ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism detecting alteration of septate junction integrity in Drosophila epithelial cells
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Abstract
Barrier functions of proliferative epithelia are constantly challenged by mechanical and chemical constraints. How epithelia respond to and cope with disturbances of barrier functions to allow tissue integrity maintenance is poorly characterised. Cellular junctions play an important role in this process and intracellular traffic contribute to their homeostasis. Here, we reveal that, in Drosophila pupal notum , alteration of the bi- or tricellular septate junctions (SJs) triggers a mechanism with two prominent outcomes. On one hand, there is an increase in the levels of E-cadherin, F-actin, and non-muscle myosin II in the plane of adherens junctions. On the other hand, β-integrin/Vinculin-positive cell contacts are reinforced along the lateral and basal membranes. We found that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, alters ESCRT-III/Vps32/Shrub distribution, reduces degradation and instead favours recycling of SJ components, an effect that extends to other recycled transmembrane protein cargoes including Crumbs, its effector β-Heavy Spectrin Karst, and β-integrin. We propose a mechanism by which epithelial cells, upon sensing alterations of the SJ, reroute the function of Shrub to adjust the balance of degradation/recycling of junctional cargoes and thereby compensate for barrier junction defects to maintain epithelial integrity.
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Referee #3
Evidence, reproducibility and clarity
This is an interesting manuscript that explores how epithelial cells respond to genetically induced disruption of occluding junction formation. To ask how epithelial integrity is maintained under these conditions, the authors investigated the developing pupal epidermis in Drosophila, where they used genetic mosaic techniques to induce patches of mutant tissue lacking selected components of bicellular or tricellular septate junctions (SJs), respectively. They show that occluding junction defects result in elevated levels of E-Cadherin, F-actin, and activated myosin II at adherens junctions (AJs) in the mutant tissue, suggesting that …
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Referee #3
Evidence, reproducibility and clarity
This is an interesting manuscript that explores how epithelial cells respond to genetically induced disruption of occluding junction formation. To ask how epithelial integrity is maintained under these conditions, the authors investigated the developing pupal epidermis in Drosophila, where they used genetic mosaic techniques to induce patches of mutant tissue lacking selected components of bicellular or tricellular septate junctions (SJs), respectively. They show that occluding junction defects result in elevated levels of E-Cadherin, F-actin, and activated myosin II at adherens junctions (AJs) in the mutant tissue, suggesting that epithelial cells sense breaches in barrier integrity and respond by reinforcing adhesion and actomyosin contractility. Consistent with this idea, the authors find mechanosensitive proteins (Ajuba, Vinculin) enriched at AJs in the mutant cells, and show that new cell-cell interfaces after cytokinesis are shortened in cells lacking the tricellular SJ (tSJ) component Aka. Moreover, aka mutant cells accumulate beta-Integrin, F-actin and vinculin on their basal side, suggesting that upon disruption of tSJs cells increase matrix adhesion by forming focal adhesions (although the authors did not address whether these structures are bona fide focal adhesions that connect to ECM). The authors go on to ask how disruption of SJs is sensed and translated into enhanced adhesion and contractility. Previous work (Pannen et al. eLife 2020) established that the ESCRT complex is required for retromer-dependent delivery of SJ components to their correct membrane destination, and that loss of ESCRT function leads to disruption of SJs. Building on this and their own earlier work, the authors show that SJ defects are accompanied by enlarged ESCRT III (Shrub:GFP)-positive structures, elevated numbers of HRS-positive vesicles, and accumulation of polyubiquitinated proteins. The latter effect upon SJ disruption was reminiscent of Shrub/ESCRTIII loss of function, leading authors to propose that modulation of ESCRT activity prevents SJ protein degradation in favor of SJ protein recycling. Such a scenario could be expected to result in elevated SJ protein levels at the plasma membrane, but whether this is the case is not addressed in the paper. Instead, the authors switch here to analyzing effects of shrub RNAi on the apical determinant Crumbs, which accumulates at or near AJs in cells lacking bSJ (nrv2) or tSJ (aka) components, consistent with reduced degradation and/or increased recycling of Crumbs protein. Finally, they show that clusters of beta-integrin (Mys), associated with vinculin and F-actin, appear on the basal side of aka-depleted cells, leading the authors to conclude that SJ-defective cells reinforce their adhesion to the ECM, perhaps to prevent extrusion from the epithelium. While the appearance of Mys clusters on the basal side is convincingly demonstrated, I don´t see evidence for apical focal adhesions, as depicted in the cartoon in Fig. 7. If focal adhesion-like structures exist on the apical side, to what kind of ECM molecules should they attach there?
Overall, the manuscript describes interesting new findings that are well documented and should be of interest to a broad audience of cell and developmental biologists. However, the following questions and technical issues remain to be addressed before the manuscript will be ready for publication.
Major comments:
The title refers to a "mechanism sensing paracellular diffusion barrier alteration", and in the discussion (line 325) authors state that "loss of bSJs and tSJs by altering the paracellular diffusion barrier triggers an ESCRT-dependent response...". However, no experiments to assess paracellular barrier function (epithelial permeability) are shown in the paper, and it is not clear that the ESCRT-dependent responses described here are triggered by altered barrier function per se, as stated by the authors, or by changes in other SJ-dependent parameters, such as cell adhesion or intra-membrane mobility of lipids and proteins. Statements about paracellular barrier alteration should be rephrased accordingly.
Altered epithelial barrier function will likely influence osmoregulation via changes in organismal hormonal status and gene expression, which may contribute to the phenotypes described here. How much time passed between induction of mutant clones and phenotypic analysis? The authors should discuss these aspects, and consider that effects of altered barrier function will depend on the distribution and size of clones with defective SJs.
In the discussion the authors speculate about a "sensing" mechanism based on (hypothetical) altered membrane lipid composition upon loss of SJs. However, such effects would not explain how altered barrier function per se (epithelial permeability) would be sensed by cells, as stated in the title and throughout the text. Please explain.
How Shrb/ESCRTIII activity could be "redirected" or "modulated" by disruption of SJs remains unclear. Can the authors briefly outline possible mechanisms for modulation of ESCRT activity?
The presentation of fluorescence intensity data in a rescaled ("standardized") format is uncommon and non-intuitive, as it obscures the true scale (fold-changes) and variation of the data. Also, if data were plotted as a range from 0 to 10, as stated in Materials & Methods, it is not clear why in all graphs (except for a single datapoint in Fig. 5C'?) values start at 1, not at 0. Highest values appear to cluster at 10 and lowest values at 1, suggesting these represent saturated or clipped signals, respectively. Were these datapoints taken into consideration for calculating mean values? Authors need to explain exactly how the analysis was done. Why was this type of representation chosen, and why should it be more appropriate than showing regular normalized data?
Authors should explain why they jump between different mutant (aka, nrv2) and RNAi (aka, cora nrv2, nrxIV) conditions and different Gal4 driver lines (pnr-Gal4, sca-Gal4) to disrupt SJ integrity. The basis for choosing these different conditions is not always clear and makes results difficult to compare.
The TEM images shown in Fig. 1A are difficult to interpret, because plasma membrane is barely visible. The images do not seem to contribute much and can be removed from the paper.
The position of mutant clones is marked by absence of nuclear RFP (Fig. 1B and elsewhere), but drawings of clone boundaries (Fig. 1B) do not match with the pattern of RFP-positive/ -negative nuclei (Fig. 1B'), presumably because different optical sections are shown in Fig. 1B and and B'. This is confusing and needs to be explained.
Minor comments:
Line 102: "We recently reported that defects at tricellular Septate Junctions (tSJs) are always accompanied by bicellular Septate Junctions (bSJs) defects". Authors may want to mention that in embryonic and larval epithelia lacking tricellular SJs, bicellular SJs assemble initially, but appear to degenerate during later development (Hildebrand et al. 2015, Byri et al. 2015).
Line 192 remove "another".
Line 194: % enrichment and fold enrichment are used; stick to one way.
Line 259 and elsewhere: Crb "activation" vs. accumulation or mislocalization. What do the authors mean by Crb "activation"?
Line 346: "FK2 protein": the FK2 antibody does not detect a particular protein, but the polyubiquitin modification, presumably on many different proteins.
Line 444: "Also, the observed changes at apical level might be mostly due to direct effects." I don´t see experimental evidence to support that the observed changes are mostly due to direct effects. Rephrase or remove.
Information on how mutant clones were induced needs to be included in Materials and Methods.
Results referred to as "not shown" should be shown, or corresponding statements be removed from the paper.
The text needs to be carefully checked for grammatical and typographical errors.
Significance
How epithelial cells cope with disruptions of occluding junctions without losing tissue integrity is an important question with far-reaching implications for understanding epithelial biology and disease. This work makes a significant contribution here by carefully describing the interrelationship between disruption of occluding junctions and possible compensatory mechanisms at the level of adherens junctions.
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Referee #2
Evidence, reproducibility and clarity
Septate junctions provide the barrier function in insect tissues, serving as analogs to the vertebrate tight junctions. Here the authors explore an interesting question-how do epithelial tissues respond to loss of barrier function in vivo. They use a powerful and well-studied system, the Drosophila pupal notum, which allows them to bring powerful genetic tools to bear and use state of the art imaging. Their data are lovely and carefully quantified. Together, they reveal some significant surprises. 1. Disrupting septate junctions leads to elevated accumulation of adherens junction proteins and myosin, and reduced apical area. 2. …
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Referee #2
Evidence, reproducibility and clarity
Septate junctions provide the barrier function in insect tissues, serving as analogs to the vertebrate tight junctions. Here the authors explore an interesting question-how do epithelial tissues respond to loss of barrier function in vivo. They use a powerful and well-studied system, the Drosophila pupal notum, which allows them to bring powerful genetic tools to bear and use state of the art imaging. Their data are lovely and carefully quantified. Together, they reveal some significant surprises. 1. Disrupting septate junctions leads to elevated accumulation of adherens junction proteins and myosin, and reduced apical area. 2. Disrupting septate junctions led to accumulation of many ESCRT-0-positive vesicles and of enlarged ESCRTIII vesicles. 3. Disrupting septate junctions led to elevated accumulation of Crumbs apically and of integrin-based focal adhesions basally. These observations are well supported by the data and in the results section conclusions are carefully drawn. I had some relatively minor comments outlined below about the results. My only significant suggestion concerns the Abstract and Discussion. The Abstract includes a statement that goes well beyond the data shown, and the Discussion is sometimes hard to follow. With these issues corrected, this will provide important new insights for cell and developmental biologists.
- The Abstract states: "We report that the weakening of SJ integrity, caused by the depletion of bi- or tricellular SJ components, reduces ESCRT-III/Vps32/Shrub-dependent degradation and promotes instead Retromer-dependent recycling of SJ components." This is too strong, as the role of the retromer, while plausible, is not directly tested. It's fine to speculate about this in the Discussion but drawing a conclusion like this in the Abstract is unwarranted.
- Similarly, the title suggests that "ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism sensing paracellular diffusion barrier alteration". They show that knocking down septate junctions alters localization of vesicle trafficking machinery, and that it leads to alterations in apparent recycling of cargo, but do they ever really assess whether these changes are ESCRT-III-dependent? Wouldn't this require knocking down ESCRT-III in cells with defects in septate junctions? There was a lot of data in this paper and perhaps I missed it but was this experiment done? I am not suggesting they do it, but that they temper this conclusion if not.
- The authors assessed "poly-ubiquitinylated proteins aggregates appearance, marked using anti-FK2" . They need to define FK2-what does it detect.
- Fig 4-is this a clone, and are we far from the boundary? Make this clearer
- The authors state: "Despite these apparent similarities, we noticed that, in contrast to Shrub depletion, NrxIV did not accumulate in enlarged intracellular compartments upon Cora depletion" Could the authors reference a Figure here?
- The authors state: "Hence, if both Shrub and bSJ/tSJ defects lead to Crumb enhanced signals" It might be better to say "altered" as they then point out the differences.
- I found the Discussion challenging to follow. Rather than focusing on the core observations, it addresses many, not very well-connected speculative possibilities, and in my opinion, will be challenging for most readers to follow. I would encourage the authors to revisit it from top-to-bottom.
Referees cross-commenting
I think we largely agree that the authors present important data, but that certain points need to be better explained or more clearly documented. While Reviewer 1 is correct that adding context about the basolateral polarity proteins would be helpful, I do not feel as strongly about this as a deficit. The authors did not manipulate Scrib, Dlg or Lgl, and i think their polarity functions may be distinct from those of the more "structural" septate junction proteins analyzed here.
Significance
Septate junctions provide the barrier function in insect tissues, serving as analogs to the vertebrate tight junctions. Here the authors explore an interesting question-how do epithelial tissues respond to loss of barrier function in vivo. They use a powerful and well-studied system, the Drosophila pupal notum, which allows them to bring powerful genetic tools to bear and use state of the art imaging. Their data are lovely and carefully quantified. Together, they reveal some significant surprises. 1. Disrupting septate junctions leads to elevated accumulation of adherens junction proteins and myosin, and reduced apical area. 2. Disrupting septate junctions led to accumulation of many ESCRT-0-positive vesicles and of enlarged ESCRTIII vesicles. 3. Disrupting septate junctions led to elevated accumulation of Crumbs apically and of integrin-based focal adhesions basally. These observations are well supported by the data and in the results section conclusions are carefully drawn. I had some relatively minor comments outlined below about the results. My only significant suggestion concerns the Abstract and Discussion. The Abstract includes a statement that goes well beyond the data shown, and the Discussion is sometimes hard to follow. With these issues corrected, this will provide important new insights for cell and developmental biologists.
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Referee #1
Evidence, reproducibility and clarity
This paper investigates the cellular response of Drosophila epithelia cells in the notum to damage to septate junctions. They find that disruption of tri- and bi-cellular septate junctions (SJ) integrity alters the distribution of adherens junction (AJ) components including E-cadherin, Myosin-II and others. Loss of SJ increases levels of AJ proteins. They then show that loss of the tri-cellular junction protein Anakonda alters the adhesive and the mechanical properties of the epithelia. They showed Myo-II activation was increased, however laser ablation/recoil studies did not reveal a change in local membrane tensions. Changes in …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #1
Evidence, reproducibility and clarity
This paper investigates the cellular response of Drosophila epithelia cells in the notum to damage to septate junctions. They find that disruption of tri- and bi-cellular septate junctions (SJ) integrity alters the distribution of adherens junction (AJ) components including E-cadherin, Myosin-II and others. Loss of SJ increases levels of AJ proteins. They then show that loss of the tri-cellular junction protein Anakonda alters the adhesive and the mechanical properties of the epithelia. They showed Myo-II activation was increased, however laser ablation/recoil studies did not reveal a change in local membrane tensions. Changes in membrane tension were observed by quantifying cell divisions in bicellular septate junction (bSJ) mutants. Building on previous work, they show that defects in SJs lead to ESCRT complex defects, and that loss of tricellular or bicellular septate junction components increase apical-medial Crumbs localization and triggers assembly of focal adhesion contacts. Together, these results show that alterations in SJ structures result in apparently compensatory increases in Crbs and focal adhesion-based intercellular adhesions that is mediated by the ESCRT complex.
Major comments:
- Title, abstract and paper body: e.g. title "ESCRT-III-dependent adhesive and mechanical changes are triggered by a mechanism sensing paracellular diffusion barrier alteration in Drosophila epithelial cells"
- The paper is completely focused on the septate junctions as a paracellular diffusion barrier. However, many of the septate junction components, including Scribble, Dlg, and Lgl, have well documented (if poorly understood) basolateral polarity functions, and considering that septate junctions contain 15 or more cell-cell adhesion proteins, they are also likely to have a adhesive/structural function in addition to paracellular barrier and polarity functions. There is no attempt in the paper to consider or disentangle these multiple roles. Indeed, the introduction and discussion consider the vertebrate tight junction as the analogue of the insect septate junctions when a better view would be that the septate junction is a combination of the claudin-based barrier function of the vertebrate tight junction and the vertebrate basolateral polarity proteins Scribble, Dlg and Lgl that localize similarly and presumably have a function similar to the Drosophila basolateral polarity/SJ proteins for which they are named. Moreover, there are no experiments in the paper to address whether the relevant parameter being sensed in SJ defects is loss of the paracellular barrier, loss of cell adhesion/contact/structure or disruption of the polarity function of the SJ complex. Notably, there aren't any experiments in the paper that test paracellular barrier function. This criticism does not in any way reduce the importance of the paper or the results, but to avoid presenting an overly simplistic and probably misleading view of the cellular processes in play, a more comprehensive discussion of SJs is in order.
- line 245: "We propose that it is the Shrub activity that is being modified upon SJ alteration, preventing SJ component degradation in favour of SJ component recycling."
line 288, "Thus, as proposed above for Nrx-IV, these data further suggest a hijacking of Shrub activity toward recycling components upon alteration of SJ integrity."
Model in Fig. 7 Arrows showing increased SJ protein delivery in right bottom panel, but decreased bicellular SJ complex formation in the left bottom panel.
The authors demonstrate that in bicellular SJ mutants, there is increased accumulation of Crb, adherens junction components, focal adhesion components, and in the text and in the model in Fig 7 focus on the upregulation of recycling activity. However, as indicated by the reduced bSJs in the left bottom panel in Fig 7, and in the reduced Nrx levels in 3C' and in the text in lines 351-53, the levels of most septate junction proteins drop in the absence of any of 15+ bicellular septate junction mutants. Previously the authors should that reduction of tricellular septate junction proteins increased levels of septate junction proteins in bicellular junctions which the authors translate to increased delivery of "SJ components" to the membrane in SJ mutants as shown in Fig 7 bottom right panel and stated in lines 245 and 288. But the data in the paper, which is consistent with statements on lines 351-353 saying that bicellular SJ mutations cause a general reduction of SJ protein levels, suggests either a more nuanced role of recycling such that Crbs and other proteins show increased recycling in bicellular SJ mutants, but biclellular SJ proteins show decreased recycling, or an alternative scenario in that the SJ proteins are recycled more in a SJ mutant, like Crb is, but SJ proteins don't form stable complexes which leads to their modification that targets them for destruction despite being recycled more. Regardless of the actual explanation, I think readers will be confused by the statements in the current version of the paper about upregulation of recycling activity but apparent reduction of SJ proteins. The authors should address this issue with appropriate changes to text and the model figure.
Minor comments:
- The assumption in the paper is that the changes in protein levels result from changes in recycling of the proteins. However, it would be nice to rule out transcriptional regulation. Has anyone established smFISH in the notum that would allow quantification of Crb or other marker RNA to show that there is not increased accumulation of the Crb RNA in the SJ mutant backgrounds?
- line 58. SJ are only the functional equivalent of tight junctions for paracellular barrier function. SJ have basolateral polarity function that correspond to basolateral polarity proteins in vertebrates, whereas vertebrate TJs are associated with apical complexes. In addition, the mechanical properties of SJ and TJ are probably wildly different since the SJ is a much more elaborate structure with many more cell-cell adhesion proteins than TJs. I feel the presented over-simplification do not adequately inform the reader about alternative functions and therefore hypotheses about the data in the paper.
- lines 120-121 , Figure 1A-A'. Please quantify the relative frequency of holes observed in the EM sections. Is it every tricellular junction or 1 in 100? Is WT statistically different than mutant?
- line 126-127 (data not shown). Does EMBO allow data not shown? Just checking current rules.
- lines 134-135. "We observed similar results upon loss of Gli and M6". Is this data not shown? I couldn't find it. Please either reference a figure or note as "data not shown" if that is allowed.
- line 319 "We propose that the disruption of SJ barrier in the ...", also line 326 . I suggest the use of "SJ complex" instead of SJ barrier or paracellular diffusion barrier, otherwise the authors need to provide some evidence or rationale that it is the barrier function of the SJ that is triggering the recycling changes rather than the disruption of the polarity or adhesive/structural functions of the SJs.
- line 341 "Our work shows that a part of the sensing mechanism involves the ESCRT machinery."
I think that the ESCRT machinery is better described as part of a response mechanism to SJ defects than as a "sensor". I don't think the paper presents any evidence that the ESCRT machinery is part of the sensing mechanism for SJ defects. There is lots of evidence that the ESCRT machinery is modified by SJ defects, but that supports a role as part of the response machinery, not as the sensor that directly detects SJ defects.
Significance
The topic and results of the paper will be of interest to a wide range of the cell biology community including those studying epithelial integrity, junctions, polarity and endocytic trafficking. The results break new ground in looking at the dynamic relationships between junctional complexes. This paper is generally well written, with the exception of the major comments below which, and the experiments well done. Overall a very interesting paper that is appropriate for a top tier journal.
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