Biology subjects

Welsh, T. J.

Publications and source records attributed to Welsh, T. J..

3 recordsLinked to original sources

bioRxiv2020

Rapid characterisation of protein phase behaviour using droplet microfluidics

The assembly of intracellular proteins into biomolecular condensates is a fundamental process underlying the organisation of intracellular space and the regulation of many cellular processes. Mapping and characterising phase behaviour of biomolecules is essential to understand the mechanisms of condensate assembly, and to develop therapeutic strategies targeting biomolecular condensate systems. A central concept for characterising phase-separating systems is the phase diagram. Phase diagrams are typically built from numerous individual measurements sampling different parts of the parameter space. However, even when performed in microwell plate format, this process is slow, low throughput and requires significant sample consumption. To address this challenge, we present here a combinatorial droplet microfluidic platform, termed PhaseScan, for rapid and high-resolution acquisition of multidimensional biomolecular phase diagrams. Using this platform, we characterise the phase behaviour of a wide range of systems under a variety of conditions and demonstrate that this approach allows the quantitative characterisation of the effect of small molecules on biomolecular phase transitions.

biophysics↗

bioRxiv2020

Reentrant liquid condensate phase of proteins is stabilized by hydrophobic and non-ionic interactions

Many cellular proteins demix spontaneously from solution to form liquid condensates. These phase-separated systems have wide-ranging roles in health and disease. Elucidating the molecular driving forces underlying liquid-liquid phase separation (LLPS) is therefore a key objective for understanding biological function and malfunction. Here we show that proteins implicated in cellular LLPS, including FUS, TDP-43, Brd4, Sox2, and Annexin A11, which form condensates at low salt concentrations, can reenter a phase-separated regime at high salt concentrations. By bringing together experiments and simulations, we demonstrate that phase separation in the high-salt regime is driven by hydrophobic and non-ionic interactions, and is mechanistically distinct from the low-salt regime, where condensates are additionally stabilized by electrostatic forces. Our work thus provides a new view on the cooperation of hydrophobicity and non-ionic interactions as non-specific driving forces for the condensation process, with important implications for aberrant function, druggability, and material properties of biomolecular condensates.

biophysics↗

bioRxiv2020

Single particle zeta-potential measurements reveal the role of electrostatics in protein condensate stability

Liquid-liquid phase separation underlies the formation of biological condensates. Physically, such systems are microemulsions which have a general propensity to fuse and coalesce; however, many condensates persist as independent droplets inside cells. This stability is crucial for their functioning, but the physicochemical mechanisms that control the emulsion stability of condensates remain poorly understood. Here, by combining single-condensate zeta potential measurements, optical microscopy, tweezer experiments, and multiscale molecular modelling, we investigate how the forces that sustain condensates impact their stability against fusion. By comparing PR25:PolyU and FUS condensates, we show that a higher condensate surface charge correlates with a lower fusion propensity, and that this behavior can be inferred from their zeta potentials. We reveal that overall stabilization against fusion stems from a combination of repulsive forces between condensates and the effects that surface electrostatics have on lowering surface tension, thus shedding light on the molecular determinants of condensate coalescence.

biophysics↗