FUS Phase Separation in Stressed Cells

Protein aggregation is a neurodegenerative diseases hallmark. Nevertheless, phase separation of disease-associated proteins can occur as part of normal cellular physiology. This can be seen in cellular response to stress, whereas stress-granules (liquid-like cellular compartments) are formed in the cytoplasm, and disassemble upon stress elimination. There are increasing evidence that aggregation-prone Ribonucleoproteins might convert, with time, from a liquid to an aggregated state, which is accelerated by disease-associated mutations. This emphasizes the necessity to define the mechanisms that govern the dynamics of SGs and condensations.
We are addressing these key questions, with respect to FUS; a stress-granules-forming, prion-like protein which contains intrinsically disordered domains, and mutations in which are causative of ALS. To that end, we are first characterizing how different stress conditions affect the morphology, localization and dynamics of WT FUS condensates. We are screening a variety of stress conditions, using live-cell-microscopy, to follow FUS-YFP condensates assembly, and subsequent disassembly during recovery. Subsequent image analysis is used to calculate the number of condensates, their morphology, localization and kinetics.
Interestingly, when comparing different stress conditions in HEK293T cells, we observed different types of FUS condensates. For instance, condensates resulted from osmotic stress (sorbitol) start at the nucleus and, with time, spread to the cytoplasm. Additionally, their morphology differs from condensates resulting from KCl stress. Current characterization of these condensates includes their co-localization with established marker proteins, such as G3BP1 and TIA1 for classical stress-granules.