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      Analysis of the myosinII-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation

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          Abstract

          Focal adhesions (FAs) undergo myosinII-mediated maturation wherein they grow and change composition to modulate integrin signaling for cell migration, growth and differentiation. To determine how FA composition is modulated by myosinII activity, we performed proteomic analysis of isolated FAs and compared protein abundance in FAs from cells with and without myosinII inhibition. We identified FA 905 proteins, 459 of which changed in FA abundance with myosinII inhibition, defining the myosinII-responsive FA proteome. FA abundance of 73% of proteins was enhanced by contractility, including those involved in Rho-mediated FA maturation and endocytosis- and calpain-dependent FA disassembly. 27% of proteins, including those involved in Rac-mediated lamellipodial protrusion, were enriched in FA by myosinII inhibition, establishing for the first time negative regulation of FA protein recruitment by contractility. We focused on the Rac guanine nucleotide exchange factor, β-PIX, documenting its role in negative regulation of FA maturation and promotion of lamellipodial protrusion, FA turnover to drive cell migration.

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          Most cited references 52

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          Large-scale analysis of the yeast proteome by multidimensional protein identification technology.

          We describe a largely unbiased method for rapid and large-scale proteome analysis by multidimensional liquid chromatography, tandem mass spectrometry, and database searching by the SEQUEST algorithm, named multidimensional protein identification technology (MudPIT). MudPIT was applied to the proteome of the Saccharomyces cerevisiae strain BJ5460 grown to mid-log phase and yielded the largest proteome analysis to date. A total of 1,484 proteins were detected and identified. Categorization of these hits demonstrated the ability of this technology to detect and identify proteins rarely seen in proteome analysis, including low-abundance proteins like transcription factors and protein kinases. Furthermore, we identified 131 proteins with three or more predicted transmembrane domains, which allowed us to map the soluble domains of many of the integral membrane proteins. MudPIT is useful for proteome analysis and may be specifically applied to integral membrane proteins to obtain detailed biochemical information on this unwieldy class of proteins.
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            Local force and geometry sensing regulate cell functions.

            The shapes of eukaryotic cells and ultimately the organisms that they form are defined by cycles of mechanosensing, mechanotransduction and mechanoresponse. Local sensing of force or geometry is transduced into biochemical signals that result in cell responses even for complex mechanical parameters such as substrate rigidity and cell-level form. These responses regulate cell growth, differentiation, shape changes and cell death. Recent tissue scaffolds that have been engineered at the micro- and nanoscale level now enable better dissection of the mechanosensing, transduction and response mechanisms.
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              FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly.

              Cell migration is a complex, highly regulated process that involves the continuous formation and disassembly of adhesions (adhesion turnover). Adhesion formation takes place at the leading edge of protrusions, whereas disassembly occurs both at the cell rear and at the base of protrusions. Despite the importance of these processes in migration, the mechanisms that regulate adhesion formation and disassembly remain largely unknown. Here we develop quantitative assays to measure the rate of incorporation of molecules into adhesions and the departure of these proteins from adhesions. Using these assays, we show that kinases and adaptor molecules, including focal adhesion kinase (FAK), Src, p130CAS, paxillin, extracellular signal-regulated kinase (ERK) and myosin light-chain kinase (MLCK) are critical for adhesion turnover at the cell front, a process central to migration.
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                Author and article information

                Journal
                100890575
                21417
                Nat Cell Biol
                Nature Cell Biology
                1465-7392
                1476-4679
                23 November 2011
                20 March 2011
                April 2011
                14 February 2012
                : 13
                : 4
                : 383-393
                Affiliations
                [1 ]Cell Biology and Physiology Center, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892
                [2 ]Cell Biology, Scripps Research Institute, La Jolla CA 92037
                [3 ]Proteomics and Analytical Biochemistry Unit, Research Resources Branch, National Institute on Aging, NIH, Baltimore, MD 21224
                Author notes
                [* ]correspondence to: Clare M. Waterman ( watermancm@ 123456nhlbi.nih.gov ). John R. Yates III ( jyates@ 123456scripps.edu )
                [**]

                equal author contribution

                Article
                nihpa268281
                10.1038/ncb2216
                3279191
                21423176

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                Funding
                Funded by: National Institute of Mental Health : NIMH
                Award ID: R01 MH067880-10 || MH
                Funded by: National Center for Research Resources : NCRR
                Award ID: P41 RR011823-16 || RR
                Categories
                Article

                Cell biology

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