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1. Transfecção de linhas celulares Método transfecção 1 (Lonza Nucleofection para a eficiência de transfecção de células primárias aumentada) <ol><li> As células devem ser de passagem recente e em crescimento log fase anterior à transfecção. Lavar as células em tampão fosfato salina (PBS) e células de colheita por tripsinização. </li><li> Coletar células por centrifugação a 1500 xg por 5 minutos. </li><li> Adicionar lamínulas esterilizadas a uma placa de seis poços. Lamínulas podem ser revestidos para a adesão celular reforçada. Coloque 1,5 mL de meios de cultura (neste caso, meio modificado Dulbecco Águia (DMEM), 10% de soro fetal bovino (FBS)) em cada poço e equilibrar os meios de comunicação na incubadora. </li><li> Colher as células por tripsinização e ressuspender em um volume mínimo de tampão fosfato (PBS). </li><li> Contar as células e centrifugar a quantidade correta de ressuspensão de fornecer ~ 5x10 5 células por amostra. </li><li> Ressuspender as células com cuidado em 100 mL de solução Nucleofector temperatura ambiente por amostra. </li><li> Combine 100 L da suspensão de células com 5 mg DNA plasmidial e transferência da amostra para um Cuvette Nucleofector tomando cuidado para não incluir bolhas de ar. </li><li> Selecione o programa apropriado Nucleofector (isso pode exigir testes anteriores para estabelecer a eficiência de transfecção ótima com a mortalidade mínima). </li><li> Inserir a cubeta contendo a suspensão de células / DNA para o Titular Cuvette Nucleofector e iniciar o programa selecionado. </li><li> Após a conclusão, remova a cubeta e adicione ~ 500 mL dos meios de comunicação pré-equilibradas cultura a ele (retirado da placa de 6 poços). </li><li> Transferir imediatamente a amostra no prato 6-bem com volume final de 1,5 mL por poço. Células deve ser banhado em qualquer populações mistas ou separadamente, para os controles. </li></ol> Método 2 transfecção (Qiagen Effectene transfecção de linhas celulares) <ol><li> Prepare complexos transfecção usando o reagente Qiagen Effectene pela primeira adição de 0,8 mg de cada amostra de DNA plasmidial a 200 mL de buffer CE. </li><li> Adicionar 6,4 mL de reagente potenciador para cada amostra, misturar brevemente sacudindo o tubo e incubar por 2 minutos em temperatura ambiente. </li><li> Adicionar 20 mL de reagente Effectene a cada amostra de mistura, sacudindo o tubo e incubar por 15 minutos em temperatura ambiente. </li><li> Durante esta incubação, preparar a duas linhas de células de interesse para transfecção. Lave as células recentemente várias passagens (&lt;confluência de 80%) uma vez em tampão fosfato salino (PBS). Adicionar 1,5 mL de volta meio de crescimento fresco aquecido e equilibrado (neste caso, meio modificado Dulbecco Águia (DMEM), 10% de soro fetal bovino (FBS)). </li><li> Uma vez que os complexos de transfecção têm incubadas por 15 minutos, transferir 1,2 mL de mídia para cada amostra. Mix por pipetagem, e transferir imediatamente 700 mL ~ dos complexos a cada um dos dois poços na placa de 6 poços. Adicione gota a gota, e misture suavemente agitando o prato. Permitir que as células transfectar por pelo menos 3 horas (pode variar com o tipo de célula). </li><li> Adicionar lamínulas esterilizadas a uma placa de 6-novo poço. Lamínulas podem ser revestidos para a adesão celular reforçada. </li><li> Depois que as células têm transfectadas, lave as células duas vezes com PBS e colher as células por trysinization mínima, adicionando cerca de 100 mL de solução de tripsina a cada poço, agitar brevemente a placa de revestimento totalmente cada poço, e imediatamente aspiração fora da tripsina. Uma vez que as células têm levantado, adicionar 1,5 mL de mídia fresco. </li><li> Neste ponto, as células devem ser banhado em populações mistos ou separados (para controles) para o lamínulas estéreis. </li><li> Permitem que as células para recuperar por 12 horas. </li></ol> 2. Fusão celular <ol><li> Remova a mídia de cultura de células e lavar duas vezes com PBS. </li><li> Neste ponto, é recomendado para tratar as células com cicloheximida (100 mcg / mL) para inibir a síntese de proteínas. Permitir que as células para incubar por 2 horas e depois lave as células duas vezes com PBS. </li><li> Células fusível, expondo-os a uma solução de 50% de polietileno glicol 1000 (PEG 1000) no soro livre de DMEM para 125 segundo a temperatura ambiente. </li><li> Lavar as células com PBS para remover a solução de PEG 1000, e adicionar 2 mL recém-aquecido e media equilibrada. </li><li> Permitir que as células incubar por 18-24 horas. </li></ol> Microscopia de fluorescência <ol><li> Remova a mídia, cultura e lave as células duas vezes em PBS. </li><li> Fix células adicionando 1 mL de uma solução de paraformaldeído a 4% (em PBS) a cada poço. Agite suavemente por 15 minutos. </li><li> Lave as células fixas duas vezes em PBS. </li><li> Remova cuidadosamente a lamínulas da placa, pavio off PBS excesso. Inverter para microscópio slides carregado com uma gota de meio de montagem (90% de glicerol, 100mM Tris pH 7,5, DABCO 2% (1,4-diazabicoyclo octanagem) contendo DAPI (4 &#39;,6-diamidino-2-phenylindole). </li><li> Retire o excesso de meio de montagem das lâminas, e selar cover desliza com unha polonês. </li><li> Visualize as células através de microscopia confocal ou epifluorescência. </li></ol> 3. Resultados representante A Figura 2 mostra um experimento heterocário exemplo usando fibroblastos primários e H1299 de células não pequenas células de carcinoma de pulmão. A proteína de interesse neste experimento (Chicken Anemia Virus-VP3) exibe a localização citoplasmática principalmente em tipos de células primárias e localização nuclear em tipos de células transformadas como visualizado por microscopia de epifluorescência. A proteína é expressa como uma fusão ou GFP (pEGFP-N1 vetor, Clontech) em fibroblastos prepúcio primária (PFF) ou dsRed (dsRed-N1 vetor, Clontech) em células H1299. Amostras de controle que envolve auto-fusão (duas primeiras linhas) exibir células multinucleadas, sem qualquer alteração nos padrões de steady-state de localização. Tais mudanças poderiam ocorrer devido a sensibilidade às condições de fusão ou a formação de syncitia. Fusões heterocário (linha inferior) demonstram entrada nuclear da proteína GFP-fundidas derivadas de células-primária e co-localização com a proteína dsRed fundida em resposta à introdução de material celular transformada. O exemplo mostrado é uma fusão heterocário relativamente rara, resultante de apenas duas células, uma de cada tipo, como demonstrado pela presença de ambos os sinais fluoroprotein. Além de detectar estes tipos de transições de localização, atividade shuttling nucleocytoplasmic pode ser facilmente detectada pela presença de um fluoróforo única em ambos os núcleos. Este tipo de determinação é clara quando se examina 01:01 fusão de células e dependeria de inibição da tradução. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Figura 1" /> Figura 1. Fluxograma do método heterocário usando fibroblastos primários e H1299 não-pequenas células de carcinoma de células. O gene de interesse é clonado para produzir uma fusão em frame-a um dos dois genes da proteína fluorescente. Linhas de células são, então, transitoriamente transfectadas com tanto construir e tempo para se recuperar. Heterocário formação é induzida pelo tratamento breve com polietileno glicol (PEG), seguido por incubação adicional. Finalmente, a localização sub-celular da proteína de interesse é observado usando várias formas de microscopia de fluorescência. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Figura 2" /> Figura 2. Tráfico Nucleocytoplasmic e shuttling atividade da proteína Anemia Virus Chicken VP3 visualizado por fusão heterocário. Linha superior: Imagens representativas de auto-fusão H1299 células que expressam dsRed-VP3 linha Médio:.. Representante imagens de epifluorescência das células do prepúcio primárias de fibroblastos (PFF) expressando GFP-VP3 após a fusão PEG Linha inferior: a fusão heterocário de PFF e H1299 células. Amarelo indica co-localização de GFP e sinais dsRed. As células foram fotografadas usando um microscópio de fluorescência Leica AF6000E Leica e software de imagem.

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	<li>Belaya, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FLIPing+heterokaryons+to+analyze+nucleo-cytoplasmic+shuttling+of+yeast+proteins.">FLIPing heterokaryons to analyze nucleo-cytoplasmic shuttling of yeast proteins.</a> RNA. 12, 921-930 (2006).</li><li>Bhaumik, 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=In+vivo+target+of+a+transcriptional+activator+revealed+by+fluorescence+resonance+energy+transfer.">In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer.</a> Genes Dev. 18, 333-343 (2004).</li><li>Cohen, H. R., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+exhibits+nuclear+retention+and+exhibits+reduced+association+with+the+export+factor+TAP/NXF1.">XIST RNA exhibits nuclear retention and exhibits reduced association with the export factor TAP/NXF1.</a> Chromosoma. 116, 373-383 (2007).</li><li>Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dapper1+is+a+nucleoplasmic+shuttling+protein+that+negatively+modulates+Wnt+signaling+in+the+nucleus.">Dapper1 is a nucleoplasmic shuttling protein that negatively modulates Wnt signaling in the nucleus.</a> J. Biol. Chem. 51, 35679-3588 (2008).</li><li>Grespin, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+Hormone+Receptor+&#945;1+Follows+a+Cooperative+CRM1/Calreticulin-mediated+Nuclear+Export+Pathway.">Thyroid Hormone Receptor &#945;1 Follows a Cooperative CRM1/Calreticulin-mediated Nuclear Export Pathway.</a> J. Biol. Chem. 283, 25576-25588 (2008).</li><li>Heilman, D. W., Teodoro, J. G., Green, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptin+Nucleocytoplasmic+Shuttling+Is+Required+for+Cell+Type-Specific+Localization,+Apoptosis,+and+Recruitment+of+the+Anaphase-Promoting+Complex/Cyclosome+to+PML+Bodies.">Apoptin Nucleocytoplasmic Shuttling Is Required for Cell Type-Specific Localization, Apoptosis, and Recruitment of the Anaphase-Promoting Complex/Cyclosome to PML Bodies.</a> J. Virology. 80, 7535-7545 (2006).</li><li>Jeffries, S., Capobianco, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+by+Notch+requires+nuclear+localization.">Neoplastic transformation by Notch requires nuclear localization.</a> Mol Cell Biol. 20, 3928-3941 (2000).</li><li>Keaton, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocytoplasmic+trafficking+of+G2/M+regulators+in+yeast.">Nucleocytoplasmic trafficking of G2/M regulators in yeast.</a> Mol. Biol. Cell. 19, 4006-4018 (2008).</li><li>Ling, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear-Cytoplasmic+Shuttling+Is+Not+Required+for+the+Epstein-Barr+Virus+EBNA-LP+Transcriptional+Coactivation+Function.">Nuclear-Cytoplasmic Shuttling Is Not Required for the Epstein-Barr Virus EBNA-LP Transcriptional Coactivation Function.</a> J. Virology. 83, 7109-7116 (2009).</li><li>Lin, X. -. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RXR&#945;+acts+as+a+carrier+for+TR3+nuclear+export+in+a+9-cis+retinoic+acid-dependent+manner+in+gastric+cancer+cells.">RXR&#945; acts as a carrier for TR3 nuclear export in a 9-cis retinoic acid-dependent manner in gastric cancer cells.</a> J. Cell Science. 117, 5609-5621 (2004).</li><li>Vissinga, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Export+of+NBN+Is+Required+for+Normal+Cellular+Responses+to+Radiation.">Nuclear Export of NBN Is Required for Normal Cellular Responses to Radiation.</a> Mol Cell Biol. 29, 1000-1006 (2009).</li><li>Zimmerman, R. S., Rosenberg, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+p19Arf+localization+accompany+apoptotic+crisis+during+pre-B-cell+transformation+by+Abelson+murine+leukemia+virus.">Changes in p19Arf localization accompany apoptotic crisis during pre-B-cell transformation by Abelson murine leukemia virus.</a> J. Virol. 82, 8383-8391 (2008).</li></ol>

Não há conflitos de interesse declarados.

O protocolo heterocário apresentado aqui fornece um método eficiente e facilmente interpretável para a caracterização de células comportamento de localização de tipo específico, assim como a atividade shuttling nucleocytoplasmic. Este método aproveita a sensibilidade de análise de fluorescência, bem como a capacidade de células de imagem ao vivo e realizar estudos da dinâmica de proteínas. A técnica é facilmente adaptado para vários tipos de células diferentes e é passível de ambas as imagens de células vivas e fixas. Por exemplo, a microscopia de fluorescência invertido pode ser usado para geração de imagens ao vivo e captura de vídeo em tempo lapso. Em contraste, as células montado pode ser utilizado em qualquer microscopia de epifluorescência, para determinação do estado estacionário ou localizações mais detalhada imagem confocal de localizações discretas. Além disso, técnicas como a recuperação de fluorescência após a fotodegradação (FRAP), ou perda de fluorescência em fotobranqueamento (FLIP) pode ser usado na determinação da cinética de localização 1. A incorporação de fluoróforos alternativo no protocolo permitiria experimentos de interação de proteínas como a transferência de energia de ressonância de fluorescência (FRET), a ser realizada em conjunto 2. Vários inibidores de tráfico de celulares, tais como B leptomycin ou negativo dominante Ran constrói GTPase pode ser usada para estabelecer a dependência de importação particular ou vias de exportação 5,6,9. <ul><li> Em certos experimentos envolvendo shuttling atividade, os cuidados devem ser tomados para evitar os efeitos de localização, devido à síntese de novas proteínas. Nestes casos, os inibidores da tradução ou pontos de tempo limitado deve ser usado. No entanto, artefatos devido à inibição traducional continuam a ser uma possibilidade. Fusão de células que contêm um de cada tipo de célula são mais desejados para a interpretação clara do shuttling e comportamento de localização. Otimização do número de células e as condições de fusão (tempo e concentração) pode ser necessário para promover tais eventos de fusão favoráveis ​​01:01. </li><li> É importante notar que a otimização de determinadas etapas no protocolo serão necessários dependendo dos tipos de célula em particular usados. Aspectos do procedimento experimental, como a eficiência de fusão, a eficiência de transfecção e viabilidade após a fusão pode variar muito entre os tipos de células. Em particular, os tipos de célula primária pode ser um desafio para gerenciar devido às condições de cultura complexa e eficiências de transfecção baixo. Uso de tecnologias como o dispositivo Nucleofector Lonza pode permitir o processamento rápido de células, bem como a eficiência de transfecção aumentou dramaticamente. Além disso, os métodos de eletroporação permitir a fácil transferência de células em suspensão para as etapas de fusão que se seguem. </li></ul>

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<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
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<td>Phosphate buffered saline (PBS)</td>
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<td>Sigma-Aldrich</td>
<td>P5493</td>
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<td>SH3023602</td>
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<td>&nbsp;</td>
<td>Fisher</td>
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<td>D9542-10MG</td>
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<td>D2522-25G</td>
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<td>Nucleofector HDF kit</td>
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heterokaryon technique for analysis of cell type-specific localization

Um número significativo de proteínas são reguladas pelo tráfico ou subcelular nucleocytolasmic shuttling. Estas proteínas exibir uma gama diversificada de funções celulares, incluindo a importação nuclear / exportação de RNA e proteínas, regulação da transcrição e apoptose. Curiosamente, os principais reorganizações celulares, incluindo a divisão celular, diferenciação e transformação, muitas vezes envolvem tais atividades 3,4,8,10. O estudo detalhado destas proteínas e seus respectivos mecanismos de regulação pode ser um desafio como estímulo para essas mudanças de localização pode ser fugaz, e os próprios movimentos pode ser bastante dinâmico e difícil de controlar. Estudos envolvendo oncogênese celular, por exemplo, continuar a beneficiar do entendimento caminhos e atividades de proteínas que diferem entre normal pilhas e células transformadas 6,7,11,12. Como muitas proteínas mostrar a localização alterada durante a transformação ou como resultado de transformação, os métodos de forma eficiente caracterizar estas proteínas e as vias em que participam têm a melhorar a compreensão das oncogênese e abrir novas áreas para a droga segmentação. Aqui apresentamos um método para a análise de proteínas e tráfico shuttling atividade entre primário e transformado em células de mamíferos. Este método combina a geração de fusões heterocário com microscopia de fluorescência para fornecer um protocolo flexível que pode ser usado para detectar proteínas localizações de estado estacionário ou dinâmico. Conforme mostrado na Figura 1, dois tipos de células são separadas transitoriamente transfectadas com construções plasmídeo carregando um gene fluoroprotein ligado ao gene de interesse. Depois de expressão, as células são fundidas usando polietileno glicol, e localizações proteína pode então ser fotografada usando uma variedade de métodos. O protocolo aqui apresentado é uma abordagem fundamental para que técnicas especializadas podem ser adicionados.

Watch this Scientific Journal Video about Heterokaryon Technique for Analysis of Cell Type-specific Localization at JoVE.com

Heterokaryon Technique for Analysis of Cell Type-specific Localization

Um método flexível e eficiente para a caracterização de células localização de proteínas específicas do tipo e nucleocytoplasmic shuttling é descrito. Esta abordagem heterocário usa proteínas de fusão fluorescente marcado para localizações de proteína imagem após a fusão celular. O protocolo é passível de steady-state localizações ou mais determinações dinâmicas baseadas em imagens de células vivas.

Técnica heterocário para Análise de Localização Celular Tipo específico

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

heterokaryon-technique-for-analysis-of-cell-type-specific-localization

Research

JoVE Journal

Biology

16.3K visualizações.  Worcester Polytechnic Institute- WPI. Um método flexível e eficiente para a caracterização de células localização de proteínas específicas do tipo e nucleocytoplasmic shuttling é descrito. Esta abordagem heterocário usa proteínas de fusão fluorescente marcado para localizações de proteína imagem após a fusão celular. O protocolo é passível de steady-state localizações ou mais determinações dinâmicas baseadas em imagens de células vivas.

Vídeo: Heterokaryon Technique for Analysis of Cell Type-specific Localization

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A Neuronal e astrocitária Ensaio Co-Cultura de Análise de Conteúdo de Alta de neurotoxicidade

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Biologia

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizando Localização RNA em Xenopus Oócitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Investigando Tecido e órgãos específicos Responses Fitocromo usando FACS assistida Célula tipo Profiling expressão específica no Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Single-cell Microinjection for Cell Communication Analysis

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a hemichannel by each neighboring cell to form the gap junction. In mammalian cells, the hemichannel is formed by six connexins, monomers with four transmembrane domains and a C and N terminal within the cytoplasm. Gap junctions permit the exchange of ions, second messengers, and small metabolites. In addition, they have important roles in many forms of cellular communication within physiological processes such as synaptic transmission, heart contraction, cell growth and differentiation. We detail how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells. It is expected that in functional gap junctions, the dye will diffuse from the loaded cell to the connected cells. It is a very useful technique to study gap junctions since you can evaluate the diffusion of the fluorescence in real time. We discuss how to prepare the cells and the micropipette, how to use a micromanipulator and inject a low molecular weight fluorescent dye in an epithelial cell line.

We describe here how to perform a single-cell microinjection of Lucifer Yellow to visualize cellular communication via gap-junctions in living cells, and provide some useful tips. We expect that this paper will help everyone to evaluate the degree of cellular coupling due to functional gap junctions. Everything described here could be, in principle, adapted to other fluorescent dyes with molecular weight below 1,000 Daltons.

Microinjeção de uma única célula para análise da comunicação celular

Gap junctions are intercellular channels that allow the communication of neighboring cells. This communication depends on the contribution of a ...

Micromanipulation Techniques Allowing Analysis of Morphogenetic Dynamics and Turnover of Cytoskeletal Regulators

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how fluorescent recovery after photobleaching (FRAP) and photoactivation techniques can be used to study the spatiotemporal dynamics of proteins in subcellular locations. We also show how these techniques enable straightforward determination of various parameters linked to actin cytoskeletal regulation and cell motility. Moreover, the microinjection of cells is additionally described as an alternative treatment (potentially preceding or complementing the aforementioned photomanipulation techniques) to trigger instantaneous effects of translocated proteins on cell morphology and function. Micromanipulation such as protein injection or local application of plasma membrane-permeable drugs or cytoskeletal inhibitors can serve as powerful tool to record immediate consequences of a given treatment on cell behavior at the single cell and subcellular level. This is exemplified here by immediate induction of lamellipodial cell edge protrusion by the injection of recombinant Rac1 protein, as established a quarter-century ago. In addition, we provide a protocol for determining the turnover of enhanced green fluorescent protein (EGFP)-VASP, an actin filament polymerase prominently accumulating at lamellipodial tips of B16-F1 cells, employing FRAP and including associated data analysis and curve fitting. We also present guidelines for estimating the rates of lamellipodial actin network polymerization, as exemplified by cells expressing EGFP-tagged &#946;-actin. Finally, instructions are given for how to investigate the rates of actin monomer mobility within the cell cytoplasm, followed by actin incorporation at sites of rapid filament assembly, such as the tips of protruding lamellipodia, using photoactivation approaches. None of these protocols is&#160;restricted to components or regulators of the actin cytoskeleton, but can easily be extended to explore in analogous fashion the spatiotemporal dynamics and function of proteins in various different subcellular structures or functional contexts.

We describe how micro- and photomanipulation techniques such as FRAP and photoactivation enable the determination of motility parameters and the spatiotemporal dynamics of proteins within migrating cells. Experimental readouts include subcellular dynamics and turnover of motility regulators or of the underlying actin cytoskeleton.

Técnicas de micromanipulação, permitindo a análise da dinâmica morfogenética e rotatividade dos reguladores do citoesqueleto

Examining the spatiotemporal dynamics of proteins can reveal their functional importance in various contexts. In this article, it is discussed how ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: Epigenoma célula tipo-específicas de criação de perfil

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Assessment of Zebrafish Lens Nucleus Localization and Sutural Integrity

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for generation of transgenics for in vivo imaging. These unique capabilities make the zebrafish an ideal platform to study ocular lens development and physiology. Our recent findings that an Aquaporin-0, Aqp0a, is required for stability of the anterior lens suture, as well as for the shift of the lens nucleus to the lens center with age led us to develop tools especially suited to analyzing the properties of zebrafish lenses. Here we outline detailed methods for lens dissection that can be applied to both larval and adult lenses, to prepare them for histological analysis, immunohistochemistry and imaging. We focus on analysis of lens suture integrity and cortical cell morphology and compare data generated from dissected lenses with data obtained from in vivo imaging of lens morphology made possible by a novel transgenic zebrafish line with a genetically encoded fluorescent marker. Analysis of dissected lenses perpendicular to their optical axis allows quantification of the relative position of the lens nucleus along the anterior-posterior axis. Movement of the lens nucleus from an initial anterior position to the center is required for normal lens optics in adult zebrafish. Thus, a quantitative measure of lens nuclear position directly correlates with its optical properties.

These protocols were developed to analyze cortical lens morphology, structural integrity of the zebrafish lens sutures in fixed and live lenses and to measure the position of the zebrafish lens nucleus along the anterior-posterior axis.

Avaliação da localização do núcleo da lente de zebrafish e integridade sutural

The zebrafish is uniquely suited to genetic manipulation and in vivo imaging, making it an increasingly popular model for reverse genetic studies and for ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Perfil de expressão gênica específica do tipo de célula no fígado do mouse

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

O Método CrioAPEX para Análise de Microscopia Eletrônica da Localização da Proteína da Membrana dentro de células ultraestruturalmente preservadas

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &amp;#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

Analisando a rede de α-Actinin em Cardiomiócitos derivados do iPSC humano usando microscopia de localização de molécula única

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...