TORC1, BORC, ARL-8 cycling, and kinesin-1 drive vesiculation of cell corpse phagolysosomes

  1. Gholamreza Fazeli
  2. Roni Levin-Konigsberg
  3. Michael C Bassik
  4. Christian Stigloher
  5. Ann M Wehman

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Abstract

The dynamics of phagolysosomes leading to cargo clearance are important to provide cells with metabolites and avoid auto-immune responses, but little is known about how phagolysosomes finally resolve cell corpses and cell debris. We previously discovered that polar body phagolysosomes tubulate into small vesicles to facilitate corpse clearance within 1.5 hours in C. elegans . Here, we show that vesiculation depends on activating TORC1 through amino acid release by the solute transporter SLC-36.1. Downstream of TORC1, BLOC-1-related complex (BORC) recruits the Arf-like GTPase ARL-8 to the phagolysosome for tubulation by kinesin-1. We find that disrupting the regulated GTP-GDP cycle of ARL-8 reduces tubulation, delays corpse clearance, and mislocalizes ARL-8 away from lysosomes. We also demonstrate that mammalian phagocytes use BORC to promote phagolysosomal degradation, confirming the conserved importance of this pathway. Finally, we show that HOPS is required for rapid degradation of the small phagolysosomal vesicles. Thus, by observing single phagolysosomes over time, we identified the molecular pathway regulating phagolysosome vesiculation that promotes efficient resolution.

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  1. Version published to 10.1101/2022.02.09.479694v2 on bioRxiv
  5. Review Commons

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    Reply to the reviewers

    We would like to thank the reviewers for their positive and constructive reviews. We have already addressed their major concerns by including additional screen and control data, especially in the new Figure 7, supplemental figures 3 and 4 and new Table S1. We also detail the planned experiments that we propose to perform to address their remaining comments. Some points are mentioned in both section 2 and 3 of the revision plan.

    2. Description of the planned revisions

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

    Reviewer #1

    • Fig 4 and 5. ARL-8 is localized to lysosomes, phagolysosomes and early endosomes. How about TORC and BORC subunits?*

    We plan to acquire images of our endogenously tagged BORC subunit SAM-4::mSc on the polar body phagolysosome. We also crossed SAM-4::mSc to a GFP::RAB-5 early endosomal marker and plan to cross it to a CTNS-1::mCit lysosomal marker and will characterize their colocalization.

    To look for the localization of TORC1, we requested a recently published strain with a single-copy transgene encoding a fluorescently tagged DAF-15/RAPTOR (AK Sewell et al., iScience 2022), but there is no fluorescence visible in live embryos. We are currently staining these strains to test for any embryonic expression, but it is not uncommon for transgenes to undergo germline silencing, even single-copy transgenes. To observe TORC1 localization in early embryos, it may be necessary to create a knock-in strain which would take a couple of months to generate and another month to cross to various reporters and analyze.

    We predict that TORC1 and BORC will localize to lysosomes and phagolysosomes, consistent with previous literature (Sancak *et al. *Cell 2010, Pu et al. Dev Cell 2015).

    • Figure 6 *

    *Authors need to establish knockout cell lines by picking up single drug-resistant colonies and characterize each line by western blot or immunofluorescent microscopy. *

    We have been successful in creating stable mutant cell lines for Myrlysin and Lyspersin using CRISPR/Cas9 and are currently characterizing these lines before performing direct assays for phagolysosomal vesiculation.

    b) While the authors monitored cell growth every 3 hours for 4 days, the authors showed the result of day 4 only. Time lapse data would be useful.

    We plan to replace the RBC-overfeeding experiment with more direct evidence for phagolysosomal vesiculation (see 3c below).

    *c) The effect of KO may be because of defects in phagolysosomes. However, the authors cannot conclude "phagolysosomal vesiculation is affected in mammalian cells" or not until they directly observe the phenomena. *

    We plan to feed our newly isolated Myrlysin and Lyspersin mutant cell lines with RBCs and use a stage-based analysis at defined time points to test how the size and shape of the phagolysosomes change over time, similar to Fig. 1a-c in R Levin-Konigsberg et al. Nature Cell Biol 2019. This experiment will assess the effect of these genes directly on phagolysosomal vesiculation rather than general phagolysosome function.

    Reviewer #1 (Significance):

    This study shows the mechanism [involvement of TORC-BORC-ARL8 pathway] is conserved in phagolysosomes as well in worms. As described above, the involvement of these molecules in the mammalian phagolysosomes is not convincing at this stage.

    We plan to provide direct evidence for the involvement of BORC by using stable mutant cell lines and directly monitoring phagolysosome vesiculation, as described above.

    **Referees cross-commenting**

    *I fully agree with the reviewer #2 that identification of arl8 effectors will make this paper more attractive as I described in my original peer review. Moreover, I agree with the reviewer that genetic data in C. elegans is convincing. *

    As RNAi targeting UNC-116 disrupts embryonic development, including the birth of the polar body during meiosis, we generated a ZF1 degron-tagged UNC-116 allele to test the role of Kinesin-1 in phagolysosomal vesiculation. We are currently characterizing this strain and crossing it to polar body markers. We predict that degron-mediated degradation of UNC-116 will start during the 4-cell stage, which is after polar body birth but well before the onset of phagolysosome vesiculation. This new unc-116::mCitrine::ZF1 will provide us with a tool to more specifically test the role of UNC-116 in phagolysosome vesiculation in the context of a developing embryo.

    While reviewer #2 have not concerned about the quality of experiments, I'm still not convinced by their mammalian cell experiments. I don't have any more concerns if the authors (1) remove the mammalian study or (2) improve the quality of mammalian data. *

    We hope that our planned experiments with the isolated mutant cell lines will address the reviewer’s concern. Otherwise, we can remove the mammalian experiments.

    Reviewer #2

    **Referees cross-commenting**

    The mammalian work … assays general phagolysosome function rather than directly addressing vesiculation.

    We hope that our planned experiments with the isolated mutant cell lines will directly demonstrate a role in phagolysosomal vesiculation.

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

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

    Reviewer #1

      • It is very difficult to understand which molecules are TORC, BORC and/or BLOC subunits, which molecules are required and which are not required. ** It is very helpful if the authors include a table showing the summary of RNAi and mutant phenotypes.* We have added Table S1, summarizing our findings with different protein complexes and previous findings regarding lysosome and synaptic vesicle precursor trafficking.
    • Figure 6 ** a) There are many concerns in mammalian cell experiments. It is not clear whether the knockout procedures really work or not. … *

    We performed genotyping for the pooled lines and found high editing efficiency leading to frame-shifts in Arl8B (>96%) and BLOC1S1 (>85%), as well as Cathepsin B (>67%). These data are now included in supplementary figure S4.

    As the downstream of ARL8 remains elusive in this study, it is unclear how phagolysosomes are tubulated (Fig 6D model).

    ARL8 has been shown to interact with kinesins, dyneins, and the HOPS complex directly or through bridging molecules such as PLEKHM or RUFY proteins.

    Using the reference allele e1265 of the KIF1 ortholog UNC-104, which causes a severe paralysis phenotype, we observed no effect on phagolysosomal vesiculation (new Fig. S3A) or polar body degradation (new Fig. S3B).

    We next used the partial loss-of-function wy270 allele of the KIF5 ortholog UNC-116 but observed no effect on phagolysosomal vesiculation (new Fig. S3A). There was however a mild delay in polar body degradation (new Fig. S3B), leading us to develop a degron-tagged UNC-116 allele to be able to better analyze the role of UNC-116 in phagolysosomal biology (described in section 2).

    We have also included data using RNAi and mutant alleles to test a role for PLEKHM family proteins CUP-14 and RUB-1. We observed no effect on phagolysosomal vesiculation (new Fig. S3A) or polar body degradation (new Fig. S3B). In preliminary data (n=3), knocking down rub-1 in a cup-14 mutant background had no effect on phagolysosomal vesiculation (new Fig. S3A), but sped up polar body degradation (new Fig. S3B). These data are inconsistent with PLEKHM proteins having a role in phagolysosomal vesiculation.

    BLAST searches revealed no clear homologs for RUFY proteins in C. elegans.

    Based on reviewer #2’s suggestion, we also examined a role for the HOPS complex by knocking down the HOPS subunit VPS-41. Human VPS41 has been shown to bind ARL8b (Khatter et al JCS 2015). Treating worms with vps-41 RNAi resulted in normal phagolysosomal vesiculation (new Fig. 7A) but did result in a significant delay in polar body degradation (new Fig. 7B-C). Interestingly, disappearance of small phagolysosomal vesicles was significantly delayed (new Fig. 7D), while corpse membrane breakdown within the large phagolysosome was unaffected (new Fig. 7E). As corpse membrane breakdown depends on RAB-7-mediated fusion of lysosomes (Fazeli et al., Cell Rep 2018) and HOPS promotes lysosomal fusion (JA Nguyen & RM Yates, Front Immunol 2021), these data suggest that VPS-41 and HOPS are preferentially required for lysosome fusion to small phagolysosomal vesicles and are not necessary for lysosome fusion to the large phagolysosome.

    Thus, we screened through the known ARL-8 effectors and the identity of the downstream effector(s) of ARL-8 involved in phagolysosomal vesiculation remain elusive. As the kinesin and PLEKHM data were negative, we had opted not to include it in the original manuscript, but now discuss it in the main text and present it in Fig. 7 and Fig. S3.

    Reviewer #2

    It would be good if the authors could speculate further as to why mTORC1 is required for Arl8 activity but not recruitment, and if there are further experiments that could augment this conclusion they might be helpful.

    We added a new hypothesis to the discussion of how TORC1 might affect a downstream effector of ARL-8. Unfortunately, we have not yet been able to identify any ARL-8 effectors involved in phagolysosome vesiculation to be able to test this hypothesis experimentally.

    *The authors could consider making the study more extensive by applying their simple but elegant system to further possible players in the pathway, and in particular to test some possible Arl8 effectors. Although there is no clear orthologue of PLEKHM2 in C. elegans, both the HOPS complex and PLEKHM1 have been reported to bind to Arl8. *

    As potential SKIP/PLEKHM-related proteins that link ARL8 to kinesins, we screened two RUN and PH domain-containing proteins, CUP-14 and RUB-1, for their role in phagolysosome resolution. Phagolysosome vesiculation was normal and resolution was not delayed in cup-14(cd32) or rub-1(RNAi) mutants or after treating cup-14 mutants with rub-1 RNAi (Fig. S3A-B). These data are now discussed in the main text and included as a supplementary figure.

    To test a role for the HOPS complex, best known for its role in lysosome fusion, we tested whether RNAi knockdown of the HOPS-specific subunit VPS-41 disrupted or delayed phagolysosome vesiculation. Vps41 has been shown to bind ARL8b (Khatter et al. JCS 2015). HOPS knockdown did not affect phagolysosome vesiculation (new Fig. 7A), but significantly delayed polar body degradation (Fig. 7B-C). In particular, vps-41 knockdown delayed the degradation of phagolysosomal vesicles (Fig. 7D), probably by affecting fusion of these small vesicles to additional lysosomes. These data are now discussed in the main text and included as a supplementary figure.

    **Referees cross-commenting**

    *One approach might to drop the mammalian studies, and instead add an investigation of more Arl8 effectors in C. elegans. Hopefully, few people would doubt the relevance to mammals of studies in C. elegans on the function of well conserved proteins. *

    We hope to strengthen the relevance of our findings across species to increase the impact of our work. Therefore, we plan to replace the RBC-overfeeding experiment in Fig. 6 with direct evidence for phagolysosomal vesiculation using established cell culture assays similar to Fig. 1a-c in R Levin-Konigsberg et al. Nature Cell Biol 2019 and newly isolated BORC mutant cell lines.

    We added details on our screen for ARL-8 effectors to the manuscript, including new figures (Fig. S3 and Table S1). While the effectors involved in phagolysosomal vesiculation remain a mystery, we were able to distinguish different requirements for HOPS during corpse membrane breakdown and phagolysosomal vesicle resolution, suggesting that HOPS is critical for the lysosomal fusion of small phagolysosomal vesicles, but not the large cell corpse phagosome.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors have previously used C. elegans early development to establish an elegant model system with which to investigate process by which the content of phagolysosomes is degraded and the structure resolved. This phagocytosis plays a central role in the clearance of dead cells and pathogens and so the work is likely to be of widespread interest to those working in both C. elegans and mammalian phagocytic cells like macrophages. The authors have previously shown that the tubulation and fragmentation of the phagolysosome is important for both degradation of contents and resolution of the phagolysosome, and have identified the small GTPase Arl8 and the mTor kinase as being required. This study investigates the relationship between these components and also adds further players to the pathway. In summary, they show that there is a pathway starting with amino acid release and going through mTORC1 and then the BORC complex that is known to activate Arl8. They show that only some subunits of BORC are required (and not the related BLOC complex with which is shares some subunits), and that Arl8 needs to cycle between its GDP- and GTP-bound states to exert its effects. They also examine the membrane recruitment of Arl8 and make two interesting findings. Firstly, mTORC1 is required for Arl8 activity but not its localisation, possibly suggesting that mTORC1 is required for the activity of a critical Arl8 effector. Secondly, they find that when BORC is removed, Arl8 is recruited to endosomes, which implies the existence of second, as yet unknown, GEF for Arl8 that acts on endosomes.

    Significance

    Overall the data are clear and convincing, with many conclusions based on genetic mutations, and the results carefully quantified. There seems little required to be done to improve the experiments that are presented, although it would be good if the authors could speculate further as to why mTORC1 is required for Arl8 activity but not recruitment, and if there are further experiments that could augment this conclusion they might be helpful.

    My only substantial suggestion, is that the authors could consider making the study more extensive by applying their simple but elegant system to further possible players in the pathway, and in particular to test some possible Arl8 effectors. Although there is no clear orthologue of PLEKHM2 in C. elegans, both the HOPS complex and PLEKHM1 have been reported to bind to Arl8. Thus it would be interesting to see if either of these is required in this process and at what step. If suitable mutants are available, then hopefully these experiement would take c 2-3 months and be relatively inexpensive as they would involve worm breeding and fluorescent-microscopy.

    My expertise includes membrane traffic and small GTPases but not phagocytosis or C.elegans.

    Referees cross-commenting

    The mammalian work is rather brief but it does seem to have involved collaborators with relevant experience. Nonetheless, it assays general phagolysosome function rather than directly addressing vesiculation. One approach might to drop the mammalian studies, and instead add an investigation of more Arl8 effectors in C. elegans. Hopefully, few people would doubt the relevance to mammals of studies in C. elegans on the function of well conserved proteins.

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    Referee #1

    Evidence, reproducibility and clarity

    Summary

    Phagocytosis is fundamental to innate immunity and tissue homeostasis. C. elegans is a good system to study phagolysosomal dynamics. Here, Fazeli et al. study the molecular mechanism of phagosysosomal tubulation and vesiculation using C. elegans. The authors show the involvement of TORC in the vesiculation of phagolysosomes (Fig 1). Upstream regulatior of TORC would be amino acid release because knockdown of slc-36.1 decreased the number of fission events. BORC is also required for phagolysosome vesiculation (Fig 2). Interestingly, essential subunits are different from synaptic vesicle transport and lysosomal transport. ARL-8, a small GTPase downstream of BORC, is also essential for phagolysosome vesiculation (Fig 3). The nucleotide cycle of ARL-8 is essential for the localization of ARL-8 on phagolysosomes (Fig 4). BORC is required for the vesicular localization of ARL-8 (Fig 5). Finally the authors performed an experiment to show this pathway is conserved in mammalian macrophages (Fig 6).

    Genetic experiments in worms are solid while mammalian cell experiments are not. This reviewer thinks the authors need to improve the quality of cell line experiments.

    Major comments

    1. A lot of components are analyzed in this paper. Then, it is very difficult to understand which molecules are TORC, BORC and/or BLOC subunits, which molecules are required and which are not required. It is very helpful if the authors include a table showing the summary of RNAi and mutant phenotypes. It is very helpful if the table include lysosomal phenotypes and synaptic vesicle phenotypes as well.
    2. Fig 4 and 5. ARL-8 is localized to lysosomes, phagolysosomes and early endosomes. How about TORC and BORC subunits? Are there differences in the localization of essential subunits and non-essential subunits?
    3. Figure 6
      • a) There are many concerns in mammalian cell experiments. It is not clear whether the knockout procedures really work or not. Methods section says the authors pooled puromycin resistant cells. This is not general protocol to establish knockout cell lines. Generally, some drug resistant cells are not always complete KO. Authors need to establish knockout cell lines by picking up single drug-resistant colonies and characterize each line by western blot or immunofluorescent microscopy.
      • b) While the authors monitored cell growth every 3 hours for 4 days, the authors showed the result of day 4 only. Time lapse data would be useful.
      • c) The effect of KO may be because of defects in phagolysosomes. However the authors cannnot conclude "phagolysosomal vesiculation is affected in mammalian cells" or not until they directly observe the phenomena.

    Significance

    TORC-BORC-ARL8 pathway has been shown in lysosomal transport in mammalian cells (Pu et al, 2015, 2017). It has been shown that BORC is required for the localization of ARL8 in the case of worm synaptic vesicles and mammalian lysosomes (Pu et al, 2015; Niwa et al., 2016). This study shows the mechanism is conserved in phagolysosomes as well in worms. As described above, the involvement of these molecules in the mammalian phagolysosomes is not convincing at this stage.

    In the case of lysosomes and synaptic vesicles, the effector of ARL8 (downstream motors) has been shown (Pu et al., 2015; Niwa et al., 2016). It is interesting observation that the nucleotide cycle of ARL8 is essential for the phagolysosomal fission. However, as the downstream of ARL8 remains elusive in this study, it is unclear how phagolysosomes are tubulated (Fig 6D model).

    Referees cross-commenting

    I fully agree with the reviewer #2 that identification of arl8 effectors will make this paper more attractive as I described in my original peer review. Moreover, I agree with the reviewer that genetic data in C. elegans is convincing. While reviewer #2 have not concerned about the quality of experiments, I'm still not convinced by their mammalian cell experiments. I don't have any more concerns if the authors (1) remove the mammalian study or (2) improve the quality of mammalian data.

  8. Version published to 10.1101/2022.02.09.479694v1 on bioRxiv