Rho, a member of the Rho small G protein family, regulates the formation of 
stress fibers and focal adhesions in various types of cultured cells. We 
investigated here the actions of ROCK and mDia, both of which have been 
identified to be putative downstream target molecules of Rho, in Madin-Darby 
canine kidney cells. The dominant active mutant of RhoA induced the formation of 
parallel stress fibers and focal adhesions, whereas the dominant active mutant 
of ROCK induced the formation of stellate stress fibers and focal adhesions, and 
the dominant active mutant of mDia induced the weak formation of parallel stress 
fibers without affecting the formation of focal adhesions. In the presence of C3 
ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the 
formation of stellate stress fibers and focal adhesions, whereas the dominant 
active mutant of mDia induced only the diffuse localization of actin filaments. 
These results indicate that ROCK and mDia show distinct actions in 
reorganization of the actin cytoskeleton. The dominant negative mutant of either 
ROCK or mDia inhibited the formation of stress fibers and focal adhesions, 
indicating that both ROCK and mDia are necessary for the formation of stress 
fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK 
and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate-induced 
disassembly and reassembly, respectively, of stress fibers and focal adhesions. 
The morphologies of stress fibers and focal adhesions in the cells expressing 
both the dominant active mutants of ROCK and mDia were not identical to those 
induced by the dominant active mutant of Rho. These results indicate that at 
least ROCK and mDia cooperatively act as downstream target molecules of Rho in 
the Rho-induced reorganization of the actin cytoskeleton.