Spatiotemporal organization of coacervate microdroplets

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Abstract

Liquid–liquid phase separation (LLPS) has emerged as a new paradigm in the fields of soft matter, colloid chemistry, prebiotic chemistry, and cell biology. As phase separation is a dynamic assembly process, how to spatiotemporally regulate the assembly and disassembly of these micrometre-sized droplets, which are referred as biomolecular condensates in biology is essential for their diverse applications in various disciplines. Herein, we discuss recent advances in the spatiotemporal control of phase separation using different physical tools and external environmental stimuli in bulk solutions and living cells. Specifically, the exploration of phase transition in a compartmentalized protocellular system, which can bridge the gap between synthetic and intracellular LLPS systems, is summarized, and the challenges and future research directions are discussed.

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.

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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).

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