Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes
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Evaluation Summary:
This paper would be of interest to all researchers who work in understanding the mechanisms involved in podocyte slit diaphragm homeostasis and maintenance of the glomerular filtration barrier. The work provides substantial new insights into nephrin dynamics and the mechanisms of slit diaphragm maintenance. A series of compelling experiments depicted that dynamin-mediated endocytosis was involved in ectopic nephrin turnover and that flotillin-mediated turnover of nephrin occurred within the slit diaphragm was needed to maintain filter permeability in-vivo.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Abstract
The kidneys generate about 180 l of primary urine per day by filtration of plasma. An essential part of the filtration barrier is the slit diaphragm, a multiprotein complex containing nephrin as major component. Filter dysfunction typically manifests with proteinuria and mutations in endocytosis regulating genes were discovered as causes of proteinuria. However, it is unclear how endocytosis regulates the slit diaphragm and how the filtration barrier is maintained without either protein leakage or filter clogging. Here, we study nephrin dynamics in podocyte-like nephrocytes of Drosophila and show that selective endocytosis either by dynamin- or flotillin-mediated pathways regulates a stable yet highly dynamic architecture. Short-term manipulation of endocytic functions indicates that dynamin-mediated endocytosis of ectopic nephrin restricts slit diaphragm formation spatially while flotillin-mediated turnover of nephrin within the slit diaphragm is needed to maintain filter permeability by shedding of molecules bound to nephrin in endosomes. Since slit diaphragms cannot be studied in vitro and are poorly accessible in mouse models, this is the first analysis of their dynamics within the slit diaphragm multiprotein complex. Identification of the mechanisms of slit diaphragm maintenance will help to develop novel therapies for proteinuric renal diseases that are frequently limited to symptomatic treatment.
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Evaluation Summary:
This paper would be of interest to all researchers who work in understanding the mechanisms involved in podocyte slit diaphragm homeostasis and maintenance of the glomerular filtration barrier. The work provides substantial new insights into nephrin dynamics and the mechanisms of slit diaphragm maintenance. A series of compelling experiments depicted that dynamin-mediated endocytosis was involved in ectopic nephrin turnover and that flotillin-mediated turnover of nephrin occurred within the slit diaphragm was needed to maintain filter permeability in-vivo.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The Drosophila nephrocyte is a promising model to analyze filtration function and nephrocyte diaphragm integrity and podocyte function. In this study, the authors developed assays to examine slit diaphragm dynamics directly after short-term manipulation of endocytic functions to examine the filtration barrier's dynamics, specifically by examining nephrin. The authors demonstrated that lateral diffusion of ectopic nephrin is prevented by rapid dynamin-dependent endocytosis restricting slit diaphragm localization. In contrast, nephrin engaged within the proper slit diaphragm complex is constantly endocytosed flotillin2-dependently followed by recycling. The findings suggest that such turnover offers flexibility and cleanses the filtration barrier from adherent molecules, preserving its permeability. Their …
Reviewer #1 (Public Review):
The Drosophila nephrocyte is a promising model to analyze filtration function and nephrocyte diaphragm integrity and podocyte function. In this study, the authors developed assays to examine slit diaphragm dynamics directly after short-term manipulation of endocytic functions to examine the filtration barrier's dynamics, specifically by examining nephrin. The authors demonstrated that lateral diffusion of ectopic nephrin is prevented by rapid dynamin-dependent endocytosis restricting slit diaphragm localization. In contrast, nephrin engaged within the proper slit diaphragm complex is constantly endocytosed flotillin2-dependently followed by recycling. The findings suggest that such turnover offers flexibility and cleanses the filtration barrier from adherent molecules, preserving its permeability. Their discovery that selective and functionally distinct routes of endocytic transport of components of the slit diaphragm to maintain the barrier's architecture and permeability is remarkable and could have clinical significance.
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Reviewer #2 (Public Review):
The presented work focusses on a novel Drosophila nephrocytes in-vivo model to investigate nephrin (sns) turnover, which is known to be subjected to endocytosis. The authors present their work in a clear way with well-prepared figures. The added schemes are very helpful to visualize the observed findings.
The current version of the presented data would benefit from more convincing data to prove endocytosis in the pulse-chase experiments.
Once established this fly model could be of great potential in testing substances and proteins influencing nephrin endocytosis.
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Reviewer #3 (Public Review):
Lang et al. provide high-quality data about the maintenance and functioning of the fruit fly nephrocyte slit diaphragm, a topic that has not been investigated formerly with such sophisticated (live) imaging techniques in an in vivo model. Images and videos provided by the authors give spectacular insights into the subcellular events that contribute to diaphragm maintenance and dynamics. They created genetic tools that are suitable for monitoring the trafficking of Sns, the Drosophila ortholog of Nephrin, one of the main components of the mammalian slit diaphragm. Using nephrin-GFP, the authors are the first to follow the stability and turnover rate of this slit diaphragm component. With a variety of labeling experiments (including live imaging of diaphragm proteins, channel diffusion assay, and a live …
Reviewer #3 (Public Review):
Lang et al. provide high-quality data about the maintenance and functioning of the fruit fly nephrocyte slit diaphragm, a topic that has not been investigated formerly with such sophisticated (live) imaging techniques in an in vivo model. Images and videos provided by the authors give spectacular insights into the subcellular events that contribute to diaphragm maintenance and dynamics. They created genetic tools that are suitable for monitoring the trafficking of Sns, the Drosophila ortholog of Nephrin, one of the main components of the mammalian slit diaphragm. Using nephrin-GFP, the authors are the first to follow the stability and turnover rate of this slit diaphragm component. With a variety of labeling experiments (including live imaging of diaphragm proteins, channel diffusion assay, and a live labeling pulse/chase approach, which are all more or less novel in the field) the authors dissected the role of different endocytosis routes in slit diaphragm maintenance. They show that dynamin-mediated and lipid raft-mediated endocytosis pathways cooperate to establish the strictly organized pattern of diaphragm proteins while also enabling their dynamic turnover and filter cleaning. In their model, dynamin-mediated endocytosis restricts lateral diffusion of diaphragm proteins thus maintaining the strictly organized peripheral fingerprint-like distribution of diaphragm units, this way excluding them from the labyrinthine channels. Block of dynamin-mediated endocytosis leads to the formation of ectopic diaphragm protein deposits, while the turnover of diaphragm proteins is not inhibited based on live labeling experiments. In contrast, perturbing raft-mediated endocytosis via either cyclodextrin treatment or flotillin2 knockdown inhibits nephrin turnover without ectopic diaphragm deposits. Based on the decreased uptake of a larger endocytic tracer compared to a smaller one in the case of defective raft-mediated endocytosis, the authors propose that impaired nephrin turnover leads to clogging of the filter. They claim that this might be caused by defects in the shedding of nephrin-bound molecules that would normally occur in the endosomal system right before recycling. The model established by the authors is convincingly supported by their data. Discriminating between the lateral mobility and turnover/cleaning of diaphragm proteins is the major strength of the study, representing a conceptual novelty in the field. In addition, the new methods and tools also serve as useful resources for researchers with similar interests.
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