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.
Chang
Yuan-Chen
YC
Departments of Immunology and Cell Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Nalbant
Perihan
P
Birkenfeld
Jörg
J
Chang
Zee-Fen
ZF
Bokoch
Gary M
GM
D002454:Cell Differentiation / Q000187:drug effects; D002468:Cell Physiological Phenomena / Q000187:drug effects*; D020662:Guanine Nucleotide Exchange Factors / Q000172:deficiency / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008870:Microtubules / Q000187:drug effects* / Q000201:enzymology*; D018994:Myosin Light Chains / Q000378:metabolism; D015739:Nocodazole / Q000494:pharmacology*; D034741:RNA, Small Interfering / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
C577356:ARHGEF2 protein, human; D020662:Guanine Nucleotide Exchange Factors; D018994:Myosin Light Chains; D034741:RNA, Small Interfering; D064067:Rho Guanine Nucleotide Exchange Factors; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate; D015739:Nocodazole