Live Cell Super-Resolution Microscopy Measures Membrane-Driven Sorting of B Cell Receptor Signaling Partners

Abstract

B cells are acutely sensitive to stimulation through antigen-induced clustering of the B cell receptor (BCR). Transduction of the activation signal, which is key for the adaptive immune response, involves rearrangement of membrane-resident signaling molecules relative to clustered BCR. The details and dynamics of this re-organization have been difficult to directly observe due to the small dimensions of signaling complexes and the potentially weak or transient interactions that drive their formation. To overcome these obstacles, we have utilized live-cell super-resolution localization microscopy to simultaneously image BCR and other membrane species during antigen-induced cell activation. The sensitivity of this quantitative technique has allowed us to measure the effects of subtle forces, such as those originating from lipid compositional heterogeneity, that influence the interactions of BCR with its signaling partners. We found that minimal membrane-anchored probes that partition into ordered domains of model membranes co-localize with BCR clusters, while anchors that partition into disordered domains are excluded from clusters, suggesting that BCR clusters can nucleate local enrichment of a specific lipid composition. We are exploring the effects of membrane-driven sorting on recruitment of BCR signaling proteins to receptor clusters and how it contributes to their specific role in signal transduction. In particular, we have found that the palmitoylated transmembrane adapter proteins LAT2, LIME, and PAG, which serve distinct adapter functions in BCR signaling, are diferentially recruited to BCR clusters. This differential recruitment is due to a combination of interactions between the transmembrane domains of these proteins with the membrane and to specific protein-protein interactions. Our results suggest that the membrane influences local sorting of downstream signaling proteins around BCR clusters and provide insight on the forces driving regulation of BCR signaling.