Palmitoylation of proteolipid protein M6 promotes tricellular junction assembly in epithelia of Drosophila

  1. Raphael Schleutker
  2. Stefan Luschnig

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Abstract

Tricellular junctions (TCJs) provide essential adhesive and occluding functions at epithelial cell vertices and play key roles for tissue integrity and physiology, but how TCJs are assembled and maintained is poorly understood. In Drosophila , the transmembrane proteins Anakonda (Aka), Gliotactin (Gli), and M6 constitute tricellular occluding junctions. Aka and M6 localize in an interdependent manner to vertices and are required jointly to localize Gli, but how these proteins interact to assemble TCJs was not known. Here, we show that the tetraspan proteolipid protein M6 physically interacts with Aka and with itself. M6 is palmitoylated on a conserved juxta-membrane cysteine cluster. This modification promotes efficient vertex localization of M6 and binding to Aka, but not to itself, and becomes essential when TCJ protein levels are reduced. Abolishing M6 palmitoylation leads to delayed accumulation of M6 and Aka at vertices but does not affect the rate of TCJ growth or mobility of M6 or Aka. Our findings suggest that palmitoylation-dependent recruitment of Aka by M6 promotes initiation of TCJ assembly, while subsequent TCJ growth relies on different mechanisms independent of M6 palmitoylation.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    This article by Raphael Schleutker and Stefan Luschnig addresses the importance of S-palmitoylation of the proteolipid protein M6, one of the three components of tricellular septate junction along with Anakonda and Giotactin, in the assembly of tricellular junctions using Drosophila embryo as a model system. Using a combination of state-of-the-art genome engineering, live imaging and biochemistry, the authors demonstrated that M6 is palmitoylated in vivo, elegantly identified the cysteine residue that is palmitoylated, showed that this modification is essential for interaction with Anakonda and provided convincing evidence that palmytoylation is required for the initial assembly of tricellular junctions.

    Major comments:

    • Are the claims and the conclusions supported by the data or do they require additional experiments or analyses to support them? The claims are largely supported by the very high quality data assembled in this article. I have just a few concerns that can be resolved by modifying the text or, optionally, by carrying out additional experiments:

    1. in the summary (lane 40) and all along the article it is stated that 'Abolishing M6 palmitoylation leads to delayed accumulation of M6 and Aka at vertices but does not affect the rate of TCJ growth or mobility of M6 or Aka.'
      However, whilst the data presented convincingly demonstrate the delayed localization of GFP::M6 delta Palm at TCJ, that of Aka at TCJ is not shown. Although I think this is a reasonable hypothesis, without showing Aka localization, this claim is too strong and should be toned down, or better (optional) show the dynamics of Aka localization. Lanes 184 and 197 'indicating that effcient TCJ formation depends on M6 palmitoylation.' TCJ formation is not assessed here, what is measured is the localization of M6 at vertex. I suggest to amend the text accordingly.

    2. Fig. 5C and lane 292' Lack of M6 palmitoylation reduces, although it does not completely abolish, the interaction with Aka, ....' In Fig. 5C, GFP::M6 efficiently co-precipitates three forms of Aka with different molecular weights. The two upper bands are highly enriched in the GFP::M6 coIp. In contrast, GFP::M6 delta Palm seems to coIp only the low molecular weight form of Aka. Could the authors explain what the three forms of Aka are, and provide potential explanations or interpretations of this result?

    • Please request additional experiments only if they are essential for the conclusions. Alternatively, ask the authors to qualify their claims as preliminary or speculative, or to remove them altogether.

    • Are the data and the methods presented in such a way that they can be reproduced? The data are of very high quality and the methods sufficiently described (with appropriate references where necessary) and presented in such a way that they can be reproduced.

    • Are the experiments adequately replicated and statistical analysis adequate? Although the microscopy data are perfectly quantified and the appropriate statistical tests are used, unless I am mistaken, the number of replicates and the number of independent experiments carried out in biochemistry (Fig. 2 and Fig. 5) are not indicated.

    Minor comments:

    • Specific experimental issues that are easily addressable.

    1. The localization of M6 on living specimens, GFP::M6 enriched at the tricellular junction, differs from the localization of M6 detected by anti-M6 on fixed samples, i.e. M6 homogenous distributed at the bicellular junction, no enrichment at the tricellular junction. Please comment and possibly explain the reason for the difference in localisation. Is the anti-GFP staining on the GFP::M6 sample restricted to the bicellular junction without apparent TCJ enrichment?

    2. In Fig. S2, isoforms E and F are expressed at low level but fully rescue Gli localization but not Aka. These results are somewhat surprising if Gli localization relies on Aka and M6 localization at TCJ. Is localization of M6 at TCJ important or is it the expression of M6 that matters? Would it be possible to compare the expression levels of the different isoforms?.

    3. lane 118 'Notably, vertex enrichment varied between M6-GFP isoforms and was inversely proportional to overall signal intensity, suggesting that saturation effects upon overexpression impede vertex enrichment. Consistent with this notion, endogenous GFP::M6CA06602 showed higher vertex enrichment (7.8-fold; Fig. 1E, L) than the individual overexpressed isoforms.' To conclude that all isoforms contain the elements for vertex localization, it would be interesting to provide the level of expression (signal intensity) for all M6 isoform as well as M6deltaPAlm-GFP to appreciate the threshold above which saturation is achieved? Or better (optional) to express the different isoforms in a M6 mutant background. Could the authors exclude the possibility that the position of the GFP moiety affect the localization of M6 at TCJ?

    4. lanes 187 '...in a single spot that subsequently extends basally with a speed of 0.09 μm/min ' The images are presumably projections along the apical basal axis, so it is difficult to appreciate the apical to basal extension, perhaps an orthogonal section would help.

    • Are prior studies referenced appropriately? yes

    • Are the text and figures clear and accurate? yes

    Significance

    Provide contextual information to readers (editors and researchers) about the novelty of the study, its value for the field and the communities that might be interested.

    The following aspects are important:

    Tricellular junctions are hot spots integrating mechanical and chemical inputs that are essential to ensure epithelia homeostasis. It is therefore essential to understand how the components of tricellular junctions are located and assembled to form functional tricellular junctions. The authors brilliantly demonstrate the key role of S-palmitoylation in M6 localization and ability to interact with Aka in vivo. The fact that the role of palmitoylation appears to be conserved for the assembly of vertebrate TCJs, made up of components that are not conserved throughout evolution, indicates a fundamental function of palmitoylation in protein-protein interactions at the level of TCJs and in their vesicular trafficking. As palmitoylation is reversible, this work also raises the question of how palmitoylation is regulated in time and space to ensure the plasticity of TCJs in developing epithelia.

    • General assessment: provide a summary of the strengths and limitations of the study. What are the strongest and most important aspects? What aspects of the study should be improved or could be developed?

    • Advance: compare the study to the closest related results in the literature or highlight results reported for the first time to your knowledge; does the study extend the knowledge in the field and in which way? Describe the nature of the advance and the resulting insights (for example: conceptual, technical, clinical, mechanistic, functional,...).

    This study follows on from that showing the role of Angulin1 palmitoylation in its localization to tricellular junctions in vertebrates. The present study demonstrates the conserved nature of the role of this post-translational modification in the assembly of complex membrane structures essential for epithelial homeostasis. In addition, it demonstrates the dynamic nature and temporality of the role of palmytoylation in the early stages of recruitment of M6 to the vertex, opening up numerous hypotheses for future work at the conceptual and fonctional levels, elegantly presented in the discssion.

    • Audience: describe the type of audience ("specialized", "broad", "basic research", "translational/clinical", etc...) that will be interested or influenced by this research; how will this research be used by others; will it be of interest beyond the specific field?

    I believe this article is dedicated to a rather broad audience. Although this article may at first appear to be aimed at specialists, the findings go beyond the interest in tricellular junctions in Drosophila, since the role of palmytoylation of tricellular components appears to be conserved in vertebrates. In addition, this study will have an impact on the overall cell biology community, including membrane trafficking and the role of lipid modification additions on the subcellular dynamics of transmembrane proteins in a physiological context.

    • Please define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate. I am a developmental cell biologist, with an expertise in epithelial junctions and epithelial tissue homeostasis, using vertebrate and invertebrate model systems.
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    Referee #1

    Evidence, reproducibility and clarity

    The epithelial diffusion barrier in triangular junctions is initially formed by a protein complex of Aka, Gli and M6. Aka and M6 act upstream of Gli. GPM6a, the vertebrate homolog of M6 is palmitoylated, whose functional implications have not thoroughly been analyzed, yet. It order to better define the function of M6 and especially the role of the palmitoyl moity, the authors conducted a genetic analysis of M6 in Drosophila embryos.

    • They first establish a genetic system with defined mutants (deletion and CS mutants which are not palmitoylated), tagged protein at the genetic locus and an quantitative assay for protein enrichment at triangular junctions. Secondly they provide biochemical evidence that M6 is palmitoylated at a cluster of three conserved cysteine residues in vivo. With a palmitoylation-deficient mutant, thirdly the authors investigate the function of the palmitoyl moiety for protein localization at triangular junctions and complex formation with the other proteins at triangular junctions. The authors reveal a quantitative function of the palmitoyl moiety at triangular junctions with respect to enrichment and initial accumulation but not for later functions during growth of triangular junctions. The lower enrichment of the non-palmitoylated M6 mutant are sufficient for recruitment of Aka and Gli. Importantly, reducing Aka in combination with the non-palmitoylated mutant leads to a strong phenotype with respect to Gli localization and and leads to a genetically synthetic embryonic lethality. Fourthly, on a molecular level, the palmitoyl residue mediates binding to Aka but is not required for di/oligomerization of M6 itself as shown by immunoprecipitation from embryonic lysates.

    • Though the function of M6 acting together with Aka and Gli has been demonstrated previously, molecular details of the interactions and targeting of the proteins to triangular junctions have remained unclear. Similarly, although palmitoylation of the vertebrate homologue has been previously demonstrated, its functional implications have not been investigated in a physiological context with stringent genetics. The current study provides convincing data about the role of the palmitoylated moiety of M6. Importantly, the authors manage to differentiate a function of the palmitoyl residue in initial accumulation of M6 at triangular junctions versus maintenance. Also the authors manage to reveal an essential function of M6 palmitoylation when the dose of Aka is reduced. In summary, the study provides novel and interesting insights into the detailed molecular requirements of epithelial barrier formation. Although the quality of the data and analysis provides an argument for publication on its own, it may be noted that similar mechanisms may underlie barrier formation at triangular junction in vertebrates given the conservation of the protein components.

    Minor comments:

    1. L100: it is stated that "... is not detectable on other known TCJ components". What about Angulin-1, which is palmitoylated?

    2. L122 In my understanding all M6 isoforms contain an element which is sufficient. Not "required".

    3. L336. The allele designation "DeltaPalm" is misleading. A designation like "3xCS" would be more better because three defined cysteine residues are mutated.

    4. L329 A reference to FLYBASE is missing. Similarly not reference to stock centers are provides. To document the importance of the community services it is essential that their services are properly cited in a way that can be automatically tracked, e. g. by a literature citation.

    Significance

    In summary, the study provides novel and interesting insights into the detailed molecular requirements of epithelial barrier formation. Although the quality of the data and analysis provides an argument for publication on its own, it may be noted that similar mechanisms may underlie barrier formation at triangular junction in vertebrates given the conservation of the protein components.

  4. Version published to 10.1101/2023.09.04.556077v1 on bioRxiv