Tuning Cellular Mechanics and Motility through Genetic Manipulation of Rho GTPase and Myosin Activity

Abstract

The mechanical properties of cells such as size, shape, and stiffness have recently been recognized as important regulators of cell behavior. For example, cells cultured on small areas of extracellular matrix (ECM) grow slower than cells on large areas (Chen et al., Science 1997), and cells confined to one-dimensional lines elongate their bodies and migrate faster than cells cultured on two-dimensional substrates (Doyle et al., JCB 2009). While there are many strategies to indirectly control such behaviors through manipulation of the ECM, there is a marked lack of strategies for controlling these behaviors in more direct ways. To address this need, we have developed a genetic strategy for directly tuning the mechanical properties of cells. Specifically, we have placed genetic mutants of mechanotransductive proteins, including RhoA and myosin light chain kinase (MLCK), under a conditional promoter and introduced a single copy into glioblastoma cells using viral vectors. By expressing these proteins from a conditional promoter, we can vary their expression by simply changing the inducer concentration in the culture medium. RhoA and MLCK are known to activate myosin II, which is the motor protein responsible for force generation, and with constitutively active (CA) mutants of these proteins, we can modulate cytoskeletal architecture, force generation, and cellular stiffness in a graded fashion. Moreover, by switching expression of these proteins on and off, we can dynamically control cell spreading, migration, and ECM remodeling. We believe this strategy will serve as a valuable tool for developing quantitative relationships between intracellular signaling, cellular mechanics, and complex behaviors. Furthermore, such precise control over cell behavior could allow us to dictate how cells physically interact with their microenvironment, which would be particularly useful in tissue engineering applications that interface cells with synthetic materials.