Abstract: SA-PO206

Rap1 and Its Guanine Nucleotide Exchange Factor C3G Are Critical for Drosophila Nephrocyte Filter Function

Session Information

  • Glomerular: Cell Biology
    November 04, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Glomerular

  • 1003 Glomerular: Cell Biology

Authors

  • Dlugos, Christopher Philipp, Universitiy Hospital Münster, Münster, Germany
  • Picciotto, Cara, University Hospital Münster, Münster, Germany
  • Jeibmann, Astrid, Institute of Neuropathology, Münster, Germany
  • Krahn, Michael P., University of Muenster, Muenster, Germany
  • Wedlich-Söldner, Roland, University of Muenster, Muenster, Germany
  • Pavenstädt, Hermann, UKM Münster, Münster, Germany
  • Klämbt, Christian, University of Muenster, Muenster, Germany
  • George, Britta, Universitiy Hospital Münster, Münster, Germany
Background

Podocytes are crucial for kidney filter function. Podocyte damage is associated with a broad range of glomerular diseases with defective filter function based on aberrant intercellular junctions (slit diaphragms) and actin cytoskeletal dynamics resulting in renal failure and over time to end stage renal disease. Patients with mutations in the human gene encoding the slit diaphragm protein Nephrin do not develop functional slit diaphragms leading to a severe proteinuria. Drosophila nephrocytes form a slit diaphragm-like filtration barrier and express the Nephrin orthologue sticks-and-stones (sns). Nephrocytes represent a genetically tractable model to study Nephrin signaling in vivo. This study aims to elucidate Nephrin signal transduction to the actin cytoskeleton and focal adhesions.

Methods

To characterize the role of genes of interest in Drosophila nephrocyte filter function the secreted protein Atrial Natriuretic Factor (ANF-GFP-GFP) which is taken up by nephrocytes with functional filtration slits was ectopically expressed by the ubiquitin promoter (ubi::ANF-GFP-GFP). The sns::Gal4 transgene allowed nephrocyte-specific expression of UAS-dsRNA by crossing the respective strains. Standard immunofluorescence and transmission electron microscopy were employed to analyze nephrocyte phenotypes.

Results

Co-immunoprecipitation experiments revealed an interaction of Nephrin and guanine nucleotide exchange factor C3G in HEK lysates. Engagement of a chimeric Nephrin molecule which results in downstream signaling events led to activation of integrin beta1 in podocytes. In vivo, nephrocyte-specific knockdown of the Nephrin orthologue sns as well as C3G or roughened (mammalian Rap1) compromised filtration, shown by impaired ANF-GFP-GFP uptake into Drosophila nephrocytes. Altered localization of integrin and the integrin-associated protein talin was observed following knockdown of sns. Ultrastructurally, knockdown of either sns, C3G or roughened resulted in loss of nephrocyte filtration slits.

Conclusion

Drosophila melanogaster is an effective model system to study nephrocyte function and Nephrin signaling. C3G interacts with Nephrin and is necessary for nephrocyte function.

Funding

  • Government Support - Non-U.S.