RESUMO
Optical interrogation of voltage in deep brain locations with cellular resolution would be immensely useful for understanding how neuronal circuits process information. Here, we report ASAP3, a genetically encoded voltage indicator with 51% fluorescence modulation by physiological voltages, submillisecond activation kinetics, and full responsivity under two-photon excitation. We also introduce an ultrafast local volume excitation (ULoVE) method for kilohertz-rate two-photon sampling in vivo with increased stability and sensitivity. Combining a soma-targeted ASAP3 variant and ULoVE, we show single-trial tracking of spikes and subthreshold events for minutes in deep locations, with subcellular resolution and with repeated sampling over days. In the visual cortex, we use soma-targeted ASAP3 to illustrate cell-type-dependent subthreshold modulation by locomotion. Thus, ASAP3 and ULoVE enable high-speed optical recording of electrical activity in genetically defined neurons at deep locations during awake behavior.
Assuntos
Encéfalo/fisiologia , Proteínas Ativadoras de GTPase/genética , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Optogenética/métodos , Ritmo Teta , Vigília , Potenciais de Ação , Animais , Encéfalo/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Feminino , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Ratos , Ratos Sprague-Dawley , CorridaRESUMO
Nuclear pore complexes (NPCs) conduct nucleocytoplasmic transport through an FG domain-controlled barrier. We now explore how surface-features of a mobile species determine its NPC passage rate. Negative charges and lysines impede passage. Hydrophobic residues, certain polar residues (Cys, His), and, surprisingly, charged arginines have striking translocation-promoting effects. Favorable cation-π interactions between arginines and FG-phenylalanines may explain this apparent paradox. Application of these principles to redesign the surface of GFP resulted in variants that show a wide span of transit rates, ranging from 35-fold slower than wild-type to â¼500 times faster, with the latter outpacing even naturally occurring nuclear transport receptors (NTRs). The structure of a fast and particularly FG-specific GFPNTR variant illustrates how NTRs can expose multiple regions for binding hydrophobic FG motifs while evading non-specific aggregation. Finally, we document that even for NTR-mediated transport, the surface-properties of the "passively carried" cargo can strikingly affect the translocation rate.
Assuntos
Transporte Ativo do Núcleo Celular/fisiologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Interações Hidrofóbicas e Hidrofílicas , Microscopia Confocal , Mutagênese Sítio-Dirigida , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Domínios Proteicos , Estrutura Quaternária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Propriedades de SuperfícieRESUMO
Diverse biological systems utilize fluctuations ("noise") in gene expression to drive lineage-commitment decisions. However, once a commitment is made, noise becomes detrimental to reliable function, and the mechanisms enabling post-commitment noise suppression are unclear. Here, we find that architectural constraints on noise suppression are overcome to stabilize fate commitment. Using single-molecule and time-lapse imaging, we find that-after a noise-driven event-human immunodeficiency virus (HIV) strongly attenuates expression noise through a non-transcriptional negative-feedback circuit. Feedback is established through a serial cascade of post-transcriptional splicing, whereby proteins generated from spliced mRNAs auto-deplete their own precursor unspliced mRNAs. Strikingly, this auto-depletion circuitry minimizes noise to stabilize HIV's commitment decision, and a noise-suppression molecule promotes stabilization. This feedback mechanism for noise suppression suggests a functional role for delayed splicing in other systems and may represent a generalizable architecture of diverse homeostatic signaling circuits.
Assuntos
Retroalimentação Fisiológica , HIV-1/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , HIV-1/genética , Humanos , Células Jurkat , Modelos Biológicos , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Splicing de RNA , Imagem com Lapso de Tempo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Dopamine (DA) is a central monoamine neurotransmitter involved in many physiological and pathological processes. A longstanding yet largely unmet goal is to measure DA changes reliably and specifically with high spatiotemporal precision, particularly in animals executing complex behaviors. Here, we report the development of genetically encoded GPCR-activation-based-DA (GRABDA) sensors that enable these measurements. In response to extracellular DA, GRABDA sensors exhibit large fluorescence increases (ΔF/F0 â¼90%) with subcellular resolution, subsecond kinetics, nanomolar to submicromolar affinities, and excellent molecular specificity. GRABDA sensors can resolve a single-electrical-stimulus-evoked DA release in mouse brain slices and detect endogenous DA release in living flies, fish, and mice. In freely behaving mice, GRABDA sensors readily report optogenetically elicited nigrostriatal DA release and depict dynamic mesoaccumbens DA signaling during Pavlovian conditioning or during sexual behaviors. Thus, GRABDA sensors enable spatiotemporally precise measurements of DA dynamics in a variety of model organisms while exhibiting complex behaviors.
Assuntos
Dopamina/análise , Drosophila/metabolismo , Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Comportamento Animal , Dopamina/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neurônios/citologia , Neurônios/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/genética , Canais de Cátion TRPV/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
Embryonic cell fates are defined by transcription factors that are rapidly deployed, yet attempts to visualize these factors in vivo often fail because of slow fluorescent protein maturation. Here, we pioneer a protein tag, LlamaTag, which circumvents this maturation limit by binding mature fluorescent proteins, making it possible to visualize transcription factor concentration dynamics in live embryos. Implementing this approach in the fruit fly Drosophila melanogaster, we discovered stochastic bursts in the concentration of transcription factors that are correlated with bursts in transcription. We further used LlamaTags to show that the concentration of protein in a given nucleus heavily depends on transcription of that gene in neighboring nuclei; we speculate that this inter-nuclear signaling is an important mechanism for coordinating gene expression to delineate straight and sharp boundaries of gene expression. Thus, LlamaTags now make it possible to visualize the flow of information along the central dogma in live embryos.
Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Edição de Genes/métodos , Fatores de Transcrição/genética , Animais , Núcleo Celular/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/crescimento & desenvolvimento , Embrião não Mamífero/metabolismo , Embrião não Mamífero/patologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Fluorescência Verde/genética , Microscopia Confocal , Fatores de Transcrição/metabolismoRESUMO
Cell-penetrating peptides (CPPs) are short protein segments that can transport cargos into cells. Although CPPs are widely studied as potential drug delivery tools, their role in normal cell physiology is poorly understood. Early during infection, the L2 capsid protein of human papillomaviruses binds retromer, a cytoplasmic trafficking factor required for delivery of the incoming non-enveloped virus into the retrograde transport pathway. Here, we show that the C terminus of HPV L2 proteins contains a conserved cationic CPP that drives passage of a segment of the L2 protein through the endosomal membrane into the cytoplasm, where it binds retromer, thereby sorting the virus into the retrograde pathway for transport to the trans-Golgi network. These experiments define the cell-autonomous biological role of a CPP in its natural context and reveal how a luminal viral protein engages an essential cytoplasmic entry factor.
Assuntos
Proteínas do Capsídeo/metabolismo , Peptídeos Penetradores de Células/metabolismo , Proteínas Oncogênicas Virais/metabolismo , Sequência de Aminoácidos , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Peptídeos Penetradores de Células/química , Peptídeos Penetradores de Células/genética , Endossomos/metabolismo , Complexo de Golgi/virologia , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/fisiologia , Humanos , Mutagênese , Proteínas Oncogênicas Virais/química , Proteínas Oncogênicas Virais/genética , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Ligação Viral , Internalização do VírusRESUMO
Applying synthetic biology to engineer gut-resident microbes provides new avenues to investigate microbe-host interactions, perform diagnostics, and deliver therapeutics. Here, we describe a platform for engineering Bacteroides, the most abundant genus in the Western microbiota, which includes a process for high-throughput strain modification. We have identified a novel phage promoter and translational tuning strategy and achieved an unprecedented level of expression that enables imaging of fluorescent-protein-expressing Bacteroides stably colonizing the mouse gut. A detailed characterization of the phage promoter has provided a set of constitutive promoters that span over four logs of strength without detectable fitness burden within the gut over 14 days. These promoters function predictably over a 1,000,000-fold expression range in phylogenetically diverse Bacteroides species. With these promoters, unique fluorescent signatures were encoded to allow differentiation of six species within the gut. Fluorescent protein-based differentiation of isogenic strains revealed that priority of gut colonization determines colonic crypt occupancy.
Assuntos
Bacteroides/classificação , Bacteroides/genética , Microbioma Gastrointestinal , Engenharia Genética , Animais , Bacteroides/isolamento & purificação , Vida Livre de Germes , Proteínas de Fluorescência Verde/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
Transcription is episodic, consisting of a series of discontinuous bursts. Using live-imaging methods and quantitative analysis, we examine transcriptional bursting in living Drosophila embryos. Different developmental enhancers positioned downstream of synthetic reporter genes produce transcriptional bursts with similar amplitudes and duration but generate very different bursting frequencies, with strong enhancers producing more bursts than weak enhancers. Insertion of an insulator reduces the number of bursts and the corresponding level of gene expression, suggesting that enhancer regulation of bursting frequency is a key parameter of gene control in development. We also show that linked reporter genes exhibit coordinated bursting profiles when regulated by a shared enhancer, challenging conventional models of enhancer-promoter looping.
Assuntos
Cromossomos/metabolismo , Drosophila melanogaster/metabolismo , Elementos Facilitadores Genéticos , Transcrição Gênica , Ativação Transcricional , Animais , Drosophila melanogaster/genética , Embrião não Mamífero/metabolismo , Feminino , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Elementos Isolantes , Masculino , Regiões Promotoras GenéticasRESUMO
Genetically encoded biosensors are powerful tools to monitor cellular behavior, but the difficulty in generating appropriate reporters for chromatin factors hampers our ability to dissect epigenetic pathways. Here, we present TRACE (transgene reporters across chromatin environments), a high-throughput, genome-wide technique to generate fluorescent human reporter cell lines responsive to manipulation of epigenetic factors. By profiling GFP expression from a large pool of individually barcoded lentiviral integrants in the presence and absence of a perturbation, we identify reporters responsive to pharmacological inhibition of the histone lysine demethylase LSD1 and genetic ablation of the PRC2 subunit SUZ12. Furthermore, by manipulating the HIV-1 host factor LEDGF through targeted deletion or fusion to chromatin reader domains, we alter lentiviral integration site preferences, thus broadening the types of chromatin examined by TRACE. The phenotypic reporters generated through TRACE will allow the genetic interrogation of a broad range of epigenetic pathways, furthering our mechanistic understanding of chromatin biology.
Assuntos
Técnicas Biossensoriais , Epigênese Genética , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/genética , Lentivirus/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Montagem e Desmontagem da Cromatina , Epigenoma , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histona Desmetilases/metabolismo , Humanos , Lentivirus/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Compact and versatile CRISPR-Cas systems will enable genome engineering applications through high-efficiency delivery in a wide variety of contexts. Here, we create an efficient miniature Cas system (CasMINI) engineered from the type V-F Cas12f (Cas14) system by guide RNA and protein engineering, which is less than half the size of currently used CRISPR systems (Cas9 or Cas12a). We demonstrate that CasMINI can drive high levels of gene activation (up to thousands-fold increases), while the natural Cas12f system fails to function in mammalian cells. We show that the CasMINI system has comparable activities to Cas12a for gene activation, is highly specific, and allows robust base editing and gene editing. We expect that CasMINI can be broadly useful for cell engineering and gene therapy applications ex vivo and in vivo.
Assuntos
Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes , Engenharia de Proteínas , Ativação Transcricional , Proteínas Associadas a CRISPR/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Mutação , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismoRESUMO
Many genes are regulated by multiple enhancers that often simultaneously activate their target gene. However, how individual enhancers collaborate to activate transcription is not well understood. Here, we dissect the functions and interdependencies of five enhancer elements that together activate Fgf5 expression during exit from naive murine pluripotency. Four intergenic elements form a super-enhancer, and most of the elements contribute to Fgf5 induction at distinct time points. A fifth, poised enhancer located in the first intron contributes to Fgf5 expression at every time point by amplifying overall Fgf5 expression levels. Despite low individual enhancer activity, together these elements strongly induce Fgf5 expression in a super-additive fashion that involves strong accumulation of RNA polymerase II at the intronic enhancer. Finally, we observe a strong anti-correlation between RNA polymerase II levels at enhancers and their distance to the closest promoter, and we identify candidate elements with properties similar to the intronic enhancer.
Assuntos
Elementos Facilitadores Genéticos , Fator 5 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Murinas/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/genética , Animais , Linhagem Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Éxons , Fator 5 de Crescimento de Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histonas/genética , Histonas/metabolismo , Íntrons , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/citologia , RNA Polimerase II/metabolismo , Análise de Sequência de RNA , Transdução de Sinais , Análise de Célula Única , Transcrição Gênica , Proteína Vermelha FluorescenteRESUMO
Currently, either highly multiplexed genetic manipulations can be delivered to mammalian cells all at once or extensive engineering of gene regulatory sequences can be used to conditionally activate a few manipulations. Here, we provide proof of principle for a new system enabling multiple genetic manipulations to be executed as a preprogrammed cascade of events. The system leverages the programmability of the S. pyogenes Cas9 and is based on flexible arrangements of individual modules of activity. The basic module consists of an inactive single-guide RNA (sgRNA)-like component that is converted to an active state through the effects of another sgRNA. Modules can be arranged to bring about an algorithmic program of sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences. With the expanding diversity of available tools that use spCas9, this sgRNA-based system provides multiple levels of interfacing with mammalian cell biology.
Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , RNA Guia de Cinetoplastídeos/genética , Animais , Pareamento de Bases , Sequência de Bases , Proteína 9 Associada à CRISPR/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Conformação de Ácido Nucleico , Plasmídeos/química , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Guia de Cinetoplastídeos/química , RNA Guia de Cinetoplastídeos/metabolismo , Streptococcus pyogenes/química , Streptococcus pyogenes/enzimologiaRESUMO
Selective protein degradation by the ubiquitin-proteasome system (UPS) is involved in all cellular processes. However, the substrates and specificity of most UPS components are not well understood. Here we systematically characterized the UPS in Saccharomyces cerevisiae. Using fluorescent timers, we determined how loss of individual UPS components affects yeast proteome turnover, detecting phenotypes for 76% of E2, E3, and deubiquitinating enzymes. We exploit this dataset to gain insights into N-degron pathways, which target proteins carrying N-terminal degradation signals. We implicate Ubr1, an E3 of the Arg/N-degron pathway, in targeting mitochondrial proteins processed by the mitochondrial inner membrane protease. Moreover, we identify Ylr149c/Gid11 as a substrate receptor of the glucose-induced degradation-deficient (GID) complex, an E3 of the Pro/N-degron pathway. Our results suggest that Gid11 recognizes proteins with N-terminal threonines, expanding the specificity of the GID complex. This resource of potential substrates and relationships between UPS components enables exploring functions of selective protein degradation.
Assuntos
Proteínas Mitocondriais/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligases/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/metabolismo , Transporte Proteico , Proteólise , Proteômica/métodos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Treonina/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/classificação , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteína Vermelha FluorescenteRESUMO
The RAD51 recombinase forms nucleoprotein filaments to promote double-strand break repair, replication fork reversal, and fork stabilization. The stability of these filaments is highly regulated, as both too little and too much RAD51 activity can cause genome instability. RADX is a single-strand DNA (ssDNA) binding protein that regulates DNA replication. Here, we define its mechanism of action. We find that RADX inhibits RAD51 strand exchange and D-loop formation activities. RADX directly and selectively interacts with ATP-bound RAD51, stimulates ATP hydrolysis, and destabilizes RAD51 nucleofilaments. The RADX interaction with RAD51, in addition to its ssDNA binding capability, is required to maintain replication fork elongation rates and fork stability. Furthermore, BRCA2 can overcome the RADX-dependent RAD51 inhibition. Thus, RADX functions in opposition to BRCA2 in regulating RAD51 nucleofilament stability to ensure the right level of RAD51 function during DNA replication.
Assuntos
Proteína BRCA2/genética , Replicação do DNA , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a RNA/genética , Rad51 Recombinase/genética , Trifosfato de Adenosina/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Hidrólise , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Ligação a RNA/metabolismo , Rad51 Recombinase/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Proteína Vermelha FluorescenteRESUMO
Homologous recombination (HR) is essential for maintenance of genome integrity. Rad51 paralogs fulfill a conserved but undefined role in HR, and their mutations are associated with increased cancer risk in humans. Here, we use single-molecule imaging to reveal that the Saccharomyces cerevisiae Rad51 paralog complex Rad55-Rad57 promotes assembly of Rad51 recombinase filament through transient interactions, providing evidence that it acts like a classical molecular chaperone. Srs2 is an ATP-dependent anti-recombinase that downregulates HR by actively dismantling Rad51 filaments. Contrary to the current model, we find that Rad55-Rad57 does not physically block the movement of Srs2. Instead, Rad55-Rad57 promotes rapid re-assembly of Rad51 filaments after their disruption by Srs2. Our findings support a model in which Rad51 is in flux between free and single-stranded DNA (ssDNA)-bound states, the rate of which is controlled dynamically though the opposing actions of Rad55-Rad57 and Srs2.
Assuntos
Adenosina Trifosfatases/genética , DNA Helicases/genética , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Recombinação Homóloga , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Imagem Individual de Molécula , Proteína Vermelha FluorescenteRESUMO
Nervous systems process information by integrating the electrical activity of neurons in complex networks. This motivates the long-standing interest in using optical methods to simultaneously monitor the membrane potential of multiple genetically targeted neurons via expression of genetically encoded fluorescent voltage indicators (GEVIs) in intact neural circuits. No currently available GEVIs have demonstrated robust signals in intact brain tissue that enable reliable recording of individual electrical events simultaneously in multiple neurons. Here, we show that the recently developed "ArcLight" GEVI robustly reports both subthreshold events and action potentials in genetically targeted neurons in the intact Drosophila fruit fly brain and reveals electrical signals in neurite branches. In the same way that genetically encoded fluorescent sensors have revolutionized the study of intracellular Ca(2+) signals, ArcLight now enables optical measurement in intact neural circuits of membrane potential, the key cellular parameter that underlies neuronal information processing.
Assuntos
Drosophila melanogaster/fisiologia , Fenômenos Eletrofisiológicos , Rede Nervosa , Optogenética/métodos , Animais , Encéfalo/fisiologia , Relógios Circadianos , Drosophila melanogaster/citologia , Proteínas de Fluorescência Verde/genética , Neurônios/fisiologiaRESUMO
Fluorescent proteins are commonly used to label cells across organisms, but the unmodified forms cannot control biological activities. Using GFP-binding proteins derived from Camelid antibodies, we co-opted GFP as a scaffold for inducing formation of biologically active complexes, developing a library of hybrid transcription factors that control gene expression only in the presence of GFP or its derivatives. The modular design allows for variation in key properties such as DNA specificity, transcriptional potency, and drug dependency. Production of GFP controlled cell-specific gene expression and facilitated functional perturbations in the mouse retina and brain. Further, retrofitting existing transgenic GFP mouse and zebrafish lines for GFP-dependent transcription enabled applications such as optogenetic probing of neural circuits. This work establishes GFP as a multifunctional scaffold and opens the door to selective manipulation of diverse GFP-labeled cells across transgenic lines. This approach may also be extended to exploit other intracellular products as cell-specific scaffolds in multicellular organisms.
Assuntos
Técnicas Genéticas , Proteínas de Fluorescência Verde/metabolismo , Transcrição Gênica , Animais , Animais Geneticamente Modificados , Fenômenos Eletrofisiológicos , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição/metabolismo , Peixe-ZebraRESUMO
The fluorescent protein toolbox has revolutionized experimental biology. Despite this advance, no fluorescent proteins have been identified from vertebrates, nor has chromogenic ligand-inducible activation or clinical utility been demonstrated. Here, we report the cloning and characterization of UnaG, a fluorescent protein from Japanese eel. UnaG belongs to the fatty-acid-binding protein (FABP) family, and expression in eel is restricted to small-diameter muscle fibers. On heterologous expression in cell lines or mouse brain, UnaG produces oxygen-independent green fluorescence. Remarkably, UnaG fluorescence is triggered by an endogenous ligand, bilirubin, a membrane-permeable heme metabolite and clinical health biomarker. The holoUnaG structure at 1.2 Å revealed a biplanar coordination of bilirubin by reversible π-conjugation, and we used this high-affinity and high-specificity interaction to establish a fluorescence-based human bilirubin assay with promising clinical utility. UnaG will be the prototype for a versatile class of ligand-activated fluorescent proteins, with applications in research, medicine, and bioengineering.
Assuntos
Enguias/metabolismo , Proteínas de Peixes/genética , Proteínas de Peixes/isolamento & purificação , Proteínas de Fluorescência Verde/isolamento & purificação , Sequência de Aminoácidos , Animais , Bilirrubina/metabolismo , Clonagem Molecular , Proteínas de Peixes/química , Proteínas de Peixes/metabolismo , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Dados de Sequência Molecular , Músculos/metabolismo , Alinhamento de SequênciaRESUMO
Time-lapse fluorescence microscopy is key to unraveling biological development and function; however, living systems, by their nature, permit only limited interrogation and contain untapped information that can only be captured by more invasive methods. Deep-tissue live imaging presents a particular challenge owing to the spectral range of live-cell imaging probes/fluorescent proteins, which offer only modest optical penetration into scattering tissues. Herein, we employ convolutional neural networks to augment live-imaging data with deep-tissue images taken on fixed samples. We demonstrate that convolutional neural networks may be used to restore deep-tissue contrast in GFP-based time-lapse imaging using paired final-state datasets acquired using near-infrared dyes, an approach termed InfraRed-mediated Image Restoration (IR2). Notably, the networks are remarkably robust over a wide range of developmental times. We employ IR2 to enhance the information content of green fluorescent protein time-lapse images of zebrafish and Drosophila embryo/larval development and demonstrate its quantitative potential in increasing the fidelity of cell tracking/lineaging in developing pescoids. Thus, IR2 is poised to extend live imaging to depths otherwise inaccessible.
Assuntos
Drosophila , Peixe-Zebra , Animais , Imagem com Lapso de Tempo/métodos , Microscopia de Fluorescência , Proteínas de Fluorescência Verde/genéticaRESUMO
Despite the importance of signaling lipids, many questions remain about their function because few tools are available for charting lipid gradients in vivo. Here we generated a sphingosine 1-phosphate (S1P) reporter mouse and used this mouse to define the distribution of S1P in the spleen. Unexpectedly, the presence of blood did not serve as a predictor of the concentration of signaling-available S1P. Large areas of the red pulp had low concentrations of S1P, while S1P was sensed by cells inside the white pulp near the marginal sinus. The lipid phosphate phosphatase LPP3 maintained low S1P concentrations in the spleen and enabled efficient shuttling of marginal zone B cells. The exquisitely tight regulation of S1P availability might explain how a single lipid can simultaneously orchestrate the movements of many cells of the immune system.