Ce travail a été soutenu par une réintégration Marie Curie Grant (IRG249084). Nous remercions tous les membres du laboratoire Kurant.

Pour suivre la dynamique de l&#39;élimination des cellules apoptotiques in vivo deux populations de cellules doivent être étiquetés: les cellules phagocytaires et les cellules apoptotiques. Pour marquer des populations de cellules phagocytaires nous utilisons une ligne Drosophila contenant le marqueur simu-cytGFP, qui marque les cellules phagocytaires exclusivement dans l&#39;embryon: les macrophages, les cellules gliales et de l&#39;ectoderme 8. On peut utiliser différents marqueurs pour les cellules phagocytaires, y compris les lignes contenant un pilote Gal4 hemocyte spécifique (CRQ-Gal4) ou des cellules gliales spécifique Gal4 conducteur (repo-Gal4) et un rapporteur fluorescent génétiquement codés sous contrôle UAS (UAS-GFP). Pour surveiller les cellules apoptotiques au moment du dédouanement des cellules apoptotiques, nous utilisons différents apoptose / marqueurs de phagocytose. Annexine V (Molecular Probes) sert de marqueur précoce des cellules apoptotiques 10; Phiphilux (OncoImmunin) est un fluorogène substrat caspase-3, qui est utilisé comme un apopto tardsis marqueur, et Lysotracker (Molecular Probes) fonctionne comme un marqueur phagosome. Nous injectons ces réactifs en utilisant le système de micro-injection PicoPump PV 820. 1. Préparatifs <ol><li> Colle Heptane: Déroulez ruban adhésif double face et de mettre autant que vous le pouvez dans un flacon à scintillation, remplissez avec de l&#39;heptane, sceller la fiole avec du Parafilm et le rock pendant 24 heures. Ajouter heptane si la colle est trop épaisse. </li><li> Nous permettons aux mouches pondent sur une plaque préchauffée jus de raisin agar + levure collez pendant 2 heures, puis de transférer la plaque à 25 ° C pendant 10-12 heures (selon le stade désiré) avant la micro-injection et l&#39;heure des enregistrements de déchéance des embryons injectés. Nous recueillons des embryons à partir des plaques d&#39;agar maintenue à 25 ° C, fournissant ainsi des embryons de stade 15 ou 16 du développement. </li><li> préparation de l&#39;aiguille: Pour préparer aiguilles pour injection, nous utilisons un «instrument Sutter &#39;extracteur d&#39;aiguilles et minces filaments de verre à parois (tube capillaire FHC). La pointe du capillaire commun est rompun d&#39;un diamètre de 0,5 à 2 um. </li><li> Lamelle avec de la colle: En utilisant une pointe de pipette disperser une goutte de colle à l&#39;heptane en une ligne au milieu de la lamelle. Laissez sécher pendant quelques secondes. Préparer un peu de ces lamelles. </li></ol> 2. Préparation embryon <ol><li> Recueillir des embryons à partir de plaques d&#39;agar et de les transférer à un tamis cellulaire (SPL sciences de la vie) à l&#39;aide d&#39;un pinceau propre et l&#39;eau courante. La crépine est maintenue sur un gobelet à recueillir les eaux usées. </li><li> Lavez embryons dans la passoire de la cellule avec de l&#39;eau jusqu&#39;à ce que toute la pâte de levure est enlevée. </li><li> Sécher l&#39;excès d&#39;eau en l&#39;essuyant à l&#39;extérieur du filtre à l&#39;aide Kimwipes. </li><li> Placez le tamis cellulaire dans une boîte de Petri propre et ajouter suffisamment d&#39;eau de Javel à 50% pour couvrir les embryons dans le tamis cellulaire. Dechorionate les embryons pendant 2 min de temps en temps (2-3 fois) les remuant. </li><li> Rincez abondamment avec de l&#39;eau jusqu&#39;à ce que les embryons en vrac l&#39;odeur d&#39;eau de Javel. </li><li> Placez le tamis cellulaire dans un récipient propreBoîte de Pétri et couvrir les embryons avec de l&#39;eau pour éviter le dessèchement haut. </li><li> Avant de commencer réglage des embryons, charger 1 pl du réactif désiré en deux aiguilles (parfois une aiguille peut être bouchée). Il faut environ cinq minutes pour que le liquide atteigne la pointe de l&#39;aiguille. </li><li> Coupez un morceau rectangulaire d&#39;agar jus de raisin et le placer sur une lame de microscope. Transfert d&#39;embryons dechorionated de la boîte de Pétri à la pièce de gélose à l&#39;aide d&#39;un pinceau propre. </li><li> Sous un microscope de dissection fluorescent sélectionner correctement mis en scène embryons exprimant le marqueur GFP en utilisant un pinceau humide et placez chaque embryon près du bord de la pièce d&#39;agar-agar dans une rangée un après l&#39;autre (figure 1A). Les embryons doivent être placés avec leur face ventrale de la phagocytose d&#39;imagerie par les cellules gliales dans le système nerveux central (SNC). </li><li> Mettre en place environ 10 embryons dans une rangée. </li><li> Attacher les embryons à une lamelle recouverte au milieu d&#39;une bande de colle heptane (figure 1B, C </strong&gt;). </li><li> Placez la lamelle dans la chambre de déshydratation pendant environ 5 minutes (cela dépend de l&#39;humidité de la pièce et la quantité à injecter). </li><li> Après séchage jusqu&#39;à couvrir les embryons avec de l&#39;huile d&#39;halocarbures 700 (Sigma) pour éviter une déshydratation. </li><li> Placer la lamelle couvre-objet sur une lame de microscope, avec des embryons vers le haut. Vous pouvez mettre une goutte d&#39;eau sur la lame pour empêcher le déplacement de la lamelle. Les embryons sont maintenant prêtes pour l&#39;injection. emplacement d&#39;injection est typiquement sur le côté latéral au milieu de l&#39;embryon. </li></ol> 3. Injection d&#39;embryons <ol><li> Fixer l&#39;aiguille à un micromanipulateur. </li><li> Pour briser la pointe de l&#39;aiguille mis au bord de la lamelle dans le champ de vision et d&#39;ajuster l&#39;aiguille au même plan focal. Très déplacer soigneusement la lamelle jusqu&#39;à ce qu&#39;elle touche le bout de l&#39;aiguille et la casse. </li><li> En se concentrant sur les embryons, placez l&#39;aiguille dans le pétrole et vous assurer d&#39;obtenir une goutte de liquide à partir de l&#39;aiguille. Cela signifie que la pointe de l&#39;aiguille a étébien cassé. </li><li> En évitant de toucher le support d&#39;aiguille déplace l&#39;embryon dans l&#39;aiguille et injecter une goutte dans l&#39;embryon. Déplacer l&#39;embryon loin et passer à la suivante jusqu&#39;à ce que tous les embryons sont injectés. </li></ol> 4. Imaging Nous avons l&#39;image des embryons sur un microscope confocal inversé avec un objectif 40X ou 100X. Nous recherchons un embryon bien positionné avec le CNS dans le milieu, ce qui montre l&#39;expression de la GFP solide et un bon étiquetage des cellules apoptotiques après l&#39;injection. Pour le temps des enregistrements de déchéance d&#39;embryons vivants, nous choisissons généralement 5 ou 6 tranches confocale (2 um d&#39;épaisseur chacune). Ensuite, nous faisons une projection de 3 tranches (6 um d&#39;épaisseur) afin d&#39;observer des cellules entières. Nous utilisons des marqueurs pour les cellules apoptotiques qui sont stables et ne blanchissent pas facilement. Pour éviter GFP blanchiment nous faire des enregistrements à des intervalles de 60 sec. Dans certains cas, les embryons peuvent commencer à roulerpendant le temps d&#39;enregistrement vidéo. Par conséquent, nous prenons un coup d&#39;œil de temps en temps à l&#39;enregistrement afin d&#39;arrêter et recommencer si nécessaire.

<ol>
	<li>Kinchen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+to+die+for:+signaling+to+apoptotic+cell+removal+in+worm,+fly+and+mouse.">A model to die for: signaling to apoptotic cell removal in worm, fly and mouse.</a> Apoptosis. 15, 998-1006 (2010).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagosome+maturation:+going+through+the+acid+test.">Phagosome maturation: going through the acid test.</a> Nat. Rev. Mol. Cell Biol. 9, 781-795 (2008).</li><li>Kinchen, J. M., Ravichandran, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+signaling:+you+can+touch,+but+you+can't+eat.">Phagocytic signaling: you can touch, but you can't eat.</a> Curr. Biol. 18, 521-524 (2008).</li><li>Stuart, L. M., Ezekowitz, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytosis:+elegant+complexity.">Phagocytosis: elegant complexity.</a> Immunity. 22, 539-550 (2005).</li><li>Lauber, K., Blumenthal, S. G., Waibel, M., Wesselborg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clearance+of+apoptotic+cells:+getting+rid+of+the+corpses.">Clearance of apoptotic cells: getting rid of the corpses.</a> Mol. Cell. 14, 277-287 (2004).</li><li>Henson, P. M., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoptotic+cell+removal+in+development+and+tissue+homeostasis.">Apoptotic cell removal in development and tissue homeostasis.</a> Trends Immunol. 27, 244-250 (2006).</li><li>Kurant, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+the+CNS+clear:+glial+phagocytic+functions+in+Drosophila.">Keeping the CNS clear: glial phagocytic functions in Drosophila.</a> Glia. 59, 1304-1311 (2011).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133, 498-509 (2008).</li><li>Mergliano, J., Minden, 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=Caspase-independent+cell+engulfment+mirrors+cell+death+pattern+in+Drosophila+embryos.">Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos.</a> Development. 130, 5779-5789 (2003).</li><li>van den Eijnde, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+surface+exposure+of+phosphatidylserine+during+apoptosis+is+phylogenetically+conserved.">Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved.</a> Apoptosis. 3, 9-16 (1998).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Clairance cellulaire par apoptose est une dernière étape critique de l&#39;apoptose, qui est hautement dynamique. Par conséquent études en temps réel du processus sont d&#39;une importance capitale. Nous décrivons ici un protocole qui permet la surveillance de la clairance des cellules apoptotiques dans le développement des embryons de drosophile vivre. Dans cette procédure, deux populations de cellules doivent être marqués à l&#39;aide de marqueurs spécifiques: les cellules apoptotiques et les phag...

L&#39;élimination correcte des cellules indésirables ou aberrante par apoptose et la phagocytose ultérieure est crucial pour le développement embryonnaire ainsi que pour l&#39;homéostasie tissulaire chez l&#39;adulte. Phagocytose des cellules apoptotiques ou l&#39;autorisation cellulaire par apoptose est un processus très dynamique qui se déroule en quatre étapes: (1) le recrutement de phagocytes à la cellule apoptotique («Trouvez-moi»), (2) la reconnaissance de la cellule comme une cible pour la phagocytose ( «eat-me») et (3) l&#39;engloutissement, suivi par (4) maturation du phagosome et de la dégradation de la particule apoptotique 1-5. Il existe deux types de phagocytes: macrophages «professionnels» et les cellules dendritiques immatures, et le «non-professionnels» les cellules voisines tissu-résidents, qui sont cruciales pour le dédouanement des cellules apoptotiques pendant métazoaires développement 6,7. En développement chez la drosophile, l&#39;élimination des cellules superflues par apoptose se produit dans trois mphases Ain, d&#39;abord dans le milieu à la fin embryon, puis au milieu chrysalide, puis de nouveau au début des adultes. Au cours de l&#39;embryogenèse particules apoptotiques sont éliminés par les phagocytes «professionnels», les macrophages et les cellules gliales par ectoderme et 8,9 «non-professionnel». Dans nos précédents travaux, nous avons identifié un récepteur phagocytaire, Six-microns-sous (SIMU), qui est exprimé exclusivement dans les cellules phagocytaires cours de l&#39;embryogenèse. En utilisant le promoteur simulation, nous avons généré un marqueur spécifique des populations de cellules phagocytaires (simu-cytGFP) qui nous permet de suivre les macrophages, les cellules gliales ectoderme et simultanément dans un embryon en développement Live 8. En outre, en utilisant des marqueurs spécifiques des cellules apoptotiques dans les différentes étapes de l&#39;apoptose, nous sommes en mesure de suivre la dynamique de dégagement cellulaire par apoptose in vivo.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagent</td>
 </tr>
 <tr>
 <td>Annexin V </td>
 <td>Molecular Probes</td>
 <td>A35108</td>
 <td></td>
 </tr>
 <tr>
 <td>PhiPhiLux G2D2</td>
 <td>OncoImmunin</td>
 <td>A304R2G-5</td>
 <td></td>
 </tr>
 <tr>
 <td>LysoTracker</td>
 <td>Molecular Probes</td>
 <td>L-7528</td>
 <td></td>
 </tr>
 <tr>
 <td>Halocarbon oil 700</td>
 <td>Sigma</td>
 <td>H8898</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Material</td>
 </tr>
 <tr>
 <td>Capillary tubing</td>
 <td>FHC </td>
 <td>30-30-0</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Strainer</td>
 <td>SPL</td>
 <td>93100</td>
 <td></td>
 </tr>
 <tr>
 <td>Paintbrush</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 </tbody>
</table>

live imaging of apoptotic cell clearance during drosophila embryogenesis

L&#39;élimination correcte des cellules indésirables ou aberrante par apoptose et la phagocytose ultérieure (clairance cellulaire par apoptose) est cruciale pour le développement normal chez tous les organismes pluricellulaires. Clairance cellulaire par apoptose est un processus hautement dynamique, intimement associée à la mort cellulaire, les cellules apoptotiques unengulfed sont à peine vus in vivo dans des conditions normales. Afin de comprendre les différentes étapes de dégagement des cellules apoptotiques et de comparer les phagocytes «professionnels» - les macrophages et les cellules dendritiques de «non-professionnel» - cellules voisines tissu-résidents, l&#39;imagerie in vivo en direct du processus est extrêmement précieux. Ici, nous décrivons un protocole pour l&#39;étude de clairance des cellules apoptotiques dans les embryons de drosophile en direct. Pour suivre la dynamique des différentes étapes de la phagocytose que nous utilisons marqueurs spécifiques des cellules apoptotiques et les phagocytes. En outre, nous pouvons surveiller deux systèmes phagocyte en parallèle: les macrophages «professionnels» et «semi-profeglie ssional »dans le système nerveux central en développement (SNC). La méthode décrite ici utilise l&#39;embryon de drosophile comme un excellent modèle pour l&#39;étude en temps réel de la clairance des cellules apoptotiques.

images représentatives d&#39;un film dans lequel les cellules apoptotiques sont étiquetés avec l&#39;annexine V et cellules gliales, ectoderme et les macrophages sont étiquetés avec simu-cytGFP sont présentés dans la figure 2. Chaque image est une projection de 3 tranches de 2 um chacun. L&#39;embryon de l&#39;étape 15 est correctement positionné montrant le SNC embryonnaire dans le milieu de la glie bien étiquetés (g). Macrophages (M), qui sont pour la p...

Watch this Scientific Journal Video about Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis at JoVE.com

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Nous décrivons ici une méthode efficace pour étudier la dynamique de la clairance des cellules apoptotiques<em&gt; In vivo</em&gt;. Cette méthode emploie vivre<em&gt; Drosophila</em&gt; Embryons comme un modèle puissant pour la surveillance phagocytose des cellules apoptotiques en utilisant un marquage spécifique des cellules apoptotiques et les phagocytes.

Vivre imagerie de dédouanement cellulaire par apoptose au cours<em&gt; Drosophila</em&gt; Embryogenèse

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

live-imaging-apoptotic-cell-clearance-during-drosophila

Research

JoVE Journal

Biology

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

13.4K vues.  Technion-Israel Institute of Technology. Nous décrivons ici une méthode efficace pour étudier la dynamique de la clairance des cellules apoptotiques In vivo. Cette méthode emploie vivre Drosophila Embryons comme un modèle puissant pour la surveillance phagocytose des cellules apoptotiques en utilisant un marquage spécifique des cellules apoptotiques et les phagocytes.

Vidéo: Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Live Imaging of Glial Cell Migration in the Drosophila Eye Imaginal Disc

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons. While recent progress has been made to describe the live migration of glial cells in the developing pupal wing (1), studies of Drosophila glial cell migration have typically involved the examination of fixed tissue. Live microscopic analysis of motile cells offers the ability to examine cellular behavior throughout the migratory process, including determining the rate of and changes in direction of growth. Paired with use of genetic tools, live imaging can be used to determine more precise roles for specific genes in the process of development. Previous work by Silies et al. (2) has described the migration of glia originating from the optic stalk, a structure that connects the developing eye and brain, into the eye imaginal disc in fixed tissue. Here we outline a protocol for examining the live migration of glial cells into the Drosophila eye imaginal disc. We take advantage of a Drosophila line that expresses GFP in developing glia to follow glial cell progression in wild type and in mutant animals.

Here we describe a protocol to examine the migration of glial cells into the developing Drosophila eye using live microscopic analysis paired with GFP tagged glial cells.

Imagerie de la migration en direct des cellules gliales dans le disque Eye drosophile Imaginal

Glial cells of both vertebrate and invertebrate organisms must migrate to final target regions in order to ensheath and support associated neurons.  While ...

Recherche

Biologie

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

En direct de l&#39;imagerie Drosophila melanogaster Migrations hémocytaires embryonnaires

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

Préparation de développement et de l&#39;adulte Drosophile Cerveaux et la rétine d&#39;imagerie en temps réel

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

A Reverse Genetic Approach to Test Functional Redundancy During Embryogenesis

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In the zebrafish model, effective reverse genetic techniques have been developed using microinjection of gene-specific antisense morpholinos. Morpholinos target an mRNA through specific base-pairing and block gene function transiently by inhibiting translation or splicing for several days during embryogenesis (knockdown). However, in vertebrates such as mouse or zebrafish, some gene functions can be obscured by these approaches due to the presence of another gene that compensates for the loss. This is especially true for gene families containing sister genes that are co-expressed in the same developing tissues. In zebrafish, functional compensation can be tested in a relatively high-throughput manner, by co-injection of morpholinos that target knockdown of both genes simultaneously. Likewise, using morpholinos, a genetic interaction between any two genes can be demonstrated by knockdown of both genes together at sub-threshold levels. For example, morpholinos can be titrated such that neither individual knockdown generates a phenotype. If, under these conditions, co-injection of both morpholinos causes a phenotype, a genetic interaction is shown. Here we demonstrate how to show functional redundancy in the context of two related GATA transcription factors. GATA factors are essential for specification of cardiac progenitors, but this is revealed only by the loss of both Gata5 and Gata6. We show how to carry out microinjection experiments, validate the morpholinos, and evaluate the compensated phenotype for cardiogenesis.

Gene function can be obscured in loss-of-function experiments if there is compensation by another gene. The zebrafish model provides a relatively high-throughput means to reveal such functional redundancy in living embryos.

Une approche de génétique inverse pour tester redondance fonctionnelle pendant l&#39;embryogenèse

Gene function during embryogenesis is typically defined by loss-of-function experiments, for example by targeted mutagenesis (knockout) in the mouse. In ...

Preparing Individual Drosophila Egg Chambers for Live Imaging

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, cell differentiation and organogenesis 1. Correct preparation of the experimental samples is a crucial, often neglected, step. The goal of preparation is to ensure physiological relevance and to establish optimal imaging conditions. To maintain tissue viability, it is critical to avoid dehydration, hypoxia, overheating or medium deterioration 2. 
 The Drosophila egg chamber is a well established system for examining questions relating, but not limited, to body patterning, mRNA localization and cytoskeletal organization 3,4. For early- and mid-stage egg chambers, mounting in halocarbon oil is good for survival in that it allows free diffusion of oxygen, prevents dehydration and hypoxia and has superb optical properties for microscopy. Imaging of fluorescent proteins is possible through the introduction of transgenes into the egg chamber or physical injection of labeled RNA, protein or antibodies 5-7. For example, addition of MS2 constructs to the genome of animals enables real time observation of mRNAs in the oocyte 8. These constructs allow for in vivo labeling of mRNA through utilization of the MS2 bacteriophage RNA stem loop interaction with its coat protein 9. 
 Here, we present a protocol for the extraction of ovaries as well as isolating individual ovarioles and egg chambers from the female Drosophila. For a detailed description of Drosophila oogenesis see Allan C. Spradling (1993, reprinted 2009) 10.

Preparing Individual Drosophila Egg Chambers for Live Imaging

The Drosophila egg chamber is an excellent model for studying the mechanisms of mRNA localization. In order to capture the dynamic events that underpin the processes of localization, rapid high resolution imaging of live tissue is required. Here, we present a protocol for dissection and imaging of live samples with minimal disruption.

Préparation individuelle Drosophila Œuf Chambres pour l&#39;imagerie en direct

Live cell imaging is an important technique applied to a number of Drosophila tissues used as models to investigate topics such as axis specification, ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

Vivez l&#39;imagerie des protéines GFP-étiquetés<em&gt; Drosophila</em&gt; Les ovocytes

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

4D imagerie de l&#39;agrégation des protéines dans les cellules vivantes

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Drosophila eye is widely used as a model for studies of development and neuronal degeneration. With the powerful mitotic recombination technique, elegant genetic screens based on clonal analysis have led to the identification of signaling pathways involved in eye development and photoreceptor (PR) differentiation at larval stages. We describe here the Tomato/GFP-FLP/FRT method, which can be used for rapid clonal analysis in the eye of living adult Drosophila. Fluorescent photoreceptor cells are imaged with the cornea neutralization technique, on retinas with mosaic clones generated by flipase-mediated recombination. This method has several major advantages over classical histological sectioning of the retina: it can be used for high-throughput screening and has proved an effective method for identifying the factors regulating PR survival and function. It can be used for kinetic analyses of PR degeneration in the same living animal over several weeks, to demonstrate the requirement for specific genes for PR survival or function in the adult fly. This method is also useful for addressing cell autonomy issues in developmental mutants, such as those in which the establishment of planar cell polarity is affected.

The Tomato/GFP-FLP/FRT Method for Live Imaging of Mosaic Adult Drosophila Photoreceptor Cells

The Tomato/GFP-FLP/FRT method involves visualizing mosaic photoreceptor cells in living Drosophila. It can be used to follow individual photoreceptor cell fates in the retina for days or weeks. This method is ideal for studies of retinal degeneration and neurodegenerative diseases or photoreceptor cell development.

Le / GFP-FLP Méthode tomate / FRT pour l&#39;imagerie en direct de mosaïque adulte<em&gt; Drosophila</em&gt; cellules photoréceptrices

The Drosophila eye is widely used as a model  for studies of development and neuronal degeneration. With the powerful mitotic  recombination technique, ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

Cytologique Analyse de la spermatogenèse: Préparatifs en direct et fixes de<em&gt; Drosophila</em&gt; Testicules

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by available technologies, apoptosis is generally assumed to occur shortly thereafter. Caspases can cleave many functional and structural components to cause rapid and complete cell destruction within a few minutes. However, accumulating evidence indicates that in normal healthy cells the same caspases have other functions, presumably at lower enzymatic levels. Studies of non-apoptotic caspase activity have been hampered by difficulties with detecting low levels of caspase activity and with tracking ultimate cell fate in vivo. Here, we illustrate the use of an ultrasensitive caspase reporter, CaspaseTracker, which permanently labels cells that have experienced caspase activity in whole animals. This in vivo dual color CaspaseTracker biosensor for Drosophila melanogaster transiently expresses red fluorescent protein (RFP) to indicate recent or on-going caspase activity, and permanently expresses green fluorescent protein (GFP) in cells that have experienced caspase activity at any time in the past yet did not die. Importantly, this caspase-dependent in vivo biosensor readily reveals the presence of non-apoptotic caspase activity in the tissues of organ systems throughout the adult fly. This is demonstrated using whole mount dissections of individual flies to detect biosensor activity in healthy cells throughout the brain, gut, malpighian tubules, cardia, ovary ducts and other tissues. CaspaseTracker detects non-apoptotic caspase activity in long-lived cells, as biosensor activity is detected in adult neurons and in other tissues at least 10 days after caspase activation. This biosensor serves as an important tool to uncover the roles and molecular mechanisms of non-apoptotic caspase activity in live animals.

In Vivo Biosensor Tracks Non-apoptotic Caspase Activity in Drosophila

To detect healthy cells in whole animals that contain low levels of caspase activity, the highly sensitive biosensor designated CaspaseTracker was generated for Drosophila. Caspase-dependent biosensor activity is detected in long-lived healthy cells throughout the internal organs of adult animals reared under optimized conditions in the absence of death stimuli.

In Vivo Biocapteur Tracks apoptotique non-activité Caspase chez la drosophile

Caspases are the key mediators of apoptotic cell death via their proteolytic activity. When caspases are activated in cells to levels detectable by ...