PA ist ein Empfänger eines Stipendiums &quot;Pierre Durand&quot; von der Fakultät für Medizin der Universität Laval. Diese Arbeit wurde von der Natural Sciences and Engineering Research Council of Canada (MOP-CG095386) gestützt auf RM Die Polysom ​​Fraktionators wurde durch einen kanadischen Stiftung für Innovation Zuschuss (MOP-GF091050) bis RMR M hat einen neuen Ermittler CIHR Gehalts Auszeichnung erworben. Wir sind dankbar, dass DRS. E. Khandjian, I. Gallouzi, S. Di-Marco und A. Cammas für hilfreiche Ratschläge.

Das Protokoll folgt den Richtlinien von Laval Ethical Review Board genehmigt. 1. Herstellung von Zellkulturen und Gehirnmanipulation <ol><li> Säuger-und Drosophila-Zellen <ol><li> Wachsen HeLa-Gebärmutterhalskrebszellen und embryonale Zellen Drosophila Schneider wie von der American Type Culture Collection ist. Arbeiten mit Zellen auf einem niedrigen Gang. </li><li> Teller Zellen, um den Tag des Experiments 80% Konfluenz erreichen. Für beste Ergebnisse verwenden ~ 12 x 10 6 Zellen für jede Versuchsbedingung. Vor Extrakte für Polysom ​​Analyse, stimulieren Übersetzung durch Zugabe von frischem Vollmedium für mindestens 90 min. </li></ol></li><li> Gehirn-Trennung: Isolieren Gehirnen von Mäusen, zwischen 9 und 16 Tage nach der Geburt im Alter. Sterbehilfe beinhaltet CO 2-Inhalation nach der Narkose und Genickbruch. Nach der Tötung, zu isolieren, das ganze Gehirn und entweder den Ort, den icht bei -80 ° C oder direkt übertragen sie in kaltem PBS 1X für den sofortigen Einsatz. </li></ol> 2. Vorbereitung der Dichte Gradient-System Fraktionierung <ol><li> Waschen Sie das Schlauchsystem mit 0,1% SDS für 5 min. </li><li> Mit 70% Ethanol für 5 min Waschen Sie das Schlauchsystem, dann mit RNaseZAP Lösung für 5 min vor dem Waschen mit DEPC Wasser. </li><li> Luftpumpe, um das Rohrleitungssystem der Vorrichtung zu trocknen. </li></ol> 3. Vorbereitung der Saccharose-Gradienten <ol><li> Machen 15% und 55% Saccharose-Lösungen in 20 mM Tris-HCl pH 7,4, 1,25 mM MgCl 2, 150 mM NaCl und 1 mM DTT. </li><li> Erzeugen die lineare 15-55% Saccharose-Gradienten mit einem Isco Modell 160 Gradientenformer, wie von der Herstelleranleitung beschrieben. Es ist jedoch wichtig, um die Schaffung von Blasen oder Turbulenzen an den Steigungen zu vermeiden und steuern die TRIS Schlauchpumpe Geschwindigkeit. Halten Sie die Gradienten bei 4 ° C bis zur Verwendung. </li><li> Während der Zeit, wenn der Gradient Herstellerprogramm ausgeführt wird, kühlen die Ultrazentrifuge auf 4 ° C </li></ol> 4. Herstellung von Zell-und Maus-Gehirn-Extrakte <ol><li> Herstellung von Zellextrakten <ol><li> Platz Platte (n) auf Eis und Wasch Zellen 3x mit kaltem PBS 1X. </li><li> Ernten der Zellen (~ 12 × 10 6) in 1 ml Lysepuffer (20 mM Tris-HCl bei pH 7,4, 1,25 mM MgCl 2, 150 mM NaCl, 1 mM DTT, 1% Nonidet P40, 5 U / ml RNase-Inhibitor ergänzt mit kompletter Mini-EDTA-freie Protease-Inhibitor-Cocktail Tabletten), übertragen Sie sie auf einem Eppendorf-Röhrchen, und gut mischen, indem man sie durch ein 15x 1cc U100 Insulinspritze 28 G 1/2. Lassen Sie das Zelllysat ruhen auf Eis für 15 min. </li><li> Setzen einige Tropfen Zelllysats auf eine Folie, zelluläre Lyse durch Beobachtung unter einem Phasenkontrastmikroskop unter Verwendung eines 10X-Objektiv beurteilen. Nur Kerne sichtbar sein soll und keine Zellmembranen sichtbar Attesting für Zell-Lyse sein. Als Kontrolle beobachtenlysierten Zellen. </li><li> Klärung des Zelllysats mittels Zentrifugation bei 11.000 × g für 20 min bei 4 ° C und halten das lösliche Lysat mit den Polyribosome. </li></ol></li><li> Vorbereitung der Maus Gehirnextrakten <ol><li> Homogenisierung des gesamten Gehirns isoliert (~ 500 mg) durch 10 Hübe in einem eiskalten Dounce-Homogenisator mit 2 ml Lyse-Puffer und Klärung des Homogenats durch Zentrifugation bei 11.000 × g für 15 min bei 4 ° C. </li><li> Laden des resultierenden Überstands auf ein Kissen aus 50% Saccharose und füllen Sedimentation für 2 Stunden bei 200.000 × g unter Verwendung einer Ultrazentrifuge Rotor TH-641 bei 4 ° C. </li><li> Resuspendieren Sie das Pellet, das die schein Polyribosomen in 1 ml Lyse-Puffer und gut mischen durch Pipettieren von oben und unten. Lassen Sie die Suspension Rest auf dem Eis für 30 Minuten, bevor sie auf Steigungen. </li></ol></li></ol> 5. Einlegen der Auszüge auf Saccharose-Gradienten und Ultrazentrifugation <ol><li> Messen Sie die Konzentrationder in der cytoplasmatischen Extrakt mit einem Spektralphotometer vorhandenen RNA. 20 OD 260 Einheiten des Extraktes vorsichtig und langsam laden ~ auf die 15% bis 55% Saccharose-Gradienten. Stellen Sie sicher, dass es 2-3 mm der verfügbaren Raum an der Oberfläche des Rohres zu vermeiden, während der Zentrifugation Überlaufen der Rohre. </li><li> Zentrifugieren der Gradienten unter Verwendung einer Ultrazentrifuge für 2 h 30 min bei 230.000 x g unter Verwendung einer Ultrazentrifuge Rotor TH-641 bei 4 ° C. Beim Zentrifugieren endet, entfernen Sie die Rohre und legen sie auf Eis vorsichtig, nicht die Steigungen stören. </li></ol> 6. Fraktionierung von Cytoplasmic Extrakte für Polysomen Profiling <ol><li> Zeigen Saccharose-Gradienten auf automatischer Dichte Fraktionierung System und fahren Sie mit Fraktionierung und automatisierte Sammlung von Fraktionen (jeweils 0,5 ml), wie vom Hersteller beschrieben. </li><li> Sammeln jeder Fraktion (~ 500 &amp;mgr; l) in einzelne Eppendorf-Röhrchen mit kontinuierlicher Überwachung der Absorption bei 254 nm auf. In parallel Fraktionierung des Gradienten Betrieb wird der polyribosomal Profil auf dem Diagrammpapier bearbeiten werden. Alternativ können Sie einen Datenerfassungseinheit auf den Schreiber, eine elektronische Erfassung der Polysom ​​Profil befestigt. </li><li> Am Ende jeder Fahrt, Transfer gesammelt Eppendorfs auf Trockeneis. Zu diesem Zeitpunkt entweder Speicher gesammelten Fraktionen bei -80 ° C ausgefällt oder direkt Protein-RNA-Komplexe wie folgt. </li></ol> 7. Proteinextraktion und-analyse <ol><li> Zu jeder gesammelten Fraktion der Saccharose-Gradienten, fügen 2 Volumen 100% kaltem Ethanol und lassen RNA-Protein-Komplexe zu fällen bei -20 ° C über Nacht. </li><li> Zentrifuge jeder RNA-Protein-Niederschlag bei 11.000 × g für 20 min bei 4 ° C und dann Waschen mit 70% Ethanol. Trockene und resuspendieren den Niederschlag in der SDS-PAGE-Probenpuffer, bevor Sie fortfahren mit Western-Blot mit spezifischen Antikörpern (siehe Abbildung 2). </li></ol> 8. RNA-Extraktion und Analyse <ol><li> Man fällt RNA-Protein-Komplexe, wie in Abschnitt 7.1. Resuspendieren jeder RNA-Protein-Niederschlag in dem Lysepuffer, enthaltend 0,1% SDS. Verdauen die Proteinkomponente des Niederschlags mit 2 mg / ml Proteinase K für 30 Minuten in einem 55 ° C Wasserbad. </li><li> Extrahieren RNA-Komponenten des Niederschlags durch Zugabe von 1 Volumen &quot;Phenol: Chloroform&quot;, 2 Volumina Chloroform und 0,1 Volumen 2 M NaOAc pH 4 und Zentrifugieren bei 10.000 × g bei 4 ° C für 20 min. Ausfällung von RNA aus der resultierenden wässrigen Phase von jeder Probe über Nacht bei -20 ° C durch Zugabe von 1 Volumen Isopropanol und 0,2 ug / ul Glykogen. Spin der Niederschlag 1 Stunde lang bei 10.000 × g bei 4 ° C. Mit 70% kaltem Ethanol waschen das RNA-Pellet. </li><li> Die RNA-Pellet in einem kleinen Volumen RNAse freies Wasser. Bewerten Sie die Menge und Qualität von RNA mit dem Spektralphotometer. </li></ol>

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Die Autoren haben nichts zu offenbaren.

Die Polysomen-Profilanalyse auf Sucrosegradienten ermöglicht die Messung der Translationsinitiation durch Analysieren der Dichte von Polysomen aus Zellen oder Geweben isoliert 9,11-14. Diese Technik ist die besten (wenn nicht der einzige) Ansatz zur Initiation der Translation in vivo zu messen. Es wird verwendet, um den Status der Translations wachsenden Zellen während des Zellzyklus 15 zu überwachen, und um die Effekte von verschiedenen Arten von Stress, einschließlich viraler Infekti...

Eukaryotischen Zellen ständig stoßen eine Reihe von physiologischen und schädlichen Umweltstressbedingungen, die eine schnelle Reaktion erfordern adaptive Zelle. Zellstressantwort beinhaltet eine präzise Balance zwischen anti-und pro-Überleben Überleben wirkende Faktoren. Die Störung dieses Gleichgewichts kann irreversible Folgen führend in der Entwicklung der menschlichen Krankheiten wie Krebs und neurodegenerativen Krankheiten. Während der ersten Stufe der Reaktion auf Stress, Zellen zu aktivieren Proüberleben Wege, die die koordinierte Steuerung von Änderungen in der Genexpression auf der Ebene der mRNA-Translation beinhalten. mRNA-Translation in Eukaryonten ist ein komplexer Prozess, der koordinierte zelluläre Wechselwirkungen zwischen Translationsinitiationsfaktoren (eIFs), spezifische RNA-bindende Proteine ​​(FGE) und RNA-Moleküle 1 beinhaltet. Initiation, Elongation und Termination: mRNA-Translation ist in drei verschiedene Phasen unterteilt. Obwohl alle drei Phasen unterliegen regelRegelungsmechanismen, Mechanismen der Translationskontrolle zielen meist die Anfangsphase der Übersetzung, die auf diese Weise bildet die geschwindigkeitsbestimmende Schritt der Proteinsynthese 2. Translation Initiation ist eine hochgeordnete Prozess, der mit der Bildung der eIF2a.GTP.Met-tRNA i Met ternären Komplex und die anschließende Bindung an die 40S-Untereinheit Ribosomen, die zur Bildung des Präinitiationskomplexes beginnt. Der nächste Schritt ist die Einstellung der Präinitiationskomplexes an mRNA, die die Aktivität des Translationsinitiationsfaktoren wie eIF4F und eIF3 beinhaltet. Der so gebildete 48S Präinitiationskomplexes erfährt spezifische Konformationsänderungen, die diese Maschinen ermöglichen, scannen, die 5&#39;-untranslatierte Bereich der mRNA, bis er erkennt die Start-Codon August Die meisten der Translationsinitiationsfaktoren werden dann veröffentlicht und 60S-Untereinheiten werden rekrutiert, um eine 80S Ribosomen-Komplex bilden, der Übersetzung, eine kompetentet welchem ​​Punkt beginnt die Proteinsynthese (Abbildung 1). Mehr als ein 80S monosome kann die Übersetzung der mRNA in einer gleichen Zeit produziert so genannte Polysomen (oder Polyribosomen). Die Dichte der Polysomen auf einer mRNA spiegelt die Initiation, Elongation und Terminierungsentgelte und ist somit ein Maß für die Übersetzbarkeit einer bestimmten Transkript. Allerdings ist Polysom ​​Profil hauptsächlich verwendet, um Veränderungen in der mRNA-Translation bei der Einleitung Schritt zu beurteilen. Hier haben wir ein Proteasom-Inhibitor als Translationsinitiationsinhibitor verwendet. Die Behandlung von Krebszellen mit dieser Droge bewirkt eine Stressreaktion, gekennzeichnet durch die Aktivierung der Stresskinase HRI benannt, der die Translationsinitiationsfaktor eIF2a 3 phosphoryliert. Phosphorylierung von eIF2a ist eine der wichtigsten Ereignisse, die zur Inhibition der Translationsinitiation in Säugerzellen 4.

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			<td height="21" style="height:21px;width:279px;">Cells</td>
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			<td height="42" style="height:42px;width:279px;">HeLa cervical cancer cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">Schneider Drosophila embryonic cells</td>
			<td style="width:311px;">American Type Culture Collection (Manassas, VA; ATCC)</td>
			<td>CRL-1963</td>
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			<td></td>
			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
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			<td height="21" style="height:21px;width:279px;">Schneider&#8217;s Drosophila Medium</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>SO146-500ml</td>
			<td></td>
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			<td height="21" style="height:21px;width:279px;">DMEM</td>
			<td>Life technologies</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">FBS</td>
			<td>Fisher Scientist Scientist</td>
			<td>SH30396-03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">penicillin/streptomycin</td>
			<td>Life technologies</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Sucrose solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">D-Sucrose</td>
			<td>Fisher Scientist</td>
			<td>BP220-212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycerol</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>49767</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Blue Bromophenol</td>
			<td>Fisher Scientist</td>
			<td>B3925</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Lysis buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tris Hydrochloride</td>
			<td>Fisher Scientist</td>
			<td>BP153-500</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:279px;">MgCl2 </td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td style="width:119px;">M2670-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">NaCl</td>
			<td>Tekniscience</td>
			<td>3624-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DTT</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D 9779</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Nonidet P40 (Igepal CA-630 )</td>
			<td>MJS Biolynx</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">SDS</td>
			<td>Tekniscience</td>
			<td>4095-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">RNase inhibitor (RnaseOUT Recombinant Ribonuclease Inhibitor)</td>
			<td>Life technologies</td>
			<td>10777-019</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Antiproteases (complete, mini, EDTA free)</td>
			<td>Roche</td>
			<td>11,836,170,001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">RNA Extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteinase K</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM2542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Phenol: Chloroforme</td>
			<td>Fisher Scientist</td>
			<td style="width:119px;">BP1754I-400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Chloroforme</td>
			<td style="width:311px;">Fisher Scientist</td>
			<td style="width:119px;">C298-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Glycogen</td>
			<td>Life technologies</td>
			<td>10814-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isopropanol</td>
			<td style="width:311px;">Acros organics</td>
			<td>327270010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Antibodies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-FMRP antibody</td>
			<td colspan="2" style="width:429px;">Fournier et al., Cancer Cell International, 2010</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">anti-Ribosomal Protein L28 antibody</td>
			<td style="width:311px;">Santa Cruz Biotechnology, Inc.</td>
			<td style="width:119px;">SC-50362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Others</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Proteasome inhibitor : Bortezomib</td>
			<td style="width:311px;">LC Laboratories</td>
			<td style="width:119px;">B-1408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">DEPC (Diethylpyrocarbonate)</td>
			<td style="width:311px;">Sigma-Aldrich</td>
			<td>D5758-25ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">RNaseZAP Solution</td>
			<td>Life technologies</td>
			<td style="width:119px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="21">
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
			<td height="21" style="height:21px;width:279px;">Materials</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">T25 cell culture flask</td>
			<td style="width:311px;">Corning</td>
			<td style="width:119px;">430639</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">1cc U100 Insulin Syringe 28 G1/2</td>
			<td>Fisher Scientist</td>
			<td>148291B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Tube ultra-centrifugation, PA, 12ml</td>
			<td>Fisher Scientist</td>
			<td>FSSP9763205</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco Model 160 gradient former</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultracentrifuge Sorvall OTD Combi</td>
			<td style="width:311px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Thermo Scientific Sorvall Rotor TH-641</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;">54295</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Automated Density Fractionation System</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;">67-9000-177</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Isco UA-6 UV-vis detector</td>
			<td style="width:311px;">Teledyne Isco, Lincoln, NE, USA</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:279px;">NanoDrop 2000 UV-Vis Spectrophotometer</td>
			<td style="width:311px;">Thermo scientific</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Ultracentrifuge C 5415</td>
			<td style="width:311px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:279px;">Optical Microscope</td>
			<td style="width:311px;">Olympus CK2</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of translation initiation during stress conditions by polysome profiling

Präzise Steuerung der mRNA-Translation ist von grundlegender Bedeutung für die eukaryotische Zelle Homöostase, insbesondere in Antwort auf physiologische und pathologische Stress. Änderungen dieses Programms können das Wachstum von geschädigten Zellen, ein Markenzeichen der Krebsentwicklung oder zu vorzeitigem Zelltod wie bei neurodegenerativen Erkrankungen gesehen führen. Vieles von dem, was bekannt ist über die molekulare Grundlage für die Translationskontrolle ist von Polysomen-Analyse unter Verwendung eines Dichtegradienten Fraktionierung System erhalten. Diese Technik beruht auf der cytoplasmatischen Extrakte Ultrazentrifugation auf einem linearen Sucrose-Gradienten. Sobald der Spinn abgeschlossen ist, kann das System Fraktionierung und Quantifizierung zentrifugiert Zonen entsprechend unterschiedlichen Übersetzungs Ribosomen Populationen, wodurch eine Polysomen-Profil. Änderungen in der Polysomen-Profil sind, die Änderungen oder Fehler in Translationsinitiation, die in Reaktion auf verschiedene Arten von Stress auftreten. Diese Technik ermöglicht auch bewerten, the Rolle von spezifischen Proteinen auf Translations-Initiations-und translationale Aktivität bestimmter mRNAs zu messen. Hier beschreiben wir unser Protokoll zu Polysomprofile zur Translationsinitiation von eukaryotischen Zellen und Geweben, entweder unter normalen oder Stresswachstumsbedingungen durchführen zu beurteilen.

Wie zuvor erwähnt, ermöglicht die Polysomen Profil Analyse der Veränderungen der Translationsinitiation unter Stressbedingungen. Fig. 1 ist eine vereinfachte Ansicht, die die Translationsinitiation, wie zuvor beschrieben ist ein mehrstufiger Prozess, der eine geordnete Anordnung von Translationsinitiationskomplexen. Unter normalen Wachstumsbedingungen sind Translationsinitiationskomplexen in Polyribosome deren Erfassung durch Polysomen Profil zeugen für eine aktive Translationsinitiation (, unbehand...

Watch this Scientific Journal Video about Analysis of Translation Initiation During Stress Conditions by Polysome Profiling at JoVE.com

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Hier beschreiben wir ein Verfahren, um Veränderungen in der mRNA-Translationsinitiation eukaryontischer Zellen in Reaktion auf Stress-Bedingungen zu untersuchen. Dieses Verfahren basiert auf der Geschwindigkeit Trennung auf Saccharosegradienten übersetzen Ribosomen nicht übersetzen Ribosomen basiert.

Analyse der Translation Initiation Während Stress-Bedingungen durch Polysomprofile Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

analysis-translation-initiation-during-stress-conditions-polysome

Research

JoVE Journal

Biology

18.2K Aufrufe.  Laval University.  Hier beschreiben wir ein Verfahren, um Veränderungen in der mRNA-Translationsinitiation eukaryontischer Zellen in Reaktion auf Stress-Bedingungen zu untersuchen. Dieses Verfahren basiert auf der Geschwindigkeit Trennung auf Saccharosegradienten übersetzen Ribosomen nicht übersetzen Ribosomen basiert. 

Serie: Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Monitoring Plant Hormones During Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. Among the most severe stresses are insect herbivory, pathogen infection, and drought stress. For each of these stresses a specific set of hormones and/or combinations thereof are known to fine-tune the responses, thereby ensuring the plant's survival. The major hormones involved in the regulation of these responses are jasmonic acid (JA), salicylic acid (SA), and abscisic acid (ABA). To better understand the role of individual hormones as well as their potential interaction during these responses it is necessary to monitor changes in their abundance in a temporal as well as in a spatial fashion. For the easy, sensitive, and reproducible quantification of these and other signaling compounds we developed a method based on vapor phase extraction and gas chromatography/mass spectrometry (GC/MS) analysis (1, 2, 3, 4). After extracting these compounds from the plant tissue by acidic aqueous 1-propanol mixed with dichloromethane the carboxylic acid-containing compounds are methylated, volatilized under heat, and collected on a polymeric absorbent. After elution into a sample vial the analytes are separated by gas chromatography and detected by chemical ionization mass spectrometry. The use of appropriate internal standards then allows for the simple quantification by relating the peak areas of analyte and internal standard.

A simple method is provided that allows for the rapid extraction and analysis of multiple plant hormones from small tissue samples. The procedure uses vapor phase extraction as the solemn purification step. Samples are analyzed by GC/MS with chemical ionization that produces mainly (M+1)+ ions.

Überwachung Pflanzenhormone bei Stress Responses

Plant hormones and related signaling compounds play an important role in the regulation of plant responses to various environmental stimuli and stresses. ...

Forschung

Biologie

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikörper Profiling von Luciferase Immunpräzipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a &#8220;whole embryo ROS-detection method&#8221; for qualitative measurement of oxidative stress and ii) a &#8220;single-cell&#160;ROS detection method&#8221; for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.

Here we report a protocol to measure oxidative stress in living zebrafish embryos. This procedure allows reactive oxygen species (ROS) detection in both whole embryo tissues and single-cell populations. This protocol will accomplish both qualitative and quantitative analyses.

Analyse von oxidativem Stress in Zebrafischembryonen

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individuelle humanen embryonalen Stammzellen durch quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Polysomprofile Fraktionierung und Analyse von Säuger Translatomes auf einer Genom-weite Skala

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Bewertung der Selective mRNA Translation in Säugerzellen durch Polysomen Profilieren

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Fecal Glucocorticoid-Analyse: Nicht-invasive Monitoring-Adrenal in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Eine einfache Methode für Pflanzen Polysomen Profilieren

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analyse der Gap-Junction-abhängigen Übertragung von miRNA mit 3D-FRAP-Mikroskopie

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

In vitro Analyse der E3-Ubiquitin-Ligase-Funktion

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...