The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics
The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics
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DOI：
10.1073/pnas.1504822112
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发表时间：
2015-06-09
影响因子：
11.1
通讯作者：
Brangwynne, Clifford P.
中科院分区：
文献类型：
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作者：
Elbaum-Garfinkle, Shana;Kim, Younghoon;Brangwynne, Clifford P.
P颗粒和其他RNA/蛋白质体是无膜细胞器，可以通过细胞内相分离组装，类似于水蒸气冷凝成液滴。然而，分子驱动力和凝聚相的性质仍然知之甚少。在这里，我们表明，秀丽隐杆线虫蛋白LAF-1，在P颗粒中发现的DDX 3 RNA解旋酶，相分离成P颗粒样液滴在体外。我们采用微观流变学技术来精确测量微米级LAF-1液滴的粘弹性，揭示了盐和RNA浓度高度可调的纯粘性特性。RNA降低粘度并增加液滴内的分子动力学。单分子FRET分析表明，这种RNA流化的结果从高度动态的RNA-蛋白质相互作用，出现接近液滴相边界。我们证明了LAF-1的N-末端、富含精氨酸/甘氨酸的内在无序蛋白（IDP）结构域对于相分离和RNA-蛋白质相互作用是必要和充分的。在体内，RNAi敲低LAF-1导致早期胚胎中P颗粒的溶解，具有与体外测量的明显亚微摩尔相边界。总之，这些研究结果表明，LAF-1是重要的促进P颗粒组装，并提供洞察IDP驱动的分子相互作用引起的液相细胞器与可调性能的机制。
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.