Nano Research

, Volume 5, Issue 8, pp 565–575 | Cite as

Nanogratings of fibronectin provide an effective biochemical cue for regulating focal adhesion and cellular structure

  • Lifang Shi
  • Jie-Ren Li
  • Yi-Ping Shih
  • Su Hao Lo
  • Gang-yu LiuEmail author
Research Article


Integrin clustering, typically nanometers in dimension, is the first and an important step in integrin-mediated cellular signaling processes such as focal adhesion. Engineered nanostructures mimicking extracellular matrices (ECM) provide a new approach for investigation and regulation of this initial step and of downstream cascades of focal adhesion. This work reveals that fibronectin (Fn) nanostructures, even at a small height of 3.2 nm ± 0.5 nm, exhibit high efficacy in guiding cellular orientation and polarization. More interestingly, the Fn nanostructures also impact intracellular structures such as preferential filopodia attachment, and commensurate alignment of intracellular actin stress fibers. The impact can be rationalized by the strong and specific interaction between integrin and Fn, leading to integrin clusters and then focal adhesion assemblies following the underlying nanostructure of Fn. This guided assembly further mediates the downstream behavior, such as actin stress fiber alignment and overall cellular morphology. Our observations collectively demonstrate that engineered nanostructures of Fn provide an alternative and high efficacy biochemical cue for regulation of cellular signaling processes.


Fn nanogratings integrin focal adhesion filopodia actin stress fibers 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. [1]
    Edelman, G. M.; Crossin, K. L. Cell adhesion molecules: Implications for a molecular histology. Annu. Rev. Biochem. 1991, 60, 155–190.CrossRefGoogle Scholar
  2. [2]
    Clark, E. A.; Brugge, J. S. Integrins and signal transduction pathways: The road taken. Science 1995, 268, 233–239.CrossRefGoogle Scholar
  3. [3]
    Gumbiner, B. M. Cell adhesion: The molecular basis of tissue architecture and morphogenesis. Cell 1996, 84, 345–357.CrossRefGoogle Scholar
  4. [4]
    Cukierman, E.; Pankov, R.; Stevens, D. R.; Yamada, K. M. Taking cell-matrix adhesions to the third dimension. Science 2001, 294, 1708–1712.CrossRefGoogle Scholar
  5. [5]
    Geiger, B.; Bershadsky, A.; Pankov, R.; Yamada, K. M. Transmembrane extracellular matrix-cytoskeleton crosstalk. Nat. Rev. Mol. Cell Biol. 2001, 2, 793–805.CrossRefGoogle Scholar
  6. [6]
    Lauffenburger, D. A.; Horwitz, A. F. Cell migration: A physically integrated molecular process. Cell 1996, 84, 359–369.CrossRefGoogle Scholar
  7. [7]
    Palecek, S. P.; Loftus, J. C.; Ginsberg, M. H.; Lauffenburger, D. A.; Horwitz, A. F. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 1997, 385, 537–540.CrossRefGoogle Scholar
  8. [8]
    Hood, J. D.; Cheresh, D. A. Role of integrins in cell invasion and migration. Nat. Rev. Cancer 2002, 2, 91–100.CrossRefGoogle Scholar
  9. [9]
    Taipale, J.; Keski-Oja, J. Growth factors in the extracellular matrix. FASEB J. 1997, 11, 51–59.Google Scholar
  10. [10]
    Chen, H. Y.; Lo, S. H. Regulation of tensin-promoted cell migration by its focal adhesion binding and Src homology domain 2. Biochem. J. 2003, 70, 1039–1045.CrossRefGoogle Scholar
  11. [11]
    Lo, S. H. Focal adhesions: What’s new inside. Dev. Biol. 2006, 294, 280–291.CrossRefGoogle Scholar
  12. [12]
    Partridge, M. A.; Marcantonio, E. E. Initiation of attachment and generation of mature focal adhesions by integrin-containing filopodia in cell spreading. Mol. Biol. Cell 2006, 17, 4237–4248.CrossRefGoogle Scholar
  13. [13]
    Galbraith, C. G.; Yamada, K. M.; Galbraith, J. A. Polymerizing actin fibers position integrins primed to probe for adhesion sites. Science 2007, 315, 992–995.CrossRefGoogle Scholar
  14. [14]
    Hynes, R. O. Integrins: Bidirectional, allosteric signaling machines. Cell 2002, 110, 673–687.CrossRefGoogle Scholar
  15. [15]
    Miyamoto, S.; Akiyama, S. K.; Yamada, K. M. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 1995, 267, 883–885.CrossRefGoogle Scholar
  16. [16]
    Miyamoto, S.; Teramoto, H.; Coso, O. A.; Gutkind, J. S.; Burbelo, P. D.; Akiyama, S. K.; Yamada, K. M. Integrin function: molecular hierarchies of cytoskeletal and signaling molecules. J. Cell Biol. 1995, 131, 791–805.CrossRefGoogle Scholar
  17. [17]
    Miyamoto, S.; Teramoto, H.; Gutkind, J. S.; Yamada, K. M. Integrins can collaborate with growth factors for phosphorylation of receptor tyrosine kinases and MAP kinase activation: Roles of integrin aggregation and occupancy of receptors. J. Cell Biol. 1996, 135, 1633–1642.CrossRefGoogle Scholar
  18. [18]
    Leahy, D. J.; Aukhil, I.; Erickson, H. P. 2.0 Å crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 1996, 84, 155–164.CrossRefGoogle Scholar
  19. [19]
    Maheshwari, G.; Brown, G.; Lauffenburger, D. A.; Wells, A.; Griffith, L. G. Cell adhesion and motility depend on nanoscale RGD clustering. J. Cell Sci. 2000, 113, 1677–1686.Google Scholar
  20. [20]
    Critchley, D. R. Focal adhesions-the cytoskeletal connection. Curr. Opin. Cell Biol. 2000, 12, 133–139.CrossRefGoogle Scholar
  21. [21]
    Arnold, M.; Cavalcanti-Adam, E. A.; Glass, R.; Blümmel, J.; Eck, W.; Kantlehner, M.; Kessler, H.; Spatz, J. P. Activation of integrin function by nanopatterned adhesive interfaces. ChemPhysChem 2004, 5, 383–388.CrossRefGoogle Scholar
  22. [22]
    Geiger, B.; Spatz, J. P.; Bershadsky, A. D. Environmental sensing through focal adhesions. Nat. Rev. Mol. Cell Biol. 2009, 10, 21–33.CrossRefGoogle Scholar
  23. [23]
    Teixeira, A. I.; Abrams, G. A.; Murphy, C. J.; Nealey, P. F. Cell behavior on lithographically defined nanostructured substrates. J. Vac. Sci. Technol. B 2003, 21, 683–687.CrossRefGoogle Scholar
  24. [24]
    Loesberg, W. A.; te Riet, J.; van Delft, F. C. M. J. M.; Schön, P.; Figdor, C. G.; Speller, S.; van Loon, J. J. W. A.; Walboomers, X. F.; Jansen, J. A. The threshold at which substrate nanogroove dimensions may influence fibroblast alignment and adhesion. Biomaterials 2007, 28, 3944–3951.CrossRefGoogle Scholar
  25. [25]
    Kim, D. H.; Han, K.; Gupta, K.; Kwon, K. W.; Suh, K. Y.; Levchenko, A. Mechanosensitivity of fibroblast cell shape and movement to anisotropic substratum topography gradients. Biomaterials 2009, 30, 5433–5444.CrossRefGoogle Scholar
  26. [26]
    Oakley, C.; Jaeger, N. A. F.; Brunette, D. M. Sensitivity of fibroblasts and their cytoskeletons to substratum topographies: Topographic guidance and topographic compensation by micromachined grooves of different dimensions. Exp. Cell Res. 1997, 234, 413–424.CrossRefGoogle Scholar
  27. [27]
    Hamilton, D. W.; Oates, C. J.; Hasanzadeh, A.; Mittler, S. Migration of periodontal ligament fibroblasts on nanometric topographical patterns: Influence of filopodia and focal adhesions on contact guidance. PLoS ONE 2010, 5, e15129.CrossRefGoogle Scholar
  28. [28]
    Hamilton, D. W.; Brunette, D. M. “Gap guidance” of fibroblasts and epithelial cells by discontinuous edged surfaces. Exp. Cell Res. 2005, 309, 429–437.CrossRefGoogle Scholar
  29. [29]
    Dalby, M. J.; Gadegaard, N.; Riehle, M. O.; Wilkinson, C. D. W.; Curtis, A. S. G. Investigating filopodia sensing using arrays of defined nano — pits down to 35 nm diameter in size. Int. J. Biochem. Cell Biol. 2004, 36, 2005–2015.CrossRefGoogle Scholar
  30. [30]
    Mrksich, M.; Chen, C. S.; Xia, Y.; Dike, L. E.; Ingber, D. E.; Whitesides, G. M. Controlling cell attachment on contoured surfaces with self-assembled monolayers of alkanethiolates on gold. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 10775–10778.CrossRefGoogle Scholar
  31. [31]
    Mrksich, M.; Dike, L. E.; Tien, J.; Ingber, D. E.; Whitesides, G. M. Using microcontact printing to pattern the attachment of mammalian cells to self-assembled monolayers of alkanethiolates on transparent films of gold and silver. Exp. Cell Res. 1997, 235, 305–313.CrossRefGoogle Scholar
  32. [32]
    Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Micropatterned surfaces for control of cell shape, position, and function. Biotechnol. Prog. 1998, 14, 356–363.CrossRefGoogle Scholar
  33. [33]
    Chen, C. S.; Mrksich, M.; Huang, S.; Whitesides, G. M.; Ingber, D. E. Geometric control of cell life and death. Science 1997, 276, 1425–1428.CrossRefGoogle Scholar
  34. [34]
    Xia, N.; Thodeti, C. K.; Hunt, T. P.; Xu, Q. B.; Ho, M.; Whitesides, G. M.; Westervelt, R.; Ingber, D. E. Directional control of cell motility through focal adhesion positioning and spatial control of Rac activation. FASEB J. 2008, 22, 1649–1659.CrossRefGoogle Scholar
  35. [35]
    Théry, M.; Bornens, M. Cell shape and cell division. Curr. Opin. Cell Biol. 2006, 18, 648–657.CrossRefGoogle Scholar
  36. [36]
    Théry, M.; Pépin, A.; Dressaire, E.; Chen, Y.; Bornens, M. Cell distribution of stress fibres in response to the geometry of the adhesive environment. Cell Motil. Cytoskeleton 2006, 63, 341–355.CrossRefGoogle Scholar
  37. [37]
    Théry, M.; Racine, V.; Piel, M.; Pépin, A.; Dimitrov, A.; Chen, Y.; Sibarita, J.-B.; Bornens, M. Anisotropy of cell adhesive microenvironment governs cell internal organization and orientation of polarity. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 19771–19776.CrossRefGoogle Scholar
  38. [38]
    Théry, M. Micropatterning as a tool to decipher cell morphogenesis and functions. J. Cell Sci. 2010, 123, 4201–4213.CrossRefGoogle Scholar
  39. [39]
    Lee, S. -J.; Son, Y.; Kim, C. -H.; Choi, M. Fabrication of micro patterned fibronectin for studying adhesion and alignment behavior of human dermal fibroblasts. Macromol. Res. 2007, 15, 348–356.CrossRefGoogle Scholar
  40. [40]
    Radha, B.; Kulkarni, G. U. Dewetting assisted patterning of polystyrene by soft lithography to create nanotrenches for nanomaterial deposition. ACS Appl. Mater. Interfaces 2009, 1, 257–260.CrossRefGoogle Scholar
  41. [41]
    Meenakshi, V.; Babayan, Y.; Odom, T. W. Benchtop nanoscale patterning using soft lithography. J. Chem. Educ. 2007, 84, 1795–1798.CrossRefGoogle Scholar
  42. [42]
    Li, J. -R.; Yin, N. -N.; Liu, G. -Y. Hierarchical micro- and nanoscale structures on surfaces produced using a one-step pattern transfer process. J. Phys. Chem. Lett. 2011, 2, 289–294.CrossRefGoogle Scholar
  43. [43]
    Chen, H. Y.; Duncan, I. C.; Bozorgchami, H.; Lo, S. H. Tensin1 and a previously undocumented family member, tensin2, positively regulate cell migration. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 733–738.Google Scholar
  44. [44]
    Lehnert, D.; Wehrle-Haller, B.; David, C.; Weiland, U.; Ballestrem, C.; Imhof, B. A.; Bastmeyer, M. Cell behaviour on micropatterned substrata: Limits of extracellular matrix geometry for spreading and adhesion. J. Cell Sci. 2004, 117, 41–52.CrossRefGoogle Scholar
  45. [45]
    Mrksich, M. Using self-assembled monolayers to model the extracellular matrix. Acta Biomater. 2009, 5, 832–841.CrossRefGoogle Scholar
  46. [46]
    Iwamoto, G. K.; Winterton, L. C.; Stoker, R. S.; van Wagenen, R. A.; Andrade, J. D.; Mosher, D. F. Fibronectin adsorption detected by interfacial fluorescence J. Colloid Interf. Sci. 1985, 106, 459–464.CrossRefGoogle Scholar
  47. [47]
    Grinnell, F.; Feld, M. K. Fibronectin adsorption on hydrophilic and hydrophobic surfaces detected by antibody binding and analyzed during cell adhesion in serum-containing medium. J. Biol. Chem. 1982, 257, 4888–4893.Google Scholar
  48. [48]
    Gao, P.; Cai, Y. G. The boundary molecules in a lysozyme pattern exhibit preferential antibody binding. Langmuir 2008, 24, 10334–10339.CrossRefGoogle Scholar
  49. [49]
    Bergkvist, M.; Carlsson, J.; Oscarsson, S. Surface-dependent conformations of human plasma fibronectin adsorbed to silica, mica, and hydrophobic surfaces, studied with use of atomic force microscopy. J. Biomed. Mater. Res. Part A 2003, 64A, 349–356.CrossRefGoogle Scholar
  50. [50]
    Mogilner, A.; Rubinstein, B. The physics of filopodial protrusion. Biophys. J. 2005, 89, 782–795.CrossRefGoogle Scholar
  51. [51]
    Mattila, P. K.; Lappalainen, P. Filopodia: Molecular architecture and cellular functions. Nat. Rev. Mol. Cell Biol. 2008, 9, 446–454.CrossRefGoogle Scholar
  52. [52]
    Haga, H.; Nagayama, M.; Kawabata, K.; Ito, E.; Ushiki, T.; Sambongi, T. Time-lapse viscoelastic imaging of living fibroblasts using force modulation mode in AFM. J. Electron Microsc. 2000, 49, 473–481.CrossRefGoogle Scholar
  53. [53]
    Amano, M.; Chihara, K.; Kimura, K.; Fukata, Y.; Nakamura, N.; Matsuura, Y.; Kaibuchi, K. Formation of actin stress fibers and focal adhesions enhanced by Rho-kinase. Science 1997, 275, 1308–1311.CrossRefGoogle Scholar
  54. [54]
    Allen, W. E.; Jones, G. E.; Pollard, J. W.; Ridley, A. J. Rho, Rac and Cdc42 regulate actin organization and cell adhesion in macrophages. J. Cell Sci. 1997, 110, 707–720.Google Scholar
  55. [55]
    Defilippi, P.; Olivo, C.; Venturino, M.; Dolce, L.; Silengo, L.; Tarone, G. Actin cytoskeleton organization in response to integrin-mediated adhesion. Microsc. Res. Tech. 1999, 47, 67–78.CrossRefGoogle Scholar
  56. [56]
    Schwartz, M. A.; Shattil, S. J. Signaling networks linking integrins and rho family GTPases. Trends Biochem. Sci. 2000, 25, 388–391.CrossRefGoogle Scholar
  57. [57]
    Cox, E. A.; Sastry, S. K.; Huttenlocher, A. Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases. Mol. Biol. Cell 2001, 12, 265–277.Google Scholar
  58. [58]
    Wozniak, M. A.; Modzelewska, K.; Kwong, L.; Keely, P. J. Focal adhesion regulation of cell behavior. Biochim.Biophys. Acta-Mol. Cell Res. 2004, 1692, 103–119.CrossRefGoogle Scholar
  59. [59]
    Sastry, S. K.; Burridge, K. Focal adhesions: A nexus for intracellular signaling and cytoskeletal dynamics. Exp. Cell Res. 2000, 261, 25–36.CrossRefGoogle Scholar
  60. [60]
    Le Clainche, C.; Carlier, M. -F. Regulation of actin assembly associated with protrusion and adhesion in cell migration. Physiol. Rev. 2008, 88, 489–513.CrossRefGoogle Scholar

Copyright information

© Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Lifang Shi
    • 1
  • Jie-Ren Li
    • 1
  • Yi-Ping Shih
    • 2
  • Su Hao Lo
    • 2
  • Gang-yu Liu
    • 1
    Email author
  1. 1.Department of ChemistryUniversity of CaliforniaDavisUSA
  2. 2.Department of Biochemistry and Molecular Medicine, Center for Tissue Regeneration and RepairUniversity of CaliforniaDavis, SacramentoUSA

Personalised recommendations