Quantification Of Surface Receptor-Actin Cortex Interplay Via Two-Color High Resolution Imaging

Mounting evidence suggests that the dynamic organization of receptors in the plasma membrane (PM) is crucial for ligand binding and downstream signaling. The actin cortex (AC) is thought to play an important role in regulating cell surface receptor organization, although its role has been almost exclusively deduced via cell-wide actin perturbation experiments, which suffer from various caveats. Here we describe a novel quantitative imaging and computational analysis strategy to derive an explicit relationship between cell surface receptor mobility and clustering on the one hand, and AC turnover kinetics and filament movement on the other. As a test model system, we are applying this imaging and analysis strategy to study the cell surface receptor CD36, the movement and clustering of which in the plasma membrane are thought to depend on the AC. To monitor both CD36 and AC behavior at the same time in the same cell, we are employing TIRF-based simultaneous single molecule imaging of CD36 and fluorescent speckle microscopy of the AC, in live endothelial cells. The imaged CD36 molecules are tracked to quantify their movement and clustering state, while the actin speckles are tracked and analyzed to quantify AC turnover kinetics and actin filament movement. These CD36 and actin properties are then combined for neighboring CD36 molecules and actin speckles to derive receptor-AC inter-relationships. In parallel, to account for cellular heterogeneity, various bins based on experimental conditions and cellular phenotypes are created to further refine our observations. Our analysis thus far suggests that CD36 clustering is inversely correlated with its diffusion coefficient and neighboring actin density. Additionally, for freely moving CD36 molecules, the density of neighboring actin is a better predictor of CD36 diffusion coefficient than the speckle speeds.