Novel players and large-scale protein dynamics of BCR activation revealed by APEX2 proximity labelling of lipid rafts

Successful B cell activation, critical for high-affinity antibody production, is controlled by the B cell antigen receptor (BCR). While the main components of the BCR signalling machinery are relatively well characterised, we still lack a comprehensive protein-level view of the very dynamic multibranched cellular events triggered by antigen binding. In this study, we pioneer usage of APEX2 proximity biotinylation for studying antigen receptor signalling. APEX2 provides critical spatial and kinetic resolution, 20 nm and 1 min, to trace early protein dynamics upon antigen engagement. We identified a total of 1677 proteins to locate in the vicinity of APEX2 at the plasma membrane lipid raft domains, where the BCR translocates upon activation. Our data provides unprecedented insights into the composition of lipid raft environment in B cells and the dynamic changes triggered upon BCR cross-linking. The data provides new understanding into the behaviour of proteins known to be involved in the proximal antigen receptor signalling, and the simultaneously triggered processes, such as actin cytoskeleton remodelling and endocytosis. Interestingly, our differential enrichment analysis identified dynamic responses in various proteins that have not previously been linked to early B cell activation. We validated Golga3 and Vti1b as novel proteins responding to BCR activation, confirming that our dataset serves as a valuable tool for future studies.