An Optimized Stress Granule Detection Method: Investigation of UBQLN2 Effect on Stress Granule Formation.

Stress granules (SGs) are cytosolic ribonucleoprotein granules that form via a liquid-liquid phase separation in response to environmental stresses such as heat, oxidative, and osmotic changes. Due to the condensation of low complexity, hydrophobic regions in core SG components in these highly dynamic granules, defects in SG maintenance and formation have been linked to toxic aggregate formation in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia. However, efforts to dissect mechanisms regulating SG formation and maintenance have been limited by methods of tracking protein-SG localization. Here we describe a method for detecting and quantifying recruitment of cytosolically enriched proteins to SGs by indirect immunofluorescence microscopy. Using this method, we tracked the transient recruitment of the cytosolically enriched ubiquitin-like protein, ubiquilin 2 (UBQLN2), and a number of other factors into SGs, demonstrating its utility (Alexander et al., Proc Natl Acad Sci U S A 115:E11485-E11494, 2018).

Ubiquilin 2 modulates ALS/FTD-linked FUS-RNA complex dynamics and stress granule formation.

The ubiquitin-like protein ubiquilin 2 (UBQLN2) has been genetically and pathologically linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but its normal cellular functions are not well understood. In a search for UBQLN2-interacting proteins, we found an enrichment of stress granule (SG) components, including ALS/FTD-linked heterogeneous ribonucleoprotein fused in sarcoma (FUS). Through the use of an optimized SG detection method, we observed UBQLN2 and its interactors at SGs. A low complexity, Sti1-like repeat region in UBQLN2 was sufficient for its localization to SGs. Functionally, UBQLN2 negatively regulated SG formation. UBQLN2 increased the dynamics of FUS-RNA interaction and promoted the fluidity of FUS-RNA complexes at a single-molecule level. This solubilizing effect corresponded to a dispersal of FUS liquid droplets in vitro and a suppression of FUS SG formation in cells. ALS-linked mutations in UBQLN2 reduced its association with FUS and impaired its function in regulating FUS-RNA complex dynamics and SG formation. These results reveal a previously unrecognized role for UBQLN2 in regulating the early stages of liquid-liquid phase separation by directly modulating the fluidity of protein-RNA complexes and the dynamics of SG formation.