Nos gustaría dar las gracias al Instituto Politécnico de Worcester Departamento de Química y Bioquímica de la financiación.

1. Transfección de líneas celulares Transfección Método 1 (Nucleofection Lonza para una mayor eficiencia de la transfección de células primarias) <ol><li> Las células deben ser de reciente aprobación y registro en el crecimiento de la fase previa de la transfección. Lavar las células de fosfato (PBS) y las células de la cosecha por tripsinización. </li><li> Recoger las células por centrifugación a 1500 xg durante 5 minutos. </li><li> Añadir esterilizados cubreobjetos en una placa de 6 pocillos. Cubreobjetos se pueden revestir para la adhesión celular mejorada. Coloque 1,5 ml de medio de cultivo (en este caso la Dulbecco modificado de Eagle (DMEM), 10% de suero fetal bovino (SFB)) en cada pocillo y equilibrar los medios de comunicación en la incubadora. </li><li> Recoger las células por tripsinización y resuspender en un volumen mínimo de tampón fosfato salino (PBS). </li><li> Contar las células y centrifugar la cantidad correcta de la resuspensión de proporcionar ~ 5x10 5 células por muestra. </li><li> Resuspender las células con cuidado en una solución de 100 Nucleofector sala de l temperatura por muestra. </li><li> Combine 100 L de suspensión de células con el ADN del plásmido 5 mg y la transferencia de la muestra a una cubeta Nucleofector teniendo cuidado de no incluir burbujas de aire. </li><li> Seleccione el programa adecuado Nucleofector (esto puede requerir pruebas previas para establecer la eficiencia de transfección óptima con un mínimo de mortalidad). </li><li> Inserte la cubeta con la suspensión de células / ADN en el soporte de la cubeta Nucleofector e iniciar el programa seleccionado. </li><li> Al terminar, retire la cubeta y añadir ~ 500 l de medio de cultivo pre-equilibrio a la misma (en la placa de 6 pocillos). </li><li> Inmediatamente la transferencia de la muestra en la placa de 6 pocillos con un volumen final de 1,5 ml por pocillo. Las células deben ser plateado, ya sea en poblaciones mixtas o por separado, para los controles. </li></ol> Transfección Método 2 (Qiagen Effectene transfección de líneas celulares) <ol><li> Preparar complejos de transfección usando el reactivo de Qiagen Effectene agregando primero 0,8 g de cada muestra de ADN del plásmido a 200 l buffer CE. </li><li> Añadir 6,4 l de reactivo potenciador de cada muestra, mezclar brevemente por prender el tubo, y se incuba durante 2 minutos a temperatura ambiente. </li><li> Añadir 20 l de reactivo Effectene a cada muestra, mezclar agitando el tubo, y se incuba durante 15 minutos a temperatura ambiente. </li><li> Durante la incubación, preparar las dos líneas celulares de interés para la transfección. Lavar las células recientemente pasajes (&lt;80% de confluencia) una vez en tampón fosfato salino (PBS). Añadir 1,5 ml de nuevo medio de cultivo fresco caliente y equilibrada (en este caso la Dulbecco modificado de Eagle (DMEM), 10% de suero fetal bovino (SFB)). </li><li> Una vez que los complejos de transfección se incubaron durante 15 minutos, la transferencia de 1,2 ml de los medios de comunicación a cada muestra. Mezclar con la pipeta, y de inmediato la transferencia de unos 700 l de los complejos de cada uno de los dos pozos en la placa de 6 pocillos. Añadir gota a gota, y mezclar suavemente haciendo girar el plato. Permiten a las células a transfectar por lo menos 3 horas (puede variar según el tipo de células). </li><li> Añadir esterilizados cubreobjetos a una nueva de 6 pocillos. Cubreobjetos se pueden revestir para la adhesión celular mejorada. </li><li> Después de que las células se transfectaron, lavar las células dos veces con PBS y la cosecha de las células por trysinization mínimo mediante la adición de aproximadamente 100 l de solución de tripsina a cada pocillo, agitar brevemente la placa a plena capa cada pozo, y de inmediato la aspiración de la tripsina. Una vez que las células se han levantado, añadir 1,5 ml de medio fresco. </li><li> En este punto, las células se sembraron en la población, ya sea mezclado o por separado (para controles) en el cubreobjetos estériles. </li><li> Permiten a las células para recuperarse durante 12 horas. </li></ol> 2. La fusión de células <ol><li> Eliminar los medios de cultivo de las células y lavar dos veces con PBS. </li><li> En este punto, se recomienda para tratar las células con cicloheximida (100 mg / mL) para inhibir la síntesis de proteínas. Permitir que las células se incuba durante 2 horas y luego se lavan las células dos veces con PBS. </li><li> Células se fusionan mediante la exposición a una solución de polietileno de 50% de 1000 glicol (PEG 1000) en el suero libre de DMEM durante 125 segundos a temperatura ambiente. </li><li> Lavar las células con PBS para eliminar la solución de PEG 1000, y agregar 2 ml recién calentado y los medios de comunicación equilibrada. </li><li> Permitir que las células se incuba durante 18 a 24 horas. </li></ol> Microscopía de fluorescencia <ol><li> Eliminar los medios de cultivo, y lavar las células dos veces en PBS. </li><li> Fijar las células mediante la adición de 1 ml de una solución de paraformaldehído al 4% (en PBS) a cada pocillo. Agitar suavemente durante 15 minutos. </li><li> Lavar las células dos veces en PBS fija. </li><li> Retire con cuidado el cubreobjetos de la placa, la mecha el exceso de PBS. Invertir en portaobjetos cargados con una gota de medio de montaje (90% de glicerol, 100 mM Tris pH 7,5, el 2% DABCO (1,4-diazabicoyclo octanaje) con DAPI (4 &#39;,6-diamidino-2-phenylindole). </li><li> Retire el exceso de medio de montaje de las diapositivas, y co sellover hojas con esmalte de uñas. </li><li> Visualizar las células a través de microscopía confocal o epifluorescencia. </li></ol> 3. Resultados representante La figura 2 muestra un experimento heterocarión ejemplo, con los fibroblastos primarios y H1299 células no pequeñas de pulmón de células cancerosas. La proteína de interés en este experimento (pollo Anemia Virus-VP3) muestra la localización citoplasmática principalmente en tipos de células primarias y la localización nuclear de tipos de células transformadas como se visualiza mediante microscopía de epifluorescencia. La proteína se expresa como una fusión de cualquiera de las buenas prácticas agrarias (pEGFP-N1 vector, Clontech) en fibroblastos de prepucio primaria (FPF) o dsRed (dsRed-N1 vector, Clontech) en las células H1299. Muestras de control de la participación auto-fusión (dos filas superiores) muestran células multinucleadas sin ningún cambio en los patrones de localización de estado estacionario. Estos cambios de lo contrario podría ocurrir debido a la sensibilidad a las condiciones de la fusión o la formación de sincitios. Fusiones heterocarión (fila inferior), demuestran la entrada nuclear de la GFP-fundido primario de células derivadas de proteínas y colocalización con la proteína dsRed fusionado en respuesta a la introducción de material de células transformadas. El ejemplo que se muestra es una fusión heterocarión relativamente raras como resultado de sólo dos células, una de cada tipo, como lo demuestra la presencia de ambas señales fluoroproteínicos. Además de detectar este tipo de transiciones de localización, actividad nucleocytoplasmic ir y venir puede ser fácilmente detectado por la presencia de un solo fluoróforo en ambos núcleos. Este tipo de determinación es clara al examinar 01:01 fusiones celulares y que dependen de la inhibición de la traducción. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Figura 1" /> Figura 1. Diagrama de flujo del método heterocarión con fibroblastos primarios y H1299 no pequeñas células de carcinoma de células. El gen de interés es clonado para producir una fusión en el marco de uno de los dos genes de proteínas fluorescentes. Líneas celulares se transfectadas transitoriamente, ya sea con la construcción y les permitió recuperar. Heterocarión formación es inducida por el tratamiento breve con polietilenglicol (PEG), seguido de la incubación. Por último, la localización subcelular de la proteína de interés se observa mediante diversas formas de microscopía de fluorescencia. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Figura 2" /> Figura 2. El tráfico de ir y venir nucleocytoplasmic y la actividad de la proteína del virus de la Anemia de pollo VP3 visualizado por la fusión heterocarión. Fila superior: Imágenes representativas de la auto-fusionado H1299 células que expresan dsRed-VP3 Fila central:.. Representante imágenes de epifluorescencia de células primarias de fibroblastos de prepucio (PFF) que expresan GFP-VP3 después de la fusión PEG Fila inferior: fusión heterocarión de la FPF y las células H1299. El color amarillo indica colocalización de las buenas prácticas agrarias y las señales de dsRed. Las células fueron fotografiadas con un microscopio de fluorescencia Leica AF6000E y el software Leica imágenes.

<ol>
	<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>

El protocolo heterocarión que aquí se presenta ofrece un método eficaz y fácilmente interpretable para la caracterización del comportamiento de la localización de células de tipo específico, así como la actividad nucleocytoplasmic yendo y viniendo. Este método se aprovecha de la sensibilidad de los análisis de fluorescencia, así como la capacidad de las células de la imagen en vivo y la realización de estudios de la dinámica de las proteínas. La técnica se adapta fácilmente a diferentes tipos de células y es susceptible de dos imágenes de células vivas y fijos. Por ejemplo, la microscopía de fluorescencia invertido se puede utilizar para imágenes en vivo y captura de lapso de tiempo de vídeo. Por el contrario, las células de montaje puede ser utilizado tanto en la microscopía de epifluorescencia para la determinación del estado de equilibrio o de imagen confocal localizaciones más detallada de localizaciones discretas. Por otra parte, las técnicas como la recuperación de la fluorescencia después de photobleaching (FRAP), o la pérdida de fluorescencia en photobleaching (FLIP) se puede utilizar en la determinación de la cinética de una localización. La incorporación de fluoróforos se alternan en el protocolo permitiría a los experimentos de interacción de proteínas como la transferencia de energía de resonancia fluorescente (FRET) que se realizará en dos conciertos. Diferentes inhibidores de la trata de celulares, tales como B leptomycin o dominante negativo construye GTPasa Ran se puede utilizar para establecer la dependencia de la importación particular o vías de exportación 5,6,9. <ul><li> En algunos experimentos implican ir y venir de actividad, se debe tener cuidado para evitar los efectos de localización debido a la síntesis de nuevas proteínas. En estos casos, los inhibidores de la traducción o puntos de tiempo limitado se debe utilizar. Sin embargo, los artefactos debido a la inhibición de la traducción sigue siendo una posibilidad. Fusiones de células que contienen uno de cada tipo de células son los más deseados para una interpretación clara de ir y venir y el comportamiento de la localización. Optimización del número de células y las condiciones de la fusión (de tiempo y concentración) puede ser necesario para promover estos eventos favorables fusión 01:01. </li><li> Es importante tener en cuenta que la optimización de ciertas medidas en el protocolo, será necesario en función de los tipos de células utilizadas. Aspectos en el procedimiento experimental como la eficiencia de fusión, la eficiencia de transfección y viabilidad después de la fusión puede variar mucho entre los distintos tipos de células. En particular, los principales tipos de células pueden ser difíciles de manejar debido a las condiciones de cultivo complejos y bajas eficiencias de transfección. El uso de tecnologías tales como el dispositivo de Lonza Nucleofector puede permitir el procesamiento rápido de las células, así como aumentado dramáticamente la eficiencia de transfección. Además, los métodos de electroporación permite una fácil transferencia de células en suspensión a los pasos que siguen a la fusión. </li></ul>

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		<th>Catalogue Number</th>
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<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
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<td>Phosphate buffered saline (PBS)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
<td>&nbsp;</td>
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<td>Trypsin</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3023602</td>
<td>&nbsp;</td>
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<td>Dulbecco&#x2019;s modified Eagle&#x2019;s medium (DMEM)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH30243FS</td>
<td>High glucose</td>
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<td>paraformaldehyde</td>
<td>&nbsp;</td>
<td>FisherChemical</td>
<td>O4042-500</td>
<td>&nbsp;</td>
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<td>4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D9542-10MG</td>
<td>&nbsp;</td>
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<td>Hyclone Serum (fetal bovine)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH3008803HI</td>
<td>&nbsp;</td>
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<td>Falcon 6-well plates</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-772-1B</td>
<td>&nbsp;</td>
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<td>Polyethylene glycol 1000</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>528875-25GM</td>
<td>&nbsp;</td>
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<td>Cover slips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545-85</td>
<td>&nbsp;</td>
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<td>DABCO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2522-25G</td>
<td>&nbsp;</td>
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<td>Nucleofector HDF kit</td>
<td>&nbsp;</td>
<td>Lonza</td>
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heterokaryon technique for analysis of cell type-specific localization

Un número importante de proteínas están regulados por el tráfico subcelular o nucleocytolasmic yendo y viniendo. Estas proteínas muestran una gran variedad de funciones celulares, incluyendo la importación nuclear / exportación de ARN y proteínas, la regulación transcripcional y apoptosis. Curiosamente, los grandes reorganizaciones celulares, incluyendo la división celular, la diferenciación y la transformación, a menudo involucran actividades como 3,4,8,10. El estudio detallado de estas proteínas y sus mecanismos de regulación respectiva puede ser un reto como el estímulo para la localización de estos cambios puede ser difícil de alcanzar, y los movimientos se puede ser muy dinámico y difícil de rastrear. Los estudios que incluyen la oncogénesis celular, por ejemplo, seguir beneficiándose de las vías de entendimiento y las actividades de las proteínas que difieren entre las células normales de primaria y las células transformadas 6,7,11,12. De proteínas que muestran la localización alterado durante la transformación o como resultado de la transformación, los métodos para caracterizar de manera eficiente estas proteínas y las vías en las que participan de pie para mejorar la comprensión de la oncogénesis y abrir nuevas áreas de fármaco que se dirige. Aquí presentamos un método para el análisis de tráfico de proteínas y yendo y viniendo entre la actividad de las células de mamíferos primarios y transformados. Este método combina la generación de fusiones heterocarión con microscopía de fluorescencia para proporcionar un protocolo flexible que puede ser utilizado para detectar la proteína localizaciones en estado estable o dinámica. Como se muestra en la Figura 1, dos tipos de células separadas se transfectadas transitoriamente con las construcciones de plásmido lleva un gen fluoroproteínicos unido al gen de interés. Después de la expresión, las células se fusionan con glicol de polietileno, y localizaciones de la proteína se puede obtener imágenes con una variedad de métodos. El protocolo que aquí se presenta es un enfoque fundamental de las técnicas especializadas se pueden añadir.

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

Un método flexible y eficiente para la caracterización de la localización de la proteína tipo de células específicas y nucleocytoplasmic ir y venir se describe. Este enfoque heterocarión utiliza fluorescencia marcado con proteínas de fusión a la proteína localizaciones imagen después de la fusión celular. El protocolo es susceptible de estado estacionario localizaciones o más determinaciones dinámicas basadas en imágenes de células vivas.

Heterocarión Técnica para el Análisis de tipo de células específicas de localización

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

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JoVE Journal

Biology

16.3K vistas.  Worcester Polytechnic Institute- WPI. Un método flexible y eficiente para la caracterización de la localización de la proteína tipo de células específicas y nucleocytoplasmic ir y venir se describe. Este enfoque heterocarión utiliza fluorescencia marcado con proteínas de fusión a la proteína localizaciones imagen después de la fusión celular. El protocolo es susceptible de estado estacionario localizaciones o más determinaciones dinámicas basadas en imágenes de células vivas.

Video: 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 neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

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

Investigación

Biología

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.

Visualizar la localización del ARN en Xenopus Los ovocitos

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.

La investigación de los tejidos y órganos específicos de las respuestas fitocromo usando FACS asistida por células de tipo Perfiles de expresión específica en 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.

La microinyección de una sola célula de Análisis de Comunicación Móvil

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 micromanipulación que permite el análisis de las dinámicas morfogenéticas y facturación de los reguladores citoesqueléticos

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: Perfiles de celular-tipo-específico epigenoma

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.

Evaluación de la localización del núcleo de la lente de pez zebrafish y integridad 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.

Perfilado de expresión génica específico del tipo de celda en el hígado del ratón

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.

El método CryoAPEX para el análisis de microscopía electrónica de la localización de proteínas de membrana dentro de células ultraestructuralmente conservadas

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.

Análisis de la red α-Actinin en cardiomiocitos humanos derivados de iPSC mediante microscopía de localización de molécula única

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