Visualizing the interior architecture of focal adhesions with high-resolution traction maps.

Focal adhesions (FAs) are micron-sized protein assemblies that coordinate cell adhesion, migration, and mechanotransduction. How the many proteins within FAs are organized into force sensing and transmitting structures is poorly understood. We combined fluorescent molecular tension sensors with super-resolution light microscopy to visualize traction forces within FAs with <100 nm spatial resolution. We find that αvß3 integrin selectively localizes to high force regions. Paxillin, which is not generally considered to play a direct role in force transmission, shows a higher degree of spatial correlation with force than vinculin, talin, or α-actinin, proteins with hypothesized roles as force transducers. These observations suggest that αvß3 integrin and paxillin may play important roles in mechanotransduction.

Regulation and dynamics of force transmission at individual cell-matrix adhesion bonds.

Integrin-based adhesion complexes link the cytoskeleton to the extracellular matrix (ECM) and are central to the construction of multicellular animal tissues. How biological function emerges from the tens to thousands of proteins present within a single adhesion complex remains unclear. We used fluorescent molecular tension sensors to visualize force transmission by individual integrins in living cells. These measurements revealed an underlying functional modularity in which integrin class controlled adhesion size and ECM ligand specificity, while the number and type of connections between integrins and F-actin determined the force per individual integrin. In addition, we found that most integrins existed in a state of near-mechanical equilibrium, a result not predicted by existing models of cytoskeletal force transduction. A revised model that includes reversible cross-links within the F-actin network can account for this result and suggests one means by which cellular mechanical homeostasis can arise at the molecular level.

Integrin-mediated traction force enhances paxillin molecular associations and adhesion dynamics that increase the invasiveness of tumor cells into a three-dimensional extracellular matrix.

Metastasis requires tumor cells to navigate through a stiff stroma and squeeze through confined microenvironments. Whether tumors exploit unique biophysical properties to metastasize remains unclear. Data show that invading mammary tumor cells, when cultured in a stiffened three-dimensional extracellular matrix that recapitulates the primary tumor stroma, adopt a basal-like phenotype. Metastatic tumor cells and basal-like tumor cells exert higher integrin-mediated traction forces at the bulk and molecular levels, consistent with a motor-clutch model in which motors and clutches are both increased. Basal-like nonmalignant mammary epithelial cells also display an altered integrin adhesion molecular organization at the nanoscale and recruit a suite of paxillin-associated proteins implicated in invasion and metastasis. Phosphorylation of paxillin by Src family kinases, which regulates adhesion turnover, is similarly enhanced in the metastatic and basal-like tumor cells, fostered by a stiff matrix, and critical for tumor cell invasion in our assays. Bioinformatics reveals an unappreciated relationship between Src kinases, paxillin, and survival of breast cancer patients. Thus adoption of the basal-like adhesion phenotype may favor the recruitment of molecules that facilitate tumor metastasis to integrin-based adhesions. Analysis of the physical properties of tumor cells and integrin adhesion composition in biopsies may be predictive of patient outcome.