Non-Muscle Myosin II Is Essential for the Negative Regulation of B-Cell Receptor Signaling and B-Cell Activation

Abstract

Antigen (Ag)-triggered B-cell receptor (BCR) signaling initiates antibody responses. However, prolonged or uncontrolled BCR signaling is associated with the development of self-reactive B-cells and autoimmune diseases. We previously showed that actin-mediated B-cell contraction on Ag-presenting surfaces negatively regulates BCR signaling. Non-muscle myosin II (NMII), an actin motor, is involved in B-cell development and antibody responses by mediating B-cell migration, cytokinesis, and Ag extraction from Ag-presenting cells. However, whether and how NMII regulates humoral responses through BCR signaling remains elusive. Utilizing a B-cell-specific, partial NMIIA knockout (cIIAKO) mouse model and NMII inhibitors, this study examined the role of NMII in BCR signaling. Upon BCR binding to antibody-coated planar lipid bilayers (PLB), NMIIA was recruited to the B-cell contact membrane and formed a ring-like structure during B-cell contraction. NMII recruitment depended on phosphatidylinositol 5-phosphatase (SHIP1), an inhibitory signaling molecule. NMII inhibition by cIIAKO did not affect B-cell spreading on PLB but delayed B-cell contraction and altered BCR clustering. Surface BCR “cap” formation induced by soluble stimulation was enhanced in cIIAKO B-cells. Notably, NMII inhibition by cIIAKO and inhibitors up-regulated BCR signaling in response to both surface-associated and soluble stimulation, increasing phosphorylated tyrosine, CD79a, BLNK, and Erk and decreasing phosphorylated SHIP1. While cIIAKO did not affect B-cell development, the number of germinal center B-cells was significantly increased in unimmunized cIIAKO mice, compared to control mice. While cIIAKO mice mounted similar antibody responses when compared to control mice upon immunization, the percentages of high-affinity antibodies, Ag-specific germinal center B-cells and isotype switched B-cells were significantly lower in cIIAKO mice than in control mice. Furthermore, autoantibody levels were elevated in cIIAKO mice, compared to control mice. Collectively, our results reveal that NMII exerts a B-cell-intrinsic inhibition on BCR signaling by regulating B-cell membrane contraction and surface BCR clustering, which curtails the activation of non-specific and self-reactive B-cells.

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

“…As non-muscle myosin II (NMII) is required for B-cell contraction (Seeley-Fallen et al . 2022), we examined the relationship between the formation of inner F-actin foci and the recruitment and reorganization of NMII in the contact zone using TIRF imaging of B-cells from mice expressing a GFP-NMIIA transgene.…”

“…We previously showed that B-cell contraction after spreading on antigen-presenting surfaces promotes BCR signaling attenuation (Liu et al . 2013a; Seeley-Fallen et al . 2022).…”

“…Our recently published data show that NMII is required for B-cell contraction. Activated NMII is recruited to the B-cell contact zone in a SHIP-1-dependent manner and forms a peripheral ring surrounding the contact zone during B-cell contraction (Seeley-Fallen et al . 2022).…”

“…Surprisingly, B-cell-specific knockout of N-WASP (cNKO) but not WASP germline knockout (WKO) enhances B-cell spreading, delays B-cell contraction, and inhibits the centralization of BCR clusters at the contact zone (Liu et al 2013a) Our previously studies demonstrate that B-cell contraction is critical for BCR signaling attenuation. Delay and inhibition of B-cell contraction through deficiencies of the actin regulators N-WASP, Abp1, or NMIIA, prolongs and/or increases BCR signaling by enhancing the activation of stimulatory kinases and suppressing the activation of inhibitory phosphatases, which elevates the production of autoantibody levels in mice (Liu et al 2013a;Seeley-Fallen et al 2014;Seeley-Fallen et al 2022). N-WASP and NMIIA are not known to interact with kinases and phosphatases directly.…”

“…As non-muscle myosin II (NMII) is required for B-cell contraction(Seeley-Fallen et al 2022), we examined the relationship between the formation of inner F-actin foci and the recruitment and reorganization of NMII in the contact zone using TIRF imaging of B-cells from mice expressing a GFP-NMIIA transgene. Upon interacting with Fab'-PLB, the GFP-NMIIA MFI in the contact zone of untreated WT B-cells increased rapidly in the first minute and slowly afterward.…”