BPS2026 – Resolving the organization and dynamics of membrane-proximal actin in live cells

The actin cortex is a dynamic meshwork coupled to the plasma membrane that regulates the organization and dynamics of membrane components, influencing biochemical networks at the cell surface. Here, we use a single-molecule membrane-proximal (MP) actin probe (SM-MPAct) to observe MP actin in live cells. SM-MPAct diffuses within the plasma membrane and is immobilized upon binding to f-actin. Isolating immobile positions of single molecules enables the localization of MP actin. Using this approach, we report a relative measure of MP actin average density and visualize slow-moving MP actin structures in reconstructed images. Fast motions of MP actin are detected by tabulating temporal correlations of probe positions, revealing both the dynamics of MP f-actin and its exchange with cytoplasmic or g-actin pools. This approach reveals that MP actin organizes over a range of spatial scales that evolve in time and are sensitive to temperature and chemical perturbations. We employ SM-MPAct to capture MP actin organization and dynamics during B-cell receptor (BCR) stimulation, finding that MP actin transiently detaches from the plasma membrane prior to its colocalization with BCR clusters.