biostudies-literatureUnknownPhua DYZNHLBI NIH HHSNIGMS NIH HHSAs the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.bioRxiv : the preprint server for biology2024.05.17.594765https://www.ebi.ac.uk/biostudies/studies/S-EPMC11118565biostudies-literatureForce-activated zyxin assemblies coordinate actin nucleation and crosslinking to orchestrate stress fiber repair.PMC11118565F31 HL165906R01 GM146880Phua DYZSun XAlushin GMfalseForce-activated zyxin assemblies coordinate actin nucleation and crosslinking to orchestrate stress fiber repair.As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.2024-01-01T00:00:00Z2024 May2026-07-09T12:23:09.347Z2026-07-09T11:16:38.637ZS-EPMC111185653879841910.1101/2024.05.17.594765