Relevant Degree Programs
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Mar 2019)
Regulation of the cytoskeleton is an essential process for normal B lymphocyte development and immune system regulation. Though this biological process is important to normal function, its regulation is not completely understood. An important receptor required to initiate antigen-mediated cytoskeletal rearrangements in B lympocytes is the B cell antigen receptor (BCR). This is a multimeric protein complex that contains two signalling proteins, Igα and Igβ, which become phosphorylated after antigen engagement leading to signalling cascades which result in cytoskeletal rearrangements and differentiation. The gap junction protein connexin43 (Cx43) is widely expressed in mammalian cells, forming intercellular channels for the transfer of small molecules between adjacent cells as well as hemichannels that mediate bidirectional transport of molecules between the cell and the surrounding environment. Cx43 has recently been shown to regulate cell adhesion and migration in neurons and glioma cells, biological processes dependent on the rearrangement of the cytoskeleton, however its role in B lymphocytes remains unknown. The aim of this thesis was to determine the importance of the B cell antigen receptor (BCR) member Igα and the gap junction protein connexin43 (Cx43) to the regulation of the B cell cytoskeleton. I show here that the cytoplasmic domain of Igα was necessary for maximal BCR-mediated cytoskeletal rearrangements, and that the cytoplasmic domain of Igβ was not sufficent to conferthis phenotype. In order to determine if Cx43 was required for B cell cytoskeletal rearrangements, both loss-of and gain-of-function approaches were used. I show that Cx43 was necessary for sustained BCR, integrin and chemokine-mediated Rap1 activation, as well as B cell spreading, adhesion, motility and migration. I also identified that the C-terminal domain of Cx43 was necessary for these processes, suggesting that this may be a site where proteins which regulate the cytoskeleton are recruited to. This thesis provides the first evidence that Cx43 is essential for regulation of the B cell cytoskeleton.
Master's Student Supervision (2010-2017)
Cellular processes requiring cytoskeletal rearrangements such as adhesion, spreading, immune synapse formation, and migration are crucial for normal B-lymphocyte (B cell) development and for immune responses. Our lab has shown that the gap junction protein connexin43 (Cx43) is both necessary and sufficient for promoting B cell adhesion, B cell receptor (BCR)-mediated spreading, B cell motility and migration. The carboxyl tail (CT) of Cx43 is important for these effects and the hypothesis is that specific regions of the CT contain sites important for interactions that can influence these processes. To identify these regions, a series of deletions and point mutations in the CT of Cx43 were made. These mutated proteins were expressed in mouse B cell lines and examined for localization, cell surface expression and for their effect on supporting cell spreading in response to BCR-signaling, the latter assay being used as one ‘read-out’ for cytoskeletal rearrangements. With regard to BCR-mediated cell spreading, the data in this thesis from the Cx43 deletions showed that the amino acids in the Cx43 CT between 246 and 307 are the most important, and that amino acids between 307 and 382 are less critical. In addition, since one of the most important proximal signaling events after BCR signaling is the activation of different protein tyrosine kinases, the effects of different tyrosine mutations in the Cx43 CT were examined. Three point mutations at tyrosine (Y) 247, 265 and 267 to phenylalanine (F), and a double mutant, Y247F/Y265F, were made and their effect on BCR-mediated spreading assessed. All of these tyrosine mutations in the Cx43 CT were found to impede BCR-mediated cell spreading. These findings have helped to better define the region of the Cx43 CT critical for regulating cytoskeletal rearrangements in response to BCR signaling. Future studies will identify other important residues of the CT, with the goal to identify the importance of these sites. These results will provide novel insights into understanding basic B lymphocyte responses that contribute to immune function, and can lead to identifying potential targets for the development of therapeutics to treat immune diseases.
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.
B cells migrate throughout the body, following chemokine gradients, to search for foreign antigens. When a B cell encounters an antigen-presenting cell (APC) bearing antigens that bind its B cell receptors (BCRs), it spreads across the surface of the APC to scan for additional antigens. This spreading is followed by contraction in which BCR microclusters recruit signaling enzymes and form an immune synapse. The resulting BCR signaling initiates an activation process in which B cells differentiate into antibody-producing plasma cells, which are directed to the bone marrow by chemokines, where they survive and produce antibodies for long periods of time. Both chemokine-induced B cell migration and BCR-induced cell spreading involve changes in the cytoskeleton. The gap junction protein connexin 43 (Cx43) regulates the migration of neuronal cells during brain development and interacts with many cytoskeletal-regulating partners. Previous work in our lab showed that Cx43 is important for BCR-induced spreading and chemokine-induced B cell migration, and for sustained activation of Rap1 GTPases, master regulators of cytoskeletal organization. However, the mechanism by which Cx43 enhances BCR- and chemokine-induced Rap1 activation was not known. The goal of my project was to determine whether Cx43 modulates all BCR signaling reactions, perhaps by acting on BCR-proximal signaling components, or whether it selectively regulates the activation of Rap1. I found that Cx43 influences the activation of certain BCR signaling pathways, specifically the PLCγ pathway, while having less effect on other targets of BCR signaling. This is the first evidence that Cx43 modulates BCR signaling and provides new insights into how Cx43 contributes to BCR-induced cytoskeletal regulation. BCR signaling is also important for B lymphoma cell survival and for the negative selection processes that eliminate autoreactive B cells. Although B cell depleting drugs have been used to treat B cell malignancies and B cell-mediated autoimmune diseases, there is variable efficacy. Small molecule inhibitors might be useful alternatives or additions to B cell depletion. Although several inhibitors of B cell signaling enzymes are being clinically tested, I have developed and optimized assay conditions to enable a large-scale screen for additional inhibitors of key components of BCR signaling pathways.