Figure 11.

Myc-SGEF and RhoG stimulate macropinocytosis in NIH3T3 cells. Cells were transiently transfected overnight with expression plasmids for the indicated protein before incubation with Alexa Fluor 594-conjugated 10-kDa dextran in uptake buffer (DMEM w/0.15% BSA) for 5 min. When appropriate, cells were immunostained for the Myc epitope tag to identify expressing cells. Cells were scored as a (-) if dextran uptake was the same as untransfected cells, and as (+) or (++) as macropinocytosis increased. (A) GFP-onco-Vav2, although a strong activator of Rho GTPases, does not significantly promote dextran uptake (-), while Myc-Rac1(Q61L) stimulated uptake (+). Both Myc-SGEF and Myc-RhoG(Q61L) consistently promoted the greatest uptake of dextran (++). (B) Dextran uptake was scored for each condition from three independent coverslips according to the (-), (+), and (++) grading system. Note that stimulation of dextran uptake by SGEF required a full-length catalytically active enzyme with a functional SH3 domain.