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

## TL;DR

The paper shows how proteasomes reorganize in response to stress caused by alpha-synuclein aggregates, forming droplets that help manage protein degradation in cells.

## Contribution

The study reveals that proteasome reorganization during stress is cytoskeleton-dependent and involves a shift from global to confined motion.

## Key findings

- Proteasomes form TAADs under alpha-synuclein aggregate stress.
- TAAD formation requires cytoskeleton-dependent transport.
- Stress shifts proteasome motion to confined diffusion within TAADs.

## Abstract

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.

Proteotoxic stress induced by alpha‐synuclein aggregates drives rapid, cytoskeleton‐dependent re‐localization of mammalian proteasomes and formation of transient aggregate‐associated droplets (TAADs). Single‐molecule localization microscopy, light‐sheet imaging, nanopipette injection, single‐cell patch clamp, and single‐particle tracking reveal a constant ratio of proteasome core to regulatory particles but a stress‐dependent shift from global diffusion to confined motion and altered subcellular density, tightly regulating proteasome availability for selective proteostasis.