Figure 3. Endosomal regulator Rab5 is required for maintenance of slit diaphragms.

(A) Schematic illustrating endocytic trafficking in a simplified manner shows raft-mediated and clathrin-mediated uptake converging in the early endosome by vesicle fusion. Uptake, early endosome formation and cargo sorting are controlled by Rab5. Sorting may direct cargo either toward degradation, which is promoted by Rab7, or back toward the cell membrane by recycling pathways such as Rab11-dependent recycling. (B–B’’) Cross-sectional confocal microscopy images from nephrocytes expressing constitutively active YFP-Rab5 for 24 hr (green) show highly enlarged early endosomes that contain ectopic fly nephrin (see also magnified inset). Nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (C) Confocal images of nephrocytes with acute silencing of Rab5 for 17 hr reveals brighter sections within the lines of slit diaphragm protein in tangential sections. Lines further are blurry and focally confluent (see also magnified inset). (D) Cross-sectional images of nephrocytes with short-term silencing of Rab5 show appearance of ectopic slit diaphragm protein below the surface (compare to control Figure 3—figure supplement 1A-A"). (E–F) Tangential sections (E) and cross sections (F) of nephrocytes with slightly longer silencing of Rab5 for 24 hr stained for nephrin (Sns) and Neph1 (Kirre) reveal progressive thickening of slit diaphragms and localized breakdown of the slit diaphragms in a circumscribed area (white arrowheads). (G–G’’’) Snapshots from a movie obtained by live-cell imaging using confocal microscopy are shown. Nephrocytes expressing nephrin-GFP (heterozygously) are shown after 24 hr of acute Rab5 silencing. Increasing gaps and a progressive reduction of slit diaphragms are observed over the course of 1 hr. Cells with a mild phenotype were chosen for live-cell imaging to ensure cellular viability. The nephrin signal in tangential sections appears slightly less blurry compared to untagged nephrin. (H–H’’) Confocal microscopy images showing cross sections of nephrocytes after 24 hr of Rab5 silencing are shown. Living cells were exposed to FITC-albumin (green) for 15 min before fixation and staining for nephrin (red). Cells show significant endocytosis of FITC-albumin indicating cell viability and residual endocytic activity despite silencing of Rab5. Ectopic nephrin and FITC-albumin do not colocalize, indicating that ectopic nephrin is not found within a subcellular compartment that is also destination for recently endocytosed cargo.

Figure 3—figure supplement 1. Validation and control experiments for loss-of-function of Rab5.

(A–B’’) Shown are a cross section (A–A’’) and a tangential section (B–B’’) of a garland cell nephrocyte that expresses GAL80^ts alone, stained for Nephrin (Sns) and Neph1 (Kirre). Nuclei are marked by Hoechst 33342 in blue here and throughout the figure. (C–D’’) Rab5 stains in small vesicles at the cell periphery in control nephrocytes (C–C’’). Silencing Rab5 strongly diminishes the Rab5 signal and fly Neph1 reveals mislocalization. This indicates that short-term silencing is sufficient for a significant knockdown of Rab5. (E–F’) Nephrocytes expressing Rab5-RNAi for 24 hr were subject to transferase dUTP nick end labeling (TUNEL) staining but no specific signal from the nuclei is observed (E, compare to Hoechst 33342 in E’), indicating that cells are not apoptotic. In contrast, when silencing fly nephrin as a positive control, we observed appearance of TUNEL-positive cells (F–F’). (G–H’’) Nephrocytes with short-term expresison of a dominant negative Rab5 for 24 hr show ectopic slit diaphragm protein below the surface in cross sections (G–G’’) and blurry and confluent lines of slit diaphragm protein in tangential sections (H–H’’).