Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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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 The Role Of The Ig alpha /Ig beta Heterodimer In The Internalization And Intracellular Transport Of The B Cell Antigen Receptor(2006-06-05) Parent, Beth A; Song, Wenxia; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The B cell antigen receptor (BCR) consists of membrane-bound immunoglobulin (mIg), which binds extracellular antigen, and the Ig alpha / Ig beta heterodimer (Ig alpha / Ig beta), the signaling component. Crosslinking of BCR by multivalent antigen initiates signaling, the first step in activating B cells, and leads to rapid internalization and transport of antigen to the major histocompatibility complex (MHC) class II containing compartments (MIICs). Here antigen is degraded to peptides which are loaded onto MHC class II molecules which are transported to the surface for presentation to T helper cells. Studies indicate that BCR-triggered signaling plays a role in accelerated internalization and transport of antigen, but the mechanism is not clear. This work examines the role of the signaling component Ig alpha / Ig beta in the efficient internalization and intracellular transport of the BCR. The effect of a lack of association between Ig alpha / Ig beta and mIgM on internalization and intracellular transport of mIgM was studied using biochemical techniques and immunofluorescence microscopy. We demonstrated that a reduction of association with Ig alpha / Ig beta leads to a reduction in signaling, a defect in internalization and transport to MIICs, and a decrease in antigen presentation. Thus, physical association of mIgM with Ig alpha / Ig beta is necessary for efficient internalization and intracellular transport of mIgM. The role of the tyrosines in the immunoreceptor tyrosine-based activation motif (ITAM) of Ig alpha / Ig beta was examined. Mutation of the N-terminal tyrosine had no significant effect on BCR signaling and antigen transport. Mutation of the C-terminal tyrosine resulted in decreased signaling and internalization and a defect in transport to MIICs. Mutating both tyrosines caused a greater decrease in BCR signaling and a greater defect in internalization and transport of antigen to MIICs than the C-terminal tyrosine mutation. The results of this study indicate that Ig alpha /Ig beta, in particular the ITAM of Ig alpha, is essential for accelerated internalization and intracellular transport of BCR to the MIICs, probably by regulating these processes through signaling.