Reversible One-way Lipid Transfer at ER-Autophagosome Membrane Contact Sites via Atg2

  1. Li Hao
  2. Tomoki Midorikawa
  3. Yuta Ogasawara
  4. Takuma Tsuji
  5. Takuma Kishimoto
  6. Yutaro Hama
  7. Huichao Lang
  8. Nobuo N. Noda
  9. Kuninori Suzuki

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Abstract

Bridge-like lipid transfer proteins (LTPs) contain a repeating β-groove domain and long hydrophobic grooves that act as bridges at membrane contact sites (MCSs) to efficiently transfer lipids. Atg2 is one such bridge-like LTP essential for autophagosome formation, during which a newly synthesized isolation membrane (IM) emerges and expands through lipid supply. However, studies on Atg2-mediated lipid transfer are limited to in vitro studies due to the lack of a suitable probe for monitoring phospholipid dynamics in vivo . Here, we characterized the lipophilic dye octadecyl rhodamine B (R18), which internalizes and labels the endoplasmic reticulum (ER) in a manner that requires flippases and oxysterol-binding protein–related proteins. Using R18, we demonstrated phospholipid transfer from the ER to the IM during autophagy in vivo . Upon autophagy termination, we observed reversible phospholipid flow from the IM to the ER in response to environmental changes. Our findings highlight the critical role of bridge-like LTPs in MCS-mediated phospholipid homeostasis.

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    Reply to the reviewers

    General Statements

    We sincerely thank all three reviewers for their thoughtful and constructive feedback. Your comments were invaluable in improving the clarity and quality of our work.

    In this study, we revisit a previously overlooked lipophilic dye, demonstrating its utility for live-cell imaging that transport in a non-vesicular pathway and label autophagy related structures. Against the backdrop of increasing attention to membrane contact sites (MCSs), bridge-like lipid transfer proteins (BLTPs), and organelle biogenesis, we aim to propose the possibility of a reversible one-way phospholipid transfer activity that really takes place in living cells.

    As Reviewer #1 noted, recent cryo-EM studies (e.g., Oikawa et al.) have highlighted the importance of lipids in autophagosome formation. And there are some existed in vitro studies. However, we believe that we have to think about the consistence of simplified in vitro reconstitution and the complex real cellular environment. In addition, to our knowledge, no studies have directly tracked lipid flow dynamics over time in living cells. We believe our work contributes to this gap by combining three interesting technical approaches: (a) R18 as a lipid-tracing dye, (b) FRAP analysis on the isolation membrane, and (c) the use of Ape1 overexpression to stall autophagosome closure, enabling us to visualize reversible lipid flow in vivo. While these techniques may not appear "fancy," we hope they offer new insights that can inspire further exploration in lipid dynamics story in a real cellular environment.

    We appreciate Reviewer #2's comments on our high imaging quality and Reviewer #3's recognition of our approach as an elegant way to study lipid transfer. We have revised the manuscript accordingly and included additional explanations, figure clarifications, and planned experiments to address remaining concerns.

    As two key concerns were raised repeatedly by all reviewers, we would like to address them here:

    Regarding the concern that the evidence for reversible lipid transfer from the IM to the ER is not sufficiently strong:

    We are deeply grateful to Reviewer #2 for the insightful suggestion to compare the fluorescence recovery of the adjacent bleached ER to that of the ER-IM MCS, to exclude the possibility that recovery at the ER-IM MCS originates from nearby ER rather than from the IM. Following this suggestion, we performed a quantitative analysis using unbleached ER as a background. Interestingly, in every sample, the adjacent bleached ER consistently showed a significantly lower fluorescence recovery than the ER-IM MCS. We also used the IM as a background for normalization, the difference became even more pronounced, further supporting the idea that the adjacent ER could not be the source of the recovery signal at the ER-IM MCS. These findings strengthen our conclusion that phospholipid recovery at the MCS could be derived from the IM. The updated analysis and corresponding figure panels (Figure 5K, 5L, and 5M), along with the relevant text (lines 384-396), have been revised accordingly.

    Regarding the concern that the evidence for R18 transfer via Atg2 as a bridge-like lipid transfer protein is not sufficiently direct:

    In addition to the evidence presented in this manuscript, we have now cited our parallel study currently under revision (Sakai et al., bioRxiv 2025.05.24.655882v1), where we provide direct evidence that Atg2 indeed functions as a bridge-like lipid transfer protein, rather than a shuttle. Importantly, we also show in that study that R18 transfer requires the bridge-like structure of Atg2. This new reference has been cited in the revised manuscript, and relevant textual explanations have been added to provide further support.

    We hope that the revisions and our revision plan can address the reviewers key concerns. Please find our detailed point-by-point responses below.

    Response to the Reviewer ____#____1

    In their study, Hao and colleagues exploited the fluorescent fatty acid R18 to follow phospholipid (PL) transfer in vivo from the endoplasmic reticulum to the IM during autophagosome formation. Although the results are interesting, especially the retrograde transport of PLs, based on the provided data, additional control experiments are needed to firmly support the conclusions.

    We sincerely thank the reviewer for the positive assessment and agree that additional controls are necessary to support our conclusion. Detailed responses and corresponding revisions are provided below.

    An additional point is that the authors also study the internalization of R18 into cells and found a role of lipid flippases and oxysterol binding proteins. While this information could be useful for researchers using this dye, these analyses/findings have no specific connection with the topic of the manuscript, i.e. the PL transfer during autophagosome formation. Therefore, they must be removed.

    We thank the reviewer for the thoughtful comment. We understand the concern that the R18 internalization analysis may appear peripheral to the manuscript's main focus on phospholipid transfer during autophagosome formation. However, we respectfully believe that this section is critical for establishing the mechanistic basis as this study represents the first detailed in vivo application of R18 for tracing lipid dynamics. We believe it is interesting that R18 entry is not due to chemically passive diffusion or non-specific adsorption, but occurs through a biologically regulated, non-vesicular lipid transport pathway. This mechanistic context underpins the reliability of using R18 to monitor ER-to-IM lipid transport in the autophagy pathway.

    To improve clarity and coherence, we have added explanatory text in the Introduction and at the start of the Results section to explicitly link the internalization assay to the subsequent autophagy-related experiments (line 94-98, 185-187). We hope this helps guide the reader through the rationale and relevance of this part of the study.

    Major points:

    1. In general, the quality of the microscopy images are quite poor and this make it difficult to assert some of the authors' conclusions.

    We thank the reviewer for the feedback. To better address this concern, we would appreciate clarification regarding which specific images or figure panels were found to be of low quality. Overall, we believe the microscopy data presented are of sufficient resolution and clarity to support our main conclusions, as also noted by Reviewer #2 ("the high-quality images and FRAP experiments").

    We acknowledge that certain phenomena-such as occasional R18 labeling of the vacuole-were not clearly explained in the original manuscript. We have now included additional clarification in the results section and mentioned this limitation in the discussion (lines 170-171, 436-438), along with a note on ongoing experiments to further investigate this point.

    1. It would be important to perform some lipidomics analysis to determine in which PLs and other lipids or lipid intermediates R18 is incorporated. First, it will be important to know which the major PL species are are labelled under the conditions of the experiments done in this study. Second, the authors assume that all the R18 is exclusively incorporated into PLs and this is what they follow in their in vivo experiments. What about acyl-CoA, which has been shown to be a key player in the IM elongation (Graef lab, Cell)?

    We thank the reviewer for raising this point. However, we believe this is based on a misunderstanding of the chemical nature of R18. R18 is not a free fatty acid analog and cannot be incorporated into phospholipids or acyl-CoA via metabolic pathways. Due to its chemical structure-a bulky rhodamine headgroup attached to a long alkyl chain-it cannot undergo enzymatic conjugation or incorporation into membrane lipids. This is why we did not pursue lipidomics analysis. Instead, we focused on characterizing the biological behavior of R18 through a range of live-cell assays, including temperature and ATP dependency, involvement of flippases, OSBP proteins, and Atg2, all of which support a regulated, non-vesicular lipid transport pathway. Additionally, the AF3 structural model presented in this study is consistent with this interpretation, showing no evidence of R18 forming chemical bonds with phospholipids.

    1. Figure 1A and 1B. The authors conclude that Atg2 is involved in the lipid transfer since R18 does not localize to the PAS/ARS in the atg2KO cells. However, another possible explanation is that in those cells the IM is not formed and does not expand, and con sequetly R18 is present in low amounts not detectable by fluorescence microscopy. To support their conclusion, the authors must assess PAS-labelling with R18 in cells lacking another ATG gene in which Atg2 is still recruited to the PAS.

    We thank the reviewer for this important suggestion. As noted, the absence of R18 at the PAS in atg2Δ cells may reflect a lack of membrane formation rather than impaired lipid transfer. However, in support of our interpretation, our previous work (Hirata E, Ohya Y, Suzuki K, 2017) has shown that R18 accumulates at PAS-like structures in delipidation mutants, where the IM fails to expand but Atg2 is still recruited (please refer to the attached revision plan for further details). This suggests that the presence of Atg2, rather than the mere existence of a mature IM, contributes to R18 localization.

    To address this, we revised our statement to the more cautious: "R18 was undetectable at the PAS in atg2Δ cells," to avoid overinterpretation (lines 119-120). 4)

    1. Figure 2. As written, the paragraph this figure seems to indicate that flippases are directly involved in the translocation of R18 from the PM to the ER. As correctly indicated by the authors, flippases flip PLs, not fatty acids. Moreover, there are no PL synthesizing at the PM and thus probably R18 is not flipped upon incorporation into PL. As a result, the relevance of flippase in R18 internalization is probably indirect. This must be explained clearly to avoid confusion/misunderstandings.

    We thank the reviewer for this important clarification. We fully agree that flippases act on phospholipids, not fatty acids, and that R18 is not metabolically incorporated into phospholipids at the plasma membrane. However, our ongoing work (Rev. Figure 1) shows that R18 preferential labeling affinity for PS and PE in vivo (yeast phospholipid synthesis mutants), consistent with its flippase-dependent localization. Flippases are known to specifically flip PS and PE. While R18 itself is not enzymatically modified or incorporated into phospholipids, its membrane distribution may thus depend on the lipid environment and the activity of lipid-translocating proteins.

    Preliminary data supporting this observation are included in the "Supplementary Figures for reviewer reference only" and are not part of the public submission.

    1. A couple of manuscript has shown a (partial) role of Drs2 in autophagy. The authors must explain the discrepancy between their own results and what published, especially because they use the GFP-Atg8 processing assay, which is less sensitive than the Pho8delta60 used in the other studies.

    We thank the reviewer for raising this important point. We are aware of prior reports implicating Drs2 in autophagy and in fact discussed this work directly with the authors during the course of our experiments, who kindly provided helpful suggestions. While our GFP-Atg8 processing assay did not show significant defects upon Drs2 deletion, strain background differences may explain this discrepancy. We also appreciate the suggestion to use the Pho8Δ60 assay and plan to include it in future experiments.

    Additionally, authors should check whether the Atg2 and Atg18 proteins are present at the IM-ER membrane contact sites in the same rates after nutrient replenished than when cells are nitrogen-starved, since this complex would determine the lipid transfer dynamics at this membrane contact site.

    We thank the reviewer for the helpful suggestion. We plan to perform additional experiments to monitor Atg18 localization during the nutrient replenishment assay.

    1. Authors used a predicted Atg2 lipid-transfer mutant (Srinivasan et al, J Cel Biol, 2024), but not direct prove that this mutant is defective for this activity. As previously done for other Atg2/ATG2-related manuscripts (Osawa et al, Nat Struct Mol Biol, 2019; Valverde et al, J Cel Biol, 2019), this must be measure in vitro. Moreover, they do not show whether other known functions of Atg2 are unaffected when expressing this Atg2 mutant, e.g. formation of the IM-ER MCSs, Atg2 interaction with Atg9 and localization at the extremity of the IM...

    We thank the reviewer for this concern. The lipid-transfer-deficient Atg2 mutant used here is based on the same structural rationale as in our recent parallel study (Sakai et al., bioRxiv 2025; https://www.biorxiv.org/content/10.1101/2025.05.24.655882v1, currently under revision). In that study, we addressed whether Atg2 indeed functions as a bridge-like lipid transfer protein, and also used R18 to directly demonstrate the lipid transfer defect of this Atg2 mutant in vivo.

    We therefore believe that referencing this study provides mechanistic support for the use of this Atg2 mutant in the current manuscript. A citation and brief explanation have now been added to the revised text (line 315-316, 439-441). We also plan to perform the lipid transfer assay in vitro.

    1. The mNG-Atg8 signal is not recovered in the fluorescent recovery assays. Based on the observation that R18 signal comes back after photobleaching, authors suggest that the supply of Atg8 is not required for IM expansion. This idea is opposite to data where the levels of Atg8 and deconjugation of lipidated Atg8 determines the size of the forming autophagosomes (e.g., Xie et al, Mol Biol Cell, 2008; Nair et al, Autophagy, 2012). Similar results have also been obtained in mammalian cells (Lazarou and Mizushima results in cell lacking components of the two ubiquitin-like conjugation systems). This discrepancy requires an explanation.

    We thank the reviewer for pointing out this imprecise interpretation, and we sincerely apologize for the confusion it may have caused. We fully agree that Atg8 is essential for the expansion of the isolation membrane (IM), as supported by previous studies. In our FRAP data, mNG-Atg8 showed gradual recovery at the later timepoints, indicating that Atg8 can be replenished over time. The reason why R18 recovery appears much more rapid is likely due to the inherently fast lipid transfer activity of Atg2, the bridge-like lipid transport protein. In contrast, Atg8 signal recovery may have been delayed for two reasons: (1) slower recruitment kinetics to the IM, and (2) partial depletion of the available mNG-Atg8 protein pool due to photobleaching during the experiment.

    We have revised the relevant paragraph in the manuscript (line 326-330) to clarify these points and avoid potential misinterpretation.

    1. Although authors claim that there is a retrograde lipid transfer from the IM to the ER, based on the data, it quite difficult to extract these conclusions as they show a decrease in the lipid flow dynamics rather to an inversion of the lipid flow per se. Can the authors exclude that ER microdomains are formed at the ERES in contact with the IM, and consequently what they measure is a slow diffusion of R18-labeled lipid from other part of the ER to these ERES?

    We appreciate the reviewer's insightful comment. Indeed, we are also considering the possibility that lipid-enriched microdomains may form in the ER and contribute to complex lipid dynamics at contact sites. However, direct visualization of such domains in cells remains technically challenging, this remains one of the important directions we aim to pursue in future studies. While our current data do not allow us to definitively state that all recovered lipids originate from the IM, our FRAP experiments provide indirect yet strong support for the possibility that at least a substantial portion of the recovered lipid signal in the ER derives from the IM. Moreover, following Reviewer 2's major point No.4, we performed a direct comparison of R18 fluorescence recovery between the photobleached ER-IM MCS region and the adjacent bleachedER region (Figure 5K and 5M). Interestingly, each sample consistently showed lower fluorescence recovery in the adjacent bleached ER near the ER-IM MCS (mean = 0.20), compared to the ER-IM MCS region (mean = 0.28). To further validate this observation, we also used the IM as a background reference for normalization. This analysis revealed a more significant difference, with the adjacent bleached ER near the ER-IM MCS showing a lower recovery (mean = 0.47) than the ER-IM MCS (mean = 0.80).

    As the Reviewer2 pointed out, these results support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS is due to the signal coming from the IM, rather than from the adjacent bleached ER, which recovers more slowly and less efficiently. We have incorporated this new analysis into Figure 5, and accordingly revised the figure legend and main text (lines 384-396).

    1. The retrograde PL transfer is studied in cells overexpressing Ape1, in which IM elongation is stalled. This is a non-physiological experimental setup and consequently it is unclear whether what observed applies to normal IM/autophagosomes. This event should be shown to occur in WT cells as well.

    We thank the reviewer for this point. Indeed, it remains technically difficult to visualize lipid flow during normal IM expansion in vivo, as this process is rapid and transient. And to date, there are no reports directly addressing lipid flow in this process.

    But the Ape1 overexpression system provides a strategic advantage by temporally extending the IM elongation phase and spatially enlarging the IM, thus offering a unique opportunity to capture membrane behavior that would otherwise be transient and difficult to resolve. Importantly, this system arrests autophagosome closure, which we leveraged to investigate the potential reversibility of phospholipid transfer in a controlled and prolonged context. Without this system, it would be exceedingly difficult for reaserchers to examine the lipid flow directionality in living cells.

    Furthermore, the use of Ape1 overexpression has been widely employed in previous high-impact autophagy studies. We emphasize that our aim is to understand Atg2-mediated lipid transfer, and in this context, the Ape1 system provides a valuable and informative tool without compromising the validity of our conclusions.

    1. From the images provided, it appears that R18 also labels the vacuole. The vacuole form MCSs with the IM. Can the author exclude a passage of R18 from the vacuole to the IM?

    We thank the reviewer for the insightful comment. Our data suggest that R18 traffics from the plasma membrane to the ER, then to autophagy-related structures. Actually, following that, as we kown, autophagosomes will eventually reaches and fused with the vacuole. This explains the occasional weak R18 signals at the vacuole membrane, particularly in late-stage cells. We have revised the figure and clarified this point in the text to avoid oversimplification of R18 localization (lines 169-171, 426-428)

    Here we also added the results of our onging work (in preparation). R18 tends to accumulate in a dot-like compartment after prolonged rapamycin treatment and incubation (Rev. Figure 2). And the vacuolar labeling of R18 correlates with the degradation status of autophagosomes, rather than reverse lipid transport from the vacuole to the IM (Rev. Figure 2). Taken together, we believe that R18 transport from the vacuole back to the IM is unlikely.

    Preliminary data supporting this response are included in the "Supplementary Figures for reviewer reference only" and are not part of the public submission.

    Minor points:

    1. L66. One report has indicated that Vps13 may also play a role in the transfer of lipids from the ER to the IM (Graef lab, J. Cell Biol).

    Thank you for pointing this out. Their excellent work also suggested that the inherent lipid transfer activity of Atg2 is required for IM expansion. We have revised the sentence (lines 67-68, 312-314) and included the appropriate citation at these two places.

    1. L70. It must be indicated that IM is also called phagophore.

    We have revised the sentence (line 70-71). Thank you for pointing this out.

    1. L74. It is mentioned "Additionally, a hydrophobic cavity in the N-terminal region of Atg2 directly tethers Atg2 to the ER, particularly the ER exit site (ERES), which is considered a key hub for autophagosome biogenesis", but there is no experimental evidence supporting that Atg2 is involved in the tethering with the ERES.

    Thank you for pointing this out. We have removed the N-terminal region part and revised the sentence accordingly (line 79-81) to avoid overstatement.

    1. L90. PAS must be listed between the ARS.

    We have revised the sentence (line 97-98). Thank you for pointing this out.

    1. Upon deletion of ATG39 and ATG40, there is a pronounced reduction of mNG-Atg8 labelled with R18. This would suggest that these two ER-phagy receptors are required for the PL transfer from the ER to the IM, which is not the case as autophagy is mildly affected by the absence of them (e.g., Zhang et al, Autophagy, 2020).

    We thank the reviewer for the important comment and agree that Atg39 and Atg40 are not required for phospholipid transfer from the ER to the IM. We have revised the text (lines 155-157). We appreciate if the reviewer could provide the DOI or PubMed ID for this paper.

    1. Authors referred that "no direct evidence has been found to confirm lipid transfer at the ER-IM MCS in living cells" (lines 282-283). However, a recent paper has shown that de novo-synthesized phosphatidylcholine is incorporated from the ER to the autophagosomes and autophagic bodies (Orii et al, J Cel Biol, 2021). This reference should be mentioned in the manuscript.

    Thank you for your insightful reminder. This paper beautifully demonstrated the importance of de novo-synthesized phosphatidylcholine in autophagy using electron microscopy. We have now included its citation and brief discussion in the revised manuscript (lines 74-76, 297-298). However, we respectfully note that direct observation of lipid transfer at the ER-IM MCS in living cells still remains unproven.

    1. In lines 252-253, the sentence "R18 transport from the PM to the ER was partially impaired in osh1Δ osh2Δ, osh6Δ osh7Δ, and oshΔ osh4-1 cells (Figure S3). These results suggest that Osh proteins participate in transferring R18 from the PM to the ER" does not recapitulate what is observed in Fig. S3. Moreover, the Emr lab has generate a tertadeletion mutant in which the PM-ER MCSs are abolished. The authors could examine this mutant.

    We thank the reviewer for this helpful comment and sincerely apologize for the lack of clarity in our original description. Our conclusion was primarily based on the partial PM accumulation of R18 observed in some osh mutant strains shown in Figure S3, which motivated us to further investigate this pathway using the OSW-1 inhibitor. We have revised the corresponding text to improve the logic and clarity of this section.

    We appreciate the recommendation of the tether∆ mutant. Our preliminary tests indicate that R18 still properly labels the ER in tether∆ cells, suggesting that its localization is not due to passive diffusion at membrane contact sites, but rather involves specific transport mechanisms. As this is an initial observation, we plan to confirm the result and include it in a future revision.

    Reviewer #1 (Significance (Required)):

    General assistent: Strength: potential new system to monitor lipid flow Limitations: Indirect evidences and in the case of the retrograde transport of phospholipids, it could be an artefact of the employed experimental approach. Advance: Little advances because something in part already shown in vitro. No new mechanisms uncovered. Audience: Autophagy and membrane contact site fields.

    We sincerely thank the reviewer for the overall evaluation. We agree that our current system offers indirect but promising evidence for lipid transfer events at ER-IM contact sites in vivo. While Atg2-mediated lipid transport has been proposed in vitro, our study adds value by (1) establishing a live-cell imaging way to monitor lipid flow in a non-vesicular transport pathway, (2) proposing a model of reversible one-way lipid transfer activity, and (3) addressing whether findings from simplified in vitro reconstitution accurately reflect the dynamics in the more complex real cellular environment.

    We recognize the limitations of our current approach and plan to include additional analyses to more cautiously interpret the observed retrograde movement. Although we do not claim to identify a new mechanism, we believe our work provides an interesting framework to inspire future efforts aimed at directly probing lipid flow at membrane contact sites in vivo.

    We also sincerely appreciate the reviewer's recognition of the potential value of this system for the autophagy and membrane contact site communities.

    Response to the Reviewer ____#2

    Non-vesicular lipid transfer plays an essential role in organelle biogenesis. Compared to vesicular lipid transfer, it is faster and more efficient to maintain proper lipid levels in organelles. In this study, Hao et al. introduced a high lipophilic dye octadecyl rhodamine B (R18), which specifically labels the ER structures and autophagy-related structures in yeast and mammalian cells. They characterised its distinct lipid entry into yeast cells via lipid flippase Neo1 and Drs2 on the plasma membrane, rather than through the endocytic pathway. They then demonstrated that R18 intracellular trafficking through plasma membrane to ER depends on "box-like" lipid transfer Osh proteins. They further looked into the "bridge-like" lipid transfer protein Atg2, using R18 as a lipid probe to track lipid transfer from ER to the isolation membrane (IM) during membrane expansion and reversible lipid transfer through IM to the ER-IM membrane contact sites (MCS) when autophagy is terminated by nutrient replenishment. The authors provide an interesting model of reversible directionality of Atg2 lipid transfer during autophagy induction and termination.

    We sincerely thank the reviewer for the thoughtful and constructive summary of our work. We are grateful for the recognition of the novelty of using R18 to visualize non-vesicular lipid transfer in vivo and for highlighting the conceptual contribution of our proposed model of reversible Atg2-mediated transport during autophagy.

    In response to the reviewer's valuable suggestions, we have revised key parts of the manuscript and prepared a detailed revision plan to address the specific concerns. We truly appreciate the reviewer's insights, which have been instrumental in improving the clarity of our study.

    Major points:

    Line 299-309: The FRAP assays were interesting and well performed. The authors photobleached R18 and Atg8 signal, and found R18 fluorescence recovery but not Atg8, which suggests lipid transfer occurs between ER and the IM and faster than Atg8 lipidation process during IM expansion. These results gave clear evidence that R18 can be transferred during IM expansion. The supply of Atg8 may not be not able to track within this time frame or the recovered amount of Atg8 may not be able to visualized due to the threshold limitation with confocal microcopy. This does not imply the supply of Atg8 to the IM is not required during IM expansion. This should be clarified.

    We thank the reviewer for this valuable comment and fully agree that Atg8 is essential for IM expansion. We apologize for any ambiguity that may have suggested otherwise.

    As pointed out, the lack of mNG-Atg8 recovery in our FRAP assay likely reflects the slower turnover of lipidated Atg8, limited observation time, and photobleaching of the existing protein pool. Notably, we observed a weak but gradual signal recovery at later time points, supporting this view. We have revised the relevant paragraph in the manuscript (line 326-330) to clarify these points and avoid potential misinterpretation.

    Please clarify how the length of the IM is measured and determined in Figure 4H and Figure 5D.

    We thank the reviewer for the vaulable comment. We have now clarified the method for quantifying IM length in the revised manuscript. Specifically, we modified the Statistical Analysis section of the Methods (line 642-643).

    Line 336-342: The description of the results should be clarified. Based on Figure 5H, the authors observed a significant decrease in the mNG-Atg8 signal during photobleaching of the R18 signal.

    We thank the reviewer for pointing out the ambiguity. We have now clarified the description in the revised manuscript. The sentence has been modified (line 360-362) as follows: "To determine whether nutrient replenishment terminates autophagy, we selectively photobleached the R18 signal and monitored the R18 (photobleached) and mNG-Atg8 (without photobleaching) signal following nutrient replenishment."

    The authors photobleached ER-IM MCS and the ER region (boxed region in Figure 5J) and quantified fluorescence recovery, normalized to the IM region and an ER control. The ER control was taken from the other cell. It would be helpful to compare and analyse the fluorescence recovery of R18 in the bleached ER region near the ER-IM MCS to that in the ER-IM MCS. This would help to confirm the ER-IM MCS fluorescence recovery is due to signal coming from the IM.

    We sincerely thank the reviewer for this insightful suggestion. We have now performed the suggested comparison. Interestingly, each sample consistently showed lower fluorescence recovery in the adjacent bleached ER near the ER-IM MCS (mean = 0.20), compared to the ER-IM MCS region (mean = 0.28). To further validate this observation, we also used the IM as a background reference for normalization. This analysis revealed a more significant difference, with the adjacent bleached ER near the ER-IM MCS showing a lower recovery (mean = 0.47) than the ER-IM MCS (mean = 0.80).

    As the reviewer pointed out, these results support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS is due to the signal coming from the IM, rather than from the adjacent bleached ER, which recovers more slowly and less efficiently. We have incorporated this new analysis into Figure 5, and accordingly revised the figure legend and main text (lines 384-396). Again, we appreciate this constructive and helpful suggestion.

    In figure 5K, the autophagic structure or IM labelled by R18 seems to be maintained when the mNG-Atg8 signal decreases or dissociates from the IM. Could the authors comment on that how they interpret the termination of the prolonged IM structure and IM shrinkage?

    We thank the reviewer for this insightful observation. Based on our live-cell imaging, we speculate that following the initial dissociation of Atg8, the IM membrane undergoes a relatively slow disassembly process, potentially retracting toward the ER-IM MCS, which often localizes near ER exit sites (ERES). This suggests that IM shrinkage may proceed via Atg8-independent mechanisms. Although the precise pathway remains unclear, we occasionally observed vesiculation events during this phase, supporting the idea that membrane remodeling continues even in the absence of Atg8. In response to this comment, we have revised our manuscript to reflect these interpretations (line 494-496).

    The author has shown that Atg2Δ and Atg2LT lipid transfer mutant impair R18 labelling of autophagic structures in Figure 4C. However, the evidence supporting that R18 fluorescence recovery at ER-IM MCS is mediated by reversible Atg2 lipid transfer is not direct. It would be helpful to clarify whether Atg2 stays on the enlarged autophagic membranes when the membrane has reached to its maximum length and no longer grows.

    We thank the reviewer for this important suggestion. As noted in our response to Reviewer 1 (Major Point 8-2), clarifying whether Atg2/Atg18 remains at the ER-IM contact sites after IM expansion is indeed important for supporting the reversible lipid transfer model. We plan to monitor the localization of Atg18 during the nutrient replenishment assay.

    Minor points:

    Figure 2A "Dpm-GFP" is missing. The experiment replicates in Figure 2M should be indicated.

    We thank the reviewer for pointing out these issues. The label for "Dpm-GFP" has been added in Figure 2A, and the number of experimental replicates for Figure 2M is now indicated in the figure legend.

    Figure S2, the magenta panel should be "R18".

    We thank the reviewer for catching this labeling error. We have corrected the magenta panel label in Figure S2 to "R18" in the revised version of the figure.

    Line 341-342: "Figure 5H and 5J" should be "Figure 5H and 5I"

    We thank the reviewer for pointing out this error. The citation has been corrected from "Figure 5H and 5J" to "Figure 5H and 5I" in the revised manuscript.

    Please describe how the lipid docking model of Atg2 is generated.

    We thank the reviewer for this question. We have added a description of the modeling approach in the Methods section of the revised manuscript (lines 640-646). We also added the configuration files of AlphaFold3 to the supplementary information.

    Reviewer #2 (Significance (Required)):

    Currently, lipid probes are emerging as powerful tools to understand membrane dynamics, integrity, and the lipid-mediated cellular functions. In this manuscript, the authors performed a detailed characterisation of octadecyl rhodamine B (R18) as a potential lipid probe, which specifically labels ER and autophagic membranes. They present high quality imaging data and performed FRAP experiments to monitor the membrane dynamics and investigate the lipid transfer directionality between the ER and autophagic structure. However, the evidence of Atg2-mediated reversible lipid transfer may not be direct and sufficient. The proposed reversible lipid transfer model is interesting and provides an explanation of lipid level regulation during autophagosome formation.

    We sincerely thank the reviewer for the positive assessment of our work and for acknowledging the potential of R18 as a lipid probe, as well as the quality of our imaging and FRAP experiments. We are particularly grateful that the reviewer found the proposed model of reversible lipid transfer both interesting and relevant to the broader question of lipid regulation during autophagosome formation.

    Regarding the reviewer's concern that the evidence for Atg2-mediated reversible lipid transfer may not be sufficiently direct, we agree this is a critical point. While technical limitations currently prevent direct visualization of lipid flow reversal at single-molecule resolution in vivo, we hope our revision plan strengthen the proposed model and better convey its biological relevance, while also acknowledging the current limitations and the need for further mechanistic work.

    Response to the ____Reviewer #3

    The authors address the question of how autophagic membrane seeds expand into autophagosomes. After nucleation, IMs expand in dependence of the bridge-like lipid transfer protein Atg2, which has been shown to tether the IM to the ER. Several studies have shown in vitro evidence for direct lipid transfer by Atg2 between tethered membranes, and previous evidence has shown that the hydrophobic groove of Atg2 implicated in lipid transfer is required for autophagosome biogenesis in vivo in yeast and mammalian cells.

    In this manuscript, the authors take advantage of the dye R18, which they show accumulates mainly in the ER after a few minutes. They show specifically that the import of R18 into cells and transfer to the ER depends on the activity of flippases in the plasma membrane and OSPB-related lipid transporter. Using different sets of FRAT experiments, the authors track the fluorescence recovery of R18 in the IM, the IM-ER membrane contact site and the neighboring ER. From these experiments the authors conclude that (a) R18 is transferred to IM from the ER when IMs expand and (b) can be transferred from IMs back to the ER when autophagy is deactivated.

    The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

    We sincerely thank the reviewer for this thoughtful and encouraging summary. We appreciate the recognition of our approach using R18 to visualize lipid dynamics at ER-IM contact sites, and agree that in vivo quantitative data are critically needed to advance our understanding of autophagic membrane expansion.

    We also fully agree with the reviewer that our current study provides indirect-but conceptually informative-support for Atg2-mediated reversible one way lipid transfer. While prior in vitro studies have demonstrated the lipid transfer capability of Atg2, our goal here was to develop a live-cell system that allows the dynamic tracking of lipid flow in vivo, and to explore the possibility of reversible transport during autophagy termination. We hope our story will offer unique insights for future studies aiming to directly probe lipid transfer mechanisms in live cells.

    Regarding the reviewer's concern about the lack of direct evidence that Atg2's lipid transfer activity underlies the observed R18 dynamics, we fully acknowledge this limitation. To address this point, we would like to cite our parallel study currently under revision (Sakai et al., bioRxiv 2025.05.24.655882v1), which provides additional mechanistic evidence linking R18 dynamics to the lipid transfer function of Atg2. Further details and planned revisions are described in the responses below.

    Major points:

    (1) The authors use R18in FRAP experiments to follow its transfer from the ER into IMs. However, whether this transfer is mediated by Atg2 via its inherent lipid transfer activity remains indirect. The only evidence that implicates Atg2 directly is the observation that a lipid transfer deficient Atg2 variant fails to support IM expansion and autophagosome biogenesis. A similar full-length Atg2 mutant has previously been shown to block autophagosome formation in Dabrowski et al. 2023 in yeast, which the authors do not cite or discuss, suggesting the inherent lipid transfer activity of Atg2 is required for IM expansion. However, aside from this experiment, the mechanisms underlying R18 transfer remain unclear and, while they likely depend on or are at least partially mediated by Atg2, they may involve alternative mechanisms including vesicle transport or continuous membrane contacts. Moreover, for the assays with stalled or dissolving IM, it is essential for the authors to test whether Atg2 is still associated with these IMs. It is quite possible that Atg2 dissociates from maximally expanded or dissolving IMs, which would make their interpretation of the data very unlikely. Thus, it will be critical to provide consistent evidence that lipid transfer from the IM to the ER is mediated by Atg2. Ideally, the authors would label IM with BFP-Atg8, R18, and Atg2-GFP and perform their in vivo analysis.

    We sincerely thank the reviewer for the critical comments and valuable suggestions. To further support the link between R18 transfer and Atg2, we would like to highlight two complementary findings. As noted in our response to Reviewer 1 (Major Point 3), R18 can still label the PAS even when Atg2 is recruited but IM expansion is impaired, suggesting that R18 trafficking occurs in an Atg2-dependent manner. In addition, in our parallel study (bioRxiv, 2025.05.24.655882v1), we demonstrated that Atg2 acts as a bridge-like lipid transfer protein. Notably, when we mutated the bridge-forming region of Atg2, R18 transport to the IM was also disrupted.

    We greatly appreciate the reviewer's reminder regarding the study by Dabrowski et al., 2023, which we have now cited and discussed in the revised manuscript (lines 66-68, 312-314). Their findings that the inherent lipid transfer activity of Atg2 is required for autophagosome formation in vivo strongly reinforce our model.

    Regarding the possibility of vesicle transport, we consider this contribution minimal based on R18's preferential labeling of continuous membranes and its divergence from FM4-64 staining. As for the role of continuous membrane contacts, as also mentioned in our response to Reviewer 1, our preliminary tests indicate that R18 still properly labels the ER in tether∆ cells, suggesting that its localization is not due to passive diffusion at membrane contact sites, but rather involves specific transport mechanisms. As this is an initial observation, we plan to confirm the result and include it in a future revision.

    We also thank the reviewer for the suggestion to monitor Atg2 localization at the dissolving IM. As similarly pointed out by two other reviewers, we plan to track Atg18 during the nutrient replenishment assay.

    Finally, we appreciate the idea of triple-labeling with BFP-Atg8, R18, and Atg2-GFP. While our preliminary attempts encountered technical difficulties such as abnormal BFP-Atg8 localization and severe bleaching during long-term imaging in yeast, we plan to optimize this approach in future experiments.

    (2) Given the ER forms contact sites with many organelles using bridge-like lipid transfer proteins, how do the authors explain the preferential accumulation of R18 in ARS and not in for example PM (Fmp27), mitochondria, endosomes or vacuole (Vps13)? Why should R18 specifically transferred by Atg2 and not or to a much lower rate by Fmp27 or Vps13?

    We sincerely thank the reviewer for raising this insightful question. Indeed, we have carefully considered this point. Our data indicate that R18 labeling of autophagy-related structures (ARS) depends on Atg2, as demonstrated in the present manuscript and supported by our parallel study currently under revision (bioRxiv, 2025.05.24.655882v1).

    We speculate that the preferential accumulation of R18 in ARS may arise from structural and contextual differences among bridge-like LTPs, such as Atg2, Vps13, and Fmp27. Although all are capable of mediating lipid transfer, these proteins differ in their membrane tethering modes, cargo specificity, and spatial regulation. For example, Atg2 localizes specifically to ER-IM contact sites during autophagosome formation, where membrane expansion requires rapid lipid supply. In contrast, Vps13 and Fmp27 may function at more stable or less dynamic contacts, where lipid turnover or probe accessibility is more limited. We have added a brief discussion of this point in the revised manuscript to reflect this important consideration (lines 439-444).

    (3) Does R18 label autophagic bodies after they are formed. Could the authors add R18 after autophagic bodies have formed in atg15 or pep4 cells?

    We thank the reviewer for this excellent suggestion. To address whether R18 can label autophagic bodies post-formation, we plan to perform additional experiments by adding R18 after autophagic bodies have accumulated in atg15Δ or pep4Δ cells. This will help clarify whether R18 incorporates into pre-formed autophagic bodies or requires earlier membrane dynamics for its labeling.

    (4) Since Neo1- or OSBP-defective cells do not transfer R18 from the PM to the ER or other membranes, the authors should include these strains as controls for ER-dependent R18 transfer to ARSs.

    We thank the reviewer for this insightful suggestion. To further validate the ER-dependency of R18 transfer to autophagy-related structures, we plan to include Neo1- and OSBP-deficient strains as additional controls.

    Comments:

    The authors neglect to mention or discuss important recent literature directly related to their study:

    Schutter et al., Cell (2020); Orii et al., JCB (2021); Polyansky et al., EMBOJ (2022); Dabrowski et al., JCB (2023); Shatz et al., Dev Cell (2024)

    We sincerely thank the reviewer for pointing out these important and highly relevant studies. We apologize for our oversight in not citing them earlier. Each of these works has provided valuable insights that are directly related to and have greatly informed our current study. We have now cited and discussed these references in appropriate sections of the revised manuscript.

    Figure 1A and B: The authors need to describe how these cells were stained with R18 in the figure legend or text to help the reader to understand how these experiments were performed. Figure legends need to indicate at which time point after rapamycin treatment cells were analyzed.

    Thank you for the helpful suggestion. We have now added the corresponding information to the figure legends to clarify the staining procedure and time points.

    The authors need to clarify whether mNG-Atg8 colocalization with R18 was included for dot- and ring-like structures for WT cells as shown separately in 1A but not in 1B.

    Thank you for the comment. The quantification in Figure 1B includes both dot- and ring-like structures of mNG-Atg8 colocalized with R18 in WT cells, as shown in Figure 1A. We have now clarified this point in the revised figure legend.

    Figure 1C: The figure legend needs to describe the conditions cells were treated with and when cells were analyzed after rapamycin treatment (presumably).

    Thank you for the helpful suggestion. We have now added the corresponding information to the figure legends.

    Figure 1C: The authors should combine atg15 and pep4 deletions with atg2 or atg7 as controls in which autophagic bodies are not formed.

    Thank you for the valuable suggestion. We plan to perform these experiments that combine atg15 and pep4 deletions with atg2 or atg7 as controls.

    Figure 1E and F: R18 stains more than just the ER in the cells shown. In addition to atg39 and atg40, authors should include atg11 to inhibit all forms of selective autophagy.

    Thank you very much for the insightful comment. We agree and plan to include the atg11Δ mutant to inhibit all forms of selective autophagy.

    Figure S2A and B: The figures are mislabeled. Instead of FM4-64 it should say R18. In addition to the ER, in several images it is obvious to see R18 staining the vacuole membrane (for example Figure 2A 30 degrees) and others. Thus, the strong thresholding in S2 may give the reader an oversimplified view on R18 localization. This needs to be corrected.

    Thank you very much for pointing this out. We have corrected the labeling error in Figure S2A and B. Regarding the observation that R18 occasionally labels the vacuole membrane, we agree with the reviewer's comment. Based on our data, we believe that this signal likely reflects autophagosomes that have reached and fused with the vacuole, as expected in the later stages of autophagy. We have clarified this point in the text to avoid oversimplification of R18 localization (lines 169-171, 426-428).

    Figure 1G and H: In 1G, there are number of R18-stained patches not co-labeled by GFP-ER. What are these patches and which organelles to they represent? In 1H, given the tight association of the ER (omegasome) with forming IMs, it is difficult to discern whether R18 labels surrounding ER membrane or the IM itself. This needs to be more closely analyzed. The authors need to quantify these data similar to the yeast data.

    Thank you for the suggestion. We plan to perform additional quantification and colocalization analysis to clarify the identity of R18-positive signals in 1G and 1H.

    Figure 4A-C: A full-length PLT-deficient variant of Atg2 has been analyzed by Dabrowski et al, JCB 2023 in vivo. This work needs to be cited and discussed. The analysis needs to include punctate Atg8 structures for WT cells to exclude effects due to expansion defects.

    Thank you for the suggestion. We have now cited and discussed the work by Dabrowski et al., JCB 2023 in the revised manuscript (lines 67-68, 312-314). In addition, we have included an analysis of punctate Atg8 structures in WT cells to address the concern regarding potential expansion defects.

    Figure 4F-H: To measure the size changes in IMs, the authors would need to perform these experiments without bleaching the mNG-Atg8 signals.

    We apologize for the lack of clarity. The method for measuring IM size has now been added to the revised manuscript. In Figure 4, we note that mNG-Atg8 fluorescence actually shows a slow recovery over time. This limited recovery likely reflects both the slower turnover of Atg8 and the fact that the pre-existing Atg8 pool at the IM was partially photobleached. We have now revised the main text to clarify this point and included additional explanation (line 326-330).

    Figure 5C: The authors need to indicate the bleached areas in the mNG-Atg8 image for easier orientation. It looks to me that the area that the authors mark as IM-ER MCS is really the IM in proximity to the ER. Thus, if lipid transfer to the IM has ceased, I would not expect recovery here. If the IM-ER MCS area includes IM and the ER to similar extent, I would expect exactly what the authors show: IM does not recover while ER quickly recovers. On average, we would observe reduced recovery as shown in 5D.

    Thank you for the helpful suggestion, and we apologize for the oversight during figure preparation. We have now clearly indicated the bleached areas in the merged image in Figure 5C for better orientation. Additionally, we have carefully re-examined the defined ER-IM MCS region and confirm that the quantified area indeed corresponds to the contact site between the ER and the IM. And double checked the measurements shown in the figure remain correct.

    Figure 5L: Since mNG-Atg8 signal homogenously disappears from the IM, it is meaningless to measure size. How do the authors measure the size of something they cannot detect?

    Thank you for pointing this out. We agree with the reviewer's comment and have removed the panel from the revised version accordingly.

    Figure 5K: The authors need to show the whole bleached area overtime for the reader to be able to see where the recovered R18 signal might be coming from. Currently, it is impossible to discern whether the signal comes from the IM or from slow recovery from neighboring ER.

    We appreciate this insightful comment. To address the concern and following the suggestion from Reviewer 2 (Major Point No.4), we have now revised the figure to include an additional measurement of fluorescence recovery in the adjacent bleached ER (Figure 5K and 5M) (lines 384-396). These results further support our reversible lipid transfer model by demonstrating that fluorescence recovery at the ER-IM MCS originates from the IM, rather than from the adjacent bleached ER, which shows slower and less efficient recovery.

    We have also added time-lapse videos to the supplementary information due to space limitations in the main figure.

    Reviewer #3 (Significance (Required)):

    The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

    We sincerely thank the reviewer for this encouraging and thoughtful comment. We appreciate the recognition that our live-cell approach using a lipophilic dye provides a valuable framework to visualize lipid dynamics during autophagosome biogenesis. As the reviewer pointed out, quantitative in vivo evidence is critically needed in this field, and we hope our study contributes meaningfully toward that goal.

    We also fully acknowledge the limitation. While our current data offer indirect evidence for Atg2-mediated lipid transfer, we would like to support this by our revision plan and also our parallel study (bioRxiv, 2025.05.24.655882v1) that shows Atg2 is indeed a bridge-like LTP and R18 transfer is lost in the bridge-structure defective strain. Together, we hope these can suggest that the lipid transfer activity of Atg2 underlies the observed R18 dynamics in vivo.

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    Referee #3

    Evidence, reproducibility and clarity

    The authors address the question of how autophagic membrane seeds expand into autophagosomes. After nucleation, IMs expand in dependence of the bridge-like lipid transfer protein Atg2, which has been shown to tether the IM to the ER. Several studies have shown in vitro evidence for direct lipid transfer by Atg2 between tethered membranes, and previous evidence has shown that the hydrophobic groove of Atg2 implicated in lipid transfer is required for autophagosome biogenesis in vivo in yeast and mammalian cells.

    In this manuscript, the authors take advantage of the dye R18, which they show accumulates mainly in the ER after a few minutes. They show specifically that the import of R18 into cells and transfer to the ER depends on the activity of flippases in the plasma membrane and OSPB-related lipid transporter. Using different sets of FRAT experiments, the authors track the fluorescence recovery of R18 in the IM, the IM-ER membrane contact site and the neighboring ER. From these experiments the authors conclude that (a) R18 is transferred to IM from the ER when IMs expand and (b) can be transferred from IMs back to the ER when autophagy is deactivated.

    The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

    Major points:

    1. The authors use R18in FRAP experiments to follow its transfer from the ER into IMs. However, whether this transfer is mediated by Atg2 via its inherent lipid transfer activity remains indirect. The only evidence that implicates Atg2 directly is the observation that a lipid transfer deficient Atg2 variant fails to support IM expansion and autophagosome biogenesis. A similar full-length Atg2 mutant has previously been shown to block autophagosome formation in Dabrowski et al. 2023 in yeast, which the authors do not cite or discuss, suggesting the inherent lipid transfer activity of Atg2 is required for IM expansion. However, aside from this experiment, the mechanisms underlying R18 transfer remain unclear and, while they likely depend on or are at least partially mediated by Atg2, they may involve alternative mechanisms including vesicle transport or continuous membrane contacts. Moreover, for the assays with stalled or dissolving IM, it is essential for the authors to test whether Atg2 is still associated with these IMs. It is quite possible that Atg2 dissociates from maximally expanded or dissolving IMs, which would make their interpretation of the data very unlikely. Thus, it will be critical to provide consistent evidence that lipid transfer from the IM to the ER is mediated by Atg2. Ideally, the authors would label IM with BFP-Atg8, R18, and Atg2-GFP and perform their in vivo analysis.
    2. Given the ER forms contact sites with many organelles using bridge-like lipid transfer proteins, how do the authors explain the preferential accumulation of R18 in ARS and not in for example PM (Fmp27), mitochondria, endosomes or vacuole (Vps13)? Why should R18 specifically transferred by Atg2 and not or to a much lower rate by Fmp27 or Vps13?
    3. Does R18 label autophagic bodies after they are formed. Could the authors add R18 after autophagic bodies have formed in atg15 or pep4 cells?
    4. Since Neo1- or OSBP-defective cells do not transfer R18 from the PM to the ER or other membranes, the authors should include these strains as controls for ER-dependent R18 transfer to ARSs.

    Comments:

    The authors neglect to mention or discuss important recent literature directly related to their study:

    Schutter et al., Cell (2020); Orii et al., JCB (2021); Polyansky et al., EMBOJ (2022); Dabrowski et al., JCB (2023); Shatz et al., Dev Cell (2024)

    Figure 1A and B: The authors need to describe how these cells were stained with R18 in the figure legend or text to help the reader to understand how these experiments were performed. Figure legends need to indicate at which time point after rapamycin treatment cells were analyzed.

    The authors need to clarify whether mNG-Atg8 colocalization with R18 was included for dot- and ring-like structures for WT cells as shown separately in 1A but not in 1B.

    Figure 1C: The figure legend needs to describe the conditions cells were treated with and when cells were analyzed after rapamycin treatment (presumably).

    The authors should combine atg15 and pep4 deletions with atg2 or atg7 as controls in which autophagic bodies are not formed.

    Figure 1E and F: R18 stains more than just the ER in the cells shown. In addition to atg39 and atg40, authors should include atg11 to inhibit all forms of selective autophagy.

    Figure S2A and B: The figures are mislabeled. Instead of FM4-64 it should say R18. In addition to the ER, in several images it is obvious to see R18 staining the vacuole membrane (for example Figure 2A 30 degrees) and others. Thus, the strong thresholding in S2 may give the reader an oversimplified view on R18 localization. This needs to be corrected.

    Figure 1G and H: In 1G, there are number of R18-stained patches not co-labeled by GFP-ER. What are these patches and which organelles to they represent? In 1H, given the tight association of the ER (omegasome) with forming IMs, it is difficult to discern whether R18 labels surrounding ER membrane or the IM itself. This needs to be more closely analyzed. The authors need to quantify these data similar to the yeast data.

    Figure 4A-C: A full-length PLT-deficient variant of Atg2 has been analyzed by Dabrowski et al, JCB 2023 in vivo. This work needs to be cited and discussed. The analysis needs to include punctate Atg8 structures for WT cells to exclude effects due to expansion defects.

    Figure 4F-H: To measure the size changes in IMs, the authors would need to perform these experiments without bleaching the mNG-Atg8 signals.

    Figure 5C: The authors need to indicate the bleached areas in the mNG-Atg8 image for easier orientation. It looks to me that the area that the authors mark as IM-ER MCS is really the IM in proximity to the ER. Thus, if lipid transfer to the IM has ceased, I would not expect recovery here. If the IM-ER MCS area includes IM and the ER to similar extent, I would expect exactly what the authors show: IM does not recover while ER quickly recovers. On average, we would observe reduced recovery as shown in 5D.

    Figure 5L: Since mNG-Atg8 signal homogenously disappears from the IM, it is meaningless to measure size. How do the authors measure the size of something they cannot detect?

    Figure 5K: The authors need to show the whole bleached area overtime for the reader to be able to see where the recovered R18 signal might be coming from. Currently, it is impossible to discern whether the signal comes from the IM or from slow recovery from neighboring ER.

    Significance

    The use of a lipophilic dye to monitor lipid dynamics during IM expansion or dissolution is an elegant way to probe the mechanisms of lipid transfer across ER-IM contact sites. Quantitative in vivo data is critically needed to address this fundamental question in autophagy and contact site biology. However, the study remains limited in providing direct evidence that it is indeed the lipid transfer activity of Atg2, which underlies the R18 dynamics in IMs in vivo.

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    Referee #2

    Evidence, reproducibility and clarity

    Non-vesicular lipid transfer plays an essential role in organelle biogenesis. Compared to vesicular lipid transfer, it is faster and more efficient to maintain proper lipid levels in organelles. In this study, Hao et al. introduced a high lipophilic dye octadecyl rhodamine B (R18), which specifically labels the ER structures and autophagy-related structures in yeast and mammalian cells. They characterised its distinct lipid entry into yeast cells via lipid flippase Neo1 and Drs2 on the plasma membrane, rather than through the endocytic pathway. They then demonstrated that R18 intracellular trafficking through plasma membrane to ER depends on "box-like" lipid transfer Osh proteins. They further looked into the "bridge-like" lipid transfer protein Atg2, using R18 as a lipid probe to track lipid transfer from ER to the isolation membrane (IM) during membrane expansion and reversible lipid transfer through IM to the ER-IM membrane contact sites (MCS) when autophagy is terminated by nutrient replenishment. The authors provide an interesting model of reversible directionality of Atg2 lipid transfer during autophagy induction and termination.

    Major points:

    1. Line 299-309: The FRAP assays were interesting and well performed. The authors photobleached R18 and Atg8 signal, and found R18 fluorescence recovery but not Atg8, which suggests lipid transfer occurs between ER and the IM and faster than Atg8 lipidation process during IM expansion. These results gave clear evidence that R18 can be transferred during IM expansion. The supply of Atg8 may not be not able to track within this time frame or the recovered amount of Atg8 may not be able to visualized due to the threshold limitation with confocal microcopy. This does not imply the supply of Atg8 to the IM is not required during IM expansion. This should be clarified.
    2. Please clarify how the length of the IM is measured and determined in Figure 4H and Figure 5D.
    3. Line 336-342: The description of the results should be clarified. Based on Figure 5H, the authors observed a significant decrease in the mNG-Atg8 signal during photobleaching of the R18 signal.
    4. The authors photobleached ER-IM MCS and the ER region (boxed region in Figure 5J) and quantified fluorescence recovery, normalized to the IM region and an ER control. The ER control was taken from the other cell. It would be helpful to compare and analyse the fluorescence recovery of R18 in the bleached ER region near the ER-IM MCS to that in the ER-IM MCS. This would help to confirm the ER-IM MCS fluorescence recovery is due to signal coming from the IM.
    5. In figure 5K, the autophagic structure or IM labelled by R18 seems to be maintained when the mNG-Atg8 signal decreases or dissociates from the IM. Could the authors comment on that how they interpret the termination of the prolonged IM structure and IM shrinkage?
    6. The author has shown that Atg2Δ and Atg2LT lipid transfer mutant impair R18 labelling of autophagic structures in Figure 4C. However, the evidence supporting that R18 fluorescence recovery at ER-IM MCS is mediated by reversible Atg2 lipid transfer is not direct. It would be helpful to clarify whether Atg2 stays on the enlarged autophagic membranes when the membrane has reached to its maximum length and no longer grows.

    Minor points:

    1. Figure 2A "Dpm-GFP" is missing. The experiment replicates in Figure 2M should be indicated.
    2. Figure S2, the magenta panel should be "R18".
    3. Line 341-342: "Figure 5H and 5J" should be "Figure 5H and 5I"
    4. Please describe how the lipid docking model of Atg2 is generated.

    Significance

    Currently, lipid probes are emerging as powerful tools to understand membrane dynamics, integrity, and the lipid-mediated cellular functions. In this manuscript, the authors performed a detailed characterisation of octadecyl rhodamine B (R18) as a potential lipid probe, which specifically labels ER and autophagic membranes. They present high quality imaging data and performed FRAP experiments to monitor the membrane dynamics and investigate the lipid transfer directionality between the ER and autophagic structure. However, the evidence of Atg2-mediated reversible lipid transfer may not be direct and sufficient. The proposed reversible lipid transfer model is interesting and provides an explanation of lipid level regulation during autophagosome formation.

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    Referee #1

    Evidence, reproducibility and clarity

    In their study, Hao and colleagues exploited the fluorescent fatty acid R18 to follow phospholipid (PL) transfer in vivo from the endoplasmic reticulum to the IM during autophagosome formation. Although the results are interesting, especially the retrograde transport of PLs, based on the provided data, additional control experiments are needed to firmly support the conclusions. An additional point is that the authors also study the internalization of R18 into cells and found a role of lipid flippases and oxysterol binding proteins. While this information could be useful for researchers using this dye, these analyses/findings have no specific connection with the topic of the manuscript, i.e. the PL transfer during autophagosome formation. Therefore, they must be removed.

    Major points:

    1. In general, the quality of the microscopy images are quite poor and this make it difficult to assert some of the authors' conclusions.
    2. It would be important to perform some lipidomics analysis to determine in which PLs and other lipids or lipid intermediates R18 is incorporated. First, it will be important to know which the major PL species are are labelled under the conditions of the experiments done in this study. Second, the authors assume that all the R18 is exclusively incorporated into PLs and this is what they follow in their in vivo experiments. What about acyl-CoA, which has been shown to be a key player in the IM elongation (Graef lab, Cell)?
    3. Figure 1A and 1B. The authors conclude that Atg2 is involved in the lipid transfer since R18 does not localize to the PAS/ARS in the atg2KO cells. However, another possible explanation is that in those cells the IM is not formed and does not expand, and con sequetly R18 is present in low amounts not detectable by fluorescence microscopy. To support their conclusion, the authors must assess PAS-labelling with R18 in cells lacking another ATG gene in which Atg2 is still recruited to the PAS.
    4. Figure 2. As written, the paragraph this figure seems to indicate that flippases are directly involved in the translocation of R18 from the PM to the ER. As correctly indicated by the authors, flippases flip PLs, not fatty acids. Moreover, there are no PL synthesizing at the PM and thus probably R18 is not flipped upon incorporation into PL. As a result, the relevance of flippase in R18 internalization is probably indirect. This must be explained clearly to avoid confusion/misunderstandings.
    5. A couple of manuscript has shown a (partial) role of Drs2 in autophagy. The authors must explain the discrepancy between their own results and what published, especially because they use the GFP-Atg8 processing assay, which is less sensitive than the Pho8delta60 used in the other studies.
    6. Authors used a predicted Atg2 lipid-transfer mutant (Srinivasan et al, J Cel Biol, 2024), but not direct prove that this mutant is defective for this activity. As previously done for other Atg2/ATG2-related manuscripts (Osawa et al, Nat Struct Mol Biol, 2019; Valverde et al, J Cel Biol, 2019), this must be measure in vitro. Moreover, they do not show whether other known functions of Atg2 are unaffected when expressing this Atg2 mutant, e.g. formation of the IM-ER MCSs, Atg2 interaction with Atg9 and localization at the extremity of the IM...
    7. The mNG-Atg8 signal is not recovered in the fluorescent recovery assays. Based on the observation that R18 signal comes back after photobleaching, authors suggest that the supply of Atg8 is not required for IM expansion. This idea is opposite to data where the levels of Atg8 and deconjugation of lipidated Atg8 determines the size of the forming autophagosomes (e.g., Xie et al, Mol Biol Cell, 2008; Nair et al, Autophagy, 2012). Similar results have also been obtained in mammalian cells (Lazarou and Mizushima results in cell lacking components of the two ubiquitin-like conjugation systems). This discrepancy requires an explanation.
    8. Although authors claim that there is a retrograde lipid transfer from the IM to the ER, based on the data, it quite difficult to extract these conclusions as they show a decrease in the lipid flow dynamics rather to an inversion of the lipid flow per se. Can the authors exclude that ER microdomains are formed at the ERES in contact with the IM, and consequently what they measure is a slow diffusion of R18-labeled lipid from other part of the ER to these ERES? Additionally, authors should check whether the Atg2 and Atg18 proteins are present at the IM-ER membrane contact sites in the same rates after nutrient replenished than when cells are nitrogen-starved, since this complex would determine the lipid transfer dynamics at this membrane contact site.
    9. The retrograde PL transfer is studied in cells overexpressing Ape1, in which IM elongation is stalled. This is a non-physiological experimental setup and consequently it is unclear whether what observed applies to normal IM/autophagosomes. This event should be shown to occur in WT cells as well.
    10. From the images provided, it appears that R18 also labels the vacuole. The vacuole form MCSs with the IM. Can the author exclude a passage of R18 from the vacuole to the IM?

    Minor points:

    1. L66. One report has indicated that Vps13 may also play a role in the transfer of lipids from the ER to the IM (Graef lab, J. Cell Biol).
    2. L70. It must be indicated that IM is also called phagophore.
    3. L74. It is mentioned "Additionally, a hydrophobic cavity in the N-terminal region of Atg2 directly tethers Atg2 to the ER, particularly the ER exit site (ERES), which is considered a key hub for autophagosome biogenesis", but there is no experimental evidence supporting that Atg2 is involved in the tethering with the ERES.
    4. L90. PAS must be listed between the ARS.
    5. Upon deletion of ATG39 and ATG40, there is a pronounced reduction of mNG-Atg8 labelled with R18. This would suggest that these two ER-phagy receptors are required for the PL transfer from the ER to the IM, which is not the case as autophagy is mildly affected by the absence of them (e.g., Zhang et al, Autophagy, 2020).
    6. Authors referred that "no direct evidence has been found to confirm lipid transfer at the ER-IM MCS in living cells" (lines 282-283). However, a recent paper has shown that de novo-synthesized phosphatidylcholine is incorporated from the ER to the autophagosomes and autophagic bodies (Orii et al, J Cel Biol, 2021). This reference should be mentioned in the manuscript.
    7. In lines 252-253, the sentence "R18 transport from the PM to the ER was partially impaired in osh1Δ osh2Δ, osh6Δ osh7Δ, and oshΔ osh4-1 cells (Figure S3). These results suggest that Osh proteins participate in transferring R18 from the PM to the ER" does not recapitulate what is observed in Fig. S3. Moreover, the Emr lab has generate a tertadeletion mutant in which the PM-ER MCSs are abolished. The authors could examine this mutant.

    Significance

    General assessment:

    Strength: potential new system to monitor lipid flow Limitations: Indirect evidences and in the case of the retrograde transport of phospholipids, it could be an artefact of the employed experimental approach.

    Advance: Little advances because something in part already shown in vitro. No ne mechanisms uncovered.

    Audience: Autophagy and membrane contact site fields.

  5. Version published to 10.1101/2025.03.27.645728 on bioRxiv