Increased substrate stiffness disrupts nuclear-cytoskeletal mechanical coupling in senescent cells

The physical coupling between the nucleus and the cytoskeleton is essential for the mechanobiological adaptation of cells to mechanical cues presented by the surrounding extracellular matrix (ECM). Although aging is known to influence both cellular and ECM mechanical properties, it remains poorly understood how cellular senescence, a hallmark of aging, affects cellular mechano-adaptation. Here, we use substrate stiffness as a mechano-modulatory cue across three distinct models of fibroblasts and demonstrate that senescent cells are limited in their capacity to integrate mechanical signals of increasing stiffness. The senescent nucleus undergoes progressive actin-mediated deformation and flattening as substrate stiffness increases, until a mechanical threshold is reached that provokes a decoupling of the nucleus from the cytoskeleton. This mechanical disengagement of the nucleus on stiff substrates is accompanied by a loss in cytoskeletal organization, abnormal focal adhesion (FA) maturation, and nuclear softening. We further suggest that the loss of nuclear compression is linked to changes in the nuclear localization of the key mechanosensitive transcriptional regulator Yes-associated protein (YAP). Our findings reveal a fundamental biophysical limitation in the mechano-adaptive response of senescent cells to high-stiffness environments, conditions typically associated with advanced tissue maturation and pathological scarring, which may underlie altered nuclear mechanotransduction and contribute to their specific role in both and physiological and pathological contexts. Proposed model: Substrate stiffness induces actin-mediated nuclear flattening in senescent cells, triggering nuclear-cytoskeletal decoupling once such compression exceeds nuclear tolerance . Despite pre-existing NE defects, sen-hdFs still present actin caps on soft substrates, adopting a polarized morphology with well-defined stress fibers while the nucleus is being compressed by the perinuclear actin-cap. On soft substrates, the maximum force being applied to the nucleus is inherently limited by the matrix itself. However, as stiffness increases, excessive nuclear compression leads to nuclear decoupling, resulting in nuclear softening and disengagement from the cytoskeletal network. Grey arrows denote proposed mechanisms not yet supported by existing literature.