Acto-myosin driven functional nanoclusters of GPI-anchored proteins are generated by integrin receptor signaling

GPI-anchored protein (GPI-AP) nanoclusters are generated by cortical acto-myosin activity. While our understanding of the physical principles behind this process is emerging, the molecular machinery required for the generation of these nanoclusters is unknown. Here, we show that ligand-mediated membrane receptor signaling triggers nanocluster formation. Both soluble and surface-tethered RGD ligands bind the {beta}1-integrin receptor and activate focal adhesion and src-kinases, resulting in RhoA signaling. This cascade ultimately triggers actin-nucleation via specific formins, driving nanoclustering of both GPI-APs and a model transmembrane protein with an actin-binding domain. Integrin signaling concurrently results in talin mediated activation of vinculin. This is necessary for the coupling of the dynamic actin machinery to the inner leaflet driving GPI-AP nanoclustering. Disruption of GPI-AP nanoclustering in either GPI-anchor remodeling mutants or in cells that express vinculin mutants, provide evidence that these nanoclusters are necessary for activating cell spreading, a hallmark of integrin function.