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Characterization of the effects of Connexin43 on B cell receptor signaling responses in B-lymphocytes Mo, Victor Cheuk-Kong

Abstract

Reorganization of the cytoskeleton is crucial in orchestrating B lymphocyte development and immune responses. Our lab aims to understand the mechanisms of B cell cytoskeleton regulation. Intracellular signaling from the B cell receptor (BCR) triggers cytoskeletal changes necessary for B cell activation. Connexin43 (Cx43) is a gap junction-forming protein that, when expressed in J558µm3 Cx43-negative B cells, is sufficient to enable BCR-mediated cellular spreading, a process in which cytoskeleton remodeling drives changes in cell shape. Knockdown of Cx43 in WEHI231 B cells reduced the activation of various BCR signaling proteins, including the Rap1 GTPase, a master regulator of cytoskeleton dynamics in B cells. Since the carboxyl tail (CT) of Cx43 was important for this increased Rap1 activation, we investigated the role of the Cx43 CT in B cell cytoskeleton regulation by characterizing its effects on BCR signaling responses. Our approach assessed J558µm3 cells transfected with various Cx43 CT mutant constructs to determine the role of Cx43 in BCR signaling. We found that Rap1 can be activated by mechanical forces applied to cells, and that physical expansion of cell size by stretching in the absence of receptor stimulation caused no apparent changes to the actin cytoskeleton. Because Cx43 had no impact on these results, it likely has the most influence in BCR signaling pathway-driven cytoskeletal changes. The Cx43 CT, in particular serines 279 and 282, but not 255 and 262, were found to be important for BCR-induced tyrosine phosphorylation of cellular proteins (pTyr), indicating a differential role of specific amino acids in the Cx43 CT on BCR signaling. The phosphorylation of Ezrin / Radixin / Moesin (ERM) proteins was also decreased upon the loss of Cx43 and its CT. We speculate that the CT recruits protein partners to act with BCR signaling enzymes. Cx43 may also control cytoskeleton dynamics via regulating ERM proteins. Future studies include monitoring pTyr in additional Cx43 CT mutants to identify other residues important for BCR signaling and uncovering how Cx43 contributes to ERM phosphorylation. This work provides insight into cytoskeletal rearrangement in B cells, a process in which dysregulation underlies B cell autoimmune diseases and cancers.

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