Disruption of Synaptic Endoplasmic Reticulum Luminal Protein Containment in Drosophila Atlastin Mutants

  1. Mónica C. Quiñones-Frías
  2. Dina M. Ocken
  3. Avital Rodal

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Abstract

The endoplasmic reticulum (ER) extends throughout neurons and regulates many neuronal functions, including neurite outgrowth, neurotransmission, and synaptic plasticity. Mutations in proteins that control ER shape are linked to the neurodegenerative disorder Hereditary Spastic Paraplegia (HSP), yet the ultrastructure and dynamics of neuronal ER remain largely unexplored, especially at presynaptic terminals. Using super-resolution and live imaging in D. melanogaster larval motor neurons, we investigated ER structure at presynaptic terminals of wild-type animals and null mutants of the ER shaping protein and HSP-linked gene, Atlastin. Previous studies using an ER luminal marker reported diffuse localization at Atlastin mutant presynaptic terminals, which was attributed to ER fragmentation. However, using an ER membrane marker, we discovered that Atlastin mutant ER forms robust networks with only mild defects in structural dynamics, indicating the primary defect is functional rather than architectural. We demonstrate that Atlastin mutants progressively displace overexpressed luminal ER proteins to the cytosol during larval development, specifically at synapses, while these proteins remain correctly localized in cell bodies, axons, and muscles. This synaptic-specific displacement phenotype, previously unreported in non-neuronal cells, emphasizes the importance of studying neurons to understand HSP pathogenesis.

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    Reply to the reviewers

    Reviewer #1:

    Major comments:

    Lines 103-116 (first paragraph of the results section) describe mainly published data that is more suitable for the introduction section. It is annoying to refer to different published articles in the Results section to strengthen the results instead of showing them. The same goes for paragraphs two and three. Why mention those data in the Results section if they are already published and known?

    We have reorganized this material by moving some background information to the Introduction. Our intention was not to incorporate published data to strengthen our results, but rather to provide essential context for interpreting our findings. We have therefore left some of this foundational information in the results section to create a clear narrative flow, enabling readers to understand the basis for our experimental design and interpretations without needing to recall details from earlier paragraphs in the Introduction. For example, we considered it crucial to restate the earlier report of the BiP:sfGFP:HDEL phenotype in Atlastin mutants, since our results supporting luminal ER protein displacement contradict the previous fragmentation model.

    The following concept was in line 103 in the Results section, and is now in the introduction in lines 82-92: "Conventional light microscopy, commonly used in studies of neuronal ER structure, lacks the resolution necessary to visualize individual ER tubules in small structures, such as presynaptic terminals. The ER is highly sensitive to fixation, and live imaging experiments in neurons in vivo have been conducted on upright microscopes using water dipping objectives with a typical axial resolution limit of >300 nm, which cannot distinguish the densely packed ER tubules at presynaptic terminals (3,8,21,28,39-42). Electron microscopy offers higher resolution, but cannot be used in live samples and has typically been limited to thin 2D sampling (in which it is difficult to distinguish ER cross-sections from synaptic vesicles) (8,20,22)."

    Figure Legends-(in all Figures): The number of experimental repeats must be mentioned in the figure legends.

    This information is provided in Supplementary Table 1, which contains detailed information about the genotype, statistical analysis, and number of larvae and NMJs analyzed. If the journal requires this information in figure legends, we can move it.

    The way the figures are labeled is worrisome; supplementary figures are not ordered numerically.

    We will be happy to rename supplementary figures according to journal guidelines.

    The tubule extension in Figure 2D is not convincing. Is there a movie showing those changes? Better images are needed. It is essential to show which supplemental movie corresponds to which panel.

    We have now included a corresponding video of the same neuron used as an example of tubule extension. We also added another frame to the figure to provide further information on the tubule event we captured. (Figure 2D, Movie S10)

    This is unnecessary in the results section: "To investigate the relationship between ER structure and function at synapses, we examined mutants of Atlastin, a GTPase that regulates ER tubule fusion. Drosophila has a single homolog while mammals have three Atlastin homologs, with Atlastin-1 enriched in the brain (Rismanchi et al., 2008)."

    This information was moved to the introduction.

    "This reduction in ER membrane marker intensity has also been observed in other HSP mutants, suggesting this is a common feature of ER shaping mutants and could indicate changes in ER membrane composition, integrity, or tubule thickness (Perez-Moreno et al., 2023)." This comparison is important and should be shown in the same settings as for the Atlastin mutant rather than referring to published data.

    We agree with the reviewer that it is important to determine whether other ER-shaping proteins, besides Atlastin, also show a decrease in tdTomato:Sec61b to support our claim that this could be a common feature among ER-shaping mutants. To do this, we examined mutants of another ER-shaping protein, Reticulon 1, which regulates membrane bending and stabilization in ER tubules. These loss-of-function mutants were a gift from Dr. Cahir O'Kane at the University of Cambridge and were used in his lab's Pérez-Moreno et al., 2023 publication. We found that in our hands tdTomato:Sec61b levels were reduced in Reticulon 1 mutants, consistent with the results reported by Pérez-Moreno et al. (2023). These results are in Figure 3E-F. We also examined the synaptic distribution of the luminal ER marker, BiP:sfGFP:HDEL, in Reticulon 1 mutants to see if it is displaced to the cytosol. Notably, it remained ER-associated, unlike in Atlastin mutants. These results are in Figure 6F-G, results lines 267-270, and discussion lines 542-545.

    Does the distribution of the luminal ER marker in Figure 6F diffuse due to mislocalization or reflux after being localized to the ER and then refluxed to the cytosol as was previously shown for the ER to Cytosol signaling (ERCYS) mechanism? Could you assess other ER-luminal protein localization biochemically? It is highly recommended to look at another soluble ER-protein localization in the Atlastin mutant without overexpression, which can be an artifact.

    ER stressors can induce ERCYS, in which some luminal proteins, including PDIA3, DNAJB11, ERp29, and an eroGFP reporter, reflux by 30-70% to the cytoplasm without subsequent degradation (unlike ERAD (ER-associated degradation). This phenomenon has only previously been observed in yeast and glioblastoma tumor cells from mice and human . We believe that our work provide the first suggestion that this may occur in neurons, and particularly in a neurological disease model.

    We do not believe that the reflux phenotype for BiP:sfGFP:HDEL is due to its overexpression for two reasons: (1) we observe reflux in our neuronal Atlastin knockdown experiments, even when the levels of BiP:sfGFP:HDEL are significantly reduced artificially because of titration of the GAL4 between the RNAi and the reporter (Figure 7A), and (2) BiP:sfGFP:HDEL overexpression somewhat suppresses endogenous BiP upregulation ((Figure 10 and see Reviewer 1.10), arguing that the transgene does not induce ER stress). We included a new "limitations of the study" section to be transparent about the caveats of the BiP:sfGFP:HDEL reporter (lines 639-664).

    Identifying potential endogenous neuronal ERCYS substrates in our in vivo preparation poses several challenges. First, biochemical approaches, such as fractionation, are not possible in our complex in vivo sample because neuronal ER proteins would mix with ER from other tissues upon homogenization. Second, detecting endogenous proteins with antibodies requires fixation and permeabilization, which notoriously disrupts ER structure and even causes our reporter BiP:sfGFP:HDEL to collapse from a smooth distribution, as visualized by live imaging and FRAP, to a punctate distribution. Third, using antibodies rather than neuronally restricted transgenes makes it challenging to determine whether the signal originates from the neuron or from dense ER structures in the surrounding muscle. Fourth, some ER luminal proteins can displace as little as 30% in the ERCYS examples cited above, and the sensitivity of our imaging assays may limit our ability to detect these small changes. Finally, the limited availability of tagged transgenes and antibodies specific to Drosophila luminal ER proteins (see next paragraph) poses additional challenges. These limitations highlight the need for future studies to develop novel tools and techniques to more definitively test whether we are indeed observing ERCYS. We have included a paragraph on these future challenges in our discussion in lines 639-664. Identifying endogenous targets of ERCYS in fly neurons is a worthwhile goal, but beyond the scope of the current study. These next steps will particularly benefit from identifying the machinery involved in the reflux of our BiP:sfGFP:HDEL reporter.

    Tools we tested: We investigated several options: (1) a tagged PDI transgene (a gift from Karen Hibbard), which was not detectable at presynaptic terminals, (2) a tagged BiP (FlyORF; F000956) that did not localize to the ER, and (3) full-length endogenous BiP detected by antibody staining. We did not detect obvious reflux of endogenous BiP to the cytoplasm (Figure 9), with the caveat that in fixed samples, the BiP signal was not tightly co-localized with the ER marker even under control conditions. However, we did use this antibody to detect an increase in BiP in Atlastin mutant presynaptic terminals, indicating ER stress (see Reviewer 1.10).

    Though we have not identified endogenous targets, we believe that our studies with the exogenous reporter will be of great interest to the field, as they clarify the previously reported Atlastin phenotype and provide the first report of a new defect in a human disease animal model.

    In comparison to Summerville et al. (2016) in Figure 7, the experiment was not done in the same way. It is important to keep the same settings for comparison

    In Figure 7D-E, we compare the distribution of BiP:sfGFP:HDEL in cell bodies, axons, and muscles between controls and Atlastin mutants. To clarify the experimental approach relative to Summerville et al. (2016): while both our studies examined the same cellular compartments (cell bodies, axons and nerve terminals) using the BiP:sfGFP:HDEL reporter, we employed super-resolution Airyscan microscopy. This enhanced resolution was critical for definitively demonstrating that this is a functional rather than a structural phenotype and that ER displacement is progressive, and repeating this experiment at lower resolution as previously reported does not provide any new information. We identified two distinct distribution phenotypes in Atlastin mutants expressing BiP:sfGFP:HDEL, which were not described in the Summerville et al., 2016 paper. From our manuscript (lines 249-251): "We identified two distinct ER network phenotypes in Atlastin mutants expressing BiP:sfGFP:HDEL: "Partial loss" NMJs retained both diffuse signal and identifiable ER network structures, while "Complete loss" NMJs showed no visible ER network structures. Note that the "Complete loss" phenotype in Atlastin mutants reflects the absence of detectable luminal marker signal in organized ER structures, but not the complete absence of ER membranes, as demonstrated by our ER membrane marker tdTomato:Sec61β results."

    Does the Atlastin mutant induce the unfolded protein response and stress within the ER? It is necessary to look for UPR markers in those settings. It was shown previously that ER stress leads to protein reflux from the ER to the cytosol. Is there a difference in the ER stress markers in the presynaptic terminal?

    The reviewer suggested that Atlastin mutant synapses may exhibit ER stress. To address this, we examined levels of the ER chaperone BiP, a well-established ER stress marker whose expression increases during UPR activation. We first validated that our BiP antibody can detect changes in ER stress by feeding control larvae with 50mM DTT for 24 hours. These results are in the new Figure 10A. Note that we were unable to test sensitivity to ER stress in this way in Atlastin mutant larvae because they did not consume the DTT-treated food, as assessed by blue food coloring in the larvae's guts.

    Using this antibody, we measured baseline BiP levels at NMJs of Atlastin mutants on normal food, and found they were slightly increased compared to controls. We conclude from these experiments that Atlastin mutant synapses have mild ER stress. Notably however, Atlastin mutants co-expressing UAS-BiP:sfGFP:HDEL or UAS-tdTomato:Sec61b did not show significantly increased endogenous BiP levels, suggesting that transgene expression at least partly suppresses the mild ER stress response, even though there is extensive cytosolic displacement. These results argue (1) that the mild ER stress in Atl mutants does not strictly correlate with the reflux phenotype, and (2) that the reflux phenotype is not an artifact of overexpression-induced stress. These results are described on lines 430-436 in the results section and shown in Figure 10B-E, and their implications discussed on lines 585-598.

    We also explored another strategy to detect ER stress by assessing eIF2α phosphorylation, a key event in the Unfolded Protein Response (UPR) pathway. We obtained a phospho-eIF2α antibody (Cell Signaling; #3597) that was reported to work in Drosophila. However, when we tested this antibody by Western blot, we were unable to detect a band at the expected molecular weight for phosphorylated eIF2α, even in positive-control samples treated with DTT to induce ER stress. We therefore concluded that this antibody is not suitable for reliably detecting ER stress in our experimental system. The failure of this antibody highlights the challenges of finding robust tools to measure ER stress in Drosophila.

    It is important to add biochemical experiments to show that no fragmentation of the ER membrane occurred. It can be simply demonstrated by looking at the redox state of the ER, which would change if it were mixed with the reducing cytosol. Moreover, this can be shown by using an ER-targeted redox-sensitive fluorescent protein that is tethered to the ER membrane to follow changes in the redox state of the ER.

    The reviewer asked us to test whether the redox state of the ER is disrupted, which could indicate exchange between the cytosol and ER due to membrane rupture. As noted above, biochemical approaches such as fractionation are not possible in this in vivo sample. We attempted to address this concern by creating a UAS-Sec61β:roGFP construct, using the roGFP sequence from Igbaria et al. (2019) to monitor the ER lumen redox environment in Atlastin mutants. Since Sec61β is membrane-tethered, it should remain in the ER and not undergo reflux, making it an ideal sensor for detecting any mixing between the reducing cytosolic environment and the oxidizing ER lumen that would occur if membrane fragmentation and/or ruptures were present. We tested this approach in wild-type Drosophila S2 cells and used the Gal4-UAS binary expression system to co-express Actin-Gal4 (to drive expression of UAS constructs), UAS-Sec61β:roGFP (redox sensor), and UAS-BiP:Halo:HDEL (as a control reporter insensitive to DTT treatment).

    Our experiments showed no detectable changes in the fluorescent properties of UAS-Sec61β:roGFP following 30 min 10mM DTT treatment compared to DMSO vehicle control, including no increase in 405-nm excitation fluorescence or changes in 488nm/405nm excitation ratios. These results suggest that either the roGFP sensor requires further optimization for sensitivity in this cellular system or that additional controls and calibration steps are needed to establish the dynamic range of the assay. We believe this experiment falls beyond the scope of the current study, given the extensive optimization required. However, it represents an important future direction for testing membrane fragmentation as a mechanism underlying the phenotypes observed in Atlastin mutants. The possibility of ER integrity defects is mentioned in the discussion on lines 547-559.

    Minor comments:

    It is important to call figures by order. Figure 2C is called before 2A-B. Figure 2B is called before Figure 2A.

    The revised manuscript has all figures in order of appearance in the text.

    Figure legends (Figure 2): "The same control dataset used in E-G was used in Figure 5 and Figure 5_Supplement." Why is this relevant?

    We wanted to be transparent about reusing the same control dataset across multiple figures to avoid any appearance of data duplication. This notation clarifies that, although the data appear in different contexts (Figures 2 and 5. This version does not contain a Figure 5_Supplement), it represents the same biological samples analyzed for different parameters, ensuring readers understand that these are not independent datasets.

    Figure 4F is called before Figure-4D-E which are not called.

    We revised our manuscript and reorganized Figure 4 to ensure that all figure panels are referenced in sequential order and that panels 4D-E, which were previously not cited in the text, are now properly referenced when discussing their corresponding results.

    Figure 5B is called before the previous ones. Same for Figure 5A supplement.

    We referenced Figure 5A in lines 211-212, which precedes our discussion of Figure 5B. To clarify the figure order, we removed the early references to Figures 2D-G and Movies 7-14, which were mentioned only to indicate that we were analyzing the same dataset in different ways.

    The revised manuscript has all figures in order of appearance in the text.

    Referees cross-commenting

    I agree with the comments raised by reviewer2 and 3. Basically it is highly important to validate those data by genetic rescue. Moreover, it is essential to know the source of the displaced luminal marker to the cytosol. Is it mislocalization or it is a reflux of pre-existing protein to the cytosol after insertion to the ER. It is also recommended by me and the reviewers and me to test the endogenous protein rather than overexpression.

    We have addressed these points in our responses to the following reviewer questions:

    • Genetic rescue: Please see our responses to Reviewer 1/Question #10 and Reviewer 2/Question #1.
    • Source of displaced luminal marker: We provide some evidence addressing this in our response to Reviewer 3/Question #1.
    • Endogenous protein localization: We have examined this and detailed our findings in our responses to Reviewer 1/Question #7 and Reviewer 2/Question #6.

    Reviewer #1 (Significance (Required)):

    General assessment: This interesting paper shows that proteins can escape the ER under special conditions. However, the authors need more evidence to show that and rely less on the overexpression system, especially of BIP-GFP, which can cause proteostasis stress within the ER. Advance: The results have been oversimplified in their explanations, and some points and complexities of the study need to be addressed further to make the most of them. These are often some of the more interesting concepts in the paper. I think many points can be addressed in the text by the authors being clear and concise with their reporting. At the same time, other experiments would turn this paper from an observational one into a very interesting mechanistic one. This paper is based on previously published articles from the group and other groups, and it is a nice progression. However, as mentioned, this paper depends primarily on published data, and the novelty is somehow lost between all the comparisons to other published data instead of emphasizing that. Without a substantial mechanistic improvement, the paper would remain observatory.

    Audience: The microscopy tools can be great addition to researchers in the field to monitor protein trafficking especially Cell biologists (basic research)

    My expertise: ER homeostasis, protein trafficking, cell biology

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

    Summary The endoplasmic reticulum (ER) is a continuous organelle that extends throughout neurons to regulate fundamental processes. The analysis of ER dynamics at synaptic terminals is limited by the challenge of imaging these structures at high resolution. In this manuscript, the authors use super-resolution (~170 nm) live imaging and a combination of membrane and luminal ER markers at the Drosophila larval NMJ, an important model synapse, to investigate dynamic ER architecture in vivo. They report a detailed characterization of the presynaptic ER organization and dynamics at wild-type and GTPase Atlastin mutant NMJs. Their analysis using the ER membrane marker tdTomato:Sec61b reveals the presence of an intact ER network in Atlastin mutants. This contrasts with the apparent ER fragmentation phenotype previously reported and replicated here when using a luminal marker. Their findings instead point to the progressive displacement of luminal proteins to the cytosol in Atlastin mutants specifically at synapses. The authors propose that the disruption of ER protein dynamics at synapses is a compartment-specific ER stress response. The manuscript is well written, results are clearly presented, and experiments are technically rigorous.

    Major comments

    The baseline ER phenotypes in Atlastin mutants are mild with complete loss of ER network only observed in terminal boutons. This interesting and unexpected result should be further confirmed by genetic rescue. The authors can use a UAS rescue line previously reported in PMID: 19341724.

    We tested the UAS-Atl-myc rescue line and unfortunately found that even in wild-type neurons, overexpression of Atlastin produced strong ER organization defects that precluded the rescue experiment. Instead, to confirm the cell autonomy of the phenotype and to test it wth an independent tool, we performed a presynaptic knockdown of Atlastin by RNAi and found that BiP:sfGFP:HDEL is displaced, as observed in the Atlastin null mutant. These results are in now shown in Figure 7A-C.

    Lines 204-7: It's not clear how a greater coefficient of variation indicates that the marker is more concentrated in subsynaptic structures or what is meant by 'subsynaptic structures.'

    We added the following text to explain, in lines 181-183: "A higher CoV indicates an uneven distribution of tdTomato:Sec61β within the presynaptic terminal, with some areas showing higher concentrations than others (in contrast to the uniform, diffuse signal expected from fragmentation)." To avoid confusion with postsynaptic structures called the subsynaptic reticulum, we have removed the term "subsynaptic". The intended meaning is distinct structures found within the presynaptic terminal.

    There's a mistake in Figure 6C and the associated text. The summed percentage of the three phenotypic categories adds up to 110% for Atlastin mutants. *

    The reviewer noted that the summed percentage of the three phenotypic categories in Figure 6C adds up to 110% for Atlastin mutants, which appears to be a mathematical error. However, this is not an error, but rather a reflection of our quantification methodology, in which a single bouton can exhibit more than one type of ER dynamics per movie recorded. Our quantification counts each phenotype independently, so boutons displaying multiple phenotypes contribute to more than one category. This approach provides a more comprehensive view of the range of ER dynamics present in Atlastin mutants, as restricting the analysis to mutually exclusive categories would underrepresent the complexity of the phenotypes observed. To make this point clear, we made the following change to the text in lines 257-259: "We note that the sum of these percentages exceeds 100% because one NMJ exhibited multiple phenotypes: one branch had a complete loss, while the other branch had no phenotype. These phenotypes were counted separately."

    Figure 8: the ER looks fragmented in 1st instar controls and mutants. The authors should address this difference from more mature NMJs.

    We would like to clarify that the bulk of experiments in this manuscript (including all ER dynamics, luminal marker redistribution, and membrane marker analyses discussed throughout the Results) were performed in 3rd instar larvae, which are more mature larval NMJ preparations standard in the field. Figure 8 was included specifically to test whether the Atlastin mutant phenotype we describe throughout the paper is also detectable at an earlier developmental stage, not to replace or reinterpret our primary findings.

    Regarding the specific observation that the ER appears more fragmented in Figure 7F-H relative to the more mature NMJs shown elsewhere: this fragmentation, observed similarly in both control and Atlastin mutant 1st instar larvae, likely reflects technical challenges associated with dissecting these smaller, more delicate early-stage specimens rather than a genotype-specific effect. Because fragmentation occurred similarly in both genotypes, we could still reliably assess the redistribution of BiP:sfGFP:HDEL as our primary phenotypic readout in this experiment. We have added the following text (lines 306-309) to clarify this point: "Note that in 1st instar larvae, both normal networks in controls and residual networks in Atlastin mutants appeared more fragmented than in 3rd instar preparations, likely due to the technical challenges of dissecting these smaller, more delicate specimens. Since ER fragmentation occurred similarly in both genotypes, we could still reliably assess the redistribution of BiP:sfGFP:HDEL as our primary phenotypic readout.

    The images in figure 9B do not seem representative of the quantification in Figure 9D. Specifically, the partial loss Atlastin NMJ appears to have recovered as fully as the complete loss Atlastin NMJ.

    The images showed FRAP recovery across the entire bouton, but we photobleached only a small region within each bouton and quantified only this region. We have now added outlines to clearly delineate the specific FRAP regions that were analyzed in each image, which clarify that the partial loss Atlastin showed less recovery than the overall bouton. We have also reordered the figures to more clearly convey our message (Figure 9 is now Figure 8).

    We also made a few changes to the paragraph on lines 347-350 to clarify our experimental reasoning: "We photobleached en passant boutons using a defined region of 6.8 x 7.8 microns (dashed box in Figure 8D) to ensure that BiP:sfGFP:HDEL could recover from the ER networks surrounding the FRAP region (Movies S20-S23)."

    We also added this sentence to the figure legends of Figure 8: "The dashed boxes in (D) indicate areas that were photobleached and analyzed for recovery quantification in (E-F)."

    Optional: An overexpressed luminal marker is displaced to the cytoplasm in Atlastin mutants. It would be interesting to know and increase the significance of the findings if the same is true of endogenous luminal proteins under biological stress conditions.

    As noted in our response to Reviewer #1 suggested that Atlastin mutant synapses may exhibit ER stress. To address this, we examined levels of the ER chaperone BiP, a well-established ER stress marker whose expression increases during UPR activation. We first validated that our BiP antibody can detect changes in ER stress by feeding control larvae with 50mM DTT for 24 hours. We were unable to perform this experiment in Atlastin mutant larvae because they did not consume the DTT-treated food, as assessed by blue food coloring in the larvae's guts. These results are in Figure 10A. In the future, it will be of interest to establish a protocol to examine Atlastin mutants by feeding or treating larval fillets with DTT.

    We measured BiP levels at NMJs of Atlastin mutants and found they were slightly increased compared to controls. Atlastin mutants co-expressing UAS-BiP:sfGFP:HDEL or UAS-tdTomato:Sec61b did not show significantly increased endogenous BiP levels, suggesting that transgene expression suppresses the mild ER stress response. We conclude from these experiments that Atlastin mutant synapses have mild ER stress. These results are in Figure 10B-E).

    Optional: Applying this approach in stimulated conditions (high potassium, increased temperature) might reveal a greater activity-dependent role for Atlastin at synaptic terminals.

    This is a very interesting idea, as we have only examined synapses at rest. However, this is beyond the scope of this paper.

    Minor Comments

    Line 16: Atlastin should be italicized.

    Thank you for catching this typo. We have fixed it.

    Figure 5A: Based on the relative intensities, it appears that control and mutant images are not contrast matched but this isn't stated.

    Thank you for catching this omission. We added to the figure legend: "Control and Atlastin mutant images are not contrast matched."

    Line 822: The number of static Atlastin mutant boutons used for analysis is missing.

    Thank you for catching this omission. We have fixed this supplementary table.

    Figure 9: The blue arrows are not annotated in the figure legend.

    Thank you for catching this omission. We have fixed this figure legend.

    Reviewer #2 (Significance (Required)):

    Atlastin is linked to Hereditary Spastic Paraplegia (HSP) and this study changes our understanding of the compartment-specific impacts of its loss. This study reveals the importance of using both membrane and luminal ER markers to accurately interpret phenotypes as well as the importance of considering compartment-specific effects on ER. These findings represent significant mechanistic and conceptual advances. The lack of genetic rescue is a limitation and adding an investigation of an endogenous luminal protein under basal and stress conditions would add significantly to our understanding of Atlastin dysfunction in HSP. Notably, the in vivo imaging approach introduced here can be adapted broadly for live imaging of Drosophila larvae. Thus, this work will be of interest to both neuronal cell biologists and the wider Drosophila community. This review is based on our expertise in neuronal cell biology.

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

    In this manuscript, the authors investigate the structural dynamics of the endoplasmic reticulum (ER) in Drosophila neurons and examine the role of the ER-shaping protein Atlastin in ER morphology. Their discovery on the neuromuscular junction (NMJ)-specific contribution of Atlastin to ER integrity is intriguing and may provide valuable insights into the pathological mechanisms underlying Atlastin mutations associated with hereditary spastic paraplegia (HSP) and hereditary sensory neuropathy. The key observation on ER protein showing an aberrant cytoplasmic localisation in mutant cells appears convincing. Though this phenomenon's characterisation stays at the point of primary observation with its mechanics unclarified, establishing this new and unexpected functional rather than structural Atl effect is important and useful for the field. The observation that ER is structurally preserved in this mutant with absolute lack of Atl are also extremely useful.

    It is unclear if the cytoplasmic localisation affects an exogenous overexpressed ER marker or endogenous protein would also appear in cytoplams, the authors should consider adding an immunostaining data to test that.

    Authors offer speculations on potential reasons for the cyto localisation of the ER marker suggesting that relocation at the cell periphery specifically combined with slow clearance there is the most likely explanation (still unclear what stops the marker from spreading through the entire cell). They suggest that decrease in cotranslational translocation is unlikely as this would result in somatic accumulation of the marker. However, if the clearance in the periphery is less efficient than in soma, the accumulation there might reflect a compromised translocation. Any clarifying experiments, if practical, to directly demonstrate how ER proteins in relocates to the cytoplasm in atl mutant would help understanding better the phenomenon. For example, would proteasomal inhibition make the marker accumulate more across the cell? Authors also suggest links to ER stress. Would stress induction phenocopy the mutant?

    Reviewer #3 asked whether defective proteasomal clearance underlies the cytosolic accumulation of BiP:sfGFP:HDEL in Atlastin mutants. We addressed this directly. First, proteasome function appears intact in the mutants: baseline ubiquitinated protein levels (FK1 antibody) were comparable between control and Atlastin mutants, and MG132 treatment produced a similar increase in ubiquitination in both genotypes, confirming both antibody specificity and normal proteasome activity. We then examined BiP:sfGFP:HDEL directly. In controls, MG132 caused the marker to accumulate at axons and presynaptic terminals, showing that it is normally cleared from these compartments by the proteasome. Critically, this accumulated marker remained associated with intact ER networks: MG132 did not induce diffuse cytosolic BiP:sfGFP:HDEL in any compartment (cell bodies, axons, or presynaptic terminals), even where levels rose substantially. Thus, blocking proteasomal clearance raises ER-localized marker but does not generate the cytosolic pool seen in Atlastin mutants, indicating that impaired clearance is not sufficient to cause the displacement phenotype. We separately noted that BiP:sfGFP:HDEL was already elevated in Atlastin mutant axons without MG132, paralleling the axonal tdTomato:Sec61β accumulation in Figure 4, consistent with reduced baseline clearance specifically in mutant axons, but this does not lead to cytosolic displacement. This experiment is now shown in Figure 11, described in Results (lines 445-475), and discussed in lines 576-581.

    Minor comments:

    Line 146:

    "fast dynamics (Thank you for catching this mistake. We have corrected it.

    Fig. 2D: The data representation of "Tubule displacement" image is unclear. The ER tubule indicated by the red arrow does not seem to show any changes over time (like static). time 0 in stamp appears behind the image.

    Thank you for catching the typo. We have fixed it. Additionally, we added black arrows to highlight a tubule that is not moving, allowing the reader to compare it with the moving tubule. We also included a video of all types of ER tubule dynamics to ensure the reader can also look at the raw data (Movies S9-11).

      • Line 157-158 (and relevant method sections):

    The definition of static and dynamic boutons is ambiguous. The author should describe in more detail this point including how long they observed the structure to define the changes in ER tubule dynamics.

    We provide in the methods (lines 779-791) a detailed explanation of how we categorized boutons as dynamic or static. In addition, we added the following to explain in the results section how we defined static vs dynamic:

    Old sentence: We qualitatively categorized boutons as "static" if we observed no change in ER network structure or "dynamic" if we observed at least one change.

    New sentence in lines 143-147: "We imaged boutons for 40 sec at 0.92 sec intervals to capture ER dynamics over this observation period. Boutons were qualitatively categorized as "static" if we observed no detectable changes in ER network structure throughout the entire 40 sec imaging session, or "dynamic" if we observed at least one of the three defined dynamic events during this time window."

    Fig. 2E: What n=75 and n=29 represent is unclear, are these the number of boutons in en passant and terminal subjected for qualitative analysis?

    We removed these n values from the figure and added this information to the Supplementary Table 1, which contains detailed information about the genotype, statistical analysis, and number of larvae and NMJs analyzed.

    Fig. 2: What the qualitative analysis represents is unclear, are the points pulled from different experiments?

    The data in Fig. 2 E-F comes from movies acquired in the same experiment. The number of independent animals and NMJs imaged is described in Table 1.

    *Line 231: Regarding "...we found a small but significant reduction in dynamic boutons in Atlastin mutants (76%), ...", how do the authors assess significance. If proportion of static/dynamic ER in boutons was obtained from multiple experiments, it should be presented e.g. as in average {plus minus} standard deviation, or clarify that the proportion is representative of x independent experiments.

    The videos used for this figure were acquired from a single experiment. We use a chi-square test to determine significance relative to the "expected" distribution of dynamics types from controls, as these are categorical rather than continuous data (see PMID 31145670). Information regarding genotype, statistical analysis and number of larvae and NMJs can also be found in Supplementary Table 1.

    Line 267-269 and Fig. 6B: The author's conclusion that "Complete loss of ER network structure in NMJ of BiP:sfGFP:HDEL overexpressing Atl mutant" seem to be based on the lack of signal from luminal marker, which may be undetectable due to changes to tubular volume or marker loss to the cytoplasm, as suggested by the authors, while the membranous ER structure is intact. It would be useful to discuss this point and potentially add ER membrane-stained control.

    We agree with the reviewer that Atlastin mutants categorized as 'complete loss mutants' do not actually lack ER at synapses. We think this is an important point so we added the following to the results in lines 251-254: "Note that the "Complete loss" phenotype in Atlastin mutants reflects the absence of detectable luminal marker signal in organized ER structures, not the complete absence of ER membranes, as demonstrated by our ER membrane marker tdTomato:Sec61β results."

    We attempted to co-label the ER membrane and ER lumen, but these crosses yielded very few live larvae (in either controls or Atlastin mutants, and those that survived had severely deformed NMJs. We added Figure 6-Supplement showing the results of this experiment, and described them on lines 270-273.

    Fig. 6C: In Atl mutant, why does the total of the proportion exceed 100% (10 + 45 + 55)?

    The reviewer noted that the summed percentage of the three phenotypic categories in Figure 6C adds up to 110% for Atlastin mutants. This is not an error, but rather a reflection of our quantification methodology because a single bouton can exhibit more than one type of ER dynamics per movie recorded. Our quantification counts each phenotype independently, so boutons displaying multiple phenotypes contribute to more than one category. This approach provides a more comprehensive view of the range of ER dynamics present in Atlastin mutants, as restricting the analysis to mutually exclusive categories would underrepresent the complexity of the phenotypes observed. To make this point clear, we made the following change to the text in lines 257-259: "We note that the sum of these percentages exceeds 100% because one NMJ exhibited multiple phenotypes: one branch had a complete loss, while the other branch had no phenotype. These phenotypes were counted separately."

    Fig. 9C, line 342-344: In FRAP experiment using CD8, it seems that the Partial loss Atl mutant shows slower recovery that control. There seems to be a mismatch in triangle symbols of Partial loss Atl mutant between legend and plot (one is filled and the other is empty). This should be clarified.

    Thank you for catching this mistake. We have fixed the figure.

    fig. 10 is a clever way to verify the cytoplasmic localization of the ER marker; however, its description and annotation can be improved, and it would be stronger if 4 curves in F for mutant and controls with the trap and normal were shown.

    The reviewer suggested merging our graphs but we believe that keeping them separate is clearer.

    Line 495: Drosophila have ReepA and ReepB, but not Reep1-4. If the authors discuss their speculation based on their observation (using Drosophila), the gene names should be unified in the same species, and explain the corresponding genes to mammalian cells.

    We made the following changes to address the reviewer's concern about gene nomenclature consistency (lines 502-506): "These ER-derived vesicles are likely to involve ReepA and ReepB, the Drosophila orthologs of mammalian REEP1-4, which regulate ER vesicle formation in mammalian cells (67). Notably, while overexpression of Atlastin can regulate REEP vesicle fusion in mammalian systems (67), it is not essential for vesicle formation, suggesting similar regulatory relationships may exist between Atlastin and Reep genes in Drosophila."

    Line 548; should UPR be Unfolded Protein Response?

    Thank you for catching the typo. We have fixed it.

    Reviewer #3 (Significance (Required)):

    This study advances the understanding of how ER morphogens affect neuronal cells specifically, the lack of which limits researchers ability to comprehend the neuronal pathologies associated with ER structure-function. The observation on ER content aberrant localisation caused by the lack of key structural protein should be of a great interest for cell and neuronal biologists and researchers of the associated diseases and shows the field a new direction. Though, mechanistic details remain to be unraveled, it constitutes a fundamental, conceptual advance.

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    Referee #3

    Evidence, reproducibility and clarity

    In this manuscript, the authors investigate the structural dynamics of the endoplasmic reticulum (ER) in Drosophila neurons and examine the role of the ER-shaping protein Atlastin in ER morphology. Their discovery on the neuromuscular junction (NMJ)-specific contribution of Atlastin to ER integrity is intriguing and may provide valuable insights into the pathological mechanisms underlying Atlastin mutations associated with hereditary spastic paraplegia (HSP) and hereditary sensory neuropathy. The key observation on ER protein showing an aberrant cytoplasmic localisation in mutant cells appears convincing. Though this phenomenon's characterisation stays at the point of primary observation with its mechanics unclarified, establishing this new and unexpected functional rather than structural Atl effect is important and useful for the field. The observation that ER is structurally preserved in this mutant with absolute lack of Atl are also extremely useful.

    It is unclear if the cytoplasmic localisation affects an exogenous overexpressed ER marker or endogenous protein would also appear in cytoplams, the authors should consider adding an immunostaining data to test that.

    Authors offer speculations on potential reasons for the cyto localisation of the ER marker suggesting that relocation at the cell periphery specifically combined with slow clearance there is the most likely explanation (still unclear what stops the marker from spreading through the entire cell). They suggest that decrease in cotranslational translocation is unlikely as this would result in somatic accumulation of the marker. However, if the clearance in the periphery is less efficient than in soma, the accumulation there might reflect a compromised translocation. Any clarifying experiments, if practical, to directly demonstrate how ER proteins in relocates to the cytoplasm in atl mutant would help understanding better the phenomenon. For example, would proteasomal inhibition make the marker accumulate more across the cell? Authors also suggest links to ER stress. Would stress induction phenocopy the mutant?

    Minor comments:

    Line 146: "fast dynamics (<1 sec)" a velocity should be presented as distance/time

    Fig. 2D: The data representation of "Tubule displacement" image is unclear. The ER tubule indicated by the red arrow does not seem to show any changes over time (like static). time 0 in stamp appears behind the image.

    Line 157-158 (and relevant method sections): The definition of static and dynamic boutons is ambiguous. The author should describe in more detail this point including how long they observed the structure to define the changes in ER tubule dynamics.

    Fig. 2E: What n=75 and n=29 represent is unclear, are these the number of boutons in en passant and terminal subjected for qualitative analysis?

    Fig. 2: What the qualitative analysis represents is unclear, are the points pulled from different experiments?

    Line 231: Regarding "...we found a small but significant reduction in dynamic boutons in Atlastin mutants (76%), ...", how do the authors assess significance. If proportion of static/dynamic ER in boutons was obtained from multiple experiments, it should be presented e.g. as in average {plus minus} standard deviation, or clarify that the proportion is representative of x independent experiments.

    Line 267-269 and Fig. 6B: The author's conclusion that "Complete loss of ER network structure in NMJ of BiP:sfGFP:HDEL overexpressing Atl mutant" seem to be based on the lack of signal from luminal marker, which may be undetectable due to changes to tubular volume or marker loss to the cytoplasm, as suggested by the authors, while the membranous ER structure is intact. It would be useful to discuss this point and potentially add ER membrane-stained control.

    Fig. 6C: In Atl mutant, why does the total of the proportion exceed 100% (10 + 45 + 55)?

    Fig. 9C, line 342-344: In FRAP experiment using CD8, it seems that the Partial loss Atl mutant shows slower recovery that control. There seems to be a mismatch in triangle symbols of Partial loss Atl mutant between legend and plot (one is filled and the other is empty). This should be clarified

    fig. 10 is a clever way to verify the cytoplasmic localisatoin of the ER marker, however its description and annotation can be improved, and it would be stronger if 4 curves in F for mutant and controls with the trap and normal were shown.

    Line 495: Drosophila have ReepA and ReepB, but not Reep1-4. If the authors discuss their speculation based on their observation (using Drosophila), the gene names should be unified in the same species, and explain the corresponding genes to mammalian cells.

    Line 548; should UPR be Unfolded Protein Response?

    Significance

    This study advances the understanding of how ER morphogens affect neuronal cells specifically, the lack of which limits researchers ability to comprehend the neuronal pathologies associated with ER structure-function. The observation on ER content aberrant localisation caused by the lack of key structural protein should be of a great interest for cell and neuronal biologists and researchers of the associated diseases and shows the field a new direction. Though, mechanistic details remain to be unraveled, it constitutes a fundamental, conceptual advance.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The endoplasmic reticulum (ER) is a continuous organelle that extends throughout neurons to regulate fundamental processes. The analysis of ER dynamics at synaptic terminals is limited by the challenge of imaging these structures at high resolution. In this manuscript, the authors use super-resolution (~170 nm) live imaging and a combination of membrane and luminal ER markers at the Drosophila larval NMJ, an important model synapse, to investigate dynamic ER architecture in vivo. They report a detailed characterization of the presynaptic ER organization and dynamics at wild-type and GTPase Atlastin mutant NMJs. Their analysis using the ER membrane marker tdTomato:Sec61b reveals the presence of an intact ER network in Atlastin mutants. This contrasts with the apparent ER fragmentation phenotype previously reported and replicated here when using a luminal marker. Their findings instead point to the progressive displacement of luminal proteins to the cytosol in Atlastin mutants specifically at synapses. The authors propose that the disruption of ER protein dynamics at synapses is a compartment-specific ER stress response. The manuscript is well written, results are clearly presented, and experiments are technically rigorous.

    Major comments

    1. The baseline ER phenotypes in Atlastin mutants are mild with complete loss of ER network only observed in terminal boutons. This interesting and unexpected result should be further confirmed by genetic rescue. The authors can use a UAS rescue line previously reported in PMID: 19341724.
    2. Lines 204-7: It's not clear how a greater coefficient of variation indicates that the marker is more concentrated in subsynaptic structures or what is meant by 'subsynaptic structures.'
    3. There's a mistake in Figure 6C and the associated text. The summed percentage of the three phenotypic categories adds up to 110% for Atlastin mutants.
    4. Figure 8: the ER looks fragmented in 1st instar controls and mutants. The authors should address this difference from more mature NMJs.
    5. The images in figure 9B do not seem representative of the quantification in Figure 9D. Specifically, the partial loss Atlastin NMJ appears to have recovered as fully as the complete loss Atlastin NMJ.
    6. Optional: An overexpressed luminal marker is displaced to the cytoplasm in Atlastin mutants. It would be interesting to know and increase the significance of the findings if the same is true of endogenous luminal proteins under biological stress conditions.
    7. Optional: Applying this approach in stimulated conditions (high potassium, increased temperature) might reveal a greater activity-dependent role for Atlastin at synaptic terminals.

    Minor Comments

    1. Line 16: Atlastin should be italicized.
    2. Figure 5A: Based on the relative intensities, it appears that control and mutant images are not contrast matched but this isn't stated.
    3. Line 822: The number of static Atlastin mutant boutons used for analysis is missing.
    4. Figure 9: The blue arrows are not annotated in the figure legend.

    Significance

    Atlastin is linked to Hereditary Spastic Paraplegia (HSP) and this study changes our understanding of the compartment-specific impacts of its loss. This study reveals the importance of using both membrane and luminal ER markers to accurately interpret phenotypes as well as the importance of considering compartment-specific effects on ER. These findings represent significant mechanistic and conceptual advances. The lack of genetic rescue is a limitation and adding an investigation of an endogenous luminal protein under basal and stress conditions would add significantly to our understanding of Atlastin dysfunction in HSP. Notably, the in vivo imaging approach introduced here can be adapted broadly for live imaging of Drosophila larvae. Thus, this work will be of interest to both neuronal cell biologists and the wider Drosophila community. This review is based on our expertise in neuronal cell biology.

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    Referee #1

    Evidence, reproducibility and clarity

    In the present manuscript, the authors address an important question related to the ultrastructure and the dynamics of the ER in HSP. In contrast to previous studies, the authors here show (by using a membrane and luminal protein markers) that in the presynaptic terminals, the overexpressed BIP "mislocalizes" to the cytosol without affecting (or with minimal effect) the integrity of the ER membrane. Although they used an artificial system by overexpressing (overexpression) of BIP-sfGFP-HDEL (fused protein), the findings lack validation of the endogenous protein by biochemical and fluorescent tools.

    Concerns:

    I am worried about how the article is presented, mainly in the results section, as most of it refers to published data. The Results section is reserved for presenting new findings without external interpretation or comparison. The paper is written mainly as a comparison paper with other studies or relies on previous studies to strengthen their findings rather than coming up with novel findings. Up to figure 4, I missed the relevance of the new findings. The manuscript needs rewriting to emphasize its novelty and significance without comparing it to previous data. Moreover, the manuscript emphasizes the technology and the findings of the localization of the ER luminal proteins to the cytosol (which is not novel and was previously reported in other settings). Those two aspects were not given enough focus. Here are the main major and minor comments:

    Major comments:

    1. Lines 103-116 (first paragraph of the results section) describe mainly published data that is more suitable for the introduction section. It is annoying to refer to different published articles in the Results section to strengthen the results instead of showing them. The same goes for paragraphs two and three. Why mention those data in the Results section if they are already published and known?
    2. Figure Legends-(in all Figures): The number of experimental repeats must be mentioned in the figure legends.
    3. The way the figures are labeled is worrisome; supplementary figures are not ordered numerically.
    4. The tubule extension in Figure 2D is not convincing. Is there a movie showing those changes? Better images are needed. It is essential to show which supplemental movie corresponds to which panel.
    5. This is unnecessary in the results section: "To investigate the relationship between ER structure and function at synapses, we examined mutants of Atlastin, a GTPase that regulates ER tubule fusion. Drosophila has a single homolog while mammals have three Atlastin homologs, with Atlastin-1 enriched in the brain (Rismanchi et al., 2008)."
    6. "This reduction in ER membrane marker intensity has also been observed in other HSP mutants, suggesting this is a common feature of ER shaping mutants and could indicate changes in ER membrane composition, integrity, or tubule thickness (P.rez-Moreno et al., 2023)." This comparison is important and should be shown in the same settings as for the Atlastin mutant rather than referring to published data.
    7. Does the distribution of the luminal ER marker in Figure 6F diffuse due to mislocalization or reflux after being localized to the ER and then refluxed to the cytosol as was previously shown for the ER to Cytosol signaling (ERCYS) mechanism? Could you assess other ER-luminal protein localization biochemically? It is highly recommended to look at another soluble ER-protein localization in the Atlastin mutant without overexpression, which can be an artifact
    8. "(data not shown)" in line 288. This affects the process of judging those data.
    9. In comparison to Summerville et al. (2016) in Figure 7, the experiment was not done in the same way. It is important to keep the same settings for comparison
    10. Does the Atlastin mutant induce the unfolded protein response and stress within the ER? It is necessary to look for UPR markers in those settings. It was shown previously that ER stress leads to protein reflux from the ER to the cytosol. Is there a difference in the ER stress markers in the presynaptic terminal?
    11. It is important to add biochemical experiments to show that no fragmentation of the ER membrane occurred. It can be simply demonstrated by looking at the redox state of the ER, which would change if it were mixed with the reducing cytosol. Moreover, this can be shown by using an ER-targeted redox-sensitive fluorescent protein that is tethered to the ER membrane to follow changes in the redox state of the ER.

    Minor comments:

    1. It is important to call figures by order. Figure 2C is called before 2A-B. Figure 2B is called before Figure 2A.
    2. Figure legends (Figure 2): "The same control dataset used in E-G was used in Figure 5 and Figure 5_Supplement." Why is this relevant?
    3. Figure 4F is called before Figure-4D-E which are not called.
    4. Figure 5B is called before the previous ones. Same for Figure 5A supplement.

    Referees cross-commenting

    I agree with the comments raised by reviewer2 and 3. Basically it is highly important to validate those data by genetic rescue. Moreover, it is essential to know the source of the displaced luminal marker to the cytosol. Is it mislocalization or it is a reflux of pre-existing protein to the cytosol after insertion to the ER. It is also recommended by me and the reviewers to test the endogenous protein rather than overexpression.

    Significance

    General assessment: This interesting paper shows that proteins can escape the ER under special conditions. However, the authors need more evidence to show that and rely less on the overexpression system, especially of BIP-GFP, which can cause proteostasis stress within the ER.

    Advance: The results have been oversimplified in their explanations, and some points and complexities of the study need to be addressed further to make the most of them. These are often some of the more interesting concepts in the paper. I think many points can be addressed in the text by the authors being clear and concise with their reporting. At the same time, other experiments would turn this paper from an observational one into a very interesting mechanistic one. This paper is based on previously published articles from the group and other groups, and it is a nice progression. However, as mentioned, this paper depends primarily on published data, and the novelty is somehow lost between all the comparisons to other published data instead of emphasizing that. Without a substantial mechanistic improvement, the paper would remain observatory.

    Audience: The microscopy tools can be great addition to researchers in the field to monitor protein trafficking especially Cell biologists (basic research)

    My expertise: ER homeostasis, protein trafficking, cell biology

  5. Version published to 10.1101/2023.09.01.555994 on bioRxiv