3385_Ch17 - Chapter 17 Regulation of Cell Contractility by...

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17 © 1999 by CRC Press LLC Chapter Regulation of Cell Contractility by RhoA: Stress Fiber and Focal Adhesion Assembly Betty P. Liu, Magdalena Chrzanowska-Wodnicka, and Keith Burridge Contents I. Introduction A. Regulation of Nonmuscle Contractility B. Contractility and Focal Adhesion Assembly II. Microinjection Protocols A. Purification of Recombinant Proteins 14 VRhoA and C3 Exotransferase B. Microinjection C. Cell Culture III. Analyses of Microinjected Cells A. Immunofluorescence B. Silicone Rubber Substrata C. Cell Labeling, Myosin Immunoprecipitation, and Quantitation of Myosin Light Chain Phosphorylation IV. Future Directions Acknowledgments References
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© 1999 by CRC Press LLC I. Introduction Many cells in culture adhere to the underlying substratum via specialized regions, known as focal adhesions. These are areas where integrins, receptors for extracellular matrix (ECM) proteins, are clustered and adhesion is made to ECM components adsorbed to the surface on which the cells are growing (for reviews see References1 and 2). Focal adhesions serve to link the extracellular matrix on the outside of the cell to the actin cytoskeleton on the inside. At their cytoplasmic face, the clustered integrins within focal adhesions anchor bundles of actin filaments (stress fibers) to the membrane. Several structural proteins (talin, vinculin, and alpha-actinin) mediate this attachment of stress fibers to the cytoplasmic domains of integrins. In addition to a structural role, focal adhesions are also sites of signal transduction. Many components involved in signal transduction have been identified in focal adhesions, and signaling cascades emanate from focal adhesions in response to integrin clustering and engage- ment. 1-3 Because focal adhesions are easily visualized and studied, they provide a model for studying cell adhesion to the extracellular matrix mediated by integrins. The assembly of focal adhesions and their associated bundles of actin filaments is regulated by the GTPase RhoA. 4 How RhoA regulates the assembly of these structures has been the subject of intense investigation for several years. It was suggested many years ago that stress fibers were under isometric tension 5,6 and that the isometric tension was itself responsible for their formation. 6 This led us to investigate whether the assembly of stress fibers and focal adhesions induced by RhoA might be the result of RhoA stimulating contractility and generating tension. This is indeed what we found, 7 which has led us to propose a model for how tension may lead to the assembly of focal adhesions by clustering integrins. 2,7,8 In this chapter, after a brief review of nonmuscle contractility and its relationship to focal adhesion assembly, we describe methods for studying RhoA by microinjection, and describe assays for the contractility of cultured cells. A.
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This note was uploaded on 05/06/2010 for the course MECH. 28197 taught by Professor Dr.shafii during the Spring '10 term at Sharif University of Technology.

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3385_Ch17 - Chapter 17 Regulation of Cell Contractility by...

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