Falahati Hanieh, Wieschaus Eric
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, NJ 08544;
Proc Natl Acad Sci U S A. 2017 Feb 7;114(6):1335-1340. doi: 10.1073/pnas.1615395114. Epub 2017 Jan 23.
Membraneless organelles play a central role in the organization of protoplasm by concentrating macromolecules, which allows efficient cellular processes. Recent studies have shown that, in vitro, certain components in such organelles can assemble through phase separation. Inside the cell, however, such organelles are multicomponent, with numerous intermolecular interactions that can potentially affect the demixing properties of individual components. In addition, the organelles themselves are inherently active, and it is not clear how the active, energy-consuming processes that occur constantly within such organelles affect the phase separation behavior of the constituent macromolecules. Here, we examine the phase separation model for the formation of membraneless organelles in vivo by assessing the two features that collectively distinguish it from active assembly, namely temperature dependence and reversibility. We use a microfluidic device that allows accurate and rapid manipulation of temperature and examine the quantitative dynamics by which six different nucleolar proteins assemble into the nucleoli of Drosophila melanogaster embryos. Our results indicate that, although phase separation is the main mode of recruitment for four of the studied proteins, the assembly of the other two is irreversible and enhanced at higher temperatures, behaviors indicative of active recruitment to the nucleolus. These two subsets of components differ in their requirements for ribosomal DNA; the two actively assembling components fail to assemble in the absence of ribosomal DNA, whereas the thermodynamically driven components assemble but lose temporal and spatial precision.
无膜细胞器通过浓缩大分子在原生质组织中发挥核心作用，这使得细胞过程高效进行。最近的研究表明，在体外，此类细胞器中的某些成分可通过相分离进行组装。然而，在细胞内部，此类细胞器是多组分的，存在众多分子间相互作用，这可能会影响单个成分的相分离特性。此外，细胞器本身具有内在活性，目前尚不清楚在这些细胞器中持续发生的活跃的、耗能过程如何影响组成大分子的相分离行为。在这里，我们通过评估将其与主动组装区分开来的两个特征，即温度依赖性和可逆性，来研究体内无膜细胞器形成的相分离模型。我们使用一种微流控装置，该装置能够精确且快速地控制温度，并研究六种不同的核仁蛋白组装到黑腹果蝇胚胎核仁中的定量动力学。我们的结果表明，虽然相分离是四种被研究蛋白质募集的主要模式，但另外两种蛋白质的组装是不可逆的，并且在较高温度下会增强，这些行为表明它们是被主动募集到核仁中的。这两组成分对核糖体DNA的需求不同；在没有核糖体DNA的情况下，两种主动组装的成分无法组装，而由热力学驱动的成分能够组装，但会失去时间和空间上的精确性。
