Role of SWAP70 protein in the formation of the immune synapse and B cell activation in response to substrate stiffness
B-cells extract immobilized antigens via the immune synapse (IS), a specialized, mechanically regulated interface that integrates receptor signaling with dynamic cytoskeletal remodeling. At this interface, actin polymerization, cortical tension, and membrane trafficking cooperate to generate the forces required for antigen extraction. Although actin dynamics are known to be essential for these processes, the molecularmechanisms that organize the cytoskeletal architecture underlying B-cell mechanosensing remain incompletely understood.Here, we identify SWAP70 as a central regulator of cytoskeletal organization at the IS. We show that SWAP70 is rapidly recruited to regions of intense actin remodeling at the synaptic interface, where it forms a stable network with slower turnover than filamentous actin. Importantly, this stability depends on formin activity, indicating that SWAP70 cooperates with the linear actin filament to help stabilize the cortical cytoskeleton.Functional analyses demonstrate that SWAP70 is not required for global actin turnover but instead regulates higher-order cytoskeletal organization. Loss of SWAP70 leads to pronounced alterations in synapse architecture, including increased cell spreading, reduced actin retrograde flow, and enhanced formation of actin foci. Despite these structural changes, actin turnover, as measured by FRAP, remains largely unchanged, supporting the idea that SWAP70 primarily controls the spatial organization and mechanical integration of actin networks rather than their polymerization kinetics. These cytoskeletal defects are accompanied by major changes in IS organization. In the absence of SWAP70, B cells display increased numbers of B cell receptor (BCR) clusters and enhanced recruitment of lysosomes to the IS. Additionally, these cells upregulate the amount of clathrin-coated pits and thereby exhibit increased antigen extraction.Our findings identify SWAP70 as a key organizer of cytoskeletal architecture that constrains the mechanical and trafficking responses of B-cells during IS formation. Given that SWAP70 deficiency has been associated with autoimmune phenotypes, we propose that SWAP70-dependent regulation of cytoskeletal architecture at the IS serves as a critical checkpoint that prevents aberrant B-cell activationand helps maintain immune tolerance.