Investigating molecular mechanisms of endocytosis of the activated B cell receptor in health and disease

About This Grant

PROJECT SUMMARY (See instructions): B cell activation and antibody production is central to the human adaptive immune response to pathogens. The B cell receptor (BCR), is the primary cell surface receptor that engages with foreign antigens and initiates activation of the cell. BCR stimulation induces receptor clustering and endocytosis. Details about the molecular mechanisms that drive internalization of large antigens in B cells are unclear. Clathrin mediated endocytosis (CME) is responsible for internalization of small BCR clusters, but studies have shown that many BCR clusters can grow far beyond the size of the average clathrin coated pit. In previous studies I identified novel plasma membrane structures that are highly co-localized with large BCR clusters. These structures are smooth raised membrane invaginations that also have small clathrin lattices associated with them (cSRM structures). The overall hypothesis investigated in this research is that cSRM structures represent a unique mechanism of hybrid endocytosis that allows B cells to internalize large antigens. Overall, the aims described in this research will elucidate the molecular mechanisms of a novel form of hybrid endocytosis in B cells, and determine the role of hybrid endocytosis in contributing to disease pathogenesis in Lymphoma. During the K99 phase of this award I investigated whether hybrid endocytosis may be active in the pathogenesis of Activated B Cell Like Diffuse Large B Cell Lymphoma (ABC DLBCL). One feature of malignant B cells in ABC DLBCL is the persistence of spontaneous BCR clusters at the plasma membrane. These clusters likely contribute to the pathogenesis by perpetually amplifying positive signaling from the BCR. In the first phase of this award I examined the plasma membrane features associated with spontaneous BCR clusters using correlative light and electron microscopy. I found that spontaneous clusters in an ABC DLBCL cell line are highly correlated with cSRM structures and contain activated BCR molecules. I also completed a comprehensive analysis of endocytic structures at the plasma membrane of ABC DLBCL cell lines using live imaging analysis and super resolution correlative imaging. I compared these analyses of ABC DLBCL cell lines to control cell lines and identified characteristics of activated BCR localization and interaction with endocytic structures at the plasma membrane that are unique to ABC DLBCL cell lines. In the R00 phase of this award I will conduct studies to further generate a more clear picture of the molecular players and kinetics of protein interactions involved in hybrid endocytosis in normal cells (Aim 1) and then determine how these proteins are dysregulated in ABC DLBCL (Aim 2). In Aim 1 I will use proximity labeling to identify proteins involved in hybrid endocytosis and advanced imaging to characterize kinetics of protein interactions during endocytosis. In Aim 2 I will investigate how specific BCR mutations associated with ABC DLBCL may cause dysregulation of hybrid endocytosis and contribute to disease pathogenesis.