, Volume 52, Issue 14, pp 3389–3398 | Cite as

Mechanosensing of substrate stiffness regulates focal adhesions dynamics in cell

  • Sabato FuscoEmail author
  • Valeria Panzetta
  • Paolo A. Netti
Active Behavior in Soft Matter and Mechanobiology


Cell mechanical recognition of extracellular matrix determines the cell activities and functions. Focal adhesions are part of the cell mechanosensing machinery and, operating at the very dynamic interface between cell and extracellular matrix, can operate this recognition and trigger conformational, functional and behavioral modification of the cell. To investigate how the dynamic of assembly and disassembly of focal adhesion are influenced by the substrate mechanics we developed a novel procedure. The analysis consists of the over time tracking of focal adhesion structures in a stable cell line of NIH/3T3 expressing fluorescent pmKate2-paxillin. From collected signals and by their autocorrelation we evaluated the average lifetime and assembly rate of focal adhesion as function of substrate stiffness. Further, by signals cross-correlation we obtained information about the mechanical nature of cytoskeleton and its network. This quantitative approach to focal adhesion dynamics characterization was presented in this study as an investigation tool for cell mechanobiology.


Cell mechanosensing Extracellular matrix Stiffness Focal adhesions dynamics 



The authors acknowledge Mrs. R. Infranca for manuscript proofreading and editing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11012_2017_676_MOESM1_ESM.docx (6.8 mb)
Supplementary material 1 (DOCX 6948 kb)


  1. 1.
    Gasiorowski JZ, Murphy CJ, Nealey PF (2013) Biophysical cues and cell behavior: the big impact of little things. Annu Rev Biomed Eng 15:155–176CrossRefGoogle Scholar
  2. 2.
    Keung AJ, Kumar S, Schaffer DV (2010) Presentation counts: microenvironmental regulation of stem cells by biophysical and material cues. Annu Rev Cell Dev Biol 26:533–556CrossRefGoogle Scholar
  3. 3.
    Discher DE, Janmey P, Y-l Wang (2005) Tissue cells feel and respond to the stiffness of their substrate. Science 310:1139–1143ADSCrossRefGoogle Scholar
  4. 4.
    Lutolf MP, Gilbert PM, Blau HM (2009) Designing materials to direct stem-cell fate. Nature 462:433–441ADSCrossRefGoogle Scholar
  5. 5.
    Downing TL, Soto J, Morez C, Houssin T, Fritz A, Yuan F, Chu J, Patel S, Schaffer DV, Li S (2013) Biophysical regulation of epigenetic state and cell reprogramming. Nat Mater 12:1154–1162ADSCrossRefGoogle Scholar
  6. 6.
    Lopez J, Mouw J, Weaver V (2008) Biomechanical regulation of cell orientation and fate. Oncogene 27:6981–6993CrossRefGoogle Scholar
  7. 7.
    Lee J, Abdeen AA, Kilian KA (2014) Rewiring mesenchymal stem cell lineage specification by switching the biophysical microenvironment. Sci Rep 4:1–8Google Scholar
  8. 8.
    Charras G, Sahai E (2014) Physical influences of the extracellular environment on cell migration. Nat Rev Mol Cell Biol 15:813–824CrossRefGoogle Scholar
  9. 9.
    Vogel V, Sheetz M (2006) Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol 7:265–275CrossRefGoogle Scholar
  10. 10.
    Wang JH-C, Thampatty BP (2006) An introductory review of cell mechanobiology. Biomech Model Mechanobiol 5:1–16CrossRefGoogle Scholar
  11. 11.
    Mammoto T, Mammoto A, Ingber DE (2013) Mechanobiology and developmental control. Annu Rev Cell Dev Biol 29:27–61CrossRefGoogle Scholar
  12. 12.
    Shin J-W, Mooney DJ (2016) Improving stem cell therapeutics with mechanobiology. Cell Stem Cell 18:16–19CrossRefGoogle Scholar
  13. 13.
    Giannone G (2015) Super-resolution links vinculin localization to function in focal adhesions. Nat Cell Biol 17:845–847CrossRefGoogle Scholar
  14. 14.
    Case LB, Baird MA, Shtengel G, Campbell SL, Hess HF, Davidson MW, Waterman CM (2015) Molecular mechanism of vinculin activation and nanoscale spatial organization in focal adhesions. Nat Cell Biol 17:880–892CrossRefGoogle Scholar
  15. 15.
    Atherton P, Stutchbury B, Wang D-Y, Jethwa D, Tsang R, Meiler-Rodriguez E, Wang P, Bate N, Zent R, Barsukov IL (2015) Vinculin controls talin engagement with the actomyosin machinery. Nat Commun 6:1–12CrossRefGoogle Scholar
  16. 16.
    Schiller HB, Fässler R (2013) Mechanosensitivity and compositional dynamics of cell–matrix adhesions. EMBO Rep 14:509–519CrossRefGoogle Scholar
  17. 17.
    Parsons JT, Horwitz AR, Schwartz MA (2010) Cell adhesion: integrating cytoskeletal dynamics and cellular tension. Nat Rev Mol Cell Biol 11:633–643CrossRefGoogle Scholar
  18. 18.
    Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay I, Mahalu D, Safran S, Bershadsky A, Addadi L (2001) Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 3:466–472CrossRefGoogle Scholar
  19. 19.
    Fusco S, Panzetta V, Embrione V, Netti PA (2015) Crosstalk between focal adhesions and material mechanical properties governs cell mechanics and functions. Acta Biomater 23:63–71CrossRefGoogle Scholar
  20. 20.
    Geiger B, Spatz JP, Bershadsky AD (2009) Environmental sensing through focal adhesions. Nat Rev Mol Cell Biol 10:21–33CrossRefGoogle Scholar
  21. 21.
    Gupta M, Doss B, Lim CT, Voituriez R, Ladoux B (2016) Single cell rigidity sensing: a complex relationship between focal adhesion dynamics and large-scale actin cytoskeleton remodeling. Cell Adh Migr 10:554–567CrossRefGoogle Scholar
  22. 22.
    Kim D-H, Khatau SB, Feng Y, Walcott S, Sun SX, Longmore GD, Wirtz D (2012) Actin cap associated focal adhesions and their distinct role in cellular mechanosensing. Sci Rep 2:1–13Google Scholar
  23. 23.
    Webb DJ, Parsons JT, Horwitz AF (2002) Adhesion assembly, disassembly and turnover in migrating cells–over and over and over again. Nat Cell Biol 4:E97–E100CrossRefGoogle Scholar
  24. 24.
    Mitra SK, Hanson DA, Schlaepfer DD (2005) Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6:56–68CrossRefGoogle Scholar
  25. 25.
    Strzyz P (2016) Cell migration: recycling active integrin for adhesion reassembly. Nat Rev Mol Cell Biol 17:264Google Scholar
  26. 26.
    Einstein A (1905) Un the movement of small particles suspended in statiunary liquids required by the molecular-kinetic theory of heat. Ann Phys 17:549–560CrossRefGoogle Scholar
  27. 27.
    Tseng Y, Kole TP, Lee S-HJ, Wirtz D (2002) Local dynamics and viscoelastic properties of cell biological systems. Curr Opin Colloid Interface Sci 7:210–217CrossRefGoogle Scholar
  28. 28.
    Tseng Y, Kole TP, Wirtz D (2002) Micromechanical mapping of live cells by multiple-particle-tracking microrheology. Biophys J 83:3162–3176CrossRefGoogle Scholar
  29. 29.
    Squires TM, Mason TG (2009) Fluid mechanics of microrheology. Annu Rev Fluid Mech 42:413ADSCrossRefGoogle Scholar
  30. 30.
    Hoffman BD, Massiera G, Van Citters KM, Crocker JC (2006) The consensus mechanics of cultured mammalian cells. Proc Natl Acad Sci 103:10259–10264ADSCrossRefGoogle Scholar
  31. 31.
    Brangwynne CP, Koenderink GH, MacKintosh FC, Weitz DA (2009) Intracellular transport by active diffusion. Trends Cell Biol 19:423–427CrossRefGoogle Scholar
  32. 32.
    Chinga G, Syverud K (2007) Quantification of paper mass distributions within local picking areas. Nord Pulp Pap Res J 22:441–446CrossRefGoogle Scholar
  33. 33.
    D’Souza SE, Ginsberg MH, Plow EF (1991) Arginyl-glycyl-aspartic acid (RGD): a cell adhesion motif. Trends Biochem Sci 16:246–250CrossRefGoogle Scholar
  34. 34.
    Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491–497ADSCrossRefGoogle Scholar
  35. 35.
    Bhadriraju K, Hansen LK (2002) Extracellular matrix-and cytoskeleton-dependent changes in cell shape and stiffness. Exp Cell Res 278:92–100CrossRefGoogle Scholar
  36. 36.
    Ghosh K, Pan Z, Guan E, Ge S, Liu Y, Nakamura T, Ren X-D, Rafailovich M, Clark RA (2007) Cell adaptation to a physiologically relevant ECM mimic with different viscoelastic properties. Biomaterials 28:671–679CrossRefGoogle Scholar
  37. 37.
    Al-Rekabi Z, Pelling AE (2013) Cross talk between matrix elasticity and mechanical force regulates myoblast traction dynamics. Phys Biol 10:066003ADSCrossRefGoogle Scholar
  38. 38.
    Vasiliev JM (1984) Spreading of non-transformed and transformed cells. Biochim Biophys Acta Rev Cancer 780:21–65CrossRefGoogle Scholar
  39. 39.
    Prager-Khoutorsky M, Lichtenstein A, Krishnan R, Rajendran K, Mayo A, Kam Z, Geiger B, Bershadsky AD (2011) Fibroblast polarization is a matrix-rigidity-dependent process controlled by focal adhesion mechanosensing. Nat Cell Biol 13:1457–1465CrossRefGoogle Scholar
  40. 40.
    Plotnikov SV, Pasapera AM, Sabass B, Waterman CM (2012) Force fluctuations within focal adhesions mediate ECM-rigidity sensing to guide directed cell migration. Cell 151:1513–1527CrossRefGoogle Scholar
  41. 41.
    Oakes PW, Gardel ML (2014) Stressing the limits of focal adhesion mechanosensitivity. Curr Opin Cell Biol 30:68–73CrossRefGoogle Scholar
  42. 42.
    Albelda SM, Buck CA (1990) Integrins and other cell adhesion molecules. FASEB J 4:2868–2880Google Scholar
  43. 43.
    Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25CrossRefGoogle Scholar
  44. 44.
    Wolfenson H, Lavelin I, Geiger B (2013) Dynamic regulation of the structure and functions of integrin adhesions. Dev Cell 24:447–458CrossRefGoogle Scholar
  45. 45.
    Bouvard D, Pouwels J, De Franceschi N, Ivaska J (2013) Integrin inactivators: balancing cellular functions in vitro and in vivo. Nat Rev Mol Cell Biol 14:430–442CrossRefGoogle Scholar
  46. 46.
    Geiger B, Bershadsky A, Pankov R, Yamada KM (2001) Transmembrane crosstalk between the extracellular matrix and the cytoskeleton. Nat Rev Mol Cell Biol 2:793–805CrossRefGoogle Scholar
  47. 47.
    Gupta M, Sarangi BR, Deschamps J, Nematbakhsh Y, Callan-Jones A, Margadant F, Mège R-M, Lim CT, Voituriez R, Ladoux B (2015) Adaptive rheology and ordering of cell cytoskeleton govern matrix rigidity sensing. Nat Commun 6:1–9Google Scholar
  48. 48.
    Pellegrin S, Mellor H (2007) Actin stress fibres. J Cell Sci 120:3491–3499CrossRefGoogle Scholar
  49. 49.
    Luo Y, Xu X, Lele T, Kumar S, Ingber DE (2008) A multi-modular tensegrity model of an actin stress fiber. J Biomech 41:2379–2387CrossRefGoogle Scholar
  50. 50.
    Tojkander S, Gateva G, Lappalainen P (2012) Actin stress fibers–assembly, dynamics and biological roles. J Cell Sci 125:1855–1864CrossRefGoogle Scholar
  51. 51.
    Schliwa M, Van Blerkom J (1981) Structural interaction of cytoskeletal components. J Cell Biol 90:222–235CrossRefGoogle Scholar
  52. 52.
    Ingber DE (1993) Cellular tensegrity: defining new rules of biological design that govern the cytoskeleton. J Cell Sci 104:613Google Scholar
  53. 53.
    Saez A, Anon E, Ghibaudo M, Du Roure O, Di Meglio J, Hersen P, Silberzan P, Buguin A, Ladoux B (2010) Traction forces exerted by epithelial cell sheets. J Phys: Condens Matter 22:194119ADSGoogle Scholar
  54. 54.
    Kumar S, Maxwell IZ, Heisterkamp A, Polte TR, Lele TP, Salanga M, Mazur E, Ingber DE (2006) Viscoelastic retraction of single living stress fibers and its impact on cell shape, cytoskeletal organization, and extracellular matrix mechanics. Biophys J 90:3762–3773CrossRefGoogle Scholar
  55. 55.
    Livne A, Bouchbinder E, Geiger B (2014) Cell reorientation under cyclic stretching. Nat Commun 5:1–8CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2017

Authors and Affiliations

  • Sabato Fusco
    • 1
    Email author
  • Valeria Panzetta
    • 1
  • Paolo A. Netti
    • 1
    • 2
  1. 1.Center for Advanced Biomaterials for Health Care IIT@CRIBIstituto Italiano di TecnologiaNaplesItaly
  2. 2.Interdisciplinary Research Centre on Biomaterials, CRIB and Department of Chemical, Materials and Industrial Production EngineeringUniversity of Naples Federico IINaplesItaly

Personalised recommendations