Figure 5. Endocytic uptake and Rab11-dependent recycling are required for slit diaphragm maintenance.

(A–A’’) Stainings of Rab7-RNAi nephrocytes reveal an additional faint signal for nephrin but not for Neph1 that likely reflects accumulation of nephrin upon defective degradation. Tangential sections (insets) show a regular fingerprint-like pattern, indicating undisturbed slit diaphragm formation. Nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (B) Electron microscopy (EM) image of Rab7-RNAi nephrocyte shows normal slit diaphragms and large vesicles. (C) EM of nephrocyte expressing Rab11-RNAi reveals reduction of labyrinthine channels with multiple slits close to the cell surface (see inset) and expansion of lysosomes (red asterisks, see also magnified inset). Scale bar represents 0.2 µm. (D) FITC-albumin endocytosis as assay for nephrocyte function shows reduced uptake for Rab7-RNAi (lower panels) and Rab11-RNAi (upper panels) using Dorothy-GAL4 or prospero-GAL4 compared to the respective controls. (E) Quantitation of results from (D) in ratio to a control experiment performed in parallel (mean ± standard deviation, n=11–14 animals per genotype, p<0.0001 for Rab7-RNAi and n=9 animals per genotype p<0.0001 for Rab11-RNAi). Sidak post hoc analysis was used to correct for multiple comparisons. (F–K’’) Confocal microscopy images of tangential sections (F–F’’, H–H’’, J–J’’) and cross sections (G–G’’, I–I’’, K–K’’) of Myc-nephrin nephrocytes after live antibody labeling and 2 hr of chasing are shown for the indicated genotypes. Silencing of Rab5 at 18°C was obtained before flies were adapted to 25°C for 1 hr (F-G’’). Live labeling (green) and total stain (red) show near-complete colocalization for Rab5-RNAi (F–G’’), indicating disrupted nephrin turnover. Extensive amounts of subcortical nephrin are revealed in cross sections (G–G’’), compatible with lateral diffusion into the membrane invaginations. Cells expressing Rab7-RNAi after live antibody labeling show undisturbed nephrin turnover as the live labeled antibody is removed from the surface (H–H’’). Cross sections of Rab7-RNAi nephrocytes reveal numerous subcortical vesicles that partially show isolated signal for the live labeling, indicating the antibody disengaged from nephrin (I–I’’). Nephrocytes expressing Rab11-RNAi show strong retention of live labeled nephrin on the cell surface (J–J’’), suggesting impaired turnover. Cross sections show the antibody on the surface, but not in labyrinthine channels (K–K’’). (L) Quantitation of results from (F–K’’) expressed as ratio of the fluorescence intensity between surface and subcortical region for individual cells (mean ± standard deviation, n=11–13 animals per genotype, p<0.0001 for Rab5-RNAi, p>0.05 for Rab7-RNAi and p<0.0001 for Rab11-RNAi). (M) Schematic illustrates findings studying nephrin live labeling upon silencing of Rab5/Rab7/Rab11.

Figure 5—figure supplement 1. Validation and controls for Rab7 and Rab11.

(A–A’’) Slit diaphragms are formed regularly upon expression of dominant negative Rab7, while nephrin accumulates diffusely in the cell. Fly Neph1 is less affected than fly nephrin upon silencing of Rab7. (B–C’’) Control nephrocytes expressing prospero-GAL4 alone (B–B’’) show the regular staining pattern of fly nephrin (Sns) and Rab7. Signal of the Rab7 antibody is lost upon expression of Rab7-RNAi (C–C’’). (D–E’’) Acute silencing of Rab11 for 24 hr in nephrocytes results in coarser, wider spaced dots in cross sections (D–D’’) matching wider gaps between the lines of slit diaphragm proteins in tangential sections (E–E’’). Slit diaphragm proteins may occasionally occur independently from each other (inset in E–E’’). (F–G’’) Short-term expression of Rab11-RNAi strongly diminishes the signal derived from an antibody raised against human Rab11 (compare F–F’’ to G–G’’) suggesting an efficient knockdown.

Figure 5—figure supplement 2. Additional images for live antibody.

(A–A’’) Confocal images of control nephrocytes that express Myc-nephrin heterozygously show complete turnover, not distinguishable from cells that carry the genomed-edited locus homozygously (compare to Figure 2). (B–C’’) Confocal images of nephrocyte expressing Rab7-RNAi after live antibody labeling show subcortical vesicles that exclusively stain for the live labeled antibody (green) but not for nephrin staining, suggesting they contain antibody that is no longer coupled to nephrin (B–B’’). Tangential sections from the same cell confirm undisturbed nephrin turnover as the live labeled antibody is removed from the surface (C–C’’). Nuclei are marked by Hoechst 33342 in blue here and throughout the figure.