Environment-responsive biological matter exhibiting metastability
The MLOs, as biological assemblages, reside in a metastable material state. They can readily form under stress conditions as induced by heat12,97,98, pH change 98,99, starving98 or chemicals (e.g. sodium arsenate12,40) in living cells. For example, the exposure of HeLa cells to heat stress by heating from 37 °C to 42 °C, or to chemical stress of 1 mM sodium arsenate, can significantly induce liquid-like stress granules in both the nucleus and cytoplasm 12. Also, the starvation of yeast by glucose depletion or simply drop of cytosolic pH (to 5.7) can trigger the LLPS of polyU-binding protein (Pub1) into stress granules 98.
Liquid-like MLOs can readily reverse to dispersed state in response to a wide range of environmental stimuli including heating53,100, pH 98, ionic strength11,53, light 101,102 and enzymatic reaction, including phosphorylation 80 and proteolytic cleavage 54, which may indicate the liquid state of assembly is a metastable state. For example, heating from 10 °C to 20 °C dissolved the liquid droplets formed from N-terminal Argonaute binding domain (ABD) of TNRC6B protein in vitro 100. The phase separation of Pub1 in vitro can be dissolved with the increase of ionic strength from 187 mM to 1 mM, as well as the change of pH from 5.7 to 7.5 98. The liquid-like droplets formed from the LCD of FUS protein gradually dissolved within two hoursin vitro , via the phosphorylation at serine and threonine sites by kinase 80. Hammer et al.54 demonstrated the controllable dissolution and formation of LLPS induced by proteolytic cleavage. They reconstituted proteins from the fusion of two RGG domains of LAF-1 protein, linked by Glu-Asn-Leu-Tyr-Phe-Gln-Gly recognition sequence by tobacco etch virus (TEV) protease. This RGG-x-RGG (x=TEV cleavage site) protein forms LLPS, whilst the dissolution of liquid droplets can be triggered by TEV proteolytic cleavage of linkage of RGG domains, as was demonstratedin vitro and in living HEK293 cells. Additionally, they fused RGG-RGG with maltose-binding protein (MBP) domain via linkage of TEV cleavage site, which has been widely used as a solubility-enhancing tag to prevent phase separation of IDPs 79. This MBP-x-RGG-RGG protein remains dispersed, while the formation of liquid droplets can be triggered by proteolytic cleavage of MBP domain with the treatment of TEV protease, as was demonstrated in water-in-oil protocells in vitro .
MLOs can spontaneously transit into more stable material state over time (known as ‘maturation16,103–106), including viscous liquids 28, gels28 and amyloid-like fibrils12,42,107 (Figure 3A ). For example, the liquid droplets from 8 μM FUS protein transit into amyloid-like fibrillar structure after 6-hour incubation in vitro12. Likewise, the liquid droplets from the LLPS of p-tau protein evolve into viscoelastic liquids, gels and finally amyloid-like aggregates after 1-day incubation in vitro , and liquid droplets almost completely converted to amyloid-like aggregates after 10-day incubation 28. Parker et al. fused polypyrimidine tract-binding protein (PTB, an RNA-binding domain) with LCDs of IDPs including PUB1, LSM4, EIF4GII, TIA1 and FUS. These reconstituted proteins form LLPS in complexation with RNAs in vitro . Notably, all of these complexed droplets mature into much more stable and solid-like assemblies within 48 hours 9. This natural and spontaneous liquid-to-solid transition of materials state can be accelerated under disease-associated conditions11,12, which will be discussed in the next section.