Figure 5.

Dominant-negative dyn2(K44A) induces Rac mislocalization. NIH3T3 fibroblasts were injected with plasmids encoding GFP-tagged constitutively active V12Rac and either dyn2(WT) or dyn2(K44A). When the GFP signal indicated proper protein expression, phase contrast and GFP-V12Rac fluorescence images were taken once per minute with a videomicroscope. Arrowheads indicate typical peripheral membrane ruffles (A and B) and abnormal dorsal ruffles (E and F), respectively. Images (A, B, E, and F) are frames of time-lapse videos available as supplemental material. Bar, 20 μm. To characterize the localization of the Rac signal in the ruffles, cells were fixed, stained, evaluated with confocal microspcopy (C, D, G, and H), and z-axis reconstructions (D and H) were generated along the bars indicated in C and G. Bar (C), 30 μm; (G), 25 μm.