Spatiotemporal recruitment of the ubiquitin-specific protease USP8 directs endosome maturation

Reviewer #1 (Public Review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of the USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that the localization of USP8 to early endosomes is disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although Rabex5 is accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidence to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members of the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

Weaknesses:

- The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.

- The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient in both factors displayed similar defects in late endosomes/lysosomes. However, the authors didn't confirm whether and/or to which extent USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Reviewer #3 (Public Review):

Summary:

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Weaknesses:

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript.

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it.

Author response:

Reviewer #1 (Public Review):

Summary:

The manuscript focuses on the role of the deubiquitinating enzyme UPS-50/USP8 in endosome maturation. The authors aimed to clarify how this enzyme drives the conversion of early endosomes into late endosomes. Overall, they did achieve their aims in shedding light on the precise mechanisms by which UPS-50/USP8 regulates endosome maturation. The results support their conclusions that UPS-50 acts by disassociating RABX-5 from early endosomes to deactivate RAB-5 and by recruiting SAND-1/Mon1 to activate RAB-7. This work is commendable and will have a significant impact on the field. The methods and data presented here will be useful to the community in advancing our understanding of endosome maturation and identifying potential therapeutic targets for diseases related to endosomal dysfunction. It is worth noting that further investigation is required to fully understand the complexities of endosome maturation. However, the findings presented in this manuscript provide a solid foundation for future studies.

We thank this reviewer for the instructive suggestions and encouragement.

Strengths:

The major strengths of this work lie in the well-designed experiments used to examine the effects of UPS-50 loss. The authors employed confocal imaging to obtain a picture of the aftermath of the USP-50 loss. Their findings indicated enlarged early endosomes and MVB-like structures in cells deficient in USP-50/USP8.

We thank this reviewer for the instructive suggestions and encouragement.

Weaknesses:

Specifically, there is a need for further investigation to accurately characterize the anomalous structures detected in the ups-50 mutant. Also, the correlation between the presence of these abnormal structures and ESCRT-0 is yet to be addressed, and the current working model needs to be revised to prevent any confusion between enlarged early endosomes and MVBs.

Excellent suggestions. The EM imaging indeed revealed an increase in enlarged cellular vesicles containing various contents in usp-50 mutants. However, the detailed molecular features of these vesicles remain unclear. Therefore, we plan to utilize ESCRT components for double staining with early or late endosome markers. This will enable us to accurately characterize the anomalous structures detected in the usp-50 mutants.

Reviewer #2 (Public Review):

Summary:

In this study, the authors study how the deubiquitinase USP8 regulates endosome maturation in C. elegans and mammalian cells. The authors have isolated USP8 mutant alleles in C. elegans and used multiple in vivo reporter lines to demonstrate the impact of USP8 loss-of-function on endosome morphology and maturation. They show that in USP8 mutant cells, the early endosomes and MVB-like structures are enlarged while the late endosomes and lysosomal compartments are reduced. They elucidate that USP8 interacts with Rabx5, a guanine nucleotide exchange factor (GEF) for Rab5, and show that USP8 likely targets specific lysine residue of Rabx5 to dissociate it from early endosomes. They also find that the localization of USP8 to early endosomes is disrupted in Rabx5 mutant cells. They observe that in both Rabx5 and USP8 mutant cells, the Rab7 GEF SAND-1 puncta which likely represents late endosomes are diminished, although Rabex5 is accumulated in USP8 mutant cells. The authors provide evidence that USP8 regulates endosomal maturation in a similar fashion in mammalian cells. Based on their observations they propose that USP8 dissociates Rabex5 from early endosomes and enhances the recruitment of SAND-1 to promote endosome maturation.

We thank this reviewer for the instructive suggestions and encouragement.

Strengths:

The major highlights of this study include the direct visualization of endosome dynamics in a living multi-cellular organism, C. elegans. The high-quality images provide clear in vivo evidence to support the main conclusions. The authors have generated valuable resources to study mechanisms involved in endosome dynamics regulation in both the worm and mammalian cells, which would benefit many members of the cell biology community. The work identifies a fascinating link between USP8 and the Rab5 guanine nucleotide exchange factor Rabx5, which expands the targets and modes of action of USP8. The findings make a solid contribution toward the understanding of how endosomal trafficking is controlled.

We thank this reviewer for the instructive suggestions and encouragement.

Weaknesses:

-The authors utilized multiple fluorescent protein reporters, including those generated by themselves, to label endosomal vesicles. Although these are routine and powerful tools for studying endosomal trafficking, these results cannot tell whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion.

Good suggestion. Indeed, to test whether the endogenous proteins (Rab5, Rabex5, Rab7, etc.) are affected in the same fashion as fluorescent protein reporters, we supplemented our approach with the utilization of endogenous markers. These markers, including Rab5, RAB-5, Rabex5, RABX-5, and EEA1 for early endosomes, as well as RAB-7, Mon1a, and Mon1b for late endosomes, were instrumental in our investigations (refer to Figure 3, Figure 6, Sup Figure 4, Sup Figure 5, and Sup Figure 7). Our comprehensive analysis, employing various methodologies such as tissue-specific fused proteins, CRISPR/Cas9 knock-in, and antibody staining, consistently highlights the critical role of USP8 in early-to-late endosome conversion.

-The authors clearly demonstrated a link between USP8 and Rabx5, and they showed that cells deficient in both factors displayed similar defects in late endosomes/lysosomes. However, the authors didn't confirm whether and/or to which extent USP8 regulates endosome maturation through Rabx5. Additional genetic and molecular evidence might be required to better support their working model.

Excellent point. We plan to conduct additional genetic analyses, including the construction of double mutants between usp-50 and various rabex-5 mutations, to further elucidate the extent to which USP8 regulates endosome maturation via Rabex5.

Reviewer #3 (Public Review):

Summary:

The authors were trying to elucidate the role of USP8 in the endocytic pathway. Using C. elegans epithelial cells as a model, they observed that when USP8 function is lost, the cells have a decreased number and size in lysosomes. Since USP8 was already known to be a protein linked to ESCRT components, they looked into what role USP8 might play in connecting lysosomes and multivesicular bodies (MVB). They observed fewer ESCRT-associated vesicles but an increased number of "abnormal" enlarged vesicles when USP8 function was lost. At this specific point, it's not clear what the objective of the authors was. What would have been their hypothesis addressing whether the reduced lysosomal structures in USP8 (-) animals were linked to MVB formation? Then they observed that the abnormally enlarged vesicles, marked by the PI3P biosensor YFP-2xFYVE, are bigger but in the same number in USP8 (-) compared to wild-type animals, suggesting homotypic fusion. They confirmed this result by knocking down USP8 in a human cell line, and they observed enlarged vesicles marked by YFP-2xFYVE as well. At this point, there is quite an important issue. The use of YFP-2xFYVE to detect early endosomes requires the transfection of the cells, which has already been demonstrated to produce differences in the distribution, number, and size of PI3P-positive vesicles (doi.org/10.1080/15548627.2017.1341465). The enlarged vesicles marked by YFP-2xFYVE would not necessarily be due to the loss of UPS8. In any case, it appears relatively clear that USP8 localizes to early endosomes, and the authors claim that this localization is mediated by Rabex-5 (or Rabx-5). They finally propose that USP8 dissociates Rabx-5 from early endosomes facilitating endosome maturation.

Weaknesses:

The weaknesses of this study are, on one side, that the results are almost exclusively dependent on the overexpression of fusion proteins. While useful in the field, this strategy does not represent the optimal way to dissect a cell biology issue. On the other side, the way the authors construct the rationale for each approximation is somehow difficult to follow. Finally, the use of two models, C. elegans and a mammalian cell line, which would strengthen the observations, contributes to the difficulty in reading the manuscript.

The findings are useful but do not clearly support the idea that USP8 mediates Rab5-Rab7 exchange and endosome maturation, In contrast, they appear to be incomplete and open new questions regarding the complexity of this process and the precise role of USP8 within it.

We thank this reviewer for the insightful comments. Fluorescence-fused proteins serve as potent tools for visualizing subcellular organelles both in vivo and in live settings. Specifically, in epidermal cells of worms, the tissue-specific expression of these fused proteins is indispensable for studying organelle dynamics within living organisms. This approach is necessitated by the inherent limitations of endogenously tagged proteins, whose fluorescence signals are often weak and unsuitable for live imaging or genetic screening purposes. Acknowledging concerns raised by the reviewer regarding potential alterations in organelle morphology due to overexpression of certain fused proteins, we supplemented our approach with the utilization of endogenous markers. These markers, including Rab5, RAB-5, Rabex5, RABX-5, and EEA1 for early endosomes, as well as RAB-7, Mon1a, and Mon1b for late endosomes, were instrumental in our investigations (refer to Figure 3, Figure 6, Sup Figure 4, Sup Figure 5, and Sup Figure 7). Our comprehensive analysis, employing various methodologies such as tissue-specific fused proteins, CRISPR/Cas9 knock-in, and antibody staining, consistently highlights the critical role of USP8 in early-to-late endosome conversion. Specifically, we discovered that the recruitment of USP-50/USP8 to early endosomes is depending on Rabex5. However, instead of stabilizing Rabex5, the recruitment of USP-50/USP8 leads to its dissociation from endosomes, concomitantly facilitating the recruitment of the Rab7 GEF SAND-1/Mon1. In cells with loss-of-function mutations in usp-50/usp8, we observed enhanced RABX-5/Rabex5 signaling and mis-localization of SAND-1/Mon1 proteins from endosomes. Consequently, this disruption impairs endolysosomal trafficking, resulting in the accumulation of enlarged vesicles containing various intraluminal contents and rudimentary lysosomal structures.

Through an unbiased genetic screen, verified by cultured mammalian cell studies, we observed that loss-of-function mutations in usp-50/usp8 result in diminished lysosome/late endosomes. To elucidate the underlying mechanisms, we investigated the formation of multivesicular bodies (MVBs), a process tightly linked to USP8 function. Extensive electron microscopy (EM) analysis indicated that MVB-like structures are largely intact in usp-50 mutant cells, suggesting that USP8/USP-50 likely regulate lysosome formation through alternative pathways in addition to their roles in MVB formation and ESCRT component function. USP8 is known to regulate the endocytic trafficking and stability of numerous transmembrane proteins. Interestingly, loss-of-function mutations in usp8 often lead to the enlargement of early endosomes, yet the mechanisms underlying this phenomenon remain unclear. Given that lysosomes receive and degrade materials generated by endocytic pathways, we hypothesized that the abnormally enlarged MVB-like vesicular structures observed in usp-50 or usp8 mutant cells correspond to the enlarged vesicles coated by early endosome markers. Indeed, in the absence of usp8/usp-50, the endosomal Rab5 signal is enhanced, while early endosomes are significantly enlarged. Given that Rab5 guanine nucleotide exchange factor (GEF), Rabex5, is essential for Rab5 activation, we further investigated its dynamics. Additional analyses conducted in both worm hypodermal cells and cultured mammalian cells revealed an increase of endosomal Rabex5 in response to usp8/usp-50 loss-of-function. Live imaging studies further demonstrated active recruitment of USP8 to newly formed Rab5-positive vesicles, aligning spatiotemporally with Rabex5 regulation. Through systematic exploration of putative USP-50 binding partners on early endosomes, we identified its interaction with Rabex5. Comprehensive genetics and biochemistry experiments demonstrated that USP8 acts through K323 site de-ubiquitination to dissociate Rabex5 from early endosomes and promotes the recruitment of the Rab7 GEF SAND-1/Mon1. In summary, our study began with an unbiased genetic screen and subsequent examination of established theories, leading to the formulation of our own hypothesis. Through multifaceted approaches, we unveiled a novel function of USP8 in early-to-late endosome conversion.