Figure 7.

Defective endocytosis reduced nephrocyte function, induced protein aggregation, and increased ROS: (A–H) Function, protein aggregation, and ROS in Drosophila nephrocytes (female, 4-day-old) inhibiting (RNAi; IR) Rab5. Control, Dot > w¹¹¹⁸. (A) 10 kDa dextran (red) and albumin (green) uptake by nephrocytes. Scale bar = 25 µm. (B) Quantitation of 10 kDa dextran fluorescence in Rab4-IR nephrocytes. (C) Quantitation of albumin fluorescence in Rab5-IR nephrocyte. (D) Localization of slit diaphragm protein Polychaetoid (Pyd; red) by immunofluorescence in Drosophila nephrocytes. Imaged at nephrocyte surface. Scale bar = 1 µm. (E) Dihydroethidium (DHE; red) labels reactive oxygen species (ROS); and, DAPI (blue) was used to visualize the nuclei in nephrocytes. Scale bar = 25 µm. (F) Quantitation of DHE fluorescence in Rab5-IR nephrocytes. (G) Anti-ubiquitinylated proteins antibody, clone FK2 (green) was used to label protein aggregates in nephrocytes. Scale bar = 5 µm. Boxed areas are shown magnified to the right (scale bar: 1 μm). (H) Quantitation of FK2 fluorescence in Rab5-IR nephrocytes, relative to control. N = 30 nephrocytes from 6 flies, per group per assay. Results are presented as mean ± SD. Statistical significance (*) is defined as p < 0.05.