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      The Rho GEFs LARG and GEF-H1 regulate the mechanical response to force on integrins

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          Abstract

          How individual cells respond to mechanical forces is of considerable interest to biologists as force affects many aspects of cell behavior 1 . Application of force on integrins triggers cytoskeletal rearrangements and growth of the associated adhesion complex, resulting in increased cellular stiffness 2, 3 , also known as reinforcement 4 . While RhoA has been shown to play a role during reinforcement 3 , the molecular mechanisms that regulate its activity are unknown. By combining biochemical and biophysical approaches, we identified two guanine nucleotide exchange factors (GEFs), LARG and GEF-H1, as key molecules that regulate the cellular adaptation to force. We show that stimulation of integrins with tensional force triggers activation of these two GEFs and their recruitment to adhesion complexes. Surprisingly, activation of LARG and GEF-H1 involves distinct signaling pathways. Our results reveal that LARG is activated by the Src family tyrosine kinase Fyn, whereas GEF-H1 catalytic activity is enhanced by ERK downstream of a signaling cascade that includes FAK and Ras.

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          Most cited references30

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          Tensional homeostasis and the malignant phenotype.

          Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.
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            Cell locomotion and focal adhesions are regulated by substrate flexibility.

            Responses of cells to mechanical properties of the adhesion substrate were examined by culturing normal rat kidney epithelial and 3T3 fibroblastic cells on a collagen-coated polyacrylamide substrate that allows the flexibility to be varied while maintaining a constant chemical environment. Compared with cells on rigid substrates, those on flexible substrates showed reduced spreading and increased rates of motility or lamellipodial activity. Microinjection of fluorescent vinculin indicated that focal adhesions on flexible substrates were irregularly shaped and highly dynamic whereas those on firm substrates had a normal morphology and were much more stable. Cells on flexible substrates also contained a reduced amount of phosphotyrosine at adhesion sites. Treatment of these cells with phenylarsine oxide, a tyrosine phosphatase inhibitor, induced the formation of normal, stable focal adhesions similar to those on firm substrates. Conversely, treatment of cells on firm substrates with myosin inhibitors 2,3-butanedione monoxime or KT5926 caused the reduction of both vinculin and phosphotyrosine at adhesion sites. These results demonstrate the ability of cells to survey the mechanical properties of their surrounding environment and suggest the possible involvement of both protein tyrosine phosphorylation and myosin-generated cortical forces in this process. Such response to physical parameters likely represents an important mechanism of cellular interaction with the surrounding environment within a complex organism.
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              Focal Contacts as Mechanosensors

              The transition of cell–matrix adhesions from the initial punctate focal complexes into the mature elongated form, known as focal contacts, requires GTPase Rho activity. In particular, activation of myosin II–driven contractility by a Rho target known as Rho-associated kinase (ROCK) was shown to be essential for focal contact formation. To dissect the mechanism of Rho-dependent induction of focal contacts and to elucidate the role of cell contractility, we applied mechanical force to vinculin-containing dot-like adhesions at the cell edge using a micropipette. Local centripetal pulling led to local assembly and elongation of these structures and to their development into streak-like focal contacts, as revealed by the dynamics of green fluorescent protein–tagged vinculin or paxillin and interference reflection microscopy. Inhibition of Rho activity by C3 transferase suppressed this force-induced focal contact formation. However, constitutively active mutants of another Rho target, the formin homology protein mDia1 (Watanabe, N., T. Kato, A. Fujita, T. Ishizaki, and S. Narumiya. 1999. Nat. Cell Biol. 1:136–143), were sufficient to restore force-induced focal contact formation in C3 transferase-treated cells. Force-induced formation of the focal contacts still occurred in cells subjected to myosin II and ROCK inhibition. Thus, as long as mDia1 is active, external tension force bypasses the requirement for ROCK-mediated myosin II contractility in the induction of focal contacts. Our experiments show that integrin-containing focal complexes behave as individual mechanosensors exhibiting directional assembly in response to local force.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nat. Cell Biol.
                Nature cell biology
                1465-7392
                1476-4679
                7 April 2011
                15 May 2011
                June 2011
                01 December 2011
                : 13
                : 6
                : 722-727
                Affiliations
                [1 ] Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
                [2 ] Curriculum in Applied Sciences and Engineering, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
                [3 ] Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA
                [4 ] Lineberger Comprehensive Cancer Center, and UNC McAllister Heart Institute, University of North Carolina, Chapel Hill, North Carolina 27599, USA
                Author notes
                [6 ]Correspondence should be addressed to K.B. ( keith_burridge@ 123456med.unc.edu )
                [5]

                These authors contributed equally to this work.

                Article
                nihpa286983
                10.1038/ncb2254
                3107386
                21572419
                29c98b89-1d9b-4159-801e-450d35be6aaf

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                History
                Funding
                Funded by: National Heart, Lung, and Blood Institute : NHLBI
                Award ID: R01 HL077546-03 || HL
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM029860-29A1 || GM
                Funded by: National Institute of General Medical Sciences : NIGMS
                Award ID: R01 GM029860-28S1 || GM
                Funded by: National Institute of Biomedical Imaging and Bioengineering : NIBIB
                Award ID: P41 EB002025-23 || EB
                Categories
                Article

                Cell biology
                Cell biology

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