Microfluidic analysis of multicomponent phase separation in ubiquitin-dependent proteostasis.

本项目将开发一种微流控相显示芯片方法来定位肿瘤抑制因子SPOP及其底物在液体核体中的酶活性和组成控制。近年来，人们描述了通过液体相分离形成的各种无膜体。虽然在体外鉴定并成功地重建了许多液体分离的物体的成分，但定义物体组成和功能的一般原理很难在实验上表征。尤其是动态方面，如液-液相分离的早期和后期或滞后、分子有序化和重组动力学，以及相分离体内酶活性的量化。此外，几个相分离的物体可能共存，酶及其底物可能会分割成多个物体，这就提出了物体之间的这种重新分配如何影响功能以及如何调节的问题。这个项目将通过开发一种相位显示芯片方法来处理数千个飞升大小的生物像素，每个像素包含一个定义的组成和温度的液体相分离反应，从而克服这些实验挑战。在整个实验过程中，每个生物像素的温度和组成都可以调整，以允许可逆地询问相分离动力学及其对酶活性的影响。与传统方法相比，我们的PhaseDisplayChip将使每个相图的样品消耗减少两到三个数量级，降低到大约10微升的总体积。同时，与机器人移液进样方法相比，每个得到的相图的测量数据点的数量(像素分辨率)将增加两到三个数量级。PhaseDisplayChip方法将通过深入分析与其底物的相分离如何触发肿瘤抑制因子SPOP(斑点类型POZ蛋白)功能来验证。SPOP是cullin3-环泛素连接酶的底物接头，定位于细胞核中含有无膜体的几种底物中，在那里它介导泛素化活性。通过我们的PhaseDisplayChip的筛选分析，我们试图确定改变SPOP和底物局部化或稳定性的非平衡调节机制及其对相分离的影响。这将为底物介导的相分离如何激活SPOP和潜在的泛素连接酶提供进一步的见解，从而为实现和调节泛素依赖的蛋白平衡提供一个共同的原则。这将使我们能够筛选液相分离动力学上的小分子效应器，以逆转SPOP癌症突变中的异常相变动力学，从而形成新的治疗策略。

This project will develop a microfluidic PhaseDisplayChip method to map enzymatic activity and compositional control of tumour suppressor SPOP with its substrates in liquid nuclear bodies.Various membraneless bodies that form through liquid phase separation have been described in recent years. While many components of liquid phase separated bodies were identified and successfully reconstituted in vitro, general principles that define body composition and function are difficult to characterise experimentally. This is in particular the case for dynamic aspects, such as early and later stages or hysteresis in liquid-liquid phase separation, molecular ordering and reorganisation dynamics, as well as quantification of enzymatic activities within phase separated bodies. Furthermore, several phase-separated bodies may co-exist and enzymes and their substrates may partition into multiple bodies, posing questions as to how such redistribution between bodies affects function and how it is regulated.This project will overcome these experimental challenges, by developing a PhaseDisplayChip method to process thousands of femtoliter sized bio-pixels, each containing a liquid phase separation reaction of defined composition and temperature. Both, temperature and composition of each bio-pixel can be adjusted throughout the experiment to allow for a reversible interrogation of phase separation dynamics and their resulting effects on enzymatic activities. Compared to conventional methods, our PhaseDisplayChip will decrease sample consumption by two to three orders of magnitude down to about 10 microliters total volume per phase diagram. Simultaneously the amount of measured datapoints per resulting phase diagrams (‘pixel’-resolution) will be increased by two to three orders of magnitude when compared to robotic pipetting approaches.The PhaseDisplayChip method will be validated through indepth analysis of how phase separation with its substrates triggers tumour suppressor SPOP (Speckle-type POZ protein) function. SPOP is a substrate adaptor of the cullin3-RING ubiquitin ligase and localises into several substrate containing membraneless bodies in the nucleus, where it mediates ubiquitination activity. Through screening assays with our PhaseDisplayChip we seek to identify non-equilibrium regulatory mechanisms that change SPOP and substrate localisation or stability and their influence on phase separation. This will provide further insights into how substrate mediated phase separation can activate SPOP and potentially ubiquitin ligases in general to provide a common principle for attaining and regulating ubiquitin-dependent proteostasis. This will allow us to screen for small molecule effectors on liquid-phase separation dynamics to revert aberrant phase transition dynamics in SPOP cancer mutations, en route to novel treatment strategies.