The Rho family GEF Asef2 regulates cancer cell migration by modulating Rac activation and actomyosin contractility
Carrington, Leolene Jean
Non-muscle myosin II (MyoII) contractility is important to the regulation of many cellular processes, including migration. The small GTPase Rho has been shown to regulate MyoII contractility, but the role of other GTPases, such as Rac, in modulating contractility is not well understood. In this study, we show that activation of Rac by the guanine nucleotide exchange factor (GEF) Asef2 increases MyoII contractility to impair cell migration on two-dimensional (2D) type I collagen. Knockdown of endogenous Rac using short hairpin RNAs (shRNAs) or treatment of cells with the Rac-specific inhibitor NSC23766 results in a significant decrease in the amount of active MyoII, as determined by serine 19 (S19) phosphorylation, and negates the Asef2-promoted increase in contractility. Furthermore, treatment of cells with blebbistatin, which inhibits MyoII activity, abolishes the Asef2-mediated effect on migration. Because three-dimensional (3D) matrices more closely mimic the physiologic environment of cells, we investigated the role of Asef2 in regulating migration and MyoII activity in 3D environments. For these studies, we developed microfluidic devices that afford a controlled, reproducible platform for generating 3D matrices. Using these devices, we show that Asef2 inhibits cell migration in 3D type I collagen matrices, and treatment of cells with blebbistatin abolishes the Asef2-mediated decrease in migration. Moreover, Asef2 enhances MyoII activity as shown by increased S19 phosphorylation, and treatment with NSC23766 abolishes the Asef2-promoted increase in active MyoII. Collectively, these results indicate that Rac activation, promoted by Asef2, is critical for modulating MyoII activity and cell migration in both 2D and 3D environments.