Investigating Ordered Phase-Like Domains on the Membrane of Intact B Cells Using Super-Resolution Microscopy

[摘要] B cells lymphocytes are responsible for mounting an immune response in a variety of species. B cells are typically activated through the antigen-binding site of its B cell receptors (BCR) which are surface-expressed on the plasma membrane. The lipid composition and organization of the B cell plasma membrane has been implicated in B cell-related immune disease and highlights that plasma membrane organization plays important roles in appropriate BCR signal propagation. B cells respond to a wide variety of antigens with varying valency and mode of presentation, although the contribution of plasma membrane lipids to signaling in these different scenarios is not clearly understood.Past work has shown that BCR clustering through streptavidin, a model soluble antigen, results in the stabilization of an ordered phase-like domain capable of sorting minimal peptides and full length regulatory proteins with respect to BCR clusters.BCR can be stimulated through surface presented antigen in vivo & in vitro, and it is hypothesized that forces mediated via the surface can also impact the sorting of regulatory proteins with respect to antigen engaged BCR.The research presented in this Thesis applies quantitative two-color super-resolution fluorescence localization microscopy to measure the co-distribution of membrane-anchored peptides and proteins on the B cell surface.I engage and cluster proteins on the surface of B cells both with model soluble antigen and with antigen presented on a mobile bilayer surface.I find that BCR clusters formed through engagement with bilayer-presented antigen sort minimal peptides in a similar manner to BCR clusters stabilized with soluble antigen, indicating that these are also ordered phase-like domains. I find Lyn kinase is more strongly enriched and CD45 phosphatase is more strongly depleted from BCR clusters engaged with bilayer presented compared to soluble antigen, and that Tyrosine phosphorylation is enhanced in surface engaged BCR clusters. Additionally, I show that clusters of cholera toxin B subunit (CTxB) also stabilize environments that sort minimal peptides, and I explore how physical parameters such as temperature and domain size impact the sorting strength of these phase-like domains.Based on these results, I conclude that ordered domains contribute to the organization of regulatory proteins with respect to BCR engaged with both soluble and surface presented antigen, and that ordered domain stabilization is a general characteristic of the B cell plasma membrane and not specific to BCR clustering. I link CTxB cluster size and temperature to ordered phase-like domain sorting strength suggesting cells can tune the strength of lipid domains to modulate signaling. Findings from my Thesis could contribute to the advancement of therapies for diseases in which altered plasma membrane lipid composition impacts the functional organization proteins on the cell surface.

[发布机构] University of Michigan

[效力级别] B cell lymphocytes

[关键词] Super-resolution microscopy;B cell lymphocytes;Lipid rafts;Phase-like domains;Cholera Toxin B subunit (CTxB);Liquid ordered;Science (General);Science;Biophysics