Investigation of Membrane Domains around the B Cell Receptor using Super-Resolution Microscopy.

[摘要] B cells are a crucial component of the mammalian adaptive immune system, and the B cell receptor (BCR) has a significant impact on the cellular fate and immunological response of the B cell.When foreign or self-antigens cluster the BCR, tyrosine residues associated with the receptor are phosphorylated by membrane anchored kinases.This process is initiated by an unknown mechanism and results in the activation of multiple signaling cascades.It is hypothesized that antigen bound BCR clusters create a domain of relatively ordered plasma membrane composition, and this domain acts to induce receptor phosphorylation by recruiting Lyn and other kinases.However, it is unclear if domains exist in the cell and unknown if domains could influence the activation state of receptors.The work in this thesis develops methods for quantitatively observing membrane probe partitioning around receptor clusters and provides evidence that a domain of relatively ordered membrane composition is created by clustered B cell receptors.Observations of domain formation show remarkable consistency with a critical fluctuation model for domain formation, and this model also reproduces receptor signal initiation in response to clustering the B cell receptor.Here, I apply multicolor super-resolution fluorescent microscopy to observe the distribution of membrane anchored probes around B cell receptors, and I demonstrate that a domain of altered membrane composition is present around the receptor clusters.To accomplish this task, I develop quantitative multi-color super-resolution microscopy techniques that report on absolute probe enrichment and depletion which can be performed in live or fixed cells.Applying these techniques to the BCR reveals that simple membrane-bound peptides are sorted around BCR clusters according to their phase preference in model systems.The membrane interacting regions of the phosphatase CD45 and the kinase Lyn are also differentially sorted by domains around BCR clusters, and applying these observations to a simulation containing fluctuating domains reproduces phosphorylation in response to BCR clustering. These results support a critical fluctuation model for domain formation and suggest that protein clustering may couple to membrane domains to perform a variety of tasks.

[发布机构] University of Michigan

[效力级别] B cell receptor

[关键词] Plasma membrane;B cell receptor;Super-resolution microscopy;Critical phenomena;Microbiology and Immunology;Science;Biophysics