Nous tenons à remercier l&#39;Institut polytechnique de Worcester Département de chimie et biochimie de financement.

1. Transfection de lignées cellulaires Une méthode de transfection (Nucléofection Lonza pour une efficacité accrue de cellules primaires de transfection) <ol><li> Les cellules doivent être de passage récent et en phase exponentielle de croissance avant la transfection. Laver les cellules dans une solution saline tamponnée au phosphate (PBS) et la récolte des cellules par trypsinisation. </li><li> Recueillir les cellules par centrifugation à 1500 xg pendant 5 minutes. </li><li> Ajouter lamelles stérilisés à une plaque de 6 puits. Lamelles peuvent être revêtus d&#39;adhésion cellulaire améliorée. Placer 1,5 ml de milieux de culture (dans ce cas moyennes Eagle modifié par Dulbecco (DMEM), 10% de sérum fœtal bovin (FBS)) dans chaque puits et équilibrer les médias dans l&#39;incubateur. </li><li> Récolter les cellules par trypsinisation et remettre en suspension dans un volume minimal de tampon phosphate salin (PBS). </li><li> Compter les cellules et centrifuger la quantité correcte de la remise en suspension de fournir ~ 5x10 5 cellules par échantillon. </li><li> Resuspendre les cellules attentivement dans 100 uL solution de salle de Nucleofector température par exemple. </li><li> Combinez 100 uL de suspension cellulaire avec 5 ug d&#39;ADN plasmidique et le transfert de l&#39;échantillon dans une cuvette Nucleofector en faisant attention de ne pas inclure de bulles d&#39;air. </li><li> Sélectionnez le programme approprié Nucleofector (cela peut nécessiter des tests précédents pour établir l&#39;efficacité de transfection optimale avec une mortalité minimale). </li><li> Insérez la cuvette contenant la suspension cellulaire / ADN dans le support de cuvette Nucleofector et commencer le programme sélectionné. </li><li> Une fois terminée, retirez la cuvette et ajouter ~ 500 ul de milieu de culture pré-équilibrée à elle (sur la plaque de 6 puits). </li><li> Immédiatement transférer l&#39;échantillon dans la plaque de 6 puits avec un volume final de 1,5 ml par puits. Les cellules doivent être plaqués dans les deux populations mixtes ou séparément, pour les contrôles. </li></ol> 2 méthode de transfection (Qiagen Effectene transfection de lignées cellulaires) <ol><li> Préparer les complexes de transfection avec le réactif Qiagen Effectene en ajoutant d&#39;abord 0,8 mg de chaque échantillon d&#39;ADN plasmidique à 200 pi de tampon CE. </li><li> Ajouter 6,4 uL de réactif Enhancer pour chaque échantillon, mélanger brièvement en feuilletant le tube et incuber pendant 2 minutes à température ambiante. </li><li> Ajouter 20 uL Effectene réactif à chaque échantillon, mélanger en feuilletant le tube et incuber pendant 15 minutes à température ambiante. </li><li> Pendant cette incubation, préparer les deux lignées de cellules d&#39;intérêt pour la transfection. Laver les cellules récemment repiquées (confluent &lt;80%) une fois dans un tampon phosphate salin (PBS). Rajouter 1,5 ml de milieu frais de croissance réchauffé et équilibrée (dans ce cas de Dulbecco modifié Eagle (DMEM), 10% de sérum fœtal bovin (FBS)). </li><li> Une fois les complexes de transfection ont incubé pendant 15 minutes, transférer 1,2 ml de milieu à chaque échantillon. Mélanger à la pipette et transférer immédiatement ~ 700 uL des complexes à chacun des deux puits dans la plaque de 6 puits. Ajouter goutte à goutte, et mélanger en douceur en faisant tournoyer la plaque. Laisser les cellules à transfecter pendant au moins 3 heures (peut varier selon le type cellulaire). </li><li> Ajouter lamelles stérilisés à une nouvelle plaque de 6 puits. Lamelles peuvent être revêtus d&#39;adhésion cellulaire améliorée. </li><li> Après que les cellules transfectées ont, laver les cellules deux fois avec du PBS et la récolte des cellules par trysinization minimale en ajoutant environ 100 uL solution de trypsine dans chaque puits, agiter brièvement la plaque pour bien enrober chaque bien, et immédiatement aspiration hors de la trypsine. Une fois les cellules ont levé, ajouter 1,5 ml de milieu frais. </li><li> À ce stade, les cellules doivent être plaqués au sein des populations mixtes ou séparées (pour les contrôles) sur les lamelles stériles. </li><li> Laisser les cellules de récupérer pendant 12 heures. </li></ol> 2. Fusion cellulaire <ol><li> Retirez le support de culture des cellules et laver deux fois avec du PBS. </li><li> À ce stade, il est recommandé de traiter les cellules avec du cycloheximide (100 pg / ml) pour inhiber la synthèse protéique. Laisser les cellules à incuber pendant 2 heures puis laver les cellules deux fois avec du PBS. </li><li> Fusible cellules en les exposant à une solution de 50% de polyéthylène glycol 1000 (PEG 1000) dans du DMEM sans sérum pour 125 secondes à température ambiante. </li><li> Laver les cellules avec du PBS pour éliminer la solution de PEG 1000, et ajouter 2 ml fraîchement réchauffé et les médias équilibrée. </li><li> Laisser les cellules à incuber pendant 18 - 24 heures. </li></ol> Microscopie par fluorescence <ol><li> Retirez le support de culture, et laver les cellules deux fois dans du PBS. </li><li> Fixer les cellules en ajoutant 1 ml d&#39;une solution de paraformaldéhyde 4% (dans du PBS) à chaque puits. Agiter doucement pendant 15 minutes. </li><li> Laver les cellules deux fois en PBS fixes. </li><li> Retirez délicatement les lamelles de la plaque, mèche l&#39;excès de PBS. Inverser sur lames de microscope chargé avec une goutte de milieu de montage (90% de glycérol, 100mm Tris pH 7,5, 2% DABCO (1,4-diazabicoyclo-octane) contenant du DAPI (4 &#39;,6-diamidino-2-phénylindole). </li><li> Enlever l&#39;excès de milieu de montage des lames, et sceller la coopérationVer glisse avec des vernis à ongles. </li><li> Visualisez les cellules par microscopie confocale ou à épifluorescence. </li></ol> 3. Les résultats représentatifs La figure 2 montre une expérience hétérocaryon exemple en utilisant des fibroblastes primaires et H1299 non à petites cellules de poumon de cellules cancéreuses. La protéine d&#39;intérêt dans cette expérience (anémie poulet Virus-VP3) affiche la localisation cytoplasmique surtout dans les types de cellules primaires et de localisation nucléaire dans des types cellulaires transformées comme visualisé par microscopie à épifluorescence. La protéine est exprimée comme une fusion soit la GFP (pEGFP-N1 vecteur, Clontech) dans des fibroblastes de prépuce primaires (PFF) ou DsRed (DsRed-N1 vecteur, Clontech) dans les cellules H1299. Les échantillons de contrôle impliquant l&#39;auto-fusions (les deux premiers rangs) l&#39;affichage des cellules multinucléées, sans changement dans les modes de localisation état stable. De tels changements pourraient autrement se produire en raison de la sensibilité aux conditions de fusion ou de la formation de syncitia. Fusions hétérocaryon (rangée du bas) démontrent d&#39;entrée nucléaire de la GFP fusionné primaires de cellules dérivées de protéines et de colocalisation avec la protéine DsRed fusionnés en réponse à l&#39;introduction de matériel cellulaire transformé. L&#39;exemple montré est une fusion hétérocaryon relativement rare résultant de seulement deux cellules, une de chaque type, comme en témoigne la présence de deux signaux fluoroprotéiniques. En plus de détecter les types de transitions de localisation, de l&#39;activité navette nucléocytoplasmique peut être facilement détecté par la présence d&#39;un fluorophore unique en deux noyaux. Ce type de détermination est claire lorsqu&#39;on examine 01:01 fusions de cellules et dépendra de l&#39;inhibition de la traduction. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Figure 1" /> Figure 1. Organigramme de la méthode utilisant hétérocaryon fibroblastes primaires et H1299 non à petites cellules de carcinome cellulaire. Le gène d&#39;intérêt est cloné pour produire une fusion en phase l&#39;un des deux gènes de protéines fluorescentes. Les lignées cellulaires sont ensuite transfectées transitoirement avec soit de construire et permis de récupérer. La formation hétérocaryon est induite par un bref traitement avec du polyéthylène glycol (PEG) suivie d&#39;une incubation supplémentaire. Enfin, la localisation sub-cellulaire de la protéine d&#39;intérêt est observé en utilisant diverses formes de microscopie à fluorescence. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Figure 2" /> Figure 2. Le trafic nucléocytoplasmique et la navette d&#39;activité de l&#39;anémie poulet Virus VP3 protéines visualisées par fusion hétérocaryon. Rangée du haut: les images représentant des auto-H1299 fusionné des cellules exprimant DsRed-VP3 Rangée du milieu:.. Représentant images épifluorescence de cellules primaires de fibroblastes du prépuce (PFF) exprimant la GFP-VP3 après fusion PEG Rangée du bas: la fusion des hétérocaryon PFF et H1299 cellules. Le jaune indique colocalisation de la GFP et les signaux DsRed. Les cellules ont été imagées par un Leica AF6000E fluorescence microscope et un logiciel d&#39;imagerie Leica.

<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>

Le protocole présenté ici fournit hétérocaryon une méthode efficace et facilement interprétables pour la caractérisation du comportement localisation cellulaire de type spécifique ainsi que l&#39;activité navette nucléocytoplasmique. Cette méthode tire parti de la sensibilité de l&#39;analyse par fluorescence ainsi que la capacité de cellules de l&#39;image en direct et effectuer des études sur la dynamique des protéines. La technique est facilement adaptable pour de nombreux types cellulaires différents et se prête à la fois imagerie des cellules vivantes et fixées. Par exemple, la microscopie à fluorescence inversé peut être utilisé pour l&#39;imagerie en direct et de capture vidéo laps de temps. En revanche, les cellules montées peuvent être utilisés soit en microscopie à épifluorescence pour la détermination de l&#39;état stable localisations ou plus détaillée d&#39;imagerie confocale de localisations discrètes. Par ailleurs, des techniques telles que la récupération de fluorescence après photoblanchiment (FRAP), ou la perte de fluorescence dans le photoblanchiment (FLIP) peut être utilisé dans la détermination des cinétiques une localisation. L&#39;incorporation de fluorophores de remplacement dans le protocole permettrait à des expériences d&#39;interactions protéiques comme le transfert d&#39;énergie par résonance de fluorescence (FRET) à être menées de concert 2. Différents inhibiteurs de la traite cellulaires tels que B leptomycin ou dominant négatif Ran GTPase des constructions peuvent être utilisées pour établir la dépendance sur les importations ou les voies d&#39;exportation notamment 5,6,9. <ul><li> Dans certaines expériences impliquant la navette activité, des précautions doivent être prises pour éviter les effets de localisation due à la synthèse de nouvelles protéines. Dans ces cas, les inhibiteurs de la traduction ou des points de temps limitée doit être utilisée. Cependant, les artefacts dus à l&#39;inhibition traductionnelle rester une possibilité. Fusions de cellules contenant un de chaque type de cellule sont plus désirés pour l&#39;interprétation claire de la navette et le comportement de localisation. Optimisation du nombre de cellules et les conditions de la fusion (timing et de concentration) peuvent être nécessaires pour promouvoir ces événements de fusion favorables 01h01. </li><li> Il est important de noter que l&#39;optimisation de certaines étapes dans le protocole seront nécessaires selon les types de cellules particulières utilisées. Aspects de la procédure expérimentale de fusion tels que l&#39;efficacité, efficacité de la transfection et la viabilité après fusion peut varier considérablement entre les différents types cellulaires. En particulier, les types de cellules primaires peuvent être difficiles à gérer en raison de conditions de culture complexe et des efficacités de transfection faible. L&#39;utilisation de technologies telles que le périphérique Lonza Nucleofector peut permettre un traitement rapide des cellules ainsi que des efficacités de transfection considérablement augmenté. En outre, les méthodes électroporation permet le transfert facile des cellules en suspension pour les étapes de fusion qui suivent. </li></ul>

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<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
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<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>P5493</td>
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<td>&nbsp;</td>
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<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>
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<td>O4042-500</td>
<td>&nbsp;</td>
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<td>&nbsp;</td>
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heterokaryon technique for analysis of cell type-specific localization

Un nombre important de protéines sont réglementés par le trafic subcellulaire ou nucleocytolasmic navette. Ces protéines afficher un large éventail de fonctions cellulaires, y compris l&#39;import nucléaire / export des ARN et des protéines, la régulation transcriptionnelle, et l&#39;apoptose. Fait intéressant, grandes réorganisations cellulaires, y compris la division cellulaire, la différenciation et la transformation, impliquent souvent des activités telles 3,4,8,10. L&#39;étude détaillée de ces protéines et leurs mécanismes de régulation peut être difficile car la stimulation de ces changements de localisation peut être insaisissable, et les mouvements eux-mêmes peuvent être très dynamiques et difficiles à suivre. Les études portant sur ​​l&#39;oncogenèse cellulaire, par exemple, continuent de bénéficier de voies de compréhension et d&#39;activités de protéines qui diffèrent entre les cellules normales primaires et les cellules transformées 6,7,11,12. Comme de nombreuses protéines montrent la localisation altérée durant la transformation ou à la suite de la transformation, des méthodes pour caractériser ces protéines efficacement et les voies à laquelle ils participent debout pour améliorer la compréhension de l&#39;oncogenèse et ouvrir de nouveaux domaines pour le ciblage de médicaments. Nous présentons ici une méthode pour l&#39;analyse du trafic des protéines et l&#39;activité navettes entre le primaire et des cellules transformées de mammifères. Cette méthode combine la génération de fusions hétérocaryon par microscopie à fluorescence de fournir un protocole flexible qui peut être utilisé pour détecter les localisations des protéines état stable ou dynamique. Comme le montre la figure 1, deux types cellulaires distincts sont transitoirement transfectées avec des constructions plasmidiques portant un gène fluoroprotéiniques attaché au gène d&#39;intérêt. Après l&#39;expression, les cellules sont fusionnées à l&#39;aide de polyéthylène glycol, et des localisations de protéines peuvent ensuite être visualisés en utilisant une variété de méthodes. Le protocole présenté ici est une approche fondamentale à laquelle les techniques spécialisées peuvent être ajoutés.

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

Une méthode souple et efficace pour la caractérisation de la localisation de protéines cellulaires spécifiques au type et à la navette nucléocytoplasmique est décrite. Cette approche utilise les protéines de fusion hétérocaryon marqués à la fluorescence d&#39;localisations protéines image après la fusion cellulaire. Le protocole se prête à l&#39;état stationnaire localisations ou plusieurs déterminations dynamique basée sur l&#39;imagerie de cellules vivantes.

Technique d&#39;analyse des hétérocaryon cellulaire spécifique de type localisation

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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16.3K vues.  Worcester Polytechnic Institute- WPI. Une méthode souple et efficace pour la caractérisation de la localisation de protéines cellulaires spécifiques au type et à la navette nucléocytoplasmique est décrite. Cette approche utilise les protéines de fusion hétérocaryon marqués à la fluorescence d'localisations protéines image après la fusion cellulaire. Le protocole se prête à l'état stationnaire localisations ou plusieurs déterminations dynamique basée sur l'imagerie de cellules vi...

Vidéo: 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.

Un neuronale et astrocytes de co-culture de dosage pour l&#39;analyse du contenu haute de la neurotoxicité

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

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Biologie

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.

Visualisation de localisation de l&#39;ARN Xenopus Les ovocytes

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.

Étude des tissus et des organes spécifiques Réponses phytochrome utilisant FACS assistée type cellulaire profilage expression spécifique dans 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.

Microinjection unique cellule pour l&#39;analyse cellulaire de communication

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.

Techniques de micromanipulation permettant l’analyse de la dynamique morphogénétique et chiffre d’affaires des organismes de réglementation du cytosquelette

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 : Type-spécifiques des cellules épigénome profilage

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.

Évaluation de la localisation des noyaux de lentilles zebrafish et de l’intégrité suturale

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.

Profilage de l'expression génique spécifique au type cellulaire dans le foie de souris

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.

Méthode CryoAPEX pour l’analyse de la microscopie électronique de la localisation des protéines membranaires dans les cellules ultrastructuralement conservées

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.

Analyse du réseau α-Actinine dans les cardiomyocytes dérivés de l’iPSC à l’aide d’une microscopie de localisation à molécule unique

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