RESUMEN
In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento , Retículo Endoplásmico , Vesículas Sinápticas , Animales , Vesículas Sinápticas/metabolismo , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Retículo Endoplásmico/metabolismo , Calcio/metabolismo , Aparato de Golgi/metabolismo , Ratas , Transporte Biológico , Terminales Presinápticos/metabolismo , Sinapsinas/metabolismo , Condensados Biomoleculares/metabolismo , Proteínas del Citoesqueleto/metabolismo , Separación de FasesRESUMEN
Enhancers are distal DNA elements believed to loop and contact promoters to control gene expression. Recently, we found diffraction-sized transcriptional condensates at genes controlled by clusters of enhancers (super-enhancers). However, a direct function of endogenous condensates in controlling gene expression remains elusive. Here, we develop live-cell super-resolution and multi-color 3D-imaging approaches to investigate putative roles of endogenous condensates in the regulation of super-enhancer controlled gene Sox2. In contrast to enhancer distance, we find instead that the condensate's positional dynamics are a better predictor of gene expression. A basal gene bursting occurs when the condensate is far (>1 µm), but burst size and frequency are enhanced when the condensate moves in proximity (<1 µm). Perturbations of cohesin and local DNA elements do not prevent basal bursting but affect the condensate and its burst enhancement. We propose a three-way kissing model whereby the condensate interacts transiently with gene locus and regulatory DNA elements to control gene bursting.
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Regulación de la Expresión Génica , Factores de Transcripción SOXB1 , Súper Potenciadores , Transcripción Genética , ADN/genética , Elementos de Facilitación Genéticos , Factores de Transcripción SOXB1/genética , Animales , Ratones , Células Madre Embrionarias/metabolismo , Microscopía/métodosRESUMEN
Lysine lactylation is a post-translational modification that links cellular metabolism to protein function. Here, we find that AARS1 functions as a lactate sensor that mediates global lysine lacylation in tumor cells. AARS1 binds to lactate and catalyzes the formation of lactate-AMP, followed by transfer of lactate to the lysince acceptor residue. Proteomics studies reveal a large number of AARS1 targets, including p53 where lysine 120 and lysine 139 in the DNA binding domain are lactylated. Generation and utilization of p53 variants carrying constitutively lactylated lysine residues revealed that AARS1 lactylation of p53 hinders its liquid-liquid phase separation, DNA binding, and transcriptional activation. AARS1 expression and p53 lacylation correlate with poor prognosis among cancer patients carrying wild type p53. ß-alanine disrupts lactate binding to AARS1, reduces p53 lacylation, and mitigates tumorigenesis in animal models. We propose that AARS1 contributes to tumorigenesis by coupling tumor cell metabolism to proteome alteration.
Asunto(s)
Carcinogénesis , Ácido Láctico , Proteína p53 Supresora de Tumor , Animales , Femenino , Humanos , Ratones , Carcinogénesis/metabolismo , Carcinogénesis/genética , Línea Celular Tumoral , Ácido Láctico/metabolismo , Lisina/metabolismo , Neoplasias/metabolismo , Neoplasias/genética , Procesamiento Proteico-Postraduccional , Proteína p53 Supresora de Tumor/metabolismo , MasculinoRESUMEN
The ability of proteins and RNA to coalesce into phase-separated assemblies, such as the nucleolus and stress granules, is a basic principle in organizing membraneless cellular compartments. While the constituents of biomolecular condensates are generally well documented, the mechanisms underlying their formation under stress are only partially understood. Here, we show in yeast that covalent modification with the ubiquitin-like modifier Urm1 promotes the phase separation of a wide range of proteins. We find that the drop in cellular pH induced by stress triggers Urm1 self-association and its interaction with both target proteins and the Urm1-conjugating enzyme Uba4. Urmylation of stress-sensitive proteins promotes their deposition into stress granules and nuclear condensates. Yeast cells lacking Urm1 exhibit condensate defects that manifest in reduced stress resilience. We propose that Urm1 acts as a reversible molecular "adhesive" to drive protective phase separation of functionally critical proteins under cellular stress.
RESUMEN
Tissue folds are structural motifs critical to organ function. In the intestine, bending of a flat epithelium into a periodic pattern of folds gives rise to villi, finger-like protrusions that enable nutrient absorption. However, the molecular and mechanical processes driving villus morphogenesis remain unclear. Here, we identify an active mechanical mechanism that simultaneously patterns and folds the intestinal epithelium to initiate villus formation. At the cellular level, we find that PDGFRA+ subepithelial mesenchymal cells generate myosin II-dependent forces sufficient to produce patterned curvature in neighboring tissue interfaces. This symmetry-breaking process requires altered cell and extracellular matrix interactions that are enabled by matrix metalloproteinase-mediated tissue fluidization. Computational models, together with in vitro and in vivo experiments, revealed that these cellular features manifest at the tissue level as differences in interfacial tensions that promote mesenchymal aggregation and interface bending through a process analogous to the active dewetting of a thin liquid film.
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Matriz Extracelular , Mucosa Intestinal , Animales , Ratones , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citología , Matriz Extracelular/metabolismo , Miosina Tipo II/metabolismo , Mesodermo/metabolismo , Mesodermo/citología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/citología , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Morfogénesis , Metaloproteinasas de la Matriz/metabolismoRESUMEN
Neuroimmune interactions mediate intercellular communication and underlie critical brain functions. Microglia, CNS-resident macrophages, modulate the brain through direct physical interactions and the secretion of molecules. One such secreted factor, the complement protein C1q, contributes to complement-mediated synapse elimination in both developmental and disease models, yet brain C1q protein levels increase significantly throughout aging. Here, we report that C1q interacts with neuronal ribonucleoprotein (RNP) complexes in an age-dependent manner. Purified C1q protein undergoes RNA-dependent liquid-liquid phase separation (LLPS) in vitro, and the interaction of C1q with neuronal RNP complexes in vivo is dependent on RNA and endocytosis. Mice lacking C1q have age-specific alterations in neuronal protein synthesis in vivo and impaired fear memory extinction. Together, our findings reveal a biophysical property of C1q that underlies RNA- and age-dependent neuronal interactions and demonstrate a role of C1q in critical intracellular neuronal processes.
Asunto(s)
Envejecimiento , Encéfalo , Complemento C1q , Homeostasis , Microglía , Neuronas , Ribonucleoproteínas , Animales , Complemento C1q/metabolismo , Ratones , Microglía/metabolismo , Envejecimiento/metabolismo , Encéfalo/metabolismo , Ribonucleoproteínas/metabolismo , Neuronas/metabolismo , Ratones Endogámicos C57BL , HumanosRESUMEN
Nucleoli are multicomponent condensates defined by coexisting sub-phases. We identified distinct intrinsically disordered regions (IDRs), including acidic (D/E) tracts and K-blocks interspersed by E-rich regions, as defining features of nucleolar proteins. We show that the localization preferences of nucleolar proteins are determined by their IDRs and the types of RNA or DNA binding domains they encompass. In vitro reconstitutions and studies in cells showed how condensation, which combines binding and complex coacervation of nucleolar components, contributes to nucleolar organization. D/E tracts of nucleolar proteins contribute to lowering the pH of co-condensates formed with nucleolar RNAs in vitro. In cells, this sets up a pH gradient between nucleoli and the nucleoplasm. By contrast, juxta-nucleolar bodies, which have different macromolecular compositions, featuring protein IDRs with very different charge profiles, have pH values that are equivalent to or higher than the nucleoplasm. Our findings show that distinct compositional specificities generate distinct physicochemical properties for condensates.
Asunto(s)
Nucléolo Celular , Proteínas Nucleares , Fuerza Protón-Motriz , Nucléolo Celular/química , Núcleo Celular/química , Proteínas Nucleares/química , ARN/metabolismo , Separación de Fases , Proteínas Intrínsecamente Desordenadas/química , Animales , Xenopus laevis , Oocitos/química , Oocitos/citologíaRESUMEN
DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.
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Reparación del ADN , Poli(ADP-Ribosa) Polimerasa-1 , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Poli(ADP-Ribosa) Polimerasa-1/genética , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , HumanosRESUMEN
The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.
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Proteínas de Arabidopsis , Arabidopsis , Fosfotransferasas/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pectinas/metabolismo , Separación de Fases , Proteínas Ligadas a GPI/metabolismoRESUMEN
Components of transcriptional machinery are selectively partitioned into specific condensates, often mediated by protein disorder, yet we know little about how this specificity is achieved. Here, we show that condensates composed of the intrinsically disordered region (IDR) of MED1 selectively partition RNA polymerase II together with its positive allosteric regulators while excluding negative regulators. This selective compartmentalization is sufficient to activate transcription and is required for gene activation during a cell-state transition. The IDRs of partitioned proteins are necessary and sufficient for selective compartmentalization and require alternating blocks of charged amino acids. Disrupting this charge pattern prevents partitioning, whereas adding the pattern to proteins promotes partitioning with functional consequences for gene activation. IDRs with similar patterned charge blocks show similar partitioning and function. These findings demonstrate that disorder-mediated interactions can selectively compartmentalize specific functionally related proteins from a complex mixture of biomolecules, leading to regulation of a biochemical pathway.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , ARN Polimerasa II , Transcripción Genética , Proteínas Intrínsecamente Desordenadas/metabolismo , ARN Polimerasa II/metabolismo , Activación Transcripcional , Animales , RatonesRESUMEN
Terrestrial organisms developed circadian rhythms for adaptation to Earth's quasi-24-h rotation. Achieving precise rhythms requires diurnal oscillation of fundamental biological processes, such as rhythmic shifts in the cellular translational landscape; however, regulatory mechanisms underlying rhythmic translation remain elusive. Here, we identified mammalian ATXN2 and ATXN2L as cooperating master regulators of rhythmic translation, through oscillating phase separation in the suprachiasmatic nucleus along circadian cycles. The spatiotemporal oscillating condensates facilitate sequential initiation of multiple cycling processes, from mRNA processing to protein translation, for selective genes including core clock genes. Depleting ATXN2 or 2L induces opposite alterations to the circadian period, whereas the absence of both disrupts translational activation cycles and weakens circadian rhythmicity in mice. Such cellular defect can be rescued by wild type, but not phase-separation-defective ATXN2. Together, we revealed that oscillating translation is regulated by spatiotemporal condensation of two master regulators to achieve precise circadian rhythm in mammals.
Asunto(s)
Relojes Circadianos , Ratones , Animales , Relojes Circadianos/genética , Ritmo Circadiano/fisiología , Núcleo Supraquiasmático/metabolismo , Procesamiento Proteico-Postraduccional , MamíferosRESUMEN
Complex diseases often involve the interplay between genetic and environmental factors. Charcot-Marie-Tooth type 2 neuropathies (CMT2) are a group of genetically heterogeneous disorders, in which similar peripheral neuropathology is inexplicably caused by various mutated genes. Their possible molecular links remain elusive. Here, we found that upon environmental stress, many CMT2-causing mutant proteins adopt similar properties by entering stress granules (SGs), where they aberrantly interact with G3BP and integrate into SG pathways. For example, glycyl-tRNA synthetase (GlyRS) is translocated from the cytoplasm into SGs upon stress, where the mutant GlyRS perturbs the G3BP-centric SG network by aberrantly binding to G3BP. This disrupts SG-mediated stress responses, leading to increased stress vulnerability in motoneurons. Disrupting this aberrant interaction rescues SG abnormalities and alleviates motor deficits in CMT2D mice. These findings reveal a stress-dependent molecular link across diverse CMT2 mutants and provide a conceptual framework for understanding genetic heterogeneity in light of environmental stress.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth , Proteínas con Motivos de Reconocimiento de ARN , Gránulos de Estrés , Animales , Ratones , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/metabolismo , Enfermedad de Charcot-Marie-Tooth/patología , Citoplasma , Neuronas Motoras , Proteínas con Motivos de Reconocimiento de ARN/metabolismoRESUMEN
Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.
Asunto(s)
Ensamble y Desensamble de Cromatina , Complejos Multiproteicos , Proteínas Nucleares , Humanos , Cromatina , Proteínas de Unión al ADN/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismoRESUMEN
The mechanism that initiates autophagosome formation on the ER in multicellular organisms is elusive. Here, we showed that autophagy stimuli trigger Ca2+ transients on the outer surface of the ER membrane, whose amplitude, frequency, and duration are controlled by the metazoan-specific ER transmembrane autophagy protein EPG-4/EI24. Persistent Ca2+ transients/oscillations on the cytosolic ER surface in EI24-depleted cells cause accumulation of FIP200 autophagosome initiation complexes on the ER. This defect is suppressed by attenuating ER Ca2+ transients. Multi-modal SIM analysis revealed that Ca2+ transients on the ER trigger the formation of dynamic and fusion-prone liquid-like FIP200 puncta. Starvation-induced Ca2+ transients on lysosomes also induce FIP200 puncta that further move to the ER. Multiple FIP200 puncta on the ER, whose association depends on the ER proteins VAPA/B and ATL2/3, assemble into autophagosome formation sites. Thus, Ca2+ transients are crucial for triggering phase separation of FIP200 to specify autophagosome initiation sites in metazoans.
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Autofagosomas , Calcio , Animales , Autofagosomas/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas de Ciclo Celular/metabolismoRESUMEN
Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.
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Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Embrión no Mamífero/metabolismo , Animales , Gránulos de Ribonucleoproteínas Citoplasmáticas , Drosophila/embriología , Proteínas de Drosophila/genética , Desarrollo Embrionario , Oocitos/metabolismo , ARN/metabolismoRESUMEN
When challenged by hypertonicity, dehydrated cells must recover their volume to survive. This process requires the phosphorylation-dependent regulation of SLC12 cation chloride transporters by WNK kinases, but how these kinases are activated by cell shrinkage remains unknown. Within seconds of cell exposure to hypertonicity, WNK1 concentrates into membraneless condensates, initiating a phosphorylation-dependent signal that drives net ion influx via the SLC12 cotransporters to restore cell volume. WNK1 condensate formation is driven by its intrinsically disordered C terminus, whose evolutionarily conserved signatures are necessary for efficient phase separation and volume recovery. This disorder-encoded phase behavior occurs within physiological constraints and is activated in vivo by molecular crowding rather than changes in cell size. This allows kinase activity despite an inhibitory ionic milieu and permits cell volume recovery through condensate-mediated signal amplification. Thus, WNK kinases are physiological crowding sensors that phase separate to coordinate a cell volume rescue response.
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Proteínas Serina-Treonina Quinasas , Fosforilación , Tamaño de la CélulaRESUMEN
Biochemical processes often require spatial regulation and specific microenvironments. The general lack of organelles in bacteria limits the potential of bioengineering complex intracellular reactions. Here, we demonstrate synthetic membraneless organelles in Escherichia coli termed transcriptionally engineered addressable RNA solvent droplets (TEARS). TEARS are assembled from RNA-binding protein recruiting domains fused to poly-CAG repeats that spontaneously drive liquid-liquid phase separation from the bulk cytoplasm. Targeting TEARS with fluorescent proteins revealed multilayered structures with composition and reaction robustness governed by non-equilibrium dynamics. We show that TEARS provide organelle-like bioprocess isolation for sequestering biochemical pathways, controlling metabolic branch points, buffering mRNA translation rates, and scaffolding protein-protein interactions. We anticipate TEARS to be a simple and versatile tool for spatially controlling E. coli biochemistry. Particularly, the modular design of TEARS enables applications without expression fine-tuning, simplifying the design-build-test cycle of bioengineering.
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Escherichia coli , Orgánulos , Escherichia coli/genética , Orgánulos/metabolismo , ARN/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Solventes/análisis , Solventes/metabolismoRESUMEN
Protein aggregation is a hallmark of multiple human pathologies. Autophagy selectively degrades protein aggregates via aggrephagy. How selectivity is achieved has been elusive. Here, we identify the chaperonin subunit CCT2 as an autophagy receptor regulating the clearance of aggregation-prone proteins in the cell and the mouse brain. CCT2 associates with aggregation-prone proteins independent of cargo ubiquitination and interacts with autophagosome marker ATG8s through a non-classical VLIR motif. In addition, CCT2 regulates aggrephagy independently of the ubiquitin-binding receptors (P62, NBR1, and TAX1BP1) or chaperone-mediated autophagy. Unlike P62, NBR1, and TAX1BP1, which facilitate the clearance of protein condensates with liquidity, CCT2 specifically promotes the autophagic degradation of protein aggregates with little liquidity (solid aggregates). Furthermore, aggregation-prone protein accumulation induces the functional switch of CCT2 from a chaperone subunit to an autophagy receptor by promoting CCT2 monomer formation, which exposes the VLIR to ATG8s interaction and, therefore, enables the autophagic function.
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Chaperonina con TCP-1 , Macroautofagia , Agregado de Proteínas , Animales , Ratones , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia/fisiología , Proteínas Portadoras/metabolismo , Chaperonina con TCP-1/metabolismo , Proteína Sequestosoma-1/metabolismoRESUMEN
NLRP6 is important in host defense by inducing functional outcomes including inflammasome activation and interferon production. Here, we show that NLRP6 undergoes liquid-liquid phase separation (LLPS) upon interaction with double-stranded RNA (dsRNA) in vitro and in cells, and an intrinsically disordered poly-lysine sequence (K350-354) of NLRP6 is important for multivalent interactions, phase separation, and inflammasome activation. Nlrp6-deficient or Nlrp6K350-354A mutant mice show reduced inflammasome activation upon mouse hepatitis virus or rotavirus infection, and in steady state stimulated by intestinal microbiota, implicating NLRP6 LLPS in anti-microbial immunity. Recruitment of ASC via helical assembly solidifies NLRP6 condensates, and ASC further recruits and activates caspase-1. Lipoteichoic acid, a known NLRP6 ligand, also promotes NLRP6 LLPS, and DHX15, a helicase in NLRP6-induced interferon signaling, co-forms condensates with NLRP6 and dsRNA. Thus, LLPS of NLRP6 is a common response to ligand stimulation, which serves to direct NLRP6 to distinct functional outcomes depending on the cellular context.
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Inflamasomas/metabolismo , Virus ARN/fisiología , Receptores de Superficie Celular/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Adaptadoras de Señalización CARD/metabolismo , Hepatocitos/virología , Intestinos/virología , Proteínas Intrínsecamente Desordenadas/química , Lipopolisacáridos/metabolismo , Hígado/virología , Ratones , Polilisina/metabolismo , Unión Proteica , ARN Bicatenario/metabolismo , Receptores de Superficie Celular/química , Transducción de Señal , Ácidos Teicoicos/metabolismoRESUMEN
Engineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells.