Decker, A.; Veatch, S. L.

Membrane-proximal (MP) actin represents the subset of cortical f-actin localized within 10 nm of the plasma membrane. Here, we describe a family of single-molecule MP actin (SM-MPAct) probes that diffuse within the plasma membrane and are transiently immobilized through binding to f-actin, enabling the localization of MP actin in both time and space. These probes quantify aspects of MP actin structure, dynamics, and remodeling by analyzing probe positions using a combined single-particle tracking and correlation-function approach. This is demonstrated using chemical and physical perturbations of actin and actin-binding proteins, and by interrogating MP actin organization and dynamics in early B cell receptor (BCR) activation. Upon crosslinking of the IgM BCR, MP actin transiently remodels to increase the size of actin corals, facilitating the efficient assembly of BCR clusters and the local accumulation of MP actin. Notably, analogous remodeling is not detected in measurements using total f-actin probes, indicating that SM-MPAct is uniquely sensitive to the f-actin pool that regulates signaling processes at the plasma membrane.