Annals of Biomedical Engineering

, Volume 38, Issue 1, pp 208–222 | Cite as

Effect of Focal Adhesion Proteins on Endothelial Cell Adhesion, Motility and Orientation Response to Cyclic Strain

  • Hai Ngu
  • Yunfeng Feng
  • Lan Lu
  • Sara J. Oswald
  • Gregory D. Longmore
  • Frank C.-P. YinEmail author


Focal adhesion proteins link cell surface integrins and intracellular actin stress fibers and therefore play an important role in mechanotransduction and cell motility. When endothelial cells are subjected to cyclic mechanical strain, time-lapse imaging revealed that cells underwent significant morphological changes with their resultant long axes aligned away from the strain direction. To explore how this response is regulated by focal adhesion-associated proteins the expression levels of paxillin, focal adhesion kinase (FAK), and zyxin were knocked down using gene silencing techniques. In addition, rescue of endogenous and two mutant zyxins were used to investigate the specific role of zyxin interactions. Cells with decreased zyxin expression levels and rescue with the mutant lacking zyxin/α-actinin binding exhibited lower orientation angles after comparable times of stretching as compared to normal and control cells. However, knockdown of the expression levels of paxillin and FAK and rescue with the mutant lacking zyxin/VASP (vasodilator-stimulated phosphoprotein) binding did not significantly affect the degree of cell orientation. In addition, wound closure speed and cell–substratum adhesive strength were observed to be significantly reduced only for cells with zyxin depletion and the mutation lacking zyxin/α-actinin binding. These results suggest that zyxin and its interaction with α-actinin are important in the regulation of endothelial cell adhesive strength, motility and orientation response to mechanical stretching.


Cell stretching Cell orientation Gene silencing Zyxin Focal adhesion Time-lapse imaging 



This research was supported by a grant-in-aid from the American Heart Association (PI: FCPY).

Supplementary material

Supplementary Movie 1

Representative time-lapse movie (5 min increments) of wildtype cells during six-hours of cyclic uniaxial (horizontal direction) stretching. These are expanded views of the upper right-hand corner of the wide field shown in supplementary movie 2 and correspond to the images in Fig. 2a (AVI 21660 kb)

Supplementary Movie 2

Representative time-lapse movie (5 min increments) of wildtype cells during six-hours of cyclic uniaxial (horizontal direction) stretching (AVI 77901 kb)

Supplementary Movie 3

Representative time-lapse movie (5 min increments) of zyxin knockdown (Zn-sh1) cells during six-hours of cyclic uniaxial (horizontal direction) stretching (AVI 74883 kb)


  1. 1.
    Brown, C. M., B. Hebert, D. L. Kolin, J. Zareno, L. Whitmore, A. R. Horwitz, and P. W. Wiseman. Probing the integrin-actin linkage using high-resolution protein velocity mapping. J. Cell Sci. 119(Pt 24):5204–5214, 2006.CrossRefPubMedGoogle Scholar
  2. 2.
    Cattaruzza, M., C. Lattrich, and M. Hecker. Focal adhesion protein zyxin is a mechanosensitive modulator of gene expression in vascular smooth muscle cells. Hypertension 43(4):726–730, 2004.CrossRefPubMedGoogle Scholar
  3. 3.
    Cozens-Roberts, C., J. A. Quinn, and D. A. Lauffenberger. Receptor-mediated adhesion phenomena. Model studies with the radical-flow detachment assay. Biophys. J. 58(1):107–125, 1990.CrossRefPubMedGoogle Scholar
  4. 4.
    Crawford, A. W., and M. C. Beckerle. Purification and characterization of zyxin, an 82,000-dalton component of adherens junctions. J. Biol. Chem. 266(9):5847–5853, 1991.PubMedGoogle Scholar
  5. 5.
    Crawford, A. W., J. W. Michelsen, and M. C. Beckerle. An interaction between zyxin and alpha-actinin. J. Cell Biol. 116(6):1381–1393, 1992.CrossRefPubMedGoogle Scholar
  6. 6.
    Dickinson, R. B., and R. T. Tranquillo. Optimal estimation of cell movement indices from the statistical analysis of cell tracking data. AIChE J. 39:1995–2010, 1993.CrossRefGoogle Scholar
  7. 7.
    DiMilla, P. A., J. A. Stone, J. A. Quinn, S. M. Albelda, and D. A. Lauffenburger. Maximal migration of human smooth muscle cells on fibronectin and type iv collagen occurs at an intermediate attachment strength. J. Cell Biol. 122(3):729–737, 1993.CrossRefPubMedGoogle Scholar
  8. 8.
    Goldstein, A. S., and P. A. DiMilla. Application of fluid mechanics and kinetic models to characterize mammalian cell detachment in a radial-flow chamber. Biotech. Bioeng. 55(4):616–629, 1997.CrossRefGoogle Scholar
  9. 9.
    Goldstein, A. S., and P. A. DiMilla. Comparison of converging and diverging radial flow for measuring cell adhesion. AIChE J. 44(2):465–473, 1998.CrossRefGoogle Scholar
  10. 10.
    Hagel, M., E. L. George, A. Kim, R. Tamimi, S. L. Opitz, C. E. Turner, A. Imamoto, and S. M. Thomas. The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling. Mol. Cell. Biol. 22(3):901–915, 2002.CrossRefPubMedGoogle Scholar
  11. 11.
    Harms, B. D., G. M. Bassi, A. R. Horwitz, and D. A. Lauffenburger. Directional persistence of egf-induced cell migration is associated with stabilization of lamellipodial protrusions. Biophys. J. 88(2):1479–1488, 2005.CrossRefPubMedGoogle Scholar
  12. 12.
    Hayakawa, K., A. Hosokawa, K. Yabusaki, and T. Obinata. Orientation of smooth muscle-derived a10 cells in culture by cyclic stretching: relationship between stress fiber rearrangement and cell reorientation. Zool. Sci. 17(5):617–624, 2000.PubMedGoogle Scholar
  13. 13.
    Hoffman, L. M., C. C. Jensen, S. Kloeker, C. L. Wang, M. Yoshigi, and M. C. Beckerle. Genetic ablation of zyxin causes mena/vasp mislocalization, increased motility, and deficits in actin remodeling. J. Cell Biol. 172(5):771–782, 2006.CrossRefPubMedGoogle Scholar
  14. 14.
    Iba, T., and B. E. Sumpio. Morphological response of human endothelial cells subjected to cyclic strain in vitro. Microvasc. Res. 42(3):245–254, 1991.CrossRefPubMedGoogle Scholar
  15. 15.
    Ilic, D., Y. Furuta, S. Kanazawa, N. Takeda, K. Sobue, N. Nakatsuji, S. Nomura, J. Fujimoto, M. Okada, and T. Yamamoto. Reduced cell motility and enhanced focal adhesion contact formation in cells from fak-deficient mice. Nature 377(6549):539–544, 1995.CrossRefPubMedGoogle Scholar
  16. 16.
    Lee, N. P., D. D. Mruk, A. M. Conway, and C. Y. Cheng. Zyxin, axin, and wiskott-aldrich syndrome protein are adaptors that link the cadherin/catenin protein complex to the cytoskeleton at adherens junctions in the seminiferous epithelium of the rat testis. J. Androl. 25(2):200–215, 2004.PubMedGoogle Scholar
  17. 17.
    Li, B., and B. Trueb. Analysis of the alpha-actinin/zyxin interaction. J. Biol. Chem. 276(36):33328–33335, 2001.CrossRefPubMedGoogle Scholar
  18. 18.
    Lu, L., Y. Feng, W. J. Hucker, S. J. Oswald, G. D. Longmore, and F. C. Yin. Actin stress fiber pre-extension in human aortic endothelial cells. Cell Motil. Cytoskeleton 65(4):281–294, 2008.CrossRefPubMedGoogle Scholar
  19. 19.
    Moody, J. D., J. Grange, M. P. Ascione, D. Boothe, E. Bushnell, and M. D. Hansen. A zyxin head-tail interaction regulates zyxin-vasp complex formation. Biochem. Biophys. Res. Commun. 378(3):625–628, 2009.CrossRefPubMedGoogle Scholar
  20. 20.
    Ngu, H., L. Lu, S. J. Oswald, S. Davis, S. Nag, and F. C. Yin. Strain-induced orientation response of endothelial cells: effect of substratum adhesiveness and actin-myosin contractile level. Mol. Cell. Biomech. 5(1):69–81, 2008.PubMedGoogle Scholar
  21. 21.
    Nix, D. A., and M. C. Beckerle. Nuclear-cytoplasmic shuttling of the focal contact protein, zyxin: a potential mechanism for communication between sites of cell adhesion and the nucleus. J. Cell Biol. 138(5):1139–1147, 1997.CrossRefPubMedGoogle Scholar
  22. 22.
    Nix, D. A., J. Fradelizi, S. Bockholt, B. Menichi, D. Louvard, E. Friederich, and M. C. Beckerle. Targeting of zyxin to sites of actin membrane interaction and to the nucleus. J. Biol. Chem. 276(37):34759–34767, 2001.CrossRefPubMedGoogle Scholar
  23. 23.
    O’Neill, G. M., S. J. Fashena, and E. A. Golemis. Integrin signalling: a new cas(t) of characters enters the stage. Trends Cell Biol. 10(3):111–119, 2000.CrossRefPubMedGoogle Scholar
  24. 24.
    Owatverot, T. B., S. J. Oswald, Y. Chen, J. J. Wille, and F. C. Yin. Effect of combined cyclic stretch and fluid shear stress on endothelial cell morphological responses. J. Biomech. Eng. 127(3):374–382, 2005.CrossRefPubMedGoogle Scholar
  25. 25.
    Palecek, S. P., J. C. Loftus, M. H. Ginsberg, D. A. Lauffenburger, and A. F. Horwitz. Integrin-ligand binding properties govern cell migration speed through cell-substratum adhesiveness. Nature 385(6616):537–540, 1997.CrossRefPubMedGoogle Scholar
  26. 26.
    Peel, M. M., and P. A. DiMilla. Effect of cell–cell interactions on the observable strength of adhesion of sheets of cells. Ann. Biomed. Eng. 27(2):236–246, 1999.CrossRefPubMedGoogle Scholar
  27. 27.
    Reinhard, M., J. Zumbrunn, D. Jaquemar, M. Kuhn, U. Walter, and B. Trueb. An alpha-actinin binding site of zyxin is essential for subcellular zyxin localization and alpha-actinin recruitment. J. Biol. Chem. 274(19):13410–13418, 1999.CrossRefPubMedGoogle Scholar
  28. 28.
    Shirinsky, V. P., A. S. Antonov, K. G. Birukov, A. V. Sobolevsky, Y. A. Romanov, N. V. Kabaeva, G. N. Antonova, and V. N. Smirnov. Mechano-chemical control of human endothelium orientation and size. J. Cell Biol. 109(1):331–339, 1989.CrossRefPubMedGoogle Scholar
  29. 29.
    Wang, H. B., M. Dembo, S. K. Hanks, and Y. Wang. Focal adhesion kinase is involved in mechanosensing during fibroblast migration. Proc. Natl Acad. Sci. USA 98(20):11295–11300, 2001.CrossRefPubMedGoogle Scholar
  30. 30.
    Wang, J. H., P. Goldschmidt-Clermont, N. Moldovan, and F. C. Yin. Leukotrienes and tyrosine phosphorylation mediate stretching-induced actin cytoskeletal remodeling in endothelial cells. Cell Motil. Cytoskeleton 46(2):137–145, 2000.CrossRefPubMedGoogle Scholar
  31. 31.
    Wang, J. H., P. Goldschmidt-Clermont, and F. C. Yin. Contractility affects stress fiber remodeling and reorientation of endothelial cells subjected to cyclic mechanical stretching. Ann. Biomed. Eng. 28(10):1165–1171, 2000.CrossRefPubMedGoogle Scholar
  32. 32.
    Wille, J. J., C. M. Ambrosi, and F. C. Yin. Comparison of the effects of cyclic stretching and compression on endothelial cell morphological responses. J. Biomech. Eng. 126(5):545–551, 2004.CrossRefPubMedGoogle Scholar
  33. 33.
    Yano, H., Y. Mazaki, K. Kurokawa, S. K. Hanks, M. Matsuda, and H. Sabe. Roles played by a subset of integrin signaling molecules in cadherin-based cell–cell adhesion. J. Cell Biol. 166(2):283–295, 2004.CrossRefPubMedGoogle Scholar
  34. 34.
    Yi, J., S. Kloeker, C. C. Jensen, S. Bockholt, H. Honda, H. Hirai, and M. C. Beckerle. Members of the zyxin family of lim proteins interact with members of the p130cas family of signal transducers. J. Biol. Chem. 277(11):9580–9589, 2002.CrossRefPubMedGoogle Scholar
  35. 35.
    Yoshigi, M., L. M. Hoffman, C. C. Jensen, H. J. Yost, and M. C. Beckerle. Mechanical force mobilizes zyxin from focal adhesions to actin filaments and regulates cytoskeletal reinforcement. J. Cell Biol. 171(2):209–215, 2005.CrossRefPubMedGoogle Scholar
  36. 36.
    Yung, L. Y., R. W. Colman, and S. L. Cooper. Neutrophil adhesion on polyurethanes preadsorbed with high molecular weight kininogen. Blood 94(8):2716–2724, 1999.PubMedGoogle Scholar

Copyright information

© Biomedical Engineering Society 2009

Authors and Affiliations

  • Hai Ngu
    • 1
  • Yunfeng Feng
    • 2
  • Lan Lu
    • 1
  • Sara J. Oswald
    • 1
  • Gregory D. Longmore
    • 2
  • Frank C.-P. Yin
    • 1
    • 2
    Email author
  1. 1.Department of Biomedical EngineeringWashington University in St. LouisSt. LouisUSA
  2. 2.Department of MedicineWashington University School of MedicineSt. LouisUSA

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