Drosophila Nephrocyte Genetics Protocol

Flies were reared on standard food at room temperature, 18°C, 25°C, or 31°C as indicated. Overexpression and transgenic RNAi studies were performed using the UAS/GAL4 system (RNAi crosses grown at 25°C or 31°C). Nephrocyte indicates the subtype of garland cell nephrocytes throughout the manuscript. Stocks obtained from the Bloomington Drosophila Stock Center (BDSC) were UAS-nephrin(sns)-RNAi (#64872), UAS-Rab5-RNAi (#34832), UAS-Rab5^S43N (dominant negative) (#42704), UAS-YFP-Rab5^Q88L (constitutively active, #9774), UAS-Rab7-RNAi (#27051), UAS-YFP-Rab7^T22N (dominant negative #9778), UAS-Rab11-RNAi (#42709), UAS-flo2-RNAi (#40833), and Shibire^ts (#2248). The second UAS-flo2-RNAi (#330316) and UAS-nephrin(sns)-RNAi-2 (VDRC #109442) were provided by the Vienna Drosophila RNAi Center (VDRC), prospero-GAL4 (Weavers et al., 2009 (link)) and Dorothy-GAL4 (#6903; BDSC) were used with or without tub-GAL80^ts (#7018; BDSC) to control expression in nephrocytes. UAS-GFP-RNAi (#41553; BDSC) or wild-type (yw¹¹¹⁸) were crossed to GAL4-drivers as control.

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Lang K., Milosavljevic J., Heinkele H., Chen M., Gerstner L., Spitz D., Kayser S., Helmstädter M., Walz G., Köttgen M., Spracklen A., Poulton J, & Hermle T. (2022). Selective endocytosis controls slit diaphragm maintenance and dynamics in Drosophila nephrocytes. eLife, 11, e79037.

Publication 2022

prospero-GAL4 Dorothy-GAL4 tub-GAL80ts UAS-GFP-RNAi

Drosophila Flies Food Nephrin Rnai Transgenic

Corresponding Organization :

Other organizations : University of Freiburg, University Medical Center Freiburg, University of North Carolina at Chapel Hill

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Variable analysis

independent variables

Temperature (18°C, 25°C, or 31°C)

dependent variables

Nephrocyte function

control variables

Standard food
Room temperature

controls

Positive controls: UAS-GFP-RNAi, wild-type (yw^1118)
Negative controls: UAS-GFP-RNAi, wild-type (yw^1118) crossed to GAL4-drivers

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