Quantifying the Effect of BCR Clustering on Plasma Membrane Organization

Abstract

The B cell antigen receptor (BCR) is an integral part of the adaptive immune system that communicates binding of antigen in the extracellular environment through the plasma membrane. Antigen binding to the BCR results in phosphorylation of intracellular tyrosine activating motifs (ITAMs), which subsequently bind to and activate numerous proteins involved in BCR regulation. Interestingly, many of the proteins regulating the early stages of the BCR signaling pathway are linked to the inner leaflet by saturated lipid anchors which tend to be associated with liquid-ordered membrane phase in model membranes. Also, the BCR becomes transiently detergent resistant following antigen-induced BCR clustering, suggesting that BCR clusters become coupled to membrane order following stimulation. In this work, we aim to characterize how BCR clustering could reorganize plasma membrane lipids by quantifying co-localization of BCR with fluorescent markers of liquid-ordered and liquid-disordered phases. We utilize two-color super-resolution fluorescence localization microscopy (STORM and PALM) in live and chemically fixed CH27 B cells to simultaneously image BCR and membrane anchored proteins, and we quantify their co-clustering using correlation functions. Our results from chemically fixed cells show that proteins anchored to the plasma membrane inner leaflet through saturated acyl-chain lipid modifications exhibit increased co-localization with BCR upon antigen stimulation, whereas those without lipid modifications or those anchored through branched acyl-chain modifications are not significantly co-localized with BCR before or after stimulation. These results are contributing to our long term goal of elucidating the role of lipid mediated interactions in the regulation of BCR signaling.