Fig. 2

The endocytosis regulator, Shi, is required for nephrocyte function and localization of slit diaphragm protein. a Immunofluorescent labeling in wild type (Control) adult fly nephrocytes exhibited tight co-localization of Pyd (green) and Sns (red) at the cell margin, defining a fine and sharply continuous ring when examined by confocal microscopy (a, medial optical section). Detection of Pyd and Sns (a′–a″) at the cell surface plane showed a highly regular and fingerprint-like distribution pattern. Cell nuclei are labeled by DAPI (blue). Scale bar: 1 μm. b–b″ Immunofluorescent labeling in shi-silenced (shi-IR) adult fly nephrocytes revealed disturbed Pyd and Sns localization at the nephrocyte cell margin and were found to have accumulated intracellularly with appearance of aggregates (b). The characteristic cell surface localization had been severely disrupted, visible as dots of seemingly Pyd and Sns aggregates and overall cell surface expression of Pyd and Sns was greatly reduced (b′–b″). c–d Immunofluorescence labeling of Pyd (green) and Clc (red). In wild type (Control) nephrocytes (c–c″), Pyd localized at the cell membrane. Clc localized to vesicles adjacent to the cell membrane. In shi-silenced (shi-IR) nephrocytes (d–d″), Pyd was mostly absent at the cell membrane, but can be occationally found in extraordinary large Clathrin-coated vesicles (arrowhead). Scale bar: 1 μm