Spatiotemporal organization of coacervate microdroplets
Graphical abstract
The spatiotemporal control of LLPS in bulk solution and biological systems are summarized with the aspects of physical stimuli, chemical stimuli and structural alteration of chemical components for the phase separation. The exploration of the phase transition in compartmentalized protocellular system, that can bridge the gap between these two systems, are summarized, and the challenges and future research directions are put forward.
Introduction
Coacervation which was first observed in a system of gum Arabic and gelatin in 1929 [1], is an associative liquid–liquid phase separation (LLPS) phenomenon, leading to the formation of a dense colloidal-rich liquid phase coexisting with a diluted phase [2]. The dense phase with a relatively large content of colloidal components is called coacervate. It is generally believed that the translation entropy gain due to counterion release is the main driving force for complex coacervation when mixing a polycationic solution with polyanionic one [3,4]. The theoretical understanding of the mechanism of complex coacervation in mixtures of oppositely charged polyelectrolytes dates back to the pioneering work by Voon and Overbeek in 1957; this classical theory is commonly termed VO theory in the literature [5]. In this theory, free energy is composed of a Flory-Huggins type mixing entropy between all components and an electrostatic correlation in terms of Debye-Hückel form, with the latter playing the role of driving force. This theory qualitatively captures a variety of experimental observations on complex coacervation in bulk, such as the suppression of phase separation when the charge fraction is sufficiently weak or the salt concentration is sufficiently high [5,6]. Despite the fact that coacervation has been discovered almost a century ago, it still attracts great interest because of its wide applications in water treatment, material purification, pharmaceutical micro-encapsulations, as well as food products [7]. Coacervate microdroplets can regulate the enrichment and exclusion of biomolecular components [8, 9, 10, 11, 12], enhance the catalysis activity of ribonucleic acid [13,14], realize gene expression [15,16] and cascade enzyme reactions [17,18], and mimic the macromolecular crowding cytosol milieu [19,20]. Moreover, coacervate microdroplets have been exploited as a protocell model to mimic the essential functions of natural cells [2] or to advance our understanding of the emergence of life in primitive Earth [21].
Recently, more and more membraneless organelles, like stress granules [22, 23, 24], Balbiani bodies [25], and condensates inside cell nucleus like nucleolus, Cajal Bodies [26], PML bodies [26], and paraspeckle [26], have been found ubiquitously to be arisen from LLPS in biological systems, since Hyman's group firstly showed that germ granules (P granules) exhibited liquid-like behaviors in 2009 [27]. These coacervate analogues in living cells, such as RNA granules, RNP bodies, and nuclear bodies, can be generally referred as biomolecular condensates [28,29]. They can buffer cellular noise [30,31], act as bio-reactors for chemical reactions [32] and drug delivery systems for cancer therapeutics [33,34], promote receptor signal transduction [35], and give rise to some neurodegenerative diseases due to the aberrant phase transitions of proteins [36]. In many cases, these biomolecular condensates usually contain proteins with repeats of weakly binding interaction domains, which may bind to either RNA or complementary binding partners on other proteins, and interactions between these multivalent motifs provide main driving force for intracellular liquid–liquid phase separation [29]. However, theoretical understanding of such phase separations remains very limited.
As LLPS is a dynamic assembly process, coacervate microdroplets can readily be formed and dissolved in response to changes in concentrations of synthetic polyelectrolytes, biomolecules, or environmental conditions. Therefore, how to spatiotemporally regulate the assembly and disassembly of these micrometre-sized droplets becomes essential for their diverse applications in various disciplines. For instance, the spatiotemporal organization of coacervate microdroplets can provide insight into the origin of life on Earth [8,9,21,37,38], can be used as a model system to study the signaling pathways in protocelluar community [17,18,39], or can offer potential mechanisms for intracellular non-membranous organelles formation in a compartmentalized system [40∗, 41, 42∗∗, 43, 44]. In biology, as many intracellular non-membrane organelles are biomolecule condensates of RNA molecules and proteins [45,46], the dynamic spatiotemporal control of the assembly and disassembly of these micrometre-sized biomolecule condensates plays a key role in some important functions, such as the distribution of chromatin and organelles during cell mitosis [47, 48, 49], and they can be used to fine tune biochemical reactions and maintain cellular homeostasis [22,29].
In this paper, we review recent advances in the spatiotemporal control of coacervate microdroplets using different physical tools and discuss how to use enzymatic cascade reactions and external environmental stimuli to induce phase transition. Also, the spatiotemporal organization of biomolecular condensates in living cells under physical, chemicals stimuli and structural alteration is highlighted. Specifically, the exploration of phase transition in compartmentalized protocellular systems which can bridge the gap between these two systems is summarized, and challenges and future research directions are discussed.
Section snippets
Spatiotemporal organization of coacervate microdroplets in solution
To date, different approaches have been developed for the spatial organization of coacervate microdroplets in solutions. They can be either spatially separated in a well-defined force field or physically isolated in hydrogel. These physical isolation techniques can give rise to the spatial resolution in a population of coacervate droplets, or build boundaries between different populations of coacervate droplets. Moreover, phase transition in a population of coacervate droplets can be modulated
Spatiotemporal organization of coacervate microdroplets in compartmentalized chemical systems
Coacervation in compartmentalized chemical systems provides a defined volume, facilitating the capture of the dynamical process such as the nucleation, growth, dissolution of coacervate, as well as the associated chemical reactions [40]. Coacervate and biomolecular condensates as synthetic membraneless organelles being loaded in compartmentalized chemical system to achieve new biochemical functionalities can mimic the cell and subcellular entities in both structural and functional points [76,79
Spatiotemporal control of LLPS in living cells
Many intracellular non-membrane organelles are dynamic assemblies of RNA molecules and proteins [45,46], and can readily be formed and dissolved in response to changes in concentrations or environmental conditions. Spatiotemporal regulation of the assembly and disassembly of these micrometre-sized biomolecule condensates plays a key role in tuning biochemical reactions and maintaining cellular homeostasis [22,29], or in the distribution of chromatin and organelles during cell mitosis [47, 48, 49
Conclusions and perspectives
LLPS has emerged as a new paradigm leading to a better understanding of subcellular organization, the emergence of life in primitive Earth, and the origins of many diseases. As a dynamic assembly process, the advancement on the spatiotemporal organization of coacervate microdroplets, which are referred as biomolecular condensates in biology, can reveal the underlying molecular and biophysical principles for the phase transition process, hence further promotes their diverse applications in
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the Hundred Talents Program of Zhejiang University, the Fundamental Research Funds for the Central Universities (No. 2020FZZX001-05), and China Postdoctoral Science Foundation (No. 2020TQ0564). P. Z. also acknowledges the financial support provided by the National Natural Science Foundation of China (No. 21803011).
References (114)
- et al.
Fatty acid membrane assembly on coacervate microdroplets as a step towards a hybrid protocell model
Nat Chem
(2014) - et al.
ATPase-modulated stress granules contain a diverse proteome and substructure
Cell
(2016) - et al.
Assembly of the nuclear transcription and processing machinery: Cajal bodies (coiled bodies) and transcriptosomes
Mol Biol Cell
(1999) - et al.
Germline P granules are liquid droplets that localize by controlled dissolution/condensation
Science
(2009) - et al.
Physical principles and extant biology reveal roles for RNA-containing membraneless compartments in origins of life chemistry
Biochemistry
(2018) - et al.
Spatiotemporal control of intracellular phase transitions using light-activated optoDroplets
Cell
(2017) - et al.
Coexisting liquid phases underlie nucleolar subcompartments
Cell
(2016) - et al.
Reversible generation of coacervate droplets in an enzymatic network
Soft Matter
(2018) - et al.
Controllable protein phase separation and modular recruitment to form responsive membraneless organelles
Nat Commun
(2018) - et al.
Phosphorylation-mediated RNA/peptide complex coacervation as a model for intracellular liquid organelles
Nat Chem
(2016)
Stress-triggered phase separation is an adaptive, evolutionarily tuned response
Cell
Phase separation of a yeast prion protein promotes cellular fitness
Science
Reversible compartmentalization of de novo purine biosynthetic complexes in living cells
Science
Kruyt: coacervation (partial miscibility in colloid systems)
Proc Koninklijke Nederl Akademie Wetenschappen
Dynamic synthetic cells based on liquid–liquid phase separation
Chembiochem
Polyelectrolyte complexation
Adv Chem Phys
Recent progress in the science of complex coacervation
Soft Matter
Phase separation in polyelectrolyte solutions: theory of complex coacervation
J Cell Comp Physiol
Binodal compositions of polyelectrolyte complexes
Macromolecules
Biopolymer-based coacervates: structures, functionality and applications in food products
Curr Opin Colloid Interface Sci
Peptide-nucleotide microdroplets as a step towards a membrane-free protocell model
Nat Chem
Polymer/nucleotide droplets as bio-inspired functional micro-compartments
Soft Matter
Protein encapsulation via polypeptide complex coacervation
ACS Macro Lett
Selective uptake and refolding of globular proteins in coacervate microdroplets
Langmuir
RNA catalysis through compartmentalization
Nat Chem
Polyanion-assisted ribozyme catalysis inside complex coacervates
ACS Chem Biol
Enhanced transcription rates in membrane-free protocells formed by coacervation of cell lysate
Proc Natl Acad Sci USA
In vitro gene expression within membrane-free coacervate protocells
Chem Commun
Hydrogel-immobilized coacervate droplets as modular microreactor assemblies
Angew Chem Int Ed
Nonequilibrium spatiotemporal sensing within acoustically patterned two-Dimensional protocell arrays
ACS Cent Sci
Mimicking cellular compartmentalization in a hierarchical protocell through spontaneous spatial organization
ACS Cent Sci
Spatial organization in proteinaceous membrane-stabilized coacervate protocells
Small
Systems of creation: the emergence of life from nonliving matter
Acc Chem Res
Stress granules and cell signaling: more than just a passing phase?
Trends Biochem Sci
Chronic optogenetic induction of stress granules is cytotoxic and reveals the evolution of ALS-FTD pathology
Elife
Amyloid-like self-assembly of a cellular compartment
Cell
Biomolecular condensates: organizers of cellular biochemistry
Nat Rev Mol Cell Biol
Liquid phase condensation in cell physiology and disease
Science
Can phase separation buffer cellular noise?
Science
Phase separation provides a mechanism to reduce noise in cells
Science
Formation and functionalization of membraneless compartments in Escherichia coli
Nat Chem Biol
Partitioning of cancer therapeutics in nuclear condensates
Science
Drug delivery to solid tumors by elastin-like polypeptides
Adv Drug Deliv Rev
Phase separation of signaling molecules promotes T cell receptor signal transduction
Science
Liquid-liquid phase separation in disease
Annu Rev Genet
The origin of life
Spontaneous assembly of chemically encoded two-dimensional coacervate droplet arrays by acoustic wave patterning
Nat Commun
Spatiotemporal control of coacervate formation within liposomes
Nat Commun
pH-Controlled coacervate−membrane interactions within liposomes
ACS Nano
Dynamic spatial formation and distribution of intrinsically disordered protein droplets in macromolecularly crowded protocells
Angew Chem Int Ed
Cited by (19)
Endoskeletal coacervates with mobile-immobile duality for long-term utility
2023, Chemical Engineering JournalEngineering strategies for sustainable synthetic cells
2022, Trends in ChemistryCitation Excerpt :Multicompartmentalization is another characteristic in living systems, in which distinct subcompartments have specialized microenvironments with selectively partitioning of biomolecules. The dynamic spatiotemporal control of the assembly and disassembly of different subcompartments, such as membraneless organelles, plays a key role to regulate the localization of functional biomolecules, which can be used to control biochemical reactions and maintain cellular homeostasis [83–85]. The construction of their synthetic analogs has also been achieved in various synthetic cellular systems by encapsulating membraneless organelles into various synthetic cellular systems [56,86–91].
Rheological characterization of β-lactoglobulin/lactoferrin complex coacervates
2022, LWTCitation Excerpt :Then, the formation of soluble complexes from these primary units (building blocks) according to a not completely elucidated mechanism. The third step is the growth step with the formation of micrometric droplets characteristic of complex coacervation (Wang, Zhang, & Tian, 2021) and finally, the coalescence of these droplets with gentle LLPS into a dense phase (coacervates) and dilute phase (Jho, Yoo, Lin, Han, & Hwang, 2017). Various research works allowed progress in the understanding of the mechanisms of the complex coacervation.
Liquid-liquid phase separated microdomains of an amphiphilic graft copolymer in a surfactant-rich medium
2022, Journal of Colloid and Interface ScienceCitation Excerpt :The results here gathered expand our knowledge on self-coacervation of grafted copolymers and define the conditions in which LLPS microdomains can be obtained in a surfactant-rich medium, which can be easily implemented in the body/home care, cosmetic, and food fields. Our findings sum up on the emerging interest to effectively control the formation of LLPS microdomains [112,113] and be used as possible in vitro models to mimic membrane-less organelles of living cells [114]. In addition, the comprehension of the formation of PEG-g-PVAc LLPS microdomains also brings directional insights on how to destabilize them, providing the first step towards a future study for the triggered release of any active molecule contained within them.
Hierarchical Structuration in Protocellular System
2023, Small Methods