Designer membraneless organelles sequester native factors for control of cell behavior

Garabedian, Mikael V.; Wang, Wentao; Dabdoub, Jorge; Tong, Michelle; Caldwell, Reese M.; Benman, William; Schuster, Benjamin S.; Deiters, Alexander; Good, Matthew C.

doi:10.1038/s41589-021-00840-4

Cited by 80 publications

(85 citation statements)

References 58 publications

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“…One of the biggest challenges is spatiotemporal control over the time-programmed condensation and disassembly of the coacervate. The time-programmed phase behavior is currently available by changes in pH, temperature, ionic strength, light (UV), and more recently by enzyme-mediated catalytic activity ( Shin et al, 2017 ; Schuster et al, 2018 ; Martin et al, 2019 ; Wheeler et al, 2019 ; Garabedian et al, 2021 ). Furthermore, by converting chemical fuels, the coacervate droplet could behave like a protocell capable of self-division, making it an ideal model for approaching the dynamic complexity of living cells ( Zwicker et al, 2017 ; Donau et al, 2020 ; Karoui et al, 2021 ).…”

Section: Discussionmentioning

confidence: 99%

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

et al. 2021

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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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Section: Discussionmentioning

confidence: 99%

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

et al. 2021

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“…The above-mentioned observations on the specific localization either inside or boundary of the droplet, depending on the conformation of giant DNA molecule is expected to stimulate further studies on the function of micro phase-segregation in living cellular systems [15][16][17][18][19][20][21][22]. Recently, we have shown that red-blood cells and epithelial cells are spontaneously entrapped into DEX-rich droplets under the condition of phase-separation with DEX/PEG aqueous solution [52].…”

Section: Plos Onementioning

confidence: 99%

“…In an aqueous solution of a mixture of semiflexible and flexible polymers, similar to the usual solution conditions for cytoplasm, semiflexible polymers are depleted into a folded compact state or a segregated state by self-assembly [ 12 – 14 ]. In relation to the segregation phenomenon, or w/w phase separation in aqueous solution with multiple macromolecules, membraneless organelles, such as nucleolus, ribosomes, P-body, and stress granules, have recently been attracting considerable interest in the biological sciences [ 15 – 17 ]. The stability of these membraneless organelles, together with experimental studies to construct their models [ 17 , 18 ], have frequently been interpreted in terms of liquid-liquid phase separation [ 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

“…In relation to the segregation phenomenon, or w/w phase separation in aqueous solution with multiple macromolecules, membraneless organelles, such as nucleolus, ribosomes, P-body, and stress granules, have recently been attracting considerable interest in the biological sciences [ 15 – 17 ]. The stability of these membraneless organelles, together with experimental studies to construct their models [ 17 , 18 ], have frequently been interpreted in terms of liquid-liquid phase separation [ 19 , 20 ]. As for the stability and function of membraneless organelles, it is becoming clear that intrinsically disordered proteins (IDPs) exhibiting flexible domains play important roles in a wide variety of cellular functions [ 17 , 21 , 22 ].…”

Section: Introductionmentioning

confidence: 99%

“…The stability of these membraneless organelles, together with experimental studies to construct their models [ 17 , 18 ], have frequently been interpreted in terms of liquid-liquid phase separation [ 19 , 20 ]. As for the stability and function of membraneless organelles, it is becoming clear that intrinsically disordered proteins (IDPs) exhibiting flexible domains play important roles in a wide variety of cellular functions [ 17 , 21 , 22 ]. Such experimental observations suggest that research on the depletion effect in a cytoplasmic environment under crowded macromolecular conditions would promote the basic understanding of living systems.…”

Section: Introductionmentioning

confidence: 99%

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Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

Nishio

Yoshikawa

2021

PLoS ONE

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Background It is becoming clearer that living cells use water/water (w/w) phase separation to form membraneless organelles that exhibit various important biological functions. Currently, it is believed that the specific localization of biomacromolecules, including DNA, RNA and proteins in w/w microdroplets is closely related to their bio-activity. Despite the importance of this possible role of micro segregation, our understanding of the underlying physico-chemical mechanism is still unrefined. Further research to unveil the underlying mechanism of the localization of macromolecules in relation to their steric conformation in w/w microdroplets is needed. Principal findings Single-DNA observation of genome-size DNA (T4 GT7 bacteriophage DNA; 166kbp) by fluorescence microscopy revealed that DNAs are spontaneously incorporated into w/w microdroplets generated in a binary aqueous polymer solution with polyethylene glycol (PEG) and dextran (DEX). Interestingly, DNAs with elongated coil and shrunken conformations exhibit Brownian fluctuation inside the droplet. On the other hand, tightly packed compact globules, as well as assemblies of multiple condensed DNAs, tend to be located near the interface in the droplet. Conclusion and significance The specific localization of DNA molecules depending on their higher-order structure occurs in w/w microdroplet phase-separation solution under a binary aqueous polymer solution. Such an aqueous solution with polymers mimics the crowded conditions in living cells, where aqueous macromolecules exist at a level of 30–40 weight %. The specific positioning of DNA depending on its higher-order structure in w/w microdroplets is expected to provide novel insights into the mechanism and function of membraneless organelles and micro-segregated particles in living cells.

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Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells

et al. 2023

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Membraneless organelles are important for spatial organization of proteins and regulation of intracellular processes. Proteins can be recruited to these condensates by specific protein–protein or protein–nucleic acid interactions, which are often regulated by post‐translational modifications. However, the mechanisms behind these dynamic, affinity‐based protein recruitment events are not well understood. Here, a coacervate system that incorporates the 14‐3‐3 scaffold protein to study enzymatically regulated recruitment of 14‐3‐3‐binding proteins is presented, which mostly bind in a phosphorylation‐dependent manner. Synthetic coacervates are efficiently loaded with 14‐3‐3, and phosphorylated binding partners, such as the c‐Raf pS233/pS259 peptide (c‐Raf), show 14‐3‐3‐dependent sequestration with up to 161‐fold increase in local concentration. The c‐Raf domain is fused to green fluorescent protein (GFP‐c‐Raf) to demonstrate recruitment of proteins. In situ phosphorylation of GFP‐c‐Raf by a kinase leads to enzymatically regulated uptake. The introduction of a phosphatase into coacervates preloaded with the phosphorylated 14‐3‐3‐GFP‐c‐Raf complex results in a significant cargo efflux mediated by dephosphorylation. Finally, the general applicability of this platform to study protein–protein interactions is demonstrated by the phosphorylation‐dependent and 14‐3‐3‐mediated active reconstitution of a split‐luciferase inside artificial cells. This work presents an approach to study dynamically regulated protein recruitment in condensates, using native interaction domains.

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Designer membraneless organelles sequester native factors for control of cell behavior

Cited by 80 publications

References 58 publications

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

Enzymatic Regulation of Protein–Protein Interactions in Artificial Cells

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