Ca 2+ binding to Esyt is required to modulate membrane contact site density in Drosophila photoreceptors

  1. Vaisaly R Nath
  2. Harini Krishnan
  3. Shirish Mishra
  4. Padinjat Raghu

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

Membrane Contact Sites (MCS) between the plasma membrane (PM) and endoplasmic reticulum (ER) have been shown to regulate Ca 2+ influx into animal cells. However, the mechanisms by which cells modulate ER-PM MCS density is not understood and the role of Ca 2+ , if any, in regulating this process is not known. We report that in Drosophila photoreceptors, MCS density is dependent on the activity of the Ca 2+ permeable channels-TRP and TRPL. This regulation of MCS density by Ca 2+ is mediated by extended synaptotagmin (dEsyt), a protein localised to ER-PM MCS in photoreceptors and previously shown to regulate MCS density. We find that the Ca 2+ binding activity of dEsyt is required for its functional activity in vivo . dEsyt CaBM , a Ca 2+ non-binding mutant of dEsyt is unable to modulate MCS structure in a manner equivalent to its wild type counterpart. Further, when reconstituted in dEsyt null photoreceptors, in contrast to wild type dEsyt, dEsyt CaBM is unable to rescue ER-PM MCS density and other key phenotypes. Finally, when expressed in wild type photoreceptors, dEsyt CaBM phenocopies loss of dEsyt function. Taken together, our data supports a role for the Ca 2+ binding activity of dEsyt in regulating the ER-PM MCS density in photoreceptors thus tuning signal transduction in response to Ca 2+ influx triggered by ambient illumination.

Abstract Figure

Article activity feed

  1. Review Commons

    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

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We thank the reviewers for their insights and helpful suggestions on the manuscript. Based on these, we have prepared a revision plan for this manuscript, which is outlined below. We believe these revisions will improve the overall quality of the manuscript.

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1

    (Evidence, reproducibility and clarity (Required)):

    Summary:

    This study builds on previous work from the same group, where they use Drosophila photoreceptors as a model system to investigate the role or ER-plasma membrane contact sites in an in vivo setting. The authors recently described a role of the ER-PM contact site protein dEsyt in regulating photoreceptor function in Drosophila. In this follow-up study, they explore whether this function of dEsyt is connected Ca2+ signaling downstream of photoreceptor activation. Using a dEsyt mutant that should be unable to bind Ca2+, they find that Ca2+ to some extent is required for dEsyt localization, membrane contact site formation and photoreceptor function.

    Major comments:

    The use of photoreceptor cells in Drosophila is an elegant model system that enable studies of membrane contact sites and associated proteins in a native condition. The data presented by the authors clearly shows that these structures are important for photoreceptor function, and that dEsyt plays a role at these sites. However, this was already known from previous studies by the same group. When it comes to whether these contacts are sensing Ca2+ changes and if these changes are acting through dEsyt, which is the focus of the current manuscript, the results are unclear to me and would need to be clarified by the authors both in text and with new experiments.

    1. What is the role of cellular Ca2+ signaling in the regulation of dEsyt function? There are several aspects here that needs to be clarified. 1) How is WT dEsyt localization regulated by Ca2+? This could for example be evaluated in the mutant flies used in Fig. 1 (trpl302; trp343), where lack of light-induced Ca2+ influx would be predicted to result in a localization of dEsyt that resembles that observed for dEsytCaBM. 2) Is Ca2+ important for dEsyt localization, lipid exchange or both? The authors express a version of dEsyt with mutation made in all three C2 domains. In mammalian E-Syts, Ca2+ binding to the C2A domain is important for lipid exchange while binding to C2C (in E-Syt1) is important for interactions with lipids in the plasma membrane. Using more carefully designed mutants will allow the authors to determine how Ca2+ regulates dEsyt function in vivo. In addition, the authors must show experimentally that the mutant dEsytCaBM is unable to bind Ca2+ (could e.g. be done by acute Ca2+ changes in the cell-based model used in Fig. 3). Writing that "This transgene carrying a total of nine mutations should render the protein unable to bind calcium" (p. 6, line 173) is not sufficient.

    2. How is WT dEsyt localization regulated by Ca2+?

    We agree that further experimental evidence would be helpful in establishing the significance of cellular Ca2+ signaling in the control of dEsyt function. As suggested by the reviewer, the localization of wild type dEsyt will be examined in the mutants: norpAP24 (PLC null mutant) and *trpl302; trp343 *(protein null mutants of TRPL and TRP channels respectively) in which light induced calcium influx is eliminated. These data will be included in the revision.

    1. Is Ca2+ important for dEsyt localization, lipid exchange or both?

    We have already performed experiments to address the question of how important calcium binding to dEsyt is for lipid transport at the ER-PM interface in Drosophila photoreceptors. This results indicate a previously unexpected role for lipid exchange and will be included in the revision.

    3). Writing that "This transgene carrying a total of nine mutations should render the protein unable to bind calcium" (p. 6, line 173) is not sufficient.

    We concur with the reviewers that at present we do not have experimental data to demonstrate that dEsytCaBM can't bind Ca2+. However, as Reviewer 4 pointed out, it will be challenging to demonstrate this experimentally. A direct proof would only come from measurements of the calcium binding affinity of dEsyt (which involves protein purification that is beyond the scope of the current work). An indirect demonstration would be any cellular or in vivo experiment. In addition to the in silico analysis already included in Fig 2 C-F, we propose the following to provide additional evidence to strengthen our in silico analysis: Use AlphaFold model to demonstrate that the arrangement of the calcium binding residues in the C2 domain of dEsyt is compatible with Ca2+ binding.

    1. The localization of dEsyt shown in Fig. 3B is a bit confusing. First of all, I would recommend including markers of the ER and the plasma membrane, because without these it is difficult to make statements about the localization of dEsyt to these structures.

    As suggested, to better appreciate the localization of dEsyt in photoreceptors, we will perform colocalization of dEsyt with markers of the PM (Rhabdomere) and ER (Sub Microvillar Cisternae).

    Second, it appears that WT dEsyt localize to the reticular ER, and that the CaBM version localize to the plasma membrane. This is somewhat opposite to mammalian ESyts, where mutations that prevent Ca2+ binding either had no effect (for ESyt2) or prevented (for ESyt1) the interaction with the plasma membrane. It also appears different from the localization in vivo (Fig. 3C). Clarifying this will be important. It will also be important to connect this localization to changes in Ca2+ and not just to the localization of a mutant that may or may not be deficient in Ca2+ binding (see comment above).

    In considering this comment, we need to bear in mind the following:

    • Mammalian cells have three genes that encode for Esyt: Esyt 1, 2 and 3 whereas the Drosophila genome encodes only a single gene for Esyt.
    • In terms of sequence similarity and structure, dEsyt and hEsyt2 are very similar. However, in contrast to hEsyt2 and hEsyt3, which localize to the plasma membrane (PMID: 17360437), dEsyt acts like hEsyt1 and localizes to the ER-PM junctions.
    • A single study (PMID: 27065097) has shown that the SMP domain of Esyt1 can transfer lipids in an in vitro assay. In our studies, we have noted an unexpected function for the SMP domain of dEsyt for in vivo function as measured through phenotypes in the eye (data will be presented in the revised ms).
    • While knocking out the single dEsyt in Drosophila photoreceptor neurons results in phenotypes (Nath et.al PMID: 32716137) to date, knocking out all three Esyts in mammalian cell culture models or mice has not revealed an in vivo Bearing these points in mind it may not be reasonable to expect every observation on mammalian Esyt to be recapitulated in the fly system or vice versa.
    1. I don't fully understand the time course of events. The authors show that dEsytCaBM is mislocalized already at day 1 in dark-reared flies (Fig. 3C) but this mislocalization is not accompanied by a change in MCS density or gap distance, and consistently does not influence the localization of RDGB. The authors next expose the flies to constant light illumination to trigger Ca2+ dependent signaling, and this leads to mislocalization of RDGB, perhaps indicating changes in MCS (this is not shown). From these results it is difficult to know what the role of dEsyt is. It would be necessary to also show a control where Ca2+ signaling is not induced, e.g. a parallel dark-control (same number of days but no illumination).

    It is important to remember that even complete loss of Esyt does not result in altered MCS or mislocalization of RDGB on day 1 post eclosion. This has been published by us previously (Nath et.al PMID: 32716137). Since we show in this manuscript that dEsytCaBM exerts a dominant negative effect when expressed in wild type and phenocopies dEsytKO, one might expect expression of dEsytCaBM to also lead to altered MCS density and mislocalization of RDGB by 6D constant light.

    Bearing this in mind, we will incorporate the following data in the manuscript: Addition of MCS density in dEsytKO photoreceptors at Day1 in Figure 3C.

    • Electron Microscopy to check MCS density in Rh1>dEsytCaBM at Day 6CL with appropriate control genotypes.
    • Confocal Imaging: RDGB staining in Rh1>dEsytCaBM- Day 6CD reared flies with appropriate control genotypes- dark control where only reduced Ca2+ signaling is induced due to dark noise or spontaneous PLC activation. This is particularly important given that the authors show in Fig. 1 that preventing Ca2+ influx had a dramatic impact on MCS density even at day 1 (which is in sharp contrast to dEsytCaBM-expressing flies, that show normal morphology at day 1, which rather implies that dEsyt is not a major Ca2+ effector).

    In thinking about this comment, it is important to bear in mind the details of the experimental paradigm in use in each of the experiments while drawing comparisons between the observed results. It is to be noted that throughout the manuscript dEsytCaBM is expressed selectively in photoreceptors using the Rhodopsin enhancer which drives expression of the transgene during late eye development. By contrast, in germ line mutant strains such as trpl302;trp343 the channels are blocked throughout development. Thus the phenotypes of trpl302;trp343 might be broader than that of expressing dEsytCaBM. Therefore, mutating the calcium binding residues of dEsyt and expressing it using Rh1 enhancer at a specific developmental time window might not have the same impact on the contact site density as completely blocking the major calcium permeable channels, TRP and TRPL that is important to sustain the ongoing phototransduction cascade all through the development.

    1. The experiments done in dEsyt KO flies are important, and here the authors show that dEsyt1 could to some extent rescue all phenotypes. Some results are a bit puzzling. For example, dEsyt1CaBM localization in dEsyt1 KO flies is identical to that of WT dEsyt (Fig. 5C), which is in sharp contrast to the data shown in Fig. 3C. What is the reason for this? I would have anticipated the opposite (i.e. that in WT flies, dEsytCaBM can form dimers with endogenous dEsyt through SMP-domain interactions which may have an impact on its localization and the function of endogenous dEsyt, but that in the dEsyt KO cells, dEsytCaBM would show a different localization due to the lack of endogenous dEyt to interact with). It is important to clarify as one of the major observations here is that dEsytCaBM no longer localize to MCS. Since the CaBM version of dEsyt could rescue, to some extent, MCS density and delay photoreceptor degeneration, this implies that Ca2+ may not be required for regulation of dEsyt function or that the mutant is still able to partially bind to Ca2+.

    The localization shown in Fig 5C is not of dEsytCaBM in dEsytKO photoreceptors but the localization of RDGB in Rh1>dEsytCaBM; dEsytKO at Day 1 (Figure 5C i) and as a function of age and illumination- Day 6CL (Figure 5C ii).

    One experiment that would help the authors determining the function of dEsyt in vivo would be to use a mutant that lacks functional SMP domain (ideally also with and without mutations in the C2-domains).

    There is information available to address the question of how the lipid binding module, SMP is important to render dEsyt functional at the ER-PM interface in Drosophila photoreceptors. The same will be included in the revision.

    1. PLC activation typically couples to rapid signaling and involved hydrolysis of PIP2 and release of Ca2+ from the ER. Mammalian Esyts also require PIP2 for plasma membrane binding (through interactions with C2-domains), so constitutive PLC activity would be expected to impair ESyt localization to MCS. Here, the authors expose flies for days of constant illumination. How does this influence plasma membrane PIP2 levels and could this be of relevance for how data is interpreted?

    This is an interesting question from the reviewer. However, we would like to clarify the fact that constitutive activation of PLC is different from constant activation of PLC during illumination. Flies have robust mechanisms for controlling PLC turnover and PIP2 levels during continuous illumination and Ca2+ is a key regulator of this process; the underlying mechanisms have been described by Raghu and Hardie in multiple past papers (PMID: 11343651, PMID: 15355960). This is why, apart from adaptation, flies grown in constant light for many days do not show electrophysiological defects and neither do they undergo retinal degeneration. We will however measure the kinetics of PIP2 resynthesis in (i) wild type (Day 1 vs Day 6CD vs Day 6CL) and (ii) Control, Rh1>dEsyt and Rh1>dEsytCaBM (Day 1 vs Day6CL). This might reveal some interesting insight into the mutants.

    Do the authors know whether the CaBM mutant has reduced affinity for PIP2?

    The ability of wild type dEsyt to bind PIP2 has not been determined. We will test this and if it does so, the impact of CaBM on PIP2 binding can be tested.

    Minor comments:

    • The overexpression of WT dEsyt had a dramatic impact on MCS density and gap distance, while expression of dEsytCaBM did not. If these contacts are important for photoreceptor function, is it not surprising that such a dramatic change in photoreceptor structure was without effect on function? This should be further discussed. The establishment of more contact sites and reduction in contact site distance in Rh1>dEsyt::GFP photoreceptors is likely indicative of the proposed tethering role of the protein at the ER-PM MCS. Increase in contact site density or reduction in distance need not directly parallel to the increase in the levels of MCS proteins that are expressed at these contact sites to enhance the ongoing signal transduction. We will test this idea proposed by the reviewer and include the following data in a revision to strengthen our statement:

    • RDGB levels in control vs Rh1>dEsyt::GFP - Western blot

    • Electroretinograms from the genotypes indicated above as a functional readout of the ongoing signaling cascade.

    • PIP2 kinetics in control vs Rh1>dEsyt::GFP to understand if establishing more contact sites can enhance the replenishment of the lipid at the PM.

    1. How is quantification of MCS density and gap distance influenced by retinal degeneration (e.g. induced by dEsyt KO)?

    Wherever we have analyzed MCS density or gap distance, these experiments have been done in flies at ages prior to the onset of retinal degeneration defined as collapse of the microvilli of the rhabdomere. Therefore, our measurements of MCS density and gap in this paper are not affected by retinal degeneration.

    1. The graphical abstract is a bit confusing. It seems to suggest that changes in dEsyt is a consequence of ageing and does not show any role of this protein in photoreceptor function. I think that the abstract could be improved to more clearly highlight the findings in the manuscript. For example, it doesn't at all show the difference in localization between WT and CaBM.

    We will modify the graphical abstract.

    1. P. 5, line 135 the authors state that "The tethering and lipid transfer activity of mammalian Esyts are reported to be influenced by Ca2+". This is a massive understatement. Ca2+ is a critical regulator of Esyt function in mammalian cells.

    The statement will be modified.

    1. In figure legend 1B and C: correct µM to µm.

    Changes will be incorporated as per the suggestion.

    1. In figure legend 2A: should be red rectangles and not black rectangles.

    Changes will be incorporated as per the suggestion.

    1. In Fig. 2B: specify which isoform of human ESyt that is shown.

    Changes will be incorporated as per the suggestion.

    1. In Fig. 2C: do the authors mean D374 or D384 (as indicated in Fig. 2A)?

    Changes will be incorporated as per the suggestion; the residue is D374.

    Significance

    Light-induced signal transduction in photoreceptor cells involves Ca2+ influx and signaling and also depends on correct formation of ER-plasma membrane contact sites. In mammalian cells, the Esyts (esp. Esyt1 and Esyt2) localize to ER-PM contacts in a Ca2+-dependent manner, and the ion has dual effects in both enriching the protein at the membrane contact sites and in promoting lipid transport. Mammalian Esyts form homo- and heterodimers, and the properties of the dimers depends on their composition (PMID: 26202220). Drosophila only have one Esyt (dEsyt) which is structurally most similar to mammalian Esyt2, and the authors have previously shown how this protein is required for photoreceptor function (PMID: 32716137), although the role of Ca2+ was not investigated in that study. However, an earlier study has shown that mutations of all Ca2+-coordinating residues in dEsyt impairs protein function in Drosophila neurons (PMID: 28882990), so a similar Ca2+-dependence in the retina would be expected. The results from the present study confirm the requirement of Ca2+ signaling for dEsyt function, and extends this Ca2+-dependent regulation to also involve photoreceptor-induced Ca2+ signaling, which corroborates many other studies showing the requirement of Ca2+ signaling for the regulation of Esyt function in mammalian cells (e.g. PMID: 23791178; PMID: 27065097; PMID: 29222176; PMID: 26202220; PMID: 24183667; PMID: 30589572). As such, the results from this study represent an incremental step towards understanding Esyt function in vivo. These results would be of greatest interest to researchers working of photoreceptor function, and of some interest to a broader audience working on membrane contact sites and signal transduction. My own background is in mammalian cell biology, with a focus on lipid and Ca2+ signaling and inter-organelle communication. I have limited understanding of the model system used here (Drosophila photoreceptor cells).

    We would like to provide an alternative perspective on the reviewer’s view that “As such, the results from this study represent an incremental step towards understanding Esyt function in vivo.”

    We are well aware of the content in several studies of Esyt in mammalian cells including the ones cited by the reviewer (e.g. PMID: 23791178; PMID: 27065097; PMID: 29222176; PMID: 26202220; PMID: 24183667; PMID: 30589572). These have been cited in our manuscript. However, it is important to recognize that each of these studies is an analysis of the properties of mammalian Esyt as a molecule in the context of Ca2+. However, none of these studies addresses the key question of whether the regulation of Esyt by Ca2+ is important for cellular function or to support cell physiology. The reason for this is quite straightforward and well known in the field. To date, there is no cellular or physiological phenotype that is reported to depend on endogenous Esyt function in mammalian cellular or animal models. As an illustrative example, deletion of all three mammalian Esyt does not affect cell signalling (PMID 23791178) including Ca2+ signalling and a triple knockout of all three Esyt in mice (PMID: 27348751) has no discernable phenotype.

    By contrast, deletion of the single Esyt gene in Drosophila results in robust phenotypes in adult photoreceptors (PMID: 32716137). Using these phenotypes, in this manuscript we study the importance of Ca2+ dependent regulation of cellular functions mediated by dEsyt. Therefore, this study fills an important unfilled gap in establishing the mechanism by which dEsyt proteins regulate cellular functions in vivo, in a Ca2+ dependent manner. We respectfully ask that this not be caricatured as an incremental step.

    Reviewer #2

    Evidence, reproducibility and clarity

    Esyt is a C domain (a Ca2+ binding domain) containing protein that localizes to the ER-MCS, playing a role in ER-mitochondria tethering and lipid transfer. At the same time, proteins at the ER-MCS are well-positioned to sense changing levels of Ca2+. Previous studies reported that loss of Esyt in Drosophila causes a loss of ER-PM integrity and retinal degeneration. Here, the authors report the consequence of disrupting the Esyt C domain in Drosophila photoreceptor cells. They used in-silico strategies to identify the Ca2+ contacting residues within the C domain and generated transgenic flies containing either the wild type or the Esyt-CaBM mutants. They show that the wild type transgene rescues several Esyt KO phenotypes in the Drosophila photoreceptors. In some cases, they report dominant negative effects of Esyt-CaBM overexpression.

    This is a straightforward structure-function analysis of the Esyt C domain. Overall, the experiments are well executed. At the same time, a few aspects of the manuscript could be further improved. For example, the authors analyze multiple aspects of photoreceptor integrity. In some cases, they show that the mutant Esyt transgene shows dominant negative effects. In others, there is no evidence or even a partial function. Clarifying these points could be helpful. Below are a few specific points for the authors' consideration:

    Major Comments

    1. RDGB is a protein that localizes to the ER-MCS. Esyt-CABM-GFP expression causes RDGB mis-localization even in the presence of wild type Esyt expression, suggestive of a dominant negative effect (Fig. 4C). But Esyt CaBM-GFP expression doesn't seem to have a dominant negative effect on contact site distance (Fig. 4D). Are the authors not seeing a dominant negative effect because they didn't examine older flies? Or, is there a distinct effect of Esyt CaBM on RDGB localization and contact site distance? If there is a distinct effect, what is the reason? As the reviewer correctly mentions, we are not seeing a dominant negative effect of dEsytCaBM::GFP expression on contact site distance because we didn't examine older flies.

    Dominant negative effect of dEsytCaBM on the wild type protein is observed in all phenotypes analyzed. The contact site distance analysis shown in the paper is done on day 1 old constant dark reared flies. Contact site distance exhibited by dEsytCaBM is like that of dEsytKO photoreceptors at day 1 post eclosion. dEsyt deprived photoreceptors are comparable to its wild type counterpart at Day 1 in all aspects of phototransduction (PMID: 32716137). But as a function of age and illumination, the dEsytKO photoreceptors exhibit progressive loss in contact site integrity, followed by induction of retinal degeneration and RDGB mis-localisation (PMID: 32716137). These observations are consistent in dEsytCaBM.

    During the revision, the following experiments will be included to strengthen this statement:

    • Add the MCS density and gap distance in dEsytKO photoreceptors at Day1 in Figure 3C.
    • Electron Microscopy to check MCS density and distance in Rh1>dEsytCaBM at Day 6CL with appropriate control genotypes.

    Esyt-CABM-GFP partially rescues the Esyt KO phenotype in retinal degeneration (Fig 6). This is surprising since cellular assays in Fig 4 show a failure of Esyt-CaBM to localize to ER-MCS. The results here contrast with earlier data showing that Esyt-CABM has dominant negative effects. How will the authors interpret the results? Is it possible that Esyt-CAMB still has some residual Ca2+ binding activity? Alternatively, does this result imply that Esyt can still function (albeit at lower capacity) without binding Ca2+? Is there Esyt function unrelated to ER-MCS site maintenance when it comes to its role in retinal degeneration? A reasonable explanation is warranted.

    Partial rescue of dEsytKO phenotypes by Rh1>dEsytCaBM; dEsytKO photoreceptors indicate that apart from calcium sensing, there might be another function for dEsyt at the ER-PM interface which is yet to be discovered.

    Minor Comments:

    Figure legends refer to "SMC" (I am guessing they are referring to Sub microvillar cisternae) without defining it in the text.

    Changes will be incorporated as per the suggestion.

    Significance

    This study will be of interest to those generally interested in the ER mitochondria contact sites. The main significance here is in dissecting the role of the C-domain within the Esyt protein. The authors demonstrate a physiological role using Drosophila photoreceptors as a model.

    We thank the reviewer for appreciating the significance of our study which seeks to show the in vivo significance of the Ca2+ regulation of dEsyt for in vivo function.

    __Reviewer #3 __

    (Evidence, reproducibility and clarity (Required)):

    Summary

    In the present work, the authors explore the role of Ca2+ binding to Esyt in the regulation of ER-PM contact sites using drosophila photoreceptors as a model system. By expressing in wild type or in EsytKO flies a mutated version of dEsyt which is predicted to lose Ca2+ binding, they highlight a potential role of Ca2+ binding to Esyt in the regulation of ER-PM contact sites density and the development of rhabdomeres. The data clearly show the effect of Esyt mutant during development of photoreceptors in Drosophila. However, as discussed below, one essential missing point is the experimental proof that the mutant has indeed lost its ability to bind Ca2+, and that PIP2 binding is not perturbed.

    Major comments

    1. One major comment is the lack of experimental proof that the EsytCABM mutant is indeed unable to bind Ca2+. The MIB tool only gives a prediction and it is not sufficient to prove their statements throughout the manuscript on the requirement of Ca2+ binding for the regulation of MCS. We understand the reviewer’s comment that this manuscript does not contain experimental data demonstrating that dEsytCaBM does not bind Ca2+. However, as Reviewer 4 pointed out, it will be challenging to demonstrate this experimentally. A direct proof would likely come from measurements of the calcium binding affinity of dEsyt (which involves protein purification that is beyond the scope of this work). An indirect demonstration would be any cellular or in vivo experiment oar any additional in silico analysis. To provide additional indirect evidence to address this question, we will:
    • Use the AlphaFold model to demonstrate that the arrangement of the calcium binding residues in dEsyt is compatible with Ca2+
    • Evaluate if the wild type dEsyt is mislocalized in the photoreceptors upon eliminating the calcium entry to these specialized sensory neurons. The localization of wild type dEsyt will be examined in the mutants: norpAP24 (PLC null mutant) and *trpl302; trp343 *(protein null mutant of TRPL and TRP channels respectively) in which light induced calcium influx is eliminated. Moreover, they should check experimentally the potential differences in the capacity of EsytCABM mutant to bind PI(4,5)P2, which can potentially perturb its subcellular localization.

    As recommended by the reviewer, it is important to determine the PIP2 binding capacity of dEsytCaBM. The ability of wild type dEsyt to bind PIP2 has not been determined. We will test this and if it does so, the impact of CaBM on PIP2 binding can be tested.

    Figure 1A: the legend on the right side of the scheme is missing. On the left, RDGB and dEsyt don't associate with the PM.

    Changes will be incorporated as per the suggestion.

    line 125: the authors should describe more precisely the Trp mutant that they used.

    The text will be modified.

    Concerning the quantification of MCS density done throughout the paper, can the authors mention what they considered as an MCS, in other words, what distance they defined as the maximal distance between the ER and the PM.

    We used fixation methods that allow enhanced membrane preservation and better visualization of membranes and MCS (PMID: 2496206). Such images allowed us to quantify the fraction of SMC that are present at the base of the microvilli in each ultrathin section of a photoreceptor. The MCS is the dark stretch that can be seen at the base of the rhabdomere in each TEM image (PMID: 32716137). Contact site distance measured is the absolute distance between the visible demarcation of the PM and SMC as indicated by the yellow arrows in Figure 4D iii, vi, and ix.

    Figure 3: the localization of Esyt and EsytCABM in S2R cells and in vivo is not precisely analyzed: a co-staining with PM and ER markers should be added in order to state the localization at ER-PM MCS or at apical PM.

    As suggested, to better understand the compartmental localization of dEsyt in photoreceptors, we will use markers of PM (Rhabdomere) and ER (Sub Microvillar Cisternae) and conduct co-localization assays.

    line 181: the authors should precise in which membrane compartments Esyt is localized.

    The text will be modified.

    line 185-187: the conclusion here doesn't seem to fit the data, as the EsytCABM mutant looks enriched at ER-PM contact sites.

    As previously answered, we will remark on whether there is an enrichment of dEsytCaBM at the ER-PM contact sites following the co-localization experiment that is recommended in Q5.

    a paragraph on the production of Drosophila transgene mutants should be added to the Mat et Med section.

    The text will be added as suggested.

    considering the phenotypes observed for the EsytCABM mutant in vivo, the authors should provide an analysis of the level of expression of the exogenous proteins Esyt and EsytCABM by western blot in the different backgrounds. EsytCABM seems to be expressed at lower levels in Figure 3C.

    As per the suggestion, western blot analysis will be conducted and better representative confocal images depicting the protein levels will be added in the manuscript.

    Fig 4D: considering the perturbation of RDGB localization observed at Day 6, the authors should analyze the organization of MCS by TEM at Day 6, in addition to Day 1.

    We agree that to support the observation of RDGB mis-localization, the decrease in contact site integrity as a function of age and illumination (Day6CL) should be evaluated in Rh1>dEsytCaBM photoreceptors. The manuscript revision will include data from this experiment.

    the EsytCABM mutant exhibits strong dominant negative effects, but rescues completely or partially some of the phenotypes of Esyt KO: could the authors discuss and provide some hypothesis on this apparent discrepancy?

    We are unsure what the reviewer means by “apparent discrepancy”. When dEsytCaBM is expressed in wild type photoreceptors, it exhibits a strong dominant negative effect presumably by inhibiting the function of wild type dEsyt protein.

    dEsytKO is a protein null allele. Therefore, when dEsytCaBM is expressed in the dEsytKO background it does not exert a dominant negative effect as there is no wild type protein to interact with. The partial rescue of dEsytKO phenotypes by Rh1>dEsytCaBM; dEsytKO photoreceptors likely indicates that calcium binding is not the sole factor affecting dEsyt function at the ER-PM interface.

    lines 230-233: the sentence is not clear. I don't see any consistency between data in Figure 5B, showing only very partial rescue by EsytCABM, and the data in Figure 5C (ii) showing complete rescue of RDGB localization by EsytCABM.

    The time point (six days of continuous light exposure following eclosion) at which RDGB localization was analyzed becomes extremely important in thinking about this reviewer comment. If we look at the degeneration kinetics depicted in figure 5B, we can see that neurodegeneration begins in both dEsytKO and Rh1>dEsytCaBM on Day 8 post-eclosion; prior to which, on Day 6, RDGB is mislocalized from the base. However, in Rh1>dEsytCaBM; dEsytKO, the onset of degeneration is delayed, and the photoreceptors show intact structure until Day 8 or Day 10, and measurable retinal degeneration begins on Day 12. This may be the reason why, RDGB continues to be correctly localized in Rh1>dEsytCaBM; dEsytKO at Day 6CL.

    Figure 6D: could the authors comment the increase of MCS density observed in Esyt-GFP expressing flies.

    Esyt is proposed to function as a tether that connects the ER and PM (PMID: 23791178; PMID: 27065097; PMID: 29222176), bringing them closer together. Based on this idea, perhaps by expressing dEsyt::GFP we are drawing the membranes together thus establishing more MCS.

    on several TEM images, some pictures illustrating different conditions look very similar, as if they were serial cuts: Fig 1B (Day 1 and Day 14), Fig 4D (Rh1 and Rh1>dEsytCABM::GFP), Fig 6B Day 1 and Day 14 and Fig 6C Day 1. Could the authors check if there was a mistake with these pictures?

    The images are not taken from serial sections of the same TEM block as is evident from the arrangement of nucleus of each photoreceptor cell. As mentioned in the figure legends, all experiments are carried out using 3 independent blocks (N=3 fly heads) prepared from each genotype and 10 photoreceptors from each block/ fly retinae are used for quantification of contact site density/ contact site distance. Aside from the arrangement of the accessory cells and cellular nuclei, the TEM images will appear very similar since Drosophila photoreceptor neurons are symmetrically arranged, with around 700–800 ommatidia per eye each comprising 8 photoreceptors.

    Minor comments:

    • lines 84-88 : the sentence is not clear. Besides, the authors should precise what they mean by "extra-cellular Ca2+ influx enhance ER-PM contact sites". Which parameter exactly has been shown to be regulated by Ca2+?

    The paper by Idevall-Hagren et al. proposes that following store operated Ca2+ influx, Esyt1 translocates to ER-PM junctions and the number of ER-PM contact sites increases. Please refer to this section of the publication from *Idevall-Hagren et al. (2015) *(PMID: 26202220):

    “As detected by TIRF microscopy, the depletion of Ca2+ from the lumen of the ER occurring under these conditions led to a progressive accumulation of ER‐anchored STIM1 at the PM, where it activates Orai Ca2+ channels (Fig 4C). Subsequent addition of 1–10 mM Ca2+ to the extracellular medium, either in the absence or in the presence of SERCA inhibitors, caused a massive increase in cytosolic Ca2+ (SOCE) through the activated Ca2+ channels (Figs 4A and EV4D–G). Such increase induced a very robust translocation of E‐Syt1 to the PM (Figs 4B and EV4D–G), which, in the absence of SERCA inhibition (i.e., when a reversible inhibitor of the SERCA pump had been washed out), preceded the dissociation of STIM1 and the inactivation of SOCE (Fig 4D). Inspection of TIRF microscopy images during the manipulation showed that E‐Syt1 does not form new contacts but populates and expands contacts previously occupied by STIM1.”

    • lines 108-110: can you give the reference?

    Reference for the localization of dEsyt to ER-PM MCS is Nath et.al PMID PMID: 32716137

    Reference for the localization of TRP and TRPL at the microvillar plasma membrane: Numerous primary research papers have shown this- for example see review PMID: 11557987, PMID: 22487656

    • line 189: the authors should summarize the findings in one sentence. "Functional activity" would refer to lipid transfer.

    The text will be modified as per the suggestion.

    Reviewer #3 (Significance (Required)):

    General assessment

    The work relies on a model system that enables the exploration of the role of Esyt in vivo, in a fundamental process highly regulated during development. The data clearly show the effect of Esyt mutant during development of photoreceptors in Drosophila but as discussed before, some experimental evidences are missing to completely prove the statements.

    Advance

    This work brings new insights in the functional role of lipid transfer during development and explores how the dialog between lipid transfer and Ca2+ flux can influence MCS organization. The interesting points that could be explored in the paper are the effects of a Ca2+ influx on Esyt and EsytCABM localization, and on their lipid transfer activity.

    Audience

    This work would be of interest for the membrane contact sites community and for the Developmental biology community.

    We thank the reviewer for highlighting the significance of our work and the clarity of the data. Additional data to address the points they have raised will be provided.

    __Reviewer #4 __

    (Evidence, reproducibility and clarity (Required)):

    In this study, Nath et al., aim at understanding the role of dESyt Ca2+ binding activity on ER-PM MCS in D. melanogaster photoreceptors. Using a combination of transmission electron microscopy and fluorescence microscopy, the authors explore the ability of a dESyt mutant, supposedly unable to bind Ca2+ (based on homology with the human ortholog hESyt2), to recapitulate the function of the wild type version of the protein in establishing ER-PM MCS and modulating their density.

    Findings:

    1. MCS density depends on the activity of TRP and TRPL channels in aging photoreceptors.

    2. Mutation of dESyt Ca2+ binding residues (dEsytCaBM::GFP) leads to a gross mis-localization of the protein, even in the presence of the endogenous protein.

    3. Overexpression of the mutant affects the structure of photoreceptors upon constant illumination.

    4. After 6 days of continuous illumination, RDGB is mis-localized in cells overexpressing dEsytCaBM::GFP.

    5. Overexpressed dEsytCaBM::GFP fails to reduce the distance between ER and PM, meaning it fails to establish ER-PM contract sites, while overexpressed dEsyt::GFP show reduced MCS distance. Overexpressed dEsyt::GFP also leads to a 10% increase in MCS density compared to WT or cells expressing dEsytCaBM::GFP.

    6. dEsytCaBM::GFP is not able to rescue the light dependent retinal degeneration of dESytKO, although it slightly delays the onset, but is able to rescue RDGB localization at day 6 of constant illumination.

    7. Examining MCS density in dESytKO cells, rescues with dEsyt::GFP and dEsytCaBM::GFP show a slightly higher MCS density than dESytKO at day 1. At day 14, ER-PM MCS were non-existent in dESytKO, unchanged in dEsyt::GFP and reduced by 20% in dEsytCaBM::GFP compared to day1.

    Specific comments:

    My field of expertise is biochemistry and structural biology (including cellular cryo-electron tomography), but I have no experience with drosophila biology, so I am not able to judge the drosophila work per se.

    While I find the confocal microscopy experiments compelling, I have some reservations regarding the quantification of the TEM images (MCS distances and density) as it was done manually, and therefore, to some extent subjective, especially, when differences between conditions are in the order of 10%. I would have found the quantification more convincing if done systematically, i.e. segmenting the MCS and computationally measuring distances and densities. Otherwise, the authors could expand a little bit on how their methodology is accurate.

    As the reviewer correctly mentions, the quantification will be more convincing if done systematically, i.e. segmenting the MCS and computationally measuring distances and densities. For MCS measurements, we have experimented with the segmentation method using ImageJ and Imaris. As mentioned in the answer to Q4 of reviewer 3, we used fixation methods that allow enhanced membrane preservation and better visualization of membranes and MCS (Matsumoto‐Suzuki et al, 1989). However, this staining method does not selectively stain the ER which is part of the MCS but all the ER. Due to this, automated segmentation poses significant challenges.

    The primary drawback of the segmentation method is that, in the process of training the software to predict/detect distinct cellular compartments, it recognizes all ER membranes, including SMC as well as the ER that is not part of the MCS. As a result, the software's minimum distance calculation may be between PM and SMC or PM and generic ER, which does not help the analysis we wish to perform. Similarly, to determine the contact site distance in images with obscure ER and PM boundaries, the software uses the border it can identify—which is typically inside the rhabdomere rather than at its edge. For the contact site density measurements, software is not able to distinguish between ER and pigment granules close to the rhabdomere as the gray scale value for both these compartments are comparable.

    Advantages of manual approach:

    To account for potential effects of photoreceptor depth on contact site density and distance, we have analyzed TEM sections obtained directly from the nuclear plane of the photoreceptors to calculate both contact site density and distance. Additionally, by utilizing the freehand line tool, manual analysis enables us to define the length of each little section of the MCS and the base of the rhabdomere. The entire length of the MCS at the base is then calculated by adding each segment together. An illustration of how the manual analysis is done will be included as part of methods in the revision.

    Another point is whether the levels of expression of dESyt proteins (dESyt-GFP and dESytCABM-GFP) are comparable. In the overexpression experiments, what are the expression levels of the constructs compared to the endogenous protein? The authors should provide e.g. a Western blot.

    As per the suggestion, western blot analysis will be conducted to compare the expression levels of the constructs utilized to the endogenous protein.

    Concerning the modelling, while I do think that the identification of dESyt Ca2+ binding residues is correct (the sequence alignment is convincing and the sequence identity is very high), and that most likely the structural arrangement will be conserved, homology modelling (using MODELLER with a single reference) leads to models highly similar to the input reference (in particular when the sequence identity is very high). Therefore, rmsd will necessarily be low and the side chain arrangement of conserved residues will be identical. This is unlikely to happen, as protein structures will not be identical despite high sequence conservation. In addition, a crystal structure is a snapshot of a protein conformation that is favorable for crystal formation. It would have been more interesting to use an AlphaFold model and show that the arrangement on the residues is compatible with Ca2+ binding (i.e., the C positions are similar).

    We agree with the reviewer that the data presented to demonstrate the inability of dEsytCaBM to bind Ca2+ is inadequate as is also pointed out by other reviewers. It would be crucial to prove this using multiple approaches. As suggested AlphaFold model will be used to answer the same.

    Minor comments:

    Line 102: indicate what PI and PA stand for (I don't think that there is a need for acronyms when they are not reused in the text later on).

    Changes will be incorporated as per the suggestion.

    Line 217-219: "When the same experimental set was examined for MCS density, we discovered that the density enhanced by 10% in Rh1>dEsyt::GFP while being comparable between wild type and dEsytCaBM::GFP flies." The authors don't comment on this finding. Does that imply that increase in the protein levels leads to increase in MCS density?

    Yes. Increase in wild type dEsyt protein levels can establish more contact sites as well as reduce the contact site distance which further elucidates the protein's role in functional tethering as mentioned in line 215 as proposed by previous studies in other models (PMID: 23791178; PMID: 27065097; PMID: 29222176).

    Lines 298-302: "...implying that dEsytCaBM exerts a dominant negative effect on wild type dEsyt. One possible mechanism for the phenotypes exhibited by dEsytCaBM expression in wild type cells is suggested by the findings of a structural and mass spectrometry investigation of hEsyt2 that reveals that the SMP domain dimerizes to create a 90Å long cylinder to facilitate the transfer of lipids (Schauder et al., 2014)." It is not clear to me what the authors suggest here: because of the dimerisation between wild type and mutant, the mutant has a negative effect or that the SMP dimerization is somehow impaired in dEsytCaBM?

    SMP domain of Esyt proteins have previously been shown to dimerize (PMID: 23791178, PMID: 24847877). They are known to form either homodimers or heterodimers in mammalian system where there are three genes that code for the protein (Esyt1, 2 and 3). In Drosophila, since it is just one gene that codes for the protein, our hypothesis is that one copy of the functional wild type gene dimerizes with the CaBM mutant and thereby render the wild type gene product nonfunctional.

    Line 304-305: "...protein expression was restricted to the cell body rather than the presynaptic terminals...". I am not sure that this is correct. The fact that a protein is localizing to a compartment does not mean that its expression is restricted to that compartment (one should measure mRNA levels to conclude this).

    The statement is based on the findings made by Kikuma et al, 2017 (PMID: 28882990) when they tried to understand the role of dEsyt at the NMJs.

    In figure 1B legend, indicate what SMC stands for (the acronym should be indicated in figure 1A legend).

    The text will be added as suggested.

    In figure 2A legend Ca binding in black box but in red boxes in figure.

    Changes will be incorporated as per the suggestion.

    **Referees cross-commenting**

    I agree with the other reviewers that one of the premise of this study relies on the loss of calcium binding by the dESyt mutant and this is not experimentally proven by the authors. However, I find that this will be difficult to prove in vivo. Only measurements of dESyt calcium binding affinity would constitute a direct proof (which requires protein purification. Any in vivo or cellular experiment would be an indirect proof. I believe that based on the high sequence conservation with ESyt proteins, the calcium binding residues have been correctly identified.

    Reviewer #4 (Significance (Required)):

    ESyt proteins are known ER-PM tethers involved in lipid transfer at MCS in a Ca2+ dependent manner. Contrary to yeast and mammals, that have several ESyt orthologs, D. melanogaster has only one ESyt, making it an ideal model to study ESyt function in vivo. It has been previously shown that proper localization of ESyt at MCS depends on Ca2+ concentration: ESyts are anchors to the ER but translocate to the PM in response to elevation of Ca2+ levels in the cytosol (Fernández-Busnadiego et al., 2015). The finding that an ESyt mutant unable to bind calcium is not localized properly is therefore not surprising. The link between RDGB, a protein known to localize at MCS, and ESyt has been shown before but to my knowledge Nath et al., show for the first time that RDBG localization at MCS is directly dependent on the Ca2+ binding activity of ESyt. In addition, the authors convincingly demonstrate that the Ca2+ binding activity of dESyt is necessary to maintain the structure of aging photoreceptors.

    The main finding of this study is that the Ca2+ binding activity of dESyt regulates the density of ER-PM MCS in photoreceptors. If true (see my comment below), that would be a novel finding, although the authors don't propose any mechanistic explanation for this.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    We haven't made any changes to the manuscript yet. However, we will be able to implement the changes mentioned in the pointwise response to reviewers above.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    We feel that experiments to directly determine the calcium binding of dEsyt and the loss of this in dEsytCaBM are beyond the scope of this study. This is because of the huge work to heterologously express and purify the protein. We have proposed alternate ways to strengthen this conclusion.

  2. 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 #4

    Evidence, reproducibility and clarity

    In this study, Nath et al., aim at understanding the role of dESyt Ca2+ binding activity on ER-PM MCS in D. melanogaster photoreceptors. Using a combination of transmission electron microscopy and fluorescence microscopy, the authors explore the ability of a dESyt mutant, supposedly unable to bind Ca2+ (based on homology with the human ortholog hESyt2), to recapitulate the function of the wild type version of the protein in establishing ER-PM MCS and modulating their density.

    Findings:

    1. MCS density depends on the activity of TRP and TRPL channels in aging photoreceptors.
    2. Mutation of dESyt Ca2+ binding residues (dEsytCaBM::GFP) leads to a gross mis-localization of the protein, even in the presence of the endogenous protein.
    3. Overexpression of the mutant affects the structure of photoreceptors upon constant illumination.
    4. After 6 days of continuous illumination, RDGB is mis-localized in cells overexpressing dEsytCaBM::GFP.
    5. Overexpressed dEsytCaBM::GFP fails to reduce the distance between ER and PM, meaning it fails to establish ER-PM contract sites, while overexpressed dEsyt::GFP show reduced MCS distance. Overexpressed dEsyt::GFP also leads to a 10% increase in MCS density compared to WT or cells expressing dEsytCaBM::GFP.
    6. dEsytCaBM::GFP is not able to rescue the light dependent retinal degeneration of dESytKO, although it slightly delays the onset, but is able to rescue RDGB localization at day 6 of constant illumination.
    7. Examining MCS density in dESytKO cells, rescues with dEsyt::GFP and dEsytCaBM::GFP show a slightly higher MCS density than dESytKO at day 1. At day 14, ER-PM MCS were non-existent in dESytKO, unchanged in dEsyt::GFP and reduced by 20% in dEsytCaBM::GFP compared to day1.

    Specific comments:

    My field of expertise is biochemistry and structural biology (including cellular cryo-electron tomography), but I have no experience with drosophila biology, so I am not able to judge the drosophila work per se. While I find the confocal microscopy experiments compelling, I have some reservations regarding the quantification of the TEM images (MCS distances and density) as it was done manually, and therefore, to some extent subjective, especially, when differences between conditions are in the order of 10%. I would have found the quantification more convincing if done systematically, i.e. segmenting the MCS and computationally measuring distances and densities. Otherwise, the authors could expand a little bit on how their methodology is accurate.

    Another point is whether the levels of expression of dESyt proteins (dESyt-GFP and dESytCABM-GFP) are comparable. In the overexpression experiments, what are the expression levels of the constructs compared to the endogenous protein? The authors should provide e.g. a Western blot.

    Concerning the modelling, while I do think that the identification of dESyt Ca2+ binding residues is correct (the sequence alignment is convincing and the sequence identity is very high), and that most likely the structural arrangement will be conserved, homology modelling (using MODELLER with a single reference) leads to models highly similar to the input reference (in particular when the sequence identity is very high). Therefore, rmsd will necessarily be low and the side chain arrangement of conserved residues will be identical. This is unlikely to happen, as protein structures will not be identical despite high sequence conservation. In addition, a crystal structure is a snapshot of a protein conformation that is favorable for crystal formation. It would have been more interesting to use an AlphaFold model and show that the arrangement on the residues is compatible with Ca2+ binding (i.e., the C positions are similar).

    Minor comments:

    Line 102: indicate what PI and PA stand for (I don't think that there is a need for acronyms when they are not reused in the text later on).

    Line 217-219: "When the same experimental set was examined for MCS density, we discovered that the density enhanced by 10% in Rh1>dEsyt::GFP while being comparable between wild type and dEsytCaBM::GFP flies." The authors don't comment on this finding. Does that imply that increase in the protein levels leads to increase in MCS density?

    Lines 298-302: "...implying that dEsytCaBM exerts a dominant negative effect on wild type dEsyt. One possible mechanism for the phenotypes exhibited by dEsytCaBM expression in wild type cells is suggested by the findings of a structural and mass spectrometry investigation of hEsyt2 that reveals that the SMP domain dimerizes to create a 90Å long cylinder to facilitate the transfer of lipids (Schauder et al., 2014)." It is not clear to me what the authors suggest here: because of the dimerisation between wild type and mutant, the mutant has a negative effect or that the SMP dimerization is somehow impaired in dEsytCaBM?

    Line 304-305: "...protein expression was restricted to the cell body rather than the presynaptic terminals...". I am not sure that this is correct. The fact that a protein is localizing to a compartment does not mean that its expression is restricted to that compartment (one should measure mRNA levels to conclude this).

    In figure 1B legend, indicate what SMC stands for (the acronym should be indicated in figure 1A legend).

    In figure 2A legend Ca binding in black box but in red boxes in figure.

    Referees cross-commenting

    I agree with the other reviewers that one of the premise of this study relies on the loss of calcium binding by the dESyt mutant and this is not experimentally proven by the authors. However, I find that this will be difficult to prove in vivo. Only measurements of dESyt calcium binding affinity would constitute a direct proof (which requires protein purification. Any in vivo or cellular experiment would be an indirect proof. I believe that based on the high sequence conservation with ESyt proteins, the calcium binding residues have been correctly identified.

    Significance

    ESyt proteins are known ER-PM tethers involved in lipid transfer at MCS in a Ca2+ dependent manner. Contrary to yeast and mammals, that have several ESyt orthologs, D. melanogaster has only one ESyt, making it an ideal model to study ESyt function in vivo. It has been previously shown that proper localization of ESyt at MCS depends on Ca2+ concentration: ESyts are anchors to the ER but translocate to the PM in response to elevation of Ca2+ levels in the cytosol (Fernández-Busnadiego et al., 2015). The finding that an ESyt mutant unable to bind calcium is not localized properly is therefore not surprising. The link between RDGB, a protein known to localize at MCS, and ESyt has been shown before but to my knowledge Nath et al., show for the first time that RDBG localization at MCS is directly dependent on the Ca2+ binding activity of ESyt. In addition, the authors convincingly demonstrate that the Ca2+ binding activity of dESyt is necessary to maintain the structure of aging photoreceptors.

    The main finding of this study is that the Ca2+ binding activity of dESyt regulates the density of ER-PM MCS in photoreceptors. If true (see my comment below), that would be a novel finding, although the authors don't propose any mechanistic explanation for this.

  3. 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

    Summary

    In the present work, the authors explore the role of Ca2+ binding to Esyt in the regulation of ER-PM contact sites using drosophila photoreceptors as a model system. By expressing in wild type or in EsytKO flies a mutated version of dEsyt which is predicted to lose Ca2+ binding, they highlight a potential role of Ca2+ binding to Esyt in the regulation of ER-PM contact sites density and the development of rhabdomeres. The data clearly show the effect of Esyt mutant during development of photoreceptors in Drosophila. However, as discussed below, one essential missing point is the experimental proof that the mutant has indeed lost its ability to bind Ca2+, and that PIP2 binding is not perturbed.

    Major comments

    • One major comment is the lack of experimental proof that the EsytCABM mutant is indeed unable to bind Ca2+. The MIB tool only gives a prediction and it is not sufficient to prove their statements throughout the manuscript on the requirement of Ca2+ binding for the regulation of MCS. Moreover, they should check experimentally the potential differences in the capacity of EsytCABM mutant to bind PI(4,5)P2, which can potentially perturb its subcellular localization.
    • Figure 1A: the legend on the right side of the scheme is missing. On the left, RDGB and dEsyt don't associate with the PM.
    • line 125: the authors should describe more precisely the Trp mutant that they used.
    • concerning the quantification of MCS density done throughout the paper, can the authors mention what they considered as an MCS, in other words, what distance they defined as the maximal distance between the ER and the PM.
    • Figure 3: the localization of Esyt and EsytCABM in S2R cells and in vivo is not precisely analyzed: a co-staining with PM and ER markers should be added in order to state the localization at ER-PM MCS or at apical PM.
    • line 181: the authors should precise in which membrane compartments Esyt is localized.
    • line 185-187: the conclusion here doesn't seem to fit the data, as the EsytCABM mutant looks enriched at ER-PM contact sites.
    • a paragraph on the production of Drosophila transgene mutants should be added to the Mat et Med section.
    • considering the phenotypes observed for the EsytCABM mutant in vivo, the authors should provide an analysis of the level of expression of the exogenous proteins Esyt and EsytCABM by western blot in the different backgrounds. EsytCABM seems to be expressed at lower levels in Figure 3C.
    • Fig 4D: considering the perturbation of RDGB localization observed at Day 6, the authors should analyze the organization of MCS by TEM at Day 6, in addition to Day 1.
    • the EsytCABM mutant exhibits strong dominant negative effects, but rescues completely or partially some of the phenotypes of Esyt KO: could the authors discuss and provide some hypothesis on this apparent discrepancy?
    • lines 230-233: the sentence is not clear. I don't see any consistency between data in Figure 5B, showing only very partial rescue by EsytCABM, and the data in Figure 5C (ii) showing complete rescue of RDGB localization by EsytCABM.
    • Figure 6D: could the authors comment the increase of MCS density observed in Esyt-GFP expressing flies.
    • on several TEM images, some pictures illustrating different conditions look very similar, as if they were serial cuts: Fig 1B (Day 1 and Day 14), Fig 4D (Rh1 and Rh1>dEsytCABM::GFP), Fig 6B Day 1 and Day 14 and Fig 6C Day 1. Could the authors check if there was a mistake with these pictures?

    Minor comments:

    • lines 84-88 : the sentence is not clear. Besides, the authors should precise what they mean by "extra-cellular Ca2+ influx enhance ER-PM contact sites". Which parameter exactly has been shown to be regulated by Ca2+?
    • lines 108-110: can you give the reference?
    • line 189: the authors should summarize the findings in one sentence. "Functional activity" would refer to lipid transfer.

    Significance

    General assessment

    The work relies on a model system that enables the exploration of the role of Esyt in vivo, in a fundamental process highly regulated during development. The data clearly show the effect of Esyt mutant during development of photoreceptors in Drosophila but as discussed before, some experimental evidences are missing to completely prove the statements.

    Advance

    This work brings new insights in the functional role of lipid transfer during development and explores how the dialog between lipid transfer and Ca2+ flux can influence MCS organization. The interesting points that could be explored in the paper are the effects of a Ca2+ influx on Esyt and EsytCABM localization, and on their lipid transfer activity.

    Audience

    This work would be of interest for the membrane contact sites community and for the Developmental biology community.

  4. 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

    Esyt is a C domain (a Ca2+ binding domain) containing protein that localizes to the ER-MCS, playing a role in ER-mitochondria tethering and lipid transfer. At the same time, proteins at the ER-MCS are well-positioned to sense changing levels of Ca2+. Previous studies reported that loss of Esyt in Drosophila causes a loss of ER-PM integrity and retinal degeneration. Here, the authors report the consequence of disrupting the Esyt C domain in Drosophila photoreceptor cells. They used in-silico strategies to identify the Ca2+ contacting residues within the C domain and generated transgenic flies containing either the wild type or the Esyt-CaBM mutants. They show that the wild type transgene rescues several Esyt KO phenotypes in the Drosophila photoreceptors. In some cases, they report dominant negative effects of Esyt-CaBM overexpression.

    This is a straightforward structure-function analysis of the Esyt C domain. Overall, the experiments are well executed. At the same time, a few aspects of the manuscript could be further improved. For example, the authors analyze multiple aspects of photoreceptor integrity. In some cases, they show that the mutant Esyt transgene shows dominant negative effects. In others, there is no evidence or even a partial function. Clarifying these points could be helpful. Below are a few specific points for the authors' consideration:

    Major

    1. RDGB is a protein that localizes to the ER-MCS. Esyt-CABM-GFP expression causes RDGB mis-localization even in the presence of wild type Esyt expression, suggestive of a dominant negative effect (Fig. 4C). But Esyt CaBM-GFP expression doesn't seem to have a dominant negative effect on contact site distance (Fig. 4D). Are the authors not seeing a dominant negative effect because they didn't examine older flies? Or, is there a distinct effect of Esyt CaBM on RDGB localization and contact site distance? If there is a distinct effect, what is the reason?
    2. Esyt-CABM-GFP partially rescues the Esyt KO phenotype in retinal degeneration (Fig 6). This is surprising since cellular assays in Fig 4 show a failure of Esyt-CaBM to localize to ER-MCS. The results here contrast with earlier data showing that Esyt-CABM has dominant negative effects. How will the authors interpret the results? Is it possible that Esyt-CAMB still has some residual Ca2+ binding activity? Alternatively, does this result imply that Esyt can still function (albeit at lower capacity) without binding Ca2+? Is there Esyt function unrelated to ER-MCS site maintenance when it comes to its role in retinal degeneration? A reasonable explanation is warranted.

    Minor:

    Figure legends refer to "SMC" (I am guessing they are referring to Sub microvillar cisternae) without defining it in the text.

    Significance

    This study will be of interest to those generally interested in the ER mitochondria contact sites. The main significance here is in dissecting the role of the C-domain within the Esyt protein. The authors demonstrate a physiological role using Drosophila photoreceptors as a model.

  5. 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

    Summary:

    This study builds on previous work from the same group, where they use Drosophila photoreceptors as a model system to investigate the role or ER-plasma membrane contact sites in an in vivo setting. The authors recently described a role of the ER-PM contact site protein dEsyt in regulating photoreceptor function in Drosophila. In this follow-up study, they explore whether this function of dEsyt is connected Ca2+ signaling downstream of photoreceptor activation. Using a dEsyt mutant that should be unable to bind Ca2+, they find that Ca2+ to some extent is required for dEsyt localization, membrane contact site formation and photoreceptor function.

    Major comments:

    The use of photoreceptor cells in Drosophila is an elegant model system that enable studies of membrane contact sites and associated proteins in a native condition. The data presented by the authors clearly shows that these structures are important for photoreceptor function, and that dEsyt plays a role at these sites. However, this was already known from previous studies by the same group. When it comes to whether these contacts are sensing Ca2+ changes and if these changes are acting through dEsyt, which is the focus of the current manuscript, the results are unclear to me and would need to be clarified by the authors both in text and with new experiments.

    1. What is the role of cellular Ca2+ signaling in the regulation of dEsyt function? There are several aspects here that needs to be clarified. 1) How is WT dEsyt localization regulated by Ca2+? This could for example be evaluated in the mutant flies used in Fig. 1 (trpl302; trp343), where lack of light-induced Ca2+ influx would be predicted to result in a localization of dEsyt that resembles that observed for dEsytCaBM. 2) Is Ca2+ important for dEsyt localization, lipid exchange or both? The authors express a version of dEsyt with mutation made in all three C2 domains. In mammalian E-Syts, Ca2+ binding to the C2A domain is important for lipid exchange while binding to C2C (in E-Syt1) is important for interactions with lipids in the plasma membrane. Using more carefully designed mutants will allow the authors to determine how Ca2+ regulates dEsyt function in vivo. In addition, the authors must show experimentally that the mutant dEsytCaBM is unable to bind Ca2+ (could e.g. be done by acute Ca2+ changes in the cell-based model used in Fig. 3). Writing that "This transgene carrying a total of nine mutations should render the protein unable to bind calcium" (p. 6, line 173) is not sufficient.
    2. The localization of dEsyt shown in Fig. 3B is a bit confusing. First of all, I would recommend including markers of the ER and the plasma membrane, because without these it is difficult to make statements about the localization of dEsyt to these structures. Second, it appears that WT dEsyt localize to the reticular ER, and that the CaBM version localize to the plasma membrane. This is somewhat opposite to mammalian ESyts, where mutations that prevent Ca2+ binding either had no effect (for ESyt2) or prevented (for ESyt1) the interaction with the plasma membrane. It also appears different from the localization in vivo (Fig. 3C). Clarifying this will be important. It will also be important to connect this localization to changes in Ca2+ and not just to the localization of a mutant that may or may not be deficient in Ca2+ binding (see comment above).
    3. I don't fully understand the time course of events. The authors show that dEsytCaBM is mislocalized already at day 1 in dark-reared flies (Fig. 3C) but this mislocalization is not accompanied by a change in MCS density or gap distance, and consistently does not influence the localization of RDGB. The authors next expose the flies to constant light illumination to trigger Ca2+ dependent signaling, and this leads to mislocalization of RDGB, perhaps indicating changes in MCS (this is not shown). From these results it is difficult to know what the role of dEsyt is. It would be necessary to also show a control where Ca2+ signaling is not induced, e.g. a parallel dark-control (same number of days but no illumination). This is particularly important given that the authors show in Fig. 1 that preventing Ca2+ influx had a dramatic impact on MCS density even at day 1 (which is in sharp contrast to dEsytCaBM-expressing flies, that show normal morphology at day 1, which rather implies that dEsyt is not a major Ca2+ effector).
    4. The experiments done in dEsyt KO flies are important, and here the authors show that dEsyt1 could to some extent rescue all phenotypes. Some results are a bit puzzling. For example, dEsyt1CaBM localization in dEsyt1 KO flies is identical to that of WT dEsyt (Fig. 5C), which is in sharp contrast to the data shown in Fig. 3C. What is the reason for this? I would have anticipated the opposite (i.e. that in WT flies, dEsytCaBM can form dimers with endogenous dEsyt through SMP-domain interactions which may have an impact on its localization and the function of endogenous dEsyt, but that in the dEsyt KO cells, dEsytCaBM would show a different localization due to the lack of endogenous dEyt to interact with). It is important to clarify as one of the major observations here is that dEsytCaBM no longer localize to MCS. Since the CaBM version of dEsyt could rescue, to some extent, MCS density and delay photoreceptor degeneration, this implies that Ca2+ may not be required for regulation of dEsyt function or that the mutant is still able to partially bind to Ca2+. One experiment that would help the authors determining the function of dEsyt in vivo would be to use a mutant that lacks functional SMP domain (ideally also with and without mutations in the C2-domains).
    5. PLC activation typically couples to rapid signaling and involved hydrolysis of PIP2 and release of Ca2+ from the ER. Mammalian Esyts also require PIP2 for plasma membrane binding (through interactions with C2-domains), so constitutive PLC activity would be expected to impair ESyt localization to MCS. Here, the authors expose flies for days of constant illumination. How does this influence plasma membrane PIP2 levels and could this be of relevance for how data is interpreted? Do the authors know whether the CaBM mutant has reduced affinity for PIP2?

    Minor comments:

    1. The overexpression of WT dEsyt had a dramatic impact on MCS density and gap distance, while expression of dEsytCaBM did not. If these contacts are important for photoreceptor function, is it not surprising that such a dramatic change in photoreceptor structure was without effect on function? This should be further discussed.
    2. How is quantification of MCS density and gap distance influenced by retinal degeneration (e.g. induced by dEsyt KO)?
    3. The graphical abstract is a bit confusing. It seems to suggest that changes in dEsyt is a consequence of ageing and does not show any role of this protein in photoreceptor function. I think that the abstract could be improved to more clearly highlight the findings in the manuscript. For example, it doesn't at all show the difference in localization between WT and CaBM.
    4. P. 5, line 135 the authors state that "The tethering and lipid transfer activity of mammalian Esyts are reported to be influenced by Ca2+". This is a massive understatement. Ca2+ is a critical regulator of Esyt function in mammalian cells.
    5. In figure legend 1B and C: correct µM to µm.
    6. In figure legend 2A: should be red rectangles and not black rectangles.
    7. In Fig. 2B: specify which isoform of human ESyt that is shown.
    8. In Fig. 2C: do the authors mean D374 or D384 (as indicated in Fig. 2A)?

    Significance

    Light-induced signal transduction in photoreceptor cells involves Ca2+ influx and signaling and also depends on correct formation of ER-plasma membrane contact sites. In mammalian cells, the Esyts (esp. Esyt1 and Esyt2) localize to ER-PM contacts in a Ca2+-dependent manner, and the ion has dual effects in both enriching the protein at the membrane contact sites and in promoting lipid transport. Mammalian Esyts form homo- and heterodimers, and the properties of the dimers depends on their composition (PMID: 26202220). Drosophila only have one Esyt (dEsyt) which is structurally most similar to mammalian Esyt2, and the authors have previously shown how this protein is required for photoreceptor function (PMID: 32716137), although the role of Ca2+ was not investigated in that study. However, an earlier study has shown that mutations of all Ca2+-coordinating residues in dEsyt impairs protein function in Drosophila neurons (PMID: 28882990), so a similar Ca2+-dependence in the retina would be expected. The results from the present study confirm the requirement of Ca2+ signaling for dEsyt function, and extends this Ca2+-dependent regulation to also involve photoreceptor-induced Ca2+ signaling, which corroborates many other studies showing the requirement of Ca2+ signaling for the regulation of Esyt function in mammalian cells (e.g. PMID: 23791178; PMID: 27065097; PMID: 29222176; PMID: 26202220; PMID: 24183667; PMID: 30589572). As such, the results from this study represent an incremental step towards understanding Esyt function in vivo. These results would be of greatest interest to researchers working of photoreceptor function, and of some interest to a broader audience working on membrane contact sites and signal transduction. My own background is in mammalian cell biology, with a focus on lipid and Ca2+ signaling and inter-organelle communication. I have limited understanding of the model system used here (Drosophila photoreceptor cells).

  6. Version published to 10.1101/2024.04.05.588361 on bioRxiv