Figure 6. Differential transport through dynamin-mediated or raft-mediated endocytosis is required for slit diaphragm maintenance in nephrocytes.

(A–B’’) Confocal images of nephrocytes stained for slit diaphragm proteins carrying a temperature-sensitive variant (G141S) of shibire, the Drosophila dynamin, homozygously. The mutant protein is functional at lower temperatures but lacks function at 30°C and the animals were exposed to 30°C for 2 hr before staining. Cross sections show accumulation of subcortical slit diaphragm protein in clusters and short lines protruding from the surface (A–A’’). Tangential sections indicate a mild confluence and few brighter clusters of slit diaphragm proteins (B–B’’). (C–D’’) Confocal images of control nephrocytes treated with cylodextrin for 2 hr ex vivo to inhibit raft-mediated endocytosis show a regular staining pattern of slit diaphragm proteins in cross-sectional (C–C’’) and tangential planes (D–D’’). (E–H’’) Confocal microscopy images showing tangential sections (panels E and G) and cross sections (panels F and G) of nephrocytes carrying one copy of the genomic Myc-nephrin after live antibody labeling with 2 hr of chase period are for the indicated genotypes or interventions. Shibire^ts animals show intense nephrocyte turnover in the live labeling assay despite exposure to a temperature of 31°C for 2 hr which inhibits function of the fly dynamin during that period (E–F’’). In contrast, blocking raft-mediated endocytosis for 2 hr by cyclodextrin in control nephrocytes strongly diminishes nephrin turnover and a large amount of the live labeled antibody is retained (G–H’’). This suggests that nephrin turnover depends on raft-mediated endocytosis that occurs independent from dynamin function. The diffuse intracellular signal from live labeling was similar to control (Figure 5—figure supplement 2A). (I) Quantitation of results from (E–H’’) expressed as ratio of the fluorescence intensity between surface and subcortical region for individual cells (mean ± standard deviation, n=11–12 animals per genotype, p>0.05 for shibire^ts, and p<0.0001 for cyclodextrin treatment).

Figure 6—figure supplement 1. Validation and controls for Shibire^ts.

(A–B’’) Nephrocytes carrying the temperature-sensitive allele of shibire show a regular staining pattern at a lower temperature at which the protein remains functional in cross sections (A–A’’) and tangential sections (B–B’’). (C–C’’) A chase time of 120 min after live antibody labeling is shown for the temperature-sensitive allele of shibire with Kirre co-staining. Most of the live labeled nephrin is removed from the cell surface while Kirre indicates a severe mislocalization.