Edited by Vann Bennett, Duke University Medical Center, Durham, NC, and approved July 23, 2015 (received for review July 3, 2015)
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.
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.
↵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.
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.
