Development of a Lab-on-a-Chip for the Study of Protein Liquid-Liquid Phase Transitions

Abstract

Phase transitions into liquid-like states have recently emerged as a key process underlying intracellular organization. The cell utilizes these phase separated liquid bodies to localize reactants essential to cellular function. For example, non-membrane bound RNA and protein (RNP) bodies play a central role in RNA processing. A key component of these RNP bodies is RNA helicases. A purified RNA helicase, LAF-1, exhibits a salt and protein concentration dependent phase separation in vitro resulting in liquid-like droplets, suggesting that it can be considered a synthetic model for RNP bodies. This study investigated such synthetic RNPs with the goal of elucidating the biophysics of RNP assembly. To accomplish this, a custom microfluidic device was developed to measure the nucleation and growth kinetics of these droplets. Protein droplet nucleation and growth can be observed and controlled through the use of oil/water emulsions. My results provide a novel platform for addressing the factors that affect non-membrane bound organelle assembly. While the optimized microfluidic device developed allows for the creation and storage of aqueous droplets of phase-separated solution, we suggest ways to optimize the device for further studies. These modifications will allow for the control of confounding concentration effects, enabling mechanistic studies on protein liquid droplets to be completed.