Immobile Integrin Signaling Transit and Relay Nodes Organize Mechanosignaling through Force-Dependent Phosphorylation in Focal Adhesions

Transmembrane signaling receptors, such as integrins, organize as nanoclusters that provide several advantages, including increasing avidity, sensitivity (increasing the signal-to-noise ratio), and robustness (signaling threshold) of the signal in contrast to signaling by single receptors. Furthermore, compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, whether nanoclusters function as signaling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at subdiffraction limited resolution of ∼100 nm length scale within a focal adhesion to examine the dynamics of diverse focal adhesion proteins. We show that (i) subregions of focal adhesions are enriched in an immobile population of integrin β3 organized as nanoclusters, which (ii) in turn serve to organize nanoclusters of associated key adhesome proteins-vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signaling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct protein dynamics, which is closely correlated to their function in signaling. (iv) Long-lived nanoclusters function as signaling hubs─wherein immobile integrin nanoclusters organize phosphorylated FAK to form stable nanoclusters in close proximity to them, which are disassembled in response to inactivation signal by removal of force and in turn activation of phosphatase PTPN12. (v) Signaling takes place in response to external signals such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. We term these functional nanoclusters as integrin signaling transit and relay nodes (STARnodes). Taken together, these results demonstrate that integrin STARnodes seed signaling downstream of the integrin receptors by organizing hubs of signaling proteins (FAK, paxillin, vinculin) to relay the incoming signal intracellularly and bring about robust function.