Ligand-induced growth and compaction of CD36 nanoclusters enriched in Fyn induces Fyn signaling

Nanoclustering is an emerging organizational principle for membrane-associated proteins. The functional consequences of nanoclustering for receptor signaling remain largely unknown. Here, we applied quantitative multi-channel high-and super-resolution imaging to analyze the endothelial cell surface receptor CD36, the clustering of which upon binding to multivalent ligands, such as the anti-angiogenic factor thrombospondin-1 (TSP-1), is thought to be crucial for signaling. We found that a substantial fraction of unligated CD36 exists in nanoclusters, which not only promote TSP-1 binding but are also enriched with the downstream effector Fyn. Exposure to multivalent ligands (TSP-1 or anti-CD36 IgM) that result in larger and denser CD36 clusters activates Fyn. Conversely, pharmacological perturbations that prevent the enhancement of CD36 clustering by TSP-1 abrogate Fyn activation. In both cases, there is no detectable change in Fyn enrichment at CD36 nanoclusters. These observations reveal a crucial role for the basal organization of a receptor into nanoclusters that are enriched with the signal-transducing downstream effectors of that receptor, such that enhancement of clustering by multivalent ligands is necessary and sufficient to activate the downstream effector without the need for its de novo recruitment.