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

Nature Cell Biology volume 13, pages 383–393 (2011)Cite this article

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Abstract

Focal adhesions undergo myosin-II-mediated maturation wherein they grow and change composition to modulate integrin signalling for cell migration, growth and differentiation. To determine how focal adhesion composition is affected by myosin II activity, we performed proteomic analysis of isolated focal adhesions and compared protein abundance in focal adhesions from cells with and without myosin II inhibition. We identified 905 focal adhesion proteins, 459 of which changed in abundance with myosin II inhibition, defining the myosin-II-responsive focal adhesion proteome. The abundance of 73% of the proteins in the myosin-II-responsive focal adhesion proteome was enhanced by contractility, including proteins involved in Rho-mediated focal adhesion maturation and endocytosis- and calpain-dependent focal adhesion disassembly. During myosin II inhibition, 27% of proteins in the myosin-II-responsive focal adhesion proteome, including proteins involved in Rac-mediated lamellipodial protrusion, were enriched in focal adhesions, establishing that focal adhesion protein recruitment is also negatively regulated by contractility. We focused on the Rac guanine nucleotide exchange factor β-Pix, documenting its role in the negative regulation of focal adhesion maturation and the promotion of lamellipodial protrusion and focal adhesion turnover to drive cell migration.

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Figure 1: Development and validation of the focal adhesion isolation method.
Figure 2: Proteome of isolated focal adhesions.
Figure 3: Development of the MDR to characterize the effects of myosin II inhibition on protein abundance in isolated focal adhesions.
Figure 4: Collective modulation by myosin II of the focal adhesion abundance of proteins in common biological pathways.
Figure 5: Abundance of β-Pix in focal adhesions is negatively regulated by myosin-II-mediated maturation.
Figure 6: Effects of β-Pix on regulation of lamellipodia formation and Rac1 activation.
Figure 7: Effects of β-Pix on focal adhesion dynamics and cell migration.

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Acknowledgements

We thank J. Hildebrand (University of Pittsburgh), J. Peterson (Fox Chase Cancer Centre), M. Davidson (Florida State), R.H. Chen (Academia Sinica) and M. Beckerle (University of Utah) for reagents, the NHLBI proteomics and flow cytometry facilities, W. Shin and I. Schneider for help with imaging and analysis, and A. Aslanian. C.M.W. is supported by NHLBI, J.R.Y. by NIH P41 RR011823.

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  1. Jean-Cheng Kuo and Xuemei Han: These authors contributed equally to this work.

Authors and Affiliations

  1. Cell Biology and Physiology Centre, National Heart, Lung, and Blood Institute, NIH, Bethesda, 20892, Maryland, USA

    Jean-Cheng Kuo & Clare M. Waterman

  2. Cell Biology, Scripps Research Institute, La Jolla, 92037, California, USA

    Xuemei Han & John R. Yates III

  3. Proteomics and Analytical Biochemistry Unit, Research Resources Branch, National Institute on Aging, NIH, Baltimore, 21224, Maryland, USA

    Cheng-Te Hsiao

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J.C.K. and C.M.W designed experiments and wrote the paper, J.C.K. performed experiments, X.H. and J.R.Y performed MudPIT MS analysis and C.T.H. created the web site.

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Correspondence to John R. Yates III or Clare M. Waterman.

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Kuo, JC., Han, X., Hsiao, CT. et al. Analysis of the myosin-II-responsive focal adhesion proteome reveals a role for β-Pix in negative regulation of focal adhesion maturation. Nat Cell Biol 13, 383–393 (2011). https://doi.org/10.1038/ncb2216

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