WASP family proteins regulate the mobility of the B cell receptor during signaling activation.

Regulation of membrane receptor mobility tunes cellular response to external signals, such as in binding of B cell receptors (BCR) to antigen, which initiates signaling. However, whether BCR signaling is regulated by BCR mobility, and what factors mediate this regulation, are not well understood. Here we use single molecule imaging to examine BCR movement during signaling activation and a novel machine learning method to classify BCR trajectories into distinct diffusive states. Inhibition of actin dynamics downstream of the actin nucleating factors, Arp2/3 and formin, decreases BCR mobility. Constitutive loss or acute inhibition of the Arp2/3 regulator, N-WASP, which is associated with enhanced signaling, increases the proportion of BCR trajectories with lower diffusivity. Furthermore, loss of N-WASP reduces the diffusivity of CD19, a stimulatory co-receptor, but not that of FcγRIIB, an inhibitory co-receptor. Our results implicate a dynamic actin network in fine-tuning receptor mobility and receptor-ligand interactions for modulating B cell signaling

Mechanisms by which the actin cytoskeleton switches B cell receptor signaling from the activation to the attenuation mode

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

N-WASP-dependent branched actin polymerization attenuates B-cell receptor signaling by increasing the molecular density of receptor clusters

Antigen-induced B-cell receptor (BCR) signaling is critical for initiating and regulating B-cell activation. The actin cytoskeleton plays essential roles in BCR signaling. Upon encountering cell-surface antigens, actin-driven B-cell spreading amplifies signaling, while B-cell contraction following spreading leads to signal attenuation. However, the mechanism by which actin dynamics switch BCR signaling from amplification to attenuation is unknown. Here, we show that Arp2/3-mediated branched actin polymerization is required for mouse splenic B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the plasma membrane region contacting antigen-presenting surfaces. Actin polymerization driven by N-WASP, but not WASP, initiates these actin foci and facilitates non-muscle myosin II recruitment to the contact zone, creating actomyosin ring-like structures. B-cell contraction increases BCR molecular density in individual clusters, leading to decreased BCR phosphorylation. Increased BCR molecular density reduced levels of the stimulatory kinase Syk, the inhibitory phosphatase SHIP-1, and their phosphorylated forms in individual BCR clusters. These results suggest that N-WASP-activated Arp2/3, coordinating with myosin, generates centripetally moving foci and contractile actomyosin ring-like structures from lamellipodial networks, enabling contraction. B-cell contraction attenuates BCR signaling by pushing out both stimulatory kinases and inhibitory phosphatases from BCR clusters, providing novel insights into actin-facilitated signal attenuation