RESUMEN
The four-dimensional nucleome (4DN) consortium studies the architecture of the genome and the nucleus in space and time. We summarize progress by the consortium and highlight the development of technologies for (1) mapping genome folding and identifying roles of nuclear components and bodies, proteins, and RNA, (2) characterizing nuclear organization with time or single-cell resolution, and (3) imaging of nuclear organization. With these tools, the consortium has provided over 2,000 public datasets. Integrative computational models based on these data are starting to reveal connections between genome structure and function. We then present a forward-looking perspective and outline current aims to (1) delineate dynamics of nuclear architecture at different timescales, from minutes to weeks as cells differentiate, in populations and in single cells, (2) characterize cis-determinants and trans-modulators of genome organization, (3) test functional consequences of changes in cis- and trans-regulators, and (4) develop predictive models of genome structure and function.
Asunto(s)
Núcleo Celular , Genoma , Genoma/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Cromatina/metabolismoRESUMEN
Evolution of minimal DNA tumor virus' genomes has selected for small viral oncoproteins that hijack critical cellular protein interaction networks. The structural basis for the multiple and dominant functions of adenovirus oncoproteins has remained elusive. E4-ORF3 forms a nuclear polymer and simultaneously inactivates p53, PML, TRIM24, and MRE11/RAD50/NBS1 (MRN) tumor suppressors. We identify oligomerization mutants and solve the crystal structure of E4-ORF3. E4-ORF3 forms a dimer with a central ß core, and its structure is unrelated to known polymers or oncogenes. E4-ORF3 dimer units coassemble through reciprocal and nonreciprocal exchanges of their C-terminal tails. This results in linear and branched oligomer chains that further assemble in variable arrangements to form a polymer network that partitions the nuclear volume. E4-ORF3 assembly creates avidity-driven interactions with PML and an emergent MRN binding interface. This reveals an elegant structural solution whereby a small protein forms a multivalent matrix that traps disparate tumor suppressors.
Asunto(s)
Proteínas E4 de Adenovirus/química , Proteínas E4 de Adenovirus/metabolismo , Adenovirus Humanos/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Infecciones por Adenovirus Humanos/virología , Línea Celular , Células Cultivadas , Cristalografía por Rayos X , Humanos , Células Vegetales/virología , Pliegue de Proteína , Nicotiana/virologíaRESUMEN
Phase separation (PS) drives the formation of biomolecular condensates that are emerging biological structures involved in diverse cellular processes. Recent studies have unveiled PS-induced formation of several transcriptional factor (TF) condensates that are transcriptionally active, but how strongly PS promotes gene activation remains unclear. Here, we show that the oncogenic TF fusion Yes-associated protein 1-Mastermind like transcriptional coactivator 2 (YAP-MAML2) undergoes PS and forms liquid-like condensates that bear the hallmarks of transcriptional activity. Furthermore, we examined the contribution of PS to YAP-MAML2-mediated gene expression by developing a chemogenetic tool that dissolves TF condensates, allowing us to compare phase-separated and non-phase-separated conditions at identical YAP-MAML2 protein levels. We found that a small fraction of YAP-MAML2-regulated genes is further affected by PS, which include the canonical YAP target genes CTGF and CYR61, and other oncogenes. On the other hand, majority of YAP-MAML2-regulated genes are not affected by PS, highlighting that transcription can be activated effectively by diffuse complexes of TFs with the transcriptional machinery. Our work opens new directions in understanding the role of PS in selective modulation of gene expression, suggesting differential roles of PS in biological processes.
Asunto(s)
Separación de Fases , Transcriptoma , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , OncogenesRESUMEN
Visualizing dynamics of kinase activity in living animals is essential for mechanistic understanding of cell and developmental biology. We describe GFP-based kinase reporters that phase-separate upon kinase activation via multivalent protein-protein interactions, forming intensively fluorescent droplets. Called SPARK (separation of phases-based activity reporter of kinase), these reporters have large dynamic range (fluorescence change), high brightness, fast kinetics, and are reversible. The SPARK-based protein kinase A (PKA) reporter reveals oscillatory dynamics of PKA activities upon G protein-coupled receptor activation. The SPARK-based extracellular signal-regulated kinase (ERK) reporter unveils transient dynamics of ERK activity during tracheal metamorphosis in live Drosophila. Because of intensive brightness and simple signal pattern, SPARKs allow easy examination of kinase signaling in living animals in a qualitative way. The modular design of SPARK will facilitate development of reporters of other kinases.
Asunto(s)
Imagen Óptica/métodos , Fosfotransferasas/fisiología , Transducción de Señal/fisiología , Animales , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Drosophila , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Fosforilación , Fosfotransferasas/metabolismoRESUMEN
Focal adhesion kinase (FAK) relays integrin signaling from outside to inside cells and contributes to cell adhesion and motility. However, the spatiotemporal dynamics of FAK activity in single FAs is unclear due to the lack of a robust FAK reporter, which limits our understanding of these essential biological processes. Here we have engineered a genetically encoded FAK activity sensor, dubbed FAK-separation of phases-based activity reporter of kinase (SPARK), which visualizes endogenous FAK activity in living cells and vertebrates. Our work reveals temporal dynamics of FAK activity during FA turnover. Most importantly, our study unveils polarized FAK activity at the distal tip of newly formed single FAs in the leading edge of a migrating cell. By combining FAK-SPARK with DNA tension probes, we show that tensions applied to FAs precede FAK activation and that FAK activity is proportional to the strength of tension. These results suggest tension-induced polarized FAK activity in single FAs, advancing the mechanistic understanding of cell migration.
Asunto(s)
Adhesiones Focales , Animales , Adhesiones Focales/metabolismo , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Fosforilación , Proteína-Tirosina Quinasas de Adhesión Focal/genética , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Movimiento Celular/fisiología , Adhesión Celular/fisiologíaRESUMEN
The respiratory disease COVID-19 is caused by the coronavirus SARS-CoV-2. Here we report the discovery of ethacridine as a potent drug against SARS-CoV-2 (EC50 ~ 0.08 µM). Ethacridine was identified via high-throughput screening of an FDA-approved drug library in living cells using a fluorescence assay. Plaque assays, RT-PCR and immunofluorescence imaging at various stages of viral infection demonstrate that the main mode of action of ethacridine is through inactivation of viral particles, preventing their binding to the host cells. Consistently, ethacridine is effective in various cell types, including primary human nasal epithelial cells that are cultured in an air-liquid interface. Taken together, our work identifies a promising, potent, and new use of the old drug via a distinct mode of action for inhibiting SARS-CoV-2.
Asunto(s)
Antivirales/farmacología , Etacridina/farmacología , Inhibidores de Proteasas/farmacología , Activación Viral/efectos de los fármacos , Animales , Línea Celular , Chlorocebus aethiops , Proteasas 3C de Coronavirus/antagonistas & inhibidores , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Células Vero , Virión/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
miniSOG, developed as the first fully genetically encoded singlet oxygen photosensitiser, has found various applications in cell imaging and functional studies. Yet, miniSOG has suboptimal properties, including a low yield of singlet oxygen generation, which can nevertheless be improved tenfold upon blue light irradiation. In a previous study, we showed that this improvement was due to the photolysis of the miniSOG chromophore, flavin mononucleotide (FMN), into lumichrome, with concomitant removal of the phosphoribityl tail, thereby improving oxygen access to the alloxazine ring. We thus reasoned that a chromophore with a shorter tail would readily improve the photosensitizing properties of miniSOG. In this work, we show that the replacement of FMN by riboflavin (RF), which lacks the bulky phosphate group, significantly improves the singlet oxygen quantum yield (ΦΔ). We then proceeded to mutagenize the residues stabilizing the phosphate group of FMN to alter the chromophore specificity. We identified miniSOG-R57Q as a flavoprotein that selectively binds RF in cellulo, with a modestly improved ΦΔ. Our results show that it is possible to modify the flavin specificity of a given flavoprotein, thus providing a new option to tune its photophysical properties, including those leading to photosensitization. We also determined the structure of miniSOG-Q103L, a mutant with a much increased ΦΔ, which allowed us to postulate the existence of another access channel to FMN for molecular oxygen.
Asunto(s)
Mononucleótido de Flavina , Oxígeno Singlete , Mononucleótido de Flavina/química , Flavoproteínas/química , Oxígeno/química , Fosfatos , Riboflavina , Oxígeno Singlete/químicaRESUMEN
Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Asunto(s)
Proteínas Luminiscentes/química , Proteínas Luminiscentes/genética , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Humanos , Fitocromo/química , Ingeniería de ProteínasRESUMEN
Amyloids adopt 'cross-ß' structures composed of long, twisted fibrils with ß-strands running perpendicular to the fibril axis. Recently, a toxic peptide was proposed to form amyloid-like cross-α structures in solution, with a planar bilayer-like assembly observed in the crystal structure. Here we crystallographically characterize designed peptides that assemble into spiraling cross-α amyloid-like structures, which resemble twisted ß-amyloid fibrils. The peptides form helical dimers, stabilized by packing of small and apolar residues, and the dimers further assemble into cross-α amyloid-like fibrils with superhelical pitches ranging from 170 Å to 200 Å. When a small residue that appeared critical for packing was converted to leucine, it resulted in structural rearrangement to a helical polymer. Fluorescently tagged versions of the designed peptides form puncta in mammalian cells, which recover from photobleaching with markedly different kinetics. These structural folds could be potentially useful for directing in vivo protein assemblies with predetermined spacing and stabilities.
Asunto(s)
Amiloide/química , Péptidos/química , Cristalografía por Rayos X , Humanos , Cinética , Modelos Moleculares , Péptidos/síntesis química , Conformación ProteicaRESUMEN
A family of proteases called caspases mediate apoptosis signaling in animals. We report a GFP-based fluorogenic protease reporter, dubbed "FlipGFP", by flipping a beta strand of the GFP. Upon protease activation and cleavage, the beta strand is restored, leading to reconstitution of the GFP and fluorescence. FlipGFP-based TEV protease reporter achieves 100-fold fluorescence change. A FlipGFP-based executioner caspase reporter visualized apoptosis in live zebrafish embryos with spatiotemporal resolution. FlipGFP also visualized apoptotic cells in the midgut of Drosophila. Thus, the FlipGFP-based caspase reporter will be useful for monitoring apoptosis during animal development and for designing reporters of proteases beyond caspases. The design strategy can be further applied to a red fluorescent protein for engineering a red fluorogenic protease reporter.
Asunto(s)
Apoptosis , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Imagen Molecular , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Animales , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Conformación Proteica en Lámina betaRESUMEN
Far-red fluorescent proteins (FPs) are desirable for in vivo imaging because with these molecules less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow, and orange FPs. We developed a new class of FP from an allophycocyanin α-subunit (APCα). Native APC requires a lyase to incorporate phycocyanobilin. The evolved FP, which we named small ultra-red FP (smURFP), covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670-nm excitation-emission peaks, a large extinction coefficient (180,000 M(-1)cm(-1)) and quantum yield (18%), and photostability comparable to that of eGFP. smURFP has significantly greater BV incorporation rate and protein stability than the bacteriophytochrome (BPH) FPs. Moreover, BV supply is limited by membrane permeability, and smURFPs (but not BPH FPs) can incorporate a more membrane-permeant BV analog, making smURFP fluorescence comparable to that of FPs from jellyfish or coral. A far-red and near-infrared fluorescent cell cycle indicator was created with smURFP and a BPH FP.
Asunto(s)
Técnicas Biosensibles , Proteínas Luminiscentes/aislamiento & purificación , Ficocianina/química , Trichodesmium/metabolismo , Biliverdina/química , Ciclo Celular/fisiología , Escherichia coli/genética , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/efectos de la radiación , Mutación , Ficocianina/metabolismo , Conformación Proteica , Estabilidad Proteica , Subunidades de Proteína , Proteína Fluorescente RojaRESUMEN
Protein-protein interactions (PPIs) mediate signal transduction in cells. Small molecules that regulate PPIs are important tools for biology and biomedicine. Dynamic imaging of small molecule induced PPIs characterizes and verifies these molecules in living cells. It is thus important to develop cellular assays for dynamic visualization of small molecule induced protein-protein association and dissociation in living cells. Here we have applied a fluorophore phase transition based principle and designed a PPI assay named SPPIER (separation of phases-based protein interaction reporter). SPPIER utilizes the green fluorescent protein (GFP) and is thus genetically encoded. Upon small molecule induced PPI, SPPIER rapidly forms highly fluorescent GFP droplets in living cells. SPPIER detects immunomodulatory drug (IMiD) induced PPI between cereblon and the transcription factor Ikaros. It also detects IMiD analogue (e.g., CC-885) induced PPI between cereblon and GSPT1. Furthermore, SPPIER can visualize bifunctional molecules (e.g. PROTAC)-induced PPI between an E3 ubiquitin ligase and a target protein. Lastly, SPPIER can be modified to image small molecule induced protein-protein dissociation, such as nutlin-induced dissociation between HDM2 and p53. The intense brightness and rapid kinetics of SPPIER enable robust and dynamic visualization of PPIs in living cells.
Asunto(s)
Factor de Transcripción Ikaros/metabolismo , Péptido Hidrolasas/metabolismo , Mapas de Interacción de Proteínas , Bibliotecas de Moléculas Pequeñas/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Proteínas de Ciclo Celular , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Factor de Transcripción Ikaros/química , Factores Inmunológicos/química , Factores Inmunológicos/metabolismo , Microscopía Confocal , Proteínas Nucleares/genética , Péptido Hidrolasas/química , Péptido Hidrolasas/genética , Compuestos de Fenilurea/química , Compuestos de Fenilurea/metabolismo , Plásmidos/genética , Plásmidos/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Talidomida/análogos & derivados , Talidomida/química , Talidomida/metabolismo , Imagen de Lapso de Tiempo , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , Proteína Supresora de Tumores del Síndrome de Von Hippel-Lindau/genéticaRESUMEN
Infrared fluorescent proteins (IFPs) provide an additional color to GFP and its homologs in protein labeling. Drawing on structural analysis of the dimer interface, we identified a bacteriophytochrome in the sequence database that is monomeric in truncated form and engineered it into a naturally monomeric IFP (mIFP). We demonstrate that mIFP correctly labels proteins in live cells, Drosophila and zebrafish. It should be useful in molecular, cell and developmental biology.
Asunto(s)
Proteínas Fluorescentes Verdes/química , Rayos Infrarrojos , Secuencia de Aminoácidos , Animales , Animales Modificados Genéticamente , ADN/química , Biología Evolutiva , Drosophila melanogaster , Colorantes Fluorescentes/química , Células HeLa , Histidina/química , Humanos , Proteínas Luminiscentes/química , Ratones , Datos de Secuencia Molecular , Mutación , Neuronas/metabolismo , Plásmidos/metabolismo , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Transfección , Pez CebraRESUMEN
Fluorescence resonance energy transfer-based reporters have been widely used in imaging cell signaling; however, their in vivo application has been handicapped because of poor signal. Although fluorogenic reporters overcome this problem, no such reporter of proteases has been demonstrated for in vivo imaging. Now we have redesigned an infrared fluorescent protein so that its chromophore incorporation is regulated by protease activity. Upon protease activation, the infrared fluorogenic protease reporter becomes fluorescent with no requirement of exogenous cofactor. To demonstrate biological applications, we have designed an infrared fluorogenic executioner-caspase reporter, which reveals spatiotemporal coordination between cell apoptosis and embryonic morphogenesis, as well as dynamics of apoptosis during tumorigenesis in Drosophila. The designed scaffold may be used to engineer reporters of other proteases with specific cleavage sequence.
Asunto(s)
Apoptosis , Drosophila melanogaster/citología , Colorantes Fluorescentes/metabolismo , Genes Reporteros , Animales , Carcinogénesis/patología , Caspasas/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/citología , Desarrollo Embrionario , Células HEK293 , Humanos , Rayos Infrarrojos , Péptido Hidrolasas/metabolismo , Factores de TiempoRESUMEN
Using X-ray crystallography, continuum electrostatic calculations, and molecular dynamics simulations, we have studied the structure, protonation behavior, and dynamics of the biliverdin chromophore and its molecular environment in a series of genetically engineered infrared fluorescent proteins (IFPs) based on the chromophore-binding domain of the Deinococcus radiodurans bacteriophytochrome. Our study suggests that the experimentally observed enhancement of fluorescent properties results from the improved rigidity and planarity of the biliverdin chromophore, in particular of the first two pyrrole rings neighboring the covalent linkage to the protein. We propose that the increases in the levels of both motion and bending of the chromophore out of planarity favor the decrease in fluorescence. The chromophore-binding pocket in some of the studied proteins, in particular the weakly fluorescent parent protein, is shown to be readily accessible to water molecules from the solvent. These waters entering the chromophore region form hydrogen bond networks that affect the otherwise planar conformation of the first three rings of the chromophore. On the basis of our simulations, the enhancement of fluorescence in IFPs can be achieved either by reducing the mobility of water molecules in the vicinity of the chromophore or by limiting the interactions of the nearby protein residues with the chromophore. Finally, simulations performed at both low and neutral pH values highlight differences in the dynamics of the chromophore and shed light on the mechanism of fluorescence loss at low pH.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Luminiscentes/química , Proteínas Bacterianas/genética , Biliverdina/química , Cristalografía por Rayos X , Deinococcus/química , Deinococcus/genética , Fluorescencia , Rayos Infrarrojos , Proteínas Luminiscentes/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Fitocromo/química , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Electricidad EstáticaRESUMEN
Protein-protein interactions regulate many biological processes. Identification of interacting proteins is thus an important step toward molecular understanding of cell signaling. The aim of this study was to investigate the use of photo-generated singlet oxygen and a small molecule for proximity labeling of interacting proteins in cellular environment. The protein of interest (POI) was fused with a small singlet oxygen photosensitizer (miniSOG), which generates singlet oxygen ((1)O2) upon irradiation. The locally generated singlet oxygen then activated a biotin-conjugated thiol molecule to form a covalent bond with the proteins nearby. The labeled proteins can then be separated and subsequently identified by mass spectrometry. To demonstrate the applicability of this labeling technology, we fused the miniSOG to Skp2, an F-box protein of the SCF ubiquitin ligase, and expressed the fusion protein in mammalian cells and identified that the surface cysteine of its interacting partner Skp1 was labeled by the biotin-thiol molecule. This photoactivatable protein labeling method may find important applications including identification of weak and transient protein-protein interactions in the native cellular context, as well as spatial and temporal control of protein labeling.
Asunto(s)
Fármacos Fotosensibilizantes/metabolismo , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Oxígeno Singlete/metabolismo , Modelos Moleculares , Estructura Molecular , Procesos Fotoquímicos , Fármacos Fotosensibilizantes/química , Proteínas Quinasas Asociadas a Fase-S/químicaRESUMEN
We describe a method for light-inducible and tissue-selective cell ablation using a genetically encoded photosensitizer, miniSOG (mini singlet oxygen generator). miniSOG is a newly engineered fluorescent protein of 106 amino acids that generates singlet oxygen in quantum yield upon blue-light illumination. We transgenically expressed mitochondrially targeted miniSOG (mito-miniSOG) in Caenorhabditis elegans neurons. Upon blue-light illumination, mito-miniSOG causes rapid and effective death of neurons in a cell-autonomous manner without detectable damages to surrounding tissues. Neuronal death induced by mito-miniSOG appears to be independent of the caspase CED-3, but the clearance of the damaged cells partially depends on the phagocytic receptor CED-1, a homolog of human CD91. We show that neurons can be killed at different developmental stages. We further use this method to investigate the role of the premotor interneurons in regulating the convulsive behavior caused by a gain-of-function mutation in the neuronal acetylcholine receptor acr-2. Our findings support an instructive role for the interneuron AVB in controlling motor neuron activity and reveal an inhibitory effect of the backward premotor interneurons on the forward interneurons. In summary, the simple inducible cell ablation method reported here allows temporal and spatial control and will prove to be a useful tool in studying the function of specific cells within complex cellular contexts.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Flavoproteínas/metabolismo , Proteínas Luminiscentes/metabolismo , Neuronas/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Caspasas/genética , Caspasas/metabolismo , Muerte Celular/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Flavoproteínas/genética , Interneuronas/citología , Interneuronas/metabolismo , Interneuronas/efectos de la radiación , Luz , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Mitocondrias/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Neuronas Motoras/efectos de la radiación , Neuronas/citología , Neuronas/efectos de la radiación , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Oxígeno Singlete/metabolismo , Factores de TiempoRESUMEN
Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce "miniSOG" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.
Asunto(s)
Coloración y Etiquetado , 3,3'-Diaminobencidina/análisis , Estructuras Animales , Animales , Encéfalo/ultraestructura , Caenorhabditis elegans/química , Células , Colorantes Fluorescentes/análisis , Ratones , Microscopía Electrónica , Microscopía Fluorescente , Mitocondrias/ultraestructura , Fototropinas/análisis , Proteínas/análisisRESUMEN
Transcription factor dynamics are used to selectively engage gene regulatory programs. Biomolecular condensates have emerged as an attractive signaling substrate in this process, but the underlying mechanisms are not well-understood. Here, we probed the molecular basis of YAP signal integration through transcriptional condensates. Leveraging light-sheet single-molecule imaging and synthetic condensates, we demonstrate charge-mediated co-condensation of the transcriptional regulators YAP and Mediator into transcriptionally active condensates in stem cells. IDR sequence analysis and YAP protein engineering demonstrate that instead of the net charge, YAP signaling specificity is established through its negative charge patterning that interacts with Mediator's positive charge blocks. The mutual enhancement of YAP/Mediator co-condensation is counteracted by negative feedback from transcription, driving an adaptive transcriptional response that is well-suited for decoding dynamic inputs. Our work reveals a molecular framework for YAP condensate formation and sheds new light on the function of YAP condensates for emergent gene regulatory behavior.