Examining the Material Properties and Dynamics of Condensed RNA/Protein Phases

Abstract

Cells contain numerous membrane-less RNA/protein (RNP) bodies that appear to assemble by intracellular liquid-liquid phase separation. The properties of these condensed phase droplets are increasingly recognized as important in their physiological function, and also through the link to protein aggregation pathologies. However, there are limited techniques to measure the properties of model in vitro RNP droplet phases. In this dissertation, we introduce a microfluidic platform that drives protein droplets into a single large phase, which facilitates viscosity measurements using simultaneous passive microrheology and/or active two-phase flow analysis. We show this method is general for a variety of phase separating proteins and can be used in cases where droplets are too small for microrheology. Moreover, the ability to simultaneously perform active and passive rheology measurements enables characterizing the impact of ATP-dependent biological activity on RNP droplet properties, a key area for future research. Viscosity of RNP bodies is also predicted to affect the rate of protein diffusion, and therefore droplet function. Most studies use fluorescence recovery after photobleaching (FRAP) to estimate protein diffusion coefficients, however two commonly used models to fit FRAP data result in a factor of five difference in the measured diffusion coefficient. We suggest guidelines for determining the appropriate model and evaluate the impact of model choice on measured values. Moreover, we address how changes in experimental conditions (e.g., bleach shape) influence the measured diffusivity and demonstrate how to overcome this by selecting an appropriate dimensional model. Finally, we develop a model to describe FRAP of entire in vitro and in vivo droplet phases and compare measured diffusivities. The ability to accurately determine diffusion coefficients enables direct comparison to literature values and facilitates insights into the effect of diffusion rates on RNP body function, which is a key area for future research.