P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA-protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA-protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.liquid droplets | intracellular phase transition | intrinsically disordered proteins | RNA granules I ntracellular RNA/protein (RNP) assemblies, including germ granules, processing bodies, stress granules, and nucleoli, are key players in the regulation of gene expression (1). RNP bodies, also referred to as RNA granules, function in diverse modes of RNA processing, including splicing, degradation, and translational repression of mRNA. These ubiquitous structures lack a membrane boundary but nonetheless represent a coherent organelle composed of thousands of molecules, manifesting as microscopically visible puncta in both the cytoplasm and the nucleus.Recent studies have demonstrated the apparent liquid-like behavior of various RNP bodies (2-5) including wetting, dripping, and relaxation to spherical structures upon fusion or shearing. The assembly and disassembly of liquid-like organelles appears to be governed by a phase separation process, demonstrated by a concentration-dependent condensation/dissolution of P granules (2, 6) and the assembly and size scaling of the nucleolus (7) in the Caenorhabditis elegans embryo. Liquid phase separation has also been suggested to play a role in stress granule assembly (4) and in multivalent signaling proteins (8). These studies lend increasing support to the hypothesis that liquid phases play a central role in intracellular organization. However, the specific molecular interactions that drive phase separation and the mechanisms by which liquid properties impart cellular function remain largely unclear. P granule...

Summary Compartmentalization in cells is central to the spatial and temporal control of biochemistry. In addition to membrane-bound organelles, membrane-less compartments form partitions in cells. Increasing evidence suggests that these compartments assemble through liquid-liquid phase separation. However the spatiotemporal control of their assembly, and how they maintain distinct functional and physical identities is poorly understood. We have previously shown an RNA-binding protein with a polyQ-expansion called Whi3 is essential for the spatial patterning of cyclin and formin transcripts in cytosol. Here, we show that specific mRNAs that are known physiological targets of Whi3 drive phase separation. mRNA can alter the viscosity of droplets, their propensity to fuse, and the exchange rates of components with bulk solution. Different mRNAs impart distinct biophysical properties of droplets indicating mRNA can bring individuality to assemblies. Our findings suggest that mRNAs can encode not only genetic information, but also the biophysical properties of phase-separated compartments.

Many intracellular membraneless organelles form via phase separation of intrinsically disordered proteins (IDPs) or regions (IDRs). These include the Caenorhabditis elegans protein LAF-1, which forms P granule-like droplets in vitro. However, the role of protein disorder in phase separation and the macromolecular organization within droplets remain elusive. Here, we utilize a novel technique, ultrafast-scanning fluorescence correlation spectroscopy, to measure the molecular interactions and full coexistence curves (binodals), which quantify the protein concentration within LAF-1 droplets. The binodals of LAF-1 and its IDR display a number of unusual features, including 'high concentration' binodal arms that correspond to remarkably dilute droplets. We find that LAF-1 and other in vitro and intracellular droplets are characterized by an effective mesh size of ∼3-8 nm, which determines the size scale at which droplet properties impact molecular diffusion and permeability. These findings reveal how specific IDPs can phase separate to form permeable, low-density (semi-dilute) liquids, whose structural features are likely to strongly impact biological function.

Liquid phase separation into two or more coexisting phases has emerged as a new paradigm for understanding subcellular organization, prebiotic life, and the origins of disease. The design principles underlying biomolecular phase separation have the potential to drive the development of novel liquid-based organelles and therapeutics, however, an understanding of how individual molecules contribute to emergent material properties, and approaches to directly manipulate phase dynamics are lacking. Here, using microrheology, we demonstrate that droplets of poly-arginine coassembled with mono/polynucleotides have approximately 100 fold greater viscosity than comparable lysine droplets, both of which can be finer tuned by polymer length. We find that these amino acid-level differences can drive the formation of coexisting immiscible phases with tunable formation kinetics and can be further exploited to trigger the controlled release of droplet components. Together, this work provides a novel mechanism for leveraging sequence-level components in order to regulate droplet dynamics and multiphase coexistence.

The aggregation and deposition of normally soluble proteins is the hallmark of several devastating neurodegenerative disorders. For proteins such as tau in Alzheimer’s disease and α-synuclein in Parkinson’s disease, aggregation involves a transition from an intrinsically disordered monomer to a highly structured fiber. While understanding the role of these proteins in neurodegeneration requires elucidation of the structural basis of self-association, the conformational heterogeneity of disordered proteins makes their structural characterization inherently challenging. Here we use single molecule Förster resonance energy transfer to measure the conformational ensemble of tau in the absence and presence of heparin to identify critical conformational changes relevant to the initiation of aggregation. We find that different domains of tau display distinct conformational properties that are strongly correlated with their degree of disorder and which may relate to their roles in aggregation. Moreover, we observe that heparin binding induces a distinct two-state structural transition in tau described by a loss of long-range contacts and a concomitant compaction of the microtubule binding domain. Our results describe a conformational intermediate of tau that precedes the formation of aggregates and could serve as a target for tau-focused therapeutics.

Intrinsically disordered proteins (IDPs) are increasingly recognized for their important roles in a range of biological contexts, both in normal physiological function and in a variety of devastating human diseases. However, their structural characterization by traditional biophysical methods, for the purposes of understanding their function and dysfunction, has proved challenging. Here, we investigate the model IDPs α-Synuclein (αS) and tau, that are involved in major neurodegenerative conditions including Parkinson's and Alzheimer's diseases, using excluded volume Monte Carlo simulations constrained by pairwise distance distributions from single-molecule fluorescence measurements. Using this, to our knowledge, novel approach we find that a relatively small number of intermolecular distance constraints are sufficient to accurately determine the dimensions and polymer conformational statistics of αS and tau in solution. Moreover, this method can detect local changes in αS and tau conformations that correlate with enhanced aggregation. Constrained Monte Carlo simulations produce ensembles that are in excellent agreement both with experimental measurements on αS and tau and with all-atom, explicit solvent molecular dynamics simulations of αS, with much lower configurational sampling requirements and computational expense.

Phase separation of biomolecules leading to the formation of assemblies with distinct material properties has recently emerged as a new paradigm underlying subcellular organization. The discovery that disordered proteins, long associated with aggregation in neurodegenerative disease, are also implicated in driving liquid phase separation has galvanized significant interest in exploring the relationship between misregulated phase transitions and disease. This review summarizes recent work linking liquid phase separation to neurodegeneration, highlighting a pathological role for altered phase behavior and material properties of proteins assembled via liquid phase separation. The techniques that recent and current work in this area have deployed are also discussed, as is the potential for these discoveries to promote new research directions for investigating the molecular etiologies of neurodegenerative diseases.

Tau is a microtubule associated protein whose aggregation is implicated in a number of neurodegenerative diseases. We investigate the mechanism by which anionic lipid vesicles induce aggregation of tau in vitro using K18, a fragment of tau corresponding to the four repeats of the microtubule binding domain. Our results show that aggregation occurs when the amount of K18 bound to the lipid bilayer exceeds a critical surface density. The ratio of protein/lipid at the critical aggregation concentration is pH-dependent, as is the binding affinity. At low pH, where the protein binds with high affinity, the critical surface density is independent both of total lipid concentration as well as the fraction of anionic lipid present in the bilayer. Furthermore, the aggregates consist of both protein and vesicles and bind the beta-sheet specific dye, Thioflavin T, in the manner characteristic of pathological aggregates. Our results suggest that the lipid bilayer facilitates protein-protein interactions both by screening charges on the protein and by increasing the local protein concentration, resulting in rapid aggregation. Because anionic lipids are abundant in cellular membranes, these findings contribute to understanding tau-lipid bilayer interactions that may be relevant to disease pathology.