Figure 6.

Modulation of RhoA-dependent apical protrusions by a radixin carboxyl-terminal domain mutant. R12 cells were transfected with the RADC mutant alone (a and b), or RADC and RhoAV14 (c and d). HA-RADC localization shown in panels a and c, and F-actin as visualized by rhodamine phalloidin are shown in b and d. Note colocalization of RADC with stress fibers in absence of RhoAV14 (arrows in a and b) and localization of actin into profuse stress fibers and long apical processes containing RADC in the presence of RhoAV14 (arrows in c and d). NIH3T3 cells were transfected with RhoAV14 and full-length radixin (e) or RhoAV14 and RADC mutant (f) and then serum-starved and subjected to confocal microscopy analysis. Panels e and f represent combined optical sections of HA-radixin (e) or HA-RADC (f) immunolocalization. NIH3T3 cells were transfected with lacZ and RhoAV14 (g) or lacZ, RhoAV14, and RADC (h) and then serum-starved and subjected to double immunostaining for lacZ and another marker of RhoA-induced apical structures, CD44. CD44 immunolocalization is shown in g and h. LacZ-positive cells (i.e., transfectants) are indicated by arrows. Transfections were performed in four independent experiments. Twenty-five percent of the cells transfected with RADC and RhoAV14 displayed the extremely long apical protrusions as seen in panels c, f, and h, whereas cells transfected with RhoA alone or RhoA plus full-length radixin never showed this phenotype. These results suggest that the presence of a mutant form of radixin can directly alter the size and number of RhoA-induced apical protrusions, but that additional factors (e.g., cell cycle) may control the full expressivity of this phenotype. Bar, 10 μm.