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Orientation-specific responses to sustained uniaxial stretching in focal adhesion growth and turnover

  1. Clare M. Watermanb,1
  1. aLaboratory of Functional Molecular Imaging, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; and
  2. bCell Biology and Physiology Center, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892

  1. Edited by Terry Lechler, Duke University, Durham, NC, and accepted by the Editorial Board May 14, 2013 (received for review December 17, 2012)

Significance

Cells are mechanosensitive: a cell’s adaptive responses to mechanical inputs will determine its morphology, gene expression, and posttranslational protein modification. Focal adhesions (FAs), the integrin-containing complexes positioned between the extracellular matrix and the cell, are key in transmitting the mechanosignal to initiate these downstream adaptive responses. In this study we made the unique observation that FA and cellular morphology changes in response to sustained uniaxial stretch are orientation specific. Our results also suggest orientation-specific responses at the molecular level in individual FAs propagate up to regulate cell morphology, which may in turn mediate adaptive responses that promote tissue growth.

Abstract

Cells are mechanosensitive to extracellular matrix (ECM) deformation, which can be caused by muscle contraction or changes in hydrostatic pressure. Focal adhesions (FAs) mediate the linkage between the cell and the ECM and initiate mechanically stimulated signaling events. We developed a stretching apparatus in which cells grown on fibronectin-coated elastic substrates can be stretched and imaged live to study how FAs dynamically respond to ECM deformation. Human bone osteosarcoma epithelial cell line U2OS was transfected with GFP-paxillin as an FA marker and subjected to sustained uniaxial stretching. Two responses at different timescales were observed: rapid FA growth within seconds after stretching, and delayed FA disassembly and loss of cell polarity that occurred over tens of minutes. Rapid FA growth occurred in all cells; however, delayed responses to stretch occurred in an orientation-specific manner, specifically in cells with their long axes perpendicular to the stretching direction, but not in cells with their long axes parallel to stretch. Pharmacological treatments demonstrated that FA kinase (FAK) promotes but Src inhibits rapid FA growth, whereas FAK, Src, and calpain 2 all contribute to delayed FA disassembly and loss of polarity in cells perpendicular to stretching. Immunostaining for phospho-FAK after stretching revealed that FAK activation was maximal at 5 s after stretching, specifically in FAs oriented perpendicular to stretch. We hypothesize that orientation-specific activation of strain/stress-sensitive proteins in FAs upstream to FAK and Src promote orientation-specific responses in FA growth and disassembly that mediate polarity rearrangement in response to sustained stretch.

Footnotes

  • ↵1To whom correspondence should be addressed. E-mail: watermancm{at}nhlbi.nih.gov.

  • Author contributions: Y.C., A.P.K., and C.M.W. designed research; Y.C. and A.M.P. performed research; Y.C. contributed new reagents/analytic tools; Y.C. analyzed data; and Y.C. and C.M.W. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission. T.L. is a guest editor invited by the Editorial Board.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1221637110/-/DCSupplemental.

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