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...

The endocytic pathway of eukaryotes is essential for the internalization and trafficking of macromolecules, fluid, membranes, and membrane proteins. One of the most enigmatic aspects of this process is endocytic recycling, the return of macromolecules (often receptors) and fluid from endosomes to the plasma membrane. We have previously shown that the EH-domain protein RME-1 is a critical regulator of endocytic recycling in worms and mammals. Here we identify the RAB-10 protein as a key regulator of endocytic recycling upstream of RME-1 in polarized epithelial cells of the Caenorhabditis elegans intestine. rab-10 null mutant intestinal cells accumulate abnormally abundant RAB-5-positive early endosomes, some of which are enlarged by more than 10-fold. Conversely most RME-1-positive recycling endosomes are lost in rab-10 mutants. The abnormal early endosomes in rab-10 mutants accumulate basolaterally recycling transmembrane cargo molecules and basolaterally recycling fluid, consistent with a block in basolateral transport. These results indicate a role for RAB-10 in basolateral recycling upstream of RME-1. We found that a functional GFP-RAB-10 reporter protein is localized to endosomes and Golgi in wild-type intestinal cells consistent with a direct role for RAB-10 in this transport pathway. INTRODUCTIONEndocytosis and endocytic trafficking controls the uptake and sorting of extracellular macromolecules as well as the components of the cell membrane itself, counterbalancing secretion and allowing a complex interplay between cells and their environment that is important for a myriad of cellular activities (Brodsky et al., 2001;Maxfield and McGraw, 2004). The steps involved in the uptake and trafficking of cargo within the endosomal system have been well described, but many of the components mediating these steps at the molecular level remain to be identified (Brodsky et al., 2001;Maxfield and McGraw, 2004). Many receptors and their associated ligands cluster into clathrincoated pits, whereas other types of cargo utilize clathrinindependent uptake mechanisms (Nichols, 2003;Gesbert et al., 2004). Plasma membrane invaginations pinch off into vesicles, uncoat, and then fuse with one another and with early endosomes. In early endosomes some ligand-receptor complexes dissociate because of the reduced pH of the endosomal lumen (Mukherjee et al., 1997). Many receptors then recycle to the plasma membrane (PM) either directly or indirectly via recycling endosomes (Mukherjee et al., 1997;Maxfield and McGraw, 2004). Many ligands do not recycle but instead are transported from early to late endosomes and eventually to lysosomes for degradation (Mukherjee et al., 1997). In polarized epithelial cells such as cultured Madin-Darby canine kidney (MDCK) cells, an additional layer of complexity in the endocytic pathway contributes to formation and/or maintenance of the specialized apical and basolateral domains (Nelson and Yeaman, 2001;Mostov et al., 2003;Hoekstra et al., 2004). Both the apical and basolateral membranes deliver cargo ...

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. Here we show binding of RAB-10 to EHBP-1, a CH-domain protein, and demonstrate a requirement for EHBP-1 in RAB-10–regulated transport in both of these tissues.

This work provides the first demonstration of a requirement for an Alix/Bro1p family member in the endocytic recycling pathway in association with the recycling regulator RME-1.