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Heterogeneity of Focal Adhesions and Focal Contacts in Motile Fibroblasts

  • Aleena Gladkikh
  • Anastasia Kovaleva
  • Anna Tvorogova
  • Ivan A. Vorobjev
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1745)

Abstract

Cell-extracellular matrix (ECM) adhesion is an important property of virtually all cells in multicellular organisms. Cell-ECM adhesion studies, therefore, are very significant both for biology and medicine. Over the last three decades, biomedical studies resulted in a tremendous advance in our understanding of the molecular basis and functions of cell-ECM adhesion. Based on morphological and molecular criteria, several different types of model cell-ECM adhesion structures including focal adhesions, focal complexes, fibrillar adhesions, podosomes, and three-dimensional matrix adhesions have been described. All the subcellular structures that mediate cell-ECM adhesion are quite heterogeneous, often varying in size, shape, distribution, dynamics, and, to a certain extent, molecular constituents. The morphological “plasticity” of cell-ECM adhesion perhaps reflects the needs of cells to sense, adapt, and respond to a variety of extracellular environments. In addition, cell type (e.g., differentiation status, oncogenic transformation, etc.) often exerts marked influence on the structure of cell-ECM adhesions. Although molecular, genetic, biochemical, and structural studies provide important maps or “snapshots” of cell-ECM adhesions, the area of research that is equally valuable is to study the heterogeneity of FA subpopulations within cells. Recently time-lapse observations on the FA dynamics become feasible, and behavior of individual FA gives additional information on cell-ECM interactions. Here we describe a robust method of labeling of FA using plasmids with fluorescent markers for paxillin and vinculin and quantifying the morphological and dynamical parameters of FA.

Keywords

Focal adhesions Fluorescence microscopy Paxillin Vinculin Image processing Heterogeneity 

References

  1. 1.
    Rooney C, White G, Nazgiewicz A, Woodcock SA, Anderson KI, Ballestrem C, Malliri A (2010) The Rac activator STEF (Tiam2) regulates cell migration by microtubule-mediated focal adhesion disassembly. EMBO Rep 11(4):292–298CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Maziveyi M, Alahari SK (2017) Cell matrix adhesions in cancer: the proteins that form the glue. Oncotarget.  10.18632/oncotarget.17265
  3. 3.
    Geiger B, Bershadsky A, Pankov R, Yamada KM (2001) Transmembrane crosstalk between the extracellular matrix–cytoskeleton crosstalk. Nat Rev Mol Cell Biol 2(11):793–805CrossRefPubMedGoogle Scholar
  4. 4.
    Wehrle-Haller B, Imhof B (2002) The inner lives of focal adhesions. Trends Cell Biol 12(8):382–389CrossRefPubMedGoogle Scholar
  5. 5.
    Larsen M, Artym VV, Green JA, Yamada KM (2006) The matrix reorganized: extracellular matrix remodeling and integrin signaling. Curr Opin Cell Biol 18:463–471CrossRefPubMedGoogle Scholar
  6. 6.
    Alexandrova AY, Arnold K, Schaub S, Vasiliev JM, Meister JJ, Bershadsky AD, Verkhovsky AB (2008) Comparative dynamics of retrograde actin flow and focal adhesions: formation of nascent adhesions triggers transition from fast to slow flow. PLoS One 3(9):e3234CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Nayal A, Webb DJ, Brown CM, Schaefer EM, Vicente-Manzanares M, Horwitz AR (2006) Paxillin phosphorylation at Ser273 localizes a GIT1-PIX-PAK complex and regulates adhesion and protrusion dynamics. J Cell Biol 173(4):587–589CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Choi CK, Margraves CH, English AE, Kihm KD (2008) Multicontrast microscopy technique to dynamically fingerprint live-cell focal contacts during exposure and replacement of a cytotoxic medium. J Biomed Optics 13(5):054069CrossRefGoogle Scholar
  9. 9.
    Mekhdjian AH, Kai F, Rubashkin MG, Prahl LS, Przybyla LM, McGregor AL, Bell ES, Barnes JM, DuFort CC, Ou G et al (2017) Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix. Mol Biol Cell 28:1467–1488CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Beningo KA, Dembo M, Kaverina I, Small JV, Wang YL (2001) Nascent focal adhesions are responsible for the generation of strong propulsive forces in migrating fibroblasts. J Cell Biol 153(4):881–888CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Vicente-Manzanares M, Horwitz AR (2011) Adhesion dynamics at a glance. J Cell Sci 124(Pt 23):3923–3927CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Webb DJ, Donais K, Whitmore LA, Thomas SM, Turner CE, Parsons JT, Horwitz AF (2004) FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat Cell Biol 6(2):154–161CrossRefPubMedGoogle Scholar
  13. 13.
    Ballestrem C, Erez N, Kirchner J, Kam Z, Bershadsky A, Geiger B (2006) Molecular mapping of tyrosine-phosphorylated proteins in focal adhesions using fluorescence resonance energy transfer. J Cell Sci 119(Pt 5):866–875CrossRefPubMedGoogle Scholar
  14. 14.
    Zaidel-Bar R, Cohen M, Addadi L, Geiger B (2004) Hierarchical assembly of cell-matrix adhesion complexes. Biochem Soc Trans 32(Pt3):416–420CrossRefPubMedGoogle Scholar
  15. 15.
    Omachi T, Ichikawa T, Kimura Y, Ueda K, Kioka N (2017) Vinculin association with actin cytoskeleton is necessary for stiffness-dependent regulation of vinculin behavior. PLoS One 12(4):e0175324CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Owen GR, Meredith DO, Gwynn I, Richards RG (2005) Focal adhesion quantification – a new assay of material biocompatibility? Rev Eur Cell Mater 9:85–96CrossRefGoogle Scholar
  17. 17.
    YL H, Lu S, Szeto KW, Sun J, Wang Y, Lasheras JC, Chien S (2014) FAK and paxillin dynamics at focal adhesions in the protrusions of migrating cells. Sci Rep 4:6024Google Scholar
  18. 18.
    Carisey A, Ballestrem C (2011) Vinculin, an adapter protein in control of cell adhesion signaling. Eur J Cell Biol 90(2-3):157–163CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Nagasato AI, Yamashita H, Matsuo M, Ueda K, Kioka N (2017) The distribution of vinculin to lipid rafts plays an important role in sensing stiffness of extracellular matrix. Biosci Biotechnol Biochem 81(6):1136–1147CrossRefPubMedGoogle Scholar
  20. 20.
    Lopez-Colome AM, Lee-Rivera I, Benavides-Hidalgo R, Lopez E (2017) Paxillin: a crossroad in pathological cell migration. J Hematol Oncol 10(1):50CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Schaller MD (2001) Paxillin: a focal adhesion-associated adaptor protein. Oncogene 20(44):6459–6472CrossRefPubMedGoogle Scholar
  22. 22.
    Humphries JD, Wang P, Streuli C, Geiger B, Humphries MJ, Ballestrem C (2007) Vinculin controls focal adhesion formation by direct interactions with talin and actin. J Cell Biol 179(5):1043–1057CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Dumbauld DW, Shin H, Gallant ND, Michael KE, Radhakrishna H, Garcia AJ (2010) Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions. J Cell Physiol 223(3):746–756PubMedPubMedCentralGoogle Scholar
  24. 24.
    Giannone G (2015) Super-resolution links vinculin localization to function in focal adhesions. Nat Cell Biol 17:845–847.  https://doi.org/10.1038/ncb3196 CrossRefPubMedGoogle Scholar
  25. 25.
    Subauste MC, Pertz O, Adamson ED, Turner CE, Junger S, Hahn KM (2004) Vinculin modulation of paxillin-FAK interactions regulates ERK to control survival and motility. J Cell Biol 165(3):371–381CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Zamir E, Geiger B (2001) Molecular complexity and dynamics of cell-matrix adhesions. J Sci 114(Pt 20):3583–3590Google Scholar
  27. 27.
    Berginski ME, Vitriol EA, Hahn KM, Gomez SM (2011) High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells. PLoS One 6(7):e22025CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Spanjaard E, Smal I, Angelopoulos N, Verlaan I, Matov A, Meijering E, Wessels L, Bos H, de Rooij J (2015) Quantitative imaging of focal adhesion dynamics and their regulation by HGF and Rap1 signaling. Exp Cell Res 330(2):382–397CrossRefPubMedGoogle Scholar
  29. 29.
    Hernandez-Varas P, Berge U, Lock JG, Stromblad S (2015) A plastic relationship between vinculin-mediated tension and adhesion complex area defines adhesion size and lifetime. Nat Commun 6:7524CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2018

Authors and Affiliations

  • Aleena Gladkikh
    • 1
  • Anastasia Kovaleva
    • 1
  • Anna Tvorogova
    • 2
  • Ivan A. Vorobjev
    • 3
  1. 1.Department of Cell Biology and Histology, School of BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  2. 2.A.N. Belozersky Institute of Physico-Chemical BiologyM.V. Lomonosov Moscow State UniversityMoscowRussia
  3. 3.Department of Biology, School of Sciences and TechnologyNazarbayev UniversityAstanaKazakhstan

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