Analyzing actin dynamics during the activation of the B cell receptor in live B cells

Actin reorganization has been shown to be important for lymphocyte activation in response to antigenic stimulation. However, methods for quantitative analysis of actin dynamics in live lymphocytes are still underdeveloped. In this study, we describe new methods to examine the actin dynamics in B cells induced by antigenic stimulation. Using the A20 B cell line expressing GFP–actin, we analyzed in real time the redistribution of F-actin and the lateral mobility of actin flow in the surface of B cells in response to soluble and/or membrane associated antigens. Using fluorescently labeled G-actin, we identified the subcellular location and quantified the level of de novo actin polymerization sites in primary B cells. Using A20 B cells expressing G-actin fused with the photoconvertible protein mEos, we examined the kinetics of actin polymerization and depolymerization at the same time. Our studies present a set of methods that are capable of quantitatively analyzing the role of actin dynamics in lymphocyte activation.

► Live-cell tracking of actin reorganization and dynamics upon BCR activation.
► Live-cell tracking of actin flow speed upon BCR activation in response to membrane antigens.
► Tracking de novo actin polymerization sites and dynamics upon BCR activation in response to membrane antigens.
► Tracking the actin depolymerization and polymerization at the same time upon BCR activation.