Significance
Plasma membrane protein organization, an important factor for shaping cellular behaviors, is influenced by cortical actin, the subset of the actin cytoskeleton near the plasma membrane. Yet it is challenging to directly and quantitatively probe this influence. Here, we developed an imaging and analysis approach that combines single-molecule imaging, fluorescent speckle microscopy and computational statistical analysis to characterize and correlate the spatiotemporal organization of plasma membrane proteins and cortical actin. Our approach revealed different relationships between different proteins and cortical actin, and highlighted the complexity of interpreting cell wide actin perturbation experiments. We expect this approach to be widely used to study the influence of cortical actin on different plasma membrane components, especially in actin-dependent processes.