The cells that constitute our bodies appropriately regulate their shape, strength, and the forces they generate in response to the stiffness and forces exerted by the external environment. However, the signaling mechanisms that underlie cellular responses to mechanical forces are complex and not fully understood. In this study, we analyzed the molecular mechanisms by which cells reorganize their actin cytoskeleton—a major component of the cytoskeleton—in response to substrate stiffness to form structures generating contractile forces. We had previously identified a protein named Solo that generates a contractile force in response to tensile forces applied to cells. Solo functions as an activator of RhoA, a small G protein, and assembles an actin cytoskeletal structure, generating contractile forces at the appropriate sites by activating RhoA. Furthermore, we searched for proteins that associate with Solo and identified leukemia-associated RhoGEF (LARG), a protein that activates RhoA in a manner similar to Solo. In this study, we focused on LARG and investigated the interaction between Solo and LARG using Madin-Darby canine kidney (MDCK) cells. We found that LARG localizes to Solo accumulation sites at near focal adhesions and that LARG enhances Solo-induced actomyosin formation. Conversely, when LARG was overexpressed in Solo-knockdown MDCK cells, the LARG induced actomyosin formation partially suppressed. These results strongly suggest that Solo regulates the localization of LARG and LARG-induced the actomyosin formation. Furthermore, we investigated the roles of Solo and LARG in the response to substrate stiffness and found that the interaction between Solo and LARG is necessary to assemble stress fibers in response to the substrate stiffness. Our study has revealed a novel molecular mechanism that cells recognize and respond to the substrate stiffness through Solo and LARG (Fig. 2).
Figure 1. MDCK cells were transfected with YFP-Solo (green) and mcherry-LARG (magenta) alone or in combination, and the effect on the subcellular localization of each and the shape of the actin cytoskeleton was observed. The arrows indicate the sites where YFP-Solo accumulates in the cells.
Figure 2. A model of the molecular mechanism by which cells regulate actin cytoskeleton remodeling in response to substrate stiffness through Solo and LARG.
