Multiscale imaging and quantitative analysis of plasma membrane protein-cortical actin interplay

The spatiotemporal organization of cell surface receptors is important for cell signaling. Cortical actin (CA), the subset of the actin cytoskeleton subjacent to the plasma membrane (PM), plays a large role in cell surface receptor organization. However, this has been shown largely through actin perturbation experiments, which raise concerns of nonspecific effects and preclude quantification of actin architecture and dynamics under unperturbed conditions. These limitations make it challenging to predict how changes in CA properties can affect receptor organization. To derive direct relationships between the architecture and dynamics of CA and the spatiotemporal organization of PM proteins, including cell surface receptors, we developed a multiscale imaging and computational analysis framework based on the integration of single-molecule imaging (SMI) of PM proteins and fluorescent speckle microscopy (FSM) of CA (combined: SMI-FSM) in the same live cell. SMI-FSM revealed differential relationships between PM proteins and CA based on the PM proteins’ actin binding ability, diffusion type, and local CA density. Combining SMI-FSM with subcellular region analysis revealed differences in CA dynamics that were predictive of differences in PM protein mobility near ruffly cell edges versus closer to the cell center. SMI-FSM also highlighted the complexity of cell-wide actin perturbation, where we found that global changes in actin properties caused by perturbation were not necessarily reflected in the CA properties near PM proteins, and that the changes in PM protein properties upon perturbation varied based on the local CA environment. Given the widespread use of SMI as a method to study the spatiotemporal organization of PM proteins and the versatility of SMI-FSM, we expect it to be widely applicable to enable future investigation of the influence of CA architecture and dynamics on different PM proteins, especially in the context of actin-dependent cellular processes.