Tropomyosin and Vinculin antagonistically mediate GPI-anchored protein nanoclustering

The plasma membrane is laterally organised into dynamic nanoscale and mesoscale domains. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) form actomyosin dependent nanoclusters at the outer leaflet of the plasma membrane via transbilayer coupling to inner leaflet lipids. These nanoclusters are nucleated downstream of integrin activation in a mechanochemical fashion and require vinculin and myosin 1 activity. Here, we identify an antagonistic relationship between vinculin and a negative regulator of myosin 1, tropomyosin, in regulating GPI-AP nanoclustering. We show vinculin mediated restoration of clustering is myosin-1 sensitive. The actin and lipid-binding vinculin tail is sufficient to restore clustering, indicating that vinculin, in particular its actin and lipid-proximal tail promote nanoclustering. Furthermore, re-expression of the non-muscle tropomyosin isoform Tpm2.1 in Tpm2-deficient MDA-MB-231 cells suppress GPI-AP nanoclustering while Tpm2.1 mutants defective in stable actin-filament association do not. Structural superposition of actin-bound vinculin tail and tropomyosin assemblies suggests that vinculin and tropomyosin engage overlapping or closely adjacent surfaces on actin filaments. Consistent with this, depletion of tropomyosin in vinculin-null cells restores GPI-AP nanoclustering. Together these results suggest that competitive interactions between tropomyosin and vinculin regulate actin availability for myosin 1 and hence contribute to the regulation of GPI-AP nanoclusters.