O pCG001 plasmídeo expressando Ubp1 foi gentilmente cedido por Rohan Baker na Curtin João Escola de Pesquisa Médica. Este trabalho foi financiado pela March of Dimes Bolsa de Investigação 1-FY2011, March of Dimes O&#39;Connor Iniciado Basil Prêmio de Pesquisa Acadêmico # 5-FY20 e NSF concessão de CARREIRA 0.746.796.

1. Fluxo de Trabalho de Engenharia tensão <ol><li> Inserir as sequências de codificação (a) uma sequência de localização da membrana, (b) uma proteína fluorescente amadurecimento rápido e (c) uma sequência de reconhecimento de protease N-terminal e para a armação com uma proteína de interesse (por exemplo, um factor de transcrição). Utilizou-se a membrana alvo Tsr-Venus repórter 2 e fundiu-o para a sequência de reconhecimento da protease de Ubiquitina (Ub) para contar o número de moléculas expressas bacteriófagos λ proteína repressora CI. Detalhes sobre como se construídas a proteína de fusão Tsr-Venus-Ub-CI, em substituição do tipo selvagem CI região codificante e que incorpora o arquétipo na E. cromossoma coli utilizando λ recombinação Red 3, estão descritos em detalhe na ref. 1. </li><li> Verifique se todas as modificações por seqüenciamento. </li><li> Transformar um plasmídeo que codifica uma protease específica para a sequência de reconhecimento usado acima para uma estirpe que expressa o repórter fluorescente e proteína de interesseem fusão traducional. Utilizou-se a protease Ubp1, que cliva imediatamente a seguir ao resíduo C-terminal da ubiquitina 4. </li></ol> 2. As células de cultura e preparar a amostra <ol><li> Escolha um E. coli colónia de interesse a partir de uma placa de agar de fresco listado em 1 ml M9 meio mínimo suplementado com 5 1X ácidos aminados do MAM e antibióticos apropriados. Incubar num agitador à temperatura desejada por tempo suficiente para alcançar OD 600&gt; 1.0 (densidade óptica a 600 nm). </li><li> Dilui-se a cultura em um meio M9 ml fresco com os antibióticos apropriados para a = 0,02 OD 600. Incubar num agitador à temperatura apropriada. Densidade celular inicial pode ser aumentada, se necessário para a baixa temperatura de crescimento. </li><li> Quando o OD 600 = 0,2-0,3 (a 37 ° C, com uma cultura inicial de células de DO600 = 0,02, isso vai levar 3-4 horas), iniciar a preparação do gel de agarose a almofada (passo 3). </li><li> As células estão prontas para imagiologia quando OD <sub&gt; 600 = 0,3-0,4. </li><li> Transferir 1 ml da cultura incubada num tubo de microcentrífuga de 1,5 ml, de centrifugação a 10000 xg durante 1 minuto numa microcentrífuga de bancada. </li><li> Descartar o sobrenadante e adicionar 1 ml de meio fresco M9 e ressuspender o pellet cuidadosamente. </li><li> Centrifugar a 10.000 xg durante 1 min. </li><li> Repita o passo 1,6 e 1,7. Descartar o sobrenadante. </li><li> Delicadamente ressuspender em 1 ml M9 mídia. As células podem ser directamente utilizados para imagiologia de microscópio ou 10 diluída - a 100 vezes para assegurar densidades celulares baixas para imagiologia timelapse. Note-se que as densidades celulares baixas são importantes para a imagiologia timelapse estendida. A câmara de imagem (passo 4) é vedada durante a experiência. Devido ao crescimento exponencial de células, as concentrações de células iniciais elevadas podem reduzir significativamente o oxigénio no interior da câmara após crescimento prolongado na almofada de gel, reduzindo a maturação da proteína fluorescente e que afectam o crescimento das células. </li></ol> 3. Preparar a solução de agarose <ol><li> Pesar 10-20 mgbaixo ponto de fusão à temperatura de agarose para um tubo de microcentrífuga de 1,5 ml. </li><li> Adicionar adequado volume médio mínimo M9 sem antibióticos para fazer uma solução de 3% de agarose. </li><li> Aquece-se a solução de agarose durante 30 min a 70 ° C para derreter, invertendo o tubo para assegurar que a solução é completamente fundida e homogénea. A almofada de gel pode ser vertido neste ponto (passo 4), ou a temperatura pode ser reduzida para 50 ° C e mantida durante várias horas para uso posterior. </li></ol> 4. Prepare Pad Gel na Câmara <ol><li> Cerca de 50 min antes da imagiologia, lavar uma corrediça Microaqueduct com água. </li><li> Lavar uma cobertura de vidro limpas (usando um método rigoroso de limpeza como o descrito no ponto 6) e Microaqueduct lâmina com água estéril e seco por sopragem com ar comprimido. </li><li> Coloque a junta de borracha na corrediça Microaqueduct modo que cubra as entradas e saídas do lado da lâmina de vidro Microaqueduct (este pode ser modificado para aplicações emque a mídia é perfundido através da amostra). Aplicar 50 ul de solução de agarose (passo 3) para o centro da lâmina Microaqueduct. </li><li> Topo da solução de agarose com a tampa de vidro limpo, seco. </li><li> Deixar a solução de agarose mantida à temperatura ambiente durante 30 min. </li><li> Enquanto isso, a preparar uma amostra de células (passo 2). </li><li> Retire cuidadosamente o vidro de cobertura da parte superior da almofada de gel. Adicionar 1,0 uL de cultura de células lavadas a parte superior da almofada de gel. Espere por ~ 2 min para a cultura a ser absorvido pela almofada de gel. É importante não esperar tanto tempo que os overdries almofada de gel, mas tempo suficiente para que as células estão devidamente aderiu à almofada de gel. O tempo ideal de espera vai variar com a temperatura e umidade. </li><li> Enquanto espera, secar outra tampa de vidro previamente limpas. </li><li> Cobrir a amostra com a tampa de vidro novo assegurando que a tampa de vidro deslizante e Microaqueduct estão bem alinhados. </li><li> Montar a temperatura da câmara de crescimento controlado seguindo as instruções do fabricante.Pode agora ser usado para imagens com microscópio (passo 5). </li></ol> 5. Imagem e Aquisição de Filmes Timelapse <ol><li> Ligar o laser e microscópio seguindo as instruções do fabricante (por exemplo, o laser pode precisar aquecido para ~ 0,5 h antes da experiência). </li><li> Bloquear a câmara montada na fase de inserção no microscópio. Definir a temperatura da câmara de crescimento e o aquecedor objectivo, a 37 ° C ou a temperatura de crescimento desejado outro. </li><li> Se imaging acima da temperatura ambiente, o foco ou almofada de gel, normalmente deriva significativamente para ~ 15 min após a mudança de temperatura inicial. Na prática, usar este tempo para encontrar regiões de interesse e modificar os scripts de imagem como necessário para uma experiência. </li><li> Encontrar as células no microscópio e armazenar a posição de cada célula após centrando-o dentro de uma região de imagem pré-definidos e alinhados. Mais do que uma célula pode ser trabalhada em cada janela de tempo de aquisição de imagem. Para timelapse imagens sobre mquaisquer gerações, assegurar que as células são inicialmente separadas imaged de outras células por pelo menos algumas centenas de ^ M, de modo que outras colónias não irá entrar na região de imagem durante o crescimento. </li><li> Ajustar a intensidade da excitação com laser, de modo que quase toda a Fotobranqueador moléculas fluorescentes dentro de 6 posições (Figura 5). </li><li> Usando um script de imagem automatizado / jornal, adquirir dados timelapse utilizando o algoritmo descrito no fluxo de trabalho experimental na Figura 3. </li></ol>

<ol>
	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

Nós não temos divulgações.

O método CoTrAC pode ser generalizado para medir a produção de outras proteínas em que N-ou C-terminais convencionais fusões de proteínas fluorescentes podem comprometer a actividade da proteína. A estratégia CoTrAC tem três vantagens únicas sobre os métodos correntes. Em primeiro lugar, co-traducional fusão assegura que uma molécula do repórter fluorescente é produzido para cada molécula da proteína de interesse, permitindo a contagem precisa da produção de proteína em tempo real. Em segundo lugar, ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Agarose (temperatura de fusão baixa) </td><td> Lonza </td><td> 50100 </td><td></td></tr><tr><td> Milli-Q H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5 × sais M9 </td><td></td><td></td><td> Seguinte receita descrita em 9 </td></tr><tr><td> Glicose 20% </td><td></td><td></td><td></td></tr><tr><td> MgSO4 </td><td></td><td></td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> 50 x solução de MEM aminoácidos </td><td> Invitrogen </td><td> 11130-051 </td><td></td></tr><tr><td> Temperatura controlada CrescimentoCâmara Adaptador de estágio </td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td> Aquecedor objetivo </td><td> Bioptechs </td><td> Modelo depende objetiva de microscópio </td><td></td></tr><tr><td> Deslize Microaqueduct </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> Micro óculos cobertura </td><td> VWR </td><td> 40CIR-1 </td><td> Pode ser difícil de fonte; também disponível a partir de Bioptechs </td></tr><tr><td> Tampa de vidro / slide junta </td><td> Bioptechs </td><td> FCS2 0,75 mm </td><td></td></tr><tr><td> Microscópio de fluorescência </td><td> Vário </td><td> Exemplo de configuração: Coherent Innova 308C Argon-ion laser, microscópio Olympus IX-81, Olympus PlanApo 100X 1,45 NA objetivo, Metamorph software </td><td> Deve ter excitação laser, automatizado fase xyz, software de automação capaz de imagem roteirizado e autofocus, óptica capazes de resolvinag únicas proteínas fluorescentes </td></tr><tr><td> EM-CCD da câmera </td><td> Vário </td><td> Exemplo de configuração: Andor ixon DU-898 </td><td> Deve ter ruído suficientemente baixo para detectar proteínas fluorescentes individuais acima fundo </td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

Descreve-se um método de fluorescência microscopia, Activation Co-translacional pela clivagem (CoTrAC) a imagem a produção de moléculas de proteína de células vivas com uma única molécula de precisão, sem perturbar a funcionalidade da proteína. Este método faz com que seja possível contar o número de moléculas de proteínas produzidas em uma célula durante sequenciais, as janelas de tempo de cinco minutos. Ela requer um microscópio de fluorescência com o laser de densidade de potência de excitação de ~ 0,5-1 kW / cm 2, o que é suficientemente sensível para detectar moléculas individuais de proteínas fluorescentes em células vivas. O repórter fluorescente usada neste método consiste em três partes: uma sequência de membrana alvo, um amadurecimento rápido, proteína fluorescente amarela e uma sequência de reconhecimento de protease. O repórter é traducionalmente fundidas com o N-terminal de uma proteína de interesse. As células são cultivadas em um estágio de microscópio de temperatura controlada. De cinco em cinco minutos, moléculas fluorescentes no interior das células são criadas imagens (e mais tarde coUnted, analisando imagens de fluorescência) e, posteriormente, Foto-descoloração, de modo que apenas as proteínas recém-convertidos são contados na medição seguinte. Imagens de fluorescência resultantes deste método podem ser analisados ​​por detecção de manchas fluorescentes em cada imagem, atribuindo-lhes a células individuais e, em seguida, a atribuição de células de linhagens de células. O número de proteínas produzidas dentro de uma janela de tempo numa dada célula é calculado dividindo-se a intensidade de fluorescência das manchas integrada com a intensidade média de moléculas fluorescentes individuais. Foi utilizado este método para medir os níveis de expressão na gama de 0-45 moléculas individuais em janelas de tempo 5 min. Este método permitiu-nos medir o ruído na expressão do repressor λ CI, e tem muitas outras aplicações potenciais em sistemas de biologia.

Os resultados típicos de uma experiência CoTrAC rastrear a produção do repressor λ CI são mostrados nas Figuras 4 e 5. Nesta experiência, foram obtidas imagens de 12 colónias em intervalos de 5 min. Em cada ponto de tempo, a colónia foi autofocused, centralizado no interior da região da imagem latente. Em seguida, a posição centrada / foco foi armazenada e uma imagem de campo claro foi adquirido. O palco foi então translocado por ~ 0,5 m ao longo do eixo z para mover a part...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Descreve-se um método de fluorescência microscopia, Activation Co-translacional pela clivagem (CoTrAC), a imagem a produção de moléculas de proteína de células vivas com uma única molécula de precisão, sem perturbar a funcionalidade da proteína. Este método tem sido utilizado para seguir as dinâmicas de expressão estocásticos de um factor de transcrição, o repressor CI λ<sup&gt; 1</sup&gt;.

Molécula única imagem da regulação gênica<em&gt; Em vivo</em&gt; Uso de ativação Cotranslational por clivagem (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

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Research

JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

9.8K visualizações.  Johns Hopkins University School of Medicine. Descreve-se um método de fluorescência microscopia, Activation Co-translacional pela clivagem (CoTrAC), a imagem a produção de moléculas de proteína de células vivas com uma única molécula de precisão, sem perturbar a funcionalidade da proteína. Este método tem sido utilizado para seguir as dinâmicas de expressão estocásticos de um factor de transcrição, o repressor CI λ 1.

Vídeo: Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Única molécula-Imaging dos Transportes Nuclear

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Biologia

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Ativação da apoptose por microinjeção citoplasmática de citocromo C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Na reconstituição in vitro da atividade T. Telomerase castaneum

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolamento de Ribossomos ligados a polipeptídeos nascentes<em&gt; In vitro</em&gt; Identificar Sites Pausa translacionais Junto mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Usando Seqüência de Detenção SECM como uma ferramenta para isolar Ribossomos Polipeptídeos encadernados

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Análise da Global RNA Síntese no nível da célula única seguinte hipóxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Demonstração de Proteolytic ativação do canal de sódio epitelial (ENAC) combinando Medidas atuais com Detecção de clivagem Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Estudar DNA Looping por Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In Vivo Rastreamento 4-Dimensional de tronco hematopoéticas e células progenitoras em adultos mouse na calota craniana de Medula Óssea

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Ensaio cinético de molécula única de alta produtividade paralela para decote de DNA específico do local

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...