Figure 5.

A surplus of SdpI and -II SH3 domain inhibits transferrin internalization in vivo. a, HeLa cells transiently transfected with SdpI SH3 domain, SdpII SH3 domain, the P434L mutant form of the SdpI SH3 domain, or the SdpI NH₂ terminus were incubated at 37°C with FITC-transferrin for 30 min, fixed, and processed for immunofluorescence microscopy. Expression of syndapin constructs was visualized with anti-Xpress antibodies, followed by a Texas red-conjugated secondary antibody. Panels show superimposition of FITC–transferrin with the staining of the epitope-tagged protein fragments. SdpI and -II SH3 domains inhibited endocytosis as evidenced by a block in accumulation of intracellular transferrin (green signal) in transfected cells (red fluorescence) compared with neighboring untransfected cells. In contrast, transfection of cells with the NH₂ terminus or the mutant SH3 domain had no effect on transferrin accumulation. b, Quantitation of the results by assessing the percentage of transferrin uptake-positive cells demonstrates that a surplus of the SH3 domain of syndapin interferes with receptor-mediated endocytosis in vivo. Whereas 97.4 ± 0.3% (n = 956) of the HeLa cells overexpressing the NH₂ terminus of SdpI and 97.0 ± 0.3% (n = 366) of cells overexpressing the P434L mutant of the SdpI SH3 domain were capable of clathrin-mediated endocytosis, only 44.4 ± 3.4% (n = 519) of the cells overexpressing the wild-type SdpI SH3 domain and 47.6 ± 4.4% (n = 450) of cells overexpressing the SdpII SH3 domain contained some internalized transferrin.