Fig. 6. Rho- and phosphorylation-independent activation of ERM proteins in MDCK II cells. (Aa,Ab) MDCK II cells were microinjected with C3/Rh-dextran (arrows in a,b). After a 30 minute incubation, cells were fixed with TCA and stained with anti-CPERM mAb (a) or fixed with formaldehyde and stained with FITC phalloidin (b). Becuase phalloidin cannot bind to actin filaments fixed with TCA, double staining with anti-CPERM mAb and phalloidin of the same specimen was impossible. Although C3 suppressed Rho activity enough to affect stress fibers (b), it showed no effects on CPERMs (a). (Ac,Ad) MDCK II cells were treated with 200 nM staurosporine for 10 minutes, then double stained with anti-CPERM mAb (c) and anti-ERM mAb, CR22 (d). Although CPERMs were mostly dephosphorylated (c), microvilli, which were sometimes elongated, remained with accumulated ERM proteins. (Ae,Af) Transmission electron micrographs of control (e) and staurosporine-treated (200 nM for 10 minutes) (f) MDCK II cells. The morphology of microvilli of staurosporine-treated MDCK II cells is normal, although they are often elongated. Bars, a,b, 10 µm; c,d, 10 µm; e,f, 0.2 µm. (B) MDCK II cells cultured in the absence (control) or presence (stauro.) of 200 nM staurosporine were homogenized and centrifuged. Equivalent amounts of supernatant (S) and pellet (P) were subjected to immunoblotting with anti-ERM pAb, TK89. Note that both in the absence and presence of staurosporine, a considerable amount of ERM protein was recovered in the insoluble (P) fraction.