SG dynamics were monitored in HeLa in vivo cells expressing N-terminal GFP-tagged TIA1 wild-type, P362L, A381T, or E384K mutants. GFP-tagged TIA1 showed the identical punctate subcellular distribution as endogenous or untagged TIA1 proteins (protein localization), and the frequency and size of SGs containing these proteins were not altered by introduction of exogenous TIA1 at this modest expression level. We observed no significant impact of the P362L, A381T, or E384K TIA1 mutations on the rates of SG assembly. By contrast, each of these disease-associated mutations resulted in significantly protracted SG disassembly, as assessed by both blinded manual counting and automated image analysis (particle size and count) (PMID:28817800). Fluorescently tagged, recombinant TIA1-EYFP was shown to self-multimerize in the presence of ZnCl₂ detected by a FRET-based assay in vitro. Using Confocal and Differential interference contrast (DIC) microscopy the droplet formation of the same fluorescently labeled protein was observed in a Zn²⁺ concentration dependent manner at physiological salt concentration. Both low and high salt (50 and 500 mM NaCl, respectively) caused a reduction in droplet size, droplet number, and rate of droplet formation emphasizing the importance of optimal electrostatic interactions to drive the formation of higher-order TIA-1 condensates. The solutions of TIA-1-EYFP developed visible turbidity that varied positively with protein concentration, zinc addition, and molecular crowding by poly-ethylene glycol (PEG) addition. Addition of DTT to simulate the reducing environment of the cell prevented the formation of TIA-1 droplets normaly induced by ZnCl₂ and eliminated the zinc dose response in the FRET assay, consistent with the idea that TIA-1 multimerization is responsive to both zinc and reduction-oxidation (redox) environment (PMID:29298433). In vitro full-length TIA1 spontaneously phase separated in the absence of any cosolute, at physiological ionic strength of 150 mM, and pH 7.5 on a temperature-sensitive way observed by DIC microscopy. The three disease related mutant of Tia1 (P362L, A381T, E384K) underwent spontaneous temperature- and concentration-dependent LLPS to create liquid droplets that at early time points were morphologically indistinguishable from liquid droplets formed by wild-type protein. In all cases a significant leftward shift in the co-existence line to a lower protein concentration was observed, indicating an increased propensity of mutant TIA1 to phase separate, due to stronger intermolecular protein-protein interactions. Using a quantitative ThT fluorescence assay it has been confirmed that disease-associated mutations in TIA1 significantly accelerated fibrillization. Fluorescence recovery after photobleaching (FRAP) measurements showed that disease-associated mutations significantly altered the dynamic exchange of TIA1 between the dense droplet phase and the light mono-disperse phase, with increased half-recovery times and a smaller overall mobile fraction. These results suggest that the mutations changed the material properties of mutant TIA1 droplets by enhancing transient, nonspecific intermolecular interactions that reduce protein mobility. This observation raises the possibility that material properties of membrane-less organelles composed of TIA1 protein in live cells, such as SGs, could be adversely affected by the disease-associated mutations (PMID:28817800). The same results were obtained in the case of the N357S mutant (PMID:29457785). The intact protein and the IDR alone precipitates by the b-isox chemical treatment. (PMID:22579281)