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Research Article

Talin determines the nanoscale architecture of focal adhesions

Jaron Liu, Yilin Wang, Wah Ing Goh, Honzhen Goh, Michelle A. Baird, Svenja Ruehland, Shijia Teo, Neil Bate, David R. Critchley, Michael W. Davidson, and View ORCID ProfilePakorn Kanchanawong
  1. aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
  2. bNational High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL 32306;
  3. cDepartment of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom;
  4. dDepartment of Biological Sciences, The Florida State University, Tallahassee, FL 32306;
  5. eDepartment of Biomedical Engineering, National University of Singapore, Republic of Singapore 117411

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PNAS September 1, 2015 112 (35) E4864-E4873; first published August 17, 2015; https://doi.org/10.1073/pnas.1512025112
Jaron Liu
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Yilin Wang
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Wah Ing Goh
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Honzhen Goh
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Michelle A. Baird
bNational High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL 32306;
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Svenja Ruehland
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Shijia Teo
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
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Neil Bate
cDepartment of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom;
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David R. Critchley
cDepartment of Biochemistry, University of Leicester, Leicester LE1 7RH, United Kingdom;
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Michael W. Davidson
bNational High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL 32306;
dDepartment of Biological Sciences, The Florida State University, Tallahassee, FL 32306;
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Pakorn Kanchanawong
aMechanobiology Institute, Singapore, National University of Singapore, Republic of Singapore 117411;
eDepartment of Biomedical Engineering, National University of Singapore, Republic of Singapore 117411
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  • ORCID record for Pakorn Kanchanawong
  • For correspondence: biekp@nus.edu.sg
  1. Edited by Vann Bennett, Duke University Medical Center, Durham, NC, and approved July 23, 2015 (received for review July 3, 2015)

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Significance

Focal adhesions (FAs) mediate cell–extracellular matrix interactions and consist of integrin receptors linked to the actin cytoskeleton via multiprotein complexes organized into nanoscale strata. In this work, we sought to determine the molecular basis of FA nanostructure. Combining superresolution microscopy and protein engineering, we demonstrate the structural role of talin in regulating the nanoscale architecture of FAs. Talin specifies the dimension of the FA core, akin to a molecular ruler, in a remarkably modular manner. Our results define the molecular geometry of the integrin–talin–actin module that comprises the key mechanical linkage within FAs and elucidate how such interactions serve to integrate multiple cellular forces at adhesion sites.

Abstract

Insight into how molecular machines perform their biological functions depends on knowledge of the spatial organization of the components, their connectivity, geometry, and organizational hierarchy. However, these parameters are difficult to determine in multicomponent assemblies such as integrin-based focal adhesions (FAs). We have previously applied 3D superresolution fluorescence microscopy to probe the spatial organization of major FA components, observing a nanoscale stratification of proteins between integrins and the actin cytoskeleton. Here we combine superresolution imaging techniques with a protein engineering approach to investigate how such nanoscale architecture arises. We demonstrate that talin plays a key structural role in regulating the nanoscale architecture of FAs, akin to a molecular ruler. Talin diagonally spans the FA core, with its N terminus at the membrane and C terminus demarcating the FA/stress fiber interface. In contrast, vinculin is found to be dispensable for specification of FA nanoscale architecture. Recombinant analogs of talin with modified lengths recapitulated its polarized orientation but altered the FA/stress fiber interface in a linear manner, consistent with its modular structure, and implicating the integrin–talin–actin complex as the primary mechanical linkage in FAs. Talin was found to be ∼97 nm in length and oriented at ∼15° relative to the plasma membrane. Our results identify talin as the primary determinant of FA nanoscale organization and suggest how multiple cellular forces may be integrated at adhesion sites.

  • superresolution microscopy
  • focal adhesions
  • talin
  • mechanobiology
  • nanoscale architecture

Footnotes

  • ↵1Present address: Microscopy Unit, A*-STAR Institute of Medical Biology, Republic of Singapore 138648.

  • ↵2J.L., Y.W., and W.I.G. contributed equally to this work.

  • ↵3Present address: Laboratory of Cell and Tissue Morphodynamics, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892.

  • ↵4Present address: Faculty of Biology, Ludwig Maximilian University of Munich, 80539 Munich, Germany.

  • ↵5To whom correspondence should be addressed. Email: biekp{at}nus.edu.sg.
  • Author contributions: J.L., W.I.G., and P.K. designed research; J.L., Y.W., W.I.G., H.G., S.T., and P.K. performed research; H.G., M.A.B., S.R., N.B., D.R.C., and M.W.D. contributed new reagents/analytic tools; J.L., Y.W., W.I.G., and P.K. analyzed data; and J.L., Y.W., W.I.G., D.R.C., and P.K. wrote the paper.

  • The authors declare no conflict of interest.

  • This article is a PNAS Direct Submission.

  • This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1512025112/-/DCSupplemental.

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Talin determines focal adhesion nanoarchitecture
Jaron Liu, Yilin Wang, Wah Ing Goh, Honzhen Goh, Michelle A. Baird, Svenja Ruehland, Shijia Teo, Neil Bate, David R. Critchley, Michael W. Davidson, Pakorn Kanchanawong
Proceedings of the National Academy of Sciences Sep 2015, 112 (35) E4864-E4873; DOI: 10.1073/pnas.1512025112

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Talin determines focal adhesion nanoarchitecture
Jaron Liu, Yilin Wang, Wah Ing Goh, Honzhen Goh, Michelle A. Baird, Svenja Ruehland, Shijia Teo, Neil Bate, David R. Critchley, Michael W. Davidson, Pakorn Kanchanawong
Proceedings of the National Academy of Sciences Sep 2015, 112 (35) E4864-E4873; DOI: 10.1073/pnas.1512025112
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