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Abstract: PO1340

Drosophila TBC1D8B Promotes Nephrin Endocytosis and Is Required for Endosomal Cargo Processing

Session Information

Category: Genetic Diseases of the Kidneys

  • 1002 Genetic Diseases of the Kidneys: Non-Cystic

Authors

  • Milosavljevic, Julian, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Lempicki, Camille, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Lang, Konrad, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Kampf, Lina Luise, Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
  • Hermle, Tobias F., Renal Division, Department of Medicine, Faculty of Medicine and Medical Center - University of Freiburg, Freiburg, Germany
Background

Mutations in TBC1D8B were recently identified as a monogenic cause of nephrotic syndrome. TBC1D8B interacts with nephrin and it was implicated as an inhibitory GAP protein for Rab11 which regulates endocytic recycling. However, the functional spectrum of TBC1D8B and its role in trafficking of nephrin remains poorly understood.

Methods

We generated and analyzed a stable genetic deletion of fly Tbc1d8b via CRISPR/Cas9. We successfully introduced a c-terminal HA-tag into the Tbc1d8b locus using microhomology-mediated end joining. For overexpression of murine Tbc1d8b-HA we further generated transgenic flies under control of GAL4/UAS. Endosomal cargo processing was assessed by sequential uptake of two tracers ex vivo.

Results

Germline expression of Cas9 and tandem Tbc1d8b-guide RNAs resulted in a stable genetic deletion spanning all functional domains while ending with a frameshift. Surprisingly, homozygous mutant animals were viable without any overt phenotype. However, analysis of the podocyte-like nephrocytes of these flies revealed mislocalization and partial loss of slit-diaphragm proteins with incomplete penetrance. This nephrocyte-restricted phenotype recapitulates the phenotype of human mutations that present exclusively with nephrotic syndrome. Overexpression of murine Tbc1d8b-HA in nephrocytes caused accumulation of endogenous fly nephrin in large vesicles while its binding partner Kirre (Neph1) was not affected. The vesicles containing fly nephrin co-localized with the late endosomal marker Rab7 and disappeared when silencing the early endosomal regulator Rab5. This suggests that mammalian Tbc1d8b specifically promotes nephrin endocytosis. To identify the endogenous subcellular localization of the fly Tbc1d8b we established a knock-in of a c-terminal HA-tag and noted partial co-localization with endosomal markers including Rab7. To investigate the functional role in the endolysosomal pathway we tracked the fate of two tracers sequentially applied ex vivo. A background of both, stable and conditional deletion of Tbc1d8b was associated with defective cargo processing.

Conclusion

Our findings implicate novel functional roles of Tbc1d8b beyond endocytic recycling: promoting nephrin endocytosis and facilitating processing of endosomal cargo.

Funding

  • Government Support – Non-U.S.