Theses and Dissertations from UMD
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Item Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode(2022) Bhanja, Anshuman; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune response is critical in fighting off invading pathogens and potentially harmful foreign substances. B cells detect antigens through the B cell receptor (BCR). The binding of cognate antigen to the BCR induces a signaling response, a critical initiation and regulatory step for B cell activation and differentiation. The actin cytoskeleton has been shown to play essential roles in BCR signaling. When encountering membrane-associated antigens, actin amplifies signaling by driving B cell spreading and BCR clustering, while promoting signal attenuation by causing B cell contraction. This signal attenuation is essential for curtailing the activation of autoreactive B cells. However, the mechanism by which the actin cytoskeleton switches BCR signaling from amplification to attenuation was unknown. My thesis research examined the mechanisms by which actin reorganization transitions B cells from spreading to contracting and B cell contraction switches BCR signaling from amplification to attenuation, using mouse splenic B cells, a functionalized planar lipid bilayer system, and total internal reflection fluorescence microscopy. Our results show that branched actin polymerized by Arp2/3 is required for B cell transition from spreading to contraction after driving B cell spreading. Ubiquitously expressed Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP), but not the haematopoietically specific WASP, activates the branched actin polymerization and generates inner actin foci from lamellipodial actin networks, by sustaining their lifetime and centripetal movement. N-WASP-dependent inner actin foci are necessary for recruiting non-muscle myosin II, creating an actomyosin ring-like structure at the periphery of the membrane contact region to drive B cell contraction. B cell contraction primarily increases the BCR molecular density in individual BCR-antigen clusters, measured by the peak fluorescence intensity. Inhibition of B cell contraction by Arp2/3 inhibitor and B cell-specific N-WASP knockout (cNKO) reduced the increasing rates of BCR molecular density. Increased molecular density caused by B cell contraction leads to decreases in the levels of phosphorylated BCR, the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. However, the levels of total Syk and SHIP-1 have a different relationship with BCR density in individual clusters: Syk does not decrease until a high threshold of BCR density, which can be achieved only by contracting B cells, but SHIP-1 consistently reduces with the increase in BCR molecular density. Inhibiting B cell contraction by cNKO reduces the molecular density of BCR clusters but does not affect the relationship of the Syk and SHIP-1 levels with BCR molecular density in clusters. Taken together, our results suggest that the actin cytoskeleton reorganizes from the lamellipodial branched actin networks to centripetally moving actin foci, enabling actomyosin ring-like structure formation, through N-WASP-activated Arp2/3. Actomyosin-mediated B cell contraction attenuates BCR signaling by increasing receptor molecular density in individual BCR clusters, which causes the dissociation of both stimulatory and inhibitory signaling molecules. My thesis research results reveal a novel negative regulatory mechanism for BCR signaling, an essential checkpoint for generating pathogen-specific and suppressing self-reactive antibody responses.Item ROLES OF PLASMA MEMBRANE WOUNDING AND REPAIR IN B CELL ANTIGEN CAPTURE AND PRESENTATION(2022) van Haaren, Jurriaan Jan Hein; Song, Wenxia W; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell-mediated humoral immune responses play a crucial role in neutralizing pathogens and unwanted foreign substances. B cells become activated upon antigen binding of their B cell receptor (BCR), and then internalize and process antigen for presentation on their MHCII for T cell recognition. acquiring T cell signaling through antigen presentation is essential for B cell differentiation into high-affinity antibody-producing cells and memory B cells. In vivo, Antigen encountered by B cells is often tightly associated with the surface of pathogens and/or antigen-presenting cells. When B cells engage surface-associated antigen, BCR signaling induces reorganization of the cytoskeleton, causing spreading and contraction of the B cell on the antigen-presenting surface. This allows the B cell to engage more antigen and gather the antigen into a central cluster for internalization. Internalization of surface-associated antigen has been shown to require myosin-generated forces and the exocytosis of lysosomal enzymes. However, the mechanism that initiates lysosomal exocytosis remains unknown.This research explored a possible mechanism for the triggering of lysosomal exocytosis of B cells during interaction with surface-associated antigen. We showed that BCR interaction with antigen tethered to beads, to planar lipid-bilayers (PLBs) or expressed on the surface of live cells causes permeabilization of the B cell plasma membrane (PM), an event that required strong BCR-antigen affinity, BCR signaling, and activation of non-muscle myosin IIA (NMIIA). Moreover, we showed that B cell PM permeabilization triggers a repair response that includes the exocytosis of lysosomes at the site of antigen interaction. Importantly, we showed that B cells undergoing PM permeabilization and subsequent repair internalize more antigen; and better activate T cells compared to unpermeabilized B cells. Thus, our research reveals a novel mechanism for B cells to capture surface-associated antigen: antigen affinity-dependent binding of the BCR indices localized B cell PM permeabilization and lysosome exocytosis as a repair response, which facilitates antigen internalization and presentation through the extracellular release of lysosomal hydrolases. In addition, we explored the molecular mechanism required for B cell PM permeabilization in response to surface-associated antigen. We showed that B cells that undergo PM permeabilization in response to PLB-associated antigen spread over the PLB at a faster rate and to a larger area in comparison to cells that remain intact. Furthermore, we showed that B cells that undergo PM permeabilization recruit more NMIIA at a faster rate, and display a higher level of NMIIA organization at the immune synapse. We additionally discovered a 2o B cell spreading and NMIIA recruitment event, approximately 25-30 minutes after antigen engagement, that facilitates B cell PM permeabilization. Thus, B cell PM permeabilization requires the engagement of a large amount of antigen through B cell spreading on the presenting surface, as well as strong NMIIA recruitment and organization at the immune synapse. This research suggests that B cell PM permeabilization in response to surface-associated antigen plays an important role in distinguishing B cells with various levels of BCR activation, providing novel insights into the mechanisms responsible for affinity differentiation during B cell activation.