoconverted cell, could some VEC also be contributed by the neighbouring endothelial cell to which the JBL is connected?

Yes, the green (non-converted) VE-cadherin can indeed originate from either of the two cells. The main point we want to make, based on our observations, is that the red (converted) VE-cadherin from the proximal junction (as defined by the ROI) does not contribute to the distal junction.

As seen for JAILs in cultured ECs, the study reveals that Arp2/3 is enhanced when JBLs form by live imaging of Arpc1b-Venus in conjunction with ZO-1 and actin. Therefore Arp2/3 likely contributes to the initial formation of the distal junction in the lamellopodium.

Inhibiting Arp2/3 with CK666 prevents JBL formation, and filopodia form instead of lamellopodia. This loss of JBLs leads to impaired EC rearrangements.

Is the effect of CK666 treatment reversible? Since only a short (30 min) treatment is used, the overall effect on the embryo would be minimal, and thus washing out CK666 might lead to JBL formation and normalized rearrangements, which would further support the role of Arp2/3.

We have performed washout experiments and find that the ectopic filopodia disappear when the inhibitor is removed. This experiment is shown in supplementary Figure 3 and supplementary Movies 12 and 13.

From the images in Figure 4d it appears that ZO-1 levels are increased in the ring after CK666 treatment. Has this been investigated, and could this overall stabilization of adhesion proteins further prevent elongation of the ring?

This is an interesting thought and we haven take a closer look. There is quite a bit of sample-to-sample variation in the ZO1 signal. The quantification (Author response image 1) indicates that there is no increase in the CK666 treated embryos on average.

Author response image 1.

To explore how the distal and proximal junctions merge, imaging of spatiotemporal imaging of Myl9 and VEC is conducted. It indicates that Myl9 is localized at the interjunctional fusion site prior to fusion. This suggests pulling forces are at play to merge the junctions, and indeed Y 27632 treatment reduces or blocks the merging of these junctions.

For this experiment, a truncated version of VEC was use,d which lacks the cytoplasmic domain. Why have the authors chosen to image this line, since lacking the cytoplasmic domain could also impair the efficiency of tension on VEC at both junction sites? This is as described in the discussion (lines 328-332).

This line was used because it labels the entire JBL protrusion more clearly. We have also included an example using the VE-cad-Venus line (supplementary Figure 4b), which shows a Myl-Cherry pattern consistent with the other examples.

Since the time-lapse movies involve high-speed imaging of rather small structures, it is understandable that these are difficult to interpret. Adding labels to indicate certain structures or proteins at essential timepoints in the movies would help the readers understand these.

We have added annotations and labels to all movies. We have also improved annotations in several figures (i.e. Figs. 1, 2, 5, 6 and 7)

Recommendations for the authors:

Reviewing Editor Comments:

Overall, the reviewers are supportive of the manuscript but identify a number of areas where the clarity of the presented data could be improved, and further quantification could be provided to strengthen your conclusions. We would encourage you to address these minor concerns as best you can and to consider the recommendations of all three reviewers when deciding how to revise your manuscript.

Reviewer #1 (Recommendations for the authors):

Lumen formation is a fundamental morphogenetic event essential for the function of all tubular organs, notably the vertebrate vascular network, where continuous and patent conduits ensure blood flow and tissue perfusion. The mechanisms by which endothelial cells organize to create and maintain luminal space have historically been categorized into two broad strategies: cell shape changes, which involve alterations in apical-basal polarity and cytoskeletal architecture, and cell rearrangements, wherein intercellular junctions and positional relationships are remodeled to form uninterrupted conduits. The study presented here focuses on the latter process, highlighting a unique morphogenetic module, junction-based lamellipodia (JBL), as the driver for endothelial rearrangements.

JBL are described as oscillating membrane protrusions emerging at endothelial junctions, operating in a ratchet-like manner to mediate convergent cell movements. This ratchet mechanism allows endothelial cells to approach each other, thereby aligning and joining local luminal segments into a continuous vascular structure. The study employs in vivo high-resolution time-lapse imaging, a technically demanding method that captures spatiotemporal dynamics of cytoskeletal and adhesion complexes during JBL activity with unprecedented detail.

The key mechanistic insight from this work is the requirement of the Arp2/3 complex, the classical nucleator of branched actin filament networks, for JBL protrusion. This implicates Arp2/3-mediated actin polymerization in pushing force generation, enabling plasma membrane advancement at junctional sites. The dependence on Arp2/3 positions JBL within the family of lamellipodia-like structures, but the junctional origin and function distinguish them from canonical, leading-edge lamellipodia seen in cell migration.

An intriguing observation is that a novel junction arises at the distal pole of a JBL. This distal junction is formed from a pool of VE-cadherin that is spatially redistributed from regions outside the initial JBL domain. The distal junction then merges with the proximal junction through a process dependent on actomyosin contractility, as was judged by Myl9 recruitment.

The alternation between pushing forces (Arp2/3-dependent JBL protrusion) and pulling forces (actomyosin-driven junction fusion) defines JBL as a bidirectional mechanical module. Inhibition of actomyosin prevents merging of proximal and distal junctions, thereby stalling lumen continuity. This two-phase system, actin-based extension followed by actomyosin-mediated constriction, ensures both elongation and maturation of endothelial arrangements, ultimately securing vascular patency.

This manuscript represents a robust and thoughtfully executed study that advances our understanding of lumen formation during vascular development. The overarching conclusions are well substantiated, and the results section provides a clear and detailed exposition of the key findings. I appreciate the explanatory movie at the end. Nevertheless, I offer several remarks for further improvement:

(1) The fluorescent images presented are visually compelling, yet lack quantitative analysis in the initial figure. Although quantification is included in Figure 3, it is advisable to incorporate this analysis into Figure 1 as well. Early presentation of quantification will help the reader to appreciate the impact and significance of the findings from the outset.

We appreciate the reviewer’s suggestion and have now added line graphs to measure the spatiotemporal intensities of the Utrophin and ZO-1 reporters in Figure 1b. These measurements demonstrate the sequence of F-actin protrusion and subsequent junctional movement. In Figure 1a, we have added a double-headed arrow which shows the overall movement of the junction towards the dorsal side of the forming DLAV.

(2) For the fluorescence images, further quantitative analysis of membrane overlap, either in terms of width or pixel overlap, would enhance the rigor of the study. Temporal quantification of overlap may provide valuable insights into the stability and reproducibility of the process across experimental replicates.

JBL are quite heterogenous with respect to size, shape and dynamics, which makes quantifications of membrane overlap (JBL size) across experimental replicates difficult. We have published some quantifications on JBL orientation and oscillation in our previous paper (Paatero et al., 2018, Nat. comm. Figures 1 and 2), which are in agreement with our current study.

(3) When referencing the role of Arp2/3, the authors employ an ArpC1b transgenic fish. The results section should thus specifically address the involvement of ArpC1b, rather than generalizing to Arp2/3. In the discussion, it would be appropriate to speculate on the potential involvement of the complete Arp2/3 complex. Notably, the use of CK is acknowledged as a broadly accepted inhibitor of actin polymerization.

As ArpC1b is a subunit of an active Arp2/3 complex (Padrick et al., 2011), we have used an ArpC1b-Venus as a readout for Arp2/3 localization. The construct has been validated before in cell culture (Law et al., 2021) as well as in zebrafish (Malchow et al., 2024) and the spatiotemporal distribution of the reporter shown to be consistent with Arp2/3 complex. We are stating this in the results section (lines 173-178) and subsequently use the term Arp2/3 to facilitate reading of the text. In the corresponding figure legends, we are maintaining the term ArpC1b. CK666 interferes with the dimerization of Arp2 and Arp3 subunits and thus prevents activity of the Arp2/3 complex.

(4) The discussion regarding JAIL versus JBL involvement remains challenging to interpret. If JAIL structures arise from the loss of cell-cell contacts, both JAIL and JBL resemble membrane protrusions and are likely governed by similar molecular mechanisms, predominantly actin polymerization and Arp2/3 activity, with probable contribution from Rac1 signaling. The precise semantic distinction between JAIL and JBL warrants further clarification, as their biological relevance may be overlapping.

We agree with the reviewer. Below we outline the reasons why lamellipodial protrusions that emanate from cell-cell junctions should not be indiscriminately called JAIL, but that JAIL and JBL constitute different cellular activities acting in different tissue contexts. We have modified the text in the Discussion (lines 348-374).

(1) JAIL have originally been described in cell culture experiments (Abu-Taha et al., 2014). According to this and subsequent papers by the same group, local dissolution of endothelial adherens junctions (i.e. downregulation of VE-cadherin) triggers the formation of lamellipodia-like structures. These protrusions eventually retract, followed by the reestablishment of EC junctions.

(2) In our in vivo studies, we observed lamellipodial protrusions during endothelial cell rearrangements, and we call these structures JBL (Paatero et al., 2018). While JBL appear very similar to JAIL in general (i.e. regulation by Arp2/3 and its localization), we also observe two critical differences. For one, JBL form while maintaining the original (proximal) junction. Moreover, a distal junction is formed at the front edge of the JBL, leading to a “double junction” configuration. In our current manuscript, we have examined the role of actomyosin contractility and find that it correlates with and is required for the merging of proximal and distal junctions during JBL cycles. These observations indicates that the proximal and distal junctions are essential components of JBL function during endothelial cell elongation and rearrangements. These salient and distinct features prompted us to adopt the term junction-based-lamellipodia (JBL), in order to differentiate them from JAIL.

(3) We like to argue that JAIL and JBL represent similar but different lamellipodia-like protrusions. JAILs are associated with the maintenance of endothelial integrity, and control permeability and trans-endothelial cell migration, as has been suggested by several publications (Cao et al., 2017; Kipcke et al., 2025; Seebach et al., 2021; Taha et al., 2014). In contrast, JBL drive cell rearrangements, by step-wise elongation of cell junctions leading to convergent cell movements.

(4) Although JAIL have also been implicated in endothelial cell migration (Cao and Schnittler, 2019; Cao et al., 2017; Seebach et al., 2021), neither junctional patterns nor junctional dynamics have been analyzed in this context. We therefore propose that JAIL and JBL are actin-based protrusions forming at endothelial cell-cell junctions, but act in different contexts to provide cell motility (JBL) or endothelial integrity (JAIL).

(5) Some of the quantification plots, specifically in figures 5d and 6c, do not display significant differences or distribution patterns. It would be beneficial to revise these graphs to clearly represent statistical significance and underlying data distributions.

Because of the spatiotemporal heterogeneity, it is difficult to perform statistical quantifications across samples. In Figure 5c/d, we have imaged/analyzed myl9-EGFP in a mosaic situation, in which only one of interacting cells expresses high levels of myl9-EGFP. This is a rare situation and we managed to image only this example. Nevertheless, it is consistent with our other expression data of myl9-reporters and also with our previous photoconversion experiments using photoconvertible UCHD (Paatero et al., 2018, Figure 4), which shows that actin-rich JBL form at the front end of the endothelial cell in the direction of junction elongation. In Figure 5d, we have quantified the average intensity of GFP signal within the region of interest. The newly added error bars indicate the standard deviation between pixel intensities within the ROI.

In Figure 6c, we have analyzed the Myl9b-mCherry intensities and find that it is redistributed during a JBL cycle. The spatial distribution is evident from the heat-map and we have not included a standard deviation. Myl9b-mCherry levels are very heterogenous and is not possible to quantify intensities across samples. We have, however, included four more examples of Myl9b-mCherry distribution in Supplementary Figure 4. The patterns observed in these samples are consistent with those in Figure 6.

(6) The observation of myosin recruitment does not inherently imply a concomitant increase in actomyosin contractile activity. The inclusion of phospho-MLC staining would considerably strengthen the evidence for enhanced actomyosin activity.

This is a good suggestion and we have extensively tried different anti-P-Myl antibodies (and protocols), but did not get them to work reliably on zebrafish embryos. We therefore rely on published work that has established the correlation between the recruitment of myosin light chain and increased actomyosin tension (Fernandez-Gonzalez et al., 2009; Munjal et al., 2015).

Reviewer #2 (Recommendations for the authors):

(1) Figure 1a is not described/mentioned in the Results.

The have corrected this (lines 102-108). We have also added measurements to better present the different dynamics of F-actin (UCHD) and ZO1 within the JBL and the relative endothelial cell movements (see Figure 1b), as suggested by reviewer#1.

(2) In Figure 3a, the authors claim that Arp2/3 is deposited at the distal side of the junction ring. While it is clear where the proximal junction is (ZO1-rich), the distal junction is less so (hardly any ZO1). It is therefore difficult to agree based on this time-lapse imaging that Arpc1b-Venus is at the distal junction. Can the authors please include panels showing merged channels and annotate where the proximal and distal junctions are?

The activation of the Arp2/3 complex and the formation of the distal junction are sequential events. We see that ArpC1b oscillates with an accumulation at the onset and during JBL protrusion. In contrast, the distal junction is formed when the protrusive activity has been stopped. One caveat of the analysis shown in Figure 3a is that our ZO1 reporters label the distal junction only very weakly – this is in particular the case for the ZO1-tdTomato knock-in. The distal junction is better visible in VE-cadherin and UCHD reporters, as shown in Figures 5 to 7.

(3) In Figures 3b and c, it is also difficult to distinguish proximal and distal junctions in these images. Please mark the boundaries in the image panels (Figure 3b) and indicate on the x-axis where the proximal and distal junctions are (Figure 3c).

In Figure 3b, we show ArpC1b-Venus and mRuby-UCHD side-by-side. This Figure demonstrates that the Arp2/3 complex maintains its position at the front of the JBL during the protrusive phase (always distal to the UCHD signal). The imaging is done at very short intervals (1/30sec), which makes it difficult to follow entire oscillations due to photo-bleaching of the ArpC1b reporter.

(4) The treatment of CK666 resulted in perturbed localization of Arpc1b-Venus. Therefore, the inhibition of junctional elongation can also be explained by the mislocalization of Arp2/3, rather than the inhibition of Arp2/3 activity at the junctions. Can the authors discuss this or perform another experiment that is more specific to manipulating Arp2/3 activity?

CK666 is a well-established inhibitor of Arp2/3. Structural and functional analyses have shown that CK666 interferes with the interaction between Arp2 and Arp3, thereby preventing the activation of the complex (Hetrick et al., 2013; Padrick et al., 2011). We therefore conclude that the phenotypes we observe in CK666 treatment are due to Arp2/3 inhibition.

It is possible that CK666 prevents ArpC1b binding to the Arp2/3 complex. However, published work suggests that ArpC1b can bind to Arp2/3 also in its inactive state (Chou et al., 2022). Thus, we can only speculate why we lose localization ArpC1b under CK666. We prefer not to do so.

(5) In Figures 5d and 6c, is the quantification of Myl9 intensity of one cell only? If so, can the authors show the dynamics of average Myl9 intensity i) between forwarding and non-forwarding JBL poles and ii) as the proximal and distal junctions merge several endothelial cells?

Figure 5c/d depicts two interacting cells, expressing different levels of Myl9a-EGFP. This is a rare experimental situation and we managed to image only this example. We quantified the average signal at both poles of the junctional ring within a region of interest. The newly added error bars indicate the standard deviation between pixel intensities within the ROI. The analysis has been done on immunofluorescent images, therefore a dynamic analysis over time is not possible.

In Figure 6c, we have analyzed the Myl9b-mCherry intensities and find that it is redistributed during a JBL cycle. The spatial distribution is evident from the heat-map and we have not included a standard deviation. Myl9b-mCherry levels are very heterogenous and is not possible to quantify intensities across samples. We have, however, included four more examples of Myl9b-mCherry distribution in Supplementary Figure 4. The patterns observed in these samples are consistent with those in Figure 6.

(6) Figure 5. The 'f' in the figure legend should be 'e' since there is no panel 'f'.

We have corrected this.

(7) Figure 7. As the boundaries for proximal and distal junctions are not always clear, especially when Cdh5 appears as clusters, how do you determine where the two junctions are in order to measure the interjunctional space? Please offer a clearer explanation in the Methods.

We have added the following in the M&M. “Junctional merging tracking Speed of junctional merge was evaluated by monitoring isolated junctional rings during DLAV formation. Inhibitor treatment Y-27632 (75 μM) or DMSO (1%) were applied 30 min before mounting. The same concentrations of chemicals were applied to the low-melting-point agarose mounting medium and the E3 medium on top of it before imaging and imaging the junctions for 10-15 min on an Olympus SpinSR spinning disc microscope. Distances were measured using Fiji software. In each frame, the interjunctional distance was defined as the maximum distance between the proximal and distal junctions. A line was manually drawn between the proximal and distal junctions in Fiji, and its length was recorded. The same proximal and distal junction landmarks were used consistently across all time points.”

(8) One would think that upon the inhibition of junctional mergence (by ROCK inhibition), actin polymerization would persist to push the distal junction forward to elongate the JBL. However, there is instead a decrease in junctional elongation (Figure 7b). Can the authors speculate why? Additionally, junction elongation can probably be achieved by continuous "pushing" of the distal junction alone (through actin polymerization). Can the authors speculate why there is a need/what is the benefit of merging proximal and distal junctions for junction elongation?

These are all very interesting questions, but they are quite complex and would require extensive and speculative answers, which is outside the scope of this study. Nevertheless, here are a few quick thoughts on these issues.

(1) When endothelial cells elongate, they have to overcome tensile forces at the junctions (generated by the subjunctional actomyosin belt). JBL are providing a tractive and deforming force, which overcomes the tensile force and thus promotes junctional elongation.

(2) The distal junction is then providing an anchor to which the actin cytoskeleton can attach. The space between proximal and distal junction becomes a compartment of local actomyosin contraction, which provides the force for the ratchet to move the proximal junction forward  junctional mergence.

(3) Thus, it is not the protrusion (pushing) itself that elongates the cell but the elongation of the junction (driven by actomyosin contraction)!

(4) The maintenance of the proximal junction is most likely needed to ensure endothelial integrity during the JBL cycles.

(5) How the frequency and the size of JBLs is regulated is not known. One possible player that might be involved is an internal clock mechanism (e.g. a feedback loop via small GTPases (such as Rac)  Arp2/3 regulation). Another possibility is that JBL size is limited by it sweeping up basally localized VE-cadherin (in cis-configuration). Increasing cell-cell adhesion (by VE-cad trans-interactions between the JBL and the underlying cell) eventually stop the protrusion. It is also possible that an cell-autonomously controlled mechanism of F-actin polymerization (actin pulses) are involved in the regulation of the JBC cycle length.

(9) The animation showing the molecular mechanism of JBL function during endothelial junction elongation (Video 25) is very helpful in understanding the dynamic coupling between junctional proteins, actomyosin cytoskeleton, and junction remodelling. However, I wonder why there are no Myosin II proteins binding to the actin bundles during the merging of proximal and distal junctions (between 0:25 and 0:28), since this is one of the main findings by the authors in this study.

Since we show two JBL cycles, we want to spread the information over both of them.

References:

Cao, J. and Schnittler, H. (2019). Putting VE-cadherin into JAIL for junction remodeling. J. Cell Sci. 132.

Cao, J., Ehling, M., März, S., Seebach, J., Tarbashevich, K., Sixta, T., Pitulescu, M. E., Werner, A. C., Flach, B., Montanez, E., et al. (2017). Polarized actin and VE-cadherin dynamics regulate junctional remodelling and cell migration during sprouting angiogenesis. Nat. Commun. 8, 1–20.

Chou, S. Z., Chatterjee, M. and Pollard, T. D. (2022). Mechanism of actin filament branch formation by Arp2/3 complex revealed by a high-resolution cryo-EM structure of the branch junction. Proc. Natl. Acad. Sci. U. S. A. 119, e2206722119.

Fernandez-Gonzalez, R., Simoes, S. de M., Röper, J. C., Eaton, S. and Zallen, J. A. (2009). Myosin II Dynamics Are Regulated by Tension in Intercalating Cells. Dev. Cell 17, 736–743.

Hetrick, B., Han, M. S., Helgeson, L. A. and Nolen, B. J. (2013). Small molecules CK-666 and CK-869 inhibit actin-related protein 2/3 complex by blocking an activating conformational change. Chem. Biol. 20, 701–712.

Kipcke, J. P., Odenthal-Schnittler, M., Aldirawi, M., Franz, J., Bojovic, V., Seebach, J. and Schnittler, H. (2025). TNF-α induces VE-cadherin-dependent gap/JAIL cycling through an intermediate state essential for neutrophil transmigration. Front. Immunol. 16,.

Law, A. L., Jalal, S., Pallett, T., Mosis, F., Guni, A., Brayford, S., Yolland, L., Marcotti, S., Levitt, J. A., Poland, S. P., et al. (2021). Nance-Horan Syndrome-like 1 protein negatively regulates Scar/WAVE-Arp2/3 activity and inhibits lamellipodia stability and cell migration. Nature Communications 2021 12:1 12, 5687-.

Malchow, J., Eberlein, J., Li, W., Hogan, B. M., Okuda, K. S. and Helker, C. S. M. (2024). Neural progenitor-derived Apelin controls tip cell behavior and vascular patterning. Sci. Adv. 10, 1174.

Munjal, A., Philippe, J. M., Munro, E. and Lecuit, T. (2015). A self-organized biomechanical network drives shape changes during tissue morphogenesis. Nature 524, 351–355.

Paatero, I., Sauteur, L., Lee, M., Lagendijk, A. K., Heutschi, D., Wiesner, C., Guzmán, C., Bieli, D., Hogan, B. M., Affolter, M., et al. (2018). Junction-based lamellipodia drive endothelial cell rearrangements in vivo via a VE-cadherin-F-actin based oscillatory cell-cell interaction. Nat. Commun. 9,.

Padrick, S. B., Doolittle, L. K., Brautigam, C. A., King, D. S. and Rosen, M. K. (2011). Arp2/3 complex is bound and activated by two WASP proteins. Proc. Natl. Acad. Sci. U. S. A. 108, E472–E479.

Sauteur, L., Krudewig, A., Herwig, L., Ehrenfeuchter, N., Lenard, A., Affolter, M. and Belting, H. G. (2014). Cdh5/VE-cadherin promotes endothelial cell interface elongation via cortical actin polymerization during angiogenic sprouting. Cell Rep. 9, 504–513.

Seebach, J., Klusmeier, N. and Schnittler, H. (2021). Autoregulatory “Multitasking” at Endothelial Cell Junctions by Junction-Associated Intermittent Lamellipodia Controls Barrier Properties. Front. Physiol. 11,.

Taha, A. A., Taha, M., Seebach, J. and Schnittler, H. J. (2014). ARP2/3-mediated junction-associated lamellipodia control VE-cadherin-based cell junction dynamics and maintain monolayer integrity. Mol. Biol. Cell 25, 245–256.

これだけで1セクションになりそうですよね。読みたいけど、深ぼると大きくなりすぎちゃうからこれだけの表にするとか？

軽量化のアプローチの手法が実務での判断ポイントに含まれていて、少し読みにくく感じました。float32 → float16 とか スカラー量子化とかいつ何を選択するか、みたいな指針があるといいのかも（だいたいGPUが対応してたり、ほとんどのVector DBで対応してるとか？）

前回のEmbeddingモデル比較表のような厚み があってもいいかなと思いました

手法・仕組み・メモリ削減の目安・どのツールでサポートされているか みたいな表があってもいいかなと思いました。

前回4月分では、uv / pip の例がここにあったので、今回もここに追記した方が良いのではと思いました。

前回のセットアップ<br /> https://terada-202605-image-search.gihyo-python-monthly.pages.dev/2026/202604#id6

Two-Towerモデルの説明も文字であって良いのではないかと思いました。後半「モダリティギャップに注意」のところである説明をこちらに持ってくる感じです

追記サンプル<br /> テキストと画像で別々のエンコーダを持ち、出力だけを同じ空間に揃えるアプローチです。

冒頭で出てくる Two-Tower モデルですが、何かという説明も冒頭部分で書いて良いのではと思いました。（冒頭部分にもコメントします

割とハイライトになり得るエピソードな気がします。冒頭の方（せめてコラムの最後くらい 筆者の環境で少量のEmbeddingテストをしたところ あたり） にあってもいいのではないかと思います

"Nessa fase da vida, boa parte das pessoas (sujeito oracional) enfrenta dificuldade de ingresso — ou reingresso — no mercado de trabalho."

Expressões partitivas: “A maioria de”, “parte de”, “metade de”

✔️ Singular ou plural (os dois corretos): • A maioria dos alunos fez A maioria dos alunos fizeram

Chapter 18. Janie and Tea cake decide to stay in their home even though a big hurricane is coming. When the storm hits and causes a flood, they have to swim for safety and Tea cake gets bitten by a dog while saving Janie.

check more annotations on abstract at acm conference paper

Indicador alterado. Considerando escola e pré-escola somente. Indicador original pede que considere escola ou creche.

Igual.

Why did you chose this over GraPhLan? - Can re-use the metacoder nextflow module from their code!?

every car?

... is Clutch Certified and has already....

more lifestyle focused rather than performance.

"If they feel it's not the right car..." bad example

remove.

can we change to "expertly serviced"

咨询公司助力大企业AI

大多数人认为大企业应建立内部AI团队，但作者认为与咨询公司的合作是Claude服务大企业的关键途径。

新公司扩展交付能力

大多数人认为现有合作伙伴网络足以满足需求，但作者认为需要新公司进一步扩展交付能力以满足快速增长的企业需求。

临床医生比工程师更懂需求

大多数人认为技术专家应主导医疗AI开发，但作者认为临床医生更清楚时间消耗和患者护理的实际需求。

小型团队创造大影响

大多数人认为大型AI项目需要庞大团队，但作者认为小型团队与客户紧密合作就能确定Claude的最大影响点。

一线人员主导AI实施

大多数人认为AI实施应由技术专家主导，但作者认为应由最贴近业务一线的人员塑造，因为他们最了解实际需求。

企业需求超出交付能力

大多数人认为企业AI需求可以通过现有模式满足，但作者认为需求远超任何单一交付模式，需要新公司扩展能力。

中小企业缺乏AI资源

大多数人认为大企业才能从AI中获益，但作者认为中小企业同样受益，只是缺乏内部资源来构建前沿部署。

+1. Alexander Winkler (2026-05-05). Nachlassinformationen aus Kalliope für Wikidata erschließen. Saxorum. https://saxorum.hypotheses.org/15723

eLife Assessment

This important study presents a technically rigorous and carefully controlled analysis of the signalling potential of cancer-associated gain-of-function Notch alleles. The work is clearly presented, and the experiments are robust, comprehensive, and well-controlled. While some data primarily establish the system or report negative findings, the comparative approach in a well-characterized model provides convincing mechanistic evidence for how these Notch variants function. This study will be of interest to researchers in both developmental and cancer biology.

Reviewer #1 (Public review):

Summary:

In their paper, Shimizu and Baron describe the signaling potential of cancer gain-of-function Notch alleles using the Drosophila Notch transfected in S2 cells. These cells do not express Notch or the ligand Dl or Dx, which are all transfected. With this simple cellular system, the authors have previously shown that it is possible to measure Notch signaling levels by using a reporter for the 3 main types of signaling outputs, basal signaling, ligand-induced signaling and ligand-independent signaling regulated by deltex. The authors proceed to test 22 cancer mutations for the above-mentioned 3 outputs. The mutation is considered a cluster in the negative regulatory region (NRR) that is composed of 3 LNR repeats wrapping around the HD domain. This arrangement shields the S2 cleavage site that starts the activation reaction.

The main findings are:

(1) Figure 1: the cell system can recapture ectopic activation of 3 existing Drosophila alleles validated in vivo.

(2) Figure 2: Some of the HD mutants do show ectopic activation that is not induced by Dl or Dx, arguing that these mutations fully expose the S2 site. Some of the HD mutants do not show ectopic activation in this system, a fact that is suggested to be related to retention in the secretory pathway.

(3) Figure 3: Some of the LNR mutants do show ectopic activation that is induced by Dl or Dx, arguing that these might partially expose the S2 site.

(4) Figure 4-6: 3 sites of the LNR3 on the surface that are involved in receptor heterodimerization, if mutated to A, are found to cause ectopic activation that is induced by Dl or Dx. This is not due to changes in their dimerization ability, and these mutants are found to be expressed at a higher level than WT, possibly due to decreased levels of protein degradation.

Strengths and Weaknesses:

The paper is very clearly written, and the experiments are robust, complete, and controlled. It is somewhat limited in scope, considering that Figure 1 and 5 could be supplementary data (setup of the system and negative data). However, the comparative approach and the controlled and well-known system allow the extraction of meaningful information in a field that has struggled to find specific anticancer approaches. In this sense, the authors contribute limited but highly valuable information.

Comments on revised version:

I reviewed the changes and response to criticism, and it seems to me that all has been reasonably addressed.

Reviewer #3 (Public review):

Summary:

This manuscript by Shimizu et al., systematically analyzes cancer-associated mutations in the Negative Regulatory Region (NRR) of Drosophila Notch to reveal diverse regulatory mechanisms with implications for cancer modelling and therapy development. The study introduces cancer-associated mutations equivalent to human NOTCH1 mutations, covering a broad spectrum across the LNR and HD domains. By linking mutant-specific mechanistic diversity to differential signaling properties, the work directly informs targeted approaches for modulating Notch activity in cancer cells. These are an exciting set of observations from S2 cells, which should be taken up further for further assessment in any physiological implications.

Strengths:

This manuscript by Shimizu et al., systematically analyzes cancer-associated mutations in the Negative Regulatory Region (NRR) of Drosophila Notch to reveal diverse regulatory mechanisms with implications for cancer modelling and therapy development. The study introduces cancer-associated mutations equivalent to human NOTCH1 mutations, covering a broad spectrum across the LNR and HD domains. The authors use rigorous phenotypic assays to classify their functional outcomes. By leveraging the S2 cell-based assay platform, the work identifies mechanistic differences between mutations that disrupt the LNR-HD interface, core HD, and LNR surface domains, enhancing understanding of Notch regulation. The discovery that certain HD and LNR-HD interface mutations (e.g., R1626Q and E1705P) in Drosophila mirror the constitutive activation and synergy with PEST deletion seen in mammalian T-ALL is nice and provides a platform for future cancer modelling. Surface-exposed LNR-C mutations were shown to increase Notch protein stability and decrease turnover, suggesting a previously unappreciated regulatory layer distinct from canonical cleavage-exposure mechanisms. By linking mutant-specific mechanistic diversity to differential signaling properties, the work directly informs targeted approaches for modulating Notch activity in cancer cells.

Weaknesses:

This is an exciting set of observations, however the work is entirely cell line based, and is the primary weakness. I list my main specific concerns herewith:

(1) The analysis is confined to Drosophila S2 cells, which may not fully recapitulate tissue or organism-level regulatory complexity observed in vivo.

(2) And perhaps for this reason too, some Drosophila HD domain mutants accumulate in the secretory pathway and do not phenocopy human T-ALL mutations. Possibly due to limitations on physiological inputs that S2 cells cannot account for or species-specific differences such as the absence of S1 cleavage. Thus, the findings may not translate directly to understanding Notch 1 function in mammalian cancer models.

(3) Also, while the manuscript highlights mechanistic variety, the functional significance of these mutations for hematopoietic malignancies or developmental contexts in live animals remains untested. Thus even though the changes are evident in Notch signaling, any impact on blood cells or hematopoiesis leading to aberrant malignancies remains to be seen.

(4) Which hematopoietic cell type, progenitor or differentiating cells, would be most sensitive to this kind of altered Notch signaling also remains unclear.

Author response:

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

Public Reviews:

Reviewer #1 (Public review):

Summary:

In their paper, Shimizu and Baron describe the signaling potential of cancer gain-of-function Notch alleles using the Drosophila Notch transfected in S2 cells. These cells do not express Notch or the ligand Dl or Dx, which are all transfected. With this simple cellular system, the authors have previously shown that it is possible to measure Notch signaling levels by using a reporter for the 3 main types of signaling outputs, basal signaling, ligand-induced signaling and ligand-independent signaling regulated by deltex. The authors proceed to test 22 cancer mutations for the above-mentioned 3 outputs. The mutation is considered a cluster in the negative regulatory region (NRR) that is composed of 3 LNR repeats wrapping around the HD domain. This arrangement shields the S2 cleavage site that starts the activation reaction.

The main findings are:

(1) Figure 1: the cell system can recapture ectopic activation of 3 existing Drosophila alleles validated in vivo.

(2) Figure 2: Some of the HD mutants do show ectopic activation that is not induced by Dl or Dx, arguing that these mutations fully expose the S2 site. Some of the HD mutants do not show ectopic activation in this system, a fact that is suggested to be related to retention in the secretory pathway.

(3) Figure 3: Some of the LNR mutants do show ectopic activation that is induced by Dl or Dx, arguing that these might partially expose the S2 site.

(4) Figure 4-6: 3 sites of the LNR3 on the surface that are involved in receptor heterodimerization, if mutated to A, are found to cause ectopic activation that is induced by Dl or Dx. This is not due to changes in their dimerization ability, and these mutants are found to be expressed at a higher level than WT, possibly due to decreased levels of protein degradation.

Strengths and Weaknesses:

The paper is very clearly written, and the experiments are robust, complete, and controlled. It is somewhat limited in scope, considering that Figure 1 and 5 could be supplementary data (setup of the system and negative data). However, the comparative approach and the controlled and well-known system allow the extraction of meaningful information in a field that has struggled to find specific anticancer approaches. In this sense, the authors contribute limited but highly valuable information.

Reviewer #2 (Public review):

Summary:

This ambitious study introduced 22 mutations corresponding to amino acid substitution mutations known to induce cancer in human Notch1, located within the Negative Regulatory Region, into the Drosophila Notch gene. It comprehensively examined their effects on activity, intracellular transport, protein levels, and stability. The results revealed that the impact of amino acid substitutions within the Negative Regulatory Region can be grouped based on their location, differing between the Heterodimerization Domain and the Lin12/Notch Repeat. These findings provide important insights into elucidating the mechanisms by which amino acid substitution mutations in human Notch1 cause leukemia and cancer.

Strengths:

In this study, the authors successfully measured the activity of amino acid-substituted Notch with high precision by effectively leveraging the advantages of their previously established experimental system. Furthermore, they clearly demonstrated ligand-dependent and Deltex-dependent properties.

Weaknesses:

Amino acid substitution mutations exhibit interesting effects depending on their position, so interest naturally turns to the mechanisms generating these differences. Unfortunately, however, elucidating these mechanisms will require considerable time in the future. Therefore, it is reasonable to conclude that questions regarding the mechanism fall outside the scope of this paper.

We thank the editors and reviewers for their initial reviews and constructive suggestions. We have revised the manuscript with some additional data contained in two additional supplementary figures and by the inclusion of additional text.

Reviewer #3 (Public review):

While this is indeed an exciting set of observations, the work is entirely cell-line-based, and is the primary reason why this approach dampens the enthusiasm for the study. The analysis is confined to Drosophila S2 cells, which may not fully recapitulate tissue or organism-level regulatory complexity observed in vivo. Some Drosophila HD domain mutants accumulate in the secretory pathway and do not phenocopy human T-ALL mutations. Possibly due to limitations on physiological inputs that S2 cells cannot account for, or species-specific differences such as the absence of S1 cleavage.

Thus, the findings may not translate directly to understanding Notch 1 function in mammalian cancer models. While the manuscript highlights mechanistic variety, the functional significance of these mutations for hematopoietic malignancies or developmental contexts in live animals remains untested. Overall, the work does not yet provide evidence for altered Notch signaling that is physiologically relevant.

S2 cells are a standard cell culture model which have been extensively used for analysing Notch signalling mechanisms and by and large are found to recapitulate the mechanisms of Notch activation and its regulation in vivo. However, we agree that it will be desirable in future work to build on our current findings by generating Notch mutants in vivo in Drosophila as the in vivo context may introduce additional nuances in the behaviour of the mutants.This can be done by overexpressing cDNA constructs in particular tissues, or more physiologically by generating endogenous gene mutations using CRISPR/Cas9 based gene editing. However, the likely outcome of the latter approach is embryo lethality due to constitutive over-activation during development. Therefore, methods of genetic manipulation need to be applied which allow the final activating mutant form to be generated in somatic clones. We feel that this would be considerable amount of additional work and is out of scope for the current study, but we look forward to developing this approach in future work.

Recommendations for the authors:

Reviewing Editor Comments:

(a) Table 1: Explain the rationale for mapping non-conserved residues between human and fly Notch; consider adding an alignment or supplementary figure.

We have added a new Supplementary figure S2 showing an alignment of Notch sequences from different species to indicate the degree of conservation at the sites chosen for our mutagenesis study. Some locations were highly conserved and some locations less so. Both conserved and non-conserved residues were included to examine how structural perturbations at equivalent positions affect signalling activity, independent of sequence conservation. In addition to the new supplementary figure, we have changed the text in the Table 1 legend to clarify.

(b) Add or discuss data connecting LNR and HD mutant expression levels with stability and degradation mechanisms.

We have added additional text in the results section referring to Fig6A/B regarding the varying Notch protein levels between the different mutants. With regard to the slower degradation kinetics of certain LNR-C mutants in Fig6 E/F, we have also added a new supplementary figure S3 which shows that mutants from the LNR/HD interface do not behave similarly to the LNR-C mutants with respect to their degradation kinetics.

(c) Some mutants, especially those retained in the secretory pathway, are insufficiently characterized. The mechanism underlying their differential trafficking and stability remains underexplored.

We have added some extra text to the discussion section which explores the issue of secretory pathway retention of HD mutants in Drosophila cells further.

(2) Figure Legends:

(a) Figure 1A - Explain the ribbon vs. space-filling representation and color coding; include a definition of the Heterodimerization Domain.

We have added extra text to the Figure 1A legend

(b) Figure 2E - Clarify mutant selection; if possible, include additional examples for consistency.

We added extra text regarding selection of mutants for study into the legend of Figure 2

(c) Figure 3-4 - Explain logic for alanine substitutions; discuss difference at residue 1570 (P vs. A).

We added the following text to the result section. “Y1532 and Y1535 are not mutated in human cancers and therefore could not be assessed through patient-derived variants. Alanine substitution provides a controlled way to probe their contribution to NRR integrity and activation sensitivity by selectively removing their side-chain interactions while preserving overall fold.” We added extra text in the discussion section regarding the differences in the outcomes of the 1570 to A and P mutations.

(d) Figure 4 - Improve resolution and legibility.

We have replaced figure 4.

(e) Figure 6C - Correct residue numbering (1563, 1566).

Thank you for spotting this. This has been corrected.

(f) Figure 6F - Include control where protein levels do not increase.

A new supplementary figure S3 has been added which included this control data.

(3) Contextual and Conceptual Framing:

(a) Incorporate the limitations of the S2 system, and delineate which mechanistic insights are likely conserved versus those that may be species- or context-specific.

We have incorporated text to discuss S2 cell limitations.

(b) The study does not test functional consequences in hematopoietic or developmental contexts. Expand the discussion to emphasize how these cell-based findings could inform future in vivo studies or mammalian cancer modeling.

thee-i-TEE-tuhs

Plato's dear friend.

thee-OD-or-uhs

Math teacher of Plato.

eLife Assessment

This manuscript offers valuable structural and mechanistic insights into the assembly of the Type II internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and the translation initiation complex, revealing a direct interaction between the IRES and the 40S ribosomal subunit. A solid experimental strategy, combining cryo-EM analysis, complementary biochemistry, and detailed structural comparisons, provides mechanistic insights into IRES-based translation initiation systems. This paper will attract researchers in cap-independent translation, host-pathogen interactions, and virology.

Reviewer #1 (Public review):

Summary:

The authors have studied how a virus (EMCV) uses its RNA (Type 2 IRES) to hijack the host's protein-making machinery. They use cryo-EM to extract structural information about the recruitment of viral Type 2 IRES to ribosomal pre-IC. The authors propose a novel interaction mechanism in which the EMCV Type 2 IRES mimics 28S rRNA and interacts with ribosomal proteins and initiator tRNA (tRNAi).

Strengths:

(1) Getting structural insights about the Type 2 IRES-based initiation is novel.

(2) The study allows a good comparison of other IRES-based initiation systems.

(3) The manuscript is well-written and clearly explains the background, methods, and results.

Comments on revised version:

I have gone through the revised manuscript by Das and Hussain along with the rebuttal comments. While the poor resolution of the ribosomal complex limits detailed analysis of the molecular interactions, addition of the luciferase reporter assay in the supplementary has enriched the paper.

Reviewer #2 (Public review):

Summary:

The field of protein translation has long sought the structure of a Type 2 Internal Ribosome Entry Site (IRES). In this work, Das and Hussain pair cryo-EM with algorithmic RNA structure prediction to present a structure of the Type 2 IRES found in Encephalomyocarditis virus (EMCV). Using medium to low resolution cryo-EM maps, they resolve the overall shape of a critical domain of this Type 2 IRES. They use algorithmic RNA prediction to model this domain onto their maps and attempt to explain previous results using this model.

Strengths:

(1) This study reveals a previously unknown/unseen binding modality used by IRESes: a direct interaction of the IRES with the initiator tRNA.

(2) Use of an IRES-associated factor to assemble and pull down an IRES bound to the small subunit of the ribosome from cellular extracts is innovative.

(3) Algorithmic modeling of RNA structure to complement medium to low resolution cryo-EM maps, as employed here, can be implemented for other RNA structures.

Comments on revised version:

Thanks to the authors for providing thorough responses to the reviewer questions and comments. I appreciate their attempts of improving overall resolution of the complex via various processing strategies that the reviewers suggested.

The authors interpretations of their cryo-EM data match those reported by Bhattacharjee et al. 2025 (EMCV-IRES 48S) and can be contextualized in the light of Velazquez et al. 2025 (poliovirus IRES-48S).

The authors' contextualization of their results with previously published studies (Discussion section lines 355-402) is satisfactory to me but can be improved.

Reviewer #3 (Public review):

Summary:

Type II IRES, such as those from encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV), mediate cap-independent translation initiation by using the full complement of eukaryotic initiation factors (eIFs), except the cap-binding protein eIF4E. The molecular details of how IRES type II interacts with the ribosome and initiation factors to promote recruitment have remained unclear. Das and Hussain used cryo-electron microscopy to determine the structure of a translation initiation complex assembled on the EMCV IRES. The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Strengths:

The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Comments on revised version:

The revised manuscript does not improve the resolution; however, the authors provide a detailed and well-reasoned rationale that directly addresses the concerns I raised about their structural interpretation. In addition, two independent preprints have been released since the initial submission. In one case, the authors report a higher-resolution, and importantly, all three studies present consistent assignments and interpretations. Together, these observations strengthen confidence in the authors' conclusions. I therefore do not have major concerns regarding the publication of this revised manuscript.

Author response:

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

eLife Assessment

This manuscript offers valuable structural and mechanistic insights into the structure and assembly of the Type II internal ribosome entry site (IRES) from encephalomyocarditis virus (EMCV) and the translation initiation complex, revealing a direct interaction between the IRES and the 40S ribosomal subunit. While a solid cryo-EM method was used, enhancing the overall resolution or adding complementary biochemical data would further improve the clarity and impact of this study. This manuscript will attract researchers in cap-independent translation, host-pathogen interactions, and virology.

We thank the editorial team for a favourable assessment and for mentioning our work as ‘valuable’. In the following sections, we have addressed the weaknesses and recommendations pointed out by the Reviewers and hope for an improvement in the description of this work.

Public Reviews:

Reviewer #1 (Public review):

Summary:

The authors have studied how a virus (EMCV) uses its RNA (Type 2 IRES) to hijack the host's protein-making machinery. They use cryo-EM to extract structural information about the recruitment of viral Type 2 IRES to ribosomal pre-IC. The authors propose a novel interaction mechanism in which the EMCV Type 2 IRES mimics 28S rRNA and interacts with ribosomal proteins and initiator tRNA (tRNAi).

Strengths:

(1) Getting structural insights about the Type 2 IRES-based initiation is novel.

(2) The study allows a good comparison of other IRES-based initiation systems.

(3) The manuscript is well-written and clearly explains the background, methods, and results.

We thank Reviewer 1 for appreciating our efforts and finding structural insights about the Type 2 IRES-based initiation presented in this study as novel.

Weaknesses:

(1) The main weakness of the work is the low resolution of the structure. This limits the possibility of data interpretation at the molecular level.

However, despite the moderate resolution of the cryo-EM reconstructions, the model fits well into the density. The analysis of the EMCV IRES-48S PIC structure is thorough and includes meaningful comparisons to previously published structures (e.g., PDB IDs - 7QP6 and 7QP7). These comparisons showed that Map B1 represents a closed conformation, in contrast to Map A in the open state (Figure 2). Additionally, the proposed 28S rRNA mimicry strategy supported by structural superposition with the 80S ribosome and sequence similarity between the I domain of the IRES and the h38 region of 28S rRNA (Fig. 4) is well-justified.

We agree that the low resolution of the map has compromised the data interpretation at the molecular level, and we thank the reviewer for appreciating our findings at this resolution. Due to the low resolution, we have reported findings for stretches or regions such as the domain I loops and stems, rather than individual nucleotides.

(2) The lack of experimental validation of the functional importance of regions like the GNRA and RAAA loops is another limitation of this study.

We agree about the lack of additional experiments other than Cryo-EM for probing the importance of regions such as GNRA and RAAA loops in this study. Previously, multiple studies have reported on the importance of GNRA and RAAA loops and we have cited them in the manuscript. The essentiality of RAAA loop for type 2 IRES was demonstrated in earlier report López de Quinto and Martínez-Salas, 1997 (Cited in manuscript). Further, the conservation of this loop across the type 2 IRES family adds to the importance of this loop (Manuscript Figure 6B). This loop and its flanking G-C stem are similar to h38 of 28S rRNA, and it appears that RAAA loop adopts a mimicry mechanism to interact with the 40S ribosomal protein- uS19, thus highlighting its importance for interaction with 40S. Experiments destabilising the G-C stem also compromise IRES activity, as shown for the case of FMDV IRES (Fernández et al 2011). Previous studies related to the mutation of the GNRA or GCGA loop in EMCV IRES have shown a deficiency in IRES activity (Roberts and Belsham, 1997; Robertson et al 1999), suggesting the importance of these regions in the viral IRES biology, and these reports are cited in the manuscript. Not only EMCV IRES, but mutation in the GUAA (representative of GNRA) loop of FMDV IRES also showed a significant reduction in IRES activity (López de Quinto and Martínez-Salas, 1997). In this work, we observe that the GCGA loop interacts with tRNA_i in the EMCV IRES-48S PIC, thus implicating the importance of this loop. Moreover, incubation of FMDV IRES with 40S ribosomes has shown a decrease in SHAPE reactivity in domain 3 apex (position 170- 200 nucleotides) (Lozano et al 2018), which corresponds to EMCV IRES domain I apex.

However, to address this concern in the revised manuscript we mutated these loops and performed luciferase assay (Supplementary figure 4 A). The results showed decreased IRES activity (Pg 10) and correlated with previous reports demonstrating the importance of these regions for overall IRES activity.

(3) Minor modifications related to data processing and biochemical studies will further validate and strengthen the findings.

(a) In the cryo-EM data section, the authors should include an image showing rejected particles during 2D classification. This would help readers understand why, despite having over 22k micrographs with sufficient particle distribution and good contrast, only a smaller number of particles were used in the final reconstruction. Additionally, employing map-sharpening tools such as Ewald sphere correction, Bayesian polishing, or reference-based motion correction might further improve the quality of the maps. Targeting high-resolution structures would be particularly informative.

We have included the image for rejected 2D classes (Author response image 1). We agree with the Reviewer’s query related to the huge number of micrographs and relatively smaller number of particles for the final reconstruction. Since the total number of micrographs (22000) is the summation of multiple datasets, prepared and collected at different times, the distribution of the particles per micrograph was not uniform in all sessions, ranging from good to poor. Among these, around 8000 micrographs have poor particle number and distribution. As a result, the number of particles per micrograph is heterogeneous across the compiled dataset, and only 237054 ribosomal particles were obtained after multiple rounds of 2D and 3D classification. Further, the final reconstruction was performed using particles obtained after masked classification for IRES and ternary complex density. Only the particles that show the best density for both IRES and ternary complex are used for this map. Another set of particles that have only a portion of IRES and NO density for ternary complex forms another map. And we have a third map with an empty 40S.

We thank the reviewer for the suggestions to improve the quality of the maps further. As suggested, we started with the processing of the data. However, during this process the common computational cluster that were using for this data processing had to be physically relocated, and unfortunately after the relocation we faced technical issues in accessing and continuing with the processing. Several attempts to resolve the issue with the help of IT team failed. Thus, we lost 3-4 months without any progress. Therefore, we used Relion on our in-house workstation to process the data files from the start, as our in-house computational resources are unequipped to run cryoSPARC processes (for large dataset due to memory limitations).

We reprocessed the datasets in Relion5 and did ‘Bayesian Processing’, for reference-based beam-induced motion correction per-particle. Post-processing, we used cryoSPARC to merge the particles and tried classifying the good ribosome particles using focus-based masked classification, as shown in Supplementary Figure 1.1. However, this processing did not improve the resolution, as Map B (containing 40S, tRNA, IRES) had an overall resolution of 4.8 Å (Author response image 2). Therefore, we would like to report the same maps as given in the initial submission.

We estimated the time to redo the entire processing using cryoSPARC on the common computational cluster, and it would take us another 3-4 months or more and we do not anticipate a massive improvement in the extra density.

Author response image 1.

The selected 2D classes and the rejected 2D classes from initial round of classification, and the final selected 2D classes, which were subjected to Ab-initio reconstruction to get the good ribosome particles.

Author response image 2.

Reprocessing of the entire dataset using Relion5 for polishing of selected particles, followed by 3D classification and refinements in cryoSPARC.

(b) The strategic modelling of different IRES domains into the density, particularly the domain into the region above the 40S head, is appreciable. However, providing the full RNA tertiary structure (RNAfold) of the EMCV IRES (nucleotides 280-905) would better explain the logic behind the model building and its molecular interpretation.

We thank the reviewer for appreciating the modelling of the domain I apex in the cryo-EM density. We tried to predict the full tertiary structure of the IRES using Alphafold3; however, inclusion of the full-length sequence from 280-905 gave models of extremely low confidence (Author response image 3), and a few domains do not abide by the secondary structure of EMCV IRES as reported in Duke et al 1992.

Author response image 3.

Prediction of tertiary structure of EMCV IRES (280-905 nucleotides) and zoomed features for each domain present in the IRES. The predicted aligned error plot for the RNA structure is shown.

We used individual domains of EMCV IRES and predicted the tertiary structure, independent of other IRES domain using Alphafold3. As a result, the confidence scores improved, and the tertiary structures also correlated with the experimentally determined EMCV IRES secondary structure (Duke et al 1992; Maloney and Joseph, 2024). Although the overall tertiary structure of EMCV IRES is lacking, recent studies were able to solve the structures of EMCV IRES domains in complex with their respective binding proteins. We superimposed the independently predicted domains D, E, and F tertiary structure on the NMR ensemble of IRES domain D to F with PTB1 (Dorn et al 2023), where the predicted domains fit in the experimental model. Similarly, we used the cryo-EM structure of domain J-K-eIF4G-eIF4A (Imai et al 2023) and found a close fit with the predicted structures. The analysis highlighted that the domain I apex serves as the best fit with the extra density with respect to architecture and fitting. This analysis is now added in the revised manuscript in Supplementary figure- 3.2.

Furthermore, 3D structural models of FMDV IRES domains 2, 3, and 4 (corresponding to EMCV IRES domains- H, I, and J-K) were predicted from SHAPE reactivity values and RNAComposer server (Figure 3, Lozano et al 2018). The predicted architecture of domain 3 apex (FMDV IRES) coincides with our domain I apex model (EMCV IRES).

(c) Although the authors compare their findings with other types of IRESs (Types 1, 3, and 4), there is no experimental validation of the functional importance of regions like the GNRA and RAAA loops. Including luciferase-based assays or mutational studies of these regions for validation of structural interpretations is strongly recommended.

We have discussed the possibility of how the other IRESs, such as type 1 and type 5, might use similar strategies as EMCV IRES to assemble the 48S PIC, given the similarity in the motif sequence and position across the viral IRESs. Like EMCV IRES, the type 1 IRES (Poliovirus, Coxsackie virus, etc.) also harbours the GNRA loop, preceded by a C-rich loop at its longest domain, known for long-range RNA-RNA interactions. The segment harbouring GNRA loop is highly conserved across the type 1 family of IRESs (Kim et al 2015). The Aichi viral IRES harbours a GNRA loop in its longest domain, that is, domain J. Deletion of the GNRA loop has compromised the IRES activity; however, substitution mutations in this region have elevated the IRES activity or remained unaltered (Yu et al 2011). We have hypothesized that these IRESs might use the GNRA motifs in their longest domain (domain IV in type 1, and domain J in Aichi virus- type 5) based on the location and architecture to that of EMCV IRES, where GNRA is present in the longest domain (I) and preceded by a C-rich loop where it can potentially mediate long-range interactions with tRNA_i, as all these IRESs require eIF2-ternary complex for the formation of 48S PIC. Parallelly, like EMCV IRES, type 1 and type 5 IRESs have the placement of this GNRA motif-containing domain before the eIF4G-binding domain. Thus, we suggest the possibility of adoption of a similar strategy by these IRESs to interact with tRNA_i during the formation of 48S PIC. During the revision of this work a preprint reported the structure of polioviral IRES-48S PIC where domain IV apex (similar to domain I apex in EMCV IRES) interacts with uS13 and uS19, and the GNRA loop directly interacts with tRNA_i during start codon recognition (Velazquez et al 2025). We hypothesize that Aichiviral IRES might use this motif to mediate long-range interactions with tRNA_i, similar to type 1 and type 2 IRESs, as all these IRESs require eIF2-ternary complex for the formation of 48S PIC.

Reviewer #2 (Public review):

Summary:

The field of protein translation has long sought the structure of a Type 2 Internal Ribosome Entry Site (IRES). In this work, Das and Hussain pair cryo-EM with algorithmic RNA structure prediction to present a structure of the Type 2 IRES found in Encephalomyocarditis virus (EMCV). Using medium to low resolution cryo-EM maps, they resolve the overall shape of a critical domain of this Type 2 IRES. They use algorithmic RNA prediction to model this domain onto their maps and attempt to explain previous results using this model.

Strengths:

(1) This study reveals a previously unknown/unseen binding modality used by IRESes: a direct interaction of the IRES with the initiator tRNA.

(2) Use of an IRES-associated factor to assemble and pull down an IRES bound to the small subunit of the ribosome from cellular extracts is innovative.

(3) Algorithmic modeling of RNA structure to complement medium to low resolution cryo-EM maps, as employed here, can be implemented for other RNA structures.

We thank Reviewer 2 for positive and encouraging comments on our work, appreciating our ‘innovative’ approach of using IRES-associated factor to assemble and pull down the IRES-bound ribosomal complex.

Weaknesses:

(1) Maps at the resolution presented prevent unambiguous modelling of the EMCV-IRES. This, combined with the lack of any biochemical data, calls into question any inferences made at the level of individual nucleotides, such as the GNRA loop and CAAA loop (Figure 4).

We understand the concerns raised by the reviewer related to the resolution of the EMCV IRES-48S PIC map. We refrained from commenting on individual nucleotides or molecular interactions in the manuscript. Instead, we discuss loops, RNA stretches or motifs that could be inferred with more confidence in the IRES density as shown in Figure 4. The EMCV IRES can directly interact with the 40S ribosome using its domain H and I (Chamond et al 2014), however, the details of this interaction were unknown. We observe that the CAAA loop of domain I apex interacts with 40S ribosome based on the placement of a portion of domain I in the cryo-EM map. This is also reflected in the SHAPE data (Chamond et al 2014-Supplementary figures 2, and 8), where a decrease in reactivity is evident in the presence of 40S ribosome. In addition, incubation of EMCV IRES with rabbit reticulocyte lysate (RRL) offered protection to domain I apex regions, which included the CAAA loop (Maloney and Joseph, 2024- Figure 4b).

Furthermore, this decrease in SHAPE reactivity pattern is evident for FMDV IRES domain 3 apex (similar to domain I in EMCV IRES) in the presence of 40S ribosome (Lozano et al 2018). Thus, these studies are consistent with the placement of IRES model in the cryo-EM map. Moreover, we performed structural analysis (mentioned above) which showed that the domain I apex serves as the best fit with the extra density with respect to architecture and fitting (Supplementary figure- 3.2).

(2) The EMCV IRES contains an upstream AUG at position 826, where the PIC can assemble (Pestova et al 1996; PMID 8943341). It is unclear if this start codon was mutated in this study. If it were not mutated, placement of AUG-834 over AUG-826 in the P-site is unexplained.

We thank the reviewer for bringing up this point, as we missed mentioning this in the initial submission. The EMCV IRES does not require scanning and directly positions the AUG-834 at the P site (Pestova et al 1996). In Pestova et al 1996, the intensity of the toeprint at AUG-834 is more intense than that of AUG-826. Further, AUG-834 lies in the Kozak context, whereas AUG-826 has a poor Kozak context, and AUG-826 codon is not in-frame with AUG-834. Therefore, the synthesis of the polypeptide requires AUG-834 at the P site. In our cryo-EM map, we observed that the tRNA_i is in a P_IN state, which indicates the recognition of the start codon, and we reasoned that it is more likely that AUG-834 is placed at the P site than AUG-826. We have mentioned this in the revised manuscript as we had NOT mutated AUG-826 (Pg 8).

(3) The claims the authors make about (i) the general overall shape and binding site of the IRES, (ii) its gross interaction with the two ribosomal proteins, (iii) the P-in state of the 48S, (iv) the rearrangement of the ternary complex are all warranted. Their claims about individual nucleotides or smaller stretches of the IRES-without any supporting biochemical data-is not warranted by the data.

We thank the reviewer for warranting major claims, and due to the low-resolution we have reported findings for stretches or regions such as the domain I loops and stems, rather than individual nucleotides. The interaction of domain I apical region with uS13, uS19, and tRNA_i is also observed the high-resolution structure of reconstituted EMCV IRES-48S PIC that was reported in a preprint while our work was under peer review process (Bhattacharjee et al 2025). Thus, the reconstituted EMCV IRES-48S PIC (Bhattacharjee et al 2025) also supports our assignment of domain I and its conserved loops, interacting with ribosome and tRNA_i.

Reviewer #3 (Public review):

Summary:

Type II IRES, such as those from encephalomyocarditis virus (EMCV) and foot-and-mouth disease virus (FMDV), mediate cap-independent translation initiation by using the full complement of eukaryotic initiation factors (eIFs), except the cap-binding protein eIF4E. The molecular details of how IRES type II interacts with the ribosome and initiation factors to promote recruitment have remained unclear. Das and Hussain used cryo-electron microscopy to determine the structure of a translation initiation complex assembled on the EMCV IRES. The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Strengths:

The structure reveals a direct interaction between the IRES and the 40S ribosomal subunit, offering mechanistic insight into how type II IRES elements recruit the ribosome.

Weaknesses:

While this reviewer acknowledges the technical challenges inherent in determining the structure of such a highly flexible complex, the overall resolution remains insufficient to fully support the authors' conclusions, particularly given that cryo-EM is the sole experimental approach presented in the manuscript.

The study is biologically significant; however, the authors should improve the resolution or include complementary biochemical validation.

We thank Reviewer 3 for acknowledging the technical challenges in this study and finding our study biologically significant. We understand the concerns related to low resolution and the requirement of complementary biochemical validation for our reported observations and interpretations in the manuscript. We tried to improve the resolution, but the improvement was not sufficient to resolve the IRES at the nucleotide level. Independently, another group has reported the same findings at a higher resolution while our work was under peer review process (Bhattacharjee et al 2025), which corroborates our structural data on EMCV IRES and its interaction with ribosome and tRNA_i in its 48S PIC stage. Further, in the revised manuscript we also present biochemical validation for GNRA and RAAA loops in EMCV IRES. We mutated these loops and performed luciferase assay (Supplementary figure 4 A). The results showed decreased IRES activity (Pg 10) and correlated with previous reports (Roberts and Belsham, 1997; López de Quinto and Martínez-Salas, 1997; Robertson et al 1999) demonstrating the importance of these regions for overall IRES activity.

Reviewing Editor Comments:

The reviewers' comments are appended. While the reviewers acknowledge the complexity associated with this system, they also raised concerns about the modeling of RNA and registering its sequence in low-resolution maps. We believe that the strength of evidence and overall impact of your study can be elevated by providing higher-resolution cryo-EM data or complementary biochemical studies and addressing reviewers' concerns.

Reviewer #2 (Recommendations for the authors):

(1) Science:

Have the authors tried a focused refinement (local refinement in cryoSPARC) using a generous mask that encloses the head and the IRES but excludes the ternary complex and the body of the 40S? This can be done with all the particles in map B (~55K) and has the possibility of improving the resolution of domain I which can be subsequently used to build a better model of the IRES. See the middle right panel, light yellow colored mask in Figure 1A in PMID 37659578 for the type of mask being suggested.

We did another round of 2D classification to eliminate any residual junk in the ~55k particle set, corresponding to Map B. Post classification, 49439 particles were selected and refined using non-uniform refinement to get Map B11. The overall resolution of Map B11 was 4.6 Å. Thereafter, we made a mask around the 40S head-IRES-tRNA on Map B11 and subjected the class for local refinement. The overall local resolution in the masked region improved to 4.5 Å (Author response image 4).

Author response image 4.

Data processing- Map B particles were 2D classified, and further junk was cleared as rejected particles. The selected particles were refined using non-uniform refinement to get Map B11, and later, a focused mask circling the head-tRNA-IRES region was used for local refinement in the region to yield map B111.

We estimated the local resolution across the focused region in Map B111 and compared this with that of Map B (Author response image 5). The local refinement shows minor improvement in the local resolution in this region, and is not sufficient to resolve the IRES density at the level of nucleotides.

Author response image 5.

Comparison of local resolution across head-IRES-tRNA in map B1 (as reported in the manuscript) and Map B111.

(2) Presentation:

(a) Please use the previously established convention of naming the domains: "domain I", "domain H", etc, instead of "I domain" or "J-K domain" while describing parts of the IRES.

We have made the changes as per the established convention.

(b) Figure 2B reports a 6.9 A distance vs. 7 A in the text. Please use ~ or approximately to keep numbers consistent.

We have used ~ symbol to suggest the approximate distance.

(c) References missing on page 15 when referring to "previously determined HCV and CrPV structures".

We have added the references (Pg 12).

(d) Please edit the text for typos and sentence structure.

The typos and sentence structure were corrected wherever necessary.

(e) Some phrases and sentences (e.g. last few sentences of the first paragraph in the discussion) could be rewritten for clarity.

Previous sentence- “The domain I of EMCV IRES is similar to domain IV of polioviral IRES (or other type 1 IRESs such as Coxsackie viral IRES) in terms of length, secondary structure, and conserved motifs (GNRA, C-rich) positioning (Fig. 6C), therefore, anticipating a similar interaction with tRNA_i, highlighting a sequestering tendency by competing with cellular mRNAs.”

Rephrased sentence- “Like EMCV IRES, the type 1 IRES (Poliovirus, Coxsackie virus, etc.) also harbours the GNRA loop, preceded by a C-rich loop at its longest domain, known for long-range RNA-RNA interactions. The segment harbouring GNRA loop is highly conserved across the type 1 family of IRESs (Kim et al 2015). The domain I of EMCV IRES is similar to domain IV of polioviral IRES or other type 1 IRESs in terms of length, secondary structure, and conserved motifs (GNRA, C-rich) positioning (Fig. 6C). Therefore, we anticipate a similar interaction of domain IV (in type 1 IRES class) with tRNA_i. Also, this interaction of IRES with tRNA_i could be a strategy by which these IRESs can sequester the tRNA_i pool in the cell, rendering them unavailable for capped cellular mRNAs.”

Reviewer #3 (Recommendations for the authors):

(1) For the revision process, the authors provided three atomic models alongside their corresponding cryo-EM density maps, including a 48S complex in closed conformation. Given this conformation, it is reasonable to interpret the structure as representing a post-start codon recognition state (late-stage initiation). However, this reviewer finds that the local resolution within the mRNA channel is insufficient to support the atomic model building as presented. The density does not allow for an unambiguous assignment of nucleotides in this region; the authors should either improve the local resolution or remove the modeled mRNA from the structure.

We understand the concern of the Reviewer. Although the mRNA density in the channel is poor, we modelled the mRNA with AUG-834 at the P site because the known biology of EMCV IRES. The EMCV IRES does not require scanning and directly positions the AUG-834 at the P site (Pestova et al 1996). In Pestova et al 1996, the intensity of the toeprint at AUG-834 is more intense than that of AUG-826. Further, AUG-834 lies in the Kozak context, whereas AUG-826 has a poor Kozak context, and AUG-826 codon is not in-frame with AUG-834. Therefore, the synthesis of the polypeptide requires AUG-834 at the P site. In our cryo-EM map, we observed that the tRNA_i is in a P_IN state, which indicates the recognition of the start codon, and we reasoned that it is very likely that AUG-834 is placed at the P site.

(2) As noted by the authors, the start codon in the EMCV IRES is positioned within a strong Kozak sequence. The nucleotide at position -3 is known to interact with eIF2α, yet, in the current model, A831 is positioned such that physical contact with eIF2α would be structurally impossible. This discrepancy raises concerns about the accuracy of the modeled eIF2α, which, like other regions of the structure, is not clearly supported by the cryo-EM density. The authors should revise the atomic model of eIF2α to ensure it is consistent with the experimental map and established molecular interactions.

In our analysis of EMCV IRES-48S PIC, we could observe eIF2α and eIF2γ in Map B and B1. However, the local resolution was low to model the entire protein with side-chains (Supplementary figure 1.2 A). So, we used rigid body fitting of eIF2α and eIF2γ (Author response image 6). From the model, we could trace the backbone of Arg55, however could not resolve the side chain. Similarly, the mRNA in the channel was modelled based on placement of AUG-834 at the P site for EMCV IRES, which enabled us to model the flanking residues, rather than at the nucleotide-level resolution. We anticipate that a higher resolution structure will be able to capture this interaction of eIF2α with mRNA nucleotide (-3), therefore refrained from commenting on this interaction in the manuscript. In the revised manuscript, we have removed the side chains of eIF2α and eIF2γ, and kept the Cα-backbone only. The map-model statistics of map B1 is updated in table 1.

Author response image 6.

(left) Fitting of eIF2α model in the map. (right) Fitting of Cα backbone of eIF2α and mRNA in the map.

(3) The authors observed additional density interacting with ribosomal proteins uS19 and uS13, and tRNA, which they tentatively assign to domain I of the IRES. Although the local resolution in this region does not allow an unambiguous assignment, the interpretation is reasonable. However, further structural and functional validation is necessary to support this assignment. The authors should improve the local resolution, either by performing focused refinement or by increasing the number of particles used in the reconstruction.

The assignment of the extra density to domain I of the IRES was based on the architecture of the density. This density allows no other IRES domain to fit in this region (Supplementary figure 3.2). We tried to improve the local resolution using focused refinement, but the resolution was insufficient to resolve the IRES at the nucleotide level. Please see the above-mentioned comments in this regard on Pg 12.

(4) Figure 5 shows a slight shift in the position of the ternary complex. Is the observed tRNA conformation compatible with the structural rearrangements required for 60S subunit joining?

During the transition of 48S PIC to 80S elongation-competent complex, there are major changes in the conformation of tRNA_i, due to the joining of eIF5B, and release of eIF2 (Petrychenko et al 2024). This joining event of eIF5B positions the tRNA_i elbow and acceptor stem towards the 40S body to aid 60S ribosomal subunit joining (Petrychenko et al 2024). However, in the context of EMCV IRES-48S PIC, we observed that the position of tRNA_i elbow and acceptor stem is towards the 40S head, and away from the body. On superimposing the human 48S PIC structure (before 60S joining), 48S-5 (PDB Id- 8PJ5- Petrychenko et al 2024), we note that tRNA_i in EMCV IRES-48S PIC is away from the canonical tRNA_i position (in contact with eIF5B). Therefore, we anticipate a change in tRNA_i conformation during eIF5B joining and eIF2 release. This hypothesis coincides with the fact that the IRES interacting with the tRNA_i elbow needs to be displaced from the position to facilitate the interaction of tRNA_i with eIF5B. Moreover, this rearrangement would also aid in 60S joining and prevent any clash with the IRES domain I. We have added this in Results selection 5 and Figure 5D.

(5) In the discussion section, the authors state: "eIF3-eIF4G interaction is dispensable for EMCV IRES-48S PIC formation, so we do not rule out the possibility that EMCV IRES may dislodge eIF3 from its position on the solvent surface as observed in the case of HCV IRES (Hashem et al, 2013)." This statement is highly speculative. Is there any experimental or structural evidence to support this proposed mechanism in the context of EMCV IRES?

Previous biochemical reports on the eIF3-eIF4G interaction suggested that eIF4G residues from 1011-1104 interact with eIF3 (Villa et al 2013). In the context of EMCV IRES, this region of eIF4G is not required to form 48S PIC on the IRES, suggesting the eIF3-eIF4G interaction is dispensable for EMCV IRES-48S PIC formation. However, the recent structure of the human canonical 48S PIC has shown that the eIF4G-HEAT1 domain can interact with eIF3 subunits c, h, and l, and that eIF4G-bound eIF4A can interact with 40S ribosomal protein eS7, thus mediating the interaction between eIF4-bound mRNA and the 43S PIC (Brito Querido et al 2024) but the known eIF3-binding region in eIF4G was not captured in the map. Although the canonical eIF3-eIF4G interaction is essential in the case of cap-dependent initiation, this interaction could be dispensable for 48S PIC formation on EMCV IRES. In case of HCV IRES-mediated initiation, eIF3 is displaced from its canonical position that facilitates the binding of HCV IRES to 40S ribosomal subunit (Hashem et al 2013). We did not see any density corresponding to eIF3 in the obtained maps. Further, we have used focused classification using a mask on the canonical eIF3 position; however, we do not see any density corresponding to eIF3 in the EMCV IRES-48S PIC complex. Therefore, we hypothesized the possibility that eIF3 might be dislodged from its canonical binding site on the 40S ribosomal subunit. However, as per the recent independent report on EMCV IRES-48S PIC, eIF3 is present in the complex (Bhattarcharjee et al 2025).

Hence, we have rephrased the existing sentence- “However, eIF3-eIF4G interaction is dispensable for EMCV IRES-48S PIC formation, so we do not rule out the possibility that EMCV IRES may dislodge eIF3 from its position on the solvent surface as observed in case of HCV IRES (Hashem et al 2013).”

Rephrased sentence- “However, the canonical eIF3-eIF4G interaction (Villa et al 2013) is dispensable for EMCV IRES-48S PIC formation (Lomakin et al 2000; Sweeney et al 2014), and we do not see any density for eIF3 even after focused classification. However, as per the recent independent report on reconstituted EMCV IRES-48S PIC, eIF3 is present in the complex at the canonical position (Bhattarcharjee et al 2025). This position of eIF3 further highlights the possibility that eIF4G-eIF4A proteins are also placed similarly to the canonical eIF3-eIF4G-eIF4A position (Brito Querido et al 2024) in context to EMCV IRES-48S PIC. Thus, placing eIF4G-domain J-K close to ES6 of 40S ribosome, which coincides with the previous hydroxyl radical cleavage assay (Yu et al 2011).”

(6) eIF4A has been shown to directly interact with eIF3 and facilitate recruitment of the 43S PIC. Does the interaction of the J-K domain with eIF4G/eIF4A, compatible with the known eIF4A-eIF3 interaction within the 43S PIC? In other words, during EMCV IRES-mediated initiation, could the eIF4A-eIF3 interaction functionally substitute for the eIF4G-eIF3 interaction?

Reports on EMCV IRES-mediated translation initiation have shown eIF4G as an essential component of 48S PIC formation (Pestova et al 1996; Lomakin et al 2000; Kolupaeva et al 2003; Sweeney et al 2014), where eIF4G directly interacts with domain J-K of IRES and eIF4A, thus enabling loading of eIF4A on the IRES. In our study, the cryo-EM map of EMCV IRES-48S PIC lacks density for eIF3 and eIF4 proteins, and locating eIF4F is challenging due to the inherent flexibility associated with the complex. Previous studies on EMCV IRES-48S PIC have mapped the location of eIF4G close to ES6 towards the platform side of the body and eIF3 using the hydroxyl radical cleavage assay (Yu et al 2011). The human 48S initiation complex structures have shown a similar location for eIF4G, which is at the mRNA exit site, contacting eIF3 (Brito Querido et al 2020; Brito Querido et al 2024). On overlapping the 18S rRNA of EMCV IRES-48S PIC to that of the human 48S PIC in closed conformation (PDB Id- 8OZ0), and further superimposing the J-K-St- eIF4G- eIF4A (PDB Id- 8HUJ) on human 48S PIC (PDB Id- 8OZ0) with respect to HEAT1 of eIF4G, the domain J-K becomes positioned at the subunit face of 40S body, close to ES6 (Author response image 7). This correlates with the previously reported position for eIF4G with respect to EMCV IRES-48S PIC (Yu et al 2011). The predicted model shows no clashes with the canonical eIF4A-eIF3/ eIF4G-eIF4A-eIF3 interaction, or with the domain J-K-eIF4G-eIF4A model. Thus, highlighting a possibly compatible interaction axis among eIF3-eIF4G-eIF4A-domain J-K of IRES.

Author response image 7.

(upper left) Location of eIF4G-eIF4A in canonical human 48S PIC (PDB Id- 8OZ0). (upper right) Superimposition of 18S rRNA from human 48S and EMCV IRES 48S. (lower left) Superimposition of Human Closed 48S PIC structure (PDB Id- 8OZ0) on EMCV IRES-48S PIC model and placement of EMCV IRES- J-K domain-HEAT1-eIF4A structure (PDB Id- 8HUJ) with respect to eIF4G-HEAT1 domain. (lower right) Predicting location of eIF3 and eIF4 proteins in EMCV IRES-48S PIC.

(7) Assuming that the additional density near the ternary complex corresponds to Domain I of the IRES and that the codon in the P site represents the EMCV AUG start codon, what is the authors' mechanistic model for EMCV IRES-mediated initiation? Specifically, how is the mRNA positioned or inserted into the 40S mRNA channel in the absence of canonical scanning? As it stands, the discussion does not sufficiently address this key aspect of the EMCV initiation mechanism.

The EMCV IRES start codon (A-834) is directly placed in the P site (Pestova et al 1996), and the captured complex harboured the initiator tRNA in P_IN state with AUG at the P site. This start codon is preceded by domains J-K-L, where the J-K domain interacts with eIF4 proteins via eIF4G1-HEAT1 domain, and L domain is 20 residues upstream of the AUG and known to interact with eIF4B (Pestova et al 1996; de Quinto et al 2001). Based on the position and binding partners for these domains, the domain L could be placed at the mRNA exit site, preceded by domain J-K, which could be placed close to eIF4G-eIF4A position on EMCV IRES 48S PIC, near expansion segment 6 (ES6). The domain J-K can interact with eIF4G, localized close to the left foot or ES6 as per previous biochemical experiments (Yu et al 2011). This suggests that position of eIF4G and eIF4A could be the same as that of cap-dependent initiation where it can interact with eIF3 core subunits as well as the IRES domain J-K and the predicted path of mRNA from the exit site can follow the path of mRNA in human closed 48S PIC (PDB Id- 8OZ0), where it interacts with eIF3 core.

Examining the path of RNA in channel from the G-825 (exit site) to C-785 (domain J-K), we found the shortest distance is ~ 173 Å. This bridge could be filled by a single-stranded stretch of 40 nucleotides. However, the presence of domain L (stem loop- residues- 782 to 810) might hinder the placement of A-834 in the P-site (Author response image 8). We anticipate that to accommodate the start codon at the P site, either the domain L stem loop is resolved, which is an energetically expensive process (free energy of the thermodynamic ensemble is -11.12 kcal/mol, predicted using RNAfold). Another way could be a change in the orientation or conformation of domain J-K such that the start codon is directly placed at the P site without resolving domain L.

Author response image 8.

(left) The shortest distance between the last fitted residue- 825th of EMCV IRES to 785th of J-K domain of IRES (keeping eIF4G position same as that of PDB Id- 8OZ0) is 173 Å. (right) Tracing the path of mRNA (red) upstream of AUG coming out of the exit site of 40S ribosome and the possible position of eIF4G on EMCV IRES-48S PIC. Addition of nucleotides between C-785 and G-825 would fill the gap. The route of predicted mRNA from the exit channel is based on the mRNA (green) exiting the channel (PDB Id- 8OZ0).

The domain I is followed by domain J-K, close to the left foot of the 40S ribosomal subunit as per previous biochemical experiments (Yu et al 2011). However, the minimum distance connecting the I domain at 601st nucleotide to 682nd nucleotide of domain J-K (at the predicted location) is ~300 Å, which might be difficult to be covered by 80 nucleotides (from 601 to 682), present as a double helical strand. We suppose there could be instances of J-K domain repositioning in the EMCV IRES-48S PIC such that the I domain apical region can contact the 40S head and simultaneously place the start codon at the P site (Author response image 9).

Author response image 9.

Rotated views of EMCV IRES domains- I apical part in contact with 40S head and tRNAi and predicted location of J-K domain in contact with eIF4G, close to the left foot of 40S (predicted from PDB Id- 8OZ0). The minimum distance connecting 601st nucleotide in I domain to 682nd nucleotide in J-K domain is 295.5 Å.

We lack any details on the other IRES domains, such as domain I lower stem, domain J-K, or L; therefore, we refrained from commenting on these in our manuscript.

(8) Supplementary Figure 1 is missing labels for the RNA ladders.

The size of the DNA ladder used is mentioned.

References:

Bhattacharjee S, Abaeva IS, Brown ZP, Arhab Y, Fallah H, Hellen CUT, Frank J, Pestova TV. The mechanism of ribosomal recruitment during translation initiation on Type 2 IRESs. bioRxiv [Preprint]. 2025 Jun 11:2025.06.11.659010. doi: 10.1101/2025.06.11.659010. PMID: 40568087; PMCID: PMC12191231.

Brito Querido J, Sokabe M, Díaz-López I, Gordiyenko Y, Fraser CS, Ramakrishnan V. The structure of a human translation initiation complex reveals two independent roles for the helicase eIF4A. Nat Struct Mol Biol. 2024 Mar;31(3):455-464. doi: 10.1038/s41594-023-01196-0. Epub 2024 Jan 29. PMID: 38287194; PMCID: PMC10948362.

Brito Querido J, Sokabe M, Kraatz S, Gordiyenko Y, Skehel JM, Fraser CS, Ramakrishnan V. Structure of a human 48S translational initiation complex. Science. 2020 Sep 4;369(6508):1220-1227. doi: 10.1126/science.aba4904. PMID: 32883864; PMCID: PMC7116333.

Chamond N, Deforges J, Ulryck N, Sargueil B. 40S recruitment in the absence of eIF4G/4A by EMCV IRES refines the model for translation initiation on the archetype of Type II IRESs. Nucleic Acids Res. 2014;42(16):10373-84. doi: 10.1093/nar/gku720. Epub 2014 Aug 26. PMID: 25159618; PMCID: PMC4176346.

Dorn G, Gmeiner C, de Vries T, Dedic E, Novakovic M, Damberger FF, Maris C, Finol E, Sarnowski CP, Kohlbrecher J, Welsh TJ, Bolisetty S, Mezzenga R, Aebersold R, Leitner A, Yulikov M, Jeschke G, Allain FH. Integrative solution structure of PTBP1-IRES complex reveals strong compaction and ordering with residual conformational flexibility. Nat Commun. 2023 Oct 13;14(1):6429. doi: 10.1038/s41467-023-42012-z. PMID: 37833274; PMCID: PMC10576089.

Duke GM, Hoffman MA, Palmenberg AC. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. J Virol. 1992 Mar;66(3):1602-9. doi: 10.1128/JVI.66.3.1602-1609.1992. PMID: 1310768; PMCID: PMC240893.

Fernández N, Fernandez-Miragall O, Ramajo J, García-Sacristán A, Bellora N, Eyras E, Briones C, Martínez-Salas E. Structural basis for the biological relevance of the invariant apical stem in IRES-mediated translation. Nucleic Acids Res. 2011 Oct;39(19):8572-85. doi: 10.1093/nar/gkr560. Epub 2011 Jul 8. PMID: 21742761; PMCID: PMC3201876.

Hashem Y, des Georges A, Dhote V, Langlois R, Liao HY, Grassucci RA, Pestova TV, Hellen CU, Frank J. Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit. Nature. 2013 Nov 28;503(7477):539-43. doi: 10.1038/nature12658. Epub 2013 Nov 3. PMID: 24185006; PMCID: PMC4106463.

Imai S, Suzuki H, Fujiyoshi Y, Shimada I. Dynamically regulated two-site interaction of viral RNA to capture host translation initiation factor. Nat Commun. 2023 Aug 28;14(1):4977. doi: 10.1038/s41467-023-40582-6. PMID: 37640715; PMCID: PMC10462655.

Kim H, Kim K, Kwon T, Kim DW, Kim SS, Kim YJ. Secondary structure conservation of the stem-loop IV sub-domain of internal ribosomal entry sites in human rhinovirus clinical isolates. Int J Infect Dis. 2015 Dec;41:21-8. doi: 10.1016/j.ijid.2015.10.015. Epub 2015 Oct 27. PMID: 26518063.

Lomakin IB, Hellen CU, Pestova TV. Physical association of eukaryotic initiation factor 4G (eIF4G) with eIF4A strongly enhances binding of eIF4G to the internal ribosomal entry site of encephalomyocarditis virus and is required for internal initiation of translation. Mol Cell Biol. 2000 Aug;20(16):6019-29. doi: 10.1128/mcb.20.16.6019-6029.2000. PMID: 10913184; PMCID: PMC86078.

López de Quinto S, Martínez-Salas E. Conserved structural motifs located in distal loops of aphthovirus internal ribosome entry site domain 3 are required for internal initiation of translation. J Virol. 1997 May;71(5):4171-5. doi: 10.1128/JVI.71.5.4171-4175.1997. PMID: 9094703; PMCID: PMC191578.

Lozano G, Francisco-Velilla R, Martinez-Salas E. Ribosome-dependent conformational flexibility changes and RNA dynamics of IRES domains revealed by differential SHAPE. Sci Rep. 2018 Apr 3;8(1):5545. doi: 10.1038/s41598-018-23845-x. PMID: 29615727; PMCID: PMC5882922.

Maloney A, Joseph S. Validating the EMCV IRES Secondary Structure with Structure-Function Analysis. Biochemistry. 2024 Jan 2;63(1):107-115. doi: 10.1021/acs.biochem.3c00579. Epub 2023 Dec 11. PMID: 38081770; PMCID: PMC10896073.

Pestova TV, Hellen CU, Shatsky IN. Canonical eukaryotic initiation factors determine initiation of translation by internal ribosomal entry. Mol Cell Biol. 1996 Dec;16(12):6859-69. doi: 10.1128/MCB.16.12.6859. PMID: 8943341; PMCID: PMC231689.

Petrychenko V, Yi SH, Liedtke D, Peng BZ, Rodnina MV, Fischer N. Structural basis for translational control by the human 48S initiation complex. Nat Struct Mol Biol. 2024 Sep 17. doi: 10.1038/s41594-024-01378-4. Epub ahead of print. PMID: 39289545.

Roberts LO, Belsham GJ. Complementation of defective picornavirus internal ribosome entry site (IRES) elements by the coexpression of fragments of the IRES. Virology. 1997 Jan 6;227(1):53-62. doi: 10.1006/viro.1996.8312. PMID: 9007058.

Robertson ME, Seamons RA, Belsham GJ. A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES. RNA. 1999 Sep;5(9):1167-79. doi: 10.1017/s1355838299990301. PMID: 10496218; PMCID: PMC1369840.

Sweeney TR, Abaeva IS, Pestova TV, Hellen CU. The mechanism of translation initiation on Type 1 picornavirus IRESs. EMBO J. 2014 Jan 7;33(1):76-92. doi: 10.1002/embj.201386124. Epub 2013 Dec 15. PMID: 24357634; PMCID: PMC3990684.

Velazquez MA, Nuthalapati SS, Hankinson J, Fominykh K, Lulla V, Sweeney TR, Hill CH. Structural and mechanistic insights into translation initiation on the enterovirus Type 1 IRES. bioRxiv [Preprint]. 2025 Oct 3: 2025.10.04.680434. doi: 10.1101/2025.10.04.680434.

Yu Y, Sweeney TR, Kafasla P, Jackson RJ, Pestova TV, Hellen CU. The mechanism of translation initiation on Aichivirus RNA mediated by a novel type of picornavirus IRES. EMBO J. 2011 Aug 26;30(21):4423-36. doi: 10.1038/emboj.2011.306. PMID: 21873976; PMCID: PMC3230369.

highlight abstract

highlight abstract

highlight abstract

highlight abstract

highlight all definitions here

highlight all definitions here

highlight all definitions here

highlight all definitions here

highlight all definitions here

highlight all definitions here

highlight all definitions here

highlight all definitions here

IMPLICATIONS

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

definitional statements (explicit or implicit) concerning intention and intentionality

examples illustrating the concept of intentionality

examples illustrating the concept of intentionality

examples illustrating the concept of intentionality

examples illustrating the concept of intentionality

examples illustrating the concept of intentionality