Abstract
Growth arrest-specific 2-like 1 protein (GAS2L1) binds both actin and microtubules through its unique structural domains: a calponin-homology (CH) domain for actin binding and a GAS2-related (GAR) domain for microtubule interaction. In this study, we demonstrate that GAS2L1 promotes stress fiber assembly, enhances focal adhesion formation, and stabilizes cytoskeletal networks against mechanical perturbation through its CH domain. Remarkably, we show that the CH domain dimerizes and induces actin filament bundling and stabilization both in cells and in vitro. The CH and GAR domains interact to form an autoinhibitory module, wherein the GAR domain suppresses CH domain dimerization and actin-bundling activity. Our findings provide novel insights into the regulatory mechanisms of GAS2L1's autoinhibition and identify the CH domain as a critical actin-bundling factor that contributes to the organization of stress fibers and focal adhesions.