The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

Abstract

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...

Tau protein liquid–liquid phase separation can initiate tau aggregation

“…Intracellular LLPS and the resulting functional membrane‐less organelles, which biophysically can be described as gels or polyelectrolyte brushes, are emerging concepts in cell biology, which are now implicated in manifold cellular functions and applications (Li et al , 2012; Aumiller et al , 2014; Elbaum‐Garfinkle et al , 2015; Jiang et al , 2015; Aguzzi & Altmeyer, 2016; Mitrea et al , 2016; Schmidt & Görlich, 2016; Schmidt & Rohatgi, 2016). For research in neurodegenerative diseases, in particular, the function and misfunction of LLPS by involved proteins—such as tau, FUS, TDP43, hnRNPA1, C9orf72 dipeptide repeats—introduces a novel exciting concept that may provide a common underlying mechanism in these diseases.…”

RNA transcription modulates phase transition-driven nuclear body assembly

“…Many nucleolar proteins, including FIB-1 and DAO-5, contain intrinsically disordered domains that are thought to promote assembly of RNA/protein droplets (2,5,8). To determine whether self-interactions between nucleolar components are sufficient for droplet assembly, we purified recombinant FIB-1 and examined its behavior in vitro.…”

“…The mechanisms by which such structures form and stably persist are not well understood. However, recent evidence suggests that these membraneless organelles, such as P granules (1,2), nucleoli (3), and stress granules (4), are liquid-phase droplets that may assemble by intracellular phase separation (5,6). This concept is supported by work on synthetic systems, including repetitive protein domains that form droplets in vitro and in the cytoplasm (7) and intrinsically disordered protein domains that show signatures of liquid-liquid phase separation when expressed in the cell nucleus (8).…”

Linear viscoelasticity of complex coacervates

“…For these materials, which are often termed "membraneless organelles," the quantity of material, as well as the need to extract such cellular granules from their native environment prevent the application of the types of large-scale SAOS measurements described thus far. However, Elbaum-Garfinkle et al, recently reported the use of microrheology techniques to coacervate-like materials formed as a result of the self-interaction of LAF-1, a DDX3 RNA helicase found in the P granules of Caenorhabditis elegans [190]. Here, confocal fluorescence microscopy was used in conjunction with particle tracking methodologies to measure the viscosity of coacervate-like droplets of LAF-1 both with and without RNA.…”