Figure 1

(A) Activated RhoA relocalizes both endogenous and transiently expressed ezrin into actin-containing dorsal protrusions. NIH 3T3 cells were transfected with 0.3 g of the RhoA.V14 expression plasmid pEF.RhoA.V14 or its parental vector EFplink (top panels), or with these plasmids together with 0.6 g of the ezrin expression plasmid pBC6-EzrinWT (lower panels). After 18 h, ezrin and F-actin were detected as described in Materials and methods. The nuclear endogenous ezrin staining is non-specific. RhoA expression was confirmed by 9E10 staining (data not shown). Each panel shows a Z stack of confocal images. (B) Relocalization of ezrin by RhoA effector loop mutants. Cells were transfected with 0.6 g of pBC6-EzrinWT as above together with 0.3 g each of expression plasmids encoding the indicated RhoA.V14 effector loop mutants. Transfected cells are indicated by arrowheads. Binding affinities of the effector loop mutants for the Rho-binding domains of ROCK, PKN and citron kinases are as follows (Sahai et al., 1998) (+, binding; -, no binding; +/-, intermediate binding): F39A: PKN-, ROCK-, CitronK-; F39V: PKN-, ROCK+, CitronK-; F39L: PKN+, ROCK+/-, CitronK-; E40L: PKN+, ROCK-, CitronK+; E40W: PKN+, ROCK-, CitronK+; E40N: PKN+, ROCK+, CitronK+; E40T: PKN+, ROCK+, CitronK+; Y42C: PKN-, ROCK+, CitronK+. The proportions of cells displaying dorsally relocalized ezrin were as follows: ezrin alone, 25%; ezrin with RhoA.V14WT, 95%; F39A, 19%; F39L, 24%; E40L, 26%; E40W; 37%; F39V, 60%; E40N, 95%; E40T, 82%; and Y42C, 84%.