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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

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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References

  1. Burridge, K., Fath, K., Kelly, T., Nuckolls, G. & Turner, C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu. Rev. Cell Biol. 4, 487–525 (1988).

    Article  CAS  Google Scholar 

  2. Hynes, R. O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

    Article  CAS  Google Scholar 

  3. Zaidel-Bar, R., Itzkovitz, S., Ma'ayan, A., Iyengar, R. & Geiger, B. Functional atlas of the integrin adhesome. Nat. Cell Biol. 9, 858–867 (2007).

    Article  CAS  Google Scholar 

  4. Zamir, E., Geiger, B. & Kam, Z. Quantitative multicolor compositional imaging resolves molecular domains in cell-matrix adhesions. PLoS One. 3, e1901 (2008).

    Article  Google Scholar 

  5. Bershadsky, A., Kozlov, M. & Geiger, B. Adhesion-mediated mechanosensitivity: a time to experiment, and a time to theorize. Curr. Opin. Cell Biol. 18, 472–481 (2006).

    Article  CAS  Google Scholar 

  6. Burridge, K. & Chrzanowska-Wodnicka, M. Focal adhesions, contractility, and signaling. Annu. Rev. Cell Dev. Biol. 12, 463–518 (1996).

    Article  CAS  Google Scholar 

  7. Chrzanowska-Wodnicka, M. & Burridge, K. Rho-stimulated contractility drives the formation of stress fibers and focal adhesions. J. Cell Biol. 133, 1403–1415 (1996).

    Article  CAS  Google Scholar 

  8. Pletjushkina, O. J. et al. Maturation of cell-substratum focal adhesions induced by depolymerization of microtubules is mediated by increased cortical tension. Cell Adhes. Commun. 5, 121–135 (1998).

    Article  CAS  Google Scholar 

  9. Riveline, D. et al. Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism. J. Cell Biol. 153, 1175–1186 (2001).

    Article  CAS  Google Scholar 

  10. Galbraith, C. G., Yamada, K. M. & Galbraith, J. A. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 315, 992–995 (2007).

    Article  CAS  Google Scholar 

  11. Vogel, V. & Sheetz, M. Local force and geometry sensing regulate cell functions. Nat. Rev. Mol. Cell Biol. 7, 265–275 (2006).

    Article  CAS  Google Scholar 

  12. Engler, A. J., Sen, S., Sweeney, H. L. & Discher, D. E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  13. Tadokoro, S. et al. Talin binding to integrin β tails: a final common step in integrin activation. Science 302, 103–106 (2003).

    Article  CAS  Google Scholar 

  14. Laukaitis, C. M., Webb, D. J., Donais, K. & Horwitz, A. F. Differential dynamics of α5 integrin, paxillin, and α-actinin during formation and disassembly of adhesions in migrating cells. J. Cell Biol. 153, 1427–1440 (2001).

    Article  CAS  Google Scholar 

  15. Webb, D. J. et al. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6, 154–161 (2004).

    Article  CAS  Google Scholar 

  16. Wiseman, P. W. et al. Spatial mapping of integrin interactions and dynamics during cell migration by image correlation microscopy. J. Cell Sci. 117, 5521–5534 (2004).

    Article  CAS  Google Scholar 

  17. Choi, C. K. et al. Actin and α-actinin orchestrate the assembly and maturation of nascent adhesions in a myosin II motor-independent manner. Nat. Cell Biol. 10, 1039–1050 (2008).

    Article  CAS  Google Scholar 

  18. Pasapera, A. M., Schneider, I. C., Rericha, E., Schlaepfer, D. D. & Waterman, C. M. Myosin II activity regulates vinculin recruitment to focal adhesions through FAK-mediated paxillin phosphorylation. J. Cell Biol. 188, 877–890 (2010).

    Article  CAS  Google Scholar 

  19. Galbraith, C. G. & Sheetz, M. P. A micromachined device provides a new bend on fibroblast traction forces. Proc. Natl Acad. Sci. USA 94, 9114–9118 (1997).

    Article  CAS  Google Scholar 

  20. Huttenlocher, A., Ginsberg, M. H. & Horwitz, A. F. Modulation of cell migration by integrin-mediated cytoskeletal linkages and ligand-binding affinity. J. Cell Biol. 134, 1551–1562 (1996).

    Article  CAS  Google Scholar 

  21. Zaidel-Bar, R., Ballestrem, C., Kam, Z. & Geiger, B. Early molecular events in the assembly of matrix adhesions at the leading edge of migrating cells. J. Cell Sci. 116, 4605–4613 (2003).

    Article  CAS  Google Scholar 

  22. Friedland, J. C., Lee, M. H. & Boettiger, D. Mechanically activated integrin switch controls α5β1 function. Science 323, 642–644 (2009).

    Article  CAS  Google Scholar 

  23. Shi, Q. & Boettiger, D. A novel mode for integrin-mediated signaling: tethering is required for phosphorylation of FAK Y397. Mol. Biol. Cell 14, 4306–4315 (2003).

    Article  CAS  Google Scholar 

  24. Ballestrem, C. et al. Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer. J. Cell Sci. 119, 866–875 (2006).

    Article  CAS  Google Scholar 

  25. Kuo, J. C., Han, X., Yates, J. R. & Waterman, C. M. in Methods in Molecular Biology (ed Shimaoka, M.) (Humana, 2010). In Press.

    Google Scholar 

  26. Washburn, M. P., Wolters, D. & Yates, J. R., III Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).

    Article  CAS  Google Scholar 

  27. Eng, J. K., McCormack, A. L. & Yates, J. R., III An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass Spectrom. 5, 976–989 (1994).

    Article  CAS  Google Scholar 

  28. Elias, J. E., Haas, W., Faherty, B. K. & Gygi, S. P. Comparative evaluation of mass spectrometry platforms used in large-scale proteomics investigations. Nat. Methods 2, 667–675 (2005).

    Article  CAS  Google Scholar 

  29. Zaidel-Bar, R. & Geiger, B. The switchable integrin adhesome. J. Cell Sci. 123, 1385–1388 (2010).

    Article  CAS  Google Scholar 

  30. Caswell, P. T., Vadrevu, S. & Norman, J. C. Integrins: masters and slaves of endocytic transport. Nat. Rev. Mol. Cell Biol. 10, 843–853 (2009).

    Article  CAS  Google Scholar 

  31. Liu, H., Sadygov, R. G. & Yates, J. R., III A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201 (2004).

    Article  CAS  Google Scholar 

  32. Paoletti, A. C. et al. Quantitative proteomic analysis of distinct mammalian Mediator complexes using normalized spectral abundance factors. Proc. Natl Acad. Sci. USA 103, 18928–18933 (2006).

    Article  CAS  Google Scholar 

  33. Marouga, R., David, S. & Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal. Bioanal. Chem. 382, 669–678 (2005).

    Article  CAS  Google Scholar 

  34. Ren, Y., Li, R., Zheng, Y. & Busch, H. Cloning and characterization of GEF-H1, a microtubule-associated guanine nucleotide exchange factor for Rac and Rho GTPases. J. Biol. Chem. 273, 34954–34960 (1998).

    Article  CAS  Google Scholar 

  35. Bai, C. Y., Ohsugi, M., Abe, Y. & Yamamoto, T. ZRP-1 controls Rho GTPase-mediated actin reorganization by localizing at cell-matrix and cell–cell adhesions. J. Cell Sci. 120, 2828–2837 (2007).

    Article  CAS  Google Scholar 

  36. Griffith, E., Coutts, A. S. & Black, D. M. RNAi knockdown of the focal adhesion protein TES reveals its role in actin stress fibre organisation. Cell Motil. Cytoskeleton 60, 140–152 (2005).

    Article  CAS  Google Scholar 

  37. Otey, C. A. & Carpen, O. α-actinin revisited: a fresh look at an old player. Cell Motil. Cytoskeleton 58, 104–111 (2004).

    Article  CAS  Google Scholar 

  38. Schroeter, M. M., Beall, B., Heid, H. W. & Chalovich, J. M. In vitro characterization of native mammalian smooth-muscle protein synaptopodin 2. Biosci. Rep. 28, 195–203 (2008).

    Article  CAS  Google Scholar 

  39. Chen, Y. et al. F-actin and myosin II binding domains in supervillin. J. Biol. Chem. 278, 46094–46106 (2003).

    Article  CAS  Google Scholar 

  40. Wulfkuhle, J. D. et al. Domain analysis of supervillin, an F-actin bundling plasma membrane protein with functional nuclear localization signals. J. Cell Sci. 112, 2125–2136 (1999).

    CAS  PubMed  Google Scholar 

  41. Schuh, M. & Ellenberg, J. A new model for asymmetric spindle positioning in mouse oocytes. Curr. Biol. 18, 1986–1992 (2008).

    Article  CAS  Google Scholar 

  42. Harris, B. Z. & Lim, W. A. Mechanism and role of PDZ domains in signaling complex assembly. J. Cell Sci. 114, 3219–3231 (2001).

    CAS  PubMed  Google Scholar 

  43. Meves, A., Stremmel, C., Gottschalk, K. & Fassler, R. The Kindlin protein family: new members to the club of focal adhesion proteins. Trends Cell Biol. 19, 504–513 (2009).

    Article  CAS  Google Scholar 

  44. Shattil, S. J., Kim, C. & Ginsberg, M. H. The final steps of integrin activation: the end game. Nat. Rev. Mol. Cell Biol. 11, 288–300 (2010).

    Article  CAS  Google Scholar 

  45. Tu, Y., Wu, S., Shi, X., Chen, K. & Wu, C. Migfilin and Mig-2 link focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113, 37–47 (2003).

    Article  CAS  Google Scholar 

  46. Takahashi, H. et al. Role of interaction with vinculin in recruitment of vinexins to focal adhesions. Biochem. Biophys. Res. Commun. 336, 239–246 (2005).

    Article  CAS  Google Scholar 

  47. Franco, S. J. et al. Calpain-mediated proteolysis of talin regulates adhesion dynamics. Nat. Cell Biol. 6, 977–983 (2004).

    Article  CAS  Google Scholar 

  48. Ezratty, E. J., Bertaux, C., Marcantonio, E. E. & Gundersen, G. G. Clathrin mediates integrin endocytosis for focal adhesion disassembly in migrating cells. J. Cell Biol. 187, 733–747 (2009).

    Article  CAS  Google Scholar 

  49. ten Klooster, J. P., Jaffer, Z. M., Chernoff, J. & Hordijk, P. L. Targeting and activation of Rac1 are mediated by the exchange factor β-Pix. J. Cell Biol. 172, 759–769 (2006).

    Article  CAS  Google Scholar 

  50. Innocenti, M. et al. Mechanisms through which Sos-1 coordinates the activation of Ras and Rac. J. Cell Biol. 156, 125–136 (2002).

    Article  CAS  Google Scholar 

  51. O'Connor, K. L. & Mercurio, A. M. Protein kinase A regulates Rac and is required for the growth factor-stimulated migration of carcinoma cells. J. Biol. Chem. 276, 47895–47900 (2001).

    Article  CAS  Google Scholar 

  52. Zhong, H. et al. Subcellular dynamics of type II PKA in neurons. Neuron 62, 363–374 (2009).

    Article  CAS  Google Scholar 

  53. Rendon, B. E. et al. Regulation of human lung adenocarcinoma cell migration and invasion by macrophage migration inhibitory factor. J. Biol. Chem. 282, 29910–29918 (2007).

    Article  CAS  Google Scholar 

  54. Del Pozo, M. A. et al. Integrins regulate GTP-Rac localized effector interactions through dissociation of Rho-GDI. Nat. Cell Biol. 4, 232–239 (2002).

    Article  CAS  Google Scholar 

  55. Miki, H., Yamaguchi, H., Suetsugu, S. & Takenawa, T. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature 408, 732–735 (2000).

    Article  CAS  Google Scholar 

  56. Eden, S., Rohatgi, R., Podtelejnikov, A. V., Mann, M. & Kirschner, M. W. Mechanism of regulation of WAVE1-induced actin nucleation by Rac1 and Nck. Nature 418, 790–793 (2002).

    Article  CAS  Google Scholar 

  57. Oser, M. & Condeelis, J. The cofilin activity cycle in lamellipodia and invadopodia. J. Cell Biochem. 108, 1252–1262 (2009).

    Article  CAS  Google Scholar 

  58. Ambach, A. et al. The serine phosphatases PP1 and PP2A associate with and activate the actin-binding protein cofilin in human T lymphocytes. Eur. J. Immunol. 30, 3422–3431 (2000).

    Article  CAS  Google Scholar 

  59. Bertling, E. et al. Cyclase-associated protein 1 (CAP1) promotes cofilin-induced actin dynamics in mammalian nonmuscle cells. Mol. Biol. Cell 15, 2324–2334 (2004).

    Article  CAS  Google Scholar 

  60. Pelham, R. J. Jr & Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl Acad. Sci. USA 94, 13661–13665 (1997).

    Article  CAS  Google Scholar 

  61. Even-Ram, S. et al. Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk. Nat. Cell Biol. 9, 299–309 (2007).

    Article  CAS  Google Scholar 

  62. Vicente-Manzanares, M., Zareno, J., Whitmore, L., Choi, C. K. & Horwitz, A. F. Regulation of protrusion, adhesion dynamics, and polarity by myosins IIA and IIB in migrating cells. J. Cell Biol. 176, 573–580 (2007).

    Article  CAS  Google Scholar 

  63. Koh, C. G., Manser, E., Zhao, Z. S., Ng, C. P. & Lim, L. β1Pix, the PAK-interacting exchange factor, requires localization via a coiled-coil region to promote microvillus-like structures and membrane ruffles. J. Cell Sci. 114, 4239–4251 (2001).

    CAS  PubMed  Google Scholar 

  64. Nayal, A. et al. Paxillin phosphorylation at Ser273 localizes a GIT1–PIX–PAK complex and regulates adhesion and protrusion dynamics. J. Cell Biol. 173, 587–589 (2006).

    Article  CAS  Google Scholar 

  65. Lee, C. S., Choi, C. K., Shin, E. Y., Schwartz, M. A. & Kim, E. G. Myosin II directly binds and inhibits Dbl family guanine nucleotide exchange factors: a possible link to Rho family GTPases. J. Cell Biol. 190, 663–674 (2010).

    Article  CAS  Google Scholar 

  66. Desmarais, V., Ichetovkin, I., Condeelis, J. & Hitchcock-DeGregori, S. E. Spatial regulation of actin dynamics: a tropomyosin-free, actin-rich compartment at the leading edge. J. Cell Sci. 115, 4649–4660 (2002).

    Article  CAS  Google Scholar 

  67. Ponti, A., Machacek, M., Gupton, S. L., Waterman-Storer, C. M. & Danuser, G. Two distinct actin networks drive the protrusion of migrating cells. Science 305, 1782–1786 (2004).

    Article  CAS  Google Scholar 

  68. Bern, M., Goldberg, D., McDonald, W. H. & Yates, J. R. III Automatic quality assessment of peptide tandem mass spectra. Bioinformatics 20, i49–i54 (2004).

    Article  CAS  Google Scholar 

  69. Sadygov, R. G. et al. Code developments to improve the efficiency of automated MS/MS spectra interpretation. J. Proteome Res. 1, 211–215 (2002).

    Article  CAS  Google Scholar 

  70. Ambatipudi, K. S., Lu, B., Hagen, F. K., Melvin, J. E. & Yates, J. R. III Quantitative analysis of age specific variation in the abundance of human female parotid salivary proteins. J. Proteome Res. 8, 5093–5102 (2009).

    Article  CAS  Google Scholar 

  71. Chen, E. I. et al. Adaptation of energy metabolism in breast cancer brain metastases. Cancer Res. 67, 1472–1486 (2007).

    Article  CAS  Google Scholar 

  72. Tabb, D. L., McDonald, W. H. & Yates, J. R. III DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26 (2002).

    Article  CAS  Google Scholar 

  73. Hoffert, J. D., van Balkom, B. W., Chou, C. L. & Knepper, M. A. Application of difference gel electrophoresis to the identification of inner medullary collecting duct proteins. Am. J. Physiol. Renal Physiol. 286, F170–F179 (2004).

    Article  CAS  Google Scholar 

  74. Marouga, R., David, S. & Hawkins, E. The development of the DIGE system: 2D fluorescence difference gel analysis technology. Anal. Bioanal. Chem. 382, 669–678 (2005).

    Article  CAS  Google Scholar 

  75. Shin, W. et al. Live Cell Imaging. A laboratory Manual. (eds R. D. Goldman, J. Swedlow, and D. L. Spector). 119–138 (Cold Spring Harbor Laboratory Press, 2010).

    Google Scholar 

  76. Pelham, R. J., Jr. & Wang, Y. Cell locomotion and focal adhesions are regulated by substrate flexibility. Proc. Natl Acad. Sci. USA 94, 13661–13665 (1997).

    Article  CAS  Google Scholar 

  77. Ren, X. D. & Schwartz, M. A. Determination of GTP loading on Rho. Methods Enzymol. 325, 264–272 (2000).

    Article  CAS  Google Scholar 

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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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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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