​Abstract 

Oxidative stress is closely associated with aging, fibrosis, and cancer progression, yet how oxidative stress affects rigidity sensing remains poorly understood. Here, we employed submicron micropillar-based traction-force microscopy to investigate oxidative stress-associated mechanotransduction dysfunction in mouse embryonic fibroblasts (MEFs). Sublethal H2O2 exposure impaired stiffness-dependent cytoskeletal remodeling, focal adhesion maturation, and traction-force generation during cell spreading. Quantitative force mapping further revealed disrupted contraction-unit (CU)-associated nanoscale force coordination at the cell periphery, characterized by increased alignment of neighboring traction-force vectors and reduced opposing contractions. These defects were accompanied by fragmented mitochondrial networks and altered mitochondrial dynamics-associated gene expression. Importantly, mitofusin (MFN)-deficient MEFs phenocopied oxidative stress-induced impairments in stiffness-responsive spreading and traction-force generation, identifying mitochondrial network integrity as a critical regulator of rigidity sensing. Together, these findings establish a mechanistic link between oxidative stress, mitochondrial dynamics, and nanoscale force coordination during rigidity sensing, while highlighting the utility of submicron traction-force mechanophenotyping for quantitative analysis of oxidative stress-associated mechanobiological dysfunction.