Rho Kinase Regulates the Intracellular Micromechanical Response of Adherent Cells to Rho Activation

Abstract

Local sol-gel transitions of the cytoskeleton modulate cell shape changes, which are required for essential cellular functions, including motility and adhesion. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics; however, the mechanical behavior of living cells and the signaling pathways by which it is regulated remains largely unknown. To address this issue, we used a nanoscale sensing method, intracellular microrheology, to examine the mechanical response of the cell to activation of the small GTPase Rho. We observe that the cytoplasmic stiffness and viscosity of serum-starved Swiss 3T3 cells transiently and locally enhances upon treatment with lysophosphatidic acid, and this mechanical behavior follows a trend similar to Rho activity. Furthermore, the time-dependent activation of Rho decreases the degree of microheterogeneity of the cytoplasm. Our results reveal fundamental differences between intracellular elasticity and cellular tension and suggest a critical role for Rho kinase in the regulation of intracellular mechanics. INTRODUCTIONCytoskeletal rearrangements are closely correlated with key cellular processes such as cell shape changes during mitosis, the separation of daughter cells by the contractile ring during cytokinesis, cell-cell and cell-substrate interactions, transmembrane signaling, endocytosis, secretion, and motility (Schmidt and Hall, 1998). These processes involve the coordinated assembly, disassembly, cross-linking, and bundling of cytoskeletal filaments, which are mediated by auxiliary proteins and are believed to regulate the mechanical properties of the cell. In vitro studies using purified cytoskeletal proteins have suggested molecular mechanisms of regulation of cytoskeleton mechanics (Sato et al., 1987;Janmey et al., 1990;Pollard et al., 1992); however, the mechanical behavior of living cells and the molecular signaling pathways by which it is regulated remains largely unknown.It is well established that members of the Rho family of small GTPases play a central role in cytoskeletal assembly and architecture Van Aelst and D'Souza-Schorey, 1997;Rottner et al., 1999;Bishop and Hall, 2000). The roles of Rho proteins in controlling actin filament dynamics and network organization are fairly well understood at the biochemical level (Ballestrem et al., 2001). However, fundamental questions still exist regarding the downstream intracellular mechanical response of the cytoskeleton to Rho GTPase activation (Heidemann and Wirtz, 2004). For instance, upon activation of Rho, what are the intracellular viscoelastic properties that modulate the intracellular transport of organelles and engineered particles (Suh et al., 2003), and how does Rho activation affect the ability of the cell to resist deformation? Using a novel functional assay, intracellular microrheology (Tseng et al., 2002b), we directly measure the micromechanical response of Swiss 3T3 fibroblasts subjected to Rho activation by lysophosphatidic acid (LPA) ...

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