Dynamics of Endoplasmic Reticulum Stress Responses in Cells with Different Endoplasmic Reticulum Volumes and Senescence States

The Unfolded Protein Response (UPR) determines cell fate under endoplasmic reticulum (ER) stress, yet its temporal dynamics across different cell types and senescence states re-main poorly understood. In this study, we investigated UPR kinetics in endothelial and T-lymphoblastoid cell lines with distinct ER volumes, as well as in senescent FRSN cells undergoing ER expansion. Variations in ER volume were confirmed via confocal microscopy and flow cytometry. Using the ‘gene response time’ metric, we demonstrated experimentally and through mathematical modeling that the BiP response kinetics closely correlate with variations in ER volume. Applying this metric to presenescent FRSN cells with a significantly expanded ER, we found that their ER stress response machinery maintained a response time similar to that of exponentially growing FRSN cells, but exhibited a lower amplitude and a prolonged lag phase, leading to chronic ER stress signaling such as a protracted XBP1(S) expression profile. Scanning ion-conductance microscopy (SICM) revealed a significant decrease in the stiffness of the near-surface layer of presenescent cells, reflecting a rarefaction of the actin cortex and possibly cytoplasmic dilution. We propose that this structural remodeling could be associated with active intracellular transport impairment that weakens the temporal ‘tethering’ of the proteome to the genome upon cellular senescence. Consequently, the stress response machinery fails to scale proportionally with cell enlargement during senescence, creating a state of ‘functional dilution’. This prevents enlarged senescent cells from mounting a scaled response, resulting in prolonged stress signaling and increased vulnerability to proteotoxic stress.