High Resolution Biomolecular Condensate Phase Diagrams with a Combinatorial Microdroplet Platform

Abstract

The assembly of intracellular proteins into biomolecular condensates via liquid-liquid phase separation (LLPS) has emerged as a fundamental process underlying the organisation and regulation of cellular space and function. Physicochemical characterisation of the parameters that control and modulate phase separation is therefore essential for an improved understanding of protein phase behaviour, including for the therapeutic modulation of LLPS phenomena. A fundamental measure with which to describe protein phase behaviour in chemical space is the phase diagram. Characterisation of phase diagrams requires measuring the presence or absence of the condensed phase under a multitude of conditions and, as such, is associated with significant consumption of time and sample volume even when performed in microwell format.However, due to the rapidly increasing number of biologically and disease-relevant condensate systems, experimental techniques that enable high-throughput analysis of protein phase behaviour are required. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for the rapid and high-resolution acquisition of protein phase diagrams. Using this platform, we demonstrate characterisation of the phase behaviour of a pathologically relevant mutant of the protein fused in sarcoma (FUS) in a highly parallelised manner, with significantly improved assay throughput and reduced sample consumption. We demonstrate the capability of the platform by finding the phase boundary at which FUS transitions from a one-phase to a two-phase state as modulated by 1,6-hexanediol, and estimate the free-energy landscape of this system using Flory-Huggins theory. These results thus provide a basis for the rapid acquisition of phase diagrams through the application of microdroplet techniques and pave the way for a wide range of applications, enabling rapid characterisation of the effect of environmental conditions and coacervate species on the thermodynamics of phase separation.

Rotavirus Replication Factories Are Complex Ribonucleoprotein Condensates

“…Both rotavirus replication factories and NSP5/NSP2 condensates were rapidly and reversibly dissolved in the presence of small aliphatic alcohols, including 1,6hexanediol, as well as lower molecular weight propylene diols, corroborating their liquid-like properties. We have validated our findings by employing a combinatorial droplet microfluidic platform, termed PhaseScan 19 , to characterise the phase behaviour of the NSP5/NSP2 condensates and mapped out the phase boundary, at which they transition from a mixed one-phase, to a two-phase demixed state.…”

“…Phase diagram generation. Phase diagrams were produced using droplet microfluidics in a similar manner to that described previously 19 . Syringe pumps (neMESYS modules, Cetoni) were used to control flows of protein solutions, consisting of 22 μM NSP5 supplemented with 6.4 μM Alexa647 dye (carboxylic acid, ThermoFisher) or 8 μM Histagged NSP2 labelled with 8 μM Atto488-nitrilotriacetic acid (NTA, Sigma), and buffer (0.5 × phosphate saline buffer, PBS, pH 7.4).…”

Microfluidic characterization of macromolecular liquid–liquid phase separation

“…Knowles and coworkers adopted differential encapsulation to formulate phase separating emulsion droplets that span a wide range of solution conditions. 48 The droplets were collected and analyzed by epifluorescence microscopy for being in the one-phase or two-phase regime. In this approach, coexistence curves are not directly measured but rather inferred by scanning the solution conditions that gave rise to LLPS.…”

Microfluidic characterization of macromolecular liquid-liquid phase separation

“…Several other groups have recently used microfluidics approaches to map biomolecular phase diagrams. Knowles and coworkers adopted differential encapsulation to formulate phase separating emulsion droplets that span a wide range of solution conditions 48 . The droplets were collected and analyzed by epifluorescence microscopy for being in the one-phase or two-phase regime.…”