A dynamic interplay between chitin synthase and the proteins Expansion/Rebuf reveals that chitin polymerisation and translocation are uncoupled in Drosophila

  1. Ettore De Giorgio
  2. Panagiotis Giannios
  3. M. Lluisa Espinàs
  4. Marta Llimargas

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Chitin is a highly abundant polymer in nature and a principal component of apical extracellular matrices in insects. In addition, chitin has proved to be an excellent biomaterial with multiple applications. In spite of its importance, the molecular mechanisms of chitin biosynthesis and chitin structural diversity are not fully elucidated yet. To investigate these issues, we use Drosophila as a model. We previously showed that chitin deposition in ectodermal tissues requires the concomitant activities of the chitin synthase enzyme Kkv and the functionally interchangeable proteins Exp and Reb. Exp/Reb are conserved proteins, but their mechanism of activity during chitin deposition has not been elucidated yet. Here, we carry out a cellular and molecular analysis of chitin deposition, and we show that chitin polymerisation and chitin translocation to the extracellular space are uncoupled. We find that Kkv activity in chitin translocation, but not in polymerisation, requires the activity of Exp/Reb, and in particular of its conserved Nα-MH2 domain. The activity of Kkv in chitin polymerisation and translocation correlate with Kkv subcellular localisation, and in absence of Kkv-mediated extracellular chitin deposition, chitin accumulates intracellularly as membrane-less punctae. Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane. We propose a model in which Exp/Reb regulate the organisation of Kkv complexes at the apical membrane, which, in turn, regulates the function of Kkv in extracellular chitin translocation.

Article activity feed

  1. Version published to 10.1371/journal.pbio.3001978
  6. Review Commons

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

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time.

    We will provide the point-by-point response when we submit the full revision of our manuscript

  7. Review Commons

    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

    This paper presents an investigation of the mechanisms of how chitin is synthesized in Drosophila by investigating the chitin synthetase Kkv and two proteins related/redundant proteins that are required for chitin production Exp and Reb.

    The authors show that synthesis of nascent chitin polymers is separable from the secretion of chitin and that Ex/Reb is specifically required for chitin translocation/secretion. To understand the functions of Exp/Reb, the authors perform structure/function analyses and examine the localization of the proteins. They find that Na-MH2 domain in Exp/Reb is required for chitin translocation, and that a motif the authors name CM2 is required for Exp localization. For Kkv, they show the WGTRE domain is required for ER exit and that a coiled-coiled domain is required for KKV localization and full Kkv activity. By using live imaging and mutations that disrupt membrane trafficking, the authors show that Kkv, which is a transmembrane protein, cycles to the membrane, and like most membrane proteins, is endocytosed and transits through the endocytic system and is returned to the apical surface. Interestingly, despite being dynamically moved around the cell, chitin synthesis produces highly organized extracellular matrixes. Considering that constitutive production of chitin by Kkv everywhere in the cell would create a mess, these results underscore that regulated organized secretion/translocation of chitin is central to generating patterned extracellular matrixes (as the saying goes, "location, location, location"). Consistent with Exp/Reb being important regulators in extracellular matrix patterning, Exp/Reb not only are required for export of chitin, in the absence of Exp/Reb, the pattern of Kkv localization at the apical surface is altered. Unexpectedly however, by using super resolution microscopy the authors show that Kkv and Exp/Reb have complementary rather than matching localizations. Thus, while it is not clear exactly how Exp/Reb are regulating Kkv, they are doing something very interesting.
    Overall, this paper will be of broad interest to the cell biology and developmental biology communities, and to the translational community working to develop chitin as a commercial biopolymer. It is also generally clearly written, although I think there are some inaccuracies in the how some points are phrased. The experiments are well done, and subject to the revisions out lined below.

    Major concerns:

    • A major conclusion of the paper is that Exp/Reb are not required for chitin synthesis. On the most basic level this statement is well supported, because chitin grains are made in the cytoplasm in the Exp/Reb mutants. However, I think the field would be better served with a more nuanced consideration or the role of Reb/Exp. From the data presented, it seems that in the absence of Reb/Exp, the total amount of chitin produced is greatly reduced. I think it would be worth considering Exp/Reb, or the synthesis process in general, as having processivity or duty cycle or quality control such that in the absence of Exp/Reb while Kkv may make short chitin polymers, or occasional long polymers, the major production of chitin doesn't get going without Exp/Reb. Thinking of Reb/Exp as processivity factors in addition to export factors dramatically changes how one thinks of the proteins and the process of chitin synthesis. While these considerations can be handed with some discussion, it would be very interesting to look at the length of the chitin polymers in the Reb/Exp mutants and see if the average chain length is much reduced. This would help distinguish between Exp/Reb reving up the total number of Kkv molecules that produce chitin and Exp/Reb allowing the same number of Kkv molecules to stay active and produce much longer chitin chains. A caveat here is that I have no idea how hard this is to do, so I won't put this at the level of a required revision, but this result would significantly deepen the analysis in the paper.
    • In looking that the subcellular localization of the Kkv and Reb in regular and super resolution, the authors I think the authors missed an important, but straight forward way to gain insight into the apparent complementary distribution of Kkv and Exp/Reb. In stage 16 WT embryos, Kkv has a distinct ringed pattern that corresponds to the tanedial ridges (e.g. clearly visible in Fig. 6A and 6G). How those ridges are set up is unclear, although there are some interesting Turing-pattern models out there. One prediction might be that Exp/Reb should be in between the Kkv rings. If so, maybe Exp/Reb are key components of patterning chitin secretion to make this 3D patterned matrix? Alternatively, maybe Exp/Reb act on a smaller length scale and will match the Kkv ring pattern, just not overlapping with Kkv at the very fine scale. These are straightforward experiments and again could provide key insights into the function of Exp/Reb.
    • In general, most of the figures do not include WT or a control for comparison. This makes it hard for non-experts to assess what the effect of a mutation or condition is. For example, there are no examples of WT or Df(exp reb) in Figures 1-4. I realize this would increase the number of panels, but the paper would be more accessible if comparisons were within figures instead of comparing between main and supplementary figures and other papers.
    • To bolster the case the Exp/Reb directly regulate Kkv distribution, the authors should examine the distribution of Kkv in a catalytically null Kkv mutant, or drugs that block Kkv, or mutations in other genes required for Kkv activity to show that the altered distribution of Kkv in Exp/Reb mutants is a direct consequence of the lack of Exp/Reb rather than in indirect consequence of lack of extracellular chitin, which causes gross perturbations in the trachea. Also, are there differences in the distributions of Kkv in salivary glands with or without the presence of Exp/Reb? If Exp/Reb change the distribution of Kkv in the salivary glands, which normally do not express Kkv and presumably many other components of the chitin ECM system, this would be a powerful argument that there is a direct effect.

    Minor concerns.

    • Page 5 "These intracellular chitin punctae disappeared from stage 14, when chitin is then deposited extracellularly (Fig 1B')." Fig. 1B' is stage 15 embryos.
    • Page 5 "lead to tracheal morphogenetic defects". It would be helpful to the reader if the text or legend told the reader what they were looking for? Broken tubes? Inflated tubes? Variable tubes?
    • Fig. 1H. Main text says "co-expression of Kkv and expMH2/rebMH2 did not lead to tracheal morphogenetic defects (Fig 1H, ...". The tracheal dorsal trunk in Fig. 1H does not look WT. The legend does not state the stage, but the DT looks to have an enlarged diameter and it might be too long. Please present measurements on stage 16 trachea to confirm that there is no effect on tracheal morphology.
    • Fig. 3E there is a lot of GFP-Kkv that is not in co-localized with the KDEL marker. Can the authors clarify what compartment all the other staining is? ER?
    • Section 3.1. The authors imply that the WGTRE domain is specifically required for ER exit. However, an alternative is that absent the WGTRE domain, the protein just does not fold correctly, which would also preclude ER exit, but would be a different problem for the protein to make chitin if it isn't folded.
    • Page 15. I disagree with statement "At stage 16, control embryos showed a highly homogeneous apical distribution of Kkv in stripes, corresponding to the taenidial folds, and Kkv vesicles were largely absent (Fig 6G)." In Fig. 6G, the tandeal ring pattern is clearly visible, as are the fusion cells. If Kkv distribution were "highly homogeneous" these structures/pattern would not be visible.
    • Page 15. I also disagree with the characterization of the apical Kkv distribution in st 15 embryos. "In control embryos we detected a very uniform and homogenous pattern of apical Kkv (Fig 6I).". To my eye, the pattern is punctate and random for the clumps of stain, with the underlying beginnings of the tanidial pattern starting to be visible. The pattern appears neither uniform nor homogenous.
    • P16. The degree of order in the distribution of Kkv is overstated. The authors state that "The results of this analysis, showed that Kkv on the apical membrane, is evenly distributed following a regular pattern (Fig. 6L,L',L',M)." However, given that there is barely a visibly perceptible difference between the actual distribution of Kkv in 6L' and a calculated random distribution in 6L", and that the pattern is neither visibly even or regular, it would be more representative to say something to the effect that the analysis shows there is "underlying order" or "some degree of order" or a "non-random pattern". Visually, the key difference between 6L ' and L" is that there are fewer closely clustered Kkv dots. You could still have an uneven distribution of Kkv that maintains minimum spacing, which is a kind of ordered organization, but not one that would be assumed from the description. It would be helpful if the authors instead of just saying a "regular pattern" also stated the nature of the pattern they observe, i.e. Grid? Stripes? Minimum spacing?
    • Discussion. Another model for the role of Exp/Reb could be to bind and neutralize an inhibitor of Kkv activity. This would account complementary distribution of Kkv and Exp/Reb.
    • Fig. 6L. what tissue is being analyzed? Presumably trachea, but this should be specified as salivary glands are also mentioned in the legend.
    • Fig. 7 C models. I believe that the super resolution data is not accurately accounted for in the models. In both model 1 and model 2, Kkv and Exp/Reb are shown to be in close proximity, but the super resolution data suggests that most Kkv and Exp/Reb are separated hundreds of nanometers. Further, showing Kkv and Exp/Reb as touching was not supported by the coIP experiments, which failed to detect an interaction. It is possible that only a small fraction of Exp/Reb that is in close proximity to Kkv is active, but if so, this should be explicitly mentioned in the models to reconcile the data showing that Kkv and Exp/Reb are mostly not anywhere near each other.
    • -Image analysis. Please detail the criteria for "apical" and "basal" regions were the basis for freehand segmentation. What was counted as apical and what was basal?
    • Abstract and Introduction: The authors state that "We find that Kkv activity in chitin translocation, but not in polymerization, requires the activity of Exp/Reb, and in particular of its conserved Na-MH2 domain.", but then follow that with the statement that "Furthermore, we find that Kkv and Exp/Reb display a largely complementary pattern at the apical domain, and that Exp/Reb activity regulates the topological distribution of Kkv at the apical membrane." Many readers, will find the use of "furthermore" confusing because they will take furthermore as the about to be described data logically following the previous data, but then run headlong into the fact the Kkv and Exp/Reb show a complementary distribution, which does not obviously follow from Kkv activity requiring Exp/Reb. The authors could clarify this and highlight the interesting, unexpected and exciting nature of their results by replacing "Furthermore" with "Unexpectedly" or "Surprisingly", and emphasizing the important role of Exp/Reb in Kkv organization. Maybe something like: Unexpectedly, we find that although Kkv and Exp/Reb display largely complementary patterns at the apical domain, Exp/Reb activity nonetheless regulates the topological distribution of Kkv at the apical membrane.

    Significance

    The topic is interesting from the aspect of cell biology in terms of how a long polymer is created intracellularly, secreted and spatially organized to create a sophisticated extracellular matrix. The topic is also of general interest because chitin is central to the body plan of all insects, crustaceans and many other species, and chitin is of increasing interest as a biopolymer that could have extensive commercial uses.

    In addition to an informative structure/function analysis of the Kvv and Exp/Reb, the results identify what is, to my knowledge, the first regulator of the spatial organization of chitin sythase in insects and it unexpectedly shows a complementary pattern to the the synthase. This highlights just how little we understand about how complex extracellular matrixes are synthesized.

  8. Review Commons

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

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.

    The authors initially detected unusual intracellular chitin by overexpression of the chitin synthase kkv in tracheal cells before regular chitin deposition occurs. In addition, they recognized that the kkv gain of function mediated unusual intracellular chitin vesicles and in later stages in exp/reb mutants. These findings were the starting point of further experiments, suggesting that Kkv synthesizes chitin and that Kkv-mediated chitin deposition requires Exp/Reb activity to translocate and release chitin. Their genetic studies further show that chitin polymerization and translocation are uncoupled.

    All primary studies were tested in embryonic tracheal cells and as a proof of principle control in salivary glands which do not express chitin. Elegant rescue experiments in the mutant background showed that the Exp/Reb Nα-MH2 domains are required but not sufficient for chitin translocation and deposition and are dispensable for protein localization. Additionally, they identified another conserved domain (CM2) which is required Exp/Reb localization but not for chitin translocation. Similarly, they investigated Kkv domains by rescue experiments in kkv mutant embryos. They identified the WGTRE domain as essential for ER exit and the coiled-coil region for proper apical Kkv localization. Altogether they provide evidence that Kkv requires proper localization at the apical membrane, which is likely coordinated by Exp/Reb. This precise Kkv localization is linked with Kkv activity and chitin deposition. Therefore, this work is new and fundamental to many disciplines such as insect biology, chitin biology, cell & developmental biology, and others. Thus, the work is worth publishing but requires some changes from my point of view.

    Major Comments

    1. The authors nicely show that Kkv is able to synthesize chitin in a constitutive manner and that it can accumulate intracellularly. However, this needs some more input to understand the underlying biological sense. For example, what are the chitin vesicles' nature of early vesicles (st 13) and the unusual late (st15) chitin vesicles? Exocytosis, endocytosis or recycling? This can be clarified to understand chitin translocation and that synthesis and translocation are uncoupled. The authors tested rab5DN mutant salivary glands to exclude endocytosis. However, the chitin-positive vesicle size and amount in the rab5 mutant appear different from the control experiment, where much more intracellular chitin accumulates. Thus, it may suggest that some chitin vesicles are independent of Rab5-mediated endocytosis, others probably not. Indeed, the authors identified some KKv and some other chitin vesicles in all discussed intracellular processes; however, additionally, chitin appears to accumulate also in the cytoplasm. The authors conclude that Kkv protein might be able to polymerize chitin at all different intercellular stages, including endocytosis and degradation pathway. First, I wonder why chitin was found within membranous vesicles and, at the same time, within the cytoplasm. Second, does it make sense in the biological context when tracheal cells or other chitin-producing organs want to secrete chitin at the apical membrane while Kkv has the ability to produce chitin in all cellular areas, even in endosomes? In this context, another fundamental question concerning chitin secretion and subsequent organization could be investigated with the author's tools. Are the chitin vesicles loaded with chitin binding proteins or deacetylases that organize the formation of the nano and makro fibrillar chitin matrix in tracheal tubes? For example, previous research showed a reduced luminal accumulation of the 2A12 antigen in kkv mutants and expRNAi knockdown embryos.
    2. Observation of Extracellular kkv-GFP: does extracellular anti-GFP staining co-localize with the anti-Kkv antibody?
    3. Putative Kkv microvesicles: the authors state that extracellular GFP staining could be Kkv located in microvesicles. I wonder whether the observed extracellular GFP puncta contain a membrane or other membranous proteins.
    4. Fig.1:
    • general remarks: some images of this figure could be improved by showing the single channels of CBP to judge whether chitin is secreted and/or vesicles appear. -In addition, some images show higher magnifications, others overview only. It would be beneficial to visualize the small vesicles additionally with higher magnifications.
    • Fig.1M: This image is problematic due to the epidermal background staining. The tracheal system is hard to recognize. A single channel of Cbp ist not indicated.
    • Fig. 1P: Apical membrane marker or any cytoplasmic marker would be extremely useful to judge subcellular Exp localization in this experiment - this image is hard to compare with Exp localization in Fig S2D.

    Fig. 2O: Apical/Basal accumulation, what are the numbers at the Y-axis?

    Fig. 3F: The authors state that the WGTRE domain is required for ER exit of Kkv based on colocalization studies with KDEL and FK2. However, the study with FK2 is not convincing as immunostainings are of poor quality. The GFP construct appears not to be expressed in all tracheal cells, and moreover, the FK2 staining is faint. Thus, judging whether the protein is not ubiquitinated from the presented image is challenging. However, it does not change the key message, Kkv does not exit ER. By the way, there is a new paper showing Serca to be essential for ER exit of Kkv, which would fit the discussion of the kkv domains.

    Fig. 7 - model: First, Since the authors do not show that Kkv is part of a membranous microvesicle, I'm skeptical whether this should be part of a model that explains the shown data. Therefore, I'm asking the authors to delete it or to show it more clearly. Second, the meaning of the yellow arrowheads is not indicated. Third, the explanation in the legend is sound, but showing the two options could be improved.

    Minor comments:

    1. Missing reference: Chirin has been recognized importance in physiology (Zhao et al., 2019; Zhu et al., 2016) but also as a biomaterial (? Reference). Suggestion: DOI: 10.3390/ma15031041 (Improving Polysaccharide-Based Chitin/Chitosan-Aerogel Materials by Learning from Genetics and Molecular Biology) This paper discusses the current usage and potential of chitin as a biomaterial in many disciplines.)
    2. 3.1, second paragraph final sentence: double point

    Referees cross-commenting

    Rev#1 asks the same questions as I do. Technical questions and the idea to compare endogenous Kkv.The same is true with Rev#3. Overlapping questions concerning technical things about figure illustration and clarity of presented stainings. Altogether, the criticism will improve the manuscript

    Significance

    This paper deciphers very nicely the genetics and cellular events where and how chitin polymers become synthesized and translocated towards the apical cell membrane for further release into the extracellular space. Altogether this is fundamental work of high significance explaining how chitin is produced and released.

  9. Review Commons

    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

    In this manuscript, the authors provide data on the function of exp/reb and Kkv in chitin deposition. They show chitin polymerization and deposition are uncoupled and exp/reb are required for the deposition of the chitin by regulating the distribution of kkv at the apical membrane. However, there is no direct interaction between Kkv and Exp/reb. The functional analysis of Kkv and Exp/reb is interesting.

    The overexpression lines are used throughout the manuscript to analyze protein functions. Since ectopic expression of kkv and exp leads to chitin synthesis and deposition. Authors use this overexpression system to analyze the functional domain of kkv and Exp/reb. It is reasonable. However overexpression line might not represent the endogenous protein perfectly, it might cause some issues to answer certain questions.

    Major comments

    1. Fig. 4 Does ectopic overexpression of Kkv-GFP have the same expression pattern as the endogenous Kkv? The overexpression line may lead to ectopic expression. the colocalization of endogenous Kkv and intracellular vesicles would be more accurate.
    2. Are Kkv and Exp/reb expressed at the same time endogenously? If kkv is expressed earlier than Exp, can intracellular chitin be detected in wild-type embryos at early stages? Fig. 1b shows overexpression of Kkv at S13 has intracellular chitin (exp is not expressed at this stage).
    3. Fig. 1B no intracellular chitin is detected. Fig. 1H intracellular chitin is detected. Does Overexpression of exp-MH2 interfere with the endogenous Exp function?
    4. For measurement, some detailed info is needed, for example, what is your area of interest?

    Minor comments:

    1. Fig. 2 and Fig. 3. how do you define the region of apical and basal? An apical marker is needed here. N is the total number of embryos or the number of sections in the same embryo?
    2. Fig. 5H What is your area of interest to measure vesicles? Which tracheal segment do you measure? Some details need to be provided here.
    3. Fig. 5 what is your area of interest when you measure Kkv?

    Significance

    This work further advance the knowledge about chitin synthesis and deposition

  10. Version published to 10.1101/2022.07.21.500966 on bioRxiv