Este trabalho foi financiado por doações de DMH e TDM (subvenções NHMRC projeto). DMH é um companheiro Grimwade, financiado pelo Fundo Miegunyah.

1. Preparação de células para a rotulagem com ReAsH / Flash <ol><li> Usando métodos de cultura de células padrão para sua linha de celulares de interesse, prepare uma cultura de células aderentes diretamente em um slide de imagens ao vivo de células pronto para transfecção. </li><li> Transfectar seu plasmídeo gene TC-tagged contendo de interesse de acordo com seu método de transfecção de escolha. </li></ol> Note que é importante o uso de controles positivos e negativos para avaliar a extensão da ligação específica para as tags TC, para avaliar bleedthrough de fluorescência entre os canais ao coletar micrografias confocal. Assim, para duas cores (por exemplo, o Flash / Cherry ou ReAsH / PCP ou ReAsH / GFP combinações), certifique-se as amostras são preparadas para cores simples (proteína fluorescente por exemplo, sozinho ou se possível uma proteína TC-tagged obrigado a Flash / ReAsH mas sem fluorescentes proteína) <ol start="3"><li> Um a dois dias após a transfecção, lave as células com 300 mL pré-aquecido (a 37 ° C) HBSS. </li><li> Delicadamente mergulhe as células com um flash M (ou ReAsH) em 300 mL de HBSS pré-aquecida e 10 mM 1,2-ethanedithiol (EDT). </li></ol> É importante acrescentar EDT primeiro antes de adicionar Flash / ReAsH e fazer o buffer pouco antes de adicioná-lo para as células. Incubar por exatamente 30 min a 37 ° C em cultura de tecidos incubadora. Em nossa experiência vezes maior de incubação aumenta significativamente a fluorescência de fundo. Novas construções também devem ser otimizados para rotulagem tempo e Flash / ReAsH concentração (0,5-2 mM). <ol start="5"><li> Aspirar cuidadosamente rotulagem solução a partir de células e em seguida, substitua com 300 mL HBSS pré-aquecido + 250 M 2,3-dimercaptopropanol (BAL) por 15 min a 37 ° C. </li><li> Remover solução de lavagem por aspiração suave e substituir com 300 mL HBSS pré-aquecido. </li></ol> Após esta lavagem, as células podem ser fixadas com paraformaldeído (15 min com solução 3,2%), embora nós descobrimos que isto aumenta não-específicos de fluorescência de corantes biarsenical. Daí que geralmente a imagem de células vivas em temperatura ambiente. (Note que a fixação das células antes da rotulagem previne a ligação corante biarsenical.) 2. Imagem das células em um microscópio confocal <ol><li> No microscópio confocal, configurar os parâmetros para geração de imagens a fluoróforos individual (ver Tabela 1) e garantir que não é desprezível bleedthrough entre os canais. Isto pode ser conseguido através da verificação fluoróforos individual (em amostras de controle) contra a aquisição de cada configuração diferente fluorescentes para fluorescência. Ajustar o comprimento de onda de emissão pode ajudar a minimizar bleedthrough (embora isso também pode reduzir o sinal / ruído). </li><li> Também ajustar as configurações fotomultiplicador para que a fluorescência máxima na amostra não saturar o detector. (Isto pode ser detectado utilizando a definição de Q-LUT na Leica SP2 confocal). Uma vez que as melhores configurações de imagem são determinados, não alterar qualquer um deles entre as amostras. </li><li> Outras definições que normalmente uso (embora estes possam ser otimizados) são uma taxa de varredura de 200 Hz e recolher quatro médias de linha e um diâmetro de 1 unidade pinhole Airy. O diâmetro pinhole pode ser expandido se o sinal / ruído é um problema, no entanto, isso pode resultar em alguma perda de imagem. </li><li> Recolher as imagens confocal em formato 12-bit (ou superior), se possível. 12-bit formato de captura uma maior gama dinâmica de valores (0-4095) para cada intensidade de pixels de 8 bits (0-255). Isso é importante para assegurar o mais rico conjunto de dados é gravado, o que maximiza a qualidade da análise de dados quantitativos. </li><li> Coletar imagens para o canal proteína fluorescente (Cherry, ou GFP PCP) e também para o canal de corante biarsenical (Flash ou ReAsH) para todas as amostras. </li></ol> 3. Análise dos dados <ol><li> Instale o software ImageJ em seu computador 8. <a href="http://rsbweb.nih.gov/ij/">http://rsbweb.nih.gov/ij/</a> </li><li> Certifique-se que a sua versão do ImageJ tem os seguintes plugins: <ul><li> <a href="http://rsbweb.nih.gov/ij/plugins/file-opener.html">http://rsbweb.nih.gov/ij/plugins/file-opener.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/image_correlator.html">http://rsbweb.nih.gov/ij/plugins/image_correlator.html</a> </li><li> <a href="http://rsbweb.nih.gov/ij/plugins/lut-editor.html">http://rsbweb.nih.gov/ij/plugins/lut-editor.html</a> </li></ul></li><li> ImageJ aberta, e se estiver usando uma versão mais antiga que v1.32c, clique nas opções seguintes para habilitar várias imagens para ser aberto ao mesmo tempo (que pode ser feito mantendo o botão de controlo enquanto clica em arquivos diferentes): <img src="/files/ftp_upload/2857/2857step1.jpg" alt="Figura 1" /></li><li> Abra as imagens de interesse em ImageJ. ImageJ automaticamente definir a intensidade dos pixels máximos e mínimos que são exibidos na tela para cada imagem separadamente. Dado que diferentes imagens terá dintensidades de pixel IFERENTES, isso significa que as imagens exibidas não são comparáveis ​​como visualizados. </li><li> Para garantir que as imagens abertas são todas na mesma escala, você pode definir o fisicamente superiores e menores intensidades de pixels para ser visto (isto não vai mudar o conteúdo dos dados reais das imagens, como seria o caso de alguns outros softwares). Estes valores podem ser definidos sob o seguinte menu: <img src="/files/ftp_upload/2857/2857step2.jpg" alt="Figura 2" /></li><li> Uma abordagem alternativa é trabalhar com pilhas, o que coloca todas as imagens juntas em um único arquivo e automaticamente escala (para ver) todas as imagens na pilha para a mesma escala. A maneira mais fácil trabalhar com os dados é converter cada canal para uma pilha. Assim, para o canal proteína fluorescente converter todos a pilha, como mostrado. Você pode facilmente selecionar um canal pelo empilhamento de todas as imagens que contenham um nome comum (por exemplo, &quot;ch00&quot;) no título. <img src="/files/ftp_upload/2857/2857step3.jpg" alt="Figura 3" /></li><li> Agora converter todas as imagens abertas para 8-bit para análise. Isto realmente rescale a gama visualizada em um intervalo 0-255 (que define 8 bits). IMPORTANTE: Não poupar mais o bit 12 original (ou superior) de formato ou você vai perder conteúdo da informação. Note que alguns pacotes de software só pode exibir imagens de 8 bits de modo que este formato é útil para fazer figuras etc <img src="/files/ftp_upload/2857/2857step4.jpg" alt="Figura 4" /></li><li> Salvar uma cópia em uma pasta nova chamada &quot;8-bit convertido&quot; usando o &quot;Salvar como ...&quot; opção. </li><li> Um método para examinar a extensão da ligação corante biarsenical em uma imagem inteira é realizar um plano de correlação de pixel intensidade. Este valor parcelas cada pixel em um canal em relação ao valor do pixel correspondente em um segundo canal. Portanto, uma posição de pixel em alta PCP fluorescência também será elevada na fluorescência ReAsH se há elevada taxa de ligação. </li><li> Para analisar o pixel co-correlação, verifique se o ReAsH 8-bit e Cerulean / GFP imagens são abertas no ImageJ. </li><li> Abra o &quot;Image Correlator&quot; plugin como indicado abaixo. Selecione &quot;Image1&quot;, como a pilha ReAsH ou Flash e &quot;Image2&quot;, como a pilha de proteína fluorescente. <img src="/files/ftp_upload/2857/2857step5.jpg" alt="Figura 5" /></li><li> A pilha resultante não pode exibir qualquer informação detalhada - isto é normal. Salvar a pilha em uma nova pasta chamada &quot;Scatterplots&quot; e dar-lhe o mesmo nome de arquivo como o exemplo refere-se a (por exemplo, &quot;plot correlação Flash-cereja&quot;) </li><li> Para visualizar os dados significativa que você pode fazer uma de duas opções. Primeiro transformar os dados de modo que é em formato de log. Então visualmente redimensionar os dados da seguinte forma: <img src="/files/ftp_upload/2857/2857step6.jpg" alt="Figura 6" /></li><li> Alternativamente, você pode redimensionar os dados visualmente para mostrar apenas valores baixos (por exemplo, 1-255) e exibir os dados usando uma LUT especial. Uma LUT (Look Up Table) representa uma tabela de cores que são atribuídos a cada valor de pixel em uma imagem . Isto pode ser usado para definir um esquema de pseudo para uma imagem e é útil para a definição de certas características em uma imagem. Para criar um clique LUT personalizado no &quot;editor LUT&quot; e fazer um novo, como a mostrada (isso pode ser guardada e utilizada mais tarde). <img src="/files/ftp_upload/2857/2857step7.jpg" alt="Figura 7" /></li><li> Definir o intervalo de brilho / contraste 0-255 (como descrito na etapa 13 acima). Para &quot;travar&quot; a imagem como a mostrada na tela, a imagem pode ser convertida em formato RGB o que economiza um valor de 8 bits para cada uma das cores vermelha, verde e azul de cada pixel. <img src="/files/ftp_upload/2857/2857step8.jpg" alt="Figura 8" /> Para copiar e colar imagens para outros programas aberto pela primeira vez as imagens de 8 bits ou 16 bits. Como descrito na etapa 14 acima, atribuir um esquema de cores LUT para a imagem. Para PCP, atribuir o &quot;Ciano&quot; LUT, conforme descrito abaixo ... <img src="/files/ftp_upload/2857/2857step9.jpg" alt="Figura 9" /></li><li> Selecione a imagem e copiá-lo para a área de transferência, selecione ... <img src="/files/ftp_upload/2857/2857step10.jpg" /></li></ol> 4. Resultados representativos: O sucesso das células rotulagem com corantes biarsenical é dependente de alguns parâmetros-chave. Primeiro, o momento da marcação com corantes é crucial. Descobrimos que longos períodos de rotulagem (mais de 30 min) resulta em um alto nível de não-específica coloração de fundo. Fig. 1 mostra um resultado típico de uma forma selvagem-tipo de fragmento huntingtina (25Q) fundida ao Cerulean PCP derivados contendo um tag TC, conforme descrito anteriormente 7. Esta amostra foi manchada por 30 min com ReAsH e não há fundo mínimo na amostra sem a tag TC. Nós descobrimos que a fixação de células com fundo aumenta paraformaldeído enquanto fixação com metanol revoga a fluorescência da tag proteína fluorescente. Por isso, sempre que possível nós a imagem de células vivas. É importante notar também que a fixação préviaà rotulagem com corantes biarsenical impede sua ligação, provavelmente devido a modificações do motivo TC. Outro fator crítico para resultados consistentes é a densidade de células. Nós descobrimos que é fundamental para as células de imagem que são distribuídas livremente e também que clumping extensa pode levar a manchas irregulares dos corantes biarsenical em diferentes células. <img src="/files/ftp_upload/2857/2857fig1.jpg" alt="Figura 1" /> Figura 1. Etiquetas Tetracysteine ​​e coloração ReAsH em células vivas transfectadas com huntingtina (exon1-25Q)-Cerulean fusões. A tag TC está localizado na junção da fusão huntingtina-Cerulean (como descrito na 7). O enredo de correlação de pixel intensidade permite uma avaliação para as diferenças de ReAsH vinculativa em toda a célula e pode ser usado para mapear mudanças na ReAsH vinculativo devido à mudança conformacional ou interações ligante.

<ol>
	<li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Ann. Rev. Biochem. 67, 509-544 (1998).</li><li>Griffin, B. A., Adams, S. R., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+covalent+labeling+of+recombinant+protein+molecules+inside+live+cells.">Specific covalent labeling of recombinant protein molecules inside live cells.</a> Science. 281, 269-2672 (1998).</li><li>Uttamapinant, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorophore+ligase+for+site-specific+protein+labeling+inside+living+cells.">A fluorophore ligase for site-specific protein labeling inside living cells.</a> Proc. Natl. Acad. Sci. USA. 107, 10914-10919 (2010).</li><li>Ignatova, Z., Gierasch, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein+stability+and+aggregation+in+vivo+by+real-time+fluorescent+labeling.">Monitoring protein stability and aggregation in vivo by real-time fluorescent labeling.</a> Proc. Natl. Acad. Sci. USA. 101, 523-528 (2004).</li><li>Coleman, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+detection+of+prion+protein+with+biarsenical+labeling+and+FlAsH+fluorescence.">Conformational detection of prion protein with biarsenical labeling and FlAsH fluorescence.</a> Biochem. Biophys. Res. Commun. 380, 564-568 (2009).</li><li>Luedtke, N. W., Dexter, R. J., Fried, D. B., Schepartz, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surveying+polypeptide+and+protein+domain+conformation+and+association+with+FlASH+and+ReAsH.">Surveying polypeptide and protein domain conformation and association with FlASH and ReAsH.</a> Nat. Chem. Biol. 3, 779-784 (2007).</li><li>Ramdzan, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformation+sensors+that+distinguish+monomeric+proteins+from+oligomers+in+live+cells.">Conformation sensors that distinguish monomeric proteins from oligomers in live cells.</a> Chem. Biol. 17, 371-379 (2010).</li><li>Abramoff, M. a. g. e. l. h. a. e. s., PJ, S. J. R. a. m. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+with+ImageJ.">Image processing with ImageJ.</a> Biophotonics International. 11, 36-42 (2004).</li><li>Hearps, 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+biarsenical+dye+Lumio+exhibits+a+reduced+ability+to+specifically+detect+tetracysteine-containing+proteins+within+live+cells.">The biarsenical dye Lumio exhibits a reduced ability to specifically detect tetracysteine-containing proteins within live cells.</a> J. Fluor. 17, 593-597 (2007).</li><li>Adams, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+biarsenical+ligands+and+tetracysteine+motifs+for+protein+labeling+in+vitro+and+in+vivo:+Synthesis+and+biological+applications.">New biarsenical ligands and tetracysteine motifs for protein labeling in vitro and in vivo: Synthesis and biological applications.</a> J. Am. Chem. Soc. 124, 6063-6076 (2002).</li><li>Shaner, N. C., Steinbach, P. A., Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+choosing+fluorescent+proteins.">A guide to choosing fluorescent proteins.</a> Nat. Meth. 2, 905-909 (2005).</li></ol>

Não há conflitos de interesse declarados.

A abordagem para localização de proteínas rótulo com uma proteína fluorescente e propriedades conformacionais com um corante segundo oferece um grande potencial para o mapeamento de onde conformações diferentes de proteínas acumulam nas células e eventos que alteram a dinâmica da conformação protéica. ReAsH / Flash foi usado primeiramente como um sensor na célula para o dobramento de proteínas dos mamíferos celular proteína ácido retinóico-binding I 4. Neste exemplo, o Flash vinculado a um ...

<table border="1"><tbody><tr><th> Nome do reagente </th><th> Companhia </th><th> Número Catálogo </th><th> Comentários </th></tr><tr><td> 8-μ bem-slides </td><td> Ibidi </td><td> 80826 </td><td> Encontramos estas câmara de slides a ser particularmente útil para a cultura de células para geração de imagens. </td></tr><tr><td> TC-FLASH II na célula Detecção Tag Tetracysteine ​​Kit * * * fluorescência verde para a imagem latente em células vivas </td><td> Invitrogen </td><td> T34561 (Flash) ou T34562 (ReAsH) </td><td></td></tr><tr><td> Solução de Hanks salina balanceada </td><td> Invitrogen </td><td> 14175-103 </td><td></td></tr><tr><td> 2,3-dimercapto-1-propanol </td><td> Sigma-Aldrich </td><td> D1129-5mL </td><td></td></tr><tr><td> 1,2-Ethanedithiol </td><td> Sigma-Aldrich </td><td> 02390-25ML </td><td></td></tr></tbody></table>

reash/flash labeling and image analysis of tetracysteine sensor proteins in cells

Proteínas fluorescentes e corantes são ferramentas essenciais para o estudo da localização de proteínas, tráfico e função nas células. Enquanto que as proteínas fluorescentes, como a proteína fluorescente verde (GFP) têm sido amplamente utilizados como parceiros de fusão para proteínas para monitorar as propriedades de uma proteína de interesse 1, os desenvolvimentos recentes com etiquetas menores permitem novas funcionalidades de proteínas a ser examinada em células, tais como mudança conformacional e proteína-associação 2, 3. Um sistema de tag pequenas envolve um motivo tetracysteine ​​(CCXXCC) geneticamente inserido em uma proteína-alvo, que se liga aos corantes biarsenical, ReAsH (vermelha fluorescente) e Flash (verde fluorescente), com alta especificidade, mesmo em células vivas 2. O TC / biarsenical sistema de corante oferece muito menos restrições estérico à proteína do hospedeiro do que proteínas fluorescentes que tem possibilitado várias novas abordagens para medir a mudança conformacional e interações proteína-proteína 4-7. Recentemente, desenvolveu uma nova aplicação de tags TC como sensores de oligomerização em células que expressam mutante, que quando sofre mutação agregados nos neurônios em Huntington doença 7. Huntingtina foi etiquetado com dois corantes fluorescentes, uma uma proteína fluorescente para rastrear a localização de proteínas, e um segundo a marca TC que obriga apenas corantes biarsenical em monômeros. Assim, mudanças na co-localização entre as proteínas e reatividade corante biarsenical habilitado conteúdo oligômero submicroscópicas ser espacialmente mapeadas dentro das células. Aqui, descrevemos como rotular TC-tagged proteínas fundida a uma proteína fluorescente (Cherry, ou GFP PCP) com flash ou ReAsH viver em células de mamíferos e como quantificar a fluorescência de duas cores (Cherry / Flash, PCP / Flash ou GFP / combinações ReAsH).

Watch this Scientific Journal Video about ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells at JoVE.com

ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

O biarsenical corantes Flash e ReAsH se ligam especificamente a motivos tetracysteine ​​em proteínas e pode seletivamente rótulo proteínas em células vivas. Recentemente, esta estratégia de rotulagem tem sido usado para desenvolver sensores de conformações de proteínas diferentes ou estados oligoméricos. Descrevemos a abordagem rotulagem e métodos para analisar quantitativamente vinculativo.

ReAsH / Flash Rotulagem e Análise de Imagem Sensor de Proteínas em Células Tetracysteine

Fluorescent proteins and dyes are essential tools for the study of protein trafficking, localization and function in cells.  While fluorescent 
proteins ...

reashflash-labeling-image-analysis-tetracysteine-sensor-proteins

Research

JoVE Journal

Biology

16.9K visualizações.  Bio21 Molecular Science and Biotechnology Institute. O biarsenical corantes Flash e ReAsH se ligam especificamente a motivos tetracysteine ​​em proteínas e pode seletivamente rótulo proteínas em células vivas. Recentemente, esta estratégia de rotulagem tem sido usado para desenvolver sensores de conformações de proteínas diferentes ou estados oligoméricos. Descrevemos a abordagem rotulagem e métodos para analisar quantitativamente vinculativo.

Vídeo: ReAsH/FlAsH Labeling and Image Analysis of Tetracysteine Sensor Proteins in Cells

Robotics and Dynamic Image Analysis for Studies of Gene Expression in Plant Tissues

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods presented here utilize the green fluorescent protein (gfp) gene for continual monitoring of gene expression in the same pieces of tissues, over time. The gfp gene was placed under regulatory control of different promoters and introduced into lima bean cotyledonary tissues via particle bombardment. Cotyledons were then placed on a robotic image collection system, which consisted of a fluorescence dissecting microscope with a digital camera and a 2-dimensional robotics platform custom-designed to allow secure attachment of culture dishes. Images were collected from cotyledonary tissues every hour for 100 hours to generate expression profiles for each promoter. Each collected series of 100 images was first subjected to manual image alignment using ImageReady to make certain that GFP-expressing foci were consistently retained within selected fields of analysis. Specific regions of the series measuring 300 x 400 pixels, were then selected for further analysis to provide GFP Intensity measurements using ImageJ software. Batch images were separated into the red, green and blue channels and GFP-expressing areas were identified using the threshold feature of ImageJ. After subtracting the background fluorescence (subtraction of gray values of non-expressing pixels from every pixel) in the respective red and green channels, GFP intensity was calculated by multiplying the mean grayscale value per pixel by the total number of GFP-expressing pixels in each channel, and then adding those values for both the red and green channels. GFP Intensity values were collected for all 100 time points to yield expression profiles. Variations in GFP expression profiles resulted from differences in factors such as promoter strength, presence of a silencing suppressor, or nature of the promoter. In addition to quantification of GFP intensity, the image series were also used to generate time-lapse animations using ImageReady. Time-lapse animations revealed that the clear majority of cells displayed a relatively rapid increase in GFP expression, followed by a slow decline. Some cells occasionally displayed a sudden loss of fluorescence, which may be associated with rapid cell death. Apparent transport of GFP across the membrane and cell wall to adjacent cells was also observed. Time lapse animations provided additional information that could not otherwise be obtained using GFP Intensity profiles or single time point image collections.

We report a method for introduction, tracking and quantitative analysis of GFP expression in plant cells. This method utilizes a custom-designed robotics system for semi-continuous image collection from large numbers of samples, over time. We also demonstrate the use of ImageJ and ImageReady for analysis of image series.

Robótica e Análise de Imagem Dinâmica de Estudos de Expressão Gênica em Tecidos Vegetais

Gene expression in plant tissues is typically studied by destructive extraction of compounds from plant tissues for in vitro analyses. The methods ...

Biologia

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Examinando o Dynamics Conformacional de Proteínas de Membrana In situ Com Labeling-Site dirigido Fluorescência

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

Phenotypic Analysis and Isolation of Murine Hematopoietic Stem Cells and Lineage-committed Progenitors

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs constitute a minute cell population of pluripotent cells capable of generating all blood cell lineages for a life-time1. The molecular dissection of HSCs homeostasis in the bone marrow has important implications in hematopoiesis, oncology and regenerative medicine. We describe the labeling protocol with fluorescent antibodies and the electronic gating procedure in flow cytometry to score hematopoietic progenitor subsets and HSCs distribution in individual mice (Fig. 1). In addition, we describe a method to extensively enrich hematopoietic progenitors as well as long-term (LT) and short term (ST) reconstituting HSCs from pooled bone marrow cell suspensions by magnetic enrichment of cells expressing c-Kit. The resulting cell preparation can be used to sort selected subsets for in vitro and in vivo functional studies (Fig. 2).
Both trabecular osteoblasts2,3 and sinusoidal endothelium4 constitute functional niches supporting HSCs in the bone marrow. Several mechanisms in the osteoblastic niche, including a subset of N-cadherin+ osteoblasts3 and interaction of the receptor tyrosine kinase Tie2 expressed in HSCs with its ligand angiopoietin-15 concur in determining HSCs quiescence. "Hibernation" in the bone marrow is crucial to protect HSCs from replication and eventual exhaustion upon excessive cycling activity6. Exogenous stimuli acting on cells of the innate immune system such as Toll-like receptor ligands7 and interferon-&alpha;6 can also induce proliferation and differentiation of HSCs into lineage committed progenitors. Recently, a population of dormant mouse HSCs within the lin- c-Kit+ Sca-1+ CD150+ CD48- CD34- population has been described8. Sorting of cells based on CD34 expression from the hematopoietic progenitors-enriched cell suspension as described here allows the isolation of both quiescent self-renewing LT-HSCs and ST-HSCs9. A similar procedure based on depletion of lineage positive cells and sorting of LT-HSC with CD48 and Flk2 antibodies has been previously described10. In the present report we provide a protocol for the phenotypic characterization and ex vivo cell cycle analysis of hematopoietic progenitors, which can be useful for monitoring hematopoiesis in different physiological and pathological conditions. Moreover, we describe a FACS sorting procedure for HSCs, which can be used to define factors and mechanisms regulating their self-renewal, expansion and differentiation in cell biology and signal transduction assays as well as for transplantation.

A method to analyse the distribution of bone marrow hematopoietic progenitors in flow cytometry as well as to efficiently isolate highly purified hematopoietic stem cells (HSCs) is described. The isolation procedure is essentially based on magnetic enrichment of c-Kit+ cells and cell sorting to purify HSCs for cellular and molecular studies.

Análise fenotípica e Isolamento de Células-Tronco Hematopoéticas Murino e Lineage comprometidos Progenitores

The bone marrow is the principal site where HSCs and more mature blood cells lineage progenitors reside and differentiate in an adult organism. HSCs ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ imaging data is time consuming and subject to bias. Automated signal analysis algorithms based on region of interest (ROI) detection have been implemented for one-dimensional line scan measurements, but there is no current algorithm which integrates optimized identification and analysis of ROIs in two-dimensional image sequences. Here an algorithm for rapid acquisition and analysis of ROIs in image sequences is described. It utilizes ellipses fit to noise filtered signals in order to determine optimal ROI placement, and computes Ca2+ signal parameters of amplitude, duration and spatial spread. This algorithm was implemented as a freely available plugin for ImageJ (NIH) software. Together with analysis scripts written for the open source statistical processing software R, this approach provides a high-capacity pipeline for performing quick statistical analysis of experimental output. The authors suggest that use of this analysis protocol will lead to a more complete and unbiased characterization of physiologic Ca2+ signaling.

Automated Analysis of Dynamic Ca2+ Signals in Image Sequences

Here a novel region of interest analysis protocol based on sorting best-fit ellipses assigned to regions of positive signal within two-dimensional time lapse image sequences is demonstrated. This algorithm may enable investigators to comprehensively analyze physiological Ca2+ signals with minimal user input and bias.

Automated Análise de Ca dinâmico<sup&gt; 2 +</sup&gt; Sinais em seqüências de imagens

Intracellular Ca2+ signals are commonly studied with fluorescent Ca2+ indicator dyes and microscopy techniques. However, quantitative analysis of Ca2+ ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Rotulagem específica de seqüência de ácidos nucléicos e proteínas com Metiltransferases e cofator Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Immunofluorescence Analysis of Endogenous and Exogenous Centromere-kinetochore Proteins

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of centromere-kinetochore proteins during the cell cycle remains a fundamental tool in investigating the mechanism(s) of these proteins. Advanced imaging methods in fluorescence microscopy provide remarkable resolution of centromere-kinetochore components and allow direct observation of specific molecular components of the centromeres and kinetochores. In addition, methods of indirect immunofluorescent (IIF) staining using specific antibodies are crucial to these observations. However, despite numerous reports about IIF protocols, few discussed in detail problems of specific centromere-kinetochore proteins.1-4 Here we report optimized protocols to stain endogenous centromere-kinetochore proteins in human cells by using paraformaldehyde fixation and IIF staining. Furthermore, we report protocols to detect Flag-tagged exogenous CENP-A proteins in human cells subjected to acetone or methanol fixation. These methods are useful in detecting and quantifying endogenous centromere-kinetochore proteins and Flag-tagged CENP-A proteins, including those in human cells.

Here we report protocols to detect endogenous and exogenous centromere-kinetochore proteins in human cells and quantify these protein levels at centromeres-kinetochores by indirect immunofluorescent staining through the use of fixation (paraformaldehyde, acetone, or methanol fixation).

Proteínas Análise de imunofluorescência de endógenos e exógenos Centromere-cinetócoro

"Centromeres" and "kinetochores" refer to the site where chromosomes associate with the spindle during cell division. Direct visualization of ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Os ensaios para a degradação de proteínas em células deformadas

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Análise de suspensões celulares isoladas de tecidos sólidos por citometria de fluxo espectral

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Uma interface de usuário gráfica para rastreamento assistido por software da concentração de proteínas em protrusões celulares dinâmicas

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...