Endoplasmic Reticulum Lumen Diffusivity in Saccharomyces cerevisiae Is Upregulated by Cellular Stress Through Network Remodeling

Molecular diffusivity in the endoplasmic reticulum (ER) lumen is essential for protein folding and quality control, but the impact of cellular stress on these properties is not well understood. We used genetically encoded multimeric nanoparticles (GEMs) localized in the ER lumen and single-particle tracking to directly measure lumen diffusivity in living Saccharomyces cerevisiae under tunicamycin-induced ER stress. Tunicamycin inhibits N-linked glycosylation, leading to unfolded protein accumulation and activation of the unfolded protein response (UPR). Our results show that tunicamycin significantly increases GEM diffusivity by approximately sixfold compared to DMSO controls. To assess whether this increase results from changes in network topology rather than luminal crowding, we skeletonized ER fluorescence images and simulated particle diffusion on the resulting network graphs. Using the same input diffusivity, the tunicamycin network produced a higher effective diffusivity, consistent with experimental observations. These results indicate that UPR-driven structural remodeling, including increased branching, junction complexity, and network density, is the main factor enhancing molecular mobility during ER stress.