Summary Liquid-liquid phase separation, driven by collective interactions among multivalent and intrinsically disordered proteins, is thought to mediate the formation of membrane-less organelles in cells. Using parallel cellular and in vitro assays we show that the Nephrin intracellular domain (NICD), a disordered protein, drives intracellular phase separation via complex coacervation, whereby the negatively charged NICD co-assembles with positively charged partners to form protein-rich dense liquid droplets. Mutagenesis reveals that the driving force for phase separation depends on the overall amino acid composition and not the precise sequence of NICD. Instead, phase separation is promoted by one or more regions of high negative charge density and aromatic/hydrophobic residues that are distributed across the protein. Many disordered proteins share similar sequence characteristics with NICD, suggesting that complex coacervation may be a widely used mechanism to promote intracellular phase separation.

Members of the Wiskott-Aldrich Syndrome Protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation by the Arp2/3 complex. The WASP relative, WAVE, regulates lamellipodia formation within a 400 kDa, hetero-pentameric WAVE Regulatory Complex (WRC). The WRC is inactive toward the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. We report the 2.3 Å crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting how these signals stimulate WRC activity toward the Arp2/3 complex. Spatial proximity of the Rac binding site and a large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes.

Islet amyloid polypeptide (IAPP) contributes to the pathogenesis of type II diabetes by depositing as cytotoxic amyloid fibers in the endocrine pancreas. Fiber formation occurs with a marked conformational change from an unstructured precursor. Using real-time quantitative kinetic methods, fibrillogenesis was characterized as a function of protein, denaturant, and seed concentration. Several observations are in sharp contrast to the expectations for nucleation-dependent polymerization. First, the half-time of conversion for both de novo and seeded kinetics were found to be independent of protein concentration. Second, while elongation kinetics scale linearly with protein concentration, they are relatively insensitive to changes in the total seed concentration. Third, seeded bypass of de novo fiber formation kinetics shows a lag phase. The seeded lag phase is eliminated by a time delay before the introduction of seed to a de novo reaction. Last, conversion is highly cooperative, with the time required for 10-90% conversion occurring much faster than the lag time. At a minimum, four kinetic steps are required to describe these observations: activation, fiber independent nucleation, fiber-dependent nucleation, and elongation. Furthermore, we invoke a phase transition in which protein initially forms an off-pathway dispersion. This single construct allows us to model both the concentration independence of the de novo reaction time and the first-order concentration dependence of the elongation kinetics. Marked acceleration of this reaction by hexafluoro-2-propanol reinforces this view by altering the relative solubility of the two phases and/or by stabilizing hydrogen-bonded structures in the transition states of the reaction pathway.