Fig. 6

Ultrastructural changes in Clc- and shi-silenced nephrocytes. a–c Transmission electron microscopy (TEM) images of fly nephrocytes. Scale bar: 100 nm. a A wild type (Control) fly nephrocyte shows the typical characteristic ultrastructural features of regularly spaced slit diaphragms (black arrow) spanning the openings of lacuna channel membrane invaginations (asterisk). b Clc gene silencing (Clc-IR) eliminated the lacuna channels and slit diaphragm structures in the nephrocyte. Note the presence of many small vesicles as well as a vesicle undergoing endocytosis (arrowhead). c Slit diaphragm and lacuna channel structures were disrupted in shi-silenced (shi-IR) nephrocytes. The thickness of basement membrane increased significantly (see e). Note the presence of many Ω-shaped clathrin-coated pits at the cell membrane (white arrow). d Quantification of the number of slit diaphragms. In the TEM image, the cell membrane was divided into two 1 μm sections, then the number of slit diaphragms within each section was counted. At least seven sections were counted for each genotype. The results are presented as mean ± SD. Results were analyzed by Student’s t-test. ***P < 0.001. Clc-IR and shi-IR nephrocytes showed near total depletion of slit diaphragms. e Quantification of the thickness of the basement membrane. In the TEM image, the thickness of the basement membrane was measured in 1 μm intervals. At least ten measurements were obtained for each genotype. The results are presented as mean ± SD. Results were analyzed by Student’s t-test. ***P < 0.001. Membrane thickness in Clc-IR nephrocytes was unchanged when compared to wild type (Control). On the other hand, the basement membrane after silencing shi (shi-IR) was significantly thicker than that of wild type (Control) nephrocytes