Cortical Cytoskeletal Structures Constrain CD36 Receptor Motion at the Cell Surface to Enhance Aggregation and Signaling

Abstract

CD36 is a key receptor in human macrophages, binding to multivalent ligands such as oxidized LDL and malaria-infected red blood cells. To study the dynamics and aggregation kinetics of CD36 receptors in the plasma membrane, we used single-molecule imaging combined with single-particle tracking and mathematical modeling of individual receptor behavior. We immuno-labeled CD36 receptors with a primary Fab fragment and a Cy3-conjugated secondary Fab fragment or with quantum dots, and imaged the dorsal surface of macrophages using live-cell epifluorescence microscopy. To track the imaged CD36 receptors, we developed a single-particle tracking algorithm that detects particles with sub-pixel localization and follows dense particle fields, capturing particle association and dissociation events and recovering trajectory interruptions resulting from temporary particle disappearance (Jaqaman Nat. Methods 2008). We found that, at rest, surface-bound CD36 receptors existed as discrete multimers that contained up to six monomers. A subpopulation of the detected CD36 multimers (∼30%) moved along linear tracks that radiated from the perinuclear region. The movement required both the acto-myosin network and microtubules. Temporal multi-scale analysis of receptor frame-to-frame displacement and run-time toward and away from the perinuclear region using sampling frequencies of 10-125 Hz revealed that the linear motion of CD36 multimers was due to constrained diffusion within cytoskeleton-mediated linear corridors in the membrane. Importantly, the dimensionality reduction resulting from motion within corridors increased the probability of CD36 aggregation two-fold. Cytoskeleton perturbations that inhibited the linear motion of CD36 inhibited the uptake of its ligands and the phosphorylation resulting from CD36 cross-linking. These data provide the first direct demonstration of the functional requirements of cortical cytoskeletal structures in the regulation of receptor activation via the spatial organization of receptor motion and aggregation at the level of the plasma membrane.