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\begin{document}

\title{Metastability of Biological Matter in Liquid Phase Separation}



\author[1]{Jianhui Liu}%
\author[1]{Xiaolin  Liu}%
\author[1]{Tianfu Zhang}%
\author[1]{Jinqing  Huang}%
\author[1]{Ben Zhong  Tang}%
\author[1]{Ying  Chau}%
\affil[1]{The Hong Kong University of Science and Technology}%


\vspace{-1em}



  
  \date{June 13, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Membraneless organelles (MLOs) form via liquid-liquid phase separation
(LLPS). The liquid-like MLOs afford multifold peculiarities including
high dynamics, reversibility and responsiveness. The MLOs are typically
metastable with a tendency to undergo fast, drastic and reversible
formation and dissolution, as well as transition into more stable glassy
or gel-like states. Moreover, the alteration of metastability of LLPS is
linked with cellular pathology. Here, we review the crucial and
ubiquitous `metastability' of MLOs driven by liquid phase separation,
from multifaceted regards including energy state, molecular
interactions, molecular structure, materials state, as well as the
associations with diseases. This review can help to advance the insight
into properties and pathogenesis associated with LLPS of biological
matter.%
\end{abstract}%



\sloppy


\textbf{Review}

\textbf{Metastability of Biological Matter in Liquid Phase Separation}

Jianhui Liu\textsuperscript{1}, Xiaolin Liu\textsuperscript{2,3}, Tianfu
Zhang\textsuperscript{2,3}, Jinqing Huang\textsuperscript{2}, Ben Zhong
Tang\textsuperscript{2,3,4} and Ying Chau\textsuperscript{1,*}

Department of Chemical and Biological Engineering

The Hong Kong University of Science and Technology

Clear Water Bay, Kowloon, Hong Kong SAR, China

Department of Chemistry

The Hong Kong University of Science and Technology

Clear Water Bay, Kowloon, Hong Kong SAR, China

Hong Kong Branch of Chinese National Engineering Research Center for
Tissue Restoration and Reconstruction The Hong Kong University of
Science and Technology Clear Water Bay, Kowloon, Hong Kong SAR, China

Shenzhen Institute of Aggregate Science and Technology School of Science
and Engineering The Chinese University of Hong Kong, Shenzhen Guangdong
518172, China\selectlanguage{english}
\begin{longtable}[]{@{}llll@{}}
\toprule
Name & ORCID & Email & Twitter\tabularnewline
\midrule
\endhead
Jianhui Liu & 0000-0003-1055-7964 & jliuca@connect.ust.hk &
Jianhui\_Liu\_\tabularnewline
Xiaolin Liu & 0000-0002-6909-117X & xliuco@connect.ust.hk &
Xiaolin\_Chem\tabularnewline
Tianfu Zhang & 0000-0001-6012-8856 & tzhangay@connect.ust.hk &
tianfu\_zhang\tabularnewline
Jinqing Huang & 0000-0001-6865-8528 & jqhuang@ust.hk &
JinqingHuangLab\tabularnewline
Ben Zhong Tang & 0000-0002-0293-964X & tangbenz@ust.hk &
BenZhongTANG1\tabularnewline
Ying Chau & 0000-0003-1759-934X & keychau@ust.hk &\tabularnewline
\bottomrule
\end{longtable}

*Correspondence: keychau@ust.hk (Chau, Y.)



\textbf{Keywords}

metastable; liquid-liquid phase separation; free energy; molecular
interactions; molecular structures; biological assemblages

\textbf{Abstract}

Membraneless organelles (MLOs) form via liquid-liquid phase separation
(LLPS). The liquid-like MLOs afford multifold peculiarities including
high dynamics, reversibility and responsiveness. The MLOs are typically
metastable with a tendency to undergo fast, drastic and reversible
formation and dissolution, as well as transition into more stable glassy
or gel-like states. Moreover, the alteration of metastability of LLPS is
linked with cellular pathology. Here, we review the crucial and
ubiquitous `metastability' of MLOs driven by liquid phase separation,
from multifaceted regards including energy state, molecular
interactions, molecular structure, materials state, as well as the
associations with diseases. This review can help to advance the insight
into properties and pathogenesis associated with LLPS of biological
matter.



\textbf{Key points}

Liquid-liquid phase separation emerges as a universal tool for the
intracellular biomolecule organization, particularly via the formation
of membraneless organelles.

The liquid-like membraneless organelles afford unique properties and
have aroused wide interests among cell biologists.

Metastability is a general feature of liquid-like membraneless
organelles, which has manifested in multiple aspects including energy
state, molecular interactions, molecular structures, biological
assemblages and its association with diseases.

Understanding of metastability of biological liquid phase separation can
not only help to elucidate the formation and properties of membraneless
organelles, but also pave the way to the insight into pathogenesis.



\textbf{Metastable Biological Matter}

Cells compartmentalize to organize a myriad of biomolecules and
biochemistry hierarchically, conventionally known to form lipid bilayer
membrane-bound organelles \textsuperscript{1}.
Nonetheless,\textbf{liquid-liquid phase separation} (LLPS, see
\textbf{Glossary} ) emerges recently as another universal phenomenon for
intracellular organization, particularly via the formation of
\textbf{membraneless organelles} (MLOs) hosting \textbf{intrinsically
disordered proteins}(IDPs) as scaffolds, which have important roles in
cellular physiology and disease \textsuperscript{2--4}. The liquid-like
MLOs are biological soft matter that afford multifold peculiarities,
including high dynamics, reversibility and environmental
responsiveness\textsuperscript{5}. Notably, there is growing evidence
that `\textbf{metastability} ' is a universal and pivotal nature of
liquid-like MLOs \textsuperscript{6}. For one thing, MLOs are capable of
undergoing fast, drastic and reversible formation and dissolution events
commensurate with surrounding intracellular
environment\textsuperscript{7}. For another, the liquid state has an
inherent tendency to transit into more stable glassy or gel-like
states\textsuperscript{8}, simply over time \textsuperscript{9} or
expedited by disease-associated mutations \textsuperscript{10--13}.
Moreover, the alteration of metastable state has been revealed to be
closely tied with cellular physiology and pathology
\textsuperscript{14}, whilst the manipulation of LLPS can be promising
route towards effective treatment of diseases \textsuperscript{15}.

In this review paper, we highlight the ubiquitous yet essential
metastability of liquid phase separation of biological soft matter. We
first depict the \textbf{free energy} landscape of biological matter,
and underscore the metastable state of MLOs with liquidity. We next
discuss about the distinctive metastable molecular interactions and
molecular structures implicated in liquid-like MLOs. Through examples,
we then highlight the metastable material state of biological
assemblages formed through liquid phase separation. Lastly, we describe
how metastability provides a pivotal link between physiology and
disease.

\textbf{Metastability of energy state}

The liquid condensate state of MLOs is energetically\textbf{metastable}
, namely, resides in a local minimum of Gibbs free energy
(\textbf{Figure 1A} ). A thermodynamic system will spontaneously
minimize the free energy to attain states with higher stability. As
such, liquid condensate state can spontaneously transit to more
energetically favorable states, namely, other local minima with lower
free energy or the global minimum, simply over longer
time\textsuperscript{16--18} or expedited by disease-associated
mutations\textsuperscript{10--13,19}. For example, liquid condensates
can evolve into reversible hydrogels or irreversible amyloid-like
aggregates, which has lower free energy and higher stability.
Additionally, LLPS can be an intermediate process to facilitate
nucleation and lower the free energy of crystallization and
aggregation\textsuperscript{18,20,29--32,21--28}. To maintain the liquid
condensate state, specific quality control machineries
\textsuperscript{33} are required to stop them from spontaneous
solidification\textsuperscript{34--37}, including post-translational
modification (PTM) \textsuperscript{38,39}, RNA binding
\textsuperscript{40}, chaperon \textsuperscript{11,41--45}, hydrotrope
\textsuperscript{46}and disaggregase \textsuperscript{47}.

In cell biology, proteins are commonly hydrophilic biomacromolecules
dispersed in the state of dilute solution. How can the metastable liquid
condensates form from the dispersed solution thermodynamically?
Basically, there are two types of interactions in a dispersed protein
solution, namely, the homotypic interactions between two biomolecules
(e.g., protein or nucleic acid) and two water molecules, as well as the
heterotypic interactions between a biomolecule and a water
molecule\textsuperscript{48}. The phase transition from dispersed state
to phase-separated state can occur if the homotypic interactions are
favored over heterotypic interactions. The enthalpy and entropy change
of this phase transition can be categorized into four quadrants
(\textbf{Figure 1B} ). The formation of liquid condensates is
thermodynamically favored within two quadrants (\textbf{Figure 1B} ,
highlighted with green color), namely, when\(\Delta H\bullet\Delta S>0\). When
\(\Delta H>0\) and\(\Delta S>0\), \(\Delta G\) is only
negative at high temperature. This means the LLPS is spontaneous when
temperature is higher than a threshold value, thus exhibiting a lower
critical solution temperature (LCST) phase behavior, which has been
found in extracellular IDPs including elastin, elastin-like polypeptide
\textsuperscript{49} and histidine-rich squid beak proteins
\textsuperscript{50--52}. When\(\Delta H<0\) and \(\Delta S<0\),
\(\Delta G\) is only negative at low temperature. This means the
LLPS is spontaneous when temperature is lower than a threshold value,
thus exhibiting an upper critical solution temperature (UCST) phase
behavior, which has been found in intracellular IDPs including Ddx4
\textsuperscript{53}, LAF-1 \textsuperscript{54}, FUS
\textsuperscript{55}, TDP-43 \textsuperscript{56} and
hnRNP\textsuperscript{17} protein. By contrast, the formation of liquid
condensates is thermodynamically disfavored within two quadrants
(\textbf{Figure 1B} , highlighted with red color), namely,
when\(\Delta H\bullet\Delta S<0\). When \(\Delta H>0\) and\(\Delta S<0\),
phase separation is always thermodynamically unfavorable, and solution
remains in the dispersed state because\(\Delta G\) is always
positive. When \(\Delta H<0\) and\(\Delta S>0\), phase
separation is always thermodynamically favorable. Dispersed solution
will form irreversible phase separation spontaneously, either
liquid-solid phase separation or LLPS, as\(\Delta G\) is always
negative.

\textbf{Molecular interaction and molecular structure for metastability}

In accordance with the metastable free energy state, IDPs, as the
scaffold protein of MLOs, often harbor transient and weak molecular
interactions \textsuperscript{34}. The \textbf{low complexity
domains}(LCDs), as the domains that mediate the LLPS of IDPs, are
largely enriched with charged, polar and aromatic residues whilst
commonly devoid of hydrophobic residues \textsuperscript{22}. Weak,
multivalent and non-specific interactions, including electrostatic,
pi--pi, cation--pi and dipole--dipole interactions (between polar amino
acids), are prevalent among residues in LCDs (\textbf{Figure 2A}
)\textsuperscript{36}. The long-range electrostatic interactions among
charged blocks may facilitate the initiation of LLPS, while short range
interactions, including pi--pi, cation--pi and dipole--dipole
interactions, may mediate the multivalent contacts among weakly
interacting motifs \textsuperscript{34}. Among these molecular
interactions, cation--pi interactions are considered as the strongest,
with the free energy of binding (\(\Delta G_{\text{bind}}\)) around --3.6
kcal/mol \textsuperscript{57}. This magnitude is lower than the average
\(\Delta G_{\text{bind}}\) per residue implicated in the formation of
A\selectlanguage{greek}β\selectlanguage{english}\textsubscript{17--42} amyloids-\selectlanguage{greek}β \selectlanguage{english}protofibrils (--19.3 kcal/mol)
\textsuperscript{58}, thus suggesting molecular interactions driving the
formation of MLOs are much weaker than amyloid plaques. Compared with
static amyloids \textsuperscript{34}, LCDs harbor transient interactions
among residues with higher dynamics. This can be quantified by
fluorescence recovery after photobleaching (FRAP), commonly showing a
half time of recovery (\(t_{\frac{1}{2}}\)) on the order of seconds
(normalizing the diameter of bleaching spot to 1
\selectlanguage{greek}μ\selectlanguage{english}m)\textsuperscript{34}. Efforts have been made to shed light on the
possible reason of liquid phase formation from the molecular level,
including theory of amyloids-\selectlanguage{greek}β \selectlanguage{english}fibril formation \textsuperscript{59},
multivalent domain interaction network model \textsuperscript{60} and
theory of polymer physics \textsuperscript{22}.

IDPs lack a stable and well-defined 3D molecular
structure\textsuperscript{61--63}. IDPs are always devoid of stable
tertiary structures under physiological conditions, albeit collapsed
IDPs could harbor some stable secondary structure elements
\textsuperscript{64}. The lack of stable structure can be possibly
considered as one common and crucial feature for IDPs to form metastable
MLOs\textsuperscript{65}. The unstable conformation allows the
flexibility of IDPs as major scaffold constituents, which may contribute
to the physical fluidity of MLOs \textsuperscript{65}. The unstable
conformation allows formation of the weak and multivalent interactions,
which is a common hallmark for the interactions that contribute to
LLPS\textsuperscript{3}. The IDPs harbor `stickers-and-spacers'
structural features, wherein modules provide attractive interactions are
considered as `stickers', and flexible linkers provide no significant
attractive interactions are considered as `spacers'. The unstable
structure with `stickers-and-spacers' features allows the multivalent
presence of PTM sites \textsuperscript{66}, whilst PTMs can efficiently
alter the stability of MLOs \textsuperscript{53}.

Modular interaction domains connected by disordered linkers can mediate
multivalent interactions that drive LLPS. Rosen \emph{et al.} reported
the LLPS of multivalent signaling proteins. Neural Wiskott--Aldrich
syndrome protein (N-WASP), the actin-regulatory protein, interact with
its established biological partners NCK and phosphorylated nephrin1 to
form LLPS, wherein NCK contain three SH3 domains that can bind to the
six proline-rich motif (PRM) ligands of N-WASP \textsuperscript{67}.
Similar multivalent system were also reported in T cell receptor
signaling pathway \textsuperscript{68}, nucleophosmin (NPM1) interacting
with proteins comprising arginine-rich linear motifs and ribosomal
RNA\textsuperscript{69} and pair of polySUMO--polySIM interacting
multivalent scaffold proteins \textsuperscript{70}.

Weakly interacting motifs are prevalent in LCDs to mediate LLPS. It has
been widely known that tightly self-complementing `\textbf{steric
zipper} ' structure forms solid-like amyloid-\selectlanguage{greek}β \selectlanguage{english}plaques with hydrophobic
interfaces and high stability \textsuperscript{71--73}. By contrast,
IDPs largely host motifs that can form thermodynamically metastable
`\selectlanguage{greek}\textbf{kinked β sheets}\selectlanguage{english} ' \textsuperscript{74--77} molecular
structure,\emph{i.e.} , the archetypical {[}G/S{]}Y{[}G/S{]} motifs of
FUS protein. These motifs can form close interactions as quantified by
the structural complementarity (\(S_{c}\)) (\textbf{Table 1} ).
However, side chains cannot interdigitate across the \selectlanguage{greek}β-\selectlanguage{english}sheet interface
owing to the prevention of kinks. They thus harbor smaller buried
solvent-accessible surface area (\(A_{b}\)) and more
hydrophilic interfaces, thus exhibiting much lower stability
\textsuperscript{74} (\textbf{Figure 2B}and \textbf{Table 1} ). This is
exemplified by metastable interaction motifs in LCDs of FUS
\textsuperscript{55,78--82}, Tau\textsuperscript{83}, TDP-43
\textsuperscript{84} and hnRNP\textsuperscript{17,76,85} proteins.
Specifically, short associative peptide motifs within LCDs can form
metastable fibrils \emph{in vitro} , whilst exhibiting melting behavior
in response to mild heating, which is distinctive from stable amyloid
fibrils (\textbf{Table 2} ).Besides kinked \selectlanguage{greek}β \selectlanguage{english}sheets, other interaction
motifs that can mediate multivalent interactions were also reported,
including repeated {[}F/R{]}G and G{[}F/R{]} pair motifs of Ddx4
proteins \textsuperscript{53}, \selectlanguage{greek}α-\selectlanguage{english}helix-forming
\textsuperscript{321}AMMAAAQAAL\textsuperscript{330}motif of TDP-43
proteins \textsuperscript{86}, VPGXG (X is a guest residue except
proline that can modulate phase behaviour) motifs of elastin-like
proteins \textsuperscript{87} and GHGLY motif of histidine-rich squid
beak proteins \textsuperscript{52}. In addition, specific motifs may
hinder LLPS, \emph{i.e.} , FGDF can bind to G3BPs to block the formation
of stress granules \textsuperscript{88}.

The metastable molecular structures and phase behavior of IDPs can be
drastically altered simply by
mutations\textsuperscript{17,55,76,80,84,85,89,90} or
PTMs\textsuperscript{55,82,84} on one single residue. For example, The
phosphorylation of FUS protein by kinase at the Ser42 site drastically
altered the molecular interactions of LCDs, haltering the formation of
metastable fibrils and LLPS formation \textsuperscript{55}. This
prominent alteration of phase behavior can be attributed to disruption
of metastable kinked structure. The Ser42 site is the primary
phosphorylation site by DNA-dependent protein kinase
(DNA-PK)\textsuperscript{91}. The phosphorylation at Ser42 can
significantly disrupt the hydrogen bonds between Ser42 and Tyr38,
interfere with the interaction of mating sheet and destabilize the RAC1
interacting motif, thereby modulating the ability of FUS to undergo
LLPS. Additionally, the mutation of Ser42 to Asp (S42D) can also
remarkably depress the LLPS of LCD of FUS protein, decreasing the
critical temperature of LLPS by 5 \selectlanguage{ngerman}°C, as the mutation S42D is a change
that mimics serine phosphorylation\textsuperscript{55}.

There are two major types of phase behavior for a biological LLPS system
of interest, namely, the entropy-driven LCST phase behavior and
enthalpy-driven UCST phase behavior \textsuperscript{24}. How is the
type of phase behavior encoded in motifs of protein sequences?
Chilkoti\emph{et al.} synthesized artificial IDP-like polymers harboring
several tens of repeats of short peptide motifs \textsuperscript{87}.
They found that motifs with low-charge content and high hydrophobicity
feature tend to engender IDP-like polymer with LCST behaviour, which is
reminiscent of tropoelastins. By comparison, motifs with high-charge
content and low hydrophobicity feature tend to engender IDP-like polymer
with UCST behaviour, which is reminiscent of the dual UCST and LCST
behaviour at extremes of temperature of resilin. Furthermore, Chilkoti
\emph{et al.}found that hysteresis behavior can also be encoded and
tuned at the motif level by the precise position of an amino acid within
a motif, as well as at the macromolecule level by chain length
\textsuperscript{92}.

The unique molecular interaction and molecular structure allow
metastable MLOs with unique properties, including liquidity, high
dynamics and environmental responsiveness. Learning from nature, the
responsible domains and motifs of IDPs have also been exploited as
building blocks to design bio-inspired
materials\textsuperscript{93--95}, which have been reviewed
elsewhere\textsuperscript{96}.

\textbf{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
heat\textsuperscript{12,97,98}, pH change \textsuperscript{98,99},
starving\textsuperscript{98} or chemicals (e.g. sodium
arsenate\textsuperscript{12,40}) 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
\textsuperscript{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 \textsuperscript{98}.

Liquid-like MLOs can readily reverse to dispersed state in response to a
wide range of environmental stimuli including
heating\textsuperscript{53,100}, pH \textsuperscript{98}, ionic
strength\textsuperscript{11,53}, light \textsuperscript{101,102} and
enzymatic reaction, including phosphorylation \textsuperscript{80} and
proteolytic cleavage \textsuperscript{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 \emph{in vitro}
\textsuperscript{100}. The phase separation of Pub1 \emph{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 \textsuperscript{98}. The
liquid-like droplets formed from the LCD of FUS protein gradually
dissolved within two hours\emph{in vitro} , via the phosphorylation at
serine and threonine sites by kinase \textsuperscript{80}. Hammer
\emph{et al.}\textsuperscript{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
demonstrated\emph{in 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
\textsuperscript{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 \emph{in vitro} .

MLOs can spontaneously transit into more stable material state over time
(known as `\textbf{maturation} ' \textsuperscript{16,103--106}),
including viscous liquids \textsuperscript{28}, gels\textsuperscript{28}
and amyloid-like fibrils\textsuperscript{12,42,107} (\textbf{Figure 3A}
). For example, the liquid droplets from 8 \selectlanguage{greek}μ\selectlanguage{english}M FUS protein transit into
amyloid-like fibrillar structure after 6-hour incubation \emph{in
vitro}\textsuperscript{12}. 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 \emph{in vitro} , and
liquid droplets almost completely converted to amyloid-like aggregates
after 10-day incubation \textsuperscript{28}. Parker \emph{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 \emph{in
vitro} . Notably, all of these complexed droplets mature into much more
stable and solid-like assemblies within 48 hours \textsuperscript{9}.
This natural and spontaneous liquid-to-solid transition of materials
state can be accelerated under disease-associated
conditions\textsuperscript{11,12}, which will be discussed in the next
section.

\textbf{Metastability is associated with diseases}

It costs to form metastable LLPS in cells. The LLPS of IDPs derives from
a state of \textbf{supersaturated} compositional proteins wherein their
cellular concentrations are high relative to their
solubilities\textsuperscript{67,83,108}, a phenomenon that can drive the
aberrant protein aggregation in \textbf{neurodegenerative
diseases}\textsuperscript{108,109}. The liquid condensed phase is
intrinsically metastable and vulnerable to uncontrolled aggregation
leading to pathological consequences \textsuperscript{9,10,14,37,110}.
The metastable liquid MLOs is susceptible to alteration simply over
time\textsuperscript{9,12}, which is commonly expedited under
pathological conditions \textsuperscript{12,19,111}. The transition from
metastable MLOs to stable aggregates can further lead to the gain of
biological changes, including cancer, neurodegenerative diseases and
aging\textsuperscript{4,11,118,119,35,75,112--117}. A wide array of
factors can engender the alteration of metastability, such as PTMs,
mutations and overexpression of proteins (\textbf{Figure 3B} ), which
are reviewed below.

Loss of metastability can be triggered by aberrant
PTMs\textsuperscript{120}. George-Hyslop \emph{et al.}
\textsuperscript{11}showed hypomethylation of arginine can drive the
formation of hydrogel structure from FUS protein, which can disrupt
ribonucleoprotein (RNP) granule function and damage protein synthesis in
neuron terminals, thus suggesting a plausible mechanism for
frontotemporal lobar degeneration (FTLD), as well as other
neurodegenerative diseases. Conversely, metastability can be preserved
by specific PTMs. Fawzi \emph{et al.}\textsuperscript{85} revealed that
arginine methylation can help to maintain the metastability of hnRNPA2
protein by disrupting interactions between arginine guanidyl group and
aromatic residues, thereby pointing out a possible method for the
regulation of solid-like assemblies and modify the toxicity. Fawzi
\emph{et al.} \textsuperscript{39}investigated the impact of
phosphorylation using phosphomimetic mutations. All the twelve
serine/threonine--glutamine (S/TQ) sites of FUS protein were mutated to
glutamate--glutamine (EQ) sites. The disordered nature of LCDs was
preserved, whilst allowing the ability to form LLPS \emph{in vitro} .
Distinctively, the aggregation-prone characteristics were reduced. With
one-day orbital agitation to induce maturation of IDPs, most of the
droplets formed from wild-type FUS protein evolved into irregular
assemblies, while the droplets formed from phosphomimetic variant 12E
remained spherical \emph{in vitro} . They further demonstrated both the
aggregation propensity and cytotoxicity in yeast \emph{Saccharomyces
cerevisiae} were reduced, suggesting that maintaining metastability of
liquid-like MLOs can be an effective way to tackle neurodegeneration
caused by aberrant protein aggregation. Ferreon\emph{et al.}
\textsuperscript{121} found PTM can counteract LLPS-initiated protein
aggregation. Tau protein underwent LLPS under physiology-relevant
conditions \emph{in vitro} , whilst the liquid-to-solid transition can
be induced in the presence of heparin. The 24-hour maturation process
was visualized by morphological change into solid-like irregular
aggregates and quantified by prominent fluorescence increase of
Thioflavin T (ThT) fluorescence assay. Tau protein was also
hyperacetylated with p300 histone acetyltransferase. Distinctively,
hyperacetylated Tau protein forms much less irregular solid after
24-hour maturation. The aggregation propensity of Tau is largely
decreased by acetylation, suggesting a way to ameliorate
neurodegenerative diseases associated with toxic Tau aggregation,
including Alzheimer's disease.

Disease-associated mutations often strengthen homotypic interactions of
IDPs, thereby impairing the liquidity of MLOs, decreasing dynamics and
promoting protein aggregation \textsuperscript{112}. Neurodegenerative
diseases have been widely reported to link with aberrant transition of
MLOs. Alberti \emph{et al.} \textsuperscript{12} showed the maturation
of FUS protein liquid droplets can be drastically accelerated by
mutations associated with amyotrophic lateral sclerosis (ALS). After
8-hour maturation \emph{in vitro} , droplets formed by the LLPS of FUS
protein was mostly preserved, only demonstrating minute solidification.
By contrast, droplets from the G156E mutant of FUS protein maturated
into stable fibrous aggregates (a state without metastability) almost
completely. How is the loss of metastability linked with disease? Da
Cruz \emph{et al.} \textsuperscript{122} further clarified that
ALS/frontotemporal dementia (FTD)-linked mutant of FUS protein drives
disease by a gain of toxicity, rather than a loss of function.
Taylor\emph{et al.} \textsuperscript{19} investigated the hnRNPA1
mutant, D262V, which is associated with multisystem proteinopathy and
ALS\textsuperscript{123}. At the initial state, D262V mutant formed LLPS
comparable with wild-type hnRNPA1, demonstrating similar propensity of
droplet formation and critical melting temperature. Repeated
heating-cooling cycling was applied to both D262V mutant and wild-type
hnRNPA1. Notably, within minutes of maturation, the reversible droplet
formation of D262V mutant was concomitant with the fibrilization and
formation of irregular solid-like assemblies, which is stable,
amyloid-like and ThT-positive. By contrast, no such liquid-to-solid
conversion was observed for wild-type hnRNPA1, thus suggesting the
metastability of MLOs was impaired by the D262V mutation, which is
associated with neurodegeneration. Fawzi \emph{et
al.}\textsuperscript{85} studied the P298L and D290V mutants of hnRNPA2
protein with connections to Paget's disease and multisystem
proteinopathy (MSP), respectively. Freshly prepared wild-type hnRNPA2,
P298L and D290V mutants all formed similar spherical droplets with
liquidity and comparable dynamics, as was quantified by FRAP.
Nonetheless, P298L and D290V mutants prominently transited into stable
solid-like assemblies after 30-min maturation \emph{in vitro} , in stark
contrast to the preservation of liquid droplets of wild-type hnRNPA2 for
at least 120 mins, thereby indicating disease-associated mutations
impaired metastability of MLOs. Fawzi \emph{et al.}
\textsuperscript{86}reported A321V, an ALS variant of C-terminal domain
(CTD) of TDP-43, formed liquid droplets similar to wild-type TDP-43, as
was confirmed by turbidity, spherical shape and FRAP. After one hour
incubation \emph{in vitro} , A321V variant changed into irregular
assemblies which is distinct from the spherical shape of wild type
TDP-43. Similar phenomena have also been found for P362L mutants in the
LCD of T cell-restricted intracellular antigen-1 (TIA1)
\textsuperscript{124}, and A4V mutants in superoxide dismutase 1 (SOD1)
\textsuperscript{45}, which are associated with ALS. Taylor \emph{et
al.} \textsuperscript{118} studied the expansion of a hexanucleotide
repeat GGGGCC in C9ORF72, which is the most common cause of ALS and FTD,
presumably through the expression of toxic dipeptide repeat proteins.
They identified that arginine-containing dipeptide repeat proteins,
namely, polyGly-Arg and polyPro-Arg, can interact with LCDs of IDPs to
alter the biophysical properties of MLOs. Both polyGly-Arg and
polyPro-Arg can impair the liquidity, dynamics and metastability of MLOs
\emph{in vitro} and in living HeLa cells, as was substantiated by FRAP
and change of spherical shape. Moreover, the polyGly-Arg and polyPro-Arg
can promote the assembly of stress granules lacking metastability in
HeLa cells, which can inhibit cellular translation and increase the risk
of cell death events. Vale \emph{et al.} \textsuperscript{125} further
showed RNAs harboring CAG and CUG repeats (length 31, 47 and 66 repeats)
underwent LLPS event into droplets \emph{in vitro} , albeit rapidly
evolving into solid-like RNA hydrogels maintaining sphere-like shape.
RNAs harboring 47 CAG repeats can form liquid-like nuclear foci in
living U-2OS cells. By contrast, RNAs with 10 or 23 GGGGCC repeats forms
irregular solid-like gels \emph{in vitro} , and RNAs with 29 GGGGCC
repeats forms irregular solid-like gels in living U-2OS cells. This
report demonstrated that expansion of the GGGGCC in the C9ORF72 gene can
trigger formation of solid-like RNA gels with impaired dynamics and
metastability. As the accumulation of the repeat-containing transcripts
into aberrant RNA foci in the nucleus is a common feature in
neurodegeneration, this research suggests sequence-specific gelation of
RNAs can be one cause of neurodegenerative diseases. Hsp27 is molecular
chaperone that is vital in maintaining the metastability and dynamics of
liquid droplets of stress granules from FUS protein, whilst mutations of
chaperons can directly engender hereditary motor neuron
diseases\textsuperscript{42}. Liu \emph{et al.} \textsuperscript{42}
mutated residues (I120, H124, 126--131, I134, F136, R140 and T143) of
Hsp27 to alanine to synthesize Hsp27-A, which can largely change the
binding surface between Hsp27 and FUS protein. Hsp27 bound to FUS LCD to
preserve the liquid phase against amyloid fibril formation, whilst the
mutant Hsp27-A had little inhibitory effect on the amyloid-like
aggregation of FUS LCD, as has been quantified by the ThT fluorescence
assay. This study suggests loss of metastability can lead to diseases
through the mutations of chaperone. Besides neurodegenerative diseases,
Mittag \emph{et al.} \textsuperscript{126} investigated the impact of
cancer mutations on LLPS and functioning of MLOs. Tumor suppressor
speckle-type POZ protein (SPOP) formed liquid-like MLOs with
death-domain-associated protein (DAXX) in living HeLa cells, and CUL3
ubiquitin ligase activity is found in the MLOs formed. By contrast,
cancer mutants (W131G and F133V) of SPOP failed to co-localize and form
liquid-like MLOs with DAXX in living HeLa cells, resulting into reduced
protein ubiquitination, thus suggesting cancer mutations are associated
with loss of function of MLOs.

The overexpression of IDPs can also trigger the alteration of metastable
MLOs. Carra \emph{et al.} \textsuperscript{127} revealed overexpression
of small heat shock proteins (HSPBs) engendered aberrant MLOs in
nucleus, which can mislocalize nuclear intermediate filament protein
lamin-A/C (LMNA) and chromatin, resulting in the abnormal distribution
of LMNA and chromatin and thereby damaging the integrity and function of
nucleus.

\textbf{Concluding remarks}

Membraneless organelles are peculiar liquid-like compartments in cells.
Membraneless organelles are unique because of their multifold
metastability, including free energy, molecular interaction, molecular
structure, and materials state. What's more, the alteration of
metastability has been tightly linked to diseases. We envision that
further elucidation of metastability in liquid phase separation,
especially in the formation, maintenance and modulation of liquid-like
MLOs, can contribute to the insight into cell physiology and mechanisms
of pathogenesis (see \textbf{Outstanding Questions} ).

\textbf{Acknowledgements}

Financial support was provided by the Hong Kong Research Grant Council
(GRF 16102520 and GRF 16103517), the Hong Kong Innovation and Technology
Commission (ITC CNERC14SC01) and the Hong Kong PhD Fellowship Scheme
(for Jianhui Liu).

\textbf{Conflict of interest disclosure}

Authors have no conflict of interest to declare

\textbf{Author contributions}

The manuscript was written through contributions of all authors. All
authors have given approval to the final version of the manuscript.

\textbf{Data availability statement}

Data sharing not applicable -- no new data generated, or the article
describes entirely theoretical research.



\textbf{Glossary}

\textbf{Free energy:} In a thermodynamic system, the portion of energy
available to perform work at constant temperature. Specifically, the
Gibbs free energy is used for constant temperature and pressure, which
is true for solution-phase chemistry including biochemistry. Systems
tend to transit into a state where the free energy is lower, which is
more thermodynamically stable.

\textbf{Intrinsically disordered proteins (IDPs)} : The most common
scaffold proteins of MLOs with a strong tendency to form LLPS, which is
often concomitant with nucleic acids.

\selectlanguage{greek}\textbf{Kinked β sheets:}\selectlanguage{english} The \selectlanguage{greek}β-\selectlanguage{english}sheet with kinks that prevent side
chains from tight interdigitating across the interface. Compared with
the steric zipper structure in amyloid plaques, they interact weakly
through polar atoms and aromatic side chains, bury smaller surface
areas, and have lower binding energy.

\textbf{Liquid-liquid phase separation (LLPS):} A homogenous liquid
phase demixes into two distinct liquid phases, namely, one light phase
and one concentrated phase. This term is also widely referred as
`coacervation' when comprising macromolecular components.

\textbf{Low complexity domains (LCDs)} : Domains of IDPs with low
sequence complexity and biased composition of amino acids, namely,
typically enriched in specific polar and charged amino acids, whilst
interspersed with aromatic residues (especially tyrosine and
phenylalanine). They are also referred as `prion-like domains (PLDs)',
which is often considered as necessary and sufficient for driving LLPS
of IDPs intracellularly.

\textbf{Maturation:} A transition process of MLOs from liquid-like
droplets to solid-like aggregates, together with the loss of
metastability. This process is thermodynamically spontaneous, which can
take place simply over time or be accelerated by pathological changes.
This term has also been referred to as `hardening', `aging' or
`solidification'.

\textbf{Membraneless organelles (MLOs)} : Non-membrane-bound organelles
that usually form via liquid-liquid phase separation in cells. They have
also been referred to as many other names including biomolecular
condensates, cellular bodies, speckles, puncta and granules.

\textbf{Metastability} /\textbf{Metastable:} A system is
thermodynamically metastable when staying at a local minimum of free
energy, with a tendency to spontaneously transit into more stable state,
namely, a global minimum of free energy. The metastable nature of a
thermodynamic system is metastability.

\textbf{Neurodegenerative diseases} : A group of diseases engendered by
the progressive loss of structure and function of neurons, which is also
known as degenerative nerve diseases. Common examples include
Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease,
Huntington's disease, multiple system atrophy and frontotemporal
dementia.

\textbf{Steric zipper:} The dry and tightly self-complementing structure
formed within double \selectlanguage{greek}β-\selectlanguage{english}sheets of amyloid-like fibrils. This name was
coined by David Eisenberg's group in 2005 because the interdigitating
side chains of \selectlanguage{greek}β-\selectlanguage{english}sheets resemble the teeth of a zipper.

\textbf{Supersaturated:} A protein is supersaturated when its
concentration is higher than the solubility under normal conditions.

\textbf{Outstanding Questions}

How can membraneless organelles attain both supersaturation and high
selectivity of a plethora of biomolecules?

Is it possible to predict phase behavior of IDPs in different
environment simply from the information of sequence?

Why do `upper critical solution temperature' and `lower critical
solution temperature' phase behaviors often exist with intracellular and
extracellular intrinsically disordered proteins, respectively? Is there
any biological significance or reason?

How and to what extent can cells prevent the pathological aggregation
from metastable membraneless organelles?

Is the maturation process in cells inevitable?

How to rescue the stable amyloid-like assemblies by therapeutic
intervention? Will this be an effective pathway for the treatment of
neurodegenerative diseases and cancer?\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

Figure 1. Energy depiction. (A) Schematic illustration of landscape of
Gibbs free energy and stability associated with different possible
materials states. (B) Enthalpy and entropy change of phase transition
from dispersed state to phase-separated state. Four possible scenarios
are shown as four quadrants of an enthalpy--entropy coordinate.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image2/image2}

\end{center}
\end{figure}

Figure 2. Metastable molecular interactions and molecular structures in
LLPS. (A) Typical molecular interactions in MLOs. (B) Schematic of
steric zippers in amyloids and kinked \selectlanguage{greek}β \selectlanguage{english}sheets in IDPs. Side chains in
and out of interfaces are colored in red and green, respectively.
Peptide backbones are in grey. Water molecules (in blue circles) are
drawn for illustrating dry (hydrophobic) and hydrous (hydrophilic)
interfaces of steric zippers and kinked \selectlanguage{greek}β \selectlanguage{english}sheets, respectively.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}

Figure 3. MLOs are metastable biological assemblages associated with
disease. (A) Metastable liquid droplets can be
changeable/interchangeable to other phase states. (B) The maturation
process can be accelerated by disease-associated changes.

Table 1. Structural parameters of kinked \selectlanguage{greek}β \selectlanguage{english}sheets compared with a steric
zipper.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Proteins} & \textbf{Peptide motifs} & \(\mathbf{S}_{\mathbf{c}}\) &
\(\mathbf{A}_{\mathbf{b}}\) & \textbf{Structure formed} &
\textbf{Ref}\tabularnewline
\midrule
\endhead
FUS & \textsuperscript{37}SYSGYS\textsuperscript{42} & 0.91 & 81 &
Kinked \selectlanguage{greek}β \selectlanguage{english}sheets & \textsuperscript{74}\tabularnewline
& \textsuperscript{54}SYSSYGQS\textsuperscript{61} & 0.81 & 120 &
&\tabularnewline
& \textsuperscript{77}STGGYG\textsuperscript{82} & 0.82 & 111 &
&\tabularnewline
hnRNPA1 & \textsuperscript{243}GYNGFG\textsuperscript{248} & 0.86 & 120
& &\tabularnewline
nup98 & \textsuperscript{116}GFGNFGTS\textsuperscript{123} & 0.86 & 114
& &\tabularnewline
Amyloid-\selectlanguage{greek}β & \textsuperscript{27}NKGAII\textsuperscript{32} & 0.86 & 178
& Steric zipper &\tabularnewline
\bottomrule
\end{longtable}

\textbf{Table 2. Metastability of fibrillogenic peptides of LCDs and
amyloids.}\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
\textbf{Proteins} & \textbf{Proteins} & \textbf{Peptide motifs} &
\textbf{Metastability} & \textbf{Melting temperature of fibrils} &
\textbf{Refs}\tabularnewline
\midrule
\endhead
hnRNP & hnRNPA1 & \textsuperscript{209}GFGGNDNFG\textsuperscript{217}
(hnRAC1) & Yes & 4--25 \selectlanguage{ngerman}°C & \textsuperscript{17}\tabularnewline
& & \textsuperscript{246}GFGNDGSNF\textsuperscript{254} (hnRAC2) & &
4--37 \selectlanguage{ngerman}°C &\tabularnewline
& & \textsuperscript{260}YNDFGNY\textsuperscript{266} (hnRAC3) & & 4--25
\selectlanguage{ngerman}°C &\tabularnewline
& hnRNPA2 & \textsuperscript{227}GFGDGYNGYG\textsuperscript{236} & &
4--37 \selectlanguage{ngerman}°C &\tabularnewline
& & \textsuperscript{270}GYGGGYDNYGG\textsuperscript{280} & & 4--37 \selectlanguage{ngerman}°C
&\tabularnewline
& hnRNPDL & \textsuperscript{365}YGGDQNY\textsuperscript{371} & & 4--50
\selectlanguage{ngerman}°C &\tabularnewline
& hnRNPH & \textsuperscript{442}SDFQSN\textsuperscript{447} & & Above 50
\selectlanguage{ngerman}°C &\tabularnewline
& hnRNPK & \textsuperscript{367}SGYDYS\textsuperscript{372} & & Above 50
\selectlanguage{ngerman}°C &\tabularnewline
& hnRNPR & \textsuperscript{615}GYNNDN\textsuperscript{620} & & Above 50
\selectlanguage{ngerman}°C &\tabularnewline
FUS & FUS & \textsuperscript{37}SYSGYS\textsuperscript{42} (RAC1) & &
4--50 \selectlanguage{ngerman}°C & \textsuperscript{55}\tabularnewline
& & \textsuperscript{54}SYSSYG\textsuperscript{59} (RAC2) & & 4--20 \selectlanguage{ngerman}°C
&\tabularnewline
Amyloid-\selectlanguage{greek}β & Amyloid-\selectlanguage{greek}β & \textsuperscript{16}KLVFFA\textsuperscript{21} &
No & No melting (up to 50 \selectlanguage{ngerman}°C) & \textsuperscript{55}\tabularnewline
\bottomrule
\end{longtable}



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