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The contribution of different domains of connexin 43 to cytoskeletal rearrangements in B-lymphocytes Falk, Letitia Louise Elizabeth

Abstract

B-cells change shape in response to crosslinking of the B-cell antigen receptor (BCR). BCR signaling induces cytoskeletal rearrangements that result in cell spreading to improve antigen accumulation and B-cell activation. It has previously been shown that the gap junction protein connexin43 (Cx43) is both necessary and sufficient for BCR-mediated B-cell spreading, as well as other B-cell responses that depend upon cytoskeletal rearrangement. Since it was found that the C-terminal domain (CT) of Cx43 was required for these effects, we hypothesized that the molecular mechanism by which the CT influences BCR-mediated spreading may be due to regulation of channel permeability or alternately, by acting as an adaptor for cytoskeletal organization. To address the role of Cx43 in forming channels, we blocked channel function both pharmacologically with channel-blocking drugs and by expressing Cx43 mutant T154A in a Cx43-null plasmacytoma cell line. Treatment with channel blocking drugs did not prevent BCR-mediated B-cell spreading, and hemichannel (HC) activity was not detected in B-cells as measured by a dye-uptake assay. Thus we conclude that Cx43 influences B-cell spreading by a channel-independent mechanism and that the Cx43 CT may act as a scaffold for protein interactions. In support of this idea, the channel-blocking point mutation T154A caused B-cell spreading defects even in the absence of functional HCs. Further characterization of this mutation suggests that it impedes cell spreading due to a distorted conformation of the Cx43 CT domain. J558μm3 cells expressing Cx43 containing a point mutation of the putative Src-binding tyrosine (Y265F and Y265D) were unable to spread, highlighting the importance of a single residue of Cx43 for BCR-mediated B-cell spreading. These findings highlight the CT domain as important for Cx43’s influence on B-cell spreading, and pave the way for further experiments on the CT tail with the goal of better understanding the molecular mechanism underlying the role of Cx43 in B-cells.

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