Cell Biology & Molecular Genetics Theses and Dissertations

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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.
REGULATORY FUNCTIONS OF THE ACTIN CYTOSKELETON IN B CELL RECEPTOR SIGNALING
(2013) Liu, Chaohong; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
The binding of antigen (Ag) to the B cell receptor (BCR) induces the activation of intracellular signaling and the reorganization of the actin cytoskeleton. However, the function of actin reorganization and the mechanisms by which BCR signaling and actin reorganization is coupled have not been well studied. This thesis has investigated how BCR signaling regulates actin reorganization and how actin remodeling in turn influences BCR signalig. My studies show that the key stimulatory signaling molecule of the BCR, Bruton's tyrosine kinase (Btk), is critical for actin polymerization at the activation surface and BCR clustering and B cell spreading, events that are essential for signaling initiation and amplification. The key inhibitory signaling molecule, SH2-containing phosphatidylinositol-5 phasphatase (SHIP-1), is important for removal of F-actin from the activation surface, and actin-mediated B cell contraction and the formation of BCR central clusters. SHIP-1 suppresses actin polymerization by inhibiting Btk-dependent activation of Wiskott-Aldrich syndrome protein (WASP). These results suggest that BCR signaling can regulate B cell morphology and surface BCR clustering via modulationg actin dynamics. To understand the roles of actin reorganization in BCR signaling, I investigated the effects of gene knockout of the two actin regulators, WASP and its homolog, neuronal (N)-WASP. My results show that both WASP and N-WASP are required for optimal BCR clustering, B cell spreading, and BCR signaling, but they play distinct roles. WASP promotes actin polymerization, B cell spreading, BCR clustering, and signaling amplification, and N-WASP inhibits actin polymerization at the activation surface and promotes B cell contraction, BCR central cluster formation, and signaling attenuation. Importantly, B cell-specific N-WASP knockout causes increases in the levels of autoantibody. In addition, WASP and N-WASP negatively regulate each other, compete for Arp2/3, and are inversely regulated by Btk and SHIP-1. Taken together, these results demonstrate that the balance of stimulatory and inhibitory BCR signaling controls actin dynamics and organization through regulating the activities of WASP and N-WASP. Actin remodeling in turn amplifies BCR signaling activation or down regulation by modulating B cell morphlogy and the organization of surface BCRs.This research reveals a bidrectional feedback loop between BCR signaling and the actin cytoskeleton.