PA est récipiendaire d&#39;une bourse «Pierre Durand» de la Faculté de médecine de l&#39;Université Laval. Ce travail a été soutenu par le Conseil de recherches en génie du Canada (MOP-CG095386) en sciences naturelles et de RM Le fractionnement de polysome a été acquis par une Fondation canadienne pour l&#39;octroi de l&#39;innovation (MOP-GF091050) à RMR M est titulaire d&#39;une bourse de nouveau chercheur des salaires des IRSC. Nous sommes reconnaissants à MM. E. Khandjian, I. Gallouzi, S. Di-Marco et A. Cammas pour obtenir des conseils utiles.

Le protocole suit les lignes directrices approuvé par la commission d&#39;examen éthique de l&#39;Université Laval. 1. Préparation des cultures de cellules et de manipulation du cerveau <ol><li> Les cellules de mammifères et de Drosophila <ol><li> Cultiver des cellules HeLa du cancer du col et les cellules embryonnaires Schneider drosophile comme recommandé par l&#39;American Type Culture Collection. Travailler avec des cellules à un passage bas. </li><li> Cellules de la plaque afin d&#39;atteindre 80% de confluence le jour de l&#39;expérience. Pour de meilleurs résultats, utilisez ~ 12 x 10 6 cellules pour chaque condition expérimentale. Avant de faire des extraits pour l&#39;analyse de polysomes, stimuler traduction en ajoutant un milieu complet frais pendant au moins 90 min. </li></ol></li><li> l&#39;isolement du cerveau: Isoler les cerveaux de souris âgées entre 9 et 16 jours après la naissance. L&#39;euthanasie consiste inhalation de CO 2 après anesthésie et dislocation cervicale. Après sacrifice, isoler l&#39;ensemble du cerveau et deux endroits it à -80 ° C ou transférer, directement dans PBS froid 1X pour une utilisation immédiate. </li></ol> 2. Préparation de la densité du système de fractionnement de gradient <ol><li> Laver le système de tube avec du SDS 0,1% pendant 5 min. </li><li> Laver le système de tuyauterie avec 70% d&#39;éthanol pendant 5 minutes, puis avec RNaseZAP solution pendant 5 minutes avant de le laver avec de l&#39;eau DEPC. </li><li> Pomper de l&#39;air afin de sécher le système de tuyauterie de l&#39;appareil. </li></ol> 3. Préparation des gradients de saccharose <ol><li> Ajoutez 15% et 55% des solutions de saccharose dans 20 mM Tris-HCl pH 7,4, 1,25 mM de MgCl2, 150 mM de NaCl, et 1 mM de DTT. </li><li> Générer le gradient linéaire de saccharose à 15-55% en utilisant une Isco Modèle 160 gradient ancien, tel qu&#39;il est décrit dans les instructions du fabricant. Cependant, il est important de bien contrôler la vitesse de la pompe péristaltique TRIS, afin d&#39;éviter la création de bulles ou des turbulences dans les gradients. Maintenir les gradients à 4 ° C jusqu&#39;à utilisation. </li><li> Pendant le temps où le gradient de programme machine est en fonctionnement, refroidir la ultracentrifugation à 4 ° C. </li></ol> 4. Préparation de la cellule et de souris Extraits cerveau <ol><li> Préparation des extraits cellulaires <ol><li> Placez la plaque (s) sur les cellules de la glace et de lavage 3x avec PBS 1X froid. </li><li> Récupération des cellules (~ 12 x 10 6) dans 1 ml de tampon de lyse (20 mM Tris-HCl à pH 7,4, 1,25 mM de MgCl2, 150 mM de NaCl, 1 mM de DTT, 1% Nonidet P40, 5 inhibiteur U / RNase ml, complété avec mini-comprimés complets inhibiteur de la protéase cocktail sans EDTA), les transférer dans un tube Eppendorf, et bien mélanger en les faisant passer 15x par une seringue 1cc U100 de l&#39;insuline 28 G 1/2. Soit le lysat cellulaire reposer sur de la glace pendant 15 min. </li><li> Mettre quelques gouttes de lysat cellulaire sur une lame pour évaluer la lyse cellulaire par l&#39;observation au microscope à contraste de phase en utilisant un objectif 10X. Seuls les noyaux doivent être visibles et aucun membranes cellulaires devraient être visibles pour attester la lyse cellulaire. Comme contrôle, observercellules non lysées. </li><li> Clarifier le lysat cellulaire par centrifugation à 11 000 g pendant 20 min à 4 ° C et garder le lysat soluble contenant les polyribosomes. </li></ol></li><li> Préparation de souris Extraits cerveau <ol><li> Homogénéiser l&#39;ensemble du cerveau isolé (~ 500 mg) par 10 coups dans un homogénéiseur Dounce glacée avec 2 ml de tampon de lyse et de clarifier l&#39;homogénat par centrifugation à 11 000 xg pendant 15 min à 4 ° C. </li><li> Charger le surnageant obtenu sur un coussin de saccharose à 50% et de procéder à une sédimentation pendant 2 heures à 200 000 x g en utilisant un rotor d&#39;ultracentrifugation TH-641 à 4 ° C. </li><li> Reprendre le culot translucide contenant les polyribosomes dans 1 ml de tampon de lyse et bien mélanger par pipetage de haut en bas. Laissez la suspension reste sur la glace pendant 30 minutes avant de le charger sur des gradients. </li></ol></li></ol> 5. Chargement des extraits sur saccharose dégradés et Ultracentrifugation <ol><li> Mesurer la concentrationde l&#39;ARN présent dans l&#39;extrait cytoplasmique en utilisant un spectrophotomètre. Prudemment et lentement charger ~ 20 OD 260 unités de l&#39;extrait sur ​​le gradient de sucrose de 15% à 55%. Assurez-vous qu&#39;il n&#39;y a 2-3 mm d&#39;espace disponible à la surface du tube pour éviter le débordement des tubes pendant la centrifugation. </li><li> Centrifuger les gradients en utilisant une ultra-centrifugeuse pour 2 h 30 min à 230 000 xg l&#39;aide d&#39;un rotor d&#39;ultracentrifugation TH-641 à 4 ° C. Lorsque la centrifugation est terminé, retirez les tubes et les placer sur la glace soigneusement à ne pas perturber les gradients. </li></ol> 6. Fractionnement des extraits cytoplasmiques de polysomes profilage <ol><li> Placer des gradients de saccharose sur le système de fractionnement de la masse volumique automatisé et procéder à un fractionnement et la collecte automatisée des fractions (0,5 ml chacune), comme décrit par le fabricant. </li><li> Recueillir chaque fraction (~ 500 pi) dans des tubes Eppendorf individuelles avec un suivi continu de l&#39;absorbance à 254 nm. En parallel de l&#39;opération de fractionnement gradient, le profil de polyribosomal sera édite sur la feuille de papier. Vous pouvez également utiliser une unité d&#39;acquisition de données attachée à l&#39;enregistreur d&#39;avoir une électronique d&#39;acquisition du profil de polysome. </li><li> A la fin de chaque terme, transférer eppendorfs recueillies sur glace sèche. A cette époque, soit magasin recueilli fractions à -80 ° C ou précipiter directement complexes protéine-ARN comme suit. </li></ol> 7. Extraction et l&#39;analyse de protéines <ol><li> Pour chaque fraction recueillie de gradient de saccharose, ajouter 2 volumes d&#39;éthanol à 100% froid et de laisser des complexes ARN-protéine précipite à -20 ° C pendant une nuit. </li><li> Centrifugeuse chaque précipité d&#39;ARN-protéine à 11 000 g pendant 20 min à 4 ° C puis laver à l&#39;éthanol à 70%. Sec et remettre en suspension le précipité dans un tampon d&#39;échantillon SDS-PAGE avant de procéder au transfert de type Western en utilisant des anticorps spécifiques (voir la figure 2). </li></ol> 8. Extraction des ARN et Analyse <ol><li> Précipiter les complexes protéines-ARN comme dans la section 7.1. Remettre en suspension chaque précipité d&#39;ARN-protéine dans le tampon de lyse contenant 0,1% de SDS. Digérer le composant de protéine du précipité avec 2 mg / ml de protéinase K pendant 30 min dans un bain-marie à 55 °. </li><li> Extraire les composants de l&#39;ARN du précipité en ajoutant 1 volume de &quot;phénol: chloroforme&quot;, 2 volumes de chloroforme, et 0,1 volume de NaOAc 2 M pH 4 et centrifugation à 10 000 xg à 4 ° C pendant 20 min. Précipité l&#39;ARN de la phase aqueuse résultant de chaque échantillon pendant une nuit à -20 ° C en ajoutant 1 volume d&#39;isopropanol et 0,2 pg / pl de glycogène. Spin le précipité pendant 1 heure à 10 000 g à 4 ° C. Laver le culot d&#39;ARN avec de l&#39;éthanol froid à 70%. </li><li> Remettre en suspension le culot d&#39;ARN dans un petit volume d&#39;eau sans RNase. Évaluer la quantité et la qualité de l&#39;ARN à l&#39;aide du spectrophotomètre. </li></ol>

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Les auteurs n&#39;ont rien à révéler.

L&#39;analyse de profils de polysomes sur des gradients de saccharose permet la mesure de l&#39;initiation de traduction en analysant la densité de polysomes isolés à partir de cellules ou de tissus 9,11-14. Cette technique est la meilleure (si ce n&#39;est pas l&#39;Unique) approche pour mesurer initiation de la traduction in vivo. Il est utilisé pour surveiller l&#39;état de translation de la croissance de cellules au cours du cycle cellulaire 15, et à évaluer les effets de diffé...

Les cellules eucaryotes rencontrent constamment une gamme de conditions de stress physiologiques et environnementaux néfastes qui nécessitent une réponse cellulaire adaptative rapide. réponse au stress cellulaire implique un équilibre précis entre anti-survie et pro-survie facteurs agissant. Perturber cet équilibre peut avoir des conséquences irréversibles menant au développement de pathologies humaines telles que le cancer et les maladies neurodégénératives. Au cours de la première étape de la réaction de stress, les cellules activent des voies pro-survie qui comportent la commande coordonnée des changements dans l&#39;expression génétique au niveau de la traduction de l&#39;ARNm. traduction de l&#39;ARNm chez les eucaryotes est un processus cellulaire complexe qui implique des interactions coordonnées entre les facteurs d&#39;initiation de traduction (SIFE), des protéines de liaison d&#39;ARN spécifique (RBD), et des molécules d&#39;ARN 1. traduction de l&#39;ARNm est divisé en trois phases distinctes: initiation, l&#39;élongation et terminaison. Bien que les trois phases sont soumis à réglemécanismes mentaires, les mécanismes de contrôle de la traduction ciblent principalement la phase d&#39;initiation de la traduction, qui constitue ainsi l&#39;étape de limitation de vitesse de la synthèse des protéines 2. initiation de la traduction est un processus hautement ordonnée qui commence avec la formation de la eIF2a.GTP.Met-ARNt i Met complexe ternaire et sa liaison ultérieure de la sous-unité 40S du ribosome, ce qui conduit à la formation du complexe de pré-initiation. La prochaine étape est le recrutement du complexe de pré-initiation à l&#39;ARNm, ce qui implique l&#39;activité des facteurs d&#39;initiation de traduction tels que eIF4F et eIF3. Le complexe de pré-initiation 48S ainsi formé subit des changements conformationnels spécifiques qui permettent cette machinerie pour commencer la numérisation de la région 5 &#39;non traduite de l&#39;ARNm jusqu&#39;à ce qu&#39;il reconnaît le codon d&#39;initiation août La plupart des facteurs d&#39;initiation de traduction sont alors libérés et sous-unités 60S sont recrutés pour former un ribosome 80S complexe compétente pour la traduction, unt qui la synthèse des protéines point commence (Figure 1). Plus d&#39;un 80S monosome peut être la traduction de l&#39;ARNm même à un moment la production de soi-disant polysomes (ou polyribosomes). La densité des polysomes sur un ARNm reflète l&#39;initiation, l&#39;élongation et les tarifs de terminaison et est une mesure de la transcription d&#39;un Traduisibilité particulier ainsi. Cependant, polysome profil est principalement utilisé pour évaluer les changements dans la traduction de l&#39;ARNm à l&#39;étape d&#39;initiation. Ici, nous avons utilisé un inhibiteur de protéasome comme inhibiteur d&#39;initiation de traduction. Le traitement des cellules cancéreuses avec ce médicament induit une réponse au stress caractérisé par l&#39;activation de la kinase de stress nommé HRI qui phosphoryle le facteur d&#39;initiation de traduction eIF2a 3. La phosphorylation de eIF2a est l&#39;un des principaux événements qui ont conduit à l&#39;inhibition de l&#39;initiation de la traduction dans les cellules de mammifères 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>
			<td></td>
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			<td height="21" style="height:21px;width:279px;">Culture medium and Supplements</td>
		</tr>
		<tr height="21">
			<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>
		</tr>
		<tr height="21">
			<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

Un contrôle précis de la traduction des ARNm est fondamentale pour l&#39;homéostasie des cellules eucaryotes, en particulier en réponse à un stress physiologique et pathologique. Des modifications de ce programme peuvent conduire à la croissance des cellules endommagées, une caractéristique de développement de cancer, ou à une mort cellulaire prématurée tel que vu dans les maladies neurodégénératives. Une grande partie de ce qui est connu au sujet de la base moléculaire pour la commande de translation a été obtenue à partir de polysomes analyse en utilisant un système de fractionnement à gradient de densité. Cette technique repose sur des extraits cytoplasmiques ultracentrifugation sur un gradient de saccharose linéaire. Une fois l&#39;essorage est terminé, le système permet le fractionnement et la quantification des zones correspondant à la traduction centrifugés ribosomes populations différentes, conduisant ainsi à un profil de polysomes. Les changements dans le profil de polysomes sont indicatifs de changements ou de défauts dans l&#39;initiation de traduction qui se produisent en réponse à différents types de stress. Cette technique permet également d&#39;évaluer èmee rôle des protéines spécifiques sur initiation de la traduction, et pour mesurer l&#39;activité de traduction des ARNm spécifiques. Nous décrivons ici notre protocole pour effectuer des profils de polysomes, afin d&#39;évaluer l&#39;initiation de traduction de cellules eucaryotes et des tissus dans des conditions de croissance normales, ou soit contrainte.

Comme mentionné précédemment, polysome profil permet l&#39;analyse de l&#39;évolution de l&#39;initiation de traduction dans des conditions de stress. Figure 1 est une vue simplifiée de l&#39;initiation de la traduction qui, comme décrit plus haut est un processus en plusieurs étapes impliquant un assemblage ordonné de complexes d&#39;initiation de traduction. Dans des conditions de croissance normales, les complexes d&#39;initiation de traduction sont convertis en polyribosomes dont la détecti...

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

Ici, nous décrivons une méthode pour analyser l&#39;évolution de l&#39;initiation de la traduction de l&#39;ARNm de cellules eucaryotes en réponse à des conditions de stress. Cette méthode est basée sur la séparation de vitesse sur des gradients de saccharose de la traduction à partir de ribosomes ribosomes non translating.

Analyse de la traduction Initiation Pendant conditions de stress par polysome profilage

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 vues.  Laval University.  Ici, nous décrivons une méthode pour analyser l'évolution de l'initiation de la traduction de l'ARNm de cellules eucaryotes en réponse à des conditions de stress. Cette méthode est basée sur la séparation de vitesse sur des gradients de saccharose de la traduction à partir de ribosomes ribosomes non translating. 

Vidéo: 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.

Surveillance des hormones végétales Durant les réponses au stress

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

Recherche

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.

Profilage anticorps par immunoprécipitation Systèmes luciférase (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 du stress oxydatif dans embryons de poissons zèbres

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.

Profilage individuel embryonnaires humaines Cellules souches par RT-PCR quantitative

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.

Polysome Fractionnement et analyse des mammifères Translatomes sur une échelle de l&#39;ensemble du génome

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.

Évaluation de la sélective ARNm traduction dans les cellules de mammifères par polysome profilage

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 glucocorticoïdes Analyse: Surveillance surrénale non invasive en Equidés

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.

Une méthode facile pour des plantes polysomes 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 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 du Gap Junction-dependent Transfert de miARN avec microscopie 3D-FRAP

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 de la fonction E3 Ubiquitin Ligase

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