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Pankov R., Cuckierman E., Katz B.-Z., Matsumoto K., Lin D.C., Lin S., Hahn C., Yamada K.M. Integrin dynamics and matrix assembly: tensin-dependent translocation of α5β1 integrins promotes early fibronectin fibrillogenesis. J Cell Biol. 148:2000;1075-1090.
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This study maps the traction forces exerted by adhesions in migrating fibroblasts. The small adhesions at the cell front generate strong propulsive forces while the larger, more mature focal adhesions exert weaker forces. The traction forces generated by these small adhesions might drive cell migration.
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Beningo K.A., Dembo M., Kaverina I., Small J.V., Wang Y.-L. Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J Cell Biol. 153:2001;881-888 This study maps the traction forces exerted by adhesions in migrating fibroblasts. The small adhesions at the cell front generate strong propulsive forces while the larger, more mature focal adhesions exert weaker forces. The traction forces generated by these small adhesions might drive cell migration.
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A technique for monitoring the kinetics of tyrosine phosphorylation in adhesions is described. This is accomplished with a 'phosphotyrosine reporter' in which yellow fluorescent protein is fused to two phosphotyrosine-binding Src-homology 2 domains derived from c-Src.
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Kirchner J., Kam Z., Tzur G., Bershadsky A.D., Geiger B. Live-cell monitoring of tyrosine phosphorylation in focal adhesions following microtubule disruption. J Cell Sci. 116:2003;975-986 A technique for monitoring the kinetics of tyrosine phosphorylation in adhesions is described. This is accomplished with a 'phosphotyrosine reporter' in which yellow fluorescent protein is fused to two phosphotyrosine-binding Src-homology 2 domains derived from c-Src.
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Using GFP-β3-integrin, the small adhesions at the cell front are shown to have lower integrin densities compared with larger, more organized focal adhesions. Thus, adhesions can be distinguished based on their integrin densities. FRAP analysis shows a rapid exchange of GFP-β3-integrin in the focal adhesions with high integrin densities and a slower recovery of GFP-β3 in small adhesions with lower integrin densities.
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Ballestrem C., Hinz B., Imhof B.A., Wehrle-Haller B. Marching at the front and dragging behind: differential αVβ3-integrin turnover regulates focal adhesion behavior. J Cell Biol. 155:2001;1319-1332 Using GFP-β3-integrin, the small adhesions at the cell front are shown to have lower integrin densities compared with larger, more organized focal adhesions. Thus, adhesions can be distinguished based on their integrin densities. FRAP analysis shows a rapid exchange of GFP-β3-integrin in the focal adhesions with high integrin densities and a slower recovery of GFP-β3 in small adhesions with lower integrin densities.
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In this study, the molecular composition and function of adhesions are characterized in cells plated in three-dimensional matrices derived from tissues or cell culture. The long, slender three-dimensional matrix adhesions differ in morphology from the oval-shaped focal adhesions observed in cultured cells. Paxillin, α5β1 integrin, vinculin, FAK, activated β1 integrin and tyrosine-phosphorylated proteins are found in three-dimensional matrix adhesions, but β3 integrin is not.
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This paper describes new solvent-sensitive fluorescent dyes that can be covalently attached to proteins and used to monitor their activities in living cells. These new dyes circumvent the problems associated with FRET and offer great promise for studying protein conformational changes, ligand binding and post-translational modifications. A novel biosensor for detection of active Cdc42 using one of these new fluorescent dyes is described.
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Toutchkine A., Kraynov V., Hahn K. Solvent-sensitive dyes to report protein conformational changes in living cells. J Am Chem Soc. 125:2003;4132-4145 This paper describes new solvent-sensitive fluorescent dyes that can be covalently attached to proteins and used to monitor their activities in living cells. These new dyes circumvent the problems associated with FRET and offer great promise for studying protein conformational changes, ligand binding and post-translational modifications. A novel biosensor for detection of active Cdc42 using one of these new fluorescent dyes is described.
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The authors use a FRET-based method, termed fluorescent activation indicator for Rho protein (FLAIR), to study Rac activation during neutrophil chemotaxis. This novel technique was originally described in Kraynov et al. (2000) [31]. It can be used to quantify the spatial and temporal distribution of active Rac in migrating cells. Active Rac is found near the leading edge and in the retracting tail of motile neutrophils.
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Gardiner E.M., Pestonjamasp K.N., Bohl B.P., Chamberlain C., Hahn K.M., Bokoch G.M. Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis. Curr Biol. 12:2002;2029-2034 The authors use a FRET-based method, termed fluorescent activation indicator for Rho protein (FLAIR), to study Rac activation during neutrophil chemotaxis. This novel technique was originally described in Kraynov et al. (2000) [31]. It can be used to quantify the spatial and temporal distribution of active Rac in migrating cells. Active Rac is found near the leading edge and in the retracting tail of motile neutrophils.
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Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells
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Single-chain FRET-based probes are developed for monitoring activation of the small GTPases Rac1 and Cdc42 in living cells. Rac activity is highest immediately behind the leading edge, and Cdc42 activity is the most concentrated at the tip of the leading edge.
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Itoh R.E., Kurokawa K., Ohba Y., Yoshizaki H., Mochizuki N., Matsuda M. Activation of Rac and Cdc42 video imaged by fluorescent resonance energy transfer-based single-molecule probes in the membrane of living cells. Mol Cell Biol. 22:2002;6582-6591 Single-chain FRET-based probes are developed for monitoring activation of the small GTPases Rac1 and Cdc42 in living cells. Rac activity is highest immediately behind the leading edge, and Cdc42 activity is the most concentrated at the tip of the leading edge.
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The authors demonstrate the use of GFP as a CALI reagent, which can be locally inactivated in living cells using high-intensity light. Using this approach, illumination of GFP-α-actinin results in detachment of stress fibers from focal adhesions, whereas laser irradiation of GFP-FAK did not induce stress fiber detachment.
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Rajfur Z., Roy P., Otey C., Romer L., Jacobson K. Dissecting the link between stress fibres and focal adhesions by CALI with EGFP fusion proteins. Nat Cell Biol. 4:2002;286-293 The authors demonstrate the use of GFP as a CALI reagent, which can be locally inactivated in living cells using high-intensity light. Using this approach, illumination of GFP-α-actinin results in detachment of stress fibers from focal adhesions, whereas laser irradiation of GFP-FAK did not induce stress fiber detachment.
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Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy
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This paper describes a novel screening strategy with a cDNA library fused to GFP, which can be used to identify gene products that localize to adhesions and the cytoskeleton. The adaptor molecule GIT1, which resides in adhesions, at the leading edge and in cytoplasmic complexes, was identified using this GFP screen [18].
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Manabe R., Whitmore L., Weiss J.M., Horwitz A.R. Identification of a novel microtubule-associated protein that regulates microtubule organization and cytokinesis by using a GFP-screening strategy. Curr Biol. 12:2002;1946-1951 This paper describes a novel screening strategy with a cDNA library fused to GFP, which can be used to identify gene products that localize to adhesions and the cytoskeleton. The adaptor molecule GIT1, which resides in adhesions, at the leading edge and in cytoplasmic complexes, was identified using this GFP screen [18].
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(2002)
Curr Biol
, vol.12
, pp. 1946-1951
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Manabe, R.1
Whitmore, L.2
Weiss, J.M.3
Horwitz, A.R.4
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