Journal Article

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

Myong, Sua, Elbaum-Garfinkle, Shana, Kim, Younghoon, Brangwynne, Clifford P., Eckmann, Christian R., Chen, Carlos Chih-Hsiung, Szczepaniak, Krzysztof

2015

Overview

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. Significance Phase transitions have recently emerged as a key mechanism for intracellular organization. However, the underlying molecular interactions and nature of the resulting condensed phases are poorly understood. Here, we identify a role for LAF-1 in the liquid phase separation of P granules—RNA/protein assemblies implicated in germ-line maintenance. We adapt microrheology techniques to measure precise viscoelastic properties of LAF-1 liquid droplets. Our experiments reveal that electrostatic disordered protein interactions give rise to droplets with tunable material properties. RNA can fluidize protein droplets by decreasing the viscosity and increasing internal molecular dynamics. Our results provide insight into the mechanism by which molecular level interactions can give rise to liquid phase organelles with tunable material properties, potentially underlying biologically adaptable functions.

• Publisher: National Academy of Sciences,National Acad Sciences
• Subject: Animals / Bacteria / Biological Sciences / Caenorhabditis elegans - genetics / Caenorhabditis elegans - physiology / Caenorhabditis elegans Proteins - chemistry / Caenorhabditis elegans Proteins - physiology / droplets / Fluidization / molecular dynamics / organelles / phase transition / Proteins / Ribonucleic acid / RNA / RNA Helicases - chemistry / RNA Helicases - physiology / RNA, Helminth - chemistry / RNA-protein interactions / separation / viscoelasticity / Viscosity / Water vapor