Submicron traction-force mapping reveals mitochondrial regulation of nanoscale force coordination during rigidity sensing under oxidative stress

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 H ₂ O ₂ 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.