OCRL regulates lysosomal function and endolysosomal homeostasis in Drosophila nephrocytes

  1. Vaishali Kataria
  2. Navyashree A Ramesh
  3. Indra Sara Lama
  4. Aishwarya Venugopal
  5. Anil Vasudevan
  6. RAGHU PADINJAT

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Abstract

The OCRL gene encodes a lipid phosphatase that dephosphorylates phosphatidylinositol 4,5 bisphosphate [(PI4,5)P2]. Mutations in OCRL lead to a rare human genetic disorder, Lowe syndrome (LS) that affects the eye, kidney and brain. OCRL is widely expressed in cells and is localized to multiple organelles, including the plasma membrane, endosomes, Golgi and lysosomes. Although multiple defects in the endo-lysosomal system have been reported in OCRL depleted cells, the primary site of action of OCRL is unclear. Here we present a Drosophila nephrocyte model of LS; depletion of Drosophila OCRL (dOCRL) manifests with defects in endocytic uptake, altered endosomal compartments as well as expanded but dysfunctional lysosomes. Reconstitution of dOCRL depleted nephrocytes with a lysosome targeted version of the enzyme rescues not only the lysosomal defects but surprisingly also defects in endosomal structure and function. These findings suggest that the primary defect in LS cells is likely to be altered lysosome structure and function. Therefore, regulation of PI(4,5)P2 homeostasis at the lysosome membrane by OCRL is critical to homeostasis of the endosomal system.

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    __1. __General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We express our appreciation to all three reviewers for their detailed comments on our manuscript and for highlighting that the experiments are well designed, manuscript well written, noting the clarity of the data sets and methods of quantification and analysis.

    We thank reviewers for appreciating our work on LS using the Drosophila model and noting that the compartment-targeting approach is a potentially valuable approach. We are of the view that understanding a human disease is a challenging task that often requires the use of multiple model systems and cell types, each of which has its strengths and limitations. Sometimes, there are species and cell type specific differences in observations on cellular and molecular processes, and we believe such differences are just as informative in deciphering underlying as are commonalities across model systems.

    We are delighted that reviewers have noted conceptually important conclusions that arise from our study with implications both for understanding the sub-cellular mechanism of disease in LS but also for the understanding the overall wiring of the endo-lysosomal system (a primary role for lysosomes and feedback from late-endo-lysosomal homeostasis for regulating upstream endocytic function). Yet, we thank the reviewers for highlighting some aspects of our data that need to be strengthened to fully realize the potential of these conceptual advances. Below, we provide a pointwise response to reviewer comments and in almost all cases propose pragmatic experiments or analysis to address each point. We believe that when completed, the revised manuscript will be a substantive advance in the understanding of the cell biology of LS, in lysosomal biology and its role in the overall wiring of the endosomal system.

    __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

    In this study the authors explore the role of the Drosophila orthologue of the Lowe syndrome protein OCRL (called dOCRL) in nephrocytes, the functional equivalent of the mammalian kidney. Using genetic knockout and complementation approaches it is shown that dOCRL is required for the clearance of the toxic compound silver nitrate from nephrocytes. This effect is phenocopied by loss of Rab7 and can be rescued by a constitutively active form of Rab7, suggesting involvement of late endosomal or lysosomal compartments. This is supported by experiments showing disrupted lysosomal function upon dOCRL loss. Loss of dOCRL also causes defects earlier in the endocytic pathway, with reduced uptake of the endocytic tracers dextran and mBSA into nephrocytes, and a loss of early endosomal compartments. Interestingly, re-expression of dOCRL targeted to the lysosome rescues the defects in both endosome and lysosome function, suggesting that the defects observed are primarily due to dOCRL function at the lysosome.

    This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. There are a few things that could be addressed in a revised version which are indicated below:

    We thank Reviewer #1 for the positive assessment of our study and for the constructive suggestions

    1.) The ability of lysosome-targeted dOCRL to rescue the phenotypes is interesting. Can the authors exclude the possibility that there is residual cytosolic dOCRL in the cells that can transiently associate with other organelles e.g. early endosomes to drive the phenotypic rescue? There are experiments described in the discussion about targeting dOCRL to early endosomes not rescuing the phenotypes. Currently this is mentioned as data not shown. It should be included.

    Response: We thank the reviewer for this important comment. We have previously validated the lysosomal targeting sequence (LTS) in S2R+ cells by demonstrating its colocalization with established lysosomal markers, including LAMP1 and LysoTracker. These data confirm that the LTS directs transgenes specifically to the lysosomal compartment. We will include these validation data in the revised manuscript.

    To further exclude the possibility that dOCRL localisation to organelles other than lysosomes contributes to the observed phenotypic rescue, we will include rescue experiments using dOCRL::GFP construct specifically targeted to the early endosomal compartment. This early endosome compartment specific construct does not rescue the defects observed in dOCRLKO nephrocytes.

    In addition we will perform colocalization analyses of LTS::dOCRL::GFP with an early endosomal marker. These experiments will help confirm the specificity of LTS-mediated targeting and rule out significant localisation of the construct to the early endosomal compartment.

    2.) The results with AgNO3 are confusing. Uptake into nephrocytes is not affected by loss of dOCRL but is affected by other manipulations that disrupt endosomal trafficking (suppl table 1). Yet, loss of dOCRL affects cellular uptake of dextran and mBSA, which rely on endocytosis. More explanation needs to be provided on what we know about AgNO3 uptake into nephrocytes. Is it by endocytosis, as the table in the supplementary table would seem to indicate? In that case, why would AgNO3 uptake not be affected by dOCRL loss? Also, what underlies the clearance of AgNO3 taken up into nephrocytes? If it is already in the cells, then does it leave by exocytosis? Or does it enter and leave cells through transporters? How does dOCRL affect the clearance of AgNO3 from the cells i.e. through what mechanism? Filtration of AgNO3 is also mentioned but not fully explained. How does filtration and clearance of AgNO3 relate to the processes that occur in the mammalian kidney, where filtration of blood is performed by the glomerulus, while clearance is in the proximal tubules, which are the region affected in Lowe syndrome?

    Response: We thank the reviewer for this important comment. In the revised manuscript, we will provide a more detailed explanation of the basis of the AgNO₃ uptake and clearance assay, drawing both from our genetic screen and from previous studies of nephrocyte endocytosis. Our data support the conclusion that AgNO₃ uptake occurs through endocytic pathways. Specifically, inhibition of key endocytic regulators, including Rab5 and Shibire (Dynamin), completely abolishes AgNO₃ uptake in nephrocytes. In contrast, loss of dOCRL reduces AgNO₃ uptake but does not eliminate it entirely, indicating that endocytic activity is impaired but not fully blocked in the mutant.

    It is important to distinguish between the uptake assays used in this study. Dextran and mBSA uptake assays are ex vivo measurements that assess endocytic uptake over relatively short time periods. In contrast, the AgNO₃ uptake and clearance assay is an in vivo functional assay performed over approximately 80 hours of larval development, thereby capturing both uptake and long-term cargo processing and clearance.

    When AgNO₃ uptake is examined over shorter time intervals (10-15 hours), we observe reduced accumulation in *dOCRLKO *nephrocytes relative to controls, consistent with impaired endocytic uptake. However, as the assay progresses, defects in cargo clearance become increasingly apparent. After approximately 36 hours, AgNO₃ begins to accumulate in dOCRLKO nephrocytes, whereas it is efficiently cleared from control cells. Consequently, the mutant nephrocytes display higher AgNO₃ retention at later time points despite their reduced initial uptake. This observation is consistent with our mBSA pulse-chase experiments. While dOCRLKO nephrocytes show reduced cargo uptake at early time points, they exhibit increased retention of mBSA during extended chase periods (e.g., 40 minutes), supporting a defect in cargo degradation and/or clearance rather than enhanced uptake.

    To further strengthen these conclusions and clarify the interpretation of the AgNO₃ assay, we will include the short-term (10-hour) AgNO₃ uptake data in the revised manuscript. These additional data will help distinguish the effects of dOCRL loss on initial uptake from its effects on subsequent cargo processing and clearance.

    The precise mechanism by which AgNO₃ is cleared from nephrocytes remains incompletely understood and was one of the primary motivations for conducting our genetic screen. Given the possibility that clearance could occur through exocytosis, we included several candidate genes involved in late endosomal and lysosomal exocytic pathways in our screen. However, either because of insufficient RNAi-mediated knockdown efficiency or because depletion of these genes produced severe nephrocyte morphological defects, we were unable to definitively identify the molecular machinery responsible for AgNO₃ clearance. Nevertheless, our findings provide evidence that the late endolysosomal pathway plays an important role in this process. In particular, the ability of constitutively active Rab7 (Rab7CA) to rescue the dOCRLKO phenotype suggests that proper functioning of the late endosomal/lysosomal compartment is critical for AgNO₃ processing and clearance. While the exact mechanism remains to be established, our data are consistent with a model in which cargo clearance is mediated through late endolysosomal trafficking pathways.

    It is important to consider the unique physiology of Drosophila nephrocytes. Unlike the mammalian renal system, where filtration occurs at the glomerulus and reabsorption is performed by proximal tubule cells (PTCs), nephrocytes perform both of these functions within a single cell type. Hemolymph components are first filtered in a size-selective manner through the basement membrane and slit diaphragms and are subsequently internalised by endocytosis. The internalised cargo is then processed through the endolysosomal pathway, functionally resembling the cargo-handling and processing activities of mammalian PTCs. Because nephrocytes play a central role in maintaining hemolymph homeostasis and clearing circulating toxins, the AgNO₃ uptake and clearance assay provides a physiologically relevant functional readout of nephrocyte activity. We therefore employed this assay to assess nephrocyte function in the absence of dOCRL. The defects in AgNO₃ handling observed in dOCRLKO nephrocytes support the conclusion that dOCRL is required for efficient endolysosomal cargo processing and clearance, thereby contributing to the maintenance of nephrocyte homeostasis. We will expand this discussion in the revised manuscript to provide greater clarity regarding both the rationale for using the AgNO₃ assay and its inherent limitations.

    3.) The data overall are clear and convincing, but Fig 3C is not. There does not appear to be any reduction in mBSA signal in the dOCRL image shown.

    Response: We will replace the figure with the one representing mean values.

    4.) Discussion could be more expansive eg dOCRL versus mammalian OCRL-clathrin binding, localisation etc... Response: We thank the reviewer for their suggestion. We will include the comparison of Drosophila and mammalian OCRL in the discussion of our manuscript.

    5.) The figures need to appear in the correct order in the text. Currently they are presented in the text in a jumbled way. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

    6.) References are missing in quite a few places in the text. There are numerous instances of statements being made without the references to support them. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

    7.) There are a few minor grammatical errors. Response: We thank the reviewer for their suggestion. We will correct this in the revised manuscript.

    Reviewer #1 (Significance (Required)):

    This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. Response: We sincerely thank the reviewer for their positive evaluation of our manuscript and for their constructive feedback.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    Summary The authors establish germline and nephrocyte-specific dOCRL loss-of-function models in Drosophila. They report that dOCRL deficiency is associated with increased plasma-membrane PI(4,5)P₂, decreased plasma-membrane PI4P, reduced dextran and BSA uptake, alterations in early- and late-endosomal markers, lysosomal expansion/dysfunction, and delayed AgNO₃ clearance. They further claim that a late-endolysosomal-targeted dOCRL construct rescues AgNO₃ retention, Cathepsin L accumulation, Rabenosyn-5 staining, and BSA uptake, whereas a presumably phosphatase-dead construct fails to normalize Cathepsin L. The authors conclude that lysosomal OCRL activity is the primary requirement for endolysosomal homeostasis and that the endocytic abnormalities are secondary to lysosomal dysfunction. The Drosophila model and compartment-targeting approach are potentially valuable. However, the central conclusion is not adequately supported because the localization and biochemical activity of the targeted construct have not been established in nephrocytes, and several endocytic and lysosomal assays are interpreted beyond what they directly measure. In addition, the authors extend the relevance of these findings too far to the pathophysiology and potential treatment of Lowe syndrome in patients. The study is restricted to Drosophila nephrocytes, and no evidence is provided in mammalian proximal-tubule cells, patient-derived cells, or mammalian disease models that selective restoration of lysosomal OCRL activity is sufficient to rescue endocytosis. The conclusions should therefore remain specific to the Drosophila system, while their relevance to human Lowe syndrome requires validation in mammalian models. We thank the reviewer for their comments, constructive suggestions, and critical evaluation of our manuscript.

    Major comments

    1. Lysosomal specificity is not convincingly demonstrated. The LTS sequence is intended to target proteins to the cytosolic surface of late endosomes and lysosomes. However, its localization must be independently validated for each fusion protein and in each cell type. In Figure 5B, localization is assessed in S2R+ cells rather than nephrocytes, and by colocalization with LTS-mCherry, which contains the same targeting sequence. This approach is partly circular and does not independently establish that the labelled organelles are lysosomes. Furthermore, their morphology differs from that of Cathepsin L-, Rab7-, and LysoTracker-positive structures. The displayed LTS-dOCRL-GFP signal is not confined to LTS-mCherry-positive structures and includes substantial peripheral and non-overlapping signal. Figure 4 uses LTS-mCherry in nephrocytes, but this reporter is not colocalized with Cathepsin L, LysoTracker, or another independent lysosomal marker. The different patterns observed in Figures 4 and 5 are also difficult to compare because they were obtained in different cell types. Response:

    We thank the reviewer for this important suggestion and agree that further validation of the lysosomal targeting sequence (LTS) localisation would strengthen the study.

    We have validated the lysosomal localisation of the LTS sequence through colocalization analyses with the lysosomal markers LAMP1 and LysoTracker in Drosophila S2R+ cells. These data support the specificity of LTS-mediated lysosomal targeting, and we can incorporate these results into the revised manuscript.

    LTS colocalization with lysosomal markers in S2R+ cells

    In addition, we have performed colocalization studies of LTS::dOCRL::GFP with LTS::mCherry in other *Drosophila *cell types, including salivary gland cells and haemocytes, where we observed substantial overlap between the two markers. We can also include these data in the revised manuscript as further evidence supporting the localisation of the construct.

    Nephrocyte and hemocytes (Red arrows- hemocytes; Blue arrow- Nephrocyte)

    Salivary glands

    In nephrocytes, however, visualisation of punctate LTS::dOCRL::GFP fluorescence is technically challenging because these cells contain a highly acidic lysosomal compartment, which likely compromises GFP fluorescence stability. To address this issue, larvae were fed chloroquine (3 mg/mL) to reduce lysosomal acidity, which enabled the detection of LTS::dOCRL::GFP signals that colocalised with LTS::mCherry. A similar reduction in GFP signal specifically in nephrocytes has been observed in our studies using the mCherry::GFP::Atg8a reporter and has also been reported previously in the literature (PMID: 35805186).

    Nephrocytes in chloroquine fed larvae

    To further address the reviewer's concern, we will perform additional colocalisation analyses of LTS::mCherry with an independent lysosomal marker in nephrocytes and include these data in the revised manuscript.

    Furthermore, to exclude the possibility that the LTS sequence targets organelles other than lysosomes, we will perform colocalization studies with markers of other endomembrane compartments, including early endosomes.

    Finally, to rule out the possibility that the observed phenotypic rescue arises from dOCRL function at the plasma membrane or early endosomes rather than lysosomes, we will include rescue experiments using dOCRL constructs specifically targeted to the plasma membrane and early endosomal compartments. These studies will help establish the compartment-specific contribution of dOCRL to the observed rescue phenotype.

    To address the reviewer’s suggestion regarding the localisation of LTS-dOCRL in nephrocytes relative to independent lysosomal, Rab7-positive, and early endosomal markers, we plan to generate an LTS::dOCRL::mCherry transgenic line and use it to examine the localisation of lysosome-targeted dOCRL relative to lysosomal, Rab7-positive late endosomal, and early endosomal compartments. This strategy is intended to overcome the loss of GFP fluorescence that we observed with the LTS::dOCRL::GFP construct in nephrocytes, likely due to the highly acidic nature of the lysosomal environment. This approach is necessary since immunostaining of endogenous dOCRL is currently not feasible, as the available Drosophila dOCRL antibody does not perform reliably for immunofluorescence applications.

    However, we acknowledge that there may be technical limitations with this approach. Specifically, we are uncertain whether the LTS::dOCRL::mCherry construct will be fully compatible with immunostaining procedures. Our current LTS::mCherry reporter exhibits poor signal retention during the immunostaining workflow, and reliable fluorescence can only be observed when samples are imaged immediately after fixation. Therefore, while we will pursue this strategy, the technical feasibility of performing extensive co-localisation analyses using the mCherry-tagged construct remains to be established.

    To complement these studies, we will independently perform colocalization experiments in S2R+ cells, where imaging and immunostaining conditions are more amenable to detailed subcellular localisation analyses. These experiments will allow us to assess the localisation of LTS::dOCRL relative to lysosomal, Rab7-positive, and early endosomal markers in an independent cellular context. Overall, we will make every effort to validate the localisation of lysosome-targeted dOCRL in nephrocytes using independent compartment-specific markers and will include any resulting data in the revised manuscript.

    Most importantly, the authors do not show: (i) direct measurement of lysosomal PI(4,5)P₂ in dOCRLKO nephrocytes; (ii) reduction of lysosomal PI(4,5)P₂ by LTS-dOCRL; or (iii) measurement of plasma-membrane PI(4,5)P₂ or PI4P following LTS-dOCRL expression. The last point is particularly important because the construct appears to display non-lysosomal signal, including apparent plasma-membrane localization and puncta that do not colocalize with LTS-mCherry. The authors should therefore quantify LTS-dOCRL localization in nephrocytes relative to independent lysosomal, Rab7-positive, and early-endosomal markers; determine whether LTS-dOCRL corrects plasma-membrane PI(4,5)P₂ and PI4P; and assess lysosomal PI(4,5)P₂ in control, dOCRLKO, and LTS-dOCRL-rescued nephrocytes.

    We thank the reviewer for this valuable suggestion. We will include measurements of plasma membrane PI(4,5)P₂ and PI4P levels following expression of LTS::dOCRL. These experiments will allow us to assess whether lysosome-targeted dOCRL influences phosphoinositide homeostasis at the plasma membrane and provide additional insight into the relationship between lysosomal dOCRL function and cellular phosphoinositide regulation. With respect to lysosomal PI4P and PI(4,5)P₂ levels, these experiments are currently technically challenging in the Drosophila nephrocyte system. Simultaneous expression of phosphoinositide biosensors, compartment-specific markers, Gal4 drivers, and LTS::dOCRL transgenes in either wild-type or dOCRLKO backgrounds requires the generation of flies carrying multiple transgenic elements. In many cases, these complex genetic combinations are difficult to obtain, or maintain limiting our ability to directly quantify lysosomal phosphoinositide pools in vivo at present.

    The reduction in plasma-membrane PI4P is a major and insufficiently validated finding. The marked reduction in plasma-membrane PI4P in dOCRLKO nephrocytes is potentially important but requires substantially stronger validation. Although OCRL hydrolyses PI(4,5)P₂ to generate PI4P, a reduction in the steady-state bulk plasma-membrane PI4P pool is not a commonly established consequence of OCRL loss. Direct analysis of plasma-membrane phosphoinositides in human platelets showed that OCRL inhibition did not reduce plasma-membrane PI4P in resting or activated platelets, and no change in plasma-membrane PI4P was detected in HEK293T cells. In contrast, inhibition of PI4KIIIα strongly depleted plasma-membrane PI4P, supporting PI4KA, rather than OCRL, as the principal determinant of this pool (Bura and Jurak Begonja, 2021; PMID: 34947862). Similarly, lipidomic analysis of Lowe syndrome patient-derived cells, performed by the same group as in the manuscript under review, did not reveal a consistent reduction in total PI4P/PIP (Akhtar et al., 2022; PMID: 35023542). The best-established phosphoinositide consequence of OCRL deficiency is instead the accumulation of PI(4,5)P₂ on early endosomes, clathrin-coated intermediates, lysosomes and, in some cell types, at the plasma membrane (Vicinanza et al., 2011; Nández et al., 2014). These studies did not identify depletion of bulk plasma-membrane PI4P as a characteristic phenotype of OCRL loss. The finding reported here could therefore represent a nephrocyte-specific effect, an indirect consequence of altered phosphoinositide metabolism, or a limitation of the probe-based measurement. This issue is mechanistically central because a substantial loss of plasma-membrane PI4P could itself affect membrane identity, PI(4,5)P₂ resynthesis, and endocytosis. Moreover, the authors do not determine whether expression of dOCRL or LTS::dOCRL restores plasma-membrane PI4P and PI(4,5)P₂.

    Response:

    We thank the reviewer for this valuable insight. We recognise that alterations in PI(4,5)P₂ levels are a well-established consequence of OCRL deficiency and have been extensively studied in multiple systems. In contrast, changes in PI4P levels following OCRL loss are less commonly reported and remain relatively unexplored in the literature.

    In the present study, we quantified PI4P levels in a renal cell type, Drosophila nephrocytes. The reduction in PI4P observed in dOCRLKO nephrocytes may represent a cell type specific consequence of OCRL loss and could reflect the unique phosphoinositide dynamics of highly endocytic renal cells.

    Supporting the possibility that OCRL can influence PI4P homeostasis, a previous study (PMID: 28871046) reported that depletion of OCRL results in reduced PI4P levels in primary cilia. These findings suggest that OCRL dependent regulation of phosphoinositide composition may extend beyond PI(4,5)P₂ and may vary according to subcellular compartment and cellular context. To further substantiate our findings, we will include a rescue experiment assessing PI4P levels in the dOCRLKO background in the revised manuscript.

    The AgNO₃ data do not currently provide a robust quantitative measure of clearance In Figure 1H, the percentage of dOCRLKO nephrocytes classified as containing high AgNO₃ is greater at 42 hours than at 36 hours. Differences in initial loading, imaging conditions, cell size, or thresholding could therefore produce an apparent increase at a later time point. For this reason, the authors should provide measurements of initial AgNO₃ accumulation. Furthermore, they should explain the use of different AgNO₃ concentrations-0.003% in Figure 1 and 0.0015% in Figures 2 and 5-as this limits direct comparison between experiments.

    Response:

    We measured AgNO₃ uptake in control and *dOCRLKO *nephrocytes concurrently with the clearance assays. In addition, we will include an experiment assessing AgNO₃ uptake at the initial time point to further substantiate these findings. The imaging conditions were kept the same throughout the experiment.

    The concentration of 0.003 was used during the assay standardisation phase, in which only control and dOCRLKO nephrocytes were analysed. At this concentration, AgNO₃ did not exhibit detectable toxicity. For subsequent experiments, we reduced the concentration to 0.0015 to facilitate more accurate comparisons between control and dOCRLKO nephrocytes, as well as across additional genotypes included in the study. Importantly, control and dOCRLKO nephrocytes were included in every experimental set alongside the other genotypes. This approach ensured direct internal comparisons across experiments and minimised variability arising from experimental conditions.

    In Figure 3, the authors show reduced dextran and BSA uptake, supporting an endocytic defect. However, lower Rabenosyn-5, 2xFYVE, and Rab7 fluorescence does not demonstrate fewer or smaller endosomes. These signals may instead reflect altered protein abundance, membrane recruitment, or phosphoinositide content. The authors should therefore avoid conclusions regarding reduced endosomal compartment size or preserved endosomal maturation unless organelle number and size are directly quantified.

    Similarly, Rab7CA rescue identifies Rab7 as a genetic suppressor but does not prove that endogenous Rab7 activity is reduced or that it causes the lysosomal defect. Increased Cathepsin L staining, although diffuse and not clearly localized to intracellular puncta, together with reduced Magic Red activity, suggests accumulation of poorly functional lysosomes rather than increased lysosomal capacity. However, the authors measured Cathepsin B activity using Magic Red substrate MR-(RR)2 , rather than Cathepsin L activity using the Magic Red substrate MR-FR2.

    The use of Cathepsin L staining as a readout of lysosomal dysfunction may therefore require further characterization. Finally, the authors should test whether Rab7CA restores lysosomal activity, rather than only Cathepsin L abundance.

    Response:

    By measuring the fluorescence intensity of these markers, we observed reduced labelling intensity for Rabenosyn-5, 2xFYVE, and Rab7. We will modify this statement into the revised manuscript. We have not yet analysed the size or number of these compartments, as reliable quantification is challenging because the cells are densely populated with endosomal structures throughout the cytoplasm.

    In dOCRLKO nephrocytes, we observed an expansion of the lysosomal compartment, as indicated by increased LTS::mCherry fluorescence. To independently validate this finding, we employed an additional lysosomal marker, Cathepsin L, which showed a similar increased signal.

    To determine whether the enlarged lysosomal compartment remained functionally active, we assessed lysosomal proteolytic activity using the Magic Red Cathepsin assay. We initially tested both the Magic Red Cathepsin L and Magic Red Cathepsin B kits. However, the Magic Red Cathepsin L reagent failed to generate a detectable signal even in control nephrocytes, suggesting a technical limitation of the kit under our experimental conditions. Consequently, we proceeded with the Magic Red Cathepsin B assay. Importantly, the manufacturer's documentation indicates that, although these substrates are designed to preferentially detect specific cathepsins, they can also be cleaved by other cathepsin family members, thereby serving as a broader readout of lysosomal cathepsin activity.

    We appreciate the reviewer's suggestion regarding the functional assessment of Rab7CA mediated rescue. To address this important point, we will perform the Magic Red Cathepsin assay in the Rab7CA rescue background and include these results in the revised manuscript.

    Figure 3C measures BSA accumulation after a 15-minute incubation, whereas Figure 4I uses an 8-minute pulse followed by a 40-minute chase, showing increased intracellular staining that is interpreted as reduced degradation. Although residual BSA is higher in dOCRLKO cells after the chase, the amount present immediately after the 8-minute pulse was not measured. Consequently, the fraction cleared or degraded cannot be calculated, and the data reported in Figure 4I cannot be directly correlated with those reported in Figure 3C. To support the conclusion of defective degradation, the authors should provide a quantitative assessment of BSA levels immediately after the 8-minute pulse and at multiple chase times, with residual fluorescence expressed relative to the genotype-specific starting signal.

    The uptake pathway used by BSA in Drosophila nephrocytes also requires clarification. Maleylated BSA is a scavenger-receptor ligand in other cell types, whereas protein uptake in nephrocytes is commonly associated with Cubilin/Amnionless-dependent uptake. The manuscript does not establish which receptor or pathway mediates BSA internalization in nephrocytes.

    Response: We thank the reviewer for this valuable suggestion. Our initial analyses showed that mBSA uptake at the early time points (5, 10, and 15 minutes) was consistently reduced in dOCRLKO nephrocytes compared with controls, indicating an impairment in uptake. However, during the subsequent chase period of 40 minutes, we observed a stronger and more persistent mBSA signal in dOCRLKO nephrocytes relative to controls, suggesting reduced degradation.

    To improve the clarity of the data presentation, we will include the pulse-only uptake data (8-minute incubation) alongside the pulse-chase experiments performed at the different time points in the revised manuscript, as suggested.

    The mBSA uptake assay is mediated through Cubilin-dependent endocytosis. In support of this, we have observed that knockdown of Cubilin by RNAi completely abolishes mBSA uptake in nephrocytes, confirming the specificity of the assay. We will provide these data as supplementary evidence to further clarify the mechanistic basis of mBSA uptake.

    Statistical analyses should reflect the biological replicate structure Most imaging datasets contain many nephrocytes derived from a smaller number of animals and experimental trials. Individual cells from the same animal are not independent biological replicates. Several three-group experiments also appear to have been analysed using unpaired t-tests rather than a single ANOVA with appropriate multiple-comparison correction.

    The authors should reanalyse the data using animal- or experimental-trial means as the biological unit, or apply nested or mixed-effects models that account for cells nested within animals. N and n should be defined consistently in every figure legend. This should not require new experiments.

    Response: We thank the reviewer for this suggestion. We will reanalyse the experiments involving three-group comparisons using one-way ANOVA with appropriate multiple-comparison corrections. In addition, where measurements are obtained from multiple cells within the same animal, we will apply mixed-effects models that account for the nested data structure (cells nested within animals).

    Minor comments

    The authors should more clearly acknowledge the differences between the endocytic systems of Drosophila nephrocytes and mammalian proximal-tubule cells, rather than primarily emphasizing their similarities. Most notably, OCRL depletion affects dextran uptake in nephrocytes, whereas fluid-phase endocytosis is reportedly preserved in mammalian proximal-tubule cells. Another important and often underappreciated difference is the absence of the OCRL-related 5-phosphatase INPP5B in Drosophila. This may substantially limit the extent to which OCRL-depleted Drosophila nephrocytes can model the molecular and cellular pathophysiology of Lowe syndrome.

    Response: We thank the reviewer for their suggestion. We will include the comparison of *Drosophila *and mammalian OCRL, the limitations of the nephrocyte system in the discussion of our revised manuscript.

    The discordant results in Supplementary Table 1-complete loss of AgNO₃ uptake in the Trpml-null allele but no phenotype following Trpml RNAi-should be discussed.

    Response: One possible explanation for the observed results is the efficiency of the RNAi knockdown. While the Trpml null allele completely abolishes gene function and results in a complete loss of AgNO₃ uptake, the RNAi-mediated knockdown may not have been sufficient to reduce TRPML levels below the threshold required to produce a detectable phenotype. Consequently, residual TRPML activity in the RNAi background could account for the absence of an observable defect in AgNO₃ uptake.

    LTS-mCherry and Cathepsin L fluorescence should not be described as measures of lysosomal "size" or "expansion" unless organelle area, volume, or number is quantified. The early-endosome-targeted dOCRL experiment mentioned as "data not shown" is central to the proposed compartment-specific model. It should be presented, including localization and expression controls, or removed from the Discussion.

    Response: We will remove the lysosomal size comment from the manuscript. To strengthen the conclusions of the study, we will include the early-endosome-targeted dOCRL rescue experiment, as well as its localisation and expression control data, in the revised manuscript.

    The phosphatase-dead OCRL mutant should be assessed for functional activity by measuring PI(4,5)P₂ following mutant overexpression in OCRL-KO cells. Response: We will include this experiment in the revised manuscript.

    Reviewer #2 (Significance (Required)):

    The potentially important conceptual advance is the observation that a late-endolysosomal-targeted, catalytically active dOCRL construct can suppress not only lysosomal phenotypes but also early-endosomal marker abnormalities and endocytic cargo uptake in vivo. If the spatial specificity of the construct is demonstrated, this would support substantial feedback from late-endolysosomal homeostasis to upstream endocytic function. The role of OCRL at lysosomes and the lysosomal PI(4,5)P₂-TRPML1 pathway are already established in mammalian Lowe syndrome models. The principal potential advance of this study is therefore not the identification of lysosomal dysfunction itself, but the proposal that restoration of lysosomal OCRL activity is sufficient to rescue upstream endocytosis. At present, this conclusion remains uncertain because direct activity of the construct at the plasma membrane, early endosomes, or other non-lysosomal compartments has not been excluded. The data are compatible with an important lysosomal contribution but do not yet establish lysosomes as the unique or primary site of OCRL action. The work should be of interest to researchers studying phosphoinositides, membrane trafficking, lysosomal signalling, renal endocytosis, Drosophila nephrocytes, and Lowe/Dent disease mechanisms. Field of expertise: membrane trafficking, phosphoinositide metabolism, lysosomal biology, inherited renal tubulopathies

    Response: We think that experiments using 2xFYVE-dOCRL and dOCRL::CAAX, which selectively target dOCRL to the early endosomal and plasma membrane compartments, respectively, would be important for assessing the contribution of these compartments to the observed rescue phenotype. In response, we will include rescue experiments using these compartment-specific dOCRL constructs in the revised manuscript.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    __Summary: Progress over recent decades has established a critical role for OCRL in endosomal-lysosomal biology, yet its primary subcellular site of action has remained a central point of debate. In this manuscript, the authors utilize a Drosophila nephrocyte model of Lowe Syndrome to demonstrate that lysosome-specific expression of functional dOCRL (specifically requiring its 5-phosphatase enzymatic activity) is sufficient to rescue key cellular phenotypes. The core conceptual strength of this study lies in its proposal of a "lysosome-first" model suggesting that lysosomal dysfunction is the primary driver of disease pathology in dOCRL-depleted nephrocytes, and that upstream endocytic uptake defects occur secondary to this lysosomal failure. The manuscript is well-written, the experiment is well designed. The data are clear, with proper controls. The results nicely quantified. I only have relatively minor questions for the authors.

    We thank the reviewer for their comments, constructive suggestions, and critical evaluation of our manuscript.

    Minor comments:

    1. The authors state that "dOCRL KO larvae showed lethality at every stage of development when grown on yeast medium"; does this imply that survival rates differ when they are grown on standard media without yeast paste?

    Response: We thank the reviewer for this important point. dOCRLKO larvae exhibit lethality at the third instar larval stage when maintained on standard fly food. However, when reared on yeast paste media, the larvae survive until the pupariation stage and display significantly improved overall health compared with those grown on standard media. To ensure consistency across experimental conditions, all genotypes included in this study were reared on yeast-paste media.

    In Figure 1C and 1E, since the cell sizes differ significantly, with mutants weighing only approx. 50% of controls, it would be beneficial to normalize for cell size or area before calculating the plasma membrane to cytoplasm (PM:Cytoplasm) ratio

    Response: We thank the reviewer for their suggestion. We quantified plasma membrane and cytoplasmic fluorescence intensities by drawing line ROIs specifically across the plasma membrane and within the cytosolic region, rather than measuring the intensity of the entire cell. This approach minimises the potential confounding effects arising from differences in cell size between control and mutant nephrocytes. It is the accepted method of quantifying the levels of protein probes that localize to membranes and report the level of a lipid.

    For the benefit of the reader, it would be helpful if the figures followed a stricter chronological order; for example, Supplementary Figure 5 is referenced immediately after Figure 1.

    Response: We thank the reviewer for pointing this out. We will correct the order of the figures in the revised manuscript to ensure that they are presented in the appropriate chronological sequence.

    Figures 5E and 5F show lower levels of Rabenosyn-5. What is the proposed mechanism for this reduction in early endosomal markers?

    Response: Does the reviewer mean the figures 3E and 3F. We have seen that depleting dOCRL leads to reduced endocytosis and reduced early endosomal compartment stained by Rabenosyn-5. The precise mechanism by which loss of dOCRL leads to reduced early endosomal levels is not yet fully understood. One possibility is that this is a secondary consequence of a broader imbalance within the endolysosomal system. Impaired endocytic uptake in dOCRL deficient nephrocytes could lead to reduced delivery of cargo and membrane into the early endosomal pathway, ultimately resulting in a decrease in early endosome formation or maintenance. A similar reduction in labelling intensity of EEA1 have also been observed in the zebrafish pronephric tubule upon loss of ocrl (PMID: 25838181).

    The rescue of the clearance defect by Rab7CA implies that the accumulation of PI(4,5)P₂ on late endolysosomes might interfere with Rab7 recruitment or activation, thereby stalling the fusion and maturation processes necessary for AgNO3 clearance. A possible explanation for why Rab7CA is sufficient to bypass the need for dOCRL activity during clearance should be highlighted in the Discussion section.

    Response: We thank the reviewer for this insightful suggestion and agree that discussing the potential mechanism underlying the Rab7CA mediated rescue would strengthen the manuscript. We will include this in our revised manuscript.

    Lines 341-343 do not appear to match the referenced figure (Supplementary Figure 2H), which shows results for the Hml-GAL4 driver rather than the Sns-GAL4 driver mentioned in the text.

    Response: We thank the reviewer for identifying this error. We will correct this mistake in the revised manuscript.

    There are a few typographical errors that need correction, such as the repetition of "that" in line 405.

    Response: We thank the reviewer for identifying this error. We will correct this mistake in the revised manuscript.

    Reviewer #3 (Significance (Required)):

    Overall, this study provides a significant shift in how we might view the progression of Lowe Syndrome. By identifying the lysosome as the critical hub for dOCRL function, it opens new therapeutic avenues focused on correcting lysosomal PI(4,5)P₂ levels or acidification rather than targeting early endocytic machinery. The data is robust, the model is appropriate, and the "lysosome-first" hypothesis is a major step forward for the field of endolysosomal homeostasis. While the data are robust and the model is appropriate, the observation that lysosomal dOCRL rescues early endocytic uptake remains "surprising" and suggests a powerful homeostatic feedback loop. How lysosomal dysfunction signals back to inhibit the initial steps of endocytosis? whether through mTORC1/TFEB pathways or the depletion of recycling membrane resources, remains an intriguing question that warrants future study. This study is relevant to broader cell biology community and have future translational values.

    Response: We thank Reviewer #3 for this thoughtful and constructive comment. At present, the mechanism by which lysosomal dysfunction feeds back to inhibit the early stages of endocytosis remains unclear. We agree that understanding this connection represents an important area for future investigation.

    Nevertheless, our observations suggest that such a feedback mechanism exists in nephrocytes. In addition to the dOCRL mutant phenotype described here, we have also observed that impairment of lysosomal function through depletion of the lysosomal TRPML channel also leads to a reduction in mBSA uptake, indicating defects in early endocytic events. These findings support the existence of a feedback loop between lysosomal activity and endocytic uptake that may function to maintain endolysosomal homeostasis.

    __3. __Description of the revisions that have already been incorporated in the transferred manuscript

    Not applicable

    __4. __Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    With respect to assessing lysosomal PI4P and PI(4,5)P₂ levels, these experiments are currently technically challenging in the Drosophila nephrocyte system. Simultaneous expression of phosphoinositide biosensors, compartment-specific markers, Gal4 drivers, and LTS::dOCRL transgenes in either wild-type or dOCRLKO backgrounds requires the generation of flies carrying multiple transgenic elements. In many cases, these complex genetic combinations are difficult to obtain, or maintain limiting our ability to directly quantify lysosomal phosphoinositide pools in vivo at present. We therefore believe that there are time, resource and technical limitations in addressing this one point.

  2. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Progress over recent decades has established a critical role for OCRL in endosomal-lysosomal biology, yet its primary subcellular site of action has remained a central point of debate. In this manuscript, the authors utilize a Drosophila nephrocyte model of Lowe Syndrome to demonstrate that lysosome-specific expression of functional dOCRL (specifically requiring its 5-phosphatase enzymatic activity) is sufficient to rescue key cellular phenotypes. The core conceptual strength of this study lies in its proposal of a "lysosome-first" model suggesting that lysosomal dysfunction is the primary driver of disease pathology in dOCRL-depleted nephrocytes, and that upstream endocytic uptake defects occur secondary to this lysosomal failure. The manuscript is well-written, the experiment is well designed. The data are clear, with proper controls. The results nicely quantified. I only have relatively minor questions for the authors.

    Minor comments:

    1. The authors state that "dOCRL KO larvae showed lethality at every stage of development when grown on yeast medium"; does this imply that survival rates differ when they are grown on standard media without yeast paste?
    2. In Figure 1C and 1E, since the cell sizes differ significantly, with mutants weighing only approx. 50% of controls, it would be beneficial to normalize for cell size or area before calculating the plasma membrane to cytoplasm (PM:Cytoplasm) ratio
    3. For the benefit of the reader, it would be helpful if the figures followed a stricter chronological order; for example, Supplementary Figure 5 is referenced immediately after Figure 1.
    4. Figures 5E and 5F show lower levels of Rabenosyn-5. What is the proposed mechanism for this reduction in early endosomal markers?
    5. The rescue of the clearance defect by Rab7CA implies that the accumulation of PI(4,5)P₂ on late endolysosomes might interfere with Rab7 recruitment or activation, thereby stalling the fusion and maturation processes necessary for AgNO3 clearance. A possible explanation for why Rab7CA is sufficient to bypass the need for dOCRL activity during clearance should be highlighted in the Discussion section.
    6. Lines 341-343 do not appear to match the referenced figure (Supplementary Figure 2H), which shows results for the Hml-GAL4 driver rather than the Sns-GAL4 driver mentioned in the text.
    7. There are a few typographical errors that need correction, such as the repetition of "that" in line 405.

    Significance

    Overall, this study provides a significant shift in how we might view the progression of Lowe Syndrome. By identifying the lysosome as the critical hub for dOCRL function, it opens new therapeutic avenues focused on correcting lysosomal PI(4,5)P₂ levels or acidification rather than targeting early endocytic machinery. The data is robust, the model is appropriate, and the "lysosome-first" hypothesis is a major step forward for the field of endolysosomal homeostasis. While the data are robust and the model is appropriate, the observation that lysosomal dOCRL rescues early endocytic uptake remains "surprising" and suggests a powerful homeostatic feedback loop. How lysosomal dysfunction signals back to inhibit the initial steps of endocytosis? whether through mTORC1/TFEB pathways or the depletion of recycling membrane resources, remains an intriguing question that warrants future study. This study is relevant to broader cell biology community and have future translational values.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary

    The authors establish germline and nephrocyte-specific dOCRL loss-of-function models in Drosophila. They report that dOCRL deficiency is associated with increased plasma-membrane PI(4,5)P₂, decreased plasma-membrane PI4P, reduced dextran and BSA uptake, alterations in early- and late-endosomal markers, lysosomal expansion/dysfunction, and delayed AgNO₃ clearance. They further claim that a late-endolysosomal-targeted dOCRL construct rescues AgNO₃ retention, Cathepsin L accumulation, Rabenosyn-5 staining, and BSA uptake, whereas a presumably phosphatase-dead construct fails to normalize Cathepsin L. The authors conclude that lysosomal OCRL activity is the primary requirement for endolysosomal homeostasis and that the endocytic abnormalities are secondary to lysosomal dysfunction.

    The Drosophila model and compartment-targeting approach are potentially valuable. However, the central conclusion is not adequately supported because the localization and biochemical activity of the targeted construct have not been established in nephrocytes, and several endocytic and lysosomal assays are interpreted beyond what they directly measure. In addition, the authors extend the relevance of these findings too far to the pathophysiology and potential treatment of Lowe syndrome in patients. The study is restricted to Drosophila nephrocytes, and no evidence is provided in mammalian proximal-tubule cells, patient-derived cells, or mammalian disease models that selective restoration of lysosomal OCRL activity is sufficient to rescue endocytosis. The conclusions should therefore remain specific to the Drosophila system, while their relevance to human Lowe syndrome requires validation in mammalian models.

    Major comments

    1. Lysosomal specificity is not convincingly demonstrated. The LTS sequence is intended to target proteins to the cytosolic surface of late endosomes and lysosomes. However, its localization must be independently validated for each fusion protein and in each cell type. In Figure 5B, localization is assessed in S2R+ cells rather than nephrocytes, and by colocalization with LTS-mCherry, which contains the same targeting sequence. This approach is partly circular and does not independently establish that the labelled organelles are lysosomes. Furthermore, their morphology differs from that of Cathepsin L-, Rab7-, and LysoTracker-positive structures. The displayed LTS-dOCRL-GFP signal is not confined to LTS-mCherry-positive structures and includes substantial peripheral and non-overlapping signal. Figure 4 uses LTS-mCherry in nephrocytes, but this reporter is not colocalized with Cathepsin L, LysoTracker, or another independent lysosomal marker. The different patterns observed in Figures 4 and 5 are also difficult to compare because they were obtained in different cell types. Most importantly, the authors do not show: (i) direct measurement of lysosomal PI(4,5)P₂ in dOCRLKO nephrocytes; (ii) reduction of lysosomal PI(4,5)P₂ by LTS-dOCRL; or (iii) measurement of plasma-membrane PI(4,5)P₂ or PI4P following LTS-dOCRL expression. The last point is particularly important because the construct appears to display non-lysosomal signal, including apparent plasma-membrane localization and puncta that do not colocalize with LTS-mCherry. The authors should therefore quantify LTS-dOCRL localization in nephrocytes relative to independent lysosomal, Rab7-positive, and early-endosomal markers; determine whether LTS-dOCRL corrects plasma-membrane PI(4,5)P₂ and PI4P; and assess lysosomal PI(4,5)P₂ in control, dOCRLKO, and LTS-dOCRL-rescued nephrocytes.
    2. The reduction in plasma-membrane PI4P is a major and insufficiently validated finding. The marked reduction in plasma-membrane PI4P in dOCRLKO nephrocytes is potentially important but requires substantially stronger validation. Although OCRL hydrolyses PI(4,5)P₂ to generate PI4P, a reduction in the steady-state bulk plasma-membrane PI4P pool is not a commonly established consequence of OCRL loss. Direct analysis of plasma-membrane phosphoinositides in human platelets showed that OCRL inhibition did not reduce plasma-membrane PI4P in resting or activated platelets, and no change in plasma-membrane PI4P was detected in HEK293T cells. In contrast, inhibition of PI4KIIIα strongly depleted plasma-membrane PI4P, supporting PI4KA, rather than OCRL, as the principal determinant of this pool (Bura and Jurak Begonja, 2021; PMID: 34947862). Similarly, lipidomic analysis of Lowe syndrome patient-derived cells, performed by the same group as in the manuscript under review, did not reveal a consistent reduction in total PI4P/PIP (Akhtar et al., 2022; PMID: 35023542). The best-established phosphoinositide consequence of OCRL deficiency is instead the accumulation of PI(4,5)P₂ on early endosomes, clathrin-coated intermediates, lysosomes and, in some cell types, at the plasma membrane (Vicinanza et al., 2011; Nández et al., 2014). These studies did not identify depletion of bulk plasma-membrane PI4P as a characteristic phenotype of OCRL loss. The finding reported here could therefore represent a nephrocyte-specific effect, an indirect consequence of altered phosphoinositide metabolism, or a limitation of the probe-based measurement. This issue is mechanistically central because a substantial loss of plasma-membrane PI4P could itself affect membrane identity, PI(4,5)P₂ resynthesis, and endocytosis. Moreover, the authors do not determine whether expression of dOCRL or LTS::dOCRL restores plasma-membrane PI4P and PI(4,5)P₂.
    3. The AgNO₃ data do not currently provide a robust quantitative measure of clearance In Figure 1H, the percentage of dOCRLKO nephrocytes classified as containing high AgNO₃ is greater at 42 hours than at 36 hours. Differences in initial loading, imaging conditions, cell size, or thresholding could therefore produce an apparent increase at a later time point. For this reason, the authors should provide measurements of initial AgNO₃ accumulation. Furthermore, they should explain the use of different AgNO₃ concentrations-0.003% in Figure 1 and 0.0015% in Figures 2 and 5-as this limits direct comparison between experiments.
    4. In Figure 3, the authors show reduced dextran and BSA uptake, supporting an endocytic defect. However, lower Rabenosyn-5, 2xFYVE, and Rab7 fluorescence does not demonstrate fewer or smaller endosomes. These signals may instead reflect altered protein abundance, membrane recruitment, or phosphoinositide content. The authors should therefore avoid conclusions regarding reduced endosomal compartment size or preserved endosomal maturation unless organelle number and size are directly quantified. Similarly, Rab7CA rescue identifies Rab7 as a genetic suppressor but does not prove that endogenous Rab7 activity is reduced or that it causes the lysosomal defect. Increased Cathepsin L staining, although diffuse and not clearly localized to intracellular puncta, together with reduced Magic Red activity, suggests accumulation of poorly functional lysosomes rather than increased lysosomal capacity. However, the authors measured Cathepsin B activity using Magic Red substrate MR-(RR)2 , rather than Cathepsin L activity using the Magic Red substrate MR-FR2. The use of Cathepsin L staining as a readout of lysosomal dysfunction may therefore require further characterization. Finally, the authors should test whether Rab7CA restores lysosomal activity, rather than only Cathepsin L abundance.
    5. Figure 3C measures BSA accumulation after a 15-minute incubation, whereas Figure 4I uses an 8-minute pulse followed by a 40-minute chase, showing increased intracellular staining that is interpreted as reduced degradation. Although residual BSA is higher in dOCRLKO cells after the chase, the amount present immediately after the 8-minute pulse was not measured. Consequently, the fraction cleared or degraded cannot be calculated, and the data reported in Figure 4I cannot be directly correlated with those reported in Figure 3C. To support the conclusion of defective degradation, the authors should provide a quantitative assessment of BSA levels immediately after the 8-minute pulse and at multiple chase times, with residual fluorescence expressed relative to the genotype-specific starting signal. The uptake pathway used by BSA in Drosophila nephrocytes also requires clarification. Maleylated BSA is a scavenger-receptor ligand in other cell types, whereas protein uptake in nephrocytes is commonly associated with Cubilin/Amnionless-dependent uptake. The manuscript does not establish which receptor or pathway mediates BSA internalization in nephrocytes.
    6. Statistical analyses should reflect the biological replicate structure Most imaging datasets contain many nephrocytes derived from a smaller number of animals and experimental trials. Individual cells from the same animal are not independent biological replicates. Several three-group experiments also appear to have been analysed using unpaired t-tests rather than a single ANOVA with appropriate multiple-comparison correction. The authors should reanalyse the data using animal- or experimental-trial means as the biological unit, or apply nested or mixed-effects models that account for cells nested within animals. N and n should be defined consistently in every figure legend. This should not require new experiments.

    Minor comments

    The authors should more clearly acknowledge the differences between the endocytic systems of Drosophila nephrocytes and mammalian proximal-tubule cells, rather than primarily emphasizing their similarities. Most notably, OCRL depletion affects dextran uptake in nephrocytes, whereas fluid-phase endocytosis is reportedly preserved in mammalian proximal-tubule cells. Another important and often underappreciated difference is the absence of the OCRL-related 5-phosphatase INPP5B in Drosophila. This may substantially limit the extent to which OCRL-depleted Drosophila nephrocytes can model the molecular and cellular pathophysiology of Lowe syndrome.

    The discordant results in Supplementary Table 1-complete loss of AgNO₃ uptake in the Trpml-null allele but no phenotype following Trpml RNAi-should be discussed. LTS-mCherry and Cathepsin L fluorescence should not be described as measures of lysosomal "size" or "expansion" unless organelle area, volume, or number is quantified.

    The early-endosome-targeted dOCRL experiment mentioned as "data not shown" is central to the proposed compartment-specific model. It should be presented, including localization and expression controls, or removed from the Discussion.

    The phosphatase-dead OCRL mutant should be assessed for functional activity by measuring PI(4,5)P₂ following mutant overexpression in OCRL-KO cells.

    Significance

    The potentially important conceptual advance is the observation that a late-endolysosomal-targeted, catalytically active dOCRL construct can suppress not only lysosomal phenotypes but also early-endosomal marker abnormalities and endocytic cargo uptake in vivo. If the spatial specificity of the construct is demonstrated, this would support substantial feedback from late-endolysosomal homeostasis to upstream endocytic function. The role of OCRL at lysosomes and the lysosomal PI(4,5)P₂-TRPML1 pathway are already established in mammalian Lowe syndrome models. The principal potential advance of this study is therefore not the identification of lysosomal dysfunction itself, but the proposal that restoration of lysosomal OCRL activity is sufficient to rescue upstream endocytosis.

    At present, this conclusion remains uncertain because direct activity of the construct at the plasma membrane, early endosomes, or other non-lysosomal compartments has not been excluded. The data are compatible with an important lysosomal contribution but do not yet establish lysosomes as the unique or primary site of OCRL action. The work should be of interest to researchers studying phosphoinositides, membrane trafficking, lysosomal signalling, renal endocytosis, Drosophila nephrocytes, and Lowe/Dent disease mechanisms.

    Field of expertise: membrane trafficking, phosphoinositide metabolism, lysosomal biology, inherited renal tubulopathies

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    Referee #1

    Evidence, reproducibility and clarity

    In this study the authors explore the role of the Drosophila orthologue of the Lowe syndrome protein OCRL (called dOCRL) in nephrocytes, the functional equivalent of the mammalian kidney. Using genetic knockout and complementation approaches it is shown that dOCRL is required for the clearance of the toxic compound silver nitrate from nephrocytes. This effect is phenocopied by loss of Rab7 and can be rescued by a constitutively active form of Rab7, suggesting involvement of late endosomal or lysosomal compartments. This is supported by experiments showing disrupted lysosomal function upon dOCRL loss. Loss of dOCRL also causes defects earlier in the endocytic pathway, with reduced uptake of the endocytic tracers dextran and mBSA into nephrocytes, and a loss of early endosomal compartments. Interestingly, re-expression of dOCRL targeted to the lysosome rescues the defects in both endosome and lysosome function, suggesting that the defects observed are primarily due to dOCRL function at the lysosome.

    This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate. There are a few things that could be addressed in a revised version which are indicated below:

    1. The ability of lysosome-targeted dOCRL to rescue the phenotypes is interesting. Can the authors exclude the possibility that there is residual cytosolic dOCRL in the cells that can transiently associate with other organelles e.g. early endosomes to drive the phenotypic rescue? There are experiments described in the discussion about targeting dOCRL to early endosomes not rescuing the phenotypes. Currently this is mentioned as data not shown. It should be included.
    2. The results with AgNO3 are confusing. Uptake into nephrocytes is not affected by loss of dOCRL but is affected by other manipulations that disrupt endosomal trafficking (suppl table 1). Yet, loss of dOCRL affects cellular uptake of dextran and mBSA, which rely on endocytosis. More explanation needs to be provided on what we know about AgNO3 uptake into nephrocytes. Is it by endocytosis, as the table in the supplementary table would seem to indicate? In that case, why would AgNO3 uptake not be affected by dOCRL loss? Also, what underlies the clearance of AgNO3 taken up into nephrocytes? If it is already in the cells, then does it leave by exocytosis? Or does it enter and leave cells through transporters? How does dOCRL affect the clearance of AgNO3 from the cells i.e. through what mechanism? Filtration of AgNO3 is also mentioned but not fully explained. How does filtration and clearance of AgNO3 relate to the processes that occur in the mammalian kidney, where filtration of blood is performed by the glomerulus, while clearance is in the proximal tubules, which are the region affected in Lowe syndrome?
    3. The data overall are clear and convincing, but Fig 3C is not. There does not appear to be any reduction in mBSA signal in the dOCRL image shown.
    4. Discussion could be more expansive eg dOCRL versus mammalian OCRL-clathrin binding, localisation etc...
    5. The figures need to appear in the correct order in the text. Currently they are presented in the text in a jumbled way.
    6. References are missing in quite a few places in the text. There are numerous instances of statements being made without the references to support them.
    7. There are a few minor grammatical errors.

    Significance

    This is a very nicely done study. The data are clear and support the authors' conclusions. The means of analysis and the quantitation of the data are appropriate.

  5. Version published to 10.64898/2025.12.03.692239 on bioRxiv