Membrane domains and phosphatase exclusion produce robust signaling responses in B cells engaged with natural ligands and artificial cross-linkers presented on lipid bilayers

Abstract

B cells respond to a wide variety of antigens with varying valiancy and mode of presentation to the highly expressed B cell receptor (BCR). We previously demonstrated that clustering the IgM isotype of BCR with an artificial soluble cross-linker led to the stabilization of an ordered phase-like domain. This domain sorted minimal peptides and full-length proteins to generate local hot-spots for tyrosine phosphorylation at receptor clusters, facilitating receptor activation. BCR can also be activated through interactions with natural ligands or artificial cross-linkers presented at bilayer surfaces, where it is proposed that alternate mechanisms play important roles in receptor clustering and activation, including one mechanism involving the exclusion phosphatases due to their bulky extracellular domains.The goals of the current study are to determine if markers of membrane phases are sorted by BCR clusters formed through engagement with bilayer-presented natural ligands or cross-linkers, and to estimate the contribution that membrane phase partitioning plays in organizing regulatory proteins with respect to BCR clusters under these stimulation conditions. We use super-resolution fluorescence localization microscopy to find that BCR engagement with either a bilayer-presented natural ligand or artificial cross-linker generates ordered phase-like domains that are more robust than those observed with soluble cross-linkers. In addition, we provide evidence that interactions between regulatory proteins and BCR are partially determined through their preference for ordered membrane domains and present a minimal model of receptor activation that incorporates both ordered domains and steric exclusion mechanisms to produce a more sensitive response. Overall, this work highlights that cells are capable of integrating multiple interaction modalities to give rise to cellular functions, likely conferring flexibility and robustness to cellular responses.