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  1. Author Response:

    Reviewer #2:

    The manuscript by Podinovskaia focuses on a new method to visualize and measure endosome maturation in common cell lines by enlarging early endosomes. This was achieved by producing acute insult to the cells by ionophore treatment, leading to budding of abnormally large post Golgi vesicles that fuse with early endosomes. Endosome maturation of these enlarged endosomes containing Golgi-derived cargo (GalT) proceeding with apparently normal kinetics, ultimately leading to lysosomal delivery. Taking advantage of this assay, the authors investigate Rab5-to-Rab7 conversion, acquisition and loss of PI3P, acquisition and loss of Snx1 on apparent endosomal subdomains, interaction of early and late endosomes with Rab11-positive recycling endosomes, and lumenal pH changes. The new maturation model presented here will likely be quite useful to the field with continuing impact. The current state of the endosome field in many ways remains fragmentary, with various processes studied extensively in isolation, but with little information on their relative timing and potential interactions as endosomes mature. This new assay should help understand the relationships between these processes, some of which are investigated in this manuscript.

    Concerns:

    1. The data and conclusions related to Rab11 interaction with early endosomes in Fig 8 are not convincing. There are simply too many Rab11 endosomes in the cell to know if their short term proximity indicates meaningful interaction with the early endosomes, or if the data simply reflects random collisions of small recycling endosomes with the enlarged early endosomes. No data is presented to show that the interactions are meaningful, e.g. that recycling cargo transfer occurs during these interactions. Conclusions from this analysis are overstated.

    We now provide more evidence for the interaction of Rab11 vesicles with the enlarged endosomes. We made movies with shorter intervals (2 sec instead of 1 min) between the individual frames. These data clearly show that this is not an accidental bumping into an endosome but rather that Rab11 vesicles can circle around endosomes and stay for several minutes (Video Fig. 8A, supplement 2 and 3).

    In addition, we imaged TfR-GFP together with mApple-Rab5. These data show that TfR-GFP positive vesicles bud off from mApple-Rab5 positive endosomes and that the GFP fluorescence intensity goes down over time in enlarged endosomes. These data are consistent with recycling of TfR to the plasma membrane. Moreover, CDMPR-GFP, which cycles between the TGN and endosomes was found to be present on Rab5 negative enlarged structure, which then turned Rab5 positive, and subsequently lost the CDMPR signal. Importantly those endosomes could regain CDMPR, which we interpret as acquisition from the TGN. These data may indicate that the TGN-endosome shuttle is intact after nigericin washout (Fig. 9).

    That the TfR and CDMPR are really transported out of the enlarged endosome is also contrasted by our finding that GalT-GFP stayed in the enlarged endosome and the signal intensity did not significantly drop.

    1. Lack of information on endocytic cargo acquisition by the enlarged early endosomes: to really establish this endosome maturation model the authors would need to establish if the enlarged endosomes contain endocytosed cargo, as opposed to Golgi-derived cargo, and determine how long it takes to acquire such cargo. This could be accomplished using Tf, EGF, or perhaps dextran at early timepoints after nigericin washout.

    As described above, we now show that TfR-GFP is present in enlarged endosomes and is lost from these endosomes over time (Fig. 9A,D,G).

    Additionally, we performed experiments with dextran-Alexa647 and nanobody-tagged surface TfR to show that endocytosed material from the plasma membrane indeed reached the enlarged endosomes (Fig. 3, figure supplement 1 and 2). Quantification of TfR signal at the enlarged endosomes demonstrates that TfR acquisition by the enlarged endosome takes place as soon as the enlarged compartment becomes Rab5-positive. This was also observed with the nanobody-tagged surface TfR and endocytosed Dextran-AF647, representative examples of which are provided (Fig 3, figure supplement 1 and 2). The quantification for the latter experiments was not carried out due to the very short time range during which asynchronous Rab5 recruitment events needed to be captured after addition of nanobody/Dextran pulse-and-chase.

    1. Figure 7 - It was not convincing that data in panels F and G are different from each other.

    We agree with the reviewer that the difference between the data presented in panel F and G is not very big. These panels represent the average of many endosomes and with the averaging the differences from the individual traces get cancelled out. The process is asynchronous and thus in this case the individual traces are more telling than the averaged traces. Nevertheless, we decided to keep the average traces in the manuscript because the highlight the asynchronous nature of the process. We modified the text to make this point clear.

    1. Figure 11 - it is unclear how we can interpret this as connected to Rab conversion when even the labeled compartments at the earliest time point in the czz1 knockout have abnormally high pH, and during the time-course even the last timepoint for czz1 KO is higher than that of the earliest timepoint for WT.

    We agree that the ccz1 KO cells display higher endosomal pH than WT cells throughout the time-course.

    However, the cells in which we express the rescue plasmid of Ccz1 also have apparently less acidified endosomes, even though Ccz1 can still drive Rab conversion, and the pH dropped at an intermediate rate, when comparing rescued cells to control and ccz1 KO cells. Even in ccz1 KO cells endosomal traffic down the degradation pathway is not completely blocked, similarly to what we observed for sand-1 (-/-) in C. elegans and Mon1a/b knockdown in mammalian cells (Poteryaev et al. 2010). Acidification eventually will occur, but it is massively slowed down; the molecular basis of which is still under investigation in our lab.

    We think that in the absence of Ccz1, a condition under which Rab conversion is severely impaired, acidification cannot occur at normal rate. As pointed out by reviewers 1 and 2, the pH is already higher in the ccz1 KO cells than in the control condition. However, in the rescue condition, the YFP/CFP ratio is not that different from the knockout and yet acidification can occur at an intermediate rate. Why under rescue conditions, the YFP/CFP ratio is at a similar level compared to the KO is not entirely clear. It is conceivable that too much Ccz1 has also a negative effect. Moreover, recently it has been shown that ccz1 KO cells accumulate free cholesterol in the enlarged endosomes (Van den Boomen Nat Comm., 2020). The transient expression might be not sufficient to rescue this accumulation phenotype or other secondary effects. Nevertheless, the v-ATPase appears to maintain its function because lysosomes can acidify in ccz1 KO cells, albeit with a delay (Figure 13).

    1. Figure 12 - The criteria used to determine which GalT structures are Golgi or lysosomes seems questionable. Morphology alone is not sufficient to identify the compartments with high accuracy, especially after perturbation. Also, it is unclear to what extent GalT-CFP labels lysosomes without nigericin treatment.

    To address these issues, we co-labelled cells with lysotracker. GalT-CFP (pHlemon) and lysotracker showed a very high degree of co-localization. These data are included in the manuscript (Fig. 10B).

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  2. Evaluation Summary:

    Endosome maturation in animal cells has been challenging to characterize by microscopy because the fluorescence patterns are complex and dynamic. This study uses acute ionophore treatment to generate enlarged early endosomes, whose behavior and maturation can then be readily tracked. The results offer new insights into several phenomena, including the regulation of endosomal acidification during the maturation process.

    (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 #2 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    Podinovskaia et al report a simple method to enlarge endosomes, and possibly the TGN, using cultured transformed cells. They report that a brief treatment of the cells with the ionophore nigericin results in a rapid swelling of endosomal compartments. It is shown that, despite this, perturbation, swelled endosomes mature via rab5 to rab7 conversion. It is further shown that the kinetics of rab conversion on swelled endosomes is approximately the same as for native endosomes, suggesting that endosomes that are swelled in this manner can be used to infer functional aspects of endo-lysosomes. Using this approach, the authors find that Rab11, a regulator of a plasma membrane recycling pathway, and Snx1, a component of multiple cargo export pathways from the endosome, persists throughout endosome maturation. They report that endosome re-acidification (ie, after nigericin washout) correlates with the appearance of Rab7 of the endosome. Finally, the authors report that the TGN is reversibly swelled similarly by nigericin and that this swelled compartment receives endocytosed material.

    Overall, this is an excellent study that provides an interesting new experimental approach for investigating endosome dynamics. The validation studies convincingly demonstrate that rate of rab conversion on nigercin-swelled endosomes is similar to the rate of maturation of untreated endosomes.

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  4. Reviewer #2 (Public Review):

    The manuscript by Podinovskaia focuses on a new method to visualize and measure endosome maturation in common cell lines by enlarging early endosomes. This was achieved by producing acute insult to the cells by ionophore treatment, leading to budding of abnormally large post Golgi vesicles that fuse with early endosomes. Endosome maturation of these enlarged endosomes containing Golgi-derived cargo (GalT) proceeding with apparently normal kinetics, ultimately leading to lysosomal delivery. Taking advantage of this assay, the authors investigate Rab5-to-Rab7 conversion, acquisition and loss of PI3P, acquisition and loss of Snx1 on apparent endosomal subdomains, interaction of early and late endosomes with Rab11-positive recycling endosomes, and lumenal pH changes. The new maturation model presented here will likely be quite useful to the field with continuing impact. The current state of the endosome field in many ways remains fragmentary, with various processes studied extensively in isolation, but with little information on their relative timing and potential interactions as endosomes mature. This new assay should help understand the relationships between these processes, some of which are investigated in this manuscript.

    Concerns:

    1. The data and conclusions related to Rab11 interaction with early endosomes in Fig 8 are not convincing. There are simply too many Rab11 endosomes in the cell to know if their short term proximity indicates meaningful interaction with the early endosomes, or if the data simply reflects random collisions of small recycling endosomes with the enlarged early endosomes. No data is presented to show that the interactions are meaningful, e.g. that recycling cargo transfer occurs during these interactions. Conclusions from this analysis are overstated.

    2. Lack of information on endocytic cargo acquisition by the enlarged early endosomes: to really establish this endosome maturation model the authors would need to establish if the enlarged endosomes contain endocytosed cargo, as opposed to Golgi-derived cargo, and determine how long it takes to acquire such cargo. This could be accomplished using Tf, EGF, or perhaps dextran at early timepoints after nigericin washout.

    3. Figure 7 - It was not convincing that data in panels F and G are different from each other.

    4. Figure 11 - it is unclear how we can interpret this as connected to Rab conversion when even the labeled compartments at the earliest time point in the czz1 knockout have abnormally high pH, and during the time-course even the last timepoint for czz1 KO is higher than that of the earliest timepoint for WT.

    5. Figure 12 - The criteria used to determine which GalT structures are Golgi or lysosomes seems questionable. Morphology alone is not sufficient to identify the compartments with high accuracy, especially after perturbation. Also, it is unclear to what extent GalT-CFP labels lysosomes without nigericin treatment.

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  5. Reviewer #3 (Public Review):

    The authors describe a useful new way to track the maturation of dramatically enlarged endosomes in animal cells. Ionophore treatment is a significant perturbation, but a variety of findings indicate that the enlarged endosomes follow the normal maturation pathway. This system was coupled with additional technical advances and targeted perturbations to perform functional tests. The results reveal that recycling to the plasma membrane is uncoupled from the Rab5-to-Rab7 early-to-late conversion process, whereas acidification seems to be linked to Rab conversion.

    My sense is that this work makes a valuable contribution to our knowledge of a fascinating cell biological process. However, the manuscript would benefit from a clearer emphasis on how the data inform our views of outstanding questions.

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