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

本项目将开发一种微流控PhaseDisplayChip方法，用于绘制肿瘤抑制因子SPOP及其底物在液态核体中的酶活性和组成控制。近年来，通过液相分离形成的各种无膜体已被描述。虽然液相分离体的许多组分被鉴定并在体外成功地重构，但定义体组成和功能的一般原理难以通过实验证实。对于动态方面，例如液-液相分离的早期和后期阶段或滞后、分子有序化和重组动力学以及相分离体内酶活性的定量，情况尤其如此。此外，几个相分离的身体可能共存，酶和它们的底物可能会分成多个身体，提出了身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。本项目将通过开发PhaseDisplayChip方法来克服这些实验挑战，处理数千毫微微升大小的生物像素，每个像素都包含定义成分和温度的液相分离反应。每个生物像素的温度和组成都可以在整个实验中进行调节，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将样品消耗量降低两到三个数量级，每个相图的总体积降低到约10微升。同时，与机器人移液方法相比，每个所得相图（“像素”分辨率）的测量数据点数量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（斑点型POZ蛋白）功能来验证。SPOP是cullin 3-RING泛素连接酶的底物适配器，并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。通过使用我们的PhaseDisplayChip进行筛选分析，我们寻求确定改变SPOP和底物定位或稳定性的非平衡调节机制及其对相分离的影响。这将进一步了解底物介导的相分离如何激活SPOP和潜在的泛素连接酶，从而为获得和调节泛素依赖性蛋白质稳态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物，以逆转SPOP癌症突变中的异常相变动力学，从而获得新的治疗策略。

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.