Abstract
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 B-cell contraction. Contracting B-cells generate centripetally moving actin foci from lamellipodial F-actin networks in the B-cell 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. Furthermore, 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.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
We have made the following modifications to the manuscript: 1. We have provided new data showing that treatment with the non-muscle myosin II motor inhibitor blebbistatin, which is known to inhibit B-cell contraction but not spreading on Fab-PLB (Seeley-Fallen et al. 2022. Frontiers in Immunology), interferes with the formation of inner actin foci ring-like structures, which are associated with B-cell contraction. These results suggest that the generation of inner actin foci ring-like structure depends on the coordination between N-WASP-mediated actin polymerization and myosin contractile activity. The new data are shown in Figure 5G and H and discussed in the revised manuscript. 2. To show the variability, we displayed the data from each experiment as individual data points and utilized three colors of dots to represent three independent experiments in Figure 1C, E, G, and I, Figure 2B-G, and new Figure 5H. 3. We have added arrows to Figure 2A to indicate all identified inner F-actin foci in images. 4. In the revised Figure 4, we have provided a kymograph of a WKO B cell. 5. We have revised Figures 7-9, where we utilized square braces to indicate groups of clusters (blue points) being compared. We have also provided additional information in the figure legend and Method sections. 6. We have provided representative images for Syk, pSyk, SHIP-1, and pSHIP-1 in revised Figure 8 and 9. 7. We have modified the Discussion section to include: a. The possibility that lamellipodial retraction may be involved in increasing the molecular density of BCR clusters b. Relationship between WASP and N-WASP c. Future studies on the potential roles of N-WASP-dependent inner actin foci and actomyosin structures in BCR internalization and intracellular signaling d. The limitations of the study and how these limitations affect the data interpretation e. A new figure (Figure 10) summarizing the results presented in the manuscript as a working model.
