Figure 7.

Only full-length proteins are sufficient to induce cytoskeletal rearrangements upon overexpression. HeLa cells transiently transfected with SdpI full-length (a and b), but not the SdpI SH3 domain alone (c and d), the SdpI full-length P434L mutant (e and f), the NH₂ terminus of SdpII-l (g and h), or SdpII-s (i and j) exhibit filopodia. 48 h after transfection, cells were fixed and stained for expression of the syndapin constructs (anti-Xpress labeling; left) and filamentous actin (phalloidin–Texas red staining; right). Bar, 25 μm. k, Overexpression of syndapin full-length proteins dramatically increased the number of filopodia observed at the cell surface of HeLa cells from 2.9 ± 2.3 filopodia/100 μm in untransfected to 52.4 ± 9.9, as observed by confocal microscopy (n = 15 cells each; Σ surface length analyzed = 2,361 μm and 1,630 μm for SdpI-overexpressing and untransfected cells, respectively. l, HeLa cells transfected with plasmids encoding wild-type or mutant syndapin proteins, or fragments thereof, were classified into those exhibiting numerous filopodia or those with almost no filopodia at their cell. Thereafter, these cells were analyzed for expression of the protein encoded by the transfected construct. Bars represent the mean ± standard deviation of the percentage of cells with filopodia induction from at least three independent experiments (n > 250 for each construct). Only wild-type full-length syndapin proteins potently triggered filopodia formation. Massive filopodia induction was observed in 97.7 ± 0.8% of cells overexpressing SdpI and 86.5 ± 2.4% and 70.0 ± 2.5% of cells overexpressing the short and long splice variants of SdpII (SdpII-s and SdpII-l), respectively. Percentages of filopodia-positive in cells overexpressing truncated syndapin constructs (5.2 ± 2.7% and 6.9 ± 1.4% for the SH3 domain of SdpI and -II, respectively, and 6.4 ± 1.6%, 9.7 ± 1.5%, and 6.8 ± 4.5% for the NH₂-terminal parts of SdpI, -II-s, and -II-l, respectively) were quite similar to levels observed in untransfected cells (4.5 ± 2.2%). Even a single point mutation in the SH3 domain, P434L, abolished the ability of SdpI to trigger filopodia formation at the cell cortex (4.0 ± 1.7%; SdpIm).