Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands.
Institute for Molecules and Materials, Radboud University, Nijmegen, The Netherlands.
Methods Enzymol. 2021;646:353-389. doi: 10.1016/bs.mie.2020.06.007. Epub 2020 Jul 27.
The discovery of membraneless organelles (MLOs) formed by liquid-liquid phase separation raised many questions about the spatial organization of biomolecular processes in cells, but also offered a new tool to mimic cellular media. Since disordered and charged protein domains are often necessary for phase separation, coacervates can be used as models both to understand MLO regulation and to develop dynamic cellular-like compartments. A versatile way to turn passive coacervate droplets into active and dynamic compartments is by introducing enzymatic reactions that affect parameters relevant for complex coacervation, such as the charge and length of the components. However, these reactions strictly take place in a heterogeneous medium, and the complexity thereof is hardly addressed, making it difficult to achieve true control. In this chapter we help close this gap by describing two coacervate systems in which enzymatic reactions endow coacervate droplets with a dynamic character. We further highlight the technical challenges posed by the two-phase systems and strategies to overcome them.
无膜细胞器（MLOs）的发现引发了许多关于细胞中生物分子过程空间组织的问题，但也为模拟细胞介质提供了新工具。由于无序和带电的蛋白质结构域通常是相分离所必需的，凝聚相可以作为模型来理解 MLO 的调控，并开发具有动态类似细胞的隔室。将被动凝聚相液滴转变为主动和动态隔室的一种多功能方法是引入影响复杂凝聚相关参数的酶反应，例如组分的电荷和长度。然而，这些反应严格发生在非均相介质中，并且其复杂性难以解决，因此难以实现真正的控制。在本章中，我们通过描述两种凝聚相系统来帮助弥补这一差距，在这两种系统中，酶反应赋予凝聚相液滴以动态特性。我们进一步强调了由两相系统带来的技术挑战，并提出了克服这些挑战的策略。
