The membrane phase transition drives tunable assembly and function of B cell receptor signaling domains.

Abstract

Clustering the B cell receptor (BCR) leads to assembly of a signaling platform with a distinct protein and lipid composition, creating a local membrane environment that facilitates interactions between signaling proteins and influences downstream responses. While many studies have linked BCR activation to “lipid rafts”, the lack of a structural correlate measured in intact cells impedes using these observations in a predictive model of the immune response. We systematically compared membrane organization in cells and plasma membrane vesicles using quantitative super-resolution imaging of a panel of fluorescent membrane anchor probes. We observed agreement between probe enrichment in BCR clusters and ordered phase partitioning in membrane vesicles, indicating that membrane phase separation is an underlying mechanism for assembly of functional domains. We showed that an ordered membrane domain emerges upon BCR clustering in which lipid-mediated interactions are sufficient to alter local membrane composition. To connect domain formation back to BCR activation, we employed two strategies to modulate the contrast between the composition of BCR proximal domains and the surrounding membrane. We altered the protein composition of BCR clusters by co-ligating BCR with engineered minimal “co-receptors” that favor or oppose ordered domain formation. We also used chemical treatments that enhance or suppress membrane phase separation overall. We found that not only is the contrast of BCR cluster domains variable and influenced by the nature of the stimulus and thermodynamics of the membrane, but the extent of BCR phosphorylation also varies with domain contrast. This dependence of activation on domain contrast extends to downstream signaling events as well as to other immune receptor systems. Together, these data suggest that domain formation at BCR clusters driven by membrane phase separation provides a mechanism to produce a tunable activation response.