U2OS cells were grown on coverslips, heat shocked at 44°C for 45 min, fixed and co-stained with DAPI (blue color), and antibodies to TIA1 (red color) and PPIA (green color). In vivo, TIA1 showed a punctate localization to stress granules and co-localization with the PPIA enzyme (PMID:26544936). In vitro differential interference contrast (DIC) microscopy experiments: Full-length WT hnRNPA2 undergoes LLPS after cleavage of a solubility tag (N-terminal maltose-binding protein). At the same conditions, D290V mutant quickly forms aggregates (morphology). Over time, WT droplets fuse and grow (morphology, particle size and count) while D290V aggregates grow larger. P298L forms droplets initially but aggregates over time. Truncation of LC domain (residues 1–189, DLC) prevents both LLPS and aggregation, suggesting that the LC domain is necessary for both phase separation and aggregation. In vitro NMR experiments: NMR spectrum of phase-separated (apparent concentration 30 mM) hnRNPA2 LC domain (181-341) is highly similar to the spectrum of the monomeric peptide in dispersed phase (65 μM), indicating that the conformations that give rise to the observed LLPS resonances remain disordered. Overlay of 15N-edited one-dimensional spectra of 65 μM monomer and phase-separated state: The monomeric signals are so weak compared to the phase-separated state, they appear as a straight line. The monomeric spectrum is visible at much lower intensity than the phase-separated state, about 470 times less intense than the condensed phase signals, providing an estimated concentration in the condensed phase. NMR spin relaxation parameters R2, R1, and NOE sensitive to local motions at observable resonances of phase-separated and monomer hnRNPA2 LC domains are consistent with structural disorder but slowed motions after LLPS. Slightly lower values of R2 for dispersed-phase reference sample at lower pH suggest some contribution from water exchange to measured R2. (PMID:26544936, PMID:29358076)