Untangling the Molecular Interactions Underlying Intracellular Phase Separation Using Combined Global Sensitivity Analyses.

Liquid-liquid phase separation is an intracellular mechanism by which molecules, usually proteins and RNAs, interact and then rapidly demix from the surrounding matrix to form membrane-less compartments necessary for cellular function. Occurring in both the cytoplasm and the nucleus, properties of the resulting droplets depend on a variety of characteristics specific to the molecules involved, such as valency, density, and diffusion within the crowded environment. Capturing these complexities in a biologically relevant model is difficult. To understand the nuanced dynamics between proteins and RNAs as they interact and form droplets, as well as the impact of these interactions on the resulting droplet properties, we turn to sensitivity analysis. In this work, we examine a previously published mathematical model of two RNA species competing for the same protein-binding partner. We use the combined analyses of Morris Method and Sobol' sensitivity analysis to understand the impact of nine molecular parameters, subjected to three different initial conditions, on two observable LLPS outputs: the time of phase separation and the composition of the droplet field. Morris Method is a screening method capable of highlighting the most important parameters impacting a given output, while the variance-based Sobol' analysis can quantify both the importance of a given parameter, as well as the other model parameters it interacts with, to produce the observed phenomena. Combining these two techniques allows Morris Method to identify the most important dynamics and circumvent the large computational expense associated with Sobol', which then provides more nuanced information about parameter relationships. Together, the results of these combined methodologies highlight the complicated protein-RNA relationships underlying both the time of phase separation and the composition of the droplet field. Sobol' sensitivity analysis reveals that observed spatial and temporal dynamics are due, at least in part, to high-level interactions between multiple (3+) parameters. Ultimately, this work discourages using a single measurement to extrapolate the value of any single rate or parameter value, while simultaneously establishing a framework in which to analyze and assess the impact of these small-scale molecular interactions on large-scale droplet properties.