Gef-H1 Couples Nocodazole-Induced Microtubule Disassembly to Cell Contractility Via Rhoa

The RhoA GTPase plays a vital role in assembly of contractile actin- myosin filaments (stress fibers) and of associated focal adhesion complexes of adherent monolayer cells in culture. GEF-H1 is a microtubule- associated guanine nucleotide exchange factor that activates RhoA upon release from microtubules. The overexpression of GEF-H1 deficient in microtubule binding or treatment of HeLa cells with nocodazole to induce microtubule depolymerization results in Rho-dependent actin stress fiber formation and contractile cell morphology. However, whether GEF-H1 is required and sufficient to mediate nocodazole-induced contractility remains unclear. We establish here that siRNA-mediated depletion of GEF-H1 in HeLa cells prevents nocodazole- induced cell contraction. Furthermore, the nocodazole- induced activation of RhoA and Rho-associated kinase ( ROCK) that mediates phosphorylation of myosin regulatory light chain (MLC) is impaired in GEF-H1-depleted cells. Conversely , RhoA activation and contractility are rescued by reintroduction of siRNA-resistant GEF-H1. Our studies reveal a critical role for a GEF-H1/RhoA/ROCK/MLC signaling pathway in mediating nocodazole- induced cell contractility