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1.
Artigo em Inglês | MEDLINE | ID: mdl-31176867

RESUMO

In rice field eel (Monopterus albus), germ cell development in the developing gonad has been revealed in detail. However, it is unclear how primordial germ cells (PGCs) migrate to the somatic part of the gonad (genital ridge). This study visualized PGC migration by injecting a chimeric mRNA containing a fluorescent protein fused to the 3' untranslated region (3'UTR) of three different genes, nanos3 of zebrafish (Danio rerio) and dead end (dnd) and vasa of rice field eel. The mRNAs were injected either alone or in pairs into embryos at the one-cell stage. The results showed that mRNAs containing nanos3 and dnd 3'UTRs labeled PGCs over a wider time frame than those containing vasa 3'UTR, suggesting that nanos3 and dnd 3'UTRs are suitable for visualizing PGCs in rice field eel. Using this direct visualization method, the normal migration route of PGCs was observed from the 50%-epiboly stage to hatching stage for the first time, and the ectopic PGCs were also visualized during this period in rice field eel. These findings extend our knowledge of germ cell development, and lay a foundation for further research on the relationship between PGCs and sex differentiation, and on incubation conditions for embryos in rice field eel.


Assuntos
Células Germinativas/metabolismo , Smegmamorpha/embriologia , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Animais , Movimento Celular/genética , Movimento Celular/fisiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Células Germinativas/citologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , RNA/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Smegmamorpha/genética , Smegmamorpha/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Photochem Photobiol Sci ; 18(7): 1823-1832, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31165126

RESUMO

The fluorescence (FL) of calcium-discharged photoprotein (CaDP) can be altered by easily mutating CaDP without modifying coelenteramide (CLM), which is the decarboxylation product of coelenterazine in calcium-regulated photoprotein. The His22-Phe88-Trp92 triad (the ordering numbers of three amino acids are sorted by a crystal structure (PDB: 2F8P) of calcium-discharged obelin, i.e., CaDP-obelin) is closely related to CaDP-obelin FL, since it exists in close proximity to the 5-p-hydroxyphenyl of CLM. Therefore, it is important to thoroughly investigate how the mutations of this triad affect the emission color of CaDP-obelin FL. In this study, by mutating wild-type CaDP-obelin (WT) at the His22-Phe88-Trp92 triad, we theoretically constructed its nine mutants of separable FL colors. Through combined quantum mechanics and molecular mechanics (QM/MM) calculations and molecular dynamics (MD) simulations, the influence of the mutations of this triad on the CaDP-obelin FL was analyzed considering the H-bond effect and the charge effect. This study demonstrated that the mutations at the His22-Phe88-Trp92 triad redistribute the charges on the D-π-A molecule, CLM, change the charge transfer from the D to the (π + A) moiety, and thereby alter the FL emission. Appending more negative charges on the phenolate moiety of CLM benefits the FL redshift.


Assuntos
Cálcio/química , Proteínas Luminescentes/química , Simulação de Dinâmica Molecular , Teoria Quântica , Animais , Ligações de Hidrogênio , Hidrozoários/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutagênese Sítio-Dirigida , Conformação Proteica , Espectrometria de Fluorescência
3.
Nat Commun ; 10(1): 2181, 2019 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-31097714

RESUMO

During the blood stage of human malaria, Plasmodium falciparum parasites divide by schizogony-a process wherein components for several daughter cells are produced within a common cytoplasm and then segmentation, a synchronized cytokinesis, produces individual invasive daughters. The basal complex is hypothesized to be required for segmentation, acting as a contractile ring to establish daughter cell boundaries. Here we identify an essential component of the basal complex which we name PfCINCH. Using three-dimensional reconstructions of parasites at electron microscopy resolution, we show that while parasite organelles form and divide normally, PfCINCH-deficient parasites develop inviable conjoined daughters that contain components for multiple cells. Through biochemical evaluation of the PfCINCH-containing complex, we discover multiple previously undescribed basal complex proteins. Therefore, this work provides genetic evidence that the basal complex is required for precise segmentation and lays the groundwork for a mechanistic understanding of how the parasite contractile ring drives cell division.


Assuntos
Divisão Celular/fisiologia , Proteínas Contráteis/fisiologia , Plasmodium falciparum/fisiologia , Proteínas de Protozoários/fisiologia , Animais , Eritrócitos/parasitologia , Microscopia Intravital/métodos , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Microscopia Eletrônica de Transmissão , Plasmodium falciparum/ultraestrutura , Esquizontes/fisiologia , Imagem com Lapso de Tempo
4.
Nat Commun ; 10(1): 2312, 2019 05 24.
Artigo em Inglês | MEDLINE | ID: mdl-31127113

RESUMO

Cardioprotection by salvage of the infarct-affected myocardium is an unmet yet highly desired therapeutic goal. To develop new dedicated therapies, experimental myocardial ischemia/reperfusion (I/R) injury would require methods to simultaneously characterize extent and localization of the damage and the ensuing inflammatory responses in whole hearts over time. Here we present a three-dimensional (3D), simultaneous quantitative investigation of key I/R injury-components by combining bleaching-augmented solvent-based non-toxic clearing (BALANCE) using ethyl cinnamate (ECi) with light sheet fluorescence microscopy. This allows structural analyses of fluorescence-labeled I/R hearts with exceptional detail. We discover and 3D-quantify distinguishable acute and late vascular I/R damage zones. These contain highly localized and spatially structured neutrophil infiltrates that are modulated upon cardiac healing. Our model demonstrates that these characteristic I/R injury patterns can detect the extent of damage even days after the ischemic index event hence allowing the investigation of long-term recovery and remodeling processes.


Assuntos
Coração/diagnóstico por imagem , Imagem Tridimensional/métodos , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Miocárdio/patologia , Animais , Biópsia , Cinamatos/química , Ponte de Artéria Coronária , Modelos Animais de Doenças , Humanos , Substâncias Luminescentes/química , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/cirurgia , Miocárdio/citologia , Miocárdio/imunologia , Neutrófilos/imunologia
5.
Nat Commun ; 10(1): 2167, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-31092821

RESUMO

Ribbon synapses transmit information in sensory systems, but their development is not well understood. To test the hypothesis that ribbon assembly stabilizes nascent synapses, we performed simultaneous time-lapse imaging of fluorescently-tagged ribbons in retinal cone bipolar cells (BCs) and postsynaptic densities (PSD95-FP) of retinal ganglion cells (RGCs). Ribbons and PSD95-FP clusters were more stable when these components colocalized at synapses. However, synapse density on ON-alpha RGCs was unchanged in mice lacking ribbons (ribeye knockout). Wildtype BCs make both ribbon-containing and ribbon-free synapses with these GCs even at maturity. Ribbon assembly and cone BC-RGC synapse maintenance are thus regulated independently. Despite the absence of synaptic ribbons, RGCs continued to respond robustly to light stimuli, although quantitative examination of the responses revealed reduced frequency and contrast sensitivity.


Assuntos
Células Fotorreceptoras Retinianas Cones/fisiologia , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/genética , Proteína 4 Homóloga a Disks-Large/metabolismo , Microscopia Intravital/métodos , Luz , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência/métodos , Estimulação Luminosa , Cultura Primária de Células , Células Bipolares da Retina/fisiologia , Células Ganglionares da Retina/fisiologia , Imagem com Lapso de Tempo/métodos
6.
Curr Microbiol ; 76(7): 896-903, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115599

RESUMO

The health-promoting effects of the probiotic strain Lactobacillus rhamnosus are based on its adherence and colonization ability. However, little is known about its adhesion and colonization rates. Lactobacillus rhamnosus in mouse intestinal mucosa a mutant of the red fluorescence protein (RFP) DSred2 was used to tag L. rhamnosus to observe the adhesion and distribution of L. rhamnosus in mouse intestinal mucosa. A mutant of the red fluorescence protein (RFP) Dsred2 was used to tag L. rhamnosus to allow us to observe and distinguish it in the mouse intestine. Seven-week-old female BALB/c mice were fed once (at day 0) with an oral administration of the labeled L. rhamnosus, and the number of labeled bacteria was detected in different regions of the intestinal tract at 3 h and at day 1, 2, 3, 4, 5, 6, 7, and 15 after administration. The labeling process changed the morphology of L. rhamnosus, as it appeared after observation under the microscope, but did not change its basic probiotic properties in vitro. In vivo, labeled L. rhamnosus reached the colonization peak at the fourth day after gavage. From the distribution point of view, the number of colonization strains increased from the proximal to the distal small intestine (duodenum < jejunum < ileum) and the number of strains in the colon was less than the distal small intestine (ileum). The labeling protocol actually allowed the detection of the distribution and adhesion of this bacterium to the intestine, thus demonstrating that the health-promoting effects of this probiotic are satisfied. This study provides a scientific basis in the use of probiotics such as L. rhamnosus in functional foods.


Assuntos
Aderência Bacteriana , Intestinos/microbiologia , Lactobacillus rhamnosus/fisiologia , Proteínas Luminescentes/metabolismo , Probióticos , Animais , Contagem de Colônia Microbiana , Feminino , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Intestinos/anatomia & histologia , Intestinos/química , Lactobacillus rhamnosus/crescimento & desenvolvimento , Lactobacillus rhamnosus/metabolismo , Proteínas Luminescentes/genética , Camundongos Endogâmicos BALB C , Probióticos/administração & dosagem , Probióticos/metabolismo
7.
Analyst ; 144(12): 3765-3772, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089611

RESUMO

Investigation of the functions of insulin-secreting cells in response to glucose in single-living cells is essential for improving our knowledge on the pathogenesis of diabetes. Therefore, it is desired to develop a new convenient method that enables the direct detection of insulin secreted from single-living cells. Here, insulin-sensor-cells expressing a protein-based insulin-detecting probe immobilized on the extracellular membrane were developed to evaluate the insulin-secretion response in single-living pancreatic ß cells. The protein-based insulin-detecting probe (NαLY) was composed of a bioluminescent protein (nano-luc), the αCT segment of the insulin receptor, L1 and CR domains of the insulin receptor, and a fluorescent protein (YPet). NαLY exhibited a bioluminescence resonance energy transfer (BRET) signal in response to insulin; thus, cells of Hepa1-6 line were genetically engineered to express NαLY on the extracellular membrane. The cells were found to act as insulin-sensor-cells, exhibiting a BRET signal in response to insulin. When the insulin-sensor-cells and pancreatic ß cells (MIN6 cell line) were cocultured and stimulated with glucose, insulin-sensor-cells nearby pancreatic ß cells showed the spike-shaped BRET signal response, whereas the insulin-sensor-cells close to one pancreatic ß cell did not exhibit such signal response. However, all the insulin-sensor-cells showed a gradual increase in BRET signals, which were presumably attributed to the increase in insulin concentrations in the culture dish, confirming the function of these insulin-sensor-cells. Therefore, we demonstrated that heterogenetic insulin secretion in single-living pancreatic ß cells could be measured directly using the insulin sensor cells.


Assuntos
Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Técnicas Biossensoriais/métodos , Células Secretoras de Insulina/metabolismo , Insulina/análise , Análise de Célula Única/métodos , Animais , Linhagem Celular Tumoral , Técnicas de Cocultura/métodos , Fluorescência , Glucose/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Engenharia de Proteínas/métodos , Receptor de Insulina/genética , Receptor de Insulina/metabolismo
8.
Analyst ; 144(12): 3773-3781, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31089613

RESUMO

MDM2 is a well-known oncoprotein overexpressed in a variety of cancers, and the identification of inhibitors that disrupt the MDM2/p53 interaction is of great interest in anticancer drug development. Here we designed a platform for the facile and visualizable identification of inhibitors of MDM2 using co-expressed protein complexes of MDM2/p53. A hexahistidine-tag on MDM2 allows the binding of the protein complex to the Ni-NTA affinity resin, while the fluorescent protein fused to p53 enables the direct visualization of the interaction of p53 with MDM2. Hence, the inhibition of the MDM2/p53 interaction can be observed with the naked eye. The assay can be set up by directly loading cell lysate to the Ni-NTA affinity resin, and no chemical modification of proteins is needed. In addition to the qualitative analyses, the binding affinity of inhibitors to the MDM2 protein can be quantified by fluorescence titration. The applications of this system have been verified using small molecules and peptide inhibitors. As a proof of concept, we screened a small library using this platform. Interestingly, two types of novel inhibitors of MDM2, including cyclohexyl-triphenylamine derivatives and platinum complexes, were identified and their binding affinities were obtained. Quantitative measurements show that these new types of inhibitors demonstrate a high binding affinity (up to Kd = 51.9 nM) to MDM2.


Assuntos
Bioensaio/métodos , Proteínas Luminescentes/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Aminoácidos , Compostos de Anilina/química , Cromatografia de Afinidade/métodos , Complexos de Coordenação/química , Escherichia coli/genética , Histidina/genética , Histidina/metabolismo , Humanos , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Simulação de Acoplamento Molecular , Oligopeptídeos/genética , Oligopeptídeos/metabolismo , Peptídeos/química , Platina/química , Estudo de Prova de Conceito , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteína Supressora de Tumor p53/genética
9.
Nat Commun ; 10(1): 2271, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118423

RESUMO

Following fertilization, cortical granules exocytose ovastacin, a metalloendopeptidase that cleaves ZP2 in the zona pellucida surrounding mouse eggs to prevent additional sperm binding. Using high- and super-resolution imaging with ovastacinmCherry as a fluorescent marker, we characterize cortical granule dynamics at single granule resolution in transgenic mouse eggs. Newly-developed imaging protocols provide an unprecedented view of vesicular dynamics near the plasma membrane in mouse eggs. We discover that cortical granule anchoring in the cortex is dependent on maternal MATER and document that myosin IIA is required for biphasic trafficking to the plasma membrane. We observe local clearance of cortical actin during exocytosis and determine that pharmacologic or genetic disruption of trafficking to the plasma membrane impairs secretion of cortical granules and results in polyspermy. Thus, the regulation of cortical granule dynamics at the cortex-plasma membrane interface is critical for exocytosis and the post-fertilization block to sperm binding that ensures monospermic fertilization.


Assuntos
Grânulos Citoplasmáticos/metabolismo , Exocitose/fisiologia , Interações Espermatozoide-Óvulo/fisiologia , Zona Pelúcida/metabolismo , Animais , Antígenos/metabolismo , Membrana Celular/metabolismo , Proteínas do Ovo/metabolismo , Feminino , Microscopia Intravital , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Masculino , Metaloproteases/química , Metaloproteases/genética , Metaloproteases/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia de Fluorescência , Glicoproteínas da Zona Pelúcida/metabolismo
10.
Photochem Photobiol Sci ; 18(7): 1793-1805, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31116222

RESUMO

Light-Oxygen-Voltage (LOV) domains are conserved parts of photoreceptors in plants, bacteria and fungi that bind flavins as chromophores and detect blue light. In the past, LOV domain variants have been developed as fluorescent reporter proteins (called flavin-based fluorescent proteins; FbFPs), which due to their ability to fluoresce under anaerobic conditions, fast folding kinetics and a small size of ∼12-16 kDa are a promising reporter system for quantitative real-time analysis of biological processes. Here, we present a small thermostable flavin-based fluorescent protein CagFbFP derived from a soluble LOV domain-containing histidine kinase from the thermophilic bacterium Chloroflexus aggregans. CagFbFP is composed of 107 amino acids with a molecular weight of 11.6 kDa and consists only of the conserved LOV core domain. The protein is thermostable with a melting point of about 68 °C. It crystallizes easily and its crystals diffract to 1.07 Å. Both the crystal structure and small angle scattering data show that the protein is a dimer. Unexpectedly, glutamine 148, which in LOV photoreceptor proteins is the key residue responsible for signal transduction, occupies two conformations. Molecular dynamics simulations show that the two conformations interconvert rapidly. The crystal structure of the wild-type Chloroflexus aggregans LOV domain determined at 1.22 Å resolution confirmed the presence of two alternative conformations of the glutamine 148 side chain. Overall, this protein, due to its stability and ease of crystallization, appears to be a promising model for ultra-high resolution structural studies of LOV domains and for application as a fluorescent reporter.


Assuntos
Proteínas de Bactérias/química , Chloroflexus/metabolismo , Flavinas/química , Proteínas Luminescentes/química , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Peso Molecular , Estrutura Terciária de Proteína , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Espectrometria de Fluorescência , Temperatura de Transição , Difração de Raios X
11.
Nat Commun ; 10(1): 1901, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015409

RESUMO

Asymmetric cell division is a major mechanism generating cell diversity. As cell cycle duration varies among cells in mammalian tissue culture cells, we asked whether their division asymmetry contributes to this variability. We identify among sibling cells an outlier using hierarchical clustering on cell cycle durations of granddaughter cells obtained by lineage tracking of single histone2B-labelled MDCKs. Remarkably, divisions involving outlier cells are not uniformly distributed in lineages, as shown by permutation tests, but appear to emerge from asymmetric divisions taking place at non-stochastic levels: a parent cell influences with 95% confidence and 0.5% error the unequal partitioning of the cell cycle duration in its two progenies. Upon ninein downregulation, this variability propagation is lost, and outlier frequency and variability in cell cycle durations in lineages is reduced. As external influences are not detectable, we propose that a cell-autonomous process, possibly involved in cell specialisation, determines cell cycle duration variability.


Assuntos
Divisão Celular Assimétrica , Linhagem da Célula/genética , Proteínas do Citoesqueleto/genética , Escherichia coli/citologia , Histonas/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Rastreamento de Células/métodos , Proteínas do Citoesqueleto/metabolismo , Cães , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Histonas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Células Madin Darby de Rim Canino , Modelos Biológicos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Tempo
12.
Mol Biotechnol ; 61(6): 451-460, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30997666

RESUMO

We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.


Assuntos
Proteínas de Bactérias/genética , Clonagem Molecular/métodos , Nitrosomonas europaea/genética , Periplasma/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Nitrosomonas europaea/metabolismo , Oxirredutases N-Desmetilantes/genética , Oxirredutases N-Desmetilantes/metabolismo , Periplasma/química , Polissacarídeo-Liase/genética , Polissacarídeo-Liase/metabolismo , Sinais Direcionadores de Proteínas , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo
13.
Methods Mol Biol ; 1933: 297-303, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945194

RESUMO

RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.


Assuntos
Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Tabaco/metabolismo , Fluorescência , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Proteínas Luminescentes/genética , Microscopia Confocal , Folhas de Planta/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Tabaco/genética
14.
Nat Commun ; 10(1): 1662, 2019 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-30971684

RESUMO

Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.


Assuntos
Córtex Cerebral/diagnóstico por imagem , Imagem Tridimensional/métodos , Proteínas Luminescentes/química , Microscopia de Fluorescência por Excitação Multifotônica/métodos , Neuroimagem/métodos , Animais , Astrócitos/metabolismo , Córtex Cerebral/citologia , Cor , Feminino , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Modelos Animais , Nestina/genética , Técnicas de Rastreamento Neuroanatômico/métodos , Parvovirinae/genética , Células Piramidais/metabolismo , Transfecção
15.
BMC Res Notes ; 12(1): 219, 2019 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-30971308

RESUMO

OBJECTIVES: Little is known about the activity and dynamics of ATPase RarA in B. subtilis, proposed to act at stalled DNA replication forks due to DNA damage. We performed fluorescence microscopy time lapse experiments with a functional RarA-mVenus fusion to visualize the dynamics of RarA during conditions that generate DNA damage. DATA DESCRIPTION: In exponentially growing cells, we observed that 15% of the cells contained single RarA-mV (mVenus fluorescent fusion) foci moving throughout the entire cell between 3 min intervals. This percentage remained constant at different time points, indicating that focus formation during unperturbed growth is maintained at about a constant rate. When cells were exposed to stress conditions, the population of cells containing RarA-mV foci tripled after 60 min. Cells exposed to two DNA-damaging drugs, to 5 mM MMS or to 0.5 mM H2O2, showed a similar type of response, with RarA-mVenus foci moving more slowly than during unperturbed growth. It is likely that RarA-mV contributes to the repair of H2O2-induced lesions, and to a minor extent to MMS-induced lesions. The presence of foci in growing cells suggests that RarA also plays a role during the cell cycle, at least in a fraction of cells, possibly contributing to heterogeneity of response to DNA damage.


Assuntos
Adenosina Trifosfatases/genética , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Reparo do DNA , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica , Adenosina Trifosfatases/metabolismo , Bacillus subtilis/efeitos dos fármacos , Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Dano ao DNA , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Genes Reporter , Peróxido de Hidrogênio/farmacologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Transporte Proteico , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Imagem com Lapso de Tempo
16.
Pain ; 160(5): 1166-1174, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30913166

RESUMO

Recent studies have made significant progress in identifying distinct populations of peripheral neurons involved in itch transmission, whereas the cellular identity of spinal interneurons that contribute to itch processing is still a debate. Combining genetic and pharmacological ablation of spinal excitatory neuronal subtypes and behavioral assays, we demonstrate that spinal somatostatin-positive (SOM) excitatory interneurons transmit pruritic sensation. We found that the ablation of spinal SOM/Lbx1 (SOM) neurons caused significant attenuation of scratching responses evoked by various chemical pruritogens (chemical itch). In an attempt to identify substrates of spinal itch neural circuit, we observed that spinal SOM neurons partially overlapped with neurons expressing natriuretic peptide receptor A (Npra), the receptor of peripheral itch transmitter B-type natriuretic peptide. Spinal SOM neurons, however, did not show any overlap with itch transmission neurons expressing gastrin-releasing peptide receptor in the dorsal spinal cord, and the gastrin-releasing peptide-triggered scratching responses were intact after ablating spinal SOM neurons. Dual ablation of SOM and Npra neurons in the spinal cord reduced chemical itch responses to a greater extent than ablation of SOM or Npra neurons alone, suggesting the existence of parallel spinal pathways transmitting chemical itch. Furthermore, we showed that SOM peptide modulated itch processing through disinhibition of somatostatin receptor 2A-positive inhibitory interneuron. Together, our findings reveal a novel spinal mechanism for sensory encoding of itch perception.


Assuntos
Interneurônios/metabolismo , Prurido/induzido quimicamente , Prurido/patologia , Somatostatina/metabolismo , Medula Espinal/patologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Inibidores da Angiogênese/farmacologia , Animais , Cloroquina/toxicidade , Modelos Animais de Doenças , Técnicas In Vitro , Interneurônios/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-fos/metabolismo , Somatostatina/genética , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , p-Metoxi-N-metilfenetilamina/toxicidade , Proteínas tau/genética , Proteínas tau/metabolismo
17.
Immunity ; 50(4): 1054-1068.e3, 2019 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-30926235

RESUMO

Innate lymphoid cell (ILC) development proposes that ILC precursors (ILCPs) segregate along natural killer (NK) cell versus helper cell (ILC1, ILC2, ILC3) pathways, the latter depending on expression of Id2, Zbtb16, and Gata3. We have developed an Id2-reporter strain expressing red fluorescent protein (RFP) in the context of normal Id2 expression to re-examine ILCP phenotype and function. We show that bone-marrow ILCPs were heterogeneous and harbored extensive NK-cell potential in vivo and in vitro. By multiplexing Id2RFP with Zbtb16CreGFP and Bcl11btdTomato strains, we made a single-cell dissection of the ILCP compartment. In contrast with the current model, we have demonstrated that Id2+Zbtb16+ ILCPs included multi-potent ILCPs that retained NK-cell potential. Late-stage ILC2P and ILC3P compartments could be defined by differential Zbtb16 and Bcl11b expression. We suggest a revised model for ILC differentiation that redefines the cell-fate potential of helper-ILC-restricted Zbtb16+ ILCPs.


Assuntos
Regulação da Expressão Gênica/imunologia , Células-Tronco Hematopoéticas/citologia , Imunidade Inata , Proteína 2 Inibidora de Diferenciação/genética , Linfopoese/genética , Transferência Adotiva , Animais , Linhagem da Célula , Fator de Transcrição GATA3/biossíntese , Fator de Transcrição GATA3/genética , Fator de Transcrição GATA3/fisiologia , Genes Reporter , Células-Tronco Hematopoéticas/metabolismo , Proteína 2 Inibidora de Diferenciação/biossíntese , Células Matadoras Naturais/citologia , Proteínas Luminescentes/análise , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , Modelos Imunológicos , Proteína com Dedos de Zinco da Leucemia Promielocítica/biossíntese , Proteína com Dedos de Zinco da Leucemia Promielocítica/genética , Proteína com Dedos de Zinco da Leucemia Promielocítica/fisiologia , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/citologia , Transcrição Genética
18.
Mol Genet Genomics ; 294(4): 849-859, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30895377

RESUMO

The multigene expression system is highly attractive to co-express multiple genes or multi-subunit complex-based genes for their functional studies, and in gene therapy and visual tracking of expressed proteins. However, the current multiple gene co-expression strategies usually suffer from severe inefficiency and unbalanced expression of multiple genes. Here, we report on an improved 2A self-cleaving peptide (2A)-based multigene expression system (2A-MGES), by introducing an optimized Kozak region (Ck) and altering the gene arrangement, both of which contributed to the efficient expression of two fluorescent protein genes in silkworm. By co-expressing DsRed and EGFP genes in insect cells and silkworms, the potent Ck was first found to improve the translation efficiency of downstream genes, and the expression of the flanking genes of 2A were improved by altering the gene arrangement in 2A-MGES. Moreover, we showed that combining Ck and an optimized gene arrangement in 2A-MGES could synergistically improve the expression of genes in the cell. Further, these two flanking genes, regulated by modified 2A-MGES, were further co-expressed in the middle silk gland and secreted into the cocoon, and both achieved efficient expression in the transgenic silkworms and their cocoons. These results suggested that the modified Ck-2A-MGES will be a potent tool for multiple gene expression, for studies of their functions, and their applications in insect species.


Assuntos
Bombyx/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas Luminescentes/metabolismo , Peptídeos/genética , Animais , Animais Geneticamente Modificados , Bombyx/genética , Engenharia Genética/métodos , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Insetos/genética , Proteínas Luminescentes/genética , Proteínas Recombinantes/metabolismo , Células Sf9
19.
Int J Biol Macromol ; 130: 675-684, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30836182

RESUMO

Chromoproteins are a good source of engineered biological tools. We previously reported the development of a blue fluorescent protein, termed shBFP, which was derived from a purple chromoprotein shCP found in the sea anemone Stichodacyla haddoni. shBFP contains a Leu63-Leu64-Gly65 tri-peptide chromophore, and shows maximum excitation and emission wavelengths at 401 nm and 458 nm, along with a high quantum yield. How this chromophore endows shBFP with the unique fluorescence property in the absence of a hydroxyphenyl ring remained unclear. Here, we present the crystal structures of shCP and shBFP at 1.9- and 2.05-Šresolution, respectively. Both proteins crystallized as similar tetramers, but they are more likely to function as dimers in solution. The chromophore in shCP shows a trans-conformation and its non-planarity is similar to most other homologues. The shBFP chromophore also contains an imidazolidone moiety in its structure, but there are a smaller number of conjugated double bonds compared to shCP. Consequently, the chromophore may prefer absorbing shorter wavelength lights in the UV region, followed by the emission of blue fluorescence. These observations provide new insights into the molecular basis that correlates chromophore conformation with light absorption and fluorescence emission for the development of improved biomarkers.


Assuntos
Proteínas Luminescentes/química , Modelos Moleculares , Peptídeos/química , Conformação Proteica , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Proteínas Luminescentes/genética , Proteínas Luminescentes/isolamento & purificação , Estrutura Molecular , Anêmonas-do-Mar/genética , Análise Espectral , Relação Estrutura-Atividade
20.
Nat Protoc ; 14(4): 1084-1107, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911173

RESUMO

Bioluminescence resonance energy transfer (BRET) is a transfer of energy between a luminescence donor and a fluorescence acceptor. Because BRET occurs when the distance between the donor and acceptor is <10 nm, and its efficiency is inversely proportional to the sixth power of distance, it has gained popularity as a proximity-based assay to monitor protein-protein interactions and conformational rearrangements in live cells. In such assays, one protein of interest is fused to a bioluminescent energy donor (luciferases from Renilla reniformis or Oplophorus gracilirostris), and the other protein is fused to a fluorescent energy acceptor (such as GFP or YFP). Because the BRET donor does not require an external light source, it does not lead to phototoxicity or autofluorescence. It therefore represents an interesting alternative to fluorescence-based imaging such as FRET. However, the low signal output of BRET energy donors has limited the spatiotemporal resolution of BRET imaging. Here, we describe how recent improvements in detection devices and BRET probes can be used to markedly improve the resolution of BRET imaging, thus widening the field of BRET imaging applications. The protocol described herein involves three main stages. First, cell preparation and transfection require 3 d, including cell culture time. Second, image acquisition takes 10-120 min per sample, after an initial 60 min for microscope setup. Finally, image analysis typically takes 1-2 h. The choices of energy donor, acceptor, luminescent substrates, cameras and microscope setup, as well as acquisition modes to be used for different applications, are also discussed.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Medições Luminescentes/métodos , Imagem Óptica/métodos , Mapeamento de Interação de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Benzenoacetamidas/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Imidazóis/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , Pirazinas/metabolismo , Receptores de Vasopressinas/genética , Receptores de Vasopressinas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Renilla , Transfecção , beta-Arrestina 2/genética , beta-Arrestina 2/metabolismo
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