Biomolecular condensates undergo a generic shear-mediated liquid-to-solid transition

Abstract

A wide range of systems containing proteins have been shown to undergo liquid-liquid phase separation (LLPS) forming membraneless compartments, such as processing bodies1, germ granules2, stress granules3 and Cajal bodies4. The condensates resulting from this phase transition control essential cell functions, including mRNA regulation, cytoplasm structuring, cell signalling and embryogenesis1-4. RNA-binding Fused in Sarcoma (FUS) protein is one of the most studied systems in this context, due to its important role in neurodegenerative diseases5-7. It has recently been discovered that FUS condensates can undergo an irreversible phase transition which results in fibrous aggregate formation6. Gelation of protein condensates is generally associated with pathology. One case where liquid-to-solid transition (LST) of liquid-liquid phase separated proteins is functional, however, is that of silk spinning8,9, which is largely driven by shear, but it is not known what factors control the pathological gelation of functional condensates. Here we show that four proteins and one peptide system not related to silk, and with no function associated with fibre formation, have a strong propensity to undergo LST when exposed to even low levels of mechanical shear comparable to those found inside a living cell, once present in their liquidliquid phase separated forms. Using microfluidics to control the application of mechanical shear, we generated fibres from single protein condensates and characterized their structures and material properties as a function of shear stress. Our results inform on the molecular grammar underlying protein LST and highlight generic backbone-backbone hydrogen bonding constraints as a determining factor in governing this transition. Taken together, these observations suggest that the shear plays an important role in the irreversible phase transition of liquid-liquid phase separated droplets, shed light on the role of physical factors in driving this transition in protein aggregation related diseases, and open a new route towards artificial shear responsive biomaterials.During silk spinning in nature, the precursor protein molecules stored in liquid-liquid phase separated form undergo a LST to generate fibres mediated by mechanical shear10-13. It is increasingly recognised, however, that a wide range of other proteins can undergo LLPS inside the cell to form condensates14,15.This process is highly dynamic and crucial to cellular function2,5,16, while irreversible phase transitions lead to solid protein aggregates and can result in disease6. Yet, it remains unexplored whether functional protein condensates can also undergo a LST driven by mechanical shear.

Nanoscale profiling of evolving intermolecular interactions in ageing FUS condensates

“…Highresolution-imaging reveals a loose, mesh-like network in these assemblies, which is not consistent with the formation of highly ordered, cross-b sheet structure typically found in amyloid fibrils (28). In our hands, we attribute the formation of these structures to the shear forces from pipetting (29,30).…”