The Rab7-Epg5 and Rab39-ema modules cooperately position autophagosomes for efficient lysosomal fusions

Reviewer #1 (Public review):

Summary:

It is well known that autophagosomes/autolysosomes move along microtubules. However, because previous studies did not distinguish between autophagosomes and autolysosomes, it remains unknown whether autophagosomes begin to move after fusion with lysosomes or even before fusion. In this manuscript, the authors show, using fusion-deficient cells, that both pre-fusion autophagosomes and lysosomes can move along the MT toward the minus end. By screening motor proteins and Rabs, the authors found that autophagosomal traffic is primarily regulated by the dynein-dynactin system and can be counter-regulated by kinesins. They also show that Rab7-Epg5 and Rab39-ema interactions are important for autophagosome trafficking.

Strengths:

This study uses reliable Drosophila genetics and high-quality fluorescence microscopy. The data are properly quantified and statistically analyzed. It is a reasonable hypothesis that gathering pre-fusion autophagosomes and lysosomes in close proximity improves fusion efficiency.

Weaknesses:

(1) To distinguish autophagosomes from autolysosomes, the authors used vps16 RNAi cells, which are supposed to be fusion deficient. However, the extent to which fusion is actually inhibited by knockdown of Vps16A is not shown. The co-localization rate of Atg8 and Lamp1 should be shown (as in Figure 8). Then, after identifying pre-fusion autophagosomes and lysosomes, the localization of each should be analyzed. It is also possible that autophagosomes and lysosomes are tethered by factors other than HOPS (even if they are not fused). If this is the case, autophagosomal trafficking would be affected by the movement of lysosomes.

(2) The authors analyze autolysosomes in Figures 6 and 7. This is based on the assumption that autophagosome-lysosome fusion takes place in cells without vps16A RANi. However, even in the presence of Vps16A, both pre-fusion autophagosomes and autolysosomes should exist. This is also true in Figure 8H, where the fusion of autophagosomes and lysosomes is partially suppressed in knockdown cells of dynein, dynactin, Rab7, and Epg5. If the effect of fusion is to be examined, it is reasonable to distinguish between autophagosomes and autolysosomes and analyze only autolysosomes.

(3) In this study, only vps16a RNAi cells were used to inhibit autophagosome-lysosome fusion. However, since HOPS has many roles besides autophagosome-lysosome fusion, it would be better to confirm the conclusion by knockdown of other factors (e.g., Stx17 RNAi).

(4) Figure 8: Rab7 and Epg5 are also known to be directly involved in autophagosome-lysosome tethering/fusion. Even if the fusion rate is reduced in the absence of Rab7 and Epg5, it may not be the result of defective autophagosome movement, but may simply indicate that these molecules are required for fusion itself. How do the authors distinguish between the two possibilities?

Reviewer #2 (Public review):

Summary:

This manuscript by Boda et al. describes the results of a targeted RNAi screen in the background of Vps16A-depleted Drosophila larval fat body cells. In this background, lysosomal fusion is inhibited, allowing the authors to analyze the motility and localization specifically of autophagosomes, prior to their fusion with lysosomes to become autolysosomes. In this Vps16A-deleted background, mCherry-Atg8a-labeled autophagosomes accumulate in the perinuclear area, through an unknown mechanism.

The authors found that the depletion of multiple subunits of the dynein/dynactin complex caused an alternation of this mCherry-Atg8a localization, moving from the perinuclear region to the cell periphery. Interactions with kinesin overexpression suggest these motor proteins may compete for autophagosome binding and transport. The authors extended these findings by examining potential upstream regulators including Rab proteins and selected effectors, and they also examined effects on lysosomal movement and autolysosome size. Altogether, the results are consistent with a model in which specific Rab/effector complexes direct the movement of lysosomes and autophagosomes toward the MTOC, promoting their fusion and subsequent dispersal throughout the cell.

Strengths:

Although previous studies of the movement of autophagic vesicles have identified roles for microtubule-based transport, this study moves the field forward by distinguishing between effects on pre- and post-fusion autophagosomes, and by its characterization of the roles of specific Dynein, Dynactin, and Rab complexes in regulating movement of distinct vesicle types. Overall, the experiments are well-controlled, appropriately analyzed, and largely support the authors' conclusions.

Weaknesses:

One limitation of the study is the genetic background that serves as the basis for the screening. In addition to preventing autophagosome-lysosome fusion, disruption of Vps16A has been shown to inhibit endosomal maturation and block the trafficking of components to the lysosome from both the endosome and Golgi apparatus. Additional effects previously reported by the authors include increased autophagosome production and reduced mTOR signaling. Thus Vps16A-depleted cells have a number of endosome, lysosome, and autophagosome-related defects, with unknown downstream consequences. Additionally, the cause and significance of the perinuclear localization of autophagosomes in this background is unclear. Thus, interpretations of the observed reversal of this phenotype are difficult, and have the caveat that they may apply only to this condition, rather than to normal autophagosomes. Additional experiments to observe autophagosome movement or positioning in a more normal environment would improve the manuscript.

Specific comments

(1) Several genes have been described that when depleted lead to perinuclear accumulation of Atg8-labeled vesicles. There seems to be a correlation of this phenotype with genes required for autophagosome-lysosome fusion; however, some genes required for lysosomal fusion such as Rab2 and Arl8 apparently did not affect autophagosome positioning as reported here. Thus, it is unclear whether the perinuclear positioning of autophagosomes is truly a general response to disruption of autophagosome-lysosome fusion, or may reflect additional aspects of Vps16A/HOPS function. A few things here would help. One would be an analysis of Atg8a vesicle localization in response to the depletion of a larger set of fusion-related genes. Another would be to repeat some of the key findings of this study (effects of specific dynein, dynactin, rabs, effectors) on Atg8a localization when Syx17 is depleted, rather than Vps16A. This should generate a more autophagosome-specific fusion defect. Third, it would greatly strengthen the findings to monitor pre-fusion autophagosome localization without disrupting fusion. Such vesicles could be identified as Atg8a-positive Lamp-negative structures. The effects of dynein and rab depletion on the tracking of these structures in a post-induction time course would serve as an important validation of the authors' findings.

(2) The authors nicely show that depletion of Shot leads to relocalization of Atg8a to ectopic foci in Vps16A-depleted cells; they should confirm that this is a mislocalized ncMTOC by co-labeling Atg8a with an MTOC component such as MSP300. The effect of Shot depletion on Atg8a localization should also be analyzed in the absence of Vps16A depletion.

(3) The authors report that depletion of Dynein subunits, either alone (Figure 6) or co-depleted with Vps16A (Figure 2), leads to redistribution of mCherry-Atg8a punctae to the "cell periphery". However, only cell clones that contact an edge of the fat body tissue are shown in these figures. Furthermore, in these cells, mCherry-Atg8a punctae appear to localize only to contact-free regions of these cells, and not to internal regions of clones that share a border with adjacent cells. Thus, these vesicles would seem to be redistributed to the periphery of the fat body itself, not to the periphery of individual cells. Microtubules emanating from the perinuclear ncMTOC have been described as having a radial organization, and thus it is unclear that this redistribution of mCherry-Atg8a punctae to the fat body edge would reflect a kinesin-dependent process as suggested by the authors.

(4) To validate whether the mCherry-Atg8a structures in Vps16A-depleted cells were of autophagic origin, the authors depleted Atg8a and observed a loss of mCherry- Atg8a signal from the mosaic cells (Figure S1D, J). A more rigorous experiment would be to deplete other Atg genes (not Atg8a) and examine whether these structures persist.

(5) The authors found that only a subset of dynein, dynactin, rab, and rab effector depletions affected mCherry- Atg8a localization, leading to their suggestion that the most important factors involved in autophagosome motility have been identified here. However, this conclusion has the caveat that depletion efficiency was not examined in this study, and thus any conclusions about negative results should be more conservative.

Reviewer #3 (Public review):

Summary:

In multicellular organisms, autophagosomes are formed throughout the cytosol, while late endosomes/lysosomes are relatively confined in the perinuclear region. It is known that autophagosomes gain access to the lysosome-enriched region by microtubule-based trafficking. The mechanism by which autophagosomes move along microtubules remains incompletely understood. In this manuscript, Péter Lőrincz and colleagues investigated the mechanism driving the movement of nascent autophagosomes along the microtubule towards the non-centrosomal microtubule organizing center (ncMTOC) using the fly fat body as a model system. The authors took an approach whereby they examined autophagosome positioning in cells where autophagosome-lysosome fusion was inhibited by knocking down the HOPS subunit Vps16A. Despite being generated at random positions in the cytosol, autophagosomes accumulate around the nucleus when Vps16A is depleted. They then performed an RNA interference screen to identify the factors involved in autophagosome positioning. They found that the dynein-dynactin complex is required for the trafficking of autophagosomes toward ncMTOC. Dynein loss leads to the peripheral relocation of autophagosomes. They further revealed that a pair of small GTPases and their effectors, Rab7-Epg5 and Rab39-ema, are required for bidirectional autophagosome transport. Knockdown of these factors in Vps16a RNAi cells causes the scattering of autophagosomes throughout the cytosol.

Strengths:

The data presented in this study help us to understand the mechanism underlying the trafficking and positioning of autophagosomes.

Weaknesses:

Major concerns:

(1) The localization of EPG5 should be determined. The authors showed that EPG5 colocalizes with endogenous Rab7. Rab7 labels late endosomes and lysosomes. Previous studies in mammalian cells have shown that EPG5 is targeted to late endosomes/lysosomes by interacting with Rab7. EPG5 promotes the fusion of autophagosomes with late endosomes/lysosomes by directly recognizing LC3 on autophagosomes and also by facilitating the assembly of the SNARE complex for fusion. In Figure 5I, the EPG5/Rab7-colocalized vesicles are large and they are likely to be lysosomes/autolysosomes.

(2) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation. The authors need to verify their findings in Stx17 KO cells, as it has a relatively specific effect on the fusion of autophagosomes with late endosomes/lysosomes.

(3) Quantification should be performed in many places such as in Figure S4D for the number of FYVE-GFP labeled endosomes and in Figures S4H and S4I for the number and size of lysosomes.

(4) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gained from this study may not apply to other cell types. This needs to be discussed.

Minor concerns:

(5) Data in some panels are of low quality. For example, the mCherry-Atg8a signal in Figure 5C is hard to see; the input bands of Dhc64c in Figure 5L are smeared.

(6) In this study, both 3xmCherry-Atg8a and mCherry-Atg8a were used. Different reporters make it difficult to compare the results presented in different figures.

(7) The small autophagosomes presented in Figures such as in Figure 1D and 1E are not clear. Enlarged images should be presented.

(8) The authors showed that Epg5-9xHA coprecipitates with the endogenous dynein motor Dhc64C. Is Rab7 required for the interaction?

(9) The perinuclear lysosome localization in Epg5 KD cells has no indication that Epg5 is an autophagosome-specific adaptor.

Author Response:

Reviewer #1 (Public review):

Summary:

It is well known that autophagosomes/autolysosomes move along microtubules. However, because previous studies did not distinguish between autophagosomes and autolysosomes, it remains unknown whether autophagosomes begin to move after fusion with lysosomes or even before fusion. In this manuscript, the authors show, using fusion-deficient cells, that both pre-fusion autophagosomes and lysosomes can move along the MT toward the minus end. By screening motor proteins and Rabs, the authors found that autophagosomal traffic is primarily regulated by the dynein-dynactin system and can be counter-regulated by kinesins. They also show that Rab7-Epg5 and Rab39-ema interactions are important for autophagosome trafficking.

Strengths:

This study uses reliable Drosophila genetics and high-quality fluorescence microscopy. The data are properly quantified and statistically analyzed. It is a reasonable hypothesis that gathering pre-fusion autophagosomes and lysosomes in close proximity improves fusion efficiency.

Thank you for your positive comments and for acknowledging the strengths of our work.

Weaknesses:

(1) To distinguish autophagosomes from autolysosomes, the authors used vps16 RNAi cells, which are supposed to be fusion deficient. However, the extent to which fusion is actually inhibited by knockdown of Vps16A is not shown. The co-localization rate of Atg8 and Lamp1 should be shown (as in Figure 8). Then, after identifying pre-fusion autophagosomes and lysosomes, the localization of each should be analyzed.

Thank you for this comment. We plan to perform immunohistochemistry experiment on Vps16A KD fat body cells for mCherry and Lamp1, as in case of other panels of Figure 8. We will also analyse the distribution of each.

It is also possible that autophagosomes and lysosomes are tethered by factors other than HOPS (even if they are not fused). If this is the case, autophagosomal trafficking would be affected by the movement of lysosomes.

While we cannot exclude the possibility that autophagosomes are transported indirectly by being tethered to lysosomes. However, we find this unlikely be the case as we believe in fat cells lysosomes and autophagosomes will rapidly fuse with each other if they get close enough.

(2) The authors analyze autolysosomes in Figures 6 and 7. This is based on the assumption that autophagosome-lysosome fusion takes place in cells without vps16A RANi. However, even in the presence of Vps16A, both pre-fusion autophagosomes and autolysosomes should exist. This is also true in Figure 8H, where the fusion of autophagosomes and lysosomes is partially suppressed in knockdown cells of dynein, dynactin, Rab7, and Epg5. If the effect of fusion is to be examined, it is reasonable to distinguish between autophagosomes and autolysosomes and analyze only autolysosomes.

Thank you for your careful insights. The mCherry-Atg8a reporter we use is highly stable in autolysosomes due to the resilience of the mCherry fluorophore within these acidic, post-fusion structures, making it useful for labelling both autophagosomes and autolysosomes. Notably, the high intensity of mCherry-Atg8a within autolysosomes allows us to distinguish them from pre-fusion autophagosomes, which appear fainter and smaller, especially when accumulated in fusion-defective backgrounds (as shown in Figure 4). We therefore regard larger, brighter structures as autolysosomes.

To improve clarity, we included additional markers—endogenous Lamp1 staining (Figure 8) and Lamp1-GFP (Figure S9)—to help differentiate between autophagic structures. Lamp1-negative, mCherry-Atg8a-positive vesicles indicate pre-fusion autophagosomes, while Lamp1/mCherry-Atg8a double-positive vesicles represent autolysosomes. Additionally, Lamp1-positive, mCherry-Atg8a-negative vesicles mark lysosomes of non-autophagic origin. We appreciate your suggestion

(3) In this study, only vps16a RNAi cells were used to inhibit autophagosome-lysosome fusion. However, since HOPS has many roles besides autophagosome-lysosome fusion, it would be better to confirm the conclusion by knockdown of other factors (e.g., Stx17 RNAi).

Thank you for this suggestion. We will generate additional Drosophila lines similar to those used in our current study, substituting Syntaxin17, SNAP29 or Vamp7 RNAi for Vps16A RNAi. We will test key phenotypic hits with these new backgrounds to confirm our findings.

(4) Figure 8: Rab7 and Epg5 are also known to be directly involved in autophagosome-lysosome tethering/fusion. Even if the fusion rate is reduced in the absence of Rab7 and Epg5, it may not be the result of defective autophagosome movement, but may simply indicate that these molecules are required for fusion itself. How do the authors distinguish between the two possibilities?

Thank you for this comment. While we agree that Rab7 and Epg5 are involved in autophagosome-lysosome tethering and subsequent fusion, we believe they also play an additional role in autophagosome movement. Our hypothesis stems from the observation that the phenotypes of vps16 RNAi and rab7 or epg5 RNAi are not identical. In contrast, RNAi targeting SNARE proteins involved exclusively in fusion (Syx17, SNAP29, and Vamp7) all result in a consistent phenotype: autophagosomes accumulate around the nucleus, closely resembling the phenotype observed with vps16 depletion. This suggests that these SNAREs are specifically involved in fusion. Since Rab7 and Epg5 depletion scatters autophagosomes throughout the cytosol rather than transporting them to the nucleus, we hypothesize that this is due to impaired movement of autophagosomes. This hypothesis is further supported by our co-IP data showing that Epg5 binds to dyneins.

Reviewer #2 (Public review):

Summary:

This manuscript by Boda et al. describes the results of a targeted RNAi screen in the background of Vps16A-depleted Drosophila larval fat body cells. In this background, lysosomal fusion is inhibited, allowing the authors to analyze the motility and localization specifically of autophagosomes, prior to their fusion with lysosomes to become autolysosomes. In this Vps16A-deleted background, mCherry-Atg8a-labeled autophagosomes accumulate in the perinuclear area, through an unknown mechanism.

The authors found that the depletion of multiple subunits of the dynein/dynactin complex caused an alternation of this mCherry-Atg8a localization, moving from the perinuclear region to the cell periphery. Interactions with kinesin overexpression suggest these motor proteins may compete for autophagosome binding and transport. The authors extended these findings by examining potential upstream regulators including Rab proteins and selected effectors, and they also examined effects on lysosomal movement and autolysosome size. Altogether, the results are consistent with a model in which specific Rab/effector complexes direct the movement of lysosomes and autophagosomes toward the MTOC, promoting their fusion and subsequent dispersal throughout the cell.

Strengths:

Although previous studies of the movement of autophagic vesicles have identified roles for microtubule-based transport, this study moves the field forward by distinguishing between effects on pre- and post-fusion autophagosomes, and by its characterization of the roles of specific Dynein, Dynactin, and Rab complexes in regulating movement of distinct vesicle types. Overall, the experiments are well-controlled, appropriately analyzed, and largely support the authors' conclusions.

Thank you for your positive comments and for acknowledging the strengths of our work.

Weaknesses:

One limitation of the study is the genetic background that serves as the basis for the screening. In addition to preventing autophagosome-lysosome fusion, disruption of Vps16A has been shown to inhibit endosomal maturation and block the trafficking of components to the lysosome from both the endosome and Golgi apparatus. Additional effects previously reported by the authors include increased autophagosome production and reduced mTOR signaling. Thus Vps16A-depleted cells have a number of endosome, lysosome, and autophagosome-related defects, with unknown downstream consequences. Additionally, the cause and significance of the perinuclear localization of autophagosomes in this background is unclear. Thus, interpretations of the observed reversal of this phenotype are difficult, and have the caveat that they may apply only to this condition, rather than to normal autophagosomes. Additional experiments to observe autophagosome movement or positioning in a more normal environment would improve the manuscript.

Thank you for highlighting this limitation. We plan to conduct time-lapse imaging of live fat body tissues expressing 3xmCherry-Atg8a and GFP-Lamp1 to visualize the movement and fusion events of pre-fusion autophagosomes (3xmCherry-Atg8a positive and GFP-Lamp1 negative) and lysosomes (GFP-Lamp1 positive). We expect these vesicles to exhibit movement toward the ncMTOC, providing insight into their behaviour under more typical conditions.

Specific comments

(1) Several genes have been described that when depleted lead to perinuclear accumulation of Atg8-labeled vesicles. There seems to be a correlation of this phenotype with genes required for autophagosome-lysosome fusion; however, some genes required for lysosomal fusion such as Rab2 and Arl8 apparently did not affect autophagosome positioning as reported here. Thus, it is unclear whether the perinuclear positioning of autophagosomes is truly a general response to disruption of autophagosome-lysosome fusion, or may reflect additional aspects of Vps16A/HOPS function. A few things here would help. One would be an analysis of Atg8a vesicle localization in response to the depletion of a larger set of fusion-related genes. Another would be to repeat some of the key findings of this study (effects of specific dynein, dynactin, rabs, effectors) on Atg8a localization when Syx17 is depleted, rather than Vps16A. This should generate a more autophagosome-specific fusion defect.

Thank you for this suggestion. We will generate additional Drosophila lines similar to those used in our current study, substituting Syntaxin17, SNAP29, and Vamp7 RNAi for Vps16A RNAi. We will test key phenotypic hits with these new backgrounds to confirm our findings.

Third, it would greatly strengthen the findings to monitor pre-fusion autophagosome localization without disrupting fusion. Such vesicles could be identified as Atg8a-positive Lamp-negative structures. The effects of dynein and rab depletion on the tracking of these structures in a post-induction time course would serve as an important validation of the authors' findings.

Thank you for this helpful suggestion. We plan to conduct time-lapse experiments under various conditions (e.g., non-starved and starved at different durations) to monitor the motility of newly formed autophagosomes (3xmCherry-Atg8a positive, Lamp1 negative), allowing us to analyze their positioning dynamics without interference from fusion defects.

(2) The authors nicely show that depletion of Shot leads to relocalization of Atg8a to ectopic foci in Vps16A-depleted cells; they should confirm that this is a mislocalized ncMTOC by co-labeling Atg8a with an MTOC component such as MSP300. The effect of Shot depletion on Atg8a localization should also be analyzed in the absence of Vps16A depletion.

Thank you for this positive comment, to confirm the presence of ectopic MTOC foci in Shot KD cells, we plan to co-label with MTOC markers, including Khc-nod-LacZ, and additional reporters like Msps-mCherry, in both Vps16A-depleted and normal backgrounds.

(3) The authors report that depletion of Dynein subunits, either alone (Figure 6) or co-depleted with Vps16A (Figure 2), leads to redistribution of mCherry-Atg8a punctae to the "cell periphery". However, only cell clones that contact an edge of the fat body tissue are shown in these figures. Furthermore, in these cells, mCherry-Atg8a punctae appear to localize only to contact-free regions of these cells, and not to internal regions of clones that share a border with adjacent cells. Thus, these vesicles would seem to be redistributed to the periphery of the fat body itself, not to the periphery of individual cells. Microtubules emanating from the perinuclear ncMTOC have been described as having a radial organization, and thus it is unclear that this redistribution of mCherry-Atg8a punctae to the fat body edge would reflect a kinesin-dependent process as suggested by the authors.

Thank you for this detailed observation. Indeed, we frequently observe autophagosomes redistributing to contact-free peripheral regions upon dynein depletion, resulting in an asymmetric distribution. We believe this redistribution to be kinesin-dependent, as shown in Figure 3: kinesin overexpression scatters or shifts autophagosomes to the periphery, while kinesin/dynein double knockdown causes widespread autophagosome scattering. The simplest explanation is that, in dynein's absence, kinesins drive autophagosome movement.

Additionally, while the radial organization of the microtubule (MT) network has been documented in two independent studies that we referenced, neither study showed MT plus-ends specifically, towards which kinesins transport. It is plausible that, while the MT network appears radial and symmetrical, subtle asymmetry might influence kinesin-dependent transport in fat cells. To explore this further, we will express MT plus-end markers, such as EB1-RFP and EB1-GFP, as well as kinesin reporters like unc-104-GFP or HA-tagged kinesins.

(4) To validate whether the mCherry-Atg8a structures in Vps16A-depleted cells were of autophagic origin, the authors depleted Atg8a and observed a loss of mCherry- Atg8a signal from the mosaic cells (Figure S1D, J). A more rigorous experiment would be to deplete other Atg genes (not Atg8a) and examine whether these structures persist.

Thank you for the suggestion to further validate our reporter. We will knock down additional Atg genes, including Atg14, Atg1, Atg6, and Vps34, to confirm that the mCherry-Atg8a-positive structures in the Vps16A RNAi background are indeed of autophagic origin.

(5) The authors found that only a subset of dynein, dynactin, rab, and rab effector depletions affected mCherry- Atg8a localization, leading to their suggestion that the most important factors involved in autophagosome motility have been identified here. However, this conclusion has the caveat that depletion efficiency was not examined in this study, and thus any conclusions about negative results should be more conservative.

Thank you for this constructive feedback. We agree and will adjust our conclusions based on the negative results in the revised manuscript to account for the potential variability in depletion efficiency.

Reviewer #3 (Public review):

Summary:

In multicellular organisms, autophagosomes are formed throughout the cytosol, while late endosomes/lysosomes are relatively confined in the perinuclear region. It is known that autophagosomes gain access to the lysosome-enriched region by microtubule-based trafficking. The mechanism by which autophagosomes move along microtubules remains incompletely understood. In this manuscript, Péter Lőrincz and colleagues investigated the mechanism driving the movement of nascent autophagosomes along the microtubule towards the non-centrosomal microtubule organizing center (ncMTOC) using the fly fat body as a model system. The authors took an approach whereby they examined autophagosome positioning in cells where autophagosome-lysosome fusion was inhibited by knocking down the HOPS subunit Vps16A. Despite being generated at random positions in the cytosol, autophagosomes accumulate around the nucleus when Vps16A is depleted. They then performed an RNA interference screen to identify the factors involved in autophagosome positioning. They found that the dynein-dynactin complex is required for the trafficking of autophagosomes toward ncMTOC. Dynein loss leads to the peripheral relocation of autophagosomes. They further revealed that a pair of small GTPases and their effectors, Rab7-Epg5 and Rab39-ema, are required for bidirectional autophagosome transport. Knockdown of these factors in Vps16a RNAi cells causes the scattering of autophagosomes throughout the cytosol.

Strengths:

The data presented in this study help us to understand the mechanism underlying the trafficking and positioning of autophagosomes.

Thank you for your positive comment and for acknowledging the strengths of our work.

Major concerns:

(1) The localization of EPG5 should be determined. The authors showed that EPG5 colocalizes with endogenous Rab7. Rab7 labels late endosomes and lysosomes. Previous studies in mammalian cells have shown that EPG5 is targeted to late endosomes/lysosomes by interacting with Rab7. EPG5 promotes the fusion of autophagosomes with late endosomes/lysosomes by directly recognizing LC3 on autophagosomes and also by facilitating the assembly of the SNARE complex for fusion. In Figure 5I, the EPG5/Rab7-colocalized vesicles are large and they are likely to be lysosomes/autolysosomes.

Thank you for suggesting an improvement to our Epg5 localization data. We plan to perform triple-staining experiments with autophagy and lysosome markers, such as Atg8a and Lamp1, together with Epg5-9xHA to provide a clearer context for Epg5 localization.

(2) The experiments were performed in Vps16A RNAi KD cells. Vps16A knockdown blocks fusion of vesicles derived from the endolysosomal compartments such as fusion between lysosomes. The pleiotropic effect of Vps16A RNAi may complicate the interpretation. The authors need to verify their findings in Stx17 KO cells, as it has a relatively specific effect on the fusion of autophagosomes with late endosomes/lysosomes.

Thank you for this valuable suggestion. We will create similar Drosophila lines as used in our study but will now employ Syntaxin17, SNAP29, or Vamp7 RNAi. We will cross our most significant hits with these new lines to confirm our findings.

(3) Quantification should be performed in many places such as in Figure S4D for the number of FYVE-GFP labeled endosomes and in Figures S4H and S4I for the number and size of lysosomes.

Thank you for pointing this out, we will perform the suggested quantifications and statistics.

(4) In this study, the transport of autophagosomes is investigated in fly fat cells. In fat cells, a large number of large lipid droplets accumulate and the endomembrane systems are distinct from that in other cell types. The knowledge gained from this study may not apply to other cell types. This needs to be discussed.

Thank you for this insight. We will discuss the potential cell-type specificity of our findings in the revised manuscript. Additionally, we plan to examine the distribution of the mCherry-Atg8a reporter in the vps16A RNAi background in other cell types, such as salivary gland cells, to broaden our analysis.

Minor concerns:

(5) Data in some panels are of low quality. For example, the mCherry-Atg8a signal in Figure 5C is hard to see; the input bands of Dhc64c in Figure 5L are smeared.

Thank you for noting this. We will repeat the experiment in Figure 5C to obtain clearer images. The smeared Dhc64C input bands in Figure 5L are due to the large size of this protein, which affects its migration characteristics. We will address this in the revised manuscript.

(6) In this study, both 3xmCherry-Atg8a and mCherry-Atg8a were used. Different reporters make it difficult to compare the results presented in different figures.

Thank you for this comment. Both reporters are well-established as autophagic markers and function similarly. However, to reduce confusion, we have used only one type per figure to ensure comparability of results.

(7) The small autophagosomes presented in Figures such as in Figure 1D and 1E are not clear. Enlarged images should be presented.

Thank you for your suggestion. We will repeat these experiments and provide higher-quality, enlarged images for clarity.

(8) The authors showed that Epg5-9xHA coprecipitates with the endogenous dynein motor Dhc64C. Is Rab7 required for the interaction?

Thank you for this question. We will investigate this by co-transfecting the cells with WT and GTP- or GDP-locked Rab7 mutants (which mimic constitutively active and dominant-negative forms, respectively) with Epg5-9xHA. This will allow us to assess whether Rab7 modulates the Epg5-Dhc interaction.

(9) The perinuclear lysosome localization in Epg5 KD cells has no indication that Epg5 is an autophagosome-specific adaptor.

Thank you for this comment. We will moderate our statement regarding Epg5's role as an autophagosome-specific adaptor in the revised manuscript.