Rho and Rab Small G Proteins Coordinately Reorganize Stress Fibers and Focal Adhesions in MDCK Cells

Abstract

The Rho subfamily of the Rho small G protein family (Rho) regulates formation of stress fibers and focal adhesions in many types of cultured cells. In moving cells, dynamic and coordinate disassembly and reassembly of stress fibers and focal adhesions are observed, but the precise mechanisms in the regulation of these processes are poorly understood. We previously showed that 12-O-tetradecanoylphorbol-13-acetate (TPA) first induced disassembly of stress fibers and focal adhesions followed by their reassembly in MDCK cells. The reassembled stress fibers showed radial-like morphology that was apparently different from the original. We analyzed here the mechanisms of these TPA-induced processes. Rho inactivation and activation were necessary for the TPA-induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. Both inactivation and activation of the Rac subfamily of the Rho family (Rac) inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly. Moreover, microinjection or transient expression of Rab GDI, a regulator of all the Rab small G protein family members, inhibited the TPA-induced reassembly of stress fibers and focal adhesions but not their TPA-induced disassembly, indicating that, furthermore, activation of some Rab family members is necessary for their TPA-induced reassembly. Of the Rab family members, at least Rab5 activation was necessary for the TPA-induced reassembly of stress fibers and focal adhesions. The TPA-induced, small G protein-mediated reorganization of stress fibers and focal adhesions was closely related to the TPA-induced cell motility. These results indicate that the Rho and Rab family members coordinately regulate the TPA-induced reorganization of stress fibers and focal adhesions that may cause cell motility.

Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells

“…At 16 h, the staining of E-cadherin at the cell-cell adhesion sites became indistinct and most cells scattered (Figure 4g). These results are consistent with the previous observations (Ridley et al, 1995;Imamura et al, 1998). Stimulation of sMDCK-RacDA-1 cells with HGF/SF for 16 h caused slight spreading of the cells and weaker staining of E-cadherin at the cell-cell adhesion sites (Figure 4h).…”

“…We previously showed that TPA also induced disruption of cell-cell adhesion, followed by cell migration in MDCK cells (Takaishi et al, 1995;Imamura et al, 1998). We furthermore showed that TPA did not disrupt the cell-cell adhesion at least for 2 h stimulation in sMDCK-RacDA-1 cells (Imamura et al, 1998). We next examined the e ects of HGF/SF and TPA on cell-cell adhesion and cell migration of sMDCK-RacDA-1 and -Cdc42HsDA-1 cells for 16 h stimulation.…”

“…During migration, membrane protrusions, such as lamellipodia and ®lopodia, at the cell front, and retraction at the cell rear are externally observed, whereas dynamic reorganization of the actin cytoskeleton, such as disassembly and reassembly of stress ®bers, is internally observed (for reviews, see Huttenlocher et al, 1995;Lau enburger and Horwitz, 1996). In cultured cells, some growth factors, such as hepatocyte growth factor/scatter factor (HGF/SF) and 12-O-tetradecanoylphorbol-13-acetate (TPA), disrupt cell-cell junctions and induce cell migration (Takaishi et al, 1995;Imamura et al, 1998; for review, see Stoker and Gherardi, 1991), but their modes of action in these processes have not fully been understood.…”

“…We have recently made MDCK cell lines stably expressing a dominant active mutant of RhoA, a dominant active mutant of Racl, or a dominant negative mutant of Racl, and have shown that Rho regulates stress ®ber formation whereas Rac regulates cadherin-based cell-cell adhesion as well as the formation of lamellipodia (Takaishi et al, 1997;Imamura et al, 1998). We have moreover made MDCK cell lines stably expressing a dominant negative mutant of Rab5 or transiently expressing Rab GDI, and have shown that the Rho and Rab families coordinately regulate cell adhesion and migration (Imamura et al, 1998).…”

“…We have moreover made MDCK cell lines stably expressing a dominant negative mutant of Rab5 or transiently expressing Rab GDI, and have shown that the Rho and Rab families coordinately regulate cell adhesion and migration (Imamura et al, 1998). The Rab small G protein family is implicated in intracellular vesicle tra cking and Rab5 is involved in endocytosis (Bucci et al, 1992;Stenmark et al, 1994;for review, see Novick and Zerial, 1997).…”