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Abstract The actin cortex plays a large role in regulating the dynamic organization of cell surface receptors, which in turn regulates their signaling. However, many receptors have short intracellular domains and no known link to cortical actin. In this work, we identified the β₁-integrin subunit and several tetraspanins – CD9, CD81 and CD151 – as part of the hitherto unknown molecular link between the surface receptor CD36 and cortical actin. We found that CD36 in vascular endothelial cells is recruited into complexes/nanodomains containing these proteins, with stronger recruitment near the cell edge. Perturbing this recruitment via the mutation G12V in the N-terminal transmembrane domain of CD36 alters the dynamic organization of CD36 on the vascular endothelial cell surface and weakens its coupling to cortical actin dynamics. Moreover, perturbing this recruitment abolishes thrombospondin-1-induced CD36 signaling through the Src family kinase Fyn. Given their many interactions with other transmembrane proteins, tetraspanins and integrins may provide a ubiquitous mechanism for plasma membrane-cortical actin coupling. 

Colocalization analysis of multicolor microscopy images is a cornerstone approach in cell biology. It provides information on the localization of molecules within subcellular compartments and allows the interrogation of known molecular interactions in their cellular context. However, almost all colocalization analyses are designed for two-color images, limiting the type of information that they reveal. Here, we describe an approach, termed “conditional colocalization analysis,” for analyzing the colocalization relationships between three molecular entities in three-color microscopy images. Going beyond the question of whether colocalization is present or not, it addresses the question of whether the colocalization between two entities is influenced, positively or negatively, by their colocalization with a third entity. We benchmark the approach and showcase its application to investigate receptor-downstream adaptor colocalization relationships in the context of functionally relevant plasma membrane locations. The software for conditional colocalization analysis is available at https://github.com/kjaqaman/conditionalColoc.