The authors wish to thank Drs. Sung-wan An and Mike O&#8217;Donnell for practical advice on establishing this assay, Dr. Chih-Jen Cheng for helpful discussions on the use of ion-specific electrodes, and Dr. Chou-Long Huang for his mentorship and support. This work was supported by the National Institutes of Health (K08DK091316 to ARR) and the American Society of Nephrology Gottschalk Award to ARR.

<ol>
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The authors have nothing to disclose.

L&#39;utilisation du test Ramsay, avec électrodes spécifiques des ions, permet de mesurer les taux de sécrétion des fluides et des flux d&#39;ions dans insecte isolé Malpighi (rénales) tubules. Vingt ou plusieurs tubes peuvent être testés à la fois, ce qui permet un débit plus élevé par rapport au dosage de l&#39;individu dans les tubules in vitro microperfused. En outre, des électrodes spécifiques des ions permettent la détermination des concentrations ioniques dans le fluide sécrété in s...

<html> Rénale transport ionique épithéliale sous-tend organismal ionosphère et osmorégulation. Le Drosophila melanogaster Malpighian (rénale) tubule offre une occasion unique d&#39;étudier les mécanismes moléculaires de transport d&#39;ions épithéliale. Cela est dû à la combinaison de la génétique de la drosophile puissants, associé à l&#39;accessibilité de ses tubules rénaux à l&#39;étude physiologique. Le dosage Ramsay, nommé d&#39;après le chercheur pionnier de la technique 1, mesure les taux de sécrétion de fluides à partir de tubes de Malpighi isolées, et a été établie chez la drosophile en 1994 par Dow et ses collègues 2. Cela a ouvert la voie à d&#39;autres études utilisant la drosophile outils génétiques, telles que le système GAL4 UAS 3,4, pour définir les voies de signalisation cellulaires spécifiques régissant la sécrétion de fluide. Un exemple comprend la signalisation du calcium en réponse à une hormone peptidique 5, parmi beaucoup d&#39;autres 6,7. ve_content &quot;&gt; Une combinaison de techniques génétiques et étude physiologique classique a montré que la production d&#39;urine chez la mouche se produit par la sécrétion d&#39;un chlorure de potassium riche en fluide à partir du segment principal du tube. Ceci se produit par l&#39;intermédiaire du transépithélial sécrétion parallèle de cations, principalement K + mais aussi Na +, à travers la cellule principale, et Cl -. sécrétion à travers la cellule stellaire 8-12 La capacité de mesurer séparément transépithéliaux flux K + et Na + permet une caractérisation plus détaillée des mécanismes de transport que la mesure de la sécrétion de fluide seul. Par exemple, dans les tubules Drosophila non stimulées, la Na + / K + -ATPase ouabaïne n&#39;a aucun effet sur ​​la sécrétion de fluide 2, même lorsque son absorption dans les cellules principales est inhibée par l&#39;inhibiteur de transport anionique organique taurocholate 13. Cependant, Linton et O&#39;Donnell a montré que l&#39;ouabaïne dépolarisele potentiel de la membrane basolatérale, Na + et augmente le flux 9. Comme le montrent les résultats représentatifs, nous avons reproduit ces résultats, et montré que flux de K + est concomitante diminué de 14; l&#39;augmentation du flux de Na + et K + a diminué flux des effets opposés sur la sécrétion de fluide, de sorte qu&#39;aucun changement net de la sécrétion. Ainsi, il ya deux résolutions au «paradoxe ouabaïne», à savoir l&#39;observation initiale que ouabaïne n&#39;a aucun effet sur ​​la sécrétion de fluide dans le tubule Drosophila:. La première, dans les tubules stimulés, l&#39;effet de la ouabaïne sur la sécrétion de fluide est pas évident en raison de son absorption par le système de transport anionique organique 13; et la seconde, dans les tubules non stimulées, ouabaïne a des effets sur transepithelial Na + et K + flux opposés, entraînant aucun changement net de la sécrétion de fluide (voir résultats représentatifs et réf. 9). Par conséquent, le rôle principal de la Na + / K + ATPase dans les tubules non stimulées est de réduire la concentration intracellulaire de Na + à générer un gradient de concentration favorable pour Na + -coupled processus de transport à travers la membrane basolatérale. En effet, en mesurant séparément Na + et K + flux, nous avons démontré que les tubules manque la volée sodium-potassium-2-chlorure cotransporteur (NKCC) ont diminué K transepithelial + flux, sans diminution supplémentaire après addition ouabaïne, et aucun changement dans transepithelial Na + flux 14. Ces résultats ont appuyé notre conclusion que Na + entrant dans la cellule par l&#39;intermédiaire du NKCC est recyclée à travers la Na + / K + -ATPase. Dans un autre exemple, Ianowski et al. Observe que l&#39;abaissement de bain concentration de K + de 10 à 6 mm diminué transepithelial flux de K + et une augmentation transepithelial Na + flux dans les tubules de Rhodnius prolixus, avec aucun changement net de la sécrétion de fluide <sup&gt; 15. Les effets différentiels sur Na + et K + flux flux à travers les tubules larvaires ont également été observés dans les tubules de drosophile en réponse à divers régimes de sel 16 et dans deux espèces de moustiques en réponse à l&#39;élevage de la salinité 17. La plus grande difficulté dans la mesure de flux transépithélial d&#39;ions dans la préparation d&#39;essai Ramsay est la détermination des concentrations des ions dans le fluide sécrété. Ce défi a été relevé avec différentes solutions, y compris la flamme photometery 18, l&#39;utilisation d&#39;ions radioactifs 19 et sonde électronique longueur d&#39;onde de la spectroscopie à dispersion 20. Ces techniques nécessitent le transfert de la goutte de fluide sécrété à un instrument de mesure des concentrations d&#39;ions. Etant donné que le volume de fluide sécrété par les tubules Drosophila non stimulé est petite, typiquement environ 0,5 nl / min, ce qui pose un défi technique et introduit également erreur si une partie du fluide est sécrétéperdu au moment du transfert. En revanche, l&#39;utilisation d&#39;électrodes spécifiques des ions permet la mesure de l&#39;activité ionique (à partir de laquelle la concentration en ions peut être calculée) in situ. Le protocole actuel a été adaptée à partir de celle utilisée par les Maddrell et collègues pour mesurer transépithélial flux de K + à travers le tube Rhodnius utilisant la valinomycine comme K + ionophore 21, et décrit également l&#39;utilisation d&#39;un -butylcalix 4- tert [4] d&#39;acides arène-tétraacétique Na tétraéthyle à base d&#39;ester + électrode spécifique-ion spécifique de caractérisé par Messerli et. . 22 al. Électrodes ions spécifiques ont également été utilisés pour mesurer les concentrations d&#39;ions dans le liquide sécrété par les tubules de Malpighi dans le dosage Ramsay chez l&#39;adulte et larvaire 9,23 16 Drosophila melanogaster, la Nouvelle-Zélande Alpine Weta (Hemideina maori) 24 et 17 chez les moustiques. Ici, nous décrivons en détail l&#39;utilisation de la Ramsay commedire pour mesurer les taux de sécrétion de fluide dans les tubes de Malpighi de Drosophila melanogaster, ainsi que l&#39;utilisation d&#39;électrodes spécifiques des ions afin de déterminer les concentrations de K + et Na + dans le fluide sécrété, et donc le calcul des flux transépithélial d&#39;ions. Une vue d&#39;ensemble de l&#39;essai est fourni à la figure 1. <img alt="Figure 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Figure 1. Schéma de l&#39;Malpighian Pailles et le test Ramsay avec utilisation de l&#39;ion-spécifiques électrodes pour mesurer les concentrations d&#39;ions. Cette figure illustre la configuration pour le dosage Ramsay. (A) Pour chaque volée comporte quatre tubes, une paire de tubules antérieure et une paire de tubules postérieures, qui flottent dans la cavité abdominale entouré d&#39;hémolymphe. Dans chaque paire, les deux tubes se rejoignent à l&#39;uretère, qui se jette ensuite l&#39;urine à la jonction de l&#39;intestin moyen et hindgut. Les tubules sont aveugles-clos. L&#39;urine est généré par le segment principal de fluide sécrétant (en rouge), et coule vers l&#39;uretère et sortir dans l&#39;intestin. Après dissection, la paire de tubule est dissocié à partir de l&#39;intestin par transection l&#39;uretère. (B) La paire de tubes est ensuite transféré dans une gouttelette de solution saline dans un bain bien du plat de dosage. L&#39;un des deux tubes, appelé ici le &quot;tube d&#39;ancrage», est enroulé autour d&#39;une tige de métal et est inerte. L&#39;autre tube est le tube de sécrétion. Le segment initial (qui ne sécrètent pas fluide) et le segment principal de la tubule sécrétant restent dans la gouttelette de solution saline de baignade. Ions et mouvement de l&#39;eau de la solution saline de bain et dans la lumière des tubules du segment principal, puis se déplacent vers l&#39;uretère, comme cela se produirait in vivo. Le segment inférieur (bleu) est en dehors de la solution saline de baignade et donc inerte. Depuis l&#39;uretère est coupé, le fluide sécrété apparaît comme une gouttelette à partir de la fin de l&#39;uretère de coupe. Til agrandit gouttelette de fluide sécrété dans le temps comme la sécrétion continue, et son diamètre est mesuré en utilisant un micromètre oculaire. Une couche d&#39;huile minérale empêche l&#39;évaporation du fluide sécrété. Les électrodes spécifiques référence et d&#39;ions de mesurer la concentration en ions du fluide sécrété. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a>

<table>
	<tbody>
		<tr>
			<td>Sylgard 184 Silicone Elastomer Kit</td>
			<td>Ellsworth Adhesives</td>
			<td>http://www.ellsworth.com/dow-corning-sylgard-184-silicone-encapsulant-0-5kg-kit-clear/</td>
			<td>May be purchased from multiple distributors</td>
		</tr>
		<tr>
			<td>Petri dish, polystyrene, 100 mm x 15 mm</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Petri dish, polystyrene, 35 mm x 10 mm</td>
			<td>Corning Life Sciences</td>
			<td>Fisher 08-757-100A</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Scalpel Handle #3</td>
			<td>Fine Science Tools</td>
			<td>10003-12</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Scalpel Blades #1</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td>Specific brand is not important; use appropriate sharps precautions</td>
		</tr>
		<tr>
			<td>Needle, 30G x 1/2</td>
			<td>Becton Dickinson</td>
			<td>305106</td>
			<td>Use appropriate sharps precautions</td>
		</tr>
		<tr>
			<td>Minutien pins, black anodized, 0.15 mm</td>
			<td>Fine Science Tools</td>
			<td>26002-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereomicroscope with ocular micrometer</td>
			<td>Nikon</td>
			<td>SMZ800</td>
			<td>Specific brand is not important; this is given as an example</td>
		</tr>
		<tr>
			<td>Sheet of black stained glass, 3 mm (1/8 inch) thick</td>
			<td>Hobby shop</td>
			<td></td>
			<td>Example includes Spectrum Black Opal by Spectrum Glass (http://www.delphiglass.com/spectrum-glass/opalescent/spectrum-black-opal)</td>
		</tr>
		<tr>
			<td>Glass cutting tools (glass cutter, glass cutting pliers)</td>
			<td>Hobby shop</td>
			<td></td>
			<td>Examples include the Studio Pro Lightweight Running Pliers by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/pliers-nippers/studio-pro-lightweight-running-pliers) and the Studio Pro Brass Glass Cutter by Diamond Tech (http://www.delphiglass.com/glass-cutters-tools/glass-cutters/studio-pro-brass-glass-cutter). Use appropriate safety precautions when cutting glass</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary tube, unfilamented, GC120-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td>
			<td>Warner Instruments</td>
			<td>30-0042</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary tube, filamented, GC120F-10, OD 1.2 mm, ID 0.69 mm, length 10 cm</td>
			<td>Warner Instruments</td>
			<td>30-0044</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitric acid, 70%</td>
			<td>Sigma</td>
			<td>438073</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines. Specific brand is not important</td>
		</tr>
		<tr>
			<td>Cimarec 7 in x 7 in hotplate</td>
			<td>Fisher</td>
			<td>11675911Q</td>
			<td>Specific brand is not important; caution when heated</td>
		</tr>
		<tr>
			<td>Selectophore dichlorodimethylsilane</td>
			<td>Sigma</td>
			<td>40136-1ML</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td>
		</tr>
		<tr>
			<td>Two-step vertical pipet puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td>Other pipet pullers can be used; this is given as an example</td>
		</tr>
		<tr>
			<td>Glass petri dish, 150 mm diameter x 15 mm height</td>
			<td>Fisher</td>
			<td>08-748E</td>
			<td>Specific brand is not important; only one dish needed</td>
		</tr>
		<tr>
			<td>World Precision Instruments E210 1 mm micropipette storage jar</td>
			<td>Fisher</td>
			<td>50-821-852</td>
			<td>May be available from other distributors. Useful to have two jars. Note that although this jar is specified for 1 mm pipets, and the pipets used here are 1.2 mm, in our experience the 1 mm jar works best for the 1.2 mm pipets.</td>
		</tr>
		<tr>
			<td>Silica Gel, Tel-Tale Desiccant, indicating, 10-18 mesh</td>
			<td>Fisher</td>
			<td>S161-500</td>
			<td>Indicating silica useful for determining whether silica gel retains desiccating ability</td>
		</tr>
		<tr>
			<td>World Precision Instruments MicroFil, 34G</td>
			<td>Fisher</td>
			<td>50-821-914</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>1 ml syringe with luer lock</td>
			<td>Becton Dickinson</td>
			<td>309659</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>3 ml syringe with luer lock</td>
			<td>Becton Dickinson</td>
			<td>309657</td>
			<td>May be available from other distributors.</td>
		</tr>
		<tr>
			<td>D300 3-way stopcock with female luer lock inlet port, male luer outlet port with rotating collar and guard</td>
			<td>Cole-Parmer</td>
			<td>UX-30600-02</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Female Luer Locking Connector</td>
			<td>4 Medical Solutions</td>
			<td>ADC 9873-10</td>
			<td>Specific brand is not important; barbed end is ~4 mm at narrowest point and ~7 mm at widest point.</td>
		</tr>
		<tr>
			<td>Silicone Tubing I.D. x O.D. x Wall: 1/16 x 1/8 x 1/32 in. (1.59 x 3.18 x 0.79 mm)</td>
			<td>Fisher</td>
			<td>14-179-110</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>E-3603 tubing, I.D. x O.D.: 1/32 x 3/32 in</td>
			<td>Fisher</td>
			<td>14171208</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Modeling clay</td>
			<td></td>
			<td></td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>Selectophore potassium ionophore I, cocktail B</td>
			<td>Sigma</td>
			<td>99373</td>
			<td>CAUTION: see Material Data Safety Sheet for appropriate storage and handling guidelines</td>
		</tr>
		<tr>
			<td>Selectophore sodium ionophore X</td>
			<td>Sigma</td>
			<td>71747</td>
			<td>Sodium ionphore X = 4-tert-butylcalix[4]arene-tetraacetic acid tetraethylester</td>
		</tr>
		<tr>
			<td>Selectophore 2-nitrophenyl octyl ether</td>
			<td>Sigma</td>
			<td>73732</td>
			<td></td>
		</tr>
		<tr>
			<td>Selectophore sodium tetraphenylborate</td>
			<td>Sigma</td>
			<td>72018</td>
			<td></td>
		</tr>
		<tr>
			<td>Schneider&#39;s Drosophila medium</td>
			<td>Life Technologies</td>
			<td>21720024</td>
			<td></td>
		</tr>
		<tr>
			<td>High impedance electrometer</td>
			<td>World Precision Instruments</td>
			<td>FD223a</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder 1 mm with 45&#176; body, vented, with handle</td>
			<td>Warner Instruments</td>
			<td>64-1051</td>
			<td></td>
		</tr>
		<tr>
			<td>Microelectrode holder 1 mm with straight body, vented</td>
			<td>Warner Instruments</td>
			<td>64-1007</td>
			<td></td>
		</tr>
		<tr>
			<td>Silver wire</td>
			<td>Warner Instruments</td>
			<td>64-1318</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulators, pair</td>
			<td>Leitz</td>
			<td></td>
			<td>Various brands/models will work; this is an example</td>
		</tr>
		<tr>
			<td>Faraday cage</td>
			<td>Technical Manufacturing Corporation</td>
			<td>81-334-03</td>
			<td>This is an example; any Faraday cage will work</td>
		</tr>
		<tr>
			<td>Single gooseneck fiberoptic light</td>
			<td>Nikon</td>
			<td></td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>mineral oil</td>
			<td>Fisher</td>
			<td>BP-2629</td>
			<td>Specific brand is not important</td>
		</tr>
		<tr>
			<td>forceps, Dumont #5 with Biologie tip</td>
			<td>Fine Science Tool</td>
			<td>11295-10</td>
			<td>May be available from other distributors.</td>
		</tr>
	</tbody>
</table>

use of the ramsay assay to measure fluid secretion and ion flux rates in the drosophila melanogaster malpighian tubule

Modulation de transport d&#39;ions rénale épithéliale organismes permet de maintenir l&#39;homéostasie ionique et osmotique dans la face de la variation des conditions extérieures. Le Drosophila melanogaster Malpighian (rénale) tubule offre une occasion unique d&#39;étudier les mécanismes moléculaires de transport d&#39;ions épithéliale, en raison de la génétique puissants de cet organisme et de l&#39;accessibilité de ses tubules rénaux à l&#39;étude physiologique. Ici, nous décrivons l&#39;utilisation de l&#39;essai Ramsay pour mesurer les taux de sécrétion de liquide de mouche isolé tubules rénaux, avec l&#39;utilisation d&#39;électrodes spécifiques des ions pour mesurer les concentrations de sodium et de potassium dans le liquide sécrété. Ce test permet d&#39;étudier des liquides ioniques et transépithélial flux de ~ 20 tubes à la fois, sans la nécessité de transférer le fluide sécrété à un appareil séparé pour mesurer les concentrations d&#39;ions. Génétiquement tubules distinctes peuvent être analysées pour évaluer le rôle des gènes spécifiques dans les processus de transport. En outre, le bsaline athing peut être modifié pour examiner les effets de ses caractéristiques chimiques, ou des médicaments ou des hormones ajoutées. En résumé, cette technique permet la caractérisation moléculaire des mécanismes de base de transport epithelial d&#39;ions dans le tubule Drosophila, ainsi que la régulation de ces mécanismes de transport.

Les figures 7 et 8 démontrent que l&#39;utilisation de l&#39;essai Ramsay avec des électrodes spécifiques des ions pour mesurer les concentrations de K + et Na + peut distinguer génétiquement et pharmacologiquement distinct K + et les flux de Na +, des informations qui ne sont pas capturés par la mesure des taux de sécrétion de liquide seul. Figure La figure 7 montre que diminution de la sécrétion de fluide dans les tubes de vol port...

Watch this Scientific Journal Video about Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule at JoVE.com

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Ce protocole décrit l&#39;utilisation de l&#39;essai Ramsay pour mesurer les taux de sécrétion de liquide de isolés Malpighian (rénale) tubules de Drosophila melanogaster. En outre, l&#39;utilisation d&#39;électrodes spécifiques des ions pour mesurer les concentrations de sodium et de potassium dans le fluide sécrété, permettant le calcul de flux transépithélial d&#39;ions, est décrit.

L&#39;utilisation du test Ramsay mesurer la sécrétion de liquide et de flux d&#39;ions dans le Tarifs<em&gt; Drosophila melanogaster</em&gt; Tubule Malpighian

Modulation of renal epithelial ion transport allows organisms to maintain ionic and osmotic homeostasis in the face of varying external conditions. The ...

use-ramsay-assay-to-measure-fluid-secretion-ion-flux-rates-drosophila

Research

JoVE Journal

Biology

Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

11.7K vues.  University of Texas Southwestern Medical Center.  Ce protocole décrit l'utilisation de l'essai Ramsay pour mesurer les taux de sécrétion de liquide de isolés Malpighian (rénale) tubules de Drosophila melanogaster. En outre, l'utilisation d'électrodes spécifiques des ions pour mesurer les concentrations de sodium et de potassium dans le fluide sécrété, permettant le calcul de flux transépithélial d'ions, est décrit. 

Vidéo: Use of the Ramsay Assay to Measure Fluid Secretion and Ion Flux Rates in the Drosophila melanogaster Malpighian Tubule

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Techniques de microinjection pour l&#39;étude de la mitose dans les Drosophila melanogaster Embryon syncytial

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Recherche

Biologie

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Photobleaching essais (FRAP et FLIP) pour mesurer la dynamique des protéines de la chromatine dans Vivre cellules souches embryonnaires

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function to maintain glucose homeostasis during developmental larval stages 1,2. Studies on largely post-mitotic adult flies have revealed perturbation of glucose homeostasis as the result of genetic ablation of insulin-like peptide (ILP) producing cells (IPCs) 3. Thus, adult fruit flies hold great promise as a suitable genetic model system for metabolic disorders including type II diabetes. To further develop the fruit fly system, comparable physiological assays used to measure glucose tolerance and insulin sensitivity in mammals must be established. To this end, we have recently described a novel procedure for measuring oral glucose tolerance response in the adult fly and demonstrated the importance of adult IPCs in maintaining glucose homeostasis 4,5. Here, we have modified a previously described procedure for insulin injection 6 and combined it with a novel hemolymph extraction method to measure peripheral insulin sensitivity in the adult fly. Uniquely, our protocol allows direct physiological measurements of the adult fly's ability to dispose of a peripheral glucose load upon insulin injection, a methodology that makes it feasible to characterize insulin signaling mutants and potential interventions affecting glucose tolerance and insulin sensitivity in the adult fly.

Insulin Injection and Hemolymph Extraction to Measure Insulin Sensitivity in Adult Drosophila melanogaster

Conserved insulin signaling pathways found in the fruit fly Drosophila melanogaster make this organism a potential tool for modeling metabolic disorders including type II diabetes. To this end, it is critical to establish physiological assays to effectively measure systemic insulin action in peripheral glucose disposal in the adult fly.

Injection d&#39;insuline et d&#39;extraction hémolymphe pour mesurer la sensibilité d&#39;insuline dans les adultes Drosophila melanogaster</em

Conserved nutrient sensing mechanisms exist between mammal and fruit fly where peptides resembling mammalian insulin and glucagon, respectively function ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Mesure de la durée de vie en<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and 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  ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imagerie à travers le cas de la nymphe<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

Immunofluorescence quantitative pour mesurer la variation des taux de protéines au centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Optique pH Quantification d’intracellulaire dans l’épithélium des tubules Malpighian Drosophila melanogaster ayant un pH fluorescents génétiquement codé indicateur

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Oral Bacterial Infection and Shedding in Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic infections, there has been a recent increase of interest in the mechanisms of gut immunocompetence to pathogens, which require methods to orally infect flies. Here we present a protocol to orally expose individual flies to an opportunistic bacterial pathogen (Pseudomonas aeruginosa) and a natural bacterial pathogen of D. melanogaster (Pseudomonas entomophila). The goal of this protocol is to provide a robust method to expose male and female flies to these pathogens. We provide representative results showing survival phenotypes, microbe loads, and bacterial shedding, which is relevant for the study of heterogeneity in pathogen transmission. Finally, we confirm that Dcy mutants (lacking the protective peritrophic matrix in the gut epithelium) and Relish mutants (lacking a functional immune deficiency (IMD) pathway), show increased susceptibility to bacterial oral infection. This protocol, therefore, describes a robust method to infect flies using the oral route of infection, which can be extended to the study of a variety genetic and environmental sources of variation in gut infection outcomes and bacterial transmission.

Oral Bacterial Infection and Shedding in Drosophila melanogaster

This protocol describes methods to orally expose and infect the fruit fly Drosophila melanogaster with bacterial pathogens, and to measure the number of infectious bacteria shed following gut infection. We further describe the effect of immune mutants on fly survival following oral bacterial infection.

Oral Infection bactérienne et excrétion chez Drosophila melanogaster

The fruit fly Drosophila melanogaster is one of the best developed model systems of infection and innate immunity. While most work has focused on systemic ...

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell imaging studies. Methods for detecting RNAs have expanded greatly since seminal studies linked site-specific mRNA localization to gene expression regulation. Dynamic mRNA processes can now be visualized via approaches that detect mRNAs, coupled with microscopy set-ups that are fast enough to capture the dynamic range of molecular behavior. The molecular beacon technology is a hybridization-based approach capable of direct detection of endogenous transcripts in living cells. Molecular beacons are hairpin-shaped, internally quenched, single-nucleotide discriminating nucleic acid probes, which fluoresce only upon hybridization to a unique target sequence. When coupled with advanced fluorescence microscopy and high-resolution imaging, they enable one to perform spatial and temporal tracking of intracellular movement of mRNAs. Although this technology is the only method capable of detecting endogenous transcripts, cell biologists have not yet fully embraced this technology due to difficulties in designing such probes for live cell imaging. A new software application, PinMol, allows for enhanced and rapid design of probes best suited to efficiently hybridize to mRNA target regions within a living cell. In addition, high-resolution, real-time image acquisition and current, open source image analysis software allow for a refined data output, leading to a finer evaluation of the complexity underlying the dynamic processes involved in the mRNA&#39;s life cycle.
Here we present a comprehensive protocol for designing and delivering molecular beacons into Drosophila melanogaster egg chambers. Direct and highly specific detection and visualization of endogenous maternal mRNAs is performed via spinning disc confocal microscopy. Imaging data is processed and analyzed using object detection and tracking in Icy software to obtain details about the dynamic movement of mRNAs, which are transported and localized to specialized regions within the oocyte.

Visualizing and Tracking Endogenous mRNAs in Live Drosophila melanogaster Egg Chambers

Here, we present a protocol for the visualization, detection, analysis and tracking of endogenous mRNA trafficking in live Drosophila melanogaster egg chamber using molecular beacons, spinning disc confocal microscopy, and open-source analysis software.

Visualisation et suivi des ARNm endogènes dans Live Drosophila melanogaster Egg Chambers

Fluorescence-based imaging techniques, in combination with developments in light microscopy, have revolutionized how cell biologists conduct live cell ...