Wir möchten die Worcester Polytechnic Institute Institut für Chemie und Biochemie für die Finanzierung danken.

1. Transfektion von Zelllinien Transfektion Methode 1 (Lonza Nucleofection für erhöhte primäre Zelle Transfektionseffizienz) <ol><li> Die Zellen sind von den letzten Durchgang und in der log-Phase Wachstum vor der Transfektion werden. Wash-Zellen in phophate Kochsalzlösung (PBS) und Ernte-Zellen durch Trypsinierung. </li><li> Sammeln Sie die Zellen durch Zentrifugation bei 1500 xg für 5 Minuten. </li><li> Add sterilisiert Deckgläser mit einem 6-Well-Platte. Deckgläser kann für eine verbesserte Zelladhäsion beschichtet werden. Legen Sie 1,5 ml Kulturmedium (in diesem Fall Dulbecco modifiziertem Eagle-Medium (DMEM), 10% fötalem Rinderserum (FBS)) in jede Vertiefung und Gleichgewicht der Medien in den Inkubator. </li><li> Ernte der Zellen durch Trypsinierung und resuspendieren in einem minimalen Volumen von Phosphat-gepufferter Kochsalzlösung (PBS). </li><li> Zählen Sie die Zellen und Zentrifuge die richtige Menge der Resuspension auf ~ 5x10 5 Zellen pro Probe zu erhalten. </li><li> Resuspendieren der Zellen vorsichtig in 100 ul Raumtemperatur Nucleofector Lösung pro Probe. </li><li> Kombinieren Sie 100 ul Zellsuspension mit 5 ug Plasmid-DNA und übertragen Sie die Probe auf eine Nucleofector Cuvette man aufpassen, nicht, um Luftblasen enthalten. </li><li> Wählen Sie das entsprechende Nucleofector Program (dies kann eine frühere Prüfung erforderlich, um eine optimale Transfektionseffizienz mit minimaler Mortalität zu etablieren). </li><li> Legen Sie die Küvette mit der Zelle / DNA-Suspension in den Nucleofector Küvettenhalter und beginnen das ausgewählte Programm. </li><li> Nach der Fertigstellung, entfernen Sie die Küvette und fügen ~ 500 ul der voräquilibriert Kulturmedien, um es (aus dem 6-Well-Platte). </li><li> Unmittelbar Übertragung der Probe in die 6-well-Platte mit Endvolumen von 1,5 ml pro Vertiefung. Die Zellen sind entweder in gemischten Populationen oder separat beschichtet werden, für die Kontrollen. </li></ol> Transfektion Methode 2 (Qiagen Effectene Transfektion für die Zell-Linien) <ol><li> Bereiten Transfektionskomplexe mit dem Qiagen Effectene Reagenz, indem man zunächst 0,8 ug jedes Plasmid-DNA-Probe zu 200 ul EC-Puffer. </li><li> Add 6,4 ul-Enhancer-Reagenz zu jeder Probe, kurz mischen Dazu den Schlauch, und inkubieren für 2 Minuten bei Raumtemperatur. </li><li> Add 20 uL Effectene Reagenz zu jeder Probe, mischen durch flicking das Rohr, und Inkubation für 15 Minuten bei Raumtemperatur. </li><li> Während dieser Inkubation, bereiten sich die beiden Zelllinien von Interesse für die Transfektion. Wash kürzlich passagiert Zellen (&lt;80% Konfluenz) einmal in Phosphat-gepufferter Kochsalzlösung (PBS). Fügen Sie wieder 1,5 ml frisch aufgewärmt und äquilibriert Wachstumsmedium (in diesem Fall Dulbecco modifiziertem Eagle-Medium (DMEM), 10% fötalem Rinderserum (FBS)). </li><li> Nach der Transfektion Komplexe für 15 Minuten inkubiert, Übertragung 1,2 ml Medium zu jeder Probe. Mix durch Pipettieren und sofort in die ~ 700 ul der Komplexe zu jedem der beiden Brunnen in der 6-Well-Platte. Fügen Sie tropfenweise zugeben und durch sanft durch Schwenken der Platte. Lassen Zellen für mindestens 3 Stunden transfizieren (kann mit Zelltyp variieren). </li><li> Add sterilisiert Deckgläser, um eine neue 6-well-Platte. Deckgläser kann für eine verbesserte Zelladhäsion beschichtet werden. </li><li> Nachdem die Zellen transfiziert wurden, waschen Sie die Zellen zweimal mit PBS und Ernte der Zellen durch minimale trysinization durch Zugabe von ca. 100 ul Trypsin-Lösung in jede Vertiefung, kurz Schütteln der Platte voll Fell jede Vertiefung, und sofort Absaugen der Trypsin. Sobald die Zellen angehoben haben, fügen Sie 1,5 ml frisches Medium. </li><li> An diesem Punkt sollten die Zellen entweder in gemischten oder getrennten Populationen (für Kontrollen) auf die sterile Deckgläser plattiert werden. </li><li> Lassen Zellen für 12 Stunden zu erholen. </li></ol> 2. Zellverschmelzung <ol><li> Entfernen Kulturmedien aus den Zellen und waschen zweimal mit PBS. </li><li> An dieser Stelle empfiehlt es sich, die Zellen mit Cycloheximid (100 ug / mL) zu behandeln, um die Proteinsynthese zu hemmen. Lassen Sie die Zellen für 2 Stunden inkubiert und dann waschen Sie die Zellen zweimal mit PBS. </li><li> Fuse-Zellen indem sie zu einer Lösung von 50% Polyethylenglykol 1000 (PEG 1000) in serumfreiem DMEM für 125 Sekunden bei Raumtemperatur. </li><li> Wash-Zellen mit PBS, die PEG 1000-Lösung zu entfernen, und fügen Sie 2 mL frisch aufgewärmt und äquilibriert Medien. </li><li> Lassen Zellen für 18 inkubieren - 24 Stunden. </li></ol> Fluoreszenz-Mikroskopie <ol><li> Entfernen Sie den Kulturmedien, und waschen Sie die Zellen zweimal in PBS. </li><li> Fix-Zellen durch Zugabe von 1 ml einer 4% Paraformaldehyd-Lösung (in PBS) in jede Vertiefung. Vorsichtig für 15 Minuten rühren. </li><li> Waschen Sie die fixierten Zellen zweimal in PBS. </li><li> Entfernen Sie vorsichtig die Deckgläser von der Platte, überschüssiges PBS Docht. Invert auf Objektträger geladen mit einem Tropfen Eindeckmedium (90% Glycerin, 100 mM Tris pH 7,5, 2% DABCO (1,4-diazabicoyclo-Oktan) mit DAPI (4 &#39;,6-Diamino-2-phenylindole). </li><li> Entfernen Sie überschüssiges Eindeckmedium vom Rutschen und Dichtung cover rutscht mit Nagellack. </li><li> Visualisieren Sie die Zellen über konfokale oder Epifluoreszenzmikroskopie. </li></ol> 3. Repräsentative Ergebnisse Abbildung 2 zeigt ein Beispiel heterokaryon Experiment mit primären Fibroblasten und H1299 nicht-kleinzelligem Lungenkarzinom-Zellen. Das Protein von Interesse in diesem Experiment (Chicken Anämie Virus-VP3) zeigt in erster Linie zytoplasmatische Lokalisation im primären Zelltypen und nukleare Lokalisation in transformierten Zelltypen wie visualisiert Epifluoreszenzmikroskopie. Das Protein wird als Fusionsprotein entweder GFP (pEGFP-N1-Vektor, Clontech) in primären Vorhaut-Fibroblasten (PFF) oder dsRed (dsRed-N1-Vektor, Clontech) in H1299-Zellen exprimiert. Kontrollproben mit Selbst-Fusionen (obere zwei Zeilen) angezeigt mehrkernige Zellen ohne Veränderung der Steady-State-Lokalisierungs-Muster. Solche Veränderungen könnten sonst entstehen durch Sensibilität für Fusion Bedingungen oder die Bildung von Syncytien. Heterokaryon Fusionen (untere Reihe) zeigen nuklearen Eingabe der GFP-fusionierten primäre cell-derived Protein und Kolokalisation mit dem dsRed-Fusionsprotein in Reaktion auf die Einführung von transformierten Zellmaterial. Das gezeigte Beispiel ist eine relativ seltene heterokaryon Fusion aus nur zwei Zellen, eine von jeder Art, wie durch die Anwesenheit der beiden Fluorprotein Signale gezeigt. Neben der Erkennung dieser Art von Lokalisierungs-Übergänge, kann nukleozytoplasmatischen pendelt Aktivität leicht durch die Anwesenheit eines einzigen Fluorophor in beide Kerne erkannt werden. Dieser Typ der Bestimmung ist klar, bei der Prüfung 01.01 Zelle Fusionen und würde auf der Hemmung der Translation abhängig. <img src="/files/ftp_upload/2488/2488fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Ablaufschema der heterokaryon Methode unter Verwendung von primären Fibroblasten und H1299 non-small cell carcinoma-Zellen. Das Gen von Interesse ist geklont, um eine in-frame Fusion zu einem von zwei fluoreszierende Protein-Gene zu erzeugen. Zelllinien werden dann vorübergehend mit entweder Konstrukt transfiziert und erlaubte sich zu erholen. Heterokaryon Bildung wird durch eine kurze Behandlung mit Polyethylenglykol (PEG) durch weitere Inkubation induziert. Schließlich ist die sub-zelluläre Lokalisation des Proteins von Interesse beobachtet mit verschiedenen Formen der Fluoreszenz-Mikroskopie. <img src="/files/ftp_upload/2488/2488fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Nukleozytoplasmatischen Menschenhandel und pendelt Aktivität der Hühner Anämie Virus VP3 Protein visualisiert heterokaryon Fusion. Obere Reihe: Repräsentative Bilder selbst verschmolzen H1299 Zellen, dsRed-VP3 Mittlere Reihe:.. Vertreter Epifluoreszenz Bilder von primären Vorhaut-Fibroblasten-Zellen (PFF), die GFP-VP3 nach PEG Fusion Untere Reihe: Heterokaryon Fusion von PFF und H1299 Zellen. Gelb bedeutet Kolokalisation von GFP und DsRed-Signale. Die Zellen wurden aufgenommen mit einem Leica AF6000E Fluoreszenzmikroskop Leica und Imaging-Software.

<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>

Die heterokaryon Protokoll hier vorgestellten bietet eine effiziente und leicht interpretierbare Methode für die Charakterisierung von Zelltyp-spezifische Lokalisation Verhalten sowie nukleozytoplasmatischen pendelt Aktivität. Diese Methode nutzt die Empfindlichkeit des Fluoreszenz-Analyse sowie die Fähigkeit, Bilder lebender Zellen und führen Studien von Protein Dynamik. Die Technik ist einfach für viele verschiedene Zelltypen angepasst und ist zugänglich sowohl live als auch feste Cell Imaging. Zum Beispiel kann invertiert Fluoreszenzmikroskopie für Live-Imaging-und Zeitraffer-Video-Capture verwendet werden. Im Gegensatz dazu kann montiert Zellen entweder Epifluoreszenzmikroskopie zur Bestimmung der steady-state Lokalisierungen oder detailliertere konfokale Bildgebung von diskreten Lokalisierungen verwendet werden. Darüber hinaus können Techniken wie die Fluoreszenz-Erholung nach Ausbleichen (FRAP), oder Fluoreszenz Verlust in photobleaching (FLIP) bei der Bestimmung der Lokalisierung Kinetik 1 verwendet werden. Die Einbindung von alternativen Fluorophoren in das Protokoll wäre für Protein-Wechselwirkung Experimente wie Fluorescence Resonance Energy Transfer (FRET) in concert 2 durchgeführt werden können. Verschiedene Inhibitoren von zellulären Menschenhandel wie Leptomycin B oder dominant negative Ran GTPase Konstrukte verwendet werden, um die Abhängigkeit von bestimmten Ein-oder Ausfuhr Wege 5,6,9 etablieren. <ul><li> In bestimmten Experimenten mit Pendeln Aktivität, muss darauf geachtet werden Lokalisierung Effekte durch die Synthese neuer Proteine ​​zu vermeiden. In diesen Fällen müssen Übersetzung Inhibitoren oder zeitlich begrenzt benutzt werden können. Dennoch bleiben Artefakte durch Inhibition der Translation eine Möglichkeit. Zellfusionen mit einer jeden Zelltyp sind die meisten für eine klare Interpretation der Shuttle-und Lokalisierungs-Verhalten gewünscht. Optimierung der Zellzahl und Fusion Bedingungen (Timing und Konzentration) kann es notwendig sein, um so günstige 1.01 Fusion Veranstaltungen zu fördern. </li><li> Es ist wichtig, dass die Optimierung bestimmter Schritte in das Protokoll wird notwendig sein, abhängig von der jeweiligen Zelltypen verwendet beachten. Aspekte in der experimentellen Verfahren wie Fusion Effizienz, Transfektionseffizienz und Lebensfähigkeit nach der Fusion kann stark variieren zwischen Zelltypen. Insbesondere können primäre Zelltypen eine Herausforderung für die Verwaltung aufgrund der komplexen Kultur und niedrige Transfektionseffizienz. Einsatz von Technologien wie der Lonza Nucleofector Gerät kann für die schnelle Verarbeitung von Zellen sowie drastisch erhöht Transfektionseffizienz zu ermöglichen. Darüber hinaus ermöglichen Elektroporation Methoden für die einfache Übertragung von Zellen in Suspension zur Fusion Schritte, die folgen. </li></ul>

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		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<td>Effectene reagent</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>301425</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>Dulbecco&#x2019;s modified Eagle&#x2019;s medium (DMEM)</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH30243FS</td>
<td>High glucose</td>
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<td>paraformaldehyde</td>
<td>&nbsp;</td>
<td>FisherChemical</td>
<td>O4042-500</td>
<td>&nbsp;</td>
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<td>4&#x2019;,6-diamidino-2-phenylindole (DAPI)</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D9542-10MG</td>
<td>&nbsp;</td>
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<td>Hyclone Serum (fetal bovine)</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>SH3008803HI</td>
<td>&nbsp;</td>
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<td>Falcon 6-well plates</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>08-772-1B</td>
<td>&nbsp;</td>
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<td>Polyethylene glycol 1000</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>528875-25GM</td>
<td>&nbsp;</td>
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<td>Cover slips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>12-545-85</td>
<td>&nbsp;</td>
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<td>DABCO</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>D2522-25G</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector HDF kit</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>VPD-1001</td>
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heterokaryon technique for analysis of cell type-specific localization

Eine beträchtliche Anzahl von Proteinen durch subzelluläre Menschenhandel oder nucleocytolasmic pendelt geregelt. Diese Proteine ​​zeigen eine Vielfalt von zellulären Funktionen, einschließlich nuklearer Import / Export von RNA und Protein, Regulation der Transkription und Apoptose. Interessanterweise wichtigen zellulären Reorganisationen einschließlich Zellteilung, Differenzierung und Transformation, oft mit solchen Aktivitäten 3,4,8,10. Die detaillierte Untersuchung dieser Proteine ​​und ihre jeweiligen Regulationsmechanismen kann als Anregung für diese Lokalisierung Veränderungen können schwer zu fassen, und die Bewegungen selbst recht dynamisch und schwer zu verfolgen, kann eine Herausforderung. Studien mit zellulären Onkogenese, zum Beispiel weiterhin von Verständnis Wege-und Protein-Aktivitäten, die zwischen normalen primären Zellen und transformierten Zellen 6,7,11,12 unterscheiden profitieren. Da viele Proteine ​​zeigen veränderte Lokalisation während der Transformation oder als Ergebnis der Transformation, Methoden zur effizienten Charakterisierung dieser Proteine ​​und die Wege, in denen sie sich beteiligen, stehen für das Verständnis der Onkogenese und neue Bereiche für Drug Targeting zu verbessern. Hier präsentieren wir eine Methode zur Analyse von Protein-Handel und pendelt Aktivität zwischen Primär-und transformierter Säugetierzellen. Diese Methode kombiniert die Erzeugung von heterokaryon Fusionen mit Fluoreszenzmikroskopie zu einem flexiblen Protokoll, mit dem steady-state oder dynamischen Protein-Lokalisierungen erkennen werden können. Wie in Abbildung 1 gezeigt, sind zwei getrennte Zelltypen transient mit Plasmid-Konstrukte die mit einem Fluorprotein Gens an das Gen von Interesse transfiziert. Nach Expression werden die Zellen verschmolzen unter Verwendung von Polyethylenglykol, und Protein-Lokalisation kann dann aufgenommen mit einer Vielzahl von Methoden. Das Protokoll hier vorgestellten ist ein grundlegendes Konzept, um die speziellen Techniken hinzugefügt werden kann.

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

Eine flexible und effiziente Verfahren zur Charakterisierung von Zelltyp-spezifische Protein-Lokalisierung und nukleozytoplasmatischen pendelt beschrieben. Dieser Ansatz nutzt heterokaryon fluoreszenzmarkierten Fusionsproteinen Bild Protein Lokalisierungen nach Zellfusion. Das Protokoll ist offen für Steady-State-Lokalisierungen oder mehrere dynamische Bestimmungen auf Live Cell Imaging basiert.

Heterokaryon Technik zur Analyse von Zelltyp-spezifischen Localization

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

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

Research

JoVE Journal

Biology

16.3K Aufrufe.  Worcester Polytechnic Institute- WPI. Eine flexible und effiziente Verfahren zur Charakterisierung von Zelltyp-spezifische Protein-Lokalisierung und nukleozytoplasmatischen pendelt beschrieben. Dieser Ansatz nutzt heterokaryon fluoreszenzmarkierten Fusionsproteinen Bild Protein Lokalisierungen nach Zellfusion. Das Protokoll ist offen für Steady-State-Lokalisierungen oder mehrere dynamische Bestimmungen auf Live Cell Imaging basiert.

Serie: 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.

Eine Neuronale und Astrocyte Co-Kultur-Assay für High Content Analyse der Neurotoxizität

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

Forschung

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.

Visualizing RNA Localization in Xenopus Oozyten

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.

Untersuchung Tissue-und Organ-spezifische Phytochrom Antworten mit FACS-gestützte zelltypspezifisch Expression Profiling 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.

Einzellige Mikroinjektions für Zellkommunikationsanalyse

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.

Mikromanipulation Techniken Analyse der morphogenetischen Dynamik und Umsatz des Zytoskeletts Regulatoren

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: Cell-Type-Specific Epigenom Profilierung

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.

Bewertung von Zebrafischen Lens Nukleus-Lokalisierung und Suturenintegrität

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.

Zelltypspezifische Gene Expression Profiling in der Mausleber

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.

Die CryoAPEX-Methode zur Elektronenmikroskopie-Analyse der Membranproteinlokalisierung in ultrastrukturell konservierten Zellen

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 des α-Actinin-Netzwerks in humanen iPSC-abgeleiteten Kardiomyozyten mittels Einzelmolekül-Lokalisierungsmikroskopie

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