Nanoscale localization of proteins within focal adhesions indicates discrete functional assemblies with selective force‐dependence

Focal adhesions (FAs) are subcellular regions at the micrometer scale that link the cell to the surrounding microenvironment and control vital cell functions. However, the spatial architecture of FAs remains unclear at the nanometer scale. We used two‐color and three‐color super‐resolution stimulated emission depletion microscopy to determine the spatial distributions and co‐localization of endogenous FA components in fibroblasts. Our data indicate that adhesion proteins inside, but not outside, FAs are organized into nanometer size units of multi‐protein assemblies. The loss of contractile force reduced the nanoscale co‐localization between different types of proteins, while it increased this co‐localization between markers of the same type. This suggests that actomyosin‐dependent force exerts a nonrandom, specific, control of the localization of adhesion proteins within cell–matrix adhesions. These observations are consistent with the possibility that proteins in cell–matrix adhesions are assembled in nanoscale particles, and that force regulates the localization of the proteins therein in a protein‐specific manner. This detailed knowledge of how the organization of FA components at the nanometer scale is linked to the capacity of the cells to generate contractile forces expands our understanding of cell adhesion in health and disease.