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  1. Author Response

    Reviewer #1 (Public Review):

    The authors have previously developed a powerful time-lapse recording protocol that allows them to observe in real time the formation and degradation of phagosomes in specific phagocytic cells (C1-C3) in the developing C. elegans embryo. Using this protocol in combination with specific reporters, they find that LC3-positive vesicles fuse with phagosomes and that these vesicles are doublemembrane vesicles. Taking advantage of different genetic requirements for the formation of LAPs and autophagosomes, they furthermore provide evidence that these LC3-positive vesicles are autophagosomes. Having established that autophagosomes fuse with phagosomes, the authors demonstrate that preventing this fusion genetically (by blocking autophagosome biogenesis) results in a general engulfment defect and that this is due to a defect in the degradation of phagosomal content. The authors identify RAB-7 and the HOPS complex as necessary for autophagosome-phagosome fusion and the CED-1, CED-6, DYN-1 pathway as necessary for recruiting autophagosomes to phagosomes. Finally, the authors find that preventing autophagosome-phagosome fusion does not affect lysosomephagosome fusion thereby ruling out that the effects observed are indirect and a consequence of defects in lysosome-phagosome fusion rather than autophagosomephagosome fusion.

    This is a very rigorous and very convincing study that will have a big impact on our understanding of cell corpse engulfment and degradation. It also uncovers a novel function of autophagosomes (i.e. in cell corpse degradation). Its strength lies in the combination of time-lapse observation of specific organelles in specific cells in vivo and the use of mutations in genes that affect specific cell biological processes. There are no major weaknesses but it would have been nice to have evidence for autophagosome-phagosome fusion for example through EM images.

    We appreciate your praise of our work. We have performed the EM analysis and observed the attachment of double-membrane vesicles to the surfaces of phagosomes.

    Reviewer #2 (Public Review):

    This is an interesting manuscript from Zhou and colleagues which make several important steps forward in understanding how cell corpses are digested by phagocytes. First, they show that autophagosomes associate with nascent/growing phagosomes. Interesting, they find that many atg genes required to make autophagosomes are essential for efficient corpse clearance. These include homologs of atg13 and atg14, which in mammals are not required for the production of LAPs. The argument from the authors is that these non-LAP vesicles are double-membrane and fuse with phagosomes to contribute contents important for efficient corpse clearance. If correct, this would be a new way in which autophagosomes contribute to phagocytosis. This is a reasonable interpretation, but not the only interpretation of the data.

    Novel points include the recruitment of autophagosomes (non-LAPs presumably based on genetics) to phagosomes and the requirement for many atg genes in corpse elimination. That Ced-1 and components of this pathway drive recruitment of these vesicles is interesting. The genetics are very strong, convincing, and well done. It seems clear that these many ATG genes are playing a role in efficient disposal of corpses in phagosomes, but I do think it remains unclear how. It is not clear to me that a double-membrane autophagosome actually fuses with the phagosome. Possibly they are fusing to aid in degradation, but another possibility is that they are interacting in a way that contributes lipids for phagosome growth/expansion. What if this is a mechanism that allows phagosomes to grow their lipid membranes, rather than fusion to digest what's in the autophagosome? Atg2/9 drive the transfer of lipids from the ER to autophagosomes, that's how autophagosomes grow. It is possible that autophagosomes are intermediates that serve as lipid sources for phagosomes. Is there an autophagosome target (inside) that one could track to show actual degradation? It would be useful to discriminate these possibilities.

    We agree with Reviewer 2 that there are multiple possible mechanisms to support the functions of autophagosomes in facilitating phagosome maturation. The “phagosome growth/expansion” hypothesis is conceivable. One particular function of the increased phagosome membrane amount might be to support the extension of the transient phagosomal tubules that aids in the recruitment of lysosomal particles to phagosomes. We added it to the revised “Discussion”.

    Regarding whether there is an autophagosome target (inside) that one could track to show actual degradation, many cellular components are known to become cargos for autophagosomes. These include protein aggregates, RNA-protein (RNP) complexes, intracellular organelles, ribosomes, and lipid droplets. However, the cargos of individual autophagosomes differ a lot depending on whether the autophagy to be studied is triggered by stress, what kind of stress, and the condition of the cells. Therefore, it is assumed that the cargos of autophagosomes are not uniform. It is thus hard to predict whether and what substance(s) provided by the cargo would contribute to phagosome degradation once autophagosomes fuse to phagosomes. In C. elegans embryos, P granules are degraded inside autophagosomes. We considered tracking P granule components PGL-1 and/or PGL-3 as cargos of autophagosomes [6]. However, P granules are produced in the P4 lineage, not in the embryonic hypodermal cells that act as engulfing cells [6], and thus are not suitable candidate cargos. One candidate protein closely related to autophagosomal cargos is p62, a conserved cargo receptor for autophagosomes, which is encoded by the sqst-1 gene in C. elegans [6]. In the future investigation, we will track p62-labeled puncta and the relationship of these puncta with phagosomes.

    Based on the knowledge that in mammalian cells, among genes required for the biogenesis of autophagosomes, atg13, atg14, and ulk1 are not needed for the generation of the LAP vesicles, we predict that if the LGG+ puncta we are following in C. elegans embryos are LAP vesicles, the atg-13 or epg-8 (atg14) mutations are not supposed to affect the biogenesis of the LGG+ puncta. We observed that in atg-13 and epg-8 mutants, the existence and incorporation of the LGG+ puncta are severely diminished, indicating that these puncta are not LAP vesicles. This line of evidence, together with other evidence, allow us to propose that the LGG+ vesicles that fuse to phagosomes are canonical autophagosomes, not LAP vesicle.

    Reviewer #3 (Public Review):

    The manuscript by Peña-Ramos et al. describes a new role of autophagosomes in the maturation of phagosomes containing apoptotic corpses. The authors find that vesicles containing LGG-1 and or LGG-2 bind to and fuse with phagosomes and provide evidence that these structures are, ostensibly, double-membraned autophagosomes. They then proceeded to assess the role of such fusion events in the rate and extent of phagosome maturation, using mutants lacking components required for autophagosome formation. In addition, they provide evidence that fusion involves Rab-7 and the HOPS complex and is influenced by the presence of the phagocytic receptor CED-1. Lastly, they document that lysosome fusion with the phagosomes persists in the absence of autophagosomes.

    The findings are novel, generally clear and convincing. On the other hand, some of the interpretations are not unambiguous and, importantly, the ultimate mechanism underlying the defective maturation is not resolved or even addressed.

    By the authors' own admission, at least a fraction of the autophagosomes have acquired Rab-7 and likely fused with lysosomes. In that event, the fusing structures are autolysosomes and not necessarily double-membraned autophagosomes, as claimed. Delivery into the phagosome of the contents of the digested inner bilayer of the original autophagosome is likely to have occurred. If so, appearance of labeled contents inside the phagosome is being misinterpreted (or at least overinterpreted) to mean that the fusing structures are double-membraned, bona fide autophagosomes. The resolution of the images seems insufficient to distinguish these two possibilities. If autolysosomes are in fact the organelles fusing, how is this different from fusion of regular lysosomes? The conceptual novelty of the paper would be greatly diminished if what is being reported is homotypic fusion of two maturing (auto)phagolysosomes or fusion of a lysosomal organelle with phagosomes. Are the LGG-1/2-positive structures acidic and do they contain NUC-1?

    We appreciate Reviewer 3’s comments and questions pointing out the important question of whether it is autophagosomes or autolysosomes that fuse to phagosomes. To address the series of questions raised by Reviewer 3, we first need to clarify that the LGG+ RAB-7+ positive puncta are not necessarily autolysosomes. Recent studies have shown that autophagosomes directly acquire the Rab7 small GTPase to their surfaces. An autophagosome that is labeled with Rab7 should not be assumed to be an autophagolysosome (autolysosome). Below are the reports and our own evidence that demonstrate this point:

    Rab7 is independently recruited to the surfaces of each of the following organelles from the cytoplasm: late endosomes, lysosomes, phagosomes, and autophagosomes. Specific to autophagosomes, Gao et al (2018) [12] have shown that yeast Atg8 (LC3) recruits Rab7/Ypt7 to pre-autophagosomal structure through directly binding to Mon1-Ccz complex, the Guanine nucleotide exchange factor (GEF) for Rab7. Furthermore, the recruitment of Rab7 to the autophagosomes is necessary for the autophagosome-lysosome fusion in yeast [12]. In starved Drosophila fat cells, the PtdIns(3)P and Mon1-Ccz complex on the surfaces of autophagosomes act together to recruit Rab7 to autophagosomes [13]. Again, this recruitment step is essential for the subsequent autophagosome-lysosome fusion [13]. A conserved LC3-Mon1-Ccz1mediated recruitment of Rab7 to autophagosomes has also been reported for mammalian cells [14].

    Like in other organisms mentioned above, C. elegans RAB-7 also plays an essential role in the fusion between autophagosomes and lysosomes [1]. During the revision period, our quantitative analysis shows that in engulfing cells for C1, C2, and C3, on average 66.2% and 63.5% of LGG-1+ and LGG-2+ puncta are RAB-7+ (Fig 8 E). Remarkably, 100% of the LGG+ puncta observed on the surfaces of phagosomes prior to the fusion event are RAB-7+ (Fig 8 E), suggesting that RAB-7 plays a role in the incorporation of LGG+ puncta to phagosomes. This suggestion was confirmed by the observation that the deletion of rab-7 results in the blockage of the fusion of LGG+ puncta to phagosomes (Fig 8 G-J).

    Since RAB-7 is not a suitable lysosomal marker, to identify autolysosomes in the population of LGG+ puncta, as Reviewer 3 requested, we followed the subcellular localization of NUC-1, a lysosomal luminal DNase. In engulfing cells, on average 40.7% of the LGG-1+ and 36.5% of the LGG-2+ puncta observed on phagosomal surfaces and subsequently fuse to phagosomes are NUC-1+ (Fig 12 C), indicating that those puncta are likely autolysosomes. Considering that 100% of the LGG+ puncta found on phagosomal surfaces are RAB-7+, these results clearly indicate that an LGG+ RAB-7+ particle is not necessarily an autolysosome. Also, 59.7% and 63.5% of the LGG-1+ or LGG-2+ puncta that fuse to phagosomes, respectively, are NUC-1- and presumed to be autophagosomes that have not fused to lysosomes. The non-autolysosomal LGG+ puncta thus are likely to fuse with phagosomal membranes as double-membrane vesicles. Furthermore, we have observed double-membrane vesicles in close contact with phagosomal surfaces using the EM analysis (Fig 4).

    Regarding the acidification status of the LGG+ structure in C. elegans embryos, Manil-Segalen et al [1] used a GFP::mCherry::LGG-1 reporter to detect the acidification of LGG-1-labeled autophogosomal structures (the color turning from yellow (green + red) to red (quenching of the GFP signal in acidic pH)). They found that in 1-cell stage embryos, all LGG-1+ puncta were non-acidic, whereas later into embryonic development, some LGG-1+ puncta that moved away from the allophagic cluster became acidic. Overall, both non-acidic and acidic LGG-1+ puncta exist in the cytoplasm.

    In the original version of “Results”, we did not do a good job to emphasize that recruiting Rab7 to autophagosomes from the cytoplasm does not necessarily involve lysosomes; rather, it is a prerequisite for the fusion between autophagosomes and lysosomes. Thanks to Reviewer 3 for requesting us to quantify the percentage of LGG+ puncta that are RAB-7+ or NUC-1+, a lysosomal marker, the results of which help clarify the issue. We revised the section “The small GTPase RAB-7 is enriched on the surfaces of autophagosomes” in “Results” and added it to Fig 12C to report our quantitation results.

    In summary, as Reviewer 3 pointed out, if all the LGG+ puncta are autolysosomes, the novelty of our finding that LGG+ puncta fuse to phagosomes would be greatly diminished. However, our experimental results demonstrate that this is not the case. More than 59% of the fusion events between LGG+ puncta and phagosomes are actually fusion events occurring between the double-membrane, NUC-1- autophagosomes and phagosomes. This conclusion is further supported by the observation of double-membrane autophagosome-like structures on the surfaces of phagosomes inside gonadal sheath cells in rab-7 mutant worms (Fig 4). Another important and relevant finding is that in rab-7 null mutant embryos, numerous LGG+ puncta were observed on phagosomal surfaces (Fig 8 G-H). It was reported that the inactivation of rab-7 in C. elegans embryos blocks the formation of autolysosomes [1]. Therefore, our result indicates that in rab-7 mutants, non-autolysosomal LGG+ organelles, most likely autophagosomes, are indeed recruited to phagosomes. Together, these lines of evidence indicate that in wild-type embryos, both autophagosomes and autolysosomes fuse to phagosomes, and the amount of the LGG+ NUC-1- autophagosomes is more than 59% of the LGG+ population. Given that the membrane and lumen of autophagosomes have different components from that of lysosomes, we propose that whereas the autolysosomes could contribute lysosomal contents to phagosomes, autophagosomes and autolysosomes also deliver the substance(s) that are unique to the autophagosomal origin to phagosomes. This is one of the important novel points of this report. We further speculated a few possible substances delivered to phagosomes from autophagosomes in the revised Discussion. Please also see our response to Reviewer #2’s “Recommendations for the authors, point 6”.

    The conclusion that lysosomal fusion with phagosomes is normal is based on the quantitation of NUC-1 fluorescence acquired by the phagosomes. However, the quantitation was made relative to the fluorescence of phagosomes at time 0, when no NUC-1 is expected to be present. The validity of these measurements and comparisons between wildtypes and mutants is therefore questionable.

    The preceding comment is critical because it is generally believed that degradation of phagosomal contents is solely dependent on fusion of lysosomes that deliver degradative enzymes and make the phagosomal lumen suitably acidic for optimal function of the hydrolases. If these parameters are normal, as argued by the authors, what is preventing normal degradation from occurring? The authors invoke a mysterious molecule(s) delivered by the autophagosome as an essential component for optimal degradation. While provocative, this notion seems unfounded unless it is shown that the delivery of hydrolases and the luminal pH of the phagosome are normal, yet degradation of proteins, lipids and nucleic acids and/or their resorption is affected by the absence of the mysterious molecule(s). It is unclear whether the authors propose that the contents of autophagosomes are somehow required for hydrolase activity.

    By measuring the fold of increase of the NUC-1::mCherry signal inside the lumen of phagosomes at 60 min post the completion of engulfment over the 0 min time point (Fig 11F), we have obtained the rate of accumulation of NUC-1 inside the phagosomal lumen, not an absolute value that could represent the amount of NUC-1::mCherry molecules per unit area inside the wild-type, atg-7, and lgg mutant phagosomes. We agree that this assay displays the caveat pointed out by Reviewer 3. However, given the possible variation of the expression level of NUC-1::mCherry from embryo to embryo, our concern is that the absolute photon numbers per unit area, if not normalized, are not informative values to be compared between strains. That is why we normalized the signal intensity at Tn (n: minutes post engulfment) with that of T0min. We argue that, considering all the results reported in Fig 11 together, which include the time-lapse images (A-D), the quantification of the increase of mCherry signal inside phagosomal lumen over time (E), the fold-of-increase of the mCherry signal at T60min over T0min of multiple samples (F), and the time points when the fusion between lysosomes and phagosomes starts (G) in multiple samples of four different strains, we conclude that mutations in atg-7, lgg-1, or lgg-2 do not significantly alter the temporal pattern or the scale of the lysosome-phagosome fusion, although these results are not sufficient to prove that the absolute amounts of NUC-1::mCherry inside the lumen of phagosomes are the same in the mutant and the wild-type embryos.

    Given that autolysosomes are part of the NUC+ vesicle population that display lysosome features, why, then, in atg-7, lgg-1, and lgg-2 mutants, which suffer severe defect in the biogenesis of autophagosomes and hence the autolysosomes, the accumulation of NUC-1::mCherry signal inside the phagosomal lumen appears not to be significantly affected? This result could be due to that (1) there is a large pool of lysosomes in the cell that provides sufficient amount of lysosomes to fuse to phagosomes despite the lacking of autolysosomes, or (2) the autolysosome population is a very small portion comparing to the lysosomal pool, and our assay is not capable to detect small reduction of the lysosomal flow into phagosomes. More fundamentally, we suspect that in a cell that bears a stable lysosome pool, whether autophagosomes exist or not might not affect the overall lysosomal activity. In a cell in which autophagosomes are lacking, the same amount of NUC-1+ vesicles are still expected to fuse to phagosomes, albeit now within the NUC-1+ vesicle population, the portion of autolysosomes is greatly diminished. Take this assumption into consideration, the observation that the lack of autophagosome-phagosome fusion causes phagosome degradation defect appears to suggest that autophagosomes might provide unique materials/activities to phagosomes.

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  2. Evaluation Summary:

    This study presents evidence that autophagosomes fuse with phagosomes and that this promotes the degradation of phagocytosed cell corpses. The study also resolves controversy in the field about the question why genes involved in autophagy affect cell corpse engulfment and degradation. With some additional data to solidify the main conclusions, the work will be of interest to a broad cell biology audience.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors have previously developed a powerful time-lapse recording protocol that allows them to observe in real time the formation and degradation of phagosomes in specific phagocytic cells (C1-C3) in the developing C. elegans embryo. Using this protocol in combination with specific reporters, they find that LC3-positive vesicles fuse with phagosomes and that these vesicles are double-membrane vesicles. Taking advantage of different genetic requirements for the formation of LAPs and autophagosomes, they furthermore provide evidence that these LC3-positive vesicles are autophagosomes. Having established that autophagosomes fuse with phagosomes, the authors demonstrate that preventing this fusion genetically (by blocking autophagosome biogenesis) results in a general engulfment defect and that this is due to a defect in the degradation of phagosomal content. The authors identify RAB-7 and the HOPS complex as necessary for autophagosome-phagosome fusion and the CED-1, CED-6, DYN-1 pathway as necessary for recruiting autophagosomes to phagosomes. Finally, the authors find that preventing autophagosome-phagosome fusion does not affect lysosome-phagosome fusion thereby ruling out that the effects observed are indirect and a consequence of defects in lysosome-phagosome fusion rather than autophagosome-phagosome fusion.

    This is a very rigorous and very convincing study that will have a big impact on our understanding of cell corpse engulfment and degradation. It also uncovers a novel function of autophagosomes (i.e. in cell corpse degradation). Its strength lies in the combination of time-lapse observation of specific organelles in specific cells in vivo and the use of mutations in genes that affect specific cell biological processes. There are no major weaknesses but it would have been nice to have evidence for autophagosome-phagosome fusion for example through EM images.

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  4. Reviewer #2 (Public Review):

    This is an interesting manuscript from Zhou and colleagues which make several important steps forward in understanding how cell corpses are digested by phagocytes. First, they show that autophagosomes associate with nascent/growing phagosomes. Interesting, they find that many atg genes required to make autophagosomes are essential for efficient corpse clearance. These include homologs of atg13 and atg14, which in mammals are not required for the production of LAPs. The argument from the authors is that these non-LAP vesicles are double-membrane and fuse with phagosomes to contribute contents important for efficient corpse clearance. If correct, this would be a new way in which autophagosomes contribute to phagocytosis. This is a reasonable interpretation, but not the only interpretation of the data.

    Novel points include the recruitment of autophagosomes (non-LAPs presumably based on genetics) to phagosomes and the requirement for many atg genes in corpse elimination. That Ced-1 and components of this pathway drive recruitment of these vesicles is interesting. The genetics are very strong, convincing, and well done. It seems clear that these many ATG genes are playing a role in efficient disposal of corpses in phagosomes, but I do think it remains unclear how. It is not clear to me that a double-membrane autophagosome actually fuses with the phagosome. Possibly they are fusing to aid in degradation, but another possibility is that they are interacting in a way that contributes lipids for phagosome growth/expansion. What if this is a mechanism that allows phagosomes to grow their lipid membranes, rather than fusion to digest what's in the autophagosome? Atg2/9 drive the transfer of lipids from the ER to autophagosomes, that's how autophagosomes grow. It is possible that autophagosomes are intermediates that serve as lipid sources for phagosomes. Is there an autophagosome target (inside) that one could track to show actual degradation? It would be useful to discriminate these possibilities.

    Read the original source
    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The manuscript by Peña-Ramos et al. describes a new role of autophagosomes in the maturation of phagosomes containing apoptotic corpses. The authors find that vesicles containing LGG-1 and or LGG-2 bind to and fuse with phagosomes and provide evidence that these structures are, ostensibly, double-membraned autophagosomes. They then proceeded to assess the role of such fusion events in the rate and extent of phagosome maturation, using mutants lacking components required for autophagosome formation. In addition, they provide evidence that fusion involves Rab-7 and the HOPS complex and is influenced by the presence of the phagocytic receptor CED-1. Lastly, they document that lysosome fusion with the phagosomes persists in the absence of autophagosomes.

    The findings are novel, generally clear and convincing. On the other hand, some of the interpretations are not unambiguous and, importantly, the ultimate mechanism underlying the defective maturation is not resolved or even addressed.

    -By the authors' own admission, at least a fraction of the autophagosomes have acquired Rab-7 and likely fused with lysosomes. In that event, the fusing structures are autolysosomes and not necessarily double-membraned autophagosomes, as claimed. Delivery into the phagosome of the contents of the digested inner bilayer of the original autophagosome is likely to have occurred. If so, appearance of labeled contents inside the phagosome is being misinterpreted (or at least over-interpreted) to mean that the fusing structures are double-membraned, bona fide autophagosomes. The resolution of the images seems insufficient to distinguish these two possibilities. If autolysosomes are in fact the organelles fusing, how is this different from fusion of regular lysosomes? The conceptual novelty of the paper would be greatly diminished if what is being reported is homotypic fusion of two maturing (auto)phagolysosomes or fusion of a lysosomal organelle with phagosomes. Are the LGG-1/2-positive structures acidic and do they contain NUC-1?

    -The conclusion that lysosomal fusion with phagosomes is normal is based on the quantitation of NUC-1 fluorescence acquired by the phagosomes. However, the quantitation was made relative to the fluorescence of phagosomes at time 0, when no NUC-1 is expected to be present. The validity of these measurements and comparisons between wildtypes and mutants is therefore questionable.

    -The preceding comment is critical because it is generally believed that degradation of phagosomal contents is solely dependent on fusion of lysosomes that deliver degradative enzymes and make the phagosomal lumen suitably acidic for optimal function of the hydrolases. If these parameters are normal, as argued by the authors, what is preventing normal degradation from occurring? The authors invoke a mysterious molecule(s) delivered by the autophagosome as an essential component for optimal degradation. While provocative, this notion seems unfounded unless it is shown that the delivery of hydrolases and the luminal pH of the phagosome are normal, yet degradation of proteins, lipids and nucleic acids and/or their resorption is affected by the absence of the mysterious molecule(s). It is unclear whether the authors propose that the contents of autophagosomes are somehow required for hydrolase activity.

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