Nano-clusters of ligand-activated integrins organize immobile, signalling active, nano-clusters of phosphorylated FAK required for mechanosignaling in focal adhesions.

Transmembrane signalling receptors, such as integrins, organise as nanoclusters that are thought to provide several advantages including, increasing avidity, sensitivity (increasing the signal-to-noise ratio) and robustness (signalling above a threshold rather than activation by a single receptor) of the signal compared to signalling by single receptors. Compared to large micron-sized clusters, nanoclusters offer the advantage of rapid turnover for the disassembly of the signal. However, if nanoclusters function as signalling hubs remains poorly understood. Here, we employ fluorescence nanoscopy combined with photoactivation and photobleaching at sub-diffraction limited resolution of ~100nm 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 immobile population of integrin β3 organised as nanoclusters, which (ii) in turn serve to organise nanoclusters of associated key adhesome proteins- vinculin, focal adhesion kinase (FAK) and paxillin, demonstrating that signalling proceeds by formation of nanoclusters rather than through individual proteins. (iii) Distinct focal adhesion protein nanoclusters exhibit distinct dynamics dependent on function. (iv) long-lived nanoclusters function as signalling hubs- wherein phosphorylated FAK and paxillin formed stable nanoclusters in close proximity to immobile integrin nanoclusters which are disassembled in response to inactivation signal by phosphatase PTPN12 (v) signalling takes place in response to an external signal such as force or geometric arrangement of the nanoclusters and when the signal is removed, these nanoclusters disassemble. Taken together, these results demonstrate that signalling downstream of transmembrane receptors is organised as hubs of signalling proteins (FAK, paxillin, vinculin) seeded by nanoclusters of the transmembrane receptor (integrin).