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

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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.