Phase separation & interfacial phenomena of a model biomolecular condensate

The organization of intracellular space into distinct, membraneless, compartments is a ubiquitous feature of living organisms. It has recently become apparent that many of these biomolecular condensates may be formed by liquid-liquid phase separation. Representing (liquid-like) regions enriched in specific proteins and nucleic acid species, they partition biomolecules by providing a chemical environment distinct from the surrounding intracellular milieu. Regulation of their interfacial phenomena allows cells to localise them to specific regions of intracellular space, as well as control the kinetics of their formation and growth.
Through identifying and studying constituent proteins of biomolecular condensates, researchers have deciphered many of the sequence features necessary for phase separation. Of principal importance was the development of model biomolecular condensate systems in vitro. This has enabled rigourous physical modelling in a controlled environment, allowing investigations into the molecular specifics driving condensate formation through phase separation. In this thesis, we advance the study of biomolecular condensates, basing our research around the Ddx4N model system focusing on condensate formation and interfacial phenomena.
Ddx4N is the intrinsically disordered N-terminal tail of human ATP-dependent DEADbox RNA helicase 4, shown to be necessary and sufficient for phase separation in vitro and in cells. Since its discovery as a condensate forming protein, its sequence features have been identified in numerous other condensate associated proteins, making Ddx4N an ideal model system. First, we expand current knowledge concerning its phase separation. By investigating mutant sequences in vitro, the the role of protein net charge is revealed, whilst also emphasising the effect of phenylalanine/tyrosine on cation-π interactions. Furthermore, we show the effect of net charge is reproducible in a cellular environment, using in cell fluorescence images of exogenous Ddx4N condensates.
Our attention is then drawn to the development of an experimental pipeline affording the precise characterisation of in vitro condensate interfacial phenomena through drop shape analysis. Confocal microscopy, the Young-Laplace equation and bespoke image analysis are combined to allow measurement of capillary lengths and contact angles of condensates resting on solid surfaces. Measuring condensate contact angles using our automated technique is found to be superior to a recently reported manual method.
We then focus on the fluid-fluid interfacial tension of condensates, presenting calculations based around the theory of van der Waals, implemented through the work of Scheutjens and Fleer. Then we aimed to verify these calculations by measuring condensate capillary lengths through drop shape analysis, with a view to extracting their fluid-fluid interfacial tensions. However, we saw an unexpected increase of capillary length with condensate size. After close interrogation of the experimental technique and possible physical origins, we were unable to and an explanation for this phenomena. As such, the origin of our observed positive relationship between capillary length and condensate size remains an open question, requiring further study.
Finally, motivated by the observation that membrane-associated condensates are localised through specific interactions with membrane bound species, we decided to investigate the non-specific wetting of membrane surfaces by biomolecular condensates. Through measurement of contact angles on a supported lipid bilayer, we found condensates to be repelled by the membrane. Further measurements of condensate contact angles on hydrophilic and hydrophobic glass surfaces revealed a role for hydrophobic interactions, likely serving to repel condensates from necessarily hydrophilic headgroups of lipid bilayers. These data suggest repulsion between biomolecular condensates and membranes is responsible for the maintenance of endogenous condensates in the cytoplasm/nucleoplasm, away from intracellular membranes. Furthermore, we discover a positive line tension at the three phase contact line, with a deleterious impact on condensate wetting at the micrometer lengthscale.

Keywords: phase separation
biomolecular condensate
Advisor: Nott, TJ
Aarts, D