FUS, EWSR1, and TAF15, which constitute the FET family showed robust phase separation (particle size and count by microscopy) in the absence of crowding agents at a protein concentration of 5 μM. Although the majority of the FUS family of proteins can phase separate, only the FET proteins possess the requisite sequence features to drive phase separation at low protein concentrations and physiologically relevant salt concentrations. The measured saturation concentration of full-length FUS is 2 μM in 75 mM KCl. Surprisingly, the FUS PLD (truncated), expressed and purified from insect cells, does not drive phase separation as an independent module even for protein concentrations that are as high as 120 μM. Therefore, the saturation concentration for the PLD is almost two orders of magnitude higher than the saturation concentration for the full-length protein. The RBD alone did not phase separate. However, when the FUS PLD was mixed with the FUS RBD at a ratio of 1:1, the PLD phase separated at concentrations as low as 15 μM. Mutational studies, where all the Tyr residues of the PLD (Y-->S) and/or all the Arg residues of the RBD (R-->G) have been changed suggested that these residues are the major determinants of LLPS of FUS and that their number governs the saturation concentration of FUS both in vitro and in vivo in cells (PMID:29961577). Further mutagenesis studies implied that the driving forces for phase separation in vitro and in vivo, quantified in terms of measured saturation concentrations, follow the order: tyrosine-arginine > tyrosine-lysine > phenylalanine-arginine > phenylalanine-lysine, which shows that the selective preference for tyrosine-arginine interactions cannot be solely due to generic cation-p interactions. Instead, the specific chemical structures of the tyrosine and the arginine side chains appear to be important determinants of the complementarity of tyrosine-arginine interactions (PMID:29961577). In vivo expression of the protein in human cells, truncated to residues 1-211 (low complexity N-terminal region) led to assembly into structures with remarkably spherical morphology via liquid demixing, evidenced by GFP-tagging and fluorescence and phase-contrast microscopy. The RGG repeat region was shown to mediate poly(ADP-ribose) (PAR) binding, using in vivo expression of GFP-tagged full length protein with induced mutations in the RGG region. The number of RGG repeats determined the efficiency of PAR binding, co-localization with PAR, and hence, recruitment to sites of DNA damage. While this is not strictly required for LLPS, PAR binding serves as a nucleation event, increasing local protein concentration at sites of DNA damage. Protein localization and co-localization was followed using standard widefield, confocal, high-content microscopy and time-lapse imaging. In a cellular, in vivo context, depletion of FUS with RNA interference, and abrogation of PAR formation both led to the lack of granule formation at sites of DNA damage, observed using time-lapse bright-field and phase-contrast live-cell microscopy. In vitro cell-free measurements recombinant FUS was incubated with and without sub-stoichiometric amounts of PAR, forming spontaneous aggregates, which were consistently larger in the presence of PAR, followed by transmission electron microscopy (TEM). Consistent with in vivo results, these data provide evidence for the intrinsic ability of PAR chains to nucleate aggregation of low complexity domain-containing disordered proteins (PMID:26286827). In vitro phosphorylation of recombinant, isolated FUS LC reduces its ability to bind hydrogels formed from amyloid‐like fibrils of purified recombinant, unphosphorylated FUS LC (PMID:22579282) Both phosphorylation and phosphomimetic variants reduce the aggregation-prone/prion-like character of FUS PLD, disrupting FUS phase separation in the presence of RNA or salt and reducing FUS propensity to aggregate. Also, phosphomimetic FUS reduces aggregation in human and yeast cell models, and can ameliorate FUS-associated cytotoxicity (PMID:28790177). BAC transgene has been generated for the in vivo expression of GFP-tagged FUS after introduced into HeLa and embryonic stem (ES) cells to generate stable cell lines. Mass spectrometry was used to determine the physiological concentration of FUS in HeLa cells, that is around 2 μM in the cytoplasm and between 4 and 8 μM in the nucleus. Actinomycin D (a potent inhibitor of RNA polymerase II) treatment of FUS-GFP HeLa cells suggested that under normal conditions, FUS assembles into compartments (particle size and count) that may be associated with transcription or splicing. FUS accumulated in the cytoplasm in heat-stressed cells and coalesced into stress granules (in vivo protein localization). In vivo FUS assemblies have all the hallmarks of a liquid state: they turn over quickly; are spherical; and when they fuse, they relax into one spherical assembly. A 10% solution of either dextran or polyethylene glycol (PEG) induced the in vitro assembly of recombinant GFP-tagged FUS (10 μM) purified from insect cells (but not that of truncated FUSΔPLD) into round micrometer-sized structures (PMID:26317470). Inhibition of PARP1/2 prevented the recruitment of FUS to DNA lesions. After 8 hr, no fusion events could be detected for G156E FUS, while 81% of the wild-type FUS droplets still fused. By 12 hr, wild-type droplets also stopped fusing. This indicates that there is indeed a change in the biophysical properties of FUS droplets with time and that the properties of the G156E FUS droplets are changing more quickly than those of the wild-type FUS droplets. This is consistent with the observation that G156E has a higher propensity to aggregate. Another patient-derived mutant of FUS R244C also accelerated the conversion of FUS droplets to a fibrous state (PMID:26317470).