Vorremmo ringraziare il Worcester Polytechnic Institute Dipartimento di Chimica e Biochimica per il finanziamento.

1. Trasfezione di linee cellulari Transfection Metodo 1 (Nucleofection Lonza per una maggiore efficienza di trasfezione delle cellule primarie) <ol><li> Le cellule devono essere di passaggio recente e in crescita log fase prima di trasfezione. Lavare le cellule in soluzione salina tamponata con fosfato (PBS) e le cellule raccolta da tripsinizzazione. </li><li> Raccogliere le cellule per centrifugazione a 1500 xg per 5 minuti. </li><li> Aggiungi scivola copertura sterile per un 6-pozzetti. Scivola la copertura può essere rivestita per l&#39;adesione cellulare migliorata. Luogo 1,5 mL di terreni di coltura (in questo caso medio Dulbecco modificato Eagle (DMEM), 10% di siero fetale bovino (FBS)) in ciascun pozzetto ed equilibrare i media nell&#39;incubatrice. </li><li> Raccogliere le cellule tripsinizzazione e risospendere in un volume minimo di tampone fosfato (PBS). </li><li> Contare le celle e centrifugare la giusta quantità di risospensione di fornire ~ 5x10 5 cellule per campione. </li><li> Risospendere le cellule con cura in 100 ul soluzione Nucleofector temperatura ambiente per campione. </li><li> Combina 100 l di sospensione cellulare con 5 mg di DNA plasmidico e trasferimento del campione ad un Cuvette Nucleofector facendo attenzione a non includere bolle d&#39;aria. </li><li> Selezionare il programma appropriato Nucleofector (questo potrebbe richiedere test precedenti di stabilire l&#39;efficienza ottimale di trasfezione con mortalità minima). </li><li> Inserire la cuvetta contenente la cella / DNA sospensione nella portacuvette Nucleofector e avviare il programma selezionato. </li><li> Al termine, rimuovere la cuvetta e aggiungere circa 500 ml di pre-equilibrato terreni di coltura ad esso (tratto dal 6 pozzetti). </li><li> Trasferire immediatamente il campione in 6 pozzetti con volume finale di 1,5 mL per pozzetto. Le cellule devono essere placcato in entrambe le popolazioni miste o separatamente, per i controlli. </li></ol> Trasfezione Metodo 2 (Qiagen trasfezione Effectene per linee cellulari) <ol><li> Preparare complessi trasfezione utilizzando il reagente Qiagen Effectene in primo luogo l&#39;aggiunta di 0,8 mg di ciascun campione di DNA plasmidico a 200 microlitri di buffer CE. </li><li> Aggiungere 6,4 microlitri di reagente enhancer a ciascun campione, miscelare brevemente muovendo il tubo, e incubare per 2 minuti a temperatura ambiente. </li><li> Aggiungere 20 microlitri di reagente Effectene ad ogni mix campione, muovendo il tubo, e incubare per 15 minuti a temperatura ambiente. </li><li> Durante questa incubazione, preparare le due linee cellulari di interesse per la trasfezione. Lavare le cellule recentemente diversi passaggi (&lt;80% confluenza), una volta in tampone fosfato (PBS). Aggiungi nuovo mezzo fresco 1,5 ml di crescita equilibrata e riscaldato (in questo caso medio Dulbecco modificato Eagle (DMEM), 10% di siero fetale bovino (FBS)). </li><li> Una volta che i complessi trasfezione hanno incubato per 15 minuti, trasferire 1,2 ml di media per ogni campione. Mescolare pipettando, e trasferire immediatamente ~ 700 ml di complessi a ciascuno dei due pozzi nel 6 pozzetti. Aggiungere goccia a goccia, e mescolare delicatamente facendo roteare il piatto. Consentono alle cellule di trasfezione per almeno 3 ore (può variare con il tipo di cellula). </li><li> Aggiungi scivola copertura sterilizzati ad un nuovo cambio a 6 pozzetti. Scivola la copertura può essere rivestita per l&#39;adesione cellulare migliorata. </li><li> Dopo che le cellule sono transfettate, lavare le cellule due volte con PBS e raccogliere le cellule trysinization minimo con l&#39;aggiunta di circa 100 microlitri soluzione tripsina in ogni pozzetto, agitare brevemente la piastra a cappotto completamente ogni bene, e subito aspirando la tripsina. Una volta che le cellule hanno sollevato, aggiungere 1,5 mL di mezzi freschi. </li><li> A questo punto, le cellule devono essere placcato in popolazioni mista o separata (per i controlli) sul coprioggetto sterili. </li><li> Consentono alle cellule di recuperare per 12 ore. </li></ol> 2. Fusione cellulare <ol><li> Togliere terreni di coltura dalle cellule e lavare due volte con PBS. </li><li> A questo punto, si consiglia di trattare le cellule con cicloesimide (100 mg / ml) per inibire la sintesi proteica. Consentire alle cellule di incubare per 2 ore e poi lavare le cellule due volte con PBS. </li><li> Cellule fusibile esponendoli ad una soluzione del 50% polietilenglicole 1000 (PEG 1000) in DMEM senza siero per 125 secondi a temperatura ambiente. </li><li> Lavare le cellule con PBS per rimuovere la soluzione di PEG 1000, e aggiungere 2 mL appena scaldato e media equilibrata. </li><li> Consentono alle cellule ad incubare per 18 - 24 ore. </li></ol> Microscopia a fluorescenza <ol><li> Rimuovere i terreni di coltura, e lavare le cellule due volte in PBS. </li><li> Fissare le cellule con l&#39;aggiunta di 1 ml di una soluzione di paraformaldeide al 4% (in PBS) in ogni pozzetto. Agitare delicatamente per 15 minuti. </li><li> Lavare le cellule fissate due volte in PBS. </li><li> Rimuovere con cautela il coperchio scivola dalla piastra, stoppino in eccesso PBS. Inverti sul microscopio diapositive caricato con una goccia di mezzo di montaggio (90% glicerolo, 100mM Tris pH 7.5, 2% DABCO (1,4-diazabicoyclo-ottano) contenente DAPI (4 &#39;,6-diamidino-2-fenilindolo). </li><li> Rimuovere l&#39;eccesso di mezzo di montaggio di diapositive, e sigillare cover scivola con smalto. </li><li> Visualizzare le cellule mediante microscopia confocale o epifluorescenza. </li></ol> 3. Rappresentante Risultati La Figura 2 mostra un esperimento heterokaryon esempio utilizzando fibroblasti primari e H1299 non a piccole cellule cellule di carcinoma del polmone. La proteina di interesse in questo esperimento (Anemia di pollo Virus-VP3) mostra la localizzazione citoplasmatica principalmente in tipi di cellule primarie e di localizzazione nucleare in tipi di cellule trasformate come visualizzato mediante microscopia in epifluorescenza. La proteina è espressa come fusione di due GFP (pEGFP-N1 vettore, Clontech) nei fibroblasti prepuzio primaria (PFF) o DsRed (DsRed-N1 vettore, Clontech) nelle cellule H1299. Campioni di controllo che coinvolge auto-fusioni (prime due righe) Display cellule multinucleate con nessun cambiamento nella steady-state modelli di localizzazione. Tali cambiamenti potrebbero altrimenti verificarsi a causa di sensibilità alle condizioni fusione o la formazione di sincizi. Fusioni Heterokaryon (riga in basso) dimostrano ingresso nucleare della GFP-fuso primario derivati ​​dalle cellule e proteine ​​colocalizzazione con la DsRed-proteina fusa in risposta alla introduzione di materiale cellula trasformata. L&#39;esempio mostrato è una fusione heterokaryon relativamente rara risultante da due sole cellule, uno di ogni tipo, come dimostra la presenza di entrambi i segnali fluoroprotein. Oltre a rilevare questi tipi di transizioni di localizzazione, nucleocytoplasmic attività spola possono essere facilmente individuati dalla presenza di un fluoroforo in questi due nuclei. Questo tipo di dosaggio è chiaro in sede di esame 01:01 la fusione di cellule e dipenderà dalla inibizione della traduzione. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Figura 1" /> Figura 1. Diagramma di flusso del metodo heterokaryon utilizzando fibroblasti primari e H1299 non a piccole cellule di carcinoma. Il gene di interesse viene clonato per produrre un telaio in fusione di uno dei due geni delle proteine ​​fluorescenti. Linee cellulari sono poi transitoriamente trasfettate sia con il permesso di costruire e recuperare. Formazione Heterokaryon è indotta dal trattamento breve con polietilene glicole (PEG), seguita da incubazione ulteriormente. Infine, la localizzazione sub-cellulare della proteina di interesse viene osservata utilizzando varie forme di microscopia a fluorescenza. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Figura 2" /> Figura 2. Traffico Nucleocytoplasmic e spola attività della proteina di pollo Anemia Virus VP3 visualizzati da fusione heterokaryon. Riga superiore: immagini rappresentative di auto-H1299 fuso cellule che esprimono DsRed-VP3 fila Oriente:.. Rappresentante immagini epifluorescenza di cellule primarie di fibroblasti prepuzio (PFF) che esprime GFP-VP3 dopo la fusione PEG fila in basso: la fusione Heterokaryon di TFP e H1299 cellule. Giallo indica colocalizzazione di GFP e segnali DsRed. Le cellule sono state ripreso usando una Leica AF6000E microscopio a fluorescenza Leica e software di imaging.

<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>

Nessun conflitto di interessi dichiarati.

Il protocollo heterokaryon qui presentato fornisce un metodo efficiente e di facile interpretazione per la caratterizzazione di cellule tipo-specifici comportamenti di localizzazione e nucleocytoplasmic attività spola. Questo metodo sfrutta la sensibilità di analisi di fluorescenza e la capacità di cellule immagine dal vivo e di eseguire studi di dinamica delle proteine. La tecnica si adatta facilmente a molti tipi di cellule diverse, ed è suscettibile di produrre immagini di cellule in vivo e fissati. Per esempio, invertito microscopia a fluorescenza può essere utilizzato per l&#39;imaging dal vivo e lapse video in tempo. Al contrario, le cellule montate possono essere utilizzate sia in epifluorescenza per la determinazione dello stato stazionario localizzazioni o più dettagliate di imaging confocale di localizzazioni discreti. Inoltre, le tecniche come il recupero di fluorescenza dopo photobleaching (FRAP), o la perdita di fluorescenza in photobleaching (FLIP) può essere utilizzato nella determinazione della cinetica di localizzazione 1. L&#39;incorporazione di fluorofori si alternano nel protocollo permetterebbe di esperimenti di interazione di proteine ​​come il trasferimento di energia di risonanza di fluorescenza (FRET) da svolgere in concerto 2. Vari inibitori del traffico di cellulari come B leptomycin o dominante negativo costruisce GTPasi Ran può essere utilizzato per stabilire la dipendenza da particolari importazioni o esportazioni percorsi 5,6,9. <ul><li> In certi esperimenti che coinvolgono la spola attività, bisogna fare attenzione per evitare gli effetti di localizzazione grazie alla sintesi di nuove proteine. In questi casi, gli inibitori della traduzione o punti di tempo limitato deve essere utilizzato. Tuttavia, artefatti a causa dell&#39;inibizione traslazionale rimanere una possibilità. La fusione di cellule contenenti una di ogni tipo cellulare sono più ambite per una chiara interpretazione della spola e del comportamento di localizzazione. Ottimizzazione del numero di cellule e le condizioni di fusione (tempi e concentrazione) può essere necessario per promuovere tale favorevole eventi di fusione 1:1. </li><li> E &#39;importante notare che l&#39;ottimizzazione di alcune fasi del protocollo sarà necessario a seconda delle particolari tipi di cellule usate. Aspetti della procedura sperimentale come l&#39;efficienza di fusione, l&#39;efficienza di trasfezione e vitalità dopo la fusione può variare notevolmente tra i tipi di cellule. In particolare, i tipi di cellule primarie può essere difficile da gestire a causa delle condizioni cultura complessa e bassa efficienza di trasfezione. Utilizzo di tecnologie come il dispositivo Lonza Nucleofector può permettere per l&#39;elaborazione rapida delle cellule così come l&#39;efficienza di transfezione aumentato drammaticamente. Inoltre, i metodi di elettroporazione consente un facile trasferimento di cellule in sospensione ai passi fusione che seguono. </li></ul>

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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>
</tr>
<tr>
<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>
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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>
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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 numero significativo di proteine ​​sono regolate dal traffico subcellulare o nucleocytolasmic spola. Queste proteine ​​visualizzare una gamma diversificata di funzioni cellulari incluse l&#39;importazione nucleare / esportazione di RNA e proteine, regolazione trascrizionale, e l&#39;apoptosi. È interessante notare che importanti riorganizzazioni cellulari tra cui la divisione cellulare, differenziamento e trasformazione, spesso comportano tali attività 3,4,8,10. Lo studio dettagliato di queste proteine ​​e rispettivi meccanismi normativi può essere impegnativo come la stimolazione di questi cambiamenti di localizzazione possono essere sfuggente, ei movimenti si può essere molto dinamici e difficili da monitorare. Studi che hanno coinvolto oncogenesi cellulare, ad esempio, continueranno a beneficiare di percorsi di comprensione e di attività delle proteine ​​che differiscono tra normali cellule primarie e cellule trasformate 6,7,11,12. Come molte proteine ​​mostrano la localizzazione alterato durante la trasformazione o come risultato della trasformazione, dei metodi per caratterizzare in modo efficiente queste proteine ​​e le vie a cui partecipano stand per migliorare la comprensione di oncogenesi e aprire nuovi settori di drug targeting. Qui vi presentiamo un metodo per l&#39;analisi del traffico di proteine ​​e attività spola tra le cellule di mammifero primarie e trasformate. Questo metodo combina la generazione di fusioni heterokaryon con microscopia a fluorescenza per fornire un protocollo flessibile che può essere utilizzato per rilevare localizzazioni proteine ​​allo stato stazionario o dinamico. Come mostrato nella Figura 1, due tipi di cellule separate sono transitoriamente transfettate con costrutti plasmide recante il gene fluoroprotein collegato al gene di interesse. Dopo l&#39;espressione, le cellule si fondono con glicole polietilenico, e localizzazioni delle proteine ​​possono poi essere ripreso con una varietà di metodi. Il protocollo presentato qui è un approccio fondamentale in cui le tecniche speciali, può essere aggiunto.

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 metodo flessibile ed efficiente per la caratterizzazione di cellule tipo-specifici di localizzazione di proteine ​​e nucleocytoplasmic spola è descritto. Questo approccio heterokaryon utilizza proteine ​​di fusione fluorescenza-etichettati in modo da localizzazioni proteina immagine dopo la fusione cellulare. Il protocollo è suscettibile di steady-state localizzazioni o più determinazioni dinamici basati sull&#39;imaging cellule vive.

Tecnica Heterokaryon per l&#39;analisi di cellule tipo-specifica localizzazione

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 Views.  Worcester Polytechnic Institute- WPI. Un metodo flessibile ed efficiente per la caratterizzazione di cellule tipo-specifici di localizzazione di proteine ​​e nucleocytoplasmic spola è descritto. Questo approccio heterokaryon utilizza proteine ​​di fusione fluorescenza-etichettati in modo da localizzazioni proteina immagine dopo la fusione cellulare. Il protocollo è suscettibile di steady-state localizzazioni o più determinazioni dinamici basati sull'imaging cellule vive.

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.

Un neuronale e astrociti Co-Cultura Assay per l&#39;analisi dei contenuti elevati di neurotossicità

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

Ricerca

Biologia

Visualizing RNA Localization in Xenopus Oocytes

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

Visualizing RNA Localization in Xenopus Oocytes

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

Visualizzazione RNA localizzazione in Xenopus Gli oociti

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.

Indagare tessuti e risposte fitocromo organo-specifici utilizzando FACS-Cell-assistita di tipo profili specifici di espressione in 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.

Microiniezione cella singola per l&#39;analisi cellulare comunicazione

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.

Tecniche di micromanipolazione che consente analisi delle dinamiche morfogenetiche e fatturato dei regolatori del citoscheletro

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: Cellula-tipo-specifico Epigenome profilatura

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.

Valutazione del nucleo di lenti zebrafish localizzazione e integrità 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.

Profiling dell'espressione genica specifica del tipo di cella nel fegato del mouse

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

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

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

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

Il metodo CryoAPEX per l'analisi della microscopia elettronica della localizzazione delle proteine membrana all'interno delle cellule ultrastrutturalizzate

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.

Analisi della α-Actinin Network in Cardiomiociti derivati da iPSC umani utilizzando la microscopia di localizzazione a singola molecola

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