Abstract: FR-PO703
Flotillin Mediates Raft-Dependent Turnover of Fly Nephrin Within Slit Diaphragms in Drosophila Nephrocytes
Session Information
- Glomerular Diseases: Podocyte Biology - I
November 04, 2022 | Location: Exhibit Hall, Orange County Convention Center‚ West Building
Abstract Time: 10:00 AM - 12:00 PM
Category: Glomerular Diseases
- 1304 Glomerular Diseases: Podocyte Biology
Authors
- Lang, Konrad, Universitatsklinikum Freiburg Abteilung Innere Medizin IV Nephrologie und Allgemeinmedizin, Freiburg, Baden-Württemberg, Germany
- Milosavljevic, Julian, Universitatsklinikum Freiburg Abteilung Innere Medizin IV Nephrologie und Allgemeinmedizin, Freiburg, Baden-Württemberg, Germany
- Heinkele, Helena, Universitatsklinikum Freiburg Abteilung Innere Medizin IV Nephrologie und Allgemeinmedizin, Freiburg, Baden-Württemberg, Germany
- Helmstädter, Martin, Universitatsklinikum Freiburg Abteilung Innere Medizin IV Nephrologie und Allgemeinmedizin, Freiburg, Baden-Württemberg, Germany
- Poulton, John S., The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States
- Hermle, Tobias F., Universitatsklinikum Freiburg Abteilung Innere Medizin IV Nephrologie und Allgemeinmedizin, Freiburg, Baden-Württemberg, Germany
Background
Evidence from in vitro studies and murine knockout models support a role of endocytosis for the glomerular filtration barrier. Two endosomal regulators, GAPVD1 and TBC1D8B, were discovered as a monogenetic cause of nephrotic syndrome in humans. However, the mechanistic role of endocytosis at the slit diaphragm remains unclear. The nephrocyte model of Drosophila provides an accessible, molecularly conserved podocyte model to study slit diaphragm dynamics.
Methods
Using acute gain- and loss-of function strategies for endosomal regulators in Drosophila nephrocytes, we studied their impact on the trafficking of slit diaphragm proteins in fixed samples and live imaging. Endocytic turnover was investigated by live antibody labeling after genome editing of fly nephrin.
Results
Sns, the Drosophila ortholog of nephrin, exhibits a half-life of about two days after acute knockdown and the architecture of slit diaphragms appears stable in live imaging short term. However, live antibody labeling of fly nephrin revealed a near complete turnover after two hours, confirmed by FRAP analysis. This suggests that slit diaphragms form a stable yet very dynamic structure. Acute knockdown of Rab5, a small GTPase and key regulator of early endosomes, caused the formation of ectopic slits as well as a severely diminished Sns turnover. Silencing of the early endosomal protein Hrs phenocopied Rab5-RNAi. Diminished endocytosis further altered characteristics of the filtration barrier, selectively reducing passage of tracers close to the upper size limit. Silencing of Rab11, which promotes recycling, led to a slower turnover of Sns. Rab7, a regulator of degradation, had no overt impact on slit diaphragm formation or turnover, suggesting that degradation is dispensable for maintenance. To study the endocytic routes of entry, we acutely blocked dynamin-dependent endocytosis, which caused ectopic slits but a normal Sns turnover. Conversely, exposure to Cyclodextrin or inhibition raft-mediated flotillin endocytosis reduced the turnover significantly.
Conclusion
Dynamin-dependent endocytosis prevents the formation of ectopic slit diaphragms in nephrocytes. Flottillin-dependent endocytosis dominates turnover of nephrin within the slit diaphragm.
Funding
- Government Support – Non-U.S.