Plasma Membrane Organization Promotes CD36 Signal Transduction in Endothelial Cells

Abstract

CD36 is a multiligand scavenger receptor that ligates Thrombospondin-1 (TSP-1) at the surface of endothelial cells and induces their apoptosis. Recent evidences have shown that clustering of CD36 is necessary for signal transduction in macrophages and is regulated by the architecture of the cortical actin cytoskeleton apposed to the plasmalemma. Here, we investigated the role of the cortical actin cytoskeleton and plasma membrane nanodomains in the control of CD36 activation in endothelial cells. Stimulation with multivalent ligands (TSP-1 and anti-CD36 IgM) resulted in the downstream phosphorylation of the Src Family kinase, Fyn. Disruption of the actin cytoskeleton or removal of cholesterol blocked this activation. To gain molecular details on the rearrangement of the receptors during TSP-1 binding, we conducted superresolution approaches (based on PhotoActivated Localization Microscopy or PALM) and quantitative spatial distribution analysis. Endothelial cell lines stably expressing CD36-PAmCherry were generated for that purpose. At steady state, CD36 receptors pre-exist in small clusters (average diameter of 100 nm), in which Fyn was also present. Upon TSP-1 binding, CD36 clusters increased in size (average diameter of 140 nm) and also became denser. The average distance between CD36 molecules in these clusters was in the range of 8 nm compare to 11 nm in the control condition. F-actin depolymerization or cholesterol depletion reduced the capacity of the ligand to induce formation of larger clusters resulting in a decreased recruitment and activation of Fyn. Our data demonstrate cooperation between cholesterol-dependent domains and the cortical actin cytoskeleton in the organization of CD36 receptors and Fyn before and during TSP-1 stimulation.