RESUMO
Nuclear pore complexes (NPCs) mediate the nucleocytoplasmic transport of macromolecules. Here we provide a structure of the isolated yeast NPC in which the inner ring is resolved by cryo-EM at sub-nanometer resolution to show how flexible connectors tie together different structural and functional layers. These connectors may be targets for phosphorylation and regulated disassembly in cells with an open mitosis. Moreover, some nucleoporin pairs and transport factors have similar interaction motifs, which suggests an evolutionary and mechanistic link between assembly and transport. We provide evidence for three major NPC variants that may foreshadow functional specializations at the nuclear periphery. Cryo-electron tomography extended these studies, providing a model of the in situ NPC with a radially expanded inner ring. Our comprehensive model reveals features of the nuclear basket and central transporter, suggests a role for the lumenal Pom152 ring in restricting dilation, and highlights structural plasticity that may be required for transport.
Assuntos
Adaptação Fisiológica , Poro Nuclear/metabolismo , Saccharomyces cerevisiae/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Fluorescência , Simulação de Acoplamento Molecular , Membrana Nuclear/metabolismo , Poro Nuclear/química , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Domínios Proteicos , Reprodutibilidade dos Testes , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce "molecular signposts" that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells.
Assuntos
Membrana Celular/ultraestrutura , Microscopia Crioeletrônica , DNA/ultraestrutura , Tomografia com Microscopia Eletrônica , Animais , Aptâmeros de Nucleotídeos/química , Fenômenos Biofísicos , Linhagem Celular , Feminino , Fluorescência , Humanos , Nanopartículas/ultraestruturaRESUMO
Ligands can induce G protein-coupled receptors (GPCRs) to adopt a myriad of conformations, many of which play critical roles in determining the activation of specific signaling cascades associated with distinct functional and behavioral consequences. For example, the 5-hydroxytryptamine 2A receptor (5-HT2AR) is the target of classic hallucinogens, atypical antipsychotics, and psychoplastogens. However, currently available methods are inadequate for directly assessing 5-HT2AR conformation both in vitro and in vivo. Here, we developed psychLight, a genetically encoded fluorescent sensor based on the 5-HT2AR structure. PsychLight detects behaviorally relevant serotonin release and correctly predicts the hallucinogenic behavioral effects of structurally similar 5-HT2AR ligands. We further used psychLight to identify a non-hallucinogenic psychedelic analog, which produced rapid-onset and long-lasting antidepressant-like effects after a single administration. The advent of psychLight will enable in vivo detection of serotonin dynamics, early identification of designer drugs of abuse, and the development of 5-HT2AR-dependent non-hallucinogenic therapeutics.
Assuntos
Técnicas Biossensoriais , Drogas Desenhadas/química , Drogas Desenhadas/farmacologia , Descoberta de Drogas/métodos , Alucinógenos/química , Alucinógenos/farmacologia , Receptor 5-HT2A de Serotonina/química , Animais , Avaliação Pré-Clínica de Medicamentos/métodos , Feminino , Fluorescência , Corantes Fluorescentes/química , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fotometria , Conformação Proteica , Engenharia de Proteínas , Receptor 5-HT2A de Serotonina/genética , Receptor 5-HT2A de Serotonina/metabolismo , Serotonina/metabolismo , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
The association of nuclear DNA with histones to form chromatin is essential for temporal and spatial control of eukaryotic genomes. In this study, we examined the physical state of condensed chromatin in vitro and in vivo. Our in vitro studies demonstrate that self-association of nucleosomal arrays under a wide range of solution conditions produces supramolecular condensates in which the chromatin is physically constrained and solid-like. By measuring DNA mobility in living cells, we show that condensed chromatin also exhibits solid-like behavior in vivo. Representative heterochromatin proteins, however, display liquid-like behavior and coalesce around the solid chromatin scaffold. Importantly, euchromatin and heterochromatin show solid-like behavior even under conditions that produce limited interactions between chromatin fibers. Our results reveal that condensed chromatin exists in a solid-like state whose properties resist external forces and create an elastic gel and provides a scaffold that supports liquid-liquid phase separation of chromatin binding proteins.
Assuntos
Cromatina/metabolismo , Acetilação/efeitos dos fármacos , Animais , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cromatina/efeitos dos fármacos , Dano ao DNA , Eucromatina/metabolismo , Fluorescência , Heterocromatina/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Lasers , Camundongos , Modelos Biológicos , Concentração Osmolar , FotodegradaçãoRESUMO
The synthesis of new ribosomes begins during transcription of the rRNA and is widely assumed to follow an orderly 5' to 3' gradient. To visualize co-transcriptional assembly of ribosomal protein-RNA complexes in real time, we developed a single-molecule platform that simultaneously monitors transcription and protein association with the elongating transcript. Unexpectedly, the early assembly protein uS4 binds newly made pre-16S rRNA only transiently, likely due to non-native folding of the rRNA during transcription. Stable uS4 binding became more probable only in the presence of additional ribosomal proteins that bind upstream and downstream of protein uS4 by allowing productive assembly intermediates to form earlier. We propose that dynamic sampling of elongating RNA by multiple proteins overcomes heterogeneous RNA folding, preventing assembly bottlenecks and initiating assembly within the transcription time window. This may be a common feature of transcription-coupled RNP assembly.
Assuntos
Ribonucleoproteínas/metabolismo , Transcrição Gênica , Fluorescência , Modelos Biológicos , Ligação Proteica , Estabilidade Proteica , Precursores de RNA/biossíntese , Precursores de RNA/química , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Ribossômicas/metabolismo , Ribossomos/metabolismo , Elongação da Transcrição GenéticaRESUMO
Stem cells determine homeostasis and repair of many tissues and are increasingly recognized as functionally heterogeneous. To define the extent of-and molecular basis for-heterogeneity, we overlaid functional, transcriptional, and epigenetic attributes of hematopoietic stem cells (HSCs) at a clonal level using endogenous fluorescent tagging. Endogenous HSC had clone-specific functional attributes over time in vivo. The intra-clonal behaviors were highly stereotypic, conserved under the stress of transplantation, inflammation, and genotoxic injury, and associated with distinctive transcriptional, DNA methylation, and chromatin accessibility patterns. Further, HSC function corresponded to epigenetic configuration but not always to transcriptional state. Therefore, hematopoiesis under homeostatic and stress conditions represents the integrated action of highly heterogeneous clones of HSC with epigenetically scripted behaviors. This high degree of epigenetically driven cell autonomy among HSCs implies that refinement of the concepts of stem cell plasticity and of the stem cell niche is warranted.
Assuntos
Epigenômica , Células-Tronco Hematopoéticas/citologia , Animais , Linhagem da Célula , Células Clonais/citologia , Fluorescência , Hematopoese , Inflamação/patologia , Camundongos , Transcrição GênicaRESUMO
Regulation of mRNA translation, the process by which ribosomes decode mRNAs into polypeptides, is used to tune cellular protein levels. Currently, methods for observing the complete process of translation from single mRNAs in vivo are unavailable. Here, we report the long-term (>1 hr) imaging of single mRNAs undergoing hundreds of rounds of translation in live cells, enabling quantitative measurements of ribosome initiation, elongation, and stalling. This approach reveals a surprising heterogeneity in the translation of individual mRNAs within the same cell, including rapid and reversible transitions between a translating and non-translating state. Applying this method to the cell-cycle gene Emi1, we find strong overall repression of translation initiation by specific 5' UTR sequences, but individual mRNA molecules in the same cell can exhibit dramatically different translational efficiencies. The ability to observe translation of single mRNA molecules in live cells provides a powerful tool to study translation regulation.
Assuntos
Imagem Óptica/métodos , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , Regiões 5' não Traduzidas , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Proteínas F-Box/metabolismo , Fluorescência , Genes Reporter , Técnicas Genéticas , Proteínas de Fluorescência Verde/análise , Humanos , Proteínas Luminescentes/análise , Elongação Traducional da Cadeia Peptídica , Iniciação Traducional da Cadeia Peptídica , RNA Mensageiro/química , Ribossomos/metabolismo , Proteína Vermelha FluorescenteRESUMO
Specific binding proteins are crucial for the correct spatiotemporal expression of mRNA. To understand this process, a method is required to characterize RNA-protein interactions in single living cells with subcellular resolution. We combined endogenous single RNA and protein detection with two-photon fluorescence fluctuation analysis to measure the average number of proteins bound to mRNA at specific locations within live cells. We applied this to quantify the known binding of zipcode binding protein 1 (ZBP1) and ribosomes to ß-actin mRNA within subcellular compartments of primary fibroblasts and neurons. ZBP1-mRNA binding did not occur in nuclei, contrary to previous conclusions. ZBP1 interaction with ß-actin mRNA was enhanced perinuclearly in neurons compared to fibroblasts. Cytoplasmic ZBP1 and ribosome binding to the mRNA were anti-correlated depending on their location in the cell. These measurements support a mechanism whereby ZBP1 inhibits translation of localizing mRNA until its release from the mRNA peripherally, allowing ribosome binding.
Assuntos
Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Neurônios/metabolismo , Análise de Célula Única/métodos , Actinas/genética , Actinas/metabolismo , Animais , Células Cultivadas , Embrião de Mamíferos/citologia , Fluorescência , Camundongos , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA , Ribossomos/metabolismoRESUMO
Metabotropic glutamate receptors belong to a family of G protein-coupled receptors that are obligate dimers and possess a large extracellular ligand-binding domain that is linked via a cysteine-rich domain to their 7-transmembrane domain1. Upon activation, these receptors undergo a large conformational change to transmit the ligand binding signal from the extracellular ligand-binding domain to the G protein-coupling 7-transmembrane domain2. In this manuscript, we propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5. We present a series of structures in lipid nanodiscs, from inactive to fully active, including agonist-bound intermediate states. Further, using bulk and single-molecule fluorescence imaging, we reveal distinct receptor conformations upon allosteric modulator and G protein binding.
Assuntos
Ligantes , Domínios Proteicos , Receptor de Glutamato Metabotrópico 5 , Humanos , Regulação Alostérica/efeitos dos fármacos , Fluorescência , Modelos Moleculares , Ligação Proteica , Receptor de Glutamato Metabotrópico 5/agonistas , Receptor de Glutamato Metabotrópico 5/química , Receptor de Glutamato Metabotrópico 5/metabolismo , Imagem Individual de Molécula , Proteínas Heterotriméricas de Ligação ao GTP/metabolismoRESUMO
Cerebrospinal fluid (CSF) in the subarachnoid space around the brain has long been known to drain through the lymphatics to cervical lymph nodes1-17, but the connections and regulation have been challenging to identify. Here, using fluorescent CSF tracers in Prox1-GFP lymphatic reporter mice18, we found that the nasopharyngeal lymphatic plexus is a major hub for CSF outflow to deep cervical lymph nodes. This plexus had unusual valves and short lymphangions but no smooth-muscle coverage, whereas downstream deep cervical lymphatics had typical semilunar valves, long lymphangions and smooth muscle coverage that transported CSF to the deep cervical lymph nodes. α-Adrenergic and nitric oxide signalling in the smooth muscle cells regulated CSF drainage through the transport properties of deep cervical lymphatics. During ageing, the nasopharyngeal lymphatic plexus atrophied, but deep cervical lymphatics were not similarly altered, and CSF outflow could still be increased by adrenergic or nitric oxide signalling. Single-cell analysis of gene expression in lymphatic endothelial cells of the nasopharyngeal plexus of aged mice revealed increased type I interferon signalling and other inflammatory cytokines. The importance of evidence for the nasopharyngeal lymphatic plexus functioning as a CSF outflow hub is highlighted by its regression during ageing. Yet, the ageing-resistant pharmacological activation of deep cervical lymphatic transport towards lymph nodes can still increase CSF outflow, offering an approach for augmenting CSF clearance in age-related neurological conditions in which greater efflux would be beneficial.
Assuntos
Líquido Cefalorraquidiano , Vértebras Cervicais , Drenagem , Vasos Linfáticos , Animais , Camundongos , Envelhecimento/metabolismo , Líquido Cefalorraquidiano/metabolismo , Vértebras Cervicais/metabolismo , Células Endoteliais/metabolismo , Fluorescência , Genes Reporter , Interferon Tipo I/imunologia , Interferon Tipo I/metabolismo , Vasos Linfáticos/fisiologia , Miócitos de Músculo Liso/metabolismo , Óxido Nítrico/metabolismo , Nariz/fisiologia , Faringe/metabolismo , Receptores Adrenérgicos alfa/metabolismo , Análise de Célula Única , Transdução de SinaisRESUMO
It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.
Assuntos
Sistemas CRISPR-Cas , Hepatócitos , Neoplasias Hepáticas , Fígado , Metástase Neoplásica , Proteínas do Tecido Nervoso , Animais , Feminino , Humanos , Masculino , Camundongos , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Sistemas CRISPR-Cas/genética , Modelos Animais de Doenças , Elementos de DNA Transponíveis , Fluorescência , Hepatócitos/metabolismo , Hepatócitos/citologia , Hepatócitos/patologia , Fator 4 Semelhante a Kruppel/metabolismo , Fígado/citologia , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/prevenção & controle , Neoplasias Hepáticas/secundário , Melanoma/metabolismo , Melanoma/patologia , Metástase Neoplásica/tratamento farmacológico , Metástase Neoplásica/patologia , Metástase Neoplásica/prevenção & controle , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Semaforinas/antagonistas & inibidores , Semaforinas/metabolismoRESUMO
Mammalian bodies have more than a billion cells per cubic centimeter, which makes whole-body cell (WBC) profiling of an organism one of the ultimate challenges in biology and medicine. Recent advances in tissue-clearing technology have enabled rapid and comprehensive cellular analyses in whole organs and in the whole body by a combination of state-of-the-art technologies of optical imaging and image informatics. In this review, we focus mainly on the chemical principles in currently available techniques for tissue clearing and staining to facilitate our understanding of their underlying mechanisms. Tissue clearing is usually conducted by the following steps: (a) fixation, (b) permeabilization, (c) decolorizing, and (d) refractive index (RI) matching. To phenotype individual cells after tissue clearing, it is important to visualize genetically encoded fluorescent reporters and/or to stain tissues with fluorescent dyes, fluorescent labeled antibodies, or nucleic acid probes. Although some technical challenges remain, the chemical principles in tissue clearing and staining for WBC profiling will enable various applications, such as identifying cellular circuits across multiple organs and measuring their dynamics in stochastic and proliferative cellular processes, for example, autoimmune and malignant neoplastic diseases.
Assuntos
Células/metabolismo , Coloração e Rotulagem , Fixação de Tecidos/métodos , Animais , Fluorescência , Humanos , Permeabilidade , RefratometriaRESUMO
Understanding the structure-function relationships at cellular, circuit, and organ-wide scale requires 3D anatomical and phenotypical maps, currently unavailable for many organs across species. At the root of this knowledge gap is the absence of a method that enables whole-organ imaging. Herein, we present techniques for tissue clearing in which whole organs and bodies are rendered macromolecule-permeable and optically transparent, thereby exposing their cellular structure with intact connectivity. We describe PACT (passive clarity technique), a protocol for passive tissue clearing and immunostaining of intact organs; RIMS (refractive index matching solution), a mounting media for imaging thick tissue; and PARS (perfusion-assisted agent release in situ), a method for whole-body clearing and immunolabeling. We show that in rodents PACT, RIMS, and PARS are compatible with endogenous-fluorescence, immunohistochemistry, RNA single-molecule FISH, long-term storage, and microscopy with cellular and subcellular resolution. These methods are applicable for high-resolution, high-content mapping and phenotyping of normal and pathological elements within intact organs and bodies.
Assuntos
Células/classificação , Imageamento Tridimensional/métodos , Análise de Célula Única , Imagem Corporal Total , Animais , Encéfalo/citologia , Células/metabolismo , Fluorescência , Camundongos , Microscopia Confocal/métodos , Microscopia Eletrônica de Varredura , FenótipoRESUMO
PIEZOs are mechanosensitive ion channels that convert force into chemoelectric signals1,2 and have essential roles in diverse physiological settings3. In vitro studies have proposed that PIEZO channels transduce mechanical force through the deformation of extensive blades of transmembrane domains emanating from a central ion-conducting pore4-8. However, little is known about how these channels interact with their native environment and which molecular movements underlie activation. Here we directly observe the conformational dynamics of the blades of individual PIEZO1 molecules in a cell using nanoscopic fluorescence imaging. Compared with previous structural models of PIEZO1, we show that the blades are significantly expanded at rest by the bending stress exerted by the plasma membrane. The degree of expansion varies dramatically along the length of the blade, where decreased binding strength between subdomains can explain increased flexibility of the distal blade. Using chemical and mechanical modulators of PIEZO1, we show that blade expansion and channel activation are correlated. Our findings begin to uncover how PIEZO1 is activated in a native environment. More generally, as we reliably detect conformational shifts of single nanometres from populations of channels, we expect that this approach will serve as a framework for the structural analysis of membrane proteins through nanoscopic imaging.
Assuntos
Canais Iônicos , Membrana Celular/metabolismo , Fluorescência , Canais Iônicos/química , Canais Iônicos/metabolismo , Modelos Moleculares , Movimento , Conformação Proteica , Análise de Célula ÚnicaRESUMO
Projected responses of ocean net primary productivity to climate change are highly uncertain1. Models suggest that the climate sensitivity of phytoplankton nutrient limitation in the low-latitude Pacific Ocean plays a crucial role1-3, but this is poorly constrained by observations4. Here we show that changes in physical forcing drove coherent fluctuations in the strength of equatorial Pacific iron limitation through multiple El Niño/Southern Oscillation (ENSO) cycles, but that this was overestimated twofold by a state-of-the-art climate model. Our assessment was enabled by first using a combination of field nutrient-addition experiments, proteomics and above-water hyperspectral radiometry to show that phytoplankton physiological responses to iron limitation led to approximately threefold changes in chlorophyll-normalized phytoplankton fluorescence. We then exploited the >18-year satellite fluorescence record to quantify climate-induced nutrient limitation variability. Such synoptic constraints provide a powerful approach for benchmarking the realism of model projections of net primary productivity to climate changes.
Assuntos
Modelos Climáticos , El Niño Oscilação Sul , Ferro , Clorofila/metabolismo , Mudança Climática , Fluorescência , Ferro/metabolismo , Nutrientes/metabolismo , Oceano Pacífico , Fitoplâncton/metabolismo , Proteômica , Radiometria , Imagens de SatélitesRESUMO
The paper-folding mechanism has been widely adopted in building of reconfigurable macroscale systems because of its unique capabilities and advantages in programming variable shapes and stiffness into a structure1-5. However, it has barely been exploited in the construction of molecular-level systems owing to the lack of a suitable design principle, even though various dynamic structures based on DNA self-assembly6-9 have been developed10-23. Here we propose a method to harness the paper-folding mechanism to create reconfigurable DNA origami structures. The main idea is to build a reference, planar wireframe structure24 whose edges follow a crease pattern in paper folding so that it can be folded into various target shapes. We realized several paper-like folding and unfolding patterns using DNA strand displacement25 with high yield. Orthogonal folding, repeatable folding and unfolding, folding-based microRNA detection and fluorescence signal control were demonstrated. Stimuli-responsive folding and unfolding triggered by pH or light-source change were also possible. Moreover, by employing hierarchical assembly26 we could expand the design space and complexity of the paper-folding mechanism in a highly programmable manner. Because of its high programmability and scalability, we expect that the proposed paper-folding-based reconfiguration method will advance the development of complex molecular systems.
Assuntos
DNA , Conformação de Ácido Nucleico , DNA/química , MicroRNAs/análise , MicroRNAs/química , Fluorescência , Concentração de Íons de HidrogênioRESUMO
Photosynthesis is generally assumed to be initiated by a single photon1-3 from the Sun, which, as a weak light source, delivers at most a few tens of photons per nanometre squared per second within a chlorophyll absorption band1. Yet much experimental and theoretical work over the past 40 years has explored the events during photosynthesis subsequent to absorption of light from intense, ultrashort laser pulses2-15. Here, we use single photons to excite under ambient conditions the light-harvesting 2 (LH2) complex of the purple bacterium Rhodobacter sphaeroides, comprising B800 and B850 rings that contain 9 and 18 bacteriochlorophyll molecules, respectively. Excitation of the B800 ring leads to electronic energy transfer to the B850 ring in approximately 0.7 ps, followed by rapid B850-to-B850 energy transfer on an approximately 100-fs timescale and light emission at 850-875 nm (refs. 16-19). Using a heralded single-photon source20,21 along with coincidence counting, we establish time correlation functions for B800 excitation and B850 fluorescence emission and demonstrate that both events involve single photons. We also find that the probability distribution of the number of heralds per detected fluorescence photon supports the view that a single photon can upon absorption drive the subsequent energy transfer and fluorescence emission and hence, by extension, the primary charge separation of photosynthesis. An analytical stochastic model and a Monte Carlo numerical model capture the data, further confirming that absorption of single photons is correlated with emission of single photons in a natural light-harvesting complex.
Assuntos
Complexos de Proteínas Captadores de Luz , Fótons , Fotossíntese , Rhodobacter sphaeroides , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/metabolismo , Transferência de Energia , Complexos de Proteínas Captadores de Luz/química , Complexos de Proteínas Captadores de Luz/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/metabolismo , Fluorescência , Processos Estocásticos , Método de Monte CarloRESUMO
The honey bee is a powerful model system to probe host-gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to noninvasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular tool kit to genetically modify symbionts and reported for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium Snodgrassella alvi as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report exposure to the molecules in a dose-dependent manner through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or noninvasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host-gut microbiota research.
Assuntos
Bactérias , Microbiota , Abelhas , Animais , Bactérias/genética , Agricultura , Fezes , FluorescênciaRESUMO
Regulation of apoptosis by Bcl-2 family proteins is a paradigm for complex protein-protein and protein-membrane systems. Elucidating the molecular mechanisms of these interactions in vitro in live cells and in animal studies has been significantly enhanced by using fluorescence techniques.
Assuntos
Apoptose , Membranas Intracelulares/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Fluorescência , Humanos , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismoRESUMO
Reinforcement learning models postulate that neurons that release dopamine encode information about action and action outcome, and provide a teaching signal to striatal spiny projection neurons in the form of dopamine release1. Dopamine is thought to guide learning via dynamic and differential modulation of protein kinase A (PKA) in each class of spiny projection neuron2. However, the real-time relationship between dopamine and PKA in spiny projection neurons remains untested in behaving animals. Here we monitor the activity of dopamine-releasing neurons, extracellular levels of dopamine and net PKA activity in spiny projection neurons in the nucleus accumbens of mice during learning. We find positive and negative modulation of dopamine that evolves across training and is both necessary and sufficient to explain concurrent fluctuations in the PKA activity of spiny projection neurons. Modulations of PKA in spiny projection neurons that express type-1 and type-2 dopamine receptors are dichotomous, such that these neurons are selectively sensitive to increases and decreases, respectively, in dopamine that occur at different phases of learning. Thus, PKA-dependent pathways in each class of spiny projection neuron are asynchronously engaged by positive or negative dopamine signals during learning.