RESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Dextranos , Proteínas Bacterianas/metabolismoRESUMEN
Intracellular condensates formed through liquid-liquid phase separation (LLPS) primarily contain proteins and RNA. Recent evidence points to major contributions of RNA self-assembly in the formation of intracellular condensates. As the majority of previous studies on LLPS have focused on protein biochemistry, effects of biological RNAs on LLPS remain largely unexplored. In this study, we investigate the effects of crowding, metal ions, and RNA structure on formation of RNA condensates lacking proteins. Using bacterial riboswitches as a model system, we first demonstrate that LLPS of RNA is promoted by molecular crowding, as evidenced by formation of RNA droplets in the presence of polyethylene glycol (PEG 8K). Crowders are not essential for LLPS, however. Elevated Mg2+ concentrations promote LLPS of specific riboswitches without PEG. Calculations identify key RNA structural and sequence elements that potentiate the formation of PEG-free condensates; these calculations are corroborated by key wet-bench experiments. Based on this, we implement structure-guided design to generate condensates with novel functions including ligand binding. Finally, we show that RNA condensates help protect their RNA components from degradation by nucleases, suggesting potential biological roles for such higher-order RNA assemblies in controlling gene expression through RNA stability. By utilizing both natural and artificial RNAs, our study provides mechanistic insight into the contributions of intrinsic RNA properties and extrinsic environmental conditions to the formation and regulation of condensates comprised of RNAs.
Asunto(s)
Proteínas Bacterianas/metabolismo , Endorribonucleasas/metabolismo , Estabilidad del ARN , ARN Bacteriano/química , ARN Bacteriano/metabolismo , Riboswitch , Extracción Líquido-Líquido , Magnesio/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/aislamiento & purificaciónRESUMEN
The use of aqueous polymer-based phase separation within water-in-oil emulsion droplets provides a powerful platform for exploring the impact of compartmentalization and preferential partitioning on biologically relevant solutes. By forming an emulsion, a bulk solution is converted into a large number of chemically isolated microscale droplets. Microfluidic techniques provide an additional level of control over the formation of such systems. This enables the selective production of multiphase droplets with desired solution compositions and specific characteristics, such as solute partitioning. Here, we demonstrate control over the chemical microenvironment by adjusting the composition to increase tie line length for poly(ethylene glycol) (PEG)-dextran aqueous two-phase systems (ATPS) encapsulated within multiphase water-in-fluorocarbon oil emulsion droplets. Through rational adjustment of microfluidic parameters alone, ATPS droplets containing differing compositions could be produced during the course of a single experiment, with the produced droplets demonstrating a controllable range of tie line lengths. This provided control over partitioning behavior for biologically relevant macromolecules such that the difference in local protein concentration between adjacent phases could be rationally tuned. This work illustrates a broadly applicable technique to rationally create emulsified multiphase aqueous systems of desired compositions through the adjustment of microfluidic parameters alone, allowing for easy and rapid screening of various chemical microenvironments.
Asunto(s)
Fluorocarburos , Microfluídica , Emulsiones , Polietilenglicoles/química , Agua/químicaRESUMEN
We report the formation of coacervate-supported phospholipid membranes by hydrating a dried lipid film in the presence of coacervate droplets. Coacervate-supported membranes were characterized by fluorescence imaging, polarization, fluorescence recovery after photobleaching of labeled lipids, lipid quenching experiments, and solute uptake experiments. Our findings are consistent with the presence of lipid membranes around the coacervates, with many droplets fully coated by what appear to be continuous lipid bilayers. In contrast to traditional giant lipid vesicles formed by gentle hydration in the absence of coacervates, the coacervate-templated membrane vesicles are more uniform in size, shape, and apparent lamellarity. Due to their fully coacervate model cytoplasm, these simple artificial cells are macromolecularly crowded and can be easily pre-loaded with high concentrations of proteins or nucleic acids. Within the same population, in addition to coacervate droplets having intact lipid membrane coatings, other coacervate droplets are coated with membranes having defects or pores that permit solute entry, and some are coated with multilayered membranes. Membranes surrounding protein-based coacervate droplets provided protection from a protease added to the external solution. The simplicity of producing artificial cells having a coacervate model cytoplasm surrounded by a model membrane is at the same time interesting as a potential mechanism for prebiotic protocell formation and appealing for biotechnology. We anticipate that such structures could serve as a new type of model system for understanding interactions between intracellular phases and cell or organelle membranes, which are implicated in a growing number of processes ranging from neurotransmission to signaling.
Asunto(s)
Células Artificiales , Fosfolípidos , Membrana Celular , Membrana Dobles de Lípidos , MembranasRESUMEN
Macromolecular condensation resulting from biologically regulated liquid-liquid phase separation is emerging as a mechanism to organize intracellular space in eukaryotes, with broad implications for cell physiology and pathology. Despite their small size, bacterial cells are also organized by proteins such as FtsZ, a tubulin homolog that assembles into a ring structure precisely at the cell midpoint and is required for cytokinesis. Here, we demonstrate that FtsZ can form crowding-induced condensates, reminiscent of those observed for eukaryotic proteins. Formation of these FtsZ-rich droplets occurs when FtsZ is bound to SlmA, a spatial regulator of FtsZ that antagonizes polymerization, while also binding to specific sites on chromosomal DNA. The resulting condensates are dynamic, allowing FtsZ to undergo GTP-driven assembly to form protein fibers. They are sensitive to compartmentalization and to the presence of a membrane boundary in cell mimetic systems. This is a novel example of a bacterial nucleoprotein complex exhibiting condensation into liquid droplets, suggesting that phase separation may also play a functional role in the spatiotemporal organization of essential bacterial processes.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Citocinesis/genética , Proteínas del Citoesqueleto/genética , ADN Bacteriano/genética , Proteínas de Escherichia coli/genética , Sitios de Unión , Escherichia coli/genética , Extracción Líquido-Líquido , Unión Proteica , Multimerización de ProteínaRESUMEN
Vesicle-stabilized all-aqueous emulsion droplets are appealing as bioreactors because they provide uniform encapsulation via equilibrium partitioning without restricting diffusion in and out of the interior. These properties rely on the composition of the aqueous two-phase system (ATPS) chosen for the emulsion and the structure of the interfacial liposome layer, respectively. Here, we explore how changing the aqueous two-phase system from a standard poly(ethyleneglycol), PEG, 8 kDa/dextran 10 kDa ATPS to PEG 8 kDa/Ficoll 70 kDa or PEG 8 kDa/Na2SO4 systems impacts droplet uniformity and partitioning of a model solute (U15 oligoRNA). We also compare liposomes formed by two different methods, both of which begin with multilamellar, polydisperse vesicles formed by gentle hydration: (1) extrusion, which produced vesicles of 150 nm average diameter, and (2) vortexing, which produced vesicles of 270 nm average diameter. Our data illustrate that while droplet uniformity and stability are somewhat better for samples based on extruded vesicles, extrusion is not necessary to create functional microreactors, as emulsions stabilized with vortexed liposomes are just as effective at solute partitioning and allow diffusion across the droplet's liposome corona. This work expands the compositions possible for liposome-stabilized, all-aqueous emulsion droplet bioreactors, making them amenable to a wider range of potential reactions. Replacing the liposome extrusion step with vortexing can reduce time and cost of bioreactor production with only modest reductions in emulsion quality.
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Dextranos , Liposomas , Emulsiones , Ficoll , Polietilenglicoles , SulfatosRESUMEN
Biological systems employ liquid-liquid phase separation to localize macromolecules and processes. The properties of intracellular condensates that allow for multiple, distinct liquid compartments and the impact of their coexistence on phase composition and solute partitioning are not well understood. Here, we generate two and three coexisting macromolecule-rich liquid compartments by complex coacervation based on ion pairing in mixtures that contain two or three polyanions together with one, two, or three polycations. While in some systems polyelectrolyte order-of-addition was important to achieve coexisting liquid phases, for others it was not, suggesting that the observed multiphase droplet morphologies are energetically favorable. Polyelectrolytes were distributed across all coacervate phases, depending on the relative interactions between them, which in turn impacted partitioning of oligonucleotide and oligopeptide solutes. These results show the ease of generating multiphase coacervates and the ability to tune their partitioning properties via the polyelectrolyte sharing inherent to multiphase complex coacervate systems.
Asunto(s)
Sustancias Macromoleculares/química , Polielectrolitos/química , Colorantes Fluorescentes/química , Oligonucleótidos/química , Oligopéptidos/química , Compuestos de Quinolinio/químicaRESUMEN
Sub-wavelength chiral resonators formed from artificial structures exhibit exceedingly large chiroptical responses compared to those observed in natural media. Owing to resonant excitation, chiral near fields can be significantly enhanced for these resonators, holding great promise for developing enantioselective photonic components such as biochemical sensors based on circular dichroism (CD) and spin-dependent nonlinear imaging. In the present work, strong linear and nonlinear chiroptical responses (scattering CD > 0.15 and nonlinear differential CDs > 0.4) at visible and near infrared frequencies are reported for the first time for individual micrometer-scale plasmonic and dielectric helical structures. By leveraging dark-field spectroscopy and nonlinear optical microscopy, the circular-polarization-selective scattering behavior and nonlinear optical responses (e.g., second harmonic generation and two-photon photoluminescence) of 3D printed micro-helices with feature sizes comparable to the wavelength (total length is â¼5λ) are demonstrated. These micro-helices provide potential for readily accessible photonic platforms, facilitating an enantiomeric analysis of chiral materials. One such example is the opportunity to explore ultracompact photonic devices based on single, complex meta-atoms enabled by state-of-the-art 3D fabrication techniques.
RESUMEN
Compartmentalization by complex coacervation is important across a range of different fields including subcellular and prebiotic organization, biomedicine, food science, and personal care products. Often, lipid self-assemblies such as vesicles are also present intracellularly or in commercial formulations. A systematic understanding of how phospholipid vesicles interact with different complex coacervates could provide insight and improve control over these systems. In this manuscript, anionic phospholipid vesicles were added to a series of different complex coacervate samples in which coacervates were formed by mixing one of five polycations with one of three (poly)anions that varied in chemical structure and length. Vesicles were found to assemble at the coacervate/continuous phase interface and/or form aggregates. We report how factors such as the charge density of polyelectrolytes and the charge ratio of cationic-to-anionic moieties impact the vesicle distribution in coacervate samples. Our findings emphasize the importance of interactions between vesicles and polycations in the dilute supernatant phase for determining whether the vesicles aggregate prior to assembly at the liquid-liquid interface. The uptake of an RNA oligonucleotide (A15) was also investigated to understand the effect of these liposome coatings on diffusion into coacervate droplets. Systems in which uniform vesicle coronas assemble around coacervate droplets without restricting the entry of biomolecules such as RNAs could be of interest as bioreactors.
RESUMEN
This Perspective focuses on RNA in biological and nonbiological compartments resulting from liquid-liquid phase separation (LLPS), with an emphasis on origins of life. In extant cells, intracellular liquid condensates, many of which are rich in RNAs and intrinsically disordered proteins, provide spatial regulation of biomolecular interactions that can result in altered gene expression. Given the diversity of biogenic and abiogenic molecules that undergo LLPS, such membraneless compartments may have also played key roles in prebiotic chemistries relevant to the origins of life. The RNA World hypothesis posits that RNA may have served as both a genetic information carrier and a catalyst during the origin of life. Because of its polyanionic backbone, RNA can undergo LLPS by complex coacervation in the presence of polycations. Phase separation could provide a mechanism for concentrating monomers for RNA synthesis and selectively partition longer RNAs with enzymatic functions, thus driving prebiotic evolution. We introduce several types of LLPS that could lead to compartmentalization and discuss potential roles in template-mediated non-enzymatic polymerization of RNA and other related biomolecules, functions of ribozymes and aptamers, and benefits or penalties imparted by liquid demixing. We conclude that tiny liquid droplets may have concentrated precious biomolecules and acted as bioreactors in the RNA World.
Asunto(s)
Compartimento Celular/genética , Proteínas Intrínsecamente Desordenadas/genética , Fenómenos Físicos , ARN/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Sustancias Macromoleculares/química , Membranas/química , Origen de la Vida , Transición de Fase , ARN/químicaRESUMEN
General acid-base catalysis is a key mechanistic strategy in protein and RNA enzymes. Ribozymes use hydrated metal ions, nucleobases, and organic cofactors to carry this out. In most small ribozymes, a guanosine is positioned to participate in proton transfer with the nucleophilic 2'-OH. The unshifted pKa values for nucleobases and solvated metal ions are far from neutrality, however, and thus nonideal for general acid-base catalysis. Herein, evidence is provided for cooperative interaction in the hammerhead ribozyme among the guanine that interacts with the nucleophilic 2'-OH, G12, the -1 nucleobase C17, and Mg2+ ions. We introduce global fitting for analyzing ribozyme rate-pH data parametric in Mg2+ concentration and benchmark this method on data from the hepatitis delta virus ribozyme. We then apply global fitting to new rate-pH data for the hammerhead ribozyme using a minimal three-dimensional, four-channel cooperative model. The value for the pKa of G12 that we obtain is channel-dependent and varies from 8.1 to 9.9, shifting closest toward neutrality in the presence of two cationic species: C17H+ and a Mg2+ ion. The value for the pKa of the -1 nucleotide, C17, is increased a remarkable 3.5-5 pKa units toward neutrality. Shifting of the pKa of C17 appears to be driven by an electrostatic sandwich of C17 between carbonyl groups of the 5'-neighboring U and of G12 and involves cation-π interactions. Rate-pH profiles reveal that the major reactive channel under biological Mg2+ and pH involves a cationic C17 rather than a second metal ion. Substitution of a cationic base for a metal underscores the versatility of RNA.
Asunto(s)
Virus de la Hepatitis Delta/enzimología , ARN Catalítico/química , Catálisis , Concentración de Iones de Hidrógeno , Magnesio/química , Modelos Moleculares , Unión Proteica , Estructura Secundaria de ProteínaRESUMEN
We report the effect of topographical features on gold nanowire assemblies in a vertically applied AC electric field. Nanowires 300 nm in diameter ×2.5 µm long, and coated with â¼30 nm silica shell, were assembled in aqueous solution between top and bottom electrodes, where the bottom electrode was patterned with cylindrical dielectric posts. Assemblies were monitored in real time using optical microscopy. Dielectrophoretic and electrohydrodynamic forces were manipulated through frequency and voltage variation, organizing nanowires parallel to the field lines, i.e., standing perpendicular to the substrate surface. Field gradients around the posts were simulated and assembly behavior was experimentally evaluated as a function of patterned feature diameter and spacing. The electric field gradient was highest around these topographic features, which resulted in accumulation of vertically oriented nanowires around the post perimeters when dielectrophoresis dominated (high AC frequency) or between the posts when electrohydrodynamics dominated (low AC frequency). This general type of reconfigurable assembly, coupled with judicious choice of nanowire and post materials/dimensions, could ultimately enable new types of optical materials capable of switching between two functional states by changing the applied field conditions.
RESUMEN
Phase separation of aqueous solutions containing polyelectrolytes can lead to formation of dense, solute-rich liquid droplets referred to as coacervates, surrounded by a dilute continuous phase of much larger volume. This type of liquid-liquid phase separation is thought to help explain the appearance of polyelectrolyte-rich intracellular droplets in the cytoplasm and nucleoplasm of extant biological cells and may be relevant to protocellular compartmentalization of nucleic acids on the early Earth. Here we describe complex coacervates formed upon mixing the polycation poly(allylamine) (PAH, 15 kDa) with the anionic nucleotides adenosine 5'-mono-, di-, and triphosphate (AMP, ADP, and ATP). Droplet formation was observed over a wide range of pH and MgCl2 concentrations. The nucleotides themselves as well as Mg(2+) and RNA oligonucleotides were all extremely concentrated within the coacervates. Nucleotides present at just 2.5 mM in bulk solution had concentrations greater than 1 M inside the coacervate droplets. A solution with a total Mg(2+) concentration of 10 mM had 1-5 M Mg(2+) in the coacervates, and RNA random sequence (N54) partitioned â¼10,000-fold into the coacervates. Coacervate droplets are thus rich in nucleotides, Mg(2+), and RNA, providing a medium favorable for generating functional RNAs. Compartmentalization of nucleotides at high concentrations could have facilitated their polymerization to form oligonucleotides, which preferentially accumulate in the droplets. Locally high Mg(2+) concentrations could have aided folding and catalysis in an RNA world, making coacervate droplets an appealing platform for exploring protocellular environments.
Asunto(s)
Nucleótidos de Adenina/química , Sustancias Macromoleculares/química , Magnesio/química , Oligorribonucleótidos/química , Poliaminas/química , Concentración de Iones de HidrógenoRESUMEN
Liquid-liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length-dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.
Asunto(s)
ARN/química , Espermidina/química , Espermina/química , Liposomas Unilamelares/química , Secuencia de Aminoácidos , Células Artificiales , Recuperación de Fluorescencia tras Fotoblanqueo , Glicerofosfolípidos/química , Conformación de Ácido Nucleico , Oligorribonucleótidos/química , Péptidos/química , Transición de Fase , Temperatura de TransiciónRESUMEN
Subcellular compartmentalization of biomolecules and their reactions is common in biology and provides a general strategy for improving and/or controlling kinetics in metabolic pathways that contain multiple sequential enzymes. Enzymes can be colocalized in multiprotein complexes, on scaffolds or inside subcellular organelles. Liquid organelles formed by intracellular phase coexistence could provide an additional means of sequential enzyme colocalization. Here we use experiment and computation to explore the kinetic consequences of sequential enzyme compartmentalization into model liquid organelles in a crowded polymer solution. Two proteins of the de novo purine biosynthesis pathway, ASL (adenylosuccinate lyase, Step 8) and ATIC (5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase, Steps 9 and 10), were studied in a polyethylene glycol/dextran aqueous two-phase system. Dextran-rich phase droplets served as model liquid compartments for enzyme colocalization. In this system, which lacks any specific binding interactions between the phase-forming polymers and the enzymes, we did not observe significant rate enhancements from colocalization for the overall reaction under our experimental conditions. The experimental results were used to adapt a mathematical model to quantitatively describe the kinetics. The mathematical model was then used to explore additional, experimentally inaccessible conditions to predict when increased local concentrations of enzymes and substrates can (or cannot) be expected to yield increased rates of product formation. Our findings indicate that colocalization within these simplified model liquid organelles can lead to enhanced metabolic rates under some conditions, but that very strong partitioning into the phase that serves as the compartment is necessary. In vivo, this could be provided by specific binding affinities between components of the liquid compartment and the molecules to be localized within it.
Asunto(s)
Adenilosuccinato Liasa/metabolismo , Compartimento Celular , Transferasas de Hidroximetilo y Formilo/metabolismo , Modelos Biológicos , Complejos Multienzimáticos/metabolismo , Nucleótido Desaminasas/metabolismo , Adenilosuccinato Liasa/química , Humanos , Transferasas de Hidroximetilo y Formilo/química , Liposomas/química , Complejos Multienzimáticos/química , Nucleótido Desaminasas/químicaRESUMEN
Mineral deposition within living cells relies on control over the distribution and availability of precursors as well as the location and rates of nucleation and growth. This control is provided in large part by biomolecular chelators, which bind precursors and regulate their availability, and compartmentalization within specialized mineralizing vesicles. Biomimetic mineralization in self-assembled lipid vesicles is an attractive means of studying the mineralization process, but has proven challenging due to vesicle heterogeneity in lamellarity, contents, and size across a population, difficulties encapsulating high and uniform precursor concentrations, and the need to transport reagents across an intact lipid bilayer membrane. Here, we report the use of liposome-stabilized all-aqueous emulsion droplets as simple artificial mineralizing vesicles (AMVs). These biomimetic microreactors allow the entry of precursors while retaining a protein catalyst by equilibrium partitioning between internal and external polymer-rich phases. Small molecule chelators with intermediate binding affinity were employed to control Ca(2+) availability during CaCO3 mineralization, providing protection against liposome aggregation while allowing CaCO3 formation. Mineral deposition was limited to the AMV interior, due to localized production of CO3(2-) by compartmentalized urease. Particle formation was uniform across the entire population of AMVs, with multiple submicrometer amorphous CaCO3 particles produced in each one. The all-aqueous emulsion-based approach to biomimetic giant mineral deposition vesicles introduced here should be adaptable for enzyme-catalyzed synthesis of a wide variety of materials, by varying the metal ion, enzyme, and/or chelator.
Asunto(s)
Materiales Biomiméticos/química , Lípidos/química , Emulsiones/química , Tamaño de la Partícula , Ureasa/química , Ureasa/metabolismo , Agua/químicaRESUMEN
Ordered two-dimensional (2D) lattices were formed by assembling silica-coated solid and segmented Au nanowires between coplanar electrodes using alternating current (ac) electric fields. Dielectrophoretic forces from the ac field concentrated wires between the electrodes, with their long axis aligned parallel to the field lines. After reaching a sufficient particle density, field-induced dipolar interactions resulted in the assembly of dense 2D lattices that spanned the electrodes, a distance of at least ten wire lengths. The ends of neighboring Au wires or segments overlapped a fraction of their length to form lattice structures with a "running bond" brickwork-like pattern. The observed lattice structures were tunable in three distinct ways: (1) particle segmentation pattern, which fixed the lattice periodicity for a given field condition; (2) ac frequency, which varied lattice periodicity in real time; and (3) switching the field on/off, which converted between lattice and smectic particle organizations. Electric field simulations were performed to understand how the observed lattice periodicity depends on the assembly conditions and particle segmentation. Directed self-assembly of well-ordered 2D metallic nanowire lattices that can be designed by Au striping pattern and reconfigured by changes in field conditions could enable new types of switchable optical or electronic devices.
RESUMEN
The construction of genetically encoded cellular mimics in compartments containing organized synthetic cytosols is desirable for the development of artificial cells. Phase separated aqueous domains were placed within water-in-oil emulsion droplets in a manner compatible with transcription and translation machinery. Aqueous two-phase and three-phase systems (ATPS and A3PS) were assembled with dextran, poly(ethylene glycol), and Ficoll. Aqueous two-phase systems were capable of supporting the cell-free expression of protein within water droplets, whereas the aqueous three-phase-based system did not give rise to detectable protein synthesis. The expressed protein preferentially partitioned to the dextran-enriched phase. The system could serve as a foundation for building cellular mimics with liquid organelles.
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Aceites/química , Biosíntesis de Proteínas , Transcripción Genética , Agua/química , Sistema Libre de Células/química , Dextranos/química , Ficoll/química , Polietilenglicoles/químicaRESUMEN
Compartments within living cells create specialized microenvironments, allowing multiple reactions to be carried out simultaneously and efficiently. While some organelles are bound by a lipid bilayer, others are formed by liquid-liquid phase separation such as P-granules and nucleoli. Synthetic minimal cells are widely used to study many natural processes, including organelle formation. In this work, synthetic cells expressing artificial membrane-less organelles that inhibit translation are described. RGG-GFP-RGG, a phase-separating protein derived from Caenorhabditis elegans P-granules, is expressed by cell-free transcription and translation, forming artificial membraneless organelles that can sequester RNA and reduce protein expression in synthetic cells. The introduction of artificial membrane-less organelles creates complex microenvironments within the synthetic cell cytoplasm and functions as a tool to inhibit protein expression in synthetic cells. The engineering of compartments within synthetic cells furthers the understanding of the evolution and function of natural organelles and facilitates the creation of more complex and multifaceted synthetic lifelike systems.
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Células Artificiales , Animales , Condensados Biomoleculares , Citoplasma/metabolismo , Orgánulos/metabolismo , Proteínas/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismoRESUMEN
A variety of cellular processes use liquid-liquid phase separation (LLPS) to create functional levels of organization, but the kinetic pathways by which it proceeds remain incompletely understood. Here in real time, we monitor the dynamics of LLPS of mixtures of segregatively phase-separating polymers inside all-synthetic, giant unilamellar vesicles. After dynamically triggering phase separation, we find that the ensuing relaxation-en route to the new equilibrium-is non-trivially modulated by a dynamic interplay between the coarsening of the evolving droplet phase and the interactive membrane boundary. The membrane boundary is preferentially wetted by one of the incipient phases, dynamically arresting the progression of coarsening and deforming the membrane. When the vesicles are composed of phase-separating mixtures of common lipids, LLPS within the vesicular interior becomes coupled to the membrane's compositional degrees of freedom, producing microphase-separated membrane textures. This coupling of bulk and surface phase-separation processes suggests a physical principle by which LLPS inside living cells might be dynamically regulated and communicated to the cellular boundaries.