Die Autoren bedanken sich bei John Woolford für die erste Grundlage dieser Methode erkennen, ist TGK von NIH GM057483 und AI076245 unterstützt und PRC wird von GM77073 unterstützt.

1. Vorbereitung von 7-47% Saccharose-Gradienten <ol><li> Bereiten Saccharosegradienten 1 Tag vor der Verwendung zu ermöglichen Gradienten zu werden kontinuierlich. Machen Sie sterile 7% und 47% Saccharose-haltigen Lösungen 50mM NH 4 Cl, 50 mM Tris-Acetat pH 7,0 und 12 mm MgCl 2 und lagern bei 4 ° C 1. </li><li> Für 6 Gradienten-Mix von 7% und 47% Saccharose-Stammlösungen zu 48 mL von jeder der folgenden Saccharose-Konzentrationen zu machen. Add DTT (1M Lager) zu jedem Saccharose-Lösung auf eine Endkonzentration von 1 mM. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Endkonzentration </td><td> 7% Saccharose </td><td> 47% Saccharose </td></tr><tr><td> 7% </td><td> 48 ml </td><td> 0 </td></tr><tr><td> 17% </td><td> 36 ml </td><td> 12 mL </td></tr><tr><td> 27% </td><td> 24 mL </td><td> 24 mL </td></tr><tr><td> 37% </td><td> 12 mL </td><td> 36 ml </td></tr><tr><td> 47% </td><td> 0 </td><td> 48 ml </td></tr></tbody></table></li><li> Um den Gradienten zu gießen, legen einer Pasteurpipette zu einer 20 ml-Spritze durch die Sicherung und Abdichtung mit Parafilm oder verwenden Sie eine lange Nadel. 7 ml 7% Saccharose auf den Boden einer 1 x 3,5 &quot;Polyallomer Ultrazentrifuge Röhre. Nächste 7 ml 17% Saccharose-Lösung unter der 7%-Lösung, indem der Pasteur Pipette in der Nähe Boden des Röhrchens und Pipettieren nicht schneller als 0,3 ml / sec. Repeat mit 7 ml jeweils 27%, 37% und 47% Saccharose-Lösungen. Sie sollten beobachten, klare Linien zwischen den Schichten, was darauf hinweist, dass minimale Durchmischung hat. Shop Gradienten bei 4 ° C über Nacht aufgetreten zusammen mit den Rotoren, Flaschen und Röhren, die verwendet werden, um Proben der Ernte werden. </li></ol> 2. Vorbereitung der Hefe-Extrakt <ol><li> Wachsen Sie 125 ml Hefekultur auf eine OD 600 von 0,8-1,0. Add Cycloheximid, um die Kultur zu einer Endkonzentration von 100 ug / mL und weiter unter Schütteln bei 30 ° C für 15 min. </li><li> In der Zwischenzeit bereiten Lysepuffer mit 80 ug / ml Cycloheximid, 200 ug / mL Heparin, 0,2% DEPC und 10 mM Tris-HCl pH 7,5, 0,1 M NaCl, 30 mM MgCl 2. Halten Sie alles auf Eis von diesem Punkt an. </li><li> Gießen Sie die Kultur in einen 250 mL Zentrifuge Flasche füllen und mit Eis. Zentrifuge bei 8.000 xg (7.250 rpm in einem SLA-1500 Rotor) für 5 min bei 4 ° C. Waschen des Pellets einmal mit 10 ml eiskaltem Lysepuffer und Transfer bis 15 mL Polypropylen-Röhrchen. Zentrifuge bei 5.900 xg (6.000 rpm in einem SLA-600TC Rotor) für 3 min bei 4 ° C. Das Pellet in 0,5 ml Lysepuffer, Transfer zum 1,5 ml Röhrchen mit 0,5 ml gekühlt, gebacken acid-washed Glasperlen. Vortex die Zellen für vier 30 Sekunden-Intervallen, die Kühlung der Röhre auf Eis für 30 sec zwischen den einzelnen Intervallen. Fügen Sie ein weiteres 0,5 ml Lysepuffer in das 1,5-ml-Tube. Zentrifuge bei maximaler Geschwindigkeit in einer Mikrozentrifuge (14.000 rpm in einer Eppendorf 5417R Mikrozentrifuge) für 10 min bei 4 ° C. Überstand in ein neues 1,5 ml Reaktionsgefäß. Entfernen 10 pl in ein separates Röhrchen auf die OD 260 / OD 280-Verhältnis (siehe Schritt 3.1) zu bestimmen. Shop-Extrakte bei -80 ° C. </li></ol> 3. Aufbereitung von Extrakten aus Säugerzellen <ol><li> Für adhärente Zellen, in 100 mm-Schale auf ~ 70% Konfluenz wachsen. Sie benötigen einen 100 mm Teller pro 11 ml Gradienten (typische Ausbeute ist ~ 20 OD 260 Einheiten). Skalieren Sie die Menge linear für größere oder kleinere Steigung Größen. Für Nicht-adhärente Zellen sind etwa 1 x 10 7 Zellen pro 11 ml Gradienten benötigt. </li><li> Vor der Lyse, fügen Cycloheximid auf 100 pg / mL. Bei 37 ° C für 15 min. Transfer-Platten auf Eis und spülen Sie die Zellen zweimal in eiskaltem PBS. Alle künftigen Schritte müssen bei 0-4 ° C durchgeführt werden </li><li> Entfernen Sie alle Spuren von PBS durch Aspiration (let Platten auf einer abgewinkelten Bett aus Eis drain) und 1 ml Lysepuffer, kratzen und Transfer zum vorgekühlte 1,5 ml Tube. Lysepuffer Komponenten müssen auf den jeweiligen Zelltyp und Wachstumsbedingungen optimiert werden. Ein guter Ausgangspunkt wäre Puffer 20 mM HEPES-KOH, pH 7,4, 15 mM MgCl 2, 200 mM KCl, 1% Triton X-100 (v / v), 100 ug / ml Cycloheximid, 2 mM DTT und 1 mg / mL Heparin. Die wichtigsten Variablen sind die Konzentrationen von Magnesium und Kalium-Chlorid sowie die Aufnahme oder Ausschluss von nicht-ionischen Detergenzien zur Gewinnung von Zytoskelett-oder Membran-gebundenen Polysomen zu verbessern. Lyse-Zellen, indem sie durch 27-Gauge-Kanüle zweimal oder um 5-10 Schläge Dounce Homogenisierung mit Typ-B-Pistill. Die Spritze oder Homogenisator sollte vorgekühlte vor dem Gebrauch. </li><li> Spin bei 14.000 xg für 5 min in gekühlten Mikrozentrifugen. Transfer frischen Lysats auf die Saccharose-Gradienten (für Säugetierzellen ist dies bis in Lysepuffer fehlt Triton X-100 und Heparin gemacht, aber sonst wie in 1.2 und 1.3 beschrieben) oder Flash-Freeze in flüssigem Stickstoff für die spätere Verwendung. </li></ol&gt; 4. Zentrifugation von Gradienten <ol><li> Thaw polyribosome Lysate auf Eis. Bestimmen Sie die Konzentration der Nukleinsäure in das Lysat, indem Sie die reservierten 10 ul Probe in 1 ml Wasser und dem Lesen der OD 260 / OD 280-Verhältnis mit einem Spektralphotometer. Typische Ausbeute aus einer Hefe gezüchtet wie beschrieben beträgt 50 OD-Einheiten. </li><li> Mit einer Pipette mit der Spitze gegen die Wand der Röhre in der Nähe der Oberfläche des Gradienten, sanft Schicht 25 OD 260 Einheiten des Lysats auf der Oberseite des Gradienten gelegt. Für eine 35 ml Gradienten 500 pl Lysat wird in der Regel geladen. Lysepuffer verwendet, um sicherzustellen, alle Steigungen sind mit dem gleichen Volumen geladen werden. Geringere Mengen an Material liefern eine bessere Auflösung. Mit einer Pinzette vorsichtig niedriger die Gradienten in Eimern von einem Ausschwingrotor. </li><li> Centrifuge die Gradienten bei 100.000 xg (23.000 rpm in einem Surespin 630 Rotor) für 4 h bei 4 ° C. Dieses Protokoll kann für kleinere Volumen Gradienten angepasst werden. </li></ol> 5. Sammlung von Daten und Fraktionen <ol><li> Etwa 30 min vor der Fertigstellung der 4 Std. Spin bringen Sie die Nadel, um das Modell 184 Schlauch Piercer. Befestigen Sie den Stift, um die Online-Isco Modell UA-5 Extinktion / Fluoreszenz-Monitor. Schalten Sie den Monitor Absorption, damit eine stabile Basislinie vor der Analyse von Proben erreicht werden. Elektronische Erfassung der Polysom ​​Profil kann leicht durch die Anbringung eines DI-148U Datenerfassungseinheit (DATAQ Instruments), die Schreiber gewonnen werden. Profile können dann in Echtzeit gesammelt werden und für eine spätere Anmerkung mit dem mitgelieferten WinDaq Software. </li><li> Füllen Sie die Spritze Pumpe mit 50 ml Fluorinert mit dem Rücklauf, schnelle Einstellung. Stellen Sie sicher, dass es keine Luftblasen vorhanden. Schließen Sie den Schlauch von der Spritze, um das Rohr Piercer und kurz auf den Fluss der Fluorinert wiederum bei 3 ml / min in Fahrtrichtung, um den Schlauch von Luftblasen klar. </li><li> Legen Sie eine Verschwendung Becher oder eine Reihe von Röhren, wenn Sammeln von Fraktionen unter den Schlauch am Ende der Messzelle sammeln, um ablaufen. </li><li> Entfernen Sie vorsichtig die Polyallomer Zentrifugenröhrchen mit der Saccharose-Gradienten von Zentrifugenrotor und legen sie auf Eis (Vorform der Brunnen in das Eis mit einem Leerrohr zu stören Steigungen zu vermeiden). </li><li> Zur Analyse Zellextrakten, platzieren Sie den Gradienten Rohr auf dem Schlauch Piercer. Verbinden Sie das obere Ende des Rohres mit dem Auslass und sicher. Heben Sie den untersten Stufe der Zentrifugenröhrchen, so dass die Nadel den Boden des Röhrchens. Sobald die Nadel durch den unteren Rand des Zentrifugenröhrchen ragte hat, beginnen den Fluss der Fluorinert in Vorwärtsrichtung bei 6mL/min. </li><li> Sobald die Fluorinert wieder fließt in die Röhre, überwachen die Bewegung der Nadel auf dem Online-Absorption zu überwachen. Sobald die Nadel zu bewegen beginnt, schalten Sie auf dem Chart Bewegung zu einer Geschwindigkeit Einstellung von 60 Jahren. Der Monitor zeichnet die OD 254, beginnend mit dem freien Material von 40S ribosomalen Untereinheit Erkennung und Weiterbildung bis hin zu polyribosome Komplexe gefolgt. Wenn Sie Sammeln von Fraktionen für die RNA-oder Protein-Analyse sind, werden typischerweise 0,2 min Fraktionen genommen (1 mL). Für die RNA-Analyse sollte Fraktionen direkt in Röhrchen mit Trizol (Invitrogen), Phenol oder SDS / Proteinase K gesammelt werden, abhängig von der bevorzugten Methode der Extraktion. </li><li> Wenn das Ende des polyribosome Profil erreicht ist, schalten Sie den Strom von Fluorinert die Zentrifugenröhrchen und stoppen Sie die Tabelle Bewegung der Absorption / Fluoreszenz-Monitor. </li><li> Ziehen Sie die Fluorinert aus dem Zentrifugenröhrchen in die Spritze von Anfang an die Strömung in umgekehrter Richtung mit einer schnellen Rate darf dabei nicht Saccharose aus der Röhre zu ziehen in die Spritze. Lösen Sie die Zentrifugenröhrchen aus der Tube Piercer, senken Sie die Nadel, und entfernen Sie das Rohr. </li><li> Wiederholen Sie die Schritte 5.5 bis 5.8 für jede Sucrosegradienten. </li><li> Wenn alle Saccharosegradienten analysiert wurden, einfahren Fluorinert aus dem Schlauch zurück in die Spritze. Nehmen Sie jedes Teil Fraktionator (Rohr Piercer und Durchflusszelle) einschließlich der Ablaufschlauch an der Spitze gelegen und sofort mit Wasser. </li></ol> 6. Repräsentative Ergebnisse <img src="/files/ftp_upload/1948/1948fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Representative Spur von Ribosomen Auszug aus Hefestamm BY4741 (Matte eine his3 Δ 1 leu2 Δ 0 met15 Δ 0 ura3 Δ 0) in Gegenwart von Cycloheximid vorbereitet. Die ribosomale Extrakt wurde fraktioniert unter Verwendung eines 7 47% Saccharose-Gradienten und analysiert mit Hilfe einer Pumpe Spritze Apparat an einem UV-Detektor. Peaks mit Polyribosome und 80S, 60S und 40S Ribosomen sind angegeben.

<ol>
	<li>Baim, S. B., Pietras, D. F., Eustice, D. C., Sherman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mutation+allowing+an+mRNA+secondary+structure+diminishes+translation+of+Saccharomyces+cerevisiae+iso-1-cytochrome+C.">A mutation allowing an mRNA secondary structure diminishes translation of Saccharomyces cerevisiae iso-1-cytochrome C.</a> Mol. Cell. Biol. 5, 1839-1846 (1985).</li><li>Ripmaster, T. L., Vaughn, G. P., Woolford, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DRS1+to+DRS7,+novel+genes+required+for+ribosome+assembly+and+function+in+Saccharomyces+cerevisiae.">DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.</a> Mol. Cell. Biol. 13, 7901-7912 (1993).</li><li>Anand, M., Chakraburtty, K., Marton, M. J., Hinnebusch, A. G., Kinzy, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+interactions+between+yeast+translation+eukaryotic+elongation+factor+(eEF)+1A+and+eEF3.">Functional interactions between yeast translation eukaryotic elongation factor (eEF) 1A and eEF3.</a> J Biol Chem. 278, 6985-6991 (2003).</li><li>Serikawa, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transcriptome+and+its+translation+during+recovery+from+cell+cycle+arrest+in+Saccharomyces+cerevisiae.">The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.</a> Mol Cell Proteomics. 2, 191-204 (2003).</li><li>Ingolia, N. T., Ghaemmaghami, S., Newman, J. R., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+in+vivo+of+translation+with+nucleotide+resolution+using+ribosome+profiling.">Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome profiling.</a> Science. 324, 218-223 (2009).</li><li>Valasek, L., Szamecz, B., Hinnebusch, A. G., Nielsen, K. H. <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+stabilization+of+preinitiation+complexes+by+formaldehyde+cross-linking.">In vivo stabilization of preinitiation complexes by formaldehyde cross-linking.</a> Methods Enzymol. 429, 163-183 (2007).</li><li>Fletcher, J. E., Copeland, P. R., Driscoll, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polysome+distribution+of+phospholipid+hydroperoxide+glutathione+peroxidase+mRNA:+evidence+for+a+block+in+elongation+at+the+UGA/selenocysteine+codon.">Polysome distribution of phospholipid hydroperoxide glutathione peroxidase mRNA: evidence for a block in elongation at the UGA/selenocysteine codon.</a> Rna. 6, 1573-1584 (2000).</li><li>Ruan, H. J., Brown, C. Y., Morris, D. R., Richter, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Analysis of mRNA formation and function. , 305-321 (1997).</li><li>Martin, G. W., Berry, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selenocysteine+codons+decrease+polysome+association+on+endogenous+selenoprotein+mRNAs.">Selenocysteine codons decrease polysome association on endogenous selenoprotein mRNAs.</a> Genes Cells. 6, 121-129 (2001).</li><li>Lee, Y. Y., Cevallos, R. C., Jan, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+upstream+open+reading+frame+regulates+translation+of+GADD34+during+cellular+stresses+that+induce+eIF2alpha+phosphorylation.">An upstream open reading frame regulates translation of GADD34 during cellular stresses that induce eIF2alpha phosphorylation.</a> J Biol Chem. 284, 6661-6673 (2009).</li></ol>

Die Informationen aus dem polyribosome Profil erhalten wertvolle Einblicke in die translatorische Status der Zelle liefern. Darüber hinaus kann der Status der Montage der ribosomalen Untereinheiten sich 2 bestimmt werden. Zum Beispiel das Vorhandensein von halfmers oder 80S und größere Peaks mit einer leichten Schulter nach rechts auf das Profil zeigt eine gebundene 40S-Untereinheit erwartet 60S-Untereinheit Beitritt. Durchführung des Experiments in das Fehlen jeglicher hinzugefügt Cycloheximid oder andere Hemmer der Dehnung erlaubt die Analyse der Geschwindigkeit der ablaufen, die, ob Dehnung 3 ist verändert zeigt. Die Fraktionen selbst sind eine reiche Quelle von Reagenzien für die anschließende Bestimmung der Assoziation eines spezifischen mRNA oder Protein mit ribosomalen Subpopulationen von Nord-oder Western-Blot der Fraktionen. Die Gesamtanzahl der mRNAs mit aktiven Ribosomen verbunden sind, können über eine zugehörige Microarray-4 oder tiefer Sequenzierung 5 ermittelt werden. Die Protein-Assoziationen können auch durch die entsprechende Zugabe einer Vernetzung Reagens 6 stabilisiert werden. Polyribosome Analyse von Säugetierzellen ist auch erwähnenswert, als eine eigene Protokoll in Bezug auf die offensichtlichen Schwierigkeiten bei der Festlegung der Voraussetzungen für eine stabile Polysomen zu erhalten. Der Puffer-Systeme in der Literatur berichtet werden häufig variable 7-10, dadurch kann es zu einer Voraussetzung für die Zelltyp-spezifische Optimierung der Lyse-Puffer sein, oder es ist ebenso plausibel, dass die Puffer-System ist nicht so wichtig, wie viel Liebe zum Umgang mit frische Lysaten gehalten bei niedrigen Temperaturen. Nach unserem Kenntnisstand eine systematische Auswertung der Parameter, die von Säugetieren Polysom ​​Bildung wurde nicht berichtet.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>polyallomer ultracentrifuge tube</td>
<td>&nbsp;</td>
<td>Beckman</td>
<td>326823</td>
<td>1 x 3.5 in. (25x89mm)</td>
</tr>
<tr>
<td>Fluorinert</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>F9755</td>
<td>&nbsp;</td>
<tr>
<td>Cycloheximde</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C-7698</td>
<td>&nbsp;</td>
<tr>
<td>Heparin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3393</td>
<td>&nbsp;</td>
<tr>
<td>BY4741</td>
<td>&nbsp;</td>
<td>Open Biosystems</td>
<td>YSC1048</td>
<td>Other strains work equally well</td>
</tr>		</tbody>
		</table>
		</div>

eukaryotic polyribosome profile analysis

Protein-Synthese ist ein komplexer zellulärer Prozess, der auf vielen Ebenen reguliert wird. Zum Beispiel kann global Übersetzung in der Anfangsphase oder die Dehnung Phase durch eine Vielzahl von zellulären Belastungen wie Aminosäure Hunger oder Wachstumsfaktorentzug gehemmt werden. Alternativ kann die Übersetzung der einzelnen mRNAs von mRNA Lokalisierung oder das Vorhandensein von verwandten microRNAs reguliert werden. Studien der Proteinsynthese häufig nutzen polyribosome Analyse, um Licht auf die Mechanismen der Übersetzung Regulierung oder Mängel bei der Proteinsynthese zu vergießen. In diesem Test werden mRNA / Ribosom-Komplexen aus eukaryotischen Zellen isoliert. Eine Saccharose-Dichtegradienten trennt mRNAs gebunden an mehrere Ribosomen als Polyribosomen von mRNAs gebunden an ein einzelnes Ribosom oder monosome bekannt. Die Fraktionierung des Gradienten erlaubt die Isolierung und Quantifizierung der verschiedenen ribosomalen Bevölkerung und die damit verbundenen mRNAs oder Proteine. Unterschiede im Verhältnis von Polyribosomen zu monosomes unter definierten Bedingungen kann ein Hinweis auf Mängel in entweder Übersetzung Einleitung oder Verlängerung / Kündigung. Die Untersuchung der mRNAs in der polyribosome Fraktionen können zeigen, ob die Kohorte der einzelnen mRNAs wird Änderungen mit experimentellen Bedingungen übersetzt. Darüber hinaus können Ribosom Montage durch eine Analyse der kleinen und großen ribosomalen Untereinheit Peaks, die auch durch den Gradienten getrennt überwacht werden. In diesem Video stellen wir eine Methode zur Herstellung von Roh-ribosomalen Extrakte aus Hefezellen, die Trennung des Extrakts durch Saccharose-Gradienten und Interpretation der Ergebnisse. Dieses Verfahren ist leicht an Säugetierzellen.

Watch this Scientific Journal Video about Eukaryotic Polyribosome Profile Analysis at JoVE.com

Eukaryotic Polyribosome Profile Analysis

Dieser Artikel beschreibt ein Protokoll für die Extraktion des Übersetzens Ribosomen aus eukaryotischen Zellen. Sobald extrahiert, Ribosomen in monosomes und Polyribosomen durch Saccharose-Gradienten Fraktionierung getrennt, um verschiedene ribosomale Populationen analysiert werden. Als solche ist diese Methode der Goldstandard für die Untersuchung der Regulation der Translation.

Eukaryotische Polyribosome Profile Analysis

Protein synthesis is a complex cellular process that is regulated at many levels. For example, global translation can be inhibited at the initiation phase ...

Research

JoVE Journal

Biology

52.4K Aufrufe.  University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School. Dieser Artikel beschreibt ein Protokoll für die Extraktion des Übersetzens Ribosomen aus eukaryotischen Zellen. Sobald extrahiert, Ribosomen in monosomes und Polyribosomen durch Saccharose-Gradienten Fraktionierung getrennt, um verschiedene ribosomale Populationen analysiert werden. Als solche ist diese Methode der Goldstandard für die Untersuchung der Regulation der Translation.

Serie: Eukaryotic Polyribosome Profile Analysis

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

Bioenergetic Profile Experiment using C2C12 Myoblast Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in understanding the role of mitochondrial function in obesity, diabetes, aging, cancer, cardiovascular function and safety toxicity.
Cellular metabolism is the process of substrate uptake, such as oxygen, glucose, fatty acids, and glutamine, and subsequent energy conversion through a series of enzymatically controlled oxidation and reduction reactions. These intracellular biochemical reactions result in the production of ATP, the release of heat and chemical byproducts, such as lactate and CO2 into the extracellular environment.
Valuable insight into the physiological state of cells, and the alteration of the state of those cells, can be gained through measuring the rate of oxygen consumed by the cells, an indicator of mitochondrial respiration - the Oxygen Consumption Rate - or OCR. Cells also generate ATP through glycolysis, i.e.: the conversion of glucose to lactate, independent of oxygen. In cultured wells, lactate is the primary source of protons. Measuring the lactic acid produced indirectly via protons released into the extracellular medium surrounding the cells, which causes acidification of the medium provides the Extra-Cellular Acidification Rate - or ECAR.
In this experiment, C2C12 myoblast cells are seeded at a given density in Seahorse cell culture plates. The basal oxygen consumption (OCR) and extracellular acidification (ECAR) rates are measured to establish baseline rates. The cells are then metabolically perturbed by three additions of different compounds (in succession) that shift the bioenergetic profile of the cell.
This assay is derived from a classic experiment to assess mitochondria and serves as a framework with which to build more complex experiments aimed at understanding both physiologic and pathophysiologic function of mitochondria and to predict the ability of cells to respond to stress and/or insults.

A description of a method for profiling mitochondrial function in cells is provided. The mitochondrial profile generated provides four parameters of mitochondrial function that can be measured in one experiment: basal respiration rate, ATP-linked respiration, proton leak, and reserve capacity.

Bioenergetische Profil Experiment mit C2C12 Myoblasten Cells

The ability to measure cellular metabolism and understand mitochondrial dysfunction, has enabled scientists worldwide to advance their research in ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.

Korrelative Licht-und Elektronenmikroskopie (CLEM) als Werkzeug zur Visualisierung mikroinjiziert Moleküle und ihre Eukaryotic Sub-zellulären Targets


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Establishment of Microbial Eukaryotic Enrichment Cultures from a Chemically Stratified Antarctic Lake and Assessment of Carbon Fixation Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a chemically stratified water column and unlike other inland bodies of water, largely prevents external input of carbon and nutrients from streams. Biota are exposed to numerous environmental stresses, including year-round severe nutrient deficiency, low temperatures, extreme shade, hypersalinity, and 24-hour darkness during the winter 1. These extreme environmental conditions limit the biota in Lake Bonney almost exclusively to microorganisms 2.
Single-celled microbial eukaryotes (called "protists") are important players in global biogeochemical cycling 3 and play important ecological roles in the cycling of carbon in the dry valley lakes, occupying both primary and tertiary roles in the aquatic food web. In the dry valley aquatic food web, protists that fix inorganic carbon (autotrophy) are the major producers of organic carbon for organotrophic organisms 4, 2. Phagotrophic or heterotrophic protists capable of ingesting bacteria and smaller protists act as the top predators in the food web 5. Last, an unknown proportion of the protist population is capable of combined mixotrophic metabolism 6, 7. Mixotrophy in protists involves the ability to combine photosynthetic capability with phagotrophic ingestion of prey microorganisms. This form of mixotrophy differs from mixotrophic metabolism in bacterial species, which generally involves uptake dissolved carbon molecules. There are currently very few protist isolates from permanently ice-capped polar lakes, and studies of protist diversity and ecology in this extreme environment have been limited 8, 4, 9, 10, 5. A better understanding of protist metabolic versatility in the simple dry valley lake food web will aid in the development of models for the role of protists in the global carbon cycle.
We employed an enrichment culture approach to isolate potentially phototrophic and mixotrophic protists from Lake Bonney. Sampling depths in the water column were chosen based on the location of primary production maxima and protist phylogenetic diversity 4, 11, as well as variability in major abiotic factors affecting protist trophic modes: shallow sampling depths are limited for major nutrients, while deeper sampling depths are limited by light availability. In addition, lake water samples were supplemented with multiple types of growth media to promote the growth of a variety of phototrophic organisms.
RubisCO catalyzes the rate limiting step in the Calvin Benson Bassham (CBB) cycle, the major pathway by which autotrophic organisms fix inorganic carbon and provide organic carbon for higher trophic levels in aquatic and terrestrial food webs 12. In this study, we applied a radioisotope assay modified for filtered samples 13 to monitor maximum carboxylase activity as a proxy for carbon fixation potential and metabolic versatility in the Lake Bonney enrichment cultures.

Microbial eukaryotes are both a source of photosynthetically-derived carbon and top predatory species in permanently ice-covered Antarctic lakes. This report describes an enrichment culture approach to isolate metabolically versatile microbial eukaryotes from the Antarctic lake, Lake Bonney, and assesses inorganic carbon fixation potential using a radioisotope assay for Ribulose-1,5-bisphophate carboxylase oxygenase (RubisCO) activity.

Establishment of Microbial Eukaryotische Anreicherungskulturen aus einer chemisch geschichteten antarktischen See und Bewertung der Kohlenstoff-Fixierung Potential

Lake Bonney is one of numerous permanently ice-covered lakes located in the McMurdo Dry Valleys, Antarctica. The perennial ice cover maintains a ...

Metabolic Profile Analysis of Zebrafish Embryos

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these relationships will help to understand the underlying etiology for a range of diseases linked with mitochondrial dysfunction, such as diabetes and obesity. Recent advances in instrumentation, has enabled the monitoring of distinct parameters of mitochondrial function in cell lines or tissue explants. Here we present a method for a rapid and sensitive analysis of mitochondrial function parameters in vivo during zebrafish embryonic development using the Seahorse bioscience XF 24 extracellular flux analyser. This protocol utilizes the Islet Capture microplates where a single embryo is placed in each well, allowing measurement of bioenergetics, including: (i) basal respiration; (ii) basal mitochondrial respiration (iii) mitochondrial respiration due to ATP turnover; (iv) mitochondrial uncoupled respiration or proton leak and (iv) maximum respiration. Using this approach embryonic zebrafish respiration parameters can be compared between wild type and genetically altered embryos (mutant, gene over-expression or gene knockdown) or those manipulated pharmacologically. It is anticipated that dissemination of this protocol will provide researchers with new tools to analyse the genetic basis of metabolic disorders in vivo in this relevant vertebrate animal model.

Zebrafish represent a powerful vertebrate model that has been under-utilised for metabolic studies. Here we describe a rapid way to measure the in vivo metabolic profile of developing zebrafish that allows the comparison of different mitochondrial function parameters between genetically or pharmacologically manipulated embryos, thereby increasing the applicability of this organism.

Metabolic-Profil-Analyse von Zebrafischembryonen

A growing goal in the field of metabolism is to determine the impact of genetics on different aspects of mitochondrial function. Understanding these ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Echtzeit-Analyse von Stoffwechselprofil in<em&gt; Ex Vivo</em&gt; Maus Intestinale Crypt Organoide Kulturen

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

Genome-wide Purification of Extrachromosomal Circular DNA from Eukaryotic Cells

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs contribute to genetic variation among somatic cells in multicellular organisms and to evolution of unicellular eukaryotes. Sensitive methods for detecting eccDNA are needed to clarify how these elements affect genome stability and how environmental and biological factors&#160;induce their formation in eukaryotic cells. This video presents a sensitive eccDNA-purification method called Circle-Seq. The method encompasses column purification of circular DNA, removal of remaining linear chromosomal DNA, rolling-circle amplification of eccDNA, deep sequencing, and mapping. Extensive exonuclease treatment&#160;was&#160;required for sufficient linear chromosomal DNA degradation. The rolling-circle amplification step by &#966;29 polymerase enriched for circular DNA over linear DNA. Validation of the Circle-Seq method on three S. cerevisiae CEN.PK populations of 1010 cells detected hundreds of eccDNA profiles in sizes larger than 1 kilobase. Repeated findings of ASP3-1, COS111, CUP1, RSC30,&#160;HXT6, HXT7 genes on circular DNA in both S288c and CEN.PK suggests that DNA circularization is conserved between strains at these loci. In sum, the Circle-Seq method has broad applicability for genome-scale screening for eccDNA in eukaryotes as well as for detecting specific eccDNA types.

This paper presents a sensitive method called Circle-Seq for purifying extrachromosomal circular DNA (eccDNA). The method encompasses column purification, removal of remaining linear chromosomal DNA, rolling-circle amplification and high-throughput sequencing. Circle-Seq is applicable to genome-scale screening of eukaryotic eccDNA and studying genome instability and copy-number variation.

Genomweite Reinigung von extrachromosomale zirkuläre DNA aus eukaryotischen Zellen

Extrachromosomal circular DNAs (eccDNAs) are common genetic elements in Saccharomyces cerevisiae and are reported in other eukaryotes as well. EccDNAs ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

Laborprotokoll für genetische Darm Inhaltsanalysen der aquatischen Makroinvertebraten Using gruppenspezifische rDNA Primer

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to cells, tissues, organelles, and even whole organisms. This protocol focuses on two chemical drying methods, hexamethyldisilazane (HMDS) and t-butyl alcohol (TBA), and their application to imaging of both prokaryotic and eukaryotic organisms using SEM. In this article, we describe how to fix, wash, dehydrate, dry, mount, sputter coat, and image three types of organisms: cyanobacteria (Toxifilum mysidocida, Golenkina sp., and an unknown sp.), two euglenoids from the genus Monomorphina (M. aenigmatica and M. pseudopyrum), and the fruit fly (Drosophila melanogaster). The purpose of this protocol is to describe a fast, inexpensive, and simple method to obtain detailed information about the structure, size, and surface characteristics of specimens that can be broadly applied to a large range of organisms for morphological assessment. Successful completion of this protocol will allow others to use SEM to visualize samples by applying these techniques to their system.

Preparation of Prokaryotic and Eukaryotic Organisms Using Chemical Drying for Morphological Analysis in Scanning Electron Microscopy SEM

SEM analysis is an effective method to aid in species identification or phenotypic discrimination. This protocol describes the methods for examining specific morphological details of three representative types of organisms and would be broadly applicable to examining features of many organismal and tissue types.

Vorbereitung der prokaryotischen und eukaryotischen Organismen mit chemischen Trocknung für die morphologische Analyse in Rasterelektronenmikroskopie (SEM)

Scanning electron microscopy (SEM) is a widely available technique that has been applied to study biological specimens ranging from individual proteins to ...

Image Rendering Techniques in Postmortem Computed Tomography: Evaluation of Biological Health and Profile in Stranded Cetaceans

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.

The Hong Kong cetacean stranding response program has incorporated postmortem computed tomography, which provides valuable information on the biological health and profile of the deceased animals. This study describes 8 image rendering techniques that are essential for the identification and visualization of postmortem findings in stranded cetaceans, which will help clinicians, veterinarians and stranding response personnel worldwide to fully utilize the radiological modality.

Bild-Rendering-Techniken in der postmortalen Computertomographie: Bewertung der biologischen Gesundheit und des Profils bei gestrandeten Walen

With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, ...