Solid/liquid coexistence during aging of FUS condensates

Abstract

A wide range of macromolecules undergo phase separation, forming biomolecular condensates in living cells. These membraneless organelles are typically highly dynamic, formed in a reversible manner, and carry out important functions in biological systems. Crucially, however, a further liquid-to-solid transition of the condensates can lead to irreversible pathological aggregation and cellular dysfunction associated with the onset and development of neurodegenerative diseases. Despite the importance of this liquid-to-solid transition of proteins, the mechanism by which it is initiated in normally functional condensates is unknown. Here we show, by measuring the changes in structure, dynamics and mechanics in time and space, that FUS condensates do not uniformly convert to a solid gel, but rather that liquid and gel phases co-exist simultaneously within the same condensate, resulting in highly inhomogeneous structures. We introduce two new optical techniques, dynamic spatial mapping and reflective confocal dynamic speckle microscopy, and use these to further show that the liquid-to-solid transition is initiated at the interface between the dense phase within condensates and the dilute phase. These results reveal the importance of the spatiotemporal dimension of the liquid-to-solid transition and highlight the interface of biomolecular condensates as a key element in driving pathological protein aggregation.

Location and concentration of aromatic-rich segments dictates the percolating inter-molecular network and viscoelastic properties of ageing condensates

“…Nevertheless, neither the patterning nor the abundance of LARKS, alter the critical temperature ( T c ) below which phase separation is observed. Since high-density fluctuations driving β -sheet transitions cannot take place in non-phase-separated protein liquid phases 66 , the critical saturation concentration also remains unaffected. Strikingly, we find that proteins with tail-located LARKS exhibit higher increments in condensate density than proteins with center-located and equispaced LARKS.…”

“…This behaviour again highlights the critical importance of LARKS location and abundance in determining the viscoelastic properties of aged condensates, where three equispaced LARKS instead, would have likely led to a complete kinetically trapped network (as recently found for the single low-complexity domain of FUS 12,32 ). However, such subtle balance between liquid-like vs. partial kinetically arrested behaviour of full-FUS is what possibly gives rise to the recently reported FUS multiphase aged condensates found in vitro 66 and in silico 33 . Overall, we conclude this Section II D by acknowledging that despite the sequence domain reordering of the lowcomplexity domain in FUS significantly contributes (by a 25%) to decelerating the rate of inter-peptide β-sheet transitions, yet complete ageing inhibition is not fully achieved.…”

“…31 ) has been recently shown to drive FUS singlecomponent condensates to display multiphase architectures upon ageing 32,65,105 or upon phosphorylation 106 . Moreover, this inter-molecular imbalance is consistent with the progressive dynamical arrest of proteins within amorphous condensates observed in LARKS-containing proteins 17,33,36 such as hnRNPA1 20,62 , FUS 9,65 , TDP-43 88 , or NUP-98 54,89 among many others 10,20,28,107 . Furthermore, we apply the primitive path analysis for revealing the transformation of the condensate molecular connectivity across a possible liquid-to-gel transition.…”

Time-dependent material properties of ageing biomolecular condensates from different viscoelasticity measurements in molecular dynamics simulations

“…Furthermore, our results help to explain the recognised asphericity of aged condensates 30,183 and the emergence of irregular morphologies caused by non-ergodic droplet coalescence 74,116 reported for LCD-containing proteins such as hnRNPA1 12 , FUS 30 , TDP-43 184 , or NUP-98 175 . Remarkably, the progressive kinetic arrest of proteins within droplets in FUS (full-sequence) in combination with a severe imbalance in the intermolecular forces has been shown to drive single-component condensates to display multiphase architectures upon maturation 73,185 or upon phosphorylation 186 .…”

Surfactants or scaffolds? RNAs of different lengths exhibit heterogeneous distributions and play diverse roles in RNA-protein condensates

“…Thus, in mixed RNA condensates, a stable core containing long polyU strands is expected to have a higher viscosity, while an outer shell with short polyU strands a lower viscosity [43]. Gelification of RNA-protein condensates via fibrillation, with potential pathological implications [21, 47, 105], has been shown to be seeded at the interface due to a local increase of protein density at the surface [106]; hence, incorporating short RNAs into RNA-protein condensates might contribute to preventing their maturation because it decreases the probability of high density fluctuations at the interface.…”