Biomolecular condensates are micron-scale compartments in eukaryotic cells that lack surrounding membranes but function to concentrate proteins and nucleic acids. These condensates are involved in diverse processes, including RNA metabolism, ribosome biogenesis, the DNA damage response and signal transduction. Recent studies have shown that liquid-liquid phase separation driven by multivalent macromolecular interactions is an important organizing principle for biomolecular condensates. With this physical framework, it is now possible to explain how the assembly, composition, physical properties and biochemical and cellular functions of these important structures are regulated.

Cells are organized on length scales ranging from Angstroms to microns. However, the mechanisms by which Angstrom-scale molecular properties are translated to micron-scale macroscopic properties are not well understood. Here we show that interactions between diverse, synthetic multivalent macromolecules (including multi-domain proteins and RNA) produce sharp, liquid-liquid demixing phase separations, generating micron-sized liquid droplets in aqueous solution. This macroscopic transition corresponds to a molecular transition between small complexes and large, dynamic supramolecular polymers. The concentrations needed for phase transition are directly related to valency of the interacting species. In the case of the actin regulatory protein, neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) interacting with its established biological partners Nck and phosphorylated nephrin1, the phase transition corresponds to a sharp increase in activity toward the actin nucleation factor, Arp2/3 complex. The transition is governed by the degree of phosphorylation of nephrin, explaining how this property of the system can be controlled to regulatory effect by kinases. The widespread occurrence of multivalent systems suggests that phase transitions are likely used to spatially organize and biochemically regulate information throughout biology.

Eukaryotic cells possess numerous dynamic membrane-less organelles, RNP granules, enriched in RNA and RNA binding proteins containing disordered regions. We demonstrate that the disordered regions of key RNP granule components, and the full-length granule protein hnRNPA1, can phase separate in vitro, producing dynamic liquid droplets. Phase separation is promoted by low salt concentrations or RNA. Over time, the droplets mature to more stable states, as assessed by slowed fluorescence recovery after photobleaching and resistance to salt. Maturation often coincides with formation of fibrous structures. Different disordered domains can co-assemble into phase-separated droplets. These biophysical properties demonstrate a plausible mechanism by which interactions between disordered regions, coupled with RNA binding, could contribute to RNP granule assembly in vivo through promoting phase separation. Progression from dynamic liquids to stable fibers may be regulated to produce cellular structures with diverse physiochemical properties and functions. Misregulation could contribute to diseases involving aberrant RNA granules.

SUMMARY Cellular bodies such as P bodies and PML nuclear bodies (PML NBs) appear to be phase-separated liquids organized by multivalent interactions among proteins and RNA molecules. Although many components of various cellular bodies are known, general principles that define body composition are lacking. We modeled cellular bodies using several engineered multivalent proteins and RNA. In vitro and in cells, these scaffold molecules form phase-separated liquids that concentrate low valency client proteins. Clients partition differently depending on the ratio of scaffolds, with a sharp switch across the phase diagram diagonal. Composition can switch rapidly through changes in scaffold concentration or valency. Natural PML NBs and P bodies show analogous partitioning behavior, suggesting how their compositions could be controlled by levels of PML SUMOylation or cellular mRNA concentration, respectively. The data suggest a conceptual framework for considering the composition and control thereof of cellular bodies assembled through heterotypic multivalent interactions.

Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micron- or submicron-sized clusters. However, the functional consequences of such clustering has been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phoshophorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases, and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling.

Highlights d Chromatin undergoes liquid-liquid phase separation (LLPS) under physiologic conditions d Linker DNA length and patterning, histone H1, and acetylation modulate chromatin LLPS d Acetylated chromatin only phase separates upon binding multi-bromodomain proteins d LLPS could enable establishment and maintenance of distinct chromatin compartments

The successful design of nanofluidic devices for the manipulation of biopolymers requires an understanding of how the predictions of soft condensed matter physics scale with device dimensions. Here we present measurements of DNA extended in nanochannels and show that below a critical width roughly twice the persistence length there is a crossover in the polymer physics. DOI: 10.1103/PhysRevLett.94.196101 PACS numbers: 81.16.Nd, 82.35.Lr, 82.39.Pj Top-down approaches to nanotechnology have the potential to revolutionize biology by making possible the construction of chip-based devices that can not only detect and separate single DNA molecules by size [1-4] but also-it is hoped in the future-actually sequence at the single molecule level [5]. While a number of top-down approaches have been proposed, all these approaches have in common the confinement of DNA to nanometer scales, typically 5-200 nm. Confinement alters the statistical mechanical properties of DNA. A DNA molecule in a nanochannel will extend along the channel axis to a substantial fraction of its full contour length [1,6]. Moreover, confinement is expected to alter the Brownian dynamics of the confined molecule [1]. While the study of confined DNA is interesting from a physics perspective, it is also critical for device design, potentially leading to new applications of nanoconfinement (for example, the use of nanochannels to prestretch and stabilize DNA before threading through a nanopore [5]). Moreover, available models [7][8][9][10][11] and simulations [12,13] are unable to account for the effect of varying confinement over the entire range of scales used in nanodevices. The theory gives asymptotic results valid only in limits that are not necessarily compatible with device requirements [1].Consider a DNA molecule of contour length L, width w, and persistence length P confined to a nanochannel of width D with D less than the radius of gyration of the molecule. When D P, the molecule is free to coil in the nanochannel and the elongation is due entirely to excluded volume interactions between segments of the polymer greatly separated in position along the backbone (see Fig. 1). de Gennes developed a scaling argument for the average extension of a confined self-avoiding polymer [8,12] which was later generalized by Schaefer and Pincus to the case of a persistent self-avoiding polymer [14]. The de Gennes theory predicts an extension r that scales with D in the following way:If the aspect ratio of the channel is not unity, i.e., the width D D 1 does not equal the depth D 2 , then Eq. (1) is still valid provided that D is replaced by the geometric average of the dimensions. As the channel width drops below the persistence length, the physics is dominated not by excluded volume but by the interplay of confinement and intrinsic DNA elasticity. In the strong confinement limit D P, backfolding is energetically unfavorable and contour length is stored exclusively in deflections made by the polymer with the walls. These deflections occur on average over th...

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.