A single-molecule reporter of membrane-proximal actin detects rapid remodeling upon B cell receptor clustering

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. STATEMENT OF SIGNIFICANCEThe actin cortex provides mechanical stability to the plasma membrane and contributes to the organization and dynamics of plasma membrane components. This report presents single-molecule probes and analytical methods to characterize the density, mesh size, motion, and turnover dynamics of the portion of the actin mesh in direct contact with the plasma membrane, enabling quantitative studies of actin remodeling in plasma membrane processes. This general framework is demonstrated through quantification of actin remodeling during early B cell receptor signaling and could be applied to a broad range of cell processes.