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1. Phase Separation and Protein Partitioning in Compartmentalized Cell-Free Expression Reactions

   https://doi.org/10.1021/acs.biomac.1c00546  

   Kato, Shuzo; Garenne, David; Noireaux, Vincent; Maeda, Yusuke T. (July 2021, Biomacromolecules)

   null (Ed.)

   Liquid-liquid phase separation (LLPS) is important to control a wide range of reactions from gene expression to protein degradation in a cell-sized space. To bring a better understanding of the compatibility of such phase-separated structures with protein synthesis, we study emergent LLPS in a cell-free transcription-translation (TXTL) reaction. When the TXTL reaction composed of many proteins is concentrated, the uniformly mixed state becomes unstable, and membrane-less phases form spontaneously. This LLPS droplet formation is induced when the TXTL reaction is enclosed in water-in-oil emulsion droplets, in which water evaporates from the surface. As the emulsion droplets shrink, smaller LLPS droplets appear inside the emulsion droplets and coalesce into large phase-separated domains that partition the localization of synthesized reporter proteins. The presence of PEG in the TXTL reaction is important not only for versatile cell-free protein synthesis but also for the formation of two large domains capable of protein partitioning. Our results may shed light on the dynamic interplay of LLPS formation and cell-free protein synthesis toward the construction of synthetic organelles. 

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2. Interfacial Assembly of Bacterial Microcompartment Shell Proteins in Aqueous Multiphase Systems

   https://doi.org/10.1002/smll.202308390  

   Abeysinghe, A_A_Dharani_T; Young, Eric_J; Rowland, Andrew_T; Dunshee, Lucas_C; Urandur, Sandeep; Sullivan, Millicent_O; Kerfeld, Cheryl_A; Keating, Christine_D (December 2023, Small)

   Abstract Compartments are a fundamental feature of life, based variously on lipid membranes, protein shells, or biopolymer phase separation. Here, this combines self‐assembling bacterial microcompartment (BMC) shell proteins and liquid‐liquid phase separation (LLPS) to develop new forms of compartmentalization. It is found that BMC shell proteins assemble at the liquid‐liquid interfaces between either 1) the dextran‐rich droplets and PEG‐rich continuous phase of a poly(ethyleneglycol)(PEG)/dextran aqueous two‐phase system, or 2) the polypeptide‐rich coacervate droplets and continuous dilute phase of a polylysine/polyaspartate complex coacervate system. Interfacial protein assemblies in the coacervate system are sensitive to the ratio of cationic to anionic polypeptides, consistent with electrostatically‐driven assembly. In both systems, interfacial protein assembly competes with aggregation, with protein concentration and polycation availability impacting coating. These two LLPS systems are then combined to form a three‐phase system wherein coacervate droplets are contained within dextran‐rich phase droplets. Interfacial localization of BMC hexameric shell proteins is tunable in a three‐phase system by changing the polyelectrolyte charge ratio. The tens‐of‐micron scale BMC shell protein‐coated droplets introduced here can accommodate bioactive cargo such as enzymes or RNA and represent a new synthetic cell strategy for organizing biomimetic functionality. 

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3. Hydrophobicity of arginine leads to reentrant liquid-liquid phase separation behaviors of arginine-rich proteins

   https://doi.org/10.1038/s41467-022-35001-1  

   Hong, Yuri; Najafi, Saeed; Casey, Thomas; Shea, Joan-Emma; Han, Song-I; Hwang, Dong Soo (November 2022, Nature Communications)

   Abstract Intrinsically disordered proteins rich in cationic amino acid groups can undergo Liquid-Liquid Phase Separation (LLPS) in the presence of charge-balancing anionic counterparts. Arginine and Lysine are the two most prevalent cationic amino acids in proteins that undergo LLPS, with arginine-rich proteins observed to undergo LLPS more readily than lysine-rich proteins, a feature commonly attributed to arginine’s ability to form stronger cation-π interactions with aromatic groups. Here, we show that arginine’s ability to promote LLPS is independent of the presence of aromatic partners, and that arginine-rich peptides, but not lysine-rich peptides, display re-entrant phase behavior at high salt concentrations. We further demonstrate that the hydrophobicity of arginine is the determining factor giving rise to the reentrant phase behavior and tunable viscoelastic properties of the dense LLPS phase. Controlling arginine-induced reentrant LLPS behavior using temperature and salt concentration opens avenues for the bioengineering of stress-triggered biological phenomena and drug delivery systems. 

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4. The solid and liquid states of chromatin

   https://doi.org/10.1186/s13072-021-00424-5  

   Hansen, Jeffrey C.; Maeshima, Kazuhiro; Hendzel, Michael J. (October 2021, Epigenetics & Chromatin)

   Abstract The review begins with a concise description of the principles of phase separation. This is followed by a comprehensive section on phase separation of chromatin, in which we recount the 60 years history of chromatin aggregation studies, discuss the evidence that chromatin aggregation intrinsically is a physiologically relevant liquid–solid phase separation (LSPS) process driven by chromatin self-interaction, and highlight the recent findings that under specific solution conditions chromatin can undergo liquid–liquid phase separation (LLPS) rather than LSPS. In the next section of the review, we discuss how certain chromatin-associated proteins undergo LLPS in vitro and in vivo. Some chromatin-binding proteins undergo LLPS in purified form in near-physiological ionic strength buffers while others will do so only in the presence of DNA, nucleosomes, or chromatin. The final section of the review evaluates the solid and liquid states of chromatin in the nucleus. While chromatin behaves as an immobile solid on the mesoscale, nucleosomes are mobile on the nanoscale. We discuss how this dual nature of chromatin, which fits well the concept of viscoelasticity, contributes to genome structure, emphasizing the dominant role of chromatin self-interaction. 

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5. Liquid–liquid phase separation and viscosity within secondary organic aerosol generated from diesel fuel vapors

   https://doi.org/10.5194/acp-19-12515-2019  

   Song, Mijung; Maclean, Adrian M.; Huang, Yuanzhou; Smith, Natalie R.; Blair, Sandra L.; Laskin, Julia; Laskin, Alexander; DeRieux, Wing-Sy Wong; Li, Ying; Shiraiwa, Manabu; et al (January 2019, Atmospheric Chemistry and Physics)

   Abstract. Information on liquid–liquid phase separation (LLPS) and viscosity (ordiffusion) within secondary organic aerosol (SOA) is needed to improvepredictions of particle size, mass, reactivity, and cloud nucleatingproperties in the atmosphere. Here we report on LLPS and viscosities withinSOA generated by the photooxidation of diesel fuel vapors. Diesel fuelcontains a wide range of volatile organic compounds, and SOA generated bythe photooxidation of diesel fuel vapors may be a good proxy for SOA fromanthropogenic emissions. In our experiments, LLPS occurred over the relativehumidity (RH) range of ∼70 % to ∼100 %,resulting in an organic-rich outer phase and a water-rich inner phase. Theseresults may have implications for predicting the cloud nucleating propertiesof anthropogenic SOA since the presence of an organic-rich outer phase athigh-RH values can lower the supersaturation with respect to water requiredfor cloud droplet formation. At ≤10 % RH, the viscosity was ≥1×108 Pa s, which corresponds to roughly the viscosity of tarpitch. At 38 %–50 % RH, the viscosity was in the range of 1×108 to 3×105 Pa s. These measured viscosities areconsistent with predictions based on oxygen to carbon elemental ratio (O:C)and molar mass as well as predictions based on the number of carbon,hydrogen, and oxygen atoms. Based on the measured viscosities and theStokes–Einstein relation, at ≤10 % RH diffusion coefficients oforganics within diesel fuel SOA is ≤5.4×10-17 cm2 s−1 and the mixing time of organics within 200 nm diesel fuel SOAparticles (τmixing) is 50 h. These small diffusion coefficientsand large mixing times may be important in laboratory experiments, where SOAis often generated and studied using low-RH conditions and on timescales ofminutes to hours. At 38 %–50 % RH, the calculated organic diffusioncoefficients are in the range of 5.4×10-17 to 1.8×10-13 cm2 s−1 and calculated τmixing values arein the range of ∼0.01 h to ∼50 h. These valuesprovide important constraints for the physicochemical properties ofanthropogenic SOA. 

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