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 ‘maturation ’ 16,103–106),
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.