Nous remercions Jordan Davis, Yanyan Lin, Eric Schadler et Jimiao Zheng pour leur aide technique à cette étude. Ce travail a été soutenu par la NSF Career Grant MCB-1054962 ALA

1. Préparation grandes jus de pomme-agar plaques <ol><li> Pour faire 4 assiettes, ajouter 375 ml H 2 O, 11,25 g d&#39;agar mouche et une barre d&#39;agitation dans un ballon de 1000 ml. C&#39;est Mix A. autoclave Mix A avec le couvercle du ballon sans serrer plafonné sur un cycle de 30 min-stérilisation pour les produits liquides. </li><li> Ajouter 125 ml de jus de pomme, 12,5 g de sucre de table et d&#39;un barreau à un bécher de 500 ml. Ce mélange est B. chaleur Mix B sur une plate-forme chauffée sous agitation et maintenir la température à environ 70 ° C jusqu&#39;à ce que le passage à l&#39;autoclave de mélange A est terminée. </li><li> À la fin de Mix Un autoclave, transférer Mix B à A. Mélanger Gardez en remuant doucement le mélange combiné et le laisser refroidir pendant 15 minutes. Évitez de trop le refroidissement et la solidification de la gélose entraîné avant l&#39;ajout d&#39;agents de conservation. </li><li> Faire 10% (poids / volume) méthyle solution 4-hydroxybenzoate dans l&#39;éthanol. Ceci agira comme un agent de conservation. Ajouter 3,75 ml de la solution dans le mélange jus-agar de pomme ci-dessus. Gardez en remuant pour another 5 min. </li><li> Verser 125 mélange de jus de pomme-agar ml avec conservateur dans chaque 300 cm 2 en plastique ou une plaque de polystyrène. Laisser refroidir à température ambiante et se solidifier. Éviter la formation de bulles d&#39;air tout en versant. Remarque: Les plaques jus-agar pomme sont prêts à utiliser immédiatement. Couvrir les plaques inutilisées avec enveloppements claires et stocker à 4 ° C pendant jusqu&#39;à une semaine. </li></ol> 2. Drosophila collecte d&#39;embryons et le traitement pré-réticulation <ol><li> Chargez une cage de population à environ 40.000 à 50.000 mouches partir desquels les embryons seront collectées. </li><li> Faire deux plaques contenant chacune 125 ml de farine de maïs mélasse standard intermédiaire 12 et saupoudrer environ 4 g de levure de boulanger séchée sur chaque plaque. Placez ces plaques dans la cage de la population dans un incubateur à 25 ° pendant 48 heures pour engraisser les mouches. </li><li> Chauffez 2 jus de pomme des plaques de gélose à 25 ° C. Saupoudrer environ 1 g de levure de boulanger séchée sur chaqueplaque. Placez les deux plaques dans la cage de la population de commencer à recueillir des embryons de 0-1 h âgé. </li><li> Retirer les plaques à la fin de la collection de 1 hr. Remplacer avec 2 nouvelles plaques si un autre cycle de collecte est nécessaire. </li><li> Ajouter suffisamment d&#39;eau pour couvrir les plaques et remettre en suspension les embryons doucement avec un pinceau fin. Verser les embryons remises en suspension à travers un tamis à deux couches faites de treillis métallique inoxydable en acier. Remarque: La couche supérieure est faite de la taille des pores moyens (850 pm) maillage qui recueille une grande partie de mouches tout en laissant passer les embryons. La couche inférieure est faite de mailles fines (75 um) qui collecte des embryons tout en laissant passer la levure et de l&#39;eau. </li><li> Utilisez le pinceau fin pour transférer les embryons collectés dans un panier de collecte faite avec un tube de 50 ml et fine maille de nylon. Rincer les embryons brièvement à l&#39;eau puis épongez la maille sur des serviettes en papier pour sécher. </li><li> Plonger les embryons dans 50% de javel remuer doucement pendant 3 min. Rincer approfondiement avec de l&#39;eau pour éliminer tout agent de blanchiment. Tamponnez la maille sur des serviettes en papier pour sécher. </li><li> Plonger les embryons dechorionated dans de l&#39;isopropanol sous agitation douce pendant 15 sec ou jusqu&#39;à ce que la répartition des bouquets multi-embryon (cela ne devrait pas prendre plus de 30 secondes). Tamponnez la maille sur des serviettes en papier pour sécher. </li><li> Plonger les embryons dans l&#39;heptane pendant 5 minutes tout en utilisant une pipette pour écouter heptane sur les embryons pour les garder remis en suspension. Tamponnez la maille sur des serviettes en papier pour sécher. </li><li> Rincer les embryons avec des flux de saline tamponnée au phosphate, pH 7,4 (PBS) contenant 0,1% de Triton X-100 (PBST) pendant 3 min. </li><li> Démonter le panier de collecte et de transfert des embryons avec le maillage à un tube de 15 ml contenant 10 ml de PBST. Retourner doucement le tube pour laver les embryons de la maille. </li><li> Retirez le filet du tube. Mettre le tube dans une position verticale et laisser les embryons se déposent au fond du tube par gravité. Remarque: Environ 100-300ul d&#39;embryons est prévu pour une collection de 1 heure d&#39;une cage. </li><li> Retirez le PBST et continuer à effectuer la réticulation immédiatement. </li></ol> 3. In vivo réticulation des embryons collectés <ol><li> Préparer 0,2% de formaldehyde dans du PBST, immédiatement avant utilisation. Ajouter 10 ml de fixateur de formaldéhyde pour les embryons et incuber à 25 ° C avec agitation douce sur un agitateur rotatif pendant 10 min. Jeter fixateur utilisé à la fin de la journée. </li><li> Stopper la réaction de réticulation <ol><li> A la fin de la réticulation, que les embryons se déposent au fond du tube. </li><li> Retirer la solution de formaldéhyde. Ajouter immédiatement 10 ml de 0,25 M de glycine dans du PBST pour arrêter la réaction de réticulation. Incuber à 25 ° C avec agitation douce sur un agitateur rotatif pendant 5 min. </li><li> Laissez les embryons se déposent au fond du tube. Retirer la solution de refroidissement. Laver les embryons avec 10 ml de PBST trois fois. </li><li> Supprimer complètement le Psolution de lavage de BST à l&#39;aide d&#39;une pipette. embryons de conserver à -80 ° C jusqu&#39;à utilisation. </li></ol></li></ol> 4. Protéine préparation de l&#39;échantillon et immunoprécipitation <ol><li> Homogénéiser 500 pi embryons réticulés en utilisant un homogénéisateur Dounce dans 2 ml de tampon de lyse (0,5 M d&#39;urée, 0,01% de SDS, 2% de Triton-X 100, phénylméthanesulfonyle fluorure 2 mM [PMSF] et inhibiteur de la protéase cocktail dans PBS). Remarque: Effectuez cette et les étapes suivantes à la température ambiante, sauf indication contraire. </li><li> Transférer le lysat tubes de 1,5 ml à centrifuger et secouez-la délicatement sur une plate-forme tournante pendant 15 min à assurer une lyse approfondie. Centrifuger le lysat à 16 000 x g pendant 5 minutes pour sédimenter les débris cellulaires. Enregistrez le surnageant dans un tube de 15 ml propre. </li><li> Ajouter 40 ul de billes d&#39;agarose anti-HA suspension pour le surnageant et laisser incuber sur une plateforme rotative pendant 2,5 heures. </li><li> Sédimenter les billes par centrifugation à 2500 xg pendant 5 min. Retirer le surnageant mais laisser approximately 250 pi dans le tube. Remettre en suspension les perles dans le surnageant restant et le transférer à une colonne de centrifugation de 1,5 ml avec un filtre à pores de 10 um. </li><li> Centrifuger la colonne de centrifugation à 12 000 g pendant 10 sec et jetez l&#39;écoulement à travers. </li><li> Laver les billes en ajoutant 200 ul de tampon de lavage (0,1 M de glycine, NaCl 1 M, 1% d&#39;IGEPAL CA-630, 0,1% de Tween-20 dans du PBS). Centrifuger la colonne à 12 000 g pendant 10 sec et jeter le lavage. Répétez le lavage deux fois de plus. </li><li> Ajouter 40 ul de tampon d&#39;élution (2 mg / ml HA-peptide dans du PBS) pour les billes et incuber à 37 ° C pendant 15 minutes avec agitation douce toutes les 5 min. Centrifuger la colonne à 12 000 g pendant 10 sec et recueillir l&#39;éluat. </li><li> Confirmer l&#39;élution du complexe par analyse Western-blot en utilisant l&#39;anticorps anti-HA 13. </li></ol>

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Les auteurs n&#39;ont rien à révéler.

Le formaldehyde a été couramment utilisé comme réactif de réticulation pour identifier la protéine-protéine et des interactions protéine-acide nucléique. Sa bonne solubilité et la perméabilité membranaire de la cellule, ainsi que la compatibilité avec les procédés de spectrométrie de masse en aval, de faire un formaldéhyde agent candidat idéal pour les applications de reticulation intracellulaires 3,15-17. En particulier, il a été utilisé avec succès pour identifier les ARNm associés à...

Isolement des assemblages de protéines unités multiples et complexes d&#39;ADN-ou d&#39;ARN-protéine est effectuée pour identifier des complexes de protéines, des loci génomiques reconnu par des protéines de régulation se liant à l&#39;ADN ou des cibles d&#39;ARN des protéines de liaison d&#39;ARN. Différents procédés permettent l&#39;identification de l&#39;ensemble du génome d&#39;ADN des sites reconnus par des facteurs de transcription ou des protéines de la chromatine (ChIP-seq) 1 et les cibles d&#39;ARN associés à une protéine de liaison à l&#39;ARN donné (CLIP-seq) 2. Les bibliothèques d&#39;ADNc de l&#39;ARN dérivé ou de cibles d&#39;ADN sont ensuite profondément séquences. Ces procédés utilisent des produits chimiques ou de réticulation pour stabiliser les complexes suivie par une immunoprécipitation (IP) avec un anticorps dirigé contre un composant protéique du complexe étudié induite par les UV. Au cours du développement d&#39;un organisme, de nombreux complexes protéiques forment de manière transitoire. Par conséquent, il est essentiel d&#39;analyser la composition et le fonctionnement de ces complexes in vivo pour comprendre les mécanismes moléculaires qui contrôlent developpement. Une telle analyse in vivo serait supérieure à l&#39;approche in vitro car il est pratiquement impossible de reproduire les concentrations indigènes des éléments en interaction et de l&#39;environnement biochimique cellulaire in vitro. Ici, nous démontrons une approche in vivo que nous utilisons avec succès pour isoler grands complexes de protéines à partir d&#39;embryons de drosophile. Dans ce procédé, les complexes de protéines dans les embryons vivants sont réticulés avec une faible concentration en formaldéhyde et ensuite complexes de protéines d&#39;intérêt sont isolés par IP avec un anticorps dirigé contre un composant connu des complexes, suivie d&#39;une purification sur gel des complexes et de l&#39;analyse par spectrométrie de masse à identifier les composants complexes inconnus. Etant donné que le formaldéhyde est capable de traverser la membrane cellulaire et a une gamme de réticulation de 02.03 à 02.07 Å 3, des complexes de protéines peuvent être réticulés in vivo et les composants complexes sont susceptibles d&#39;être proches les unes des autres. Dans cet article, nousdécrire ce procédé en utilisant l&#39;isolement de Tudor (TUD) complexe de protéines, par exemple. Tud est une protéine de la lignée germinale qui est essentiel pour le développement de la lignée germinale 4-7. Cette protéine contient 11 domaines TUD connus pour interagir avec les arginines ou lysines méthylées d&#39;autres polypeptides 8-10. Auparavant, nous avons généré une ligne drosophile transgénique qui exprime HA-étiqueté Tud fonctionnelle 5 et donc, un anticorps spécifique anti-HA est utilisé pour tirer vers le bas complexe Tud après réticulation. En plus des réticulations protéine-protéine, le formaldéhyde peut générer des réticulations d&#39;acide nucléique-protéine et est utilisé dans des expériences de ChIP-seq. En outre, chez la drosophile, in vivo de réticulation avec le formaldéhyde a permis l&#39;identification d&#39;une cible d&#39;ARN de l&#39;ARN Vasa protéine hélicase 11. Bien que dans cet article, nous décrivons un procédé pour la reticulation in vivo et purification des complexes de protéines à partir d&#39;embryons de Drosophila, ce procédé peut être adapté à d&#39;autres organismes et les tissus.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

De nombreux processus cellulaires sont contrôlées par des complexes protéiques unités multiples. Souvent, ces complexes forment transitoire et nécessitent environnement natif à assembler. Par conséquent, afin d&#39;identifier ces complexes de protéines fonctionnelles, il est important de les stabiliser in vivo avant la lyse des cellules et la purification ultérieure. Ici, nous décrivons une méthode utilisée pour isoler de grands complexes de protéines de bonne foi à partir d&#39;embryons de Drosophila. Cette méthode est basée sur l&#39;embryon perméabilisation et la stabilisation des complexes à l&#39;intérieur des embryons par reticulation in vivo en utilisant une faible concentration en formaldéhyde, qui peut facilement traverser la membrane cellulaire. Par la suite, le complexe de protéine d&#39;intérêt est immunopurifié suivie d&#39;une purification sur gel et analysés par spectrométrie de masse. Nous illustrons cette méthode en utilisant la purification d&#39;un complexe protéique Tudor, qui est essentielle pour le développement de la lignée germinale. Tudor est une grosse protéine, qui contient plusieurs domaines Tudor - petitmodules qui interagissent avec arginines méthylées ou des lysines des protéines cibles. Cette méthode peut être adaptée pour l&#39;isolement des complexes de protéines natives provenant d&#39;organismes différents et des tissus.

L&#39;efficacité de la réticulation et la purification réussie du complexe protéique réticulé TUD ont été analysés par SDS-PAGE sur un 3% - 7% de gel de l&#39;étape (illustré sur la figure 1), suivie par transfert de Western (Figure 2). Le but de l&#39;utilisation de 3% - gel de l&#39;étape 7% est basé sur la séparation effective du complexe protéique réticulé Tud de la protéine restante Tud non réticulé et la concentration du complexe. S...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Complexes de protéines multi-composants jouent un rôle crucial lors de la fonction cellulaire et le développement. Ici, nous décrivons une méthode utilisée pour isoler des complexes de protéines natives à partir d&#39;embryons de Drosophila après réticulation in vivo suivie d&#39;une purification des complexes réticulés pour analyse subséquente structure-fonction.

Un In vivo Approche de réticulation pour isoler les complexes protéiques De Drosophila Embryons

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

16.6K vues.  Murray State University.  Complexes de protéines multi-composants jouent un rôle crucial lors de la fonction cellulaire et le développement. Ici, nous décrivons une méthode utilisée pour isoler des complexes de protéines natives à partir d'embryons de Drosophila après réticulation in vivo suivie d'une purification des complexes réticulés pour analyse subséquente structure-fonction. 

Vidéo: An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Préparation des cultures de neurones d&#39;embryons de drosophile Midgastrula scène

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

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Biologie

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabriqués post-Array-détecteurs (mPADs): une approche pour isoler les forces mécaniques

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Préparation d&#39;embryons pour la microscopie électronique des Drosophile Tube cardiaque embryonnaire

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Isolement du labiles complexes multi-protéiques par des In vivo Contrôlée Cellulaire réticulation et chromatographie d&#39;affinité immuno-magnétiques

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

In vivo d&#39;embryons de drosophile Centrifugation

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imagerie interactions protéine-protéine In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagerie verticale de<em&gt; Drosophila</em&gt; Embryons

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultures de cellules primaires à partir Drosophile Embryons Gastrula

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Changement de forme d&#39;imagerie cellulaire en vie Drosophile Embryons

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolement et purification de la kinésine de Drosophila Embryons

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...