Biology subjects

Joseph, J. A.

Publications and source records attributed to Joseph, J. A..

3 recordsLinked to original sources

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↗

bioRxiv2020

Oligonucleotides can act as superscaffolds that enhance liquid-liquid phase separation of intracellular mixtures

Intracellular liquid-liquid phase separation (LLPS) enables the formation of biomolecular condensates, which play a crucial role in the spatiotemporal organisation of biomolecules (proteins, oligonucleotides). While LLPS of biopolymers has been demonstrated in both experiments and computer simulations, the physical determinants governing phase separation of protein-oligonucleotide systems are not fully understood. Here, we introduce a minimal coarse-grained model to investigate concentration-dependent features of protein-oligonucleotide mixtures. We demonstrate that adding oligonucleotides to biomolecular condensates composed of oligonucleotide-binding scaffold proteins enhances LLPS; since oligonucleotides act as ultra-high-valency molecules (termed superscaffolds) that increase the molecular connectivity among scaffold proteins. Importantly, we find that oligonucleotides promote protein LLPS via a seeding-type mechanism; recruiting numerous protein molecules and reducing the thermodynamic and kinetic barriers for nucleation and phase separation. By probing the conformational properties of oligonucleotides within droplets, we show that these biopolymers can undergo phase separation-driven compaction, which may be entropic in nature. Finally, we provide a quantitative comparison between mixture composition, protein valency, and protein-oligonucleotide interaction strengths. We find that superscaffolds preferentially recruit higher valency proteins to condensates, and that multiphase immiscibility within condensates can be achieved by modulating the relative protein-oligonucleotide binding strengths. These results shed light on the roles of oligonucleotides in ribonu-cleoprotein granule formation, heterochromatin compaction, and internal structuring of the nucleolus and stress granules.

biophysics↗