Ligand-Induced Growth of CD36-Fyn Clusters Induces Signaling

Nanoclustering is emerging as a key organizational principle of membrane proteins. Using superresolution imaging, we investigated the molecular organization of the clustering-responsive receptor CD36 and its downstream effector Fyn in response to thrombospondin-1 (TSP-1), an anti-angiogenic ligand that promotes endothelial cells apoptosis. At steady state, CD36 receptors pre-exist in nanoclusters (diameter of about 100 nm). Fyn is already present in these clusters, and its N-terminal membrane targeting domain is sufficient for this association. Even if already associated, we found that Fyn only becomes activated activated upon TSP-1 binding through an enhancement of CD36-Fyn clustering, forming larger and denser clusters. These enhancements are supported by the actin cytoskeleton and the presence of plasma membrane cholesterol as their perturbation abolishes signaling. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the reorganization of the receptor-effector pair CD36-Fyn that enables signaling during TSP-1 stimulation.