Under near-physiological conditions solutions of 100 μM Ddx4-N1 (mutation, truncation) and Ddx4-YFP (fusion protein) rapidly became turbid (change in optical properties). Under identical conditions, the splicing variant Ddx4-N2 (mutation) remained soluble, revealing that alternative splicing can regulate the formation of organelles. When droplets in the turbid phase were imaged by microscopy, their morphologies and (qualitatively) their distribution of particle size (particle size and count) and time dependence mirrored that seen within cells. Using bright-field microscopy and a thermal stage,a fully dispersed solution of Ddx4N1 (pH 8.0) at 50°C was cooled at 4°C to 22°C. At 36°C, the solution became turbid and droplets were observed to condense with the change in temperature. At all ionic strengths examined, TP increased with increasing Ddx4N1 concentration (change in protein concentration) in a manner that is well predicted by Flory-Huggins theory. The Ddx4-N1-YFP fusion protein was transfected into HeLa cells, and both its expression and protein localization were monitored using fluorescence microscopy in vivo. As the intra-cellular concentration increased over time, dense micron-sized spherical bodies (morphology) were observed to form in the nucleus. A detailed analysis suggested that the number of in vivo formed droplets and their sizes (particle size and count) were limited by the quantity of free monomers. Ddx4-N1 is posttranslationally modified at multiple sites by PRMT1 in vivo and contains six predicted methylation sites. Remarkably, a few methylations per protein of this type significantly destabilized the droplets, lowering the transition temperature by 25°C. The charge clusters of DDX4 persist for approximately 8–10 residues in length and tend to contain 3–8 similarly charged residues. To determine the physical importance of this charge patterning, a Ddx4 variant was produced, Ddx4N1CS (mutation), with the same overall net charge, but in which the blocks were scrambled. In Ddx4N1CS, the regions of opposing charge are removed while simultaneously maintaining the same overall isoelectric point (PI), amino acid composition, and positions of all other residues; this construct was unable to form organelles in vitro under near-physiological conditions or in vivo. To test the physical significance of the distributed phenylalanine residues to droplet formation, a construct was produced where these nine residues were mutated to alanine (Ddx4-N1-FtoA), which was unable to induce droplet formation either in cells (in vivo) or in vitro. While double-stranded DNA was largely excluded from the droplets, the single-stranded DNA was concentrated significantly in the interior of the droplets (protein co-localization). PMID:25747659. The protein within the concentrated phase of phase-separated Ddx4 diffuses as a particle of 600-nm hydrodynamic radius dissolved in water as studied in vitro by NMR. However, NMR spectra reveal sharp resonances with chemical shifts showing Ddx4 to be intrinsically disordered. Spin relaxation measurements indicate that the backbone amides of Ddx4 have significant mobility, explaining why high-resolution spectra are observed, but motion is reduced compared with an equivalently concentrated nonphase-separating control. PMID:28894006.