Stress‐Induced Reorganization of Proteasomes Through Diffusion and Cytoskeleton‐Dependent Mechanisms

Proteasomes are abundant molecular machines distributed throughout the eukaryotic cell to facilitate protein degradation. How their dynamic localization adapts to proteostasis requirements remains an area of active study. Recent studies from the authors show that proteotoxic stress induced by alpha-synuclein aggregates triggers proteasome reorganization into foci bodies, termed transient aggregate-associated droplets (TAADs). Here, advanced imaging and biophysical techniques are combined to examine proteasome reorganization and TAAD formation. Using single-molecule localization and light-sheet microscopy, redistribution of subcellular proteasome density is quantified in response to alpha-synuclein aggregates. Interestingly, the ratio of 20S proteasome core particles capped by 19S regulatory particles (∼60%) remains constant during proteotoxic stress. Delivery of aggregates by nanopipette injection reveals that TAAD formation is cytoskeleton-dependent, suggesting that directed transport is required for proteasome reorganization. Single-cell patch clamp further shows that cytoskeleton-dependent proteasome movement is linked to cell depolarization, implying that membrane potential can directly modulate proteasome localization. Single-particle tracking analysis detects the presence of both rapid- and slow-moving proteasome populations with proteotoxic stress shifting their motion towards confined diffusion within TAADs. Together, these results demonstrate that proteasomes adopt distinct modes of motion depending on cellular requirements and become restricted upon aggregate invasion, highlighting a tightly regulated system of proteasome organization for selective proteostasis during stress.