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Open accessJournal ArticleDOI: 10.1039/D0SC06418A

Self-programmed enzyme phase separation and multiphase coacervate droplet organization.

04 Mar 2021-Chemical Science (Royal Society of Chemistry (RSC))-Vol. 12, Iss: 8, pp 2794-2802
Abstract: Membraneless organelles are phase-separated droplets that are dynamically assembled and dissolved in response to biochemical reactions in cells. Complex coacervate droplets produced by associative liquid-liquid phase separation offer a promising approach to mimic such dynamic compartmentalization. Here, we present a model for membraneless organelles based on enzyme/polyelectrolyte complex coacervates able to induce their own condensation and dissolution. We show that glucose oxidase forms coacervate droplets with a cationic polysaccharide on a narrow pH range, so that enzyme-driven monotonic pH changes regulate the emergence, growth, decay and dissolution of the droplets depending on the substrate concentration. Significantly, we demonstrate that time-programmed coacervate assembly and dissolution can be achieved in a single-enzyme system. We further exploit this self-driven enzyme phase separation to produce multiphase droplets via dynamic polyion self-sorting in the presence of a secondary coacervate phase. Taken together, our results open perspectives for the realization of programmable synthetic membraneless organelles based on self-regulated enzyme/polyelectrolyte complex coacervation.

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Topics: Coacervate (69%)
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Open accessJournal ArticleDOI: 10.1016/J.COCIS.2021.101488
Jenna K.A. Tom1, Ashok A. Deniz1Institutions (1)
Abstract: Phase transitions and coacervates play key roles in natural and synthetic soft matter. In particular, the past few years have seen a rapid expansion in studies of these phenomena in the context of dynamic cellular compartmentalization. In this brief review, we mainly focus on a few concepts and selected in vitro and cellular examples of recent developments in the areas of dynamics and multicomponent systems. Topics covered include the flexibility and conformational dynamics of polymeric species involved in phase separation, valence and non-monotonic effects, noise modulation and feedback loops, and multicomponent systems and substructure. The fundamental concepts discussed in this review are widely applicable, including in the context of cellular function and the development of materials with novel properties.

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Topics: Context (language use) (51%)

1 Citations


Open accessJournal ArticleDOI: 10.1038/S41467-021-26532-0
Andrea Testa1, Mirco Dindo2, Aleksander A. Rebane1, Babak Nasouri3  +5 moreInstitutions (4)
Abstract: Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

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Journal ArticleDOI: 10.1039/D1CC04596B
Abstract: Dynamic droplet formation via liquid-liquid phase separation (LLPS) is believed to be involved in the regulation of various biological processes. Here, a model LLPS system coupled with a sequential glycolytic enzymatic reaction was developed to reproduce the dynamic control of liquid droplets; (i) the droplets, which consist of poly-L-lysine and nucleotides, compartmentalize two different enzymes (hexokinase and glucose-6-phosphate dehydrogenase) individually, accelerating the overall reaction, and (ii) each enzymatic reaction triggers the formation, dissolution and long-term retention of the droplets by converting the scaffold nucleotides. This model system will offer a new aspect of enzymes associated with LLPS in living cells.

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Open accessJournal ArticleDOI: 10.3389/FMICB.2021.751880
Zixu Gao1, Wenchang Zhang1, Runlei Chang1, Susu Zhang1  +2 moreInstitutions (1)
Abstract: Numerous examples of microbial phase-separated biomolecular condensates have now been identified following advances in fluorescence imaging and single molecule microscopy technologies. The structure, function, and potential applications of these microbial condensates are currently receiving a great deal of attention. By neatly compartmentalizing proteins and their interactors in membrane-less organizations while maintaining free communication between these macromolecules and the external environment, microbial cells are able to achieve enhanced metabolic efficiency. Typically, these condensates also possess the ability to rapidly adapt to internal and external changes. The biological functions of several phase-separated condensates in small bacterial cells show evolutionary convergence with the biological functions of their eukaryotic paralogs. Artificial microbial membrane-less organelles are being constructed with application prospects in biocatalysis, biosynthesis, and biomedicine. In this review, we provide an overview of currently known biomolecular condensates driven by liquid-liquid phase separation (LLPS) in microbial cells, and we elaborate on their biogenesis mechanisms and biological functions. Additionally, we highlight the major challenges and future research prospects in studying microbial LLPS.

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51 results found


Journal ArticleDOI: 10.1126/SCIENCE.1172046
26 Jun 2009-Science
Abstract: In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo Localization of P granules and their physical nature remain poorly understood Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension As with other liquids, P granules rapidly dissolved and condensed Localization occurred by a biased increase in P granule condensation at the posterior This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm

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1,513 Citations


Journal ArticleDOI: 10.1146/ANNUREV-CELLBIO-100913-013325
Abstract: Cells organize many of their biochemical reactions in non-membrane compartments. Recent evidence has shown that many of these compartments are liquids that form by phase separation from the cytoplasm. Here we discuss the basic physical concepts necessary to understand the consequences of liquid-like states for biological functions.

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1,435 Citations


Open accessJournal ArticleDOI: 10.1016/J.MOLCEL.2015.01.013
05 Mar 2015-Molecular Cell
Abstract: Cells chemically isolate molecules in compartments to both facilitate and regulate their interactions. In addition to membrane-encapsulated compartments, cells can form proteinaceous and membraneless organelles, including nucleoli, Cajal and PML bodies, and stress granules. The principles that determine when and why these structures form have remained elusive. Here, we demonstrate that the disordered tails of Ddx4, a primary constituent of nuage or germ granules, form phase-separated organelles both in live cells and in vitro. These bodies are stabilized by patterned electrostatic interactions that are highly sensitive to temperature, ionic strength, arginine methylation, and splicing. Sequence determinants are used to identify proteins found in both membraneless organelles and cell adhesion. Moreover, the bodies provide an alternative solvent environment that can concentrate single-stranded DNA but largely exclude double-stranded DNA. We propose that phase separation of disordered proteins containing weakly interacting blocks is a general mechanism for forming regulated, membraneless organelles.

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Topics: Biological phase (52%), Stress granule (50%)

961 Citations


Open accessJournal ArticleDOI: 10.1016/J.CELL.2016.04.047
Marina Feric1, Nilesh Vaidya1, Tyler S. Harmon2, Diana M. Mitrea3  +5 moreInstitutions (3)
16 Jun 2016-Cell
Abstract: The nucleolus and other ribonucleoprotein (RNP) bodies are membrane-less organelles that appear to assemble through phase separation of their molecular components. However, many such RNP bodies contain internal subcompartments, and the mechanism of their formation remains unclear. Here, we combine in vivo and in vitro studies, together with computational modeling, to show that subcompartments within the nucleolus represent distinct, coexisting liquid phases. Consistent with their in vivo immiscibility, purified nucleolar proteins phase separate into droplets containing distinct non-coalescing phases that are remarkably similar to nucleoli in vivo. This layered droplet organization is caused by differences in the biophysical properties of the phases-particularly droplet surface tension-which arises from sequence-encoded features of their macromolecular components. These results suggest that phase separation can give rise to multilayered liquids that may facilitate sequential RNA processing reactions in a variety of RNP bodies. PAPERCLIP.

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921 Citations


Journal ArticleDOI: 10.1016/J.COCIS.2004.09.006
Abstract: Coacervation of proteins and anionic polysaccharides is both of practical and theoretical interest. From a large body of literature, it seems that the phase separation is mainly entropically driven, and may most probably be attributed to the delocalisation of the counter ions of the protein and the polysaccharide. The protein and polysaccharide appear to form complexes in solution, which can be viewed as new colloidal entities. These complex particles are neutral and exhibit an attractive interaction, which leads to a phase separation of the gas-liquid type in which a (very) dilute colloidal phase coexists with a very concentrated colloidal phase. In the case of strong poly-acids, usually, a precipitate is formed rather than a liquid coacervate phase. The structure of the concentrated polymer phase seems to resemble a continuous polymer phase in which the protein can diffuse around, as well as the individual polysaccharide molecules. Time scales of diffusion vary from milliseconds to days depending on the strength of the interaction. From a rheological point of view, the concentrated phase is much more viscous than elastic and the rheology resembles the behaviour of a (viscous) concentrated particle dispersion. Theoretical developments are limited probably due to the difficulty to describe the (correlated) charge distribution in the system. There is a strong interest in coacervates for the use of encapsulation. For the same reason, much attention is given to replacing the traditional gelatin by milk and plant proteins.

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Topics: Coacervate (55%), Phase (matter) (54%)

907 Citations


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