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.

Die Verwendung des Ramsay Assay zusammen mit ionenspezifischen Elektroden, ermöglicht die Messung der Fluid Sekretionsraten und Ionenströme in isolierten Insekten Malpighian (Niereninsuffizienz) Tubuli. Zwanzig oder mehr Tubuli kann zu einem Zeitpunkt untersucht werden, was einen höheren Durchsatz im Vergleich zu dem Assay in vitro einzelne microperfused Tubuli. Zusätzlich ionenspezifischen Elektroden erlauben die Bestimmung von Ionenkonzentrationen in der sekretierten Flüssigkeit in situ, die Beg...

<html> Nierenepithelzellen Ionentransport zugrunde organismal iono- und Osmoregulation. Die Drosophila melanogaster Malpighischen (Nieren) Tubulus bietet eine einmalige Gelegenheit, um die molekularen Mechanismen der epithelialen Ionentransport zu studieren. Dies ist aufgrund der Kombination der leistungsfähigen Genetik von Drosophila, gepaart mit der Zugänglichkeit der Nierentubuli auf physiologische Studie. Die Ramsay Assay, nach dem Forscher, der die Technik 1 Pionier benannt, misst Flüssigkeitssekretion Raten von isolierten Malpighischen, und wurde in Drosophila 1994 von Dow und Kollegen 2 etabliert. Dies ebnete den Weg für weitere Studien unter Verwendung von Drosophila genetische Werkzeuge, wie beispielsweise die GAL4-UAS-System 3,4, um zellspezifische Signalwege regulieren Flüssigkeitssekretion definieren. Ein Beispiel beinhaltet Calcium-Signal in Reaktion auf ein Peptidhormon, 5, unter vielen anderen 6,7. ve_content &quot;&gt; eine Kombination von genetischen Techniken und klassischen physiologischen Studie hat gezeigt, dass Urin-Generation gezeigt, in die Fliege durch die Sekretion eines Kaliumchlorid-reiche Flüssigkeit aus dem Hauptsegment der Röhrchen erfolgt. erfolgt dies durch die parallele transepithelialen Sekretion von Kationen, in erster Linie K +, sondern auch Na +, durch die Hauptzelle und Cl -. Sekrets durch die Sternzellen 8-12 Die Fähigkeit, transepithelialen K + und Na + Flüsse getrennt zu messen ermöglicht eine genauere Charakterisierung von Transportmechanismen als die Messung Flüssigkeitssekretion allein, z. B. in unstimulierten Drosophila Tubuli, die Na + / K + -ATPase-Inhibitors Ouabain hat keine Auswirkung auf Flüssigkeitssekretion 2, auch wenn ihre Aufnahme in den Hauptzellen wird durch den organischen Anionentransporter Inhibitor Taurocholat 13 gesperrt. Jedoch Linton und O&#39;Donnell zeigte, dass Ouabain depolarisiertder basolateralen Membranpotential und erhöht Na + Fluss 9. Wie in den Repräsentative Ergebnisse gezeigt, repliziert haben wir die Ergebnisse und zeigte, daß K + Flußmittel gleichzeitig um 14; die erhöhte Na + Fluss und verminderte K + Fluss haben entgegengesetzte Auswirkungen auf Flüssigkeitssekretion, was zu keiner Nettoänderung der Sekretion. So gibt es zwei Entschließungen zum &quot;Ouabain Paradox&quot;, das heißt, die anfängliche Beobachtung, dass keine Auswirkung auf Flüssigkeitssekretion im Drosophila-Röhrchen Ouabain:. Zunächst in stimulierten Tubuli, ist nicht ersichtlich, die Wirkung von Ouabain auf Flüssigkeitssekretion aufgrund seine Aufnahme durch den organischen Anionentransporter 13; und zweitens, in unstimulierten Tubuli, Ouabain hat gegenläufige Effekte auf transepithelialen Na + und K + Fluss, was zu keiner Nettoänderung der Flüssigkeitssekretion (siehe Repräsentative Ergebnisse und ref. 9). Die primäre Rolle der Na + / K + -ATPase in unstimulierten Tubuli ist es, intrazellulären Na + -Konzentration zu senken, um eine günstige Konzentrationsgradienten zu erzeugen für die Na + -gekoppelten Transportprozesse in der basolateralen Membran. Tat kann durch separates Messen Na + und K + Flüsse zeigten wir, dass Tubuli fehlt die Fliege Natrium-Kalium-2-Chlorid-Cotransporter (NKCC) haben transepithelialen K + Flusses vermindert, wobei keine weitere Abnahme nach Ouabain hinaus und keine Veränderung des transepithelialen Na + Fluss 14. Diese Ergebnisse unterstützten unsere Schlussfolgerung, dass Na + in die Zelle durch die NKCC wird durch die Na + / K + -ATPase recycelt. In einem anderen Beispiel Ianowski et al. Beobachtet, dass die Senkung Bad K + -Konzentration von 10 mM bis 6 mM verringert transepithelialen K + Flußmittel und erhöht den transepithelialen Na + Flusses in Tubuli aus Rhodnius prolixus, ohne Nettoänderung in Flüssigkeitssekretion <sup&gt; 15. Unterschiedliche Auswirkungen auf Na + und K + Flussflusses über Larven Tubuli wurden auch in Drosophila Tubuli als Reaktion auf variierende Salzdiäten 16 und in zwei Mückenarten in Reaktion auf die Aufzucht Salinität 17 beobachtet worden. Die größte Herausforderung bei der Messung des transepithelialen Ionenfluß im Ramsay Probenaufbereitung ist die Bestimmung von Ionenkonzentrationen in der sekretierten Flüssigkeit. Diese Herausforderung hat mit unterschiedlichen Lösungen, einschließlich flamm photometery 18, Verwendung von radioaktiven Ionen 19 und Elektronensonde Wellenlänge Spektroskopie 20 erfüllt sind. Diese Techniken erfordern Übertragung des sekretierten Fluidtropfen auf ein Instrument zur Messung von Ionenkonzentrationen. Da das Volumen der Flüssigkeit durch den unstimulierten Drosophila Tubulus sekretiert ist klein, typischerweise ~ 0,5 nl / min, stellt dies eine technische Herausforderung dar und führt auch Fehler, wenn einige der sekretierten Flüssigkeitverlor bei der Übertragung. Im Gegensatz dazu die Verwendung von ionenspezifischen Elektroden ermöglicht die Messung der Ionenaktivität (von dem Ionenkonzentration berechnet werden kann) in situ. Das aktuelle Protokoll wurde von dem von Maddrellschem und Kollegen zur transepithelialen K + Flusses über die Rhodnius Tubulus Messung mit Valinomycin als Ionophor K + 21 angepasst, und beschreibt auch die Verwendung eines 4-tert-Butylcalix [4] aren-tetraessigsäure Tetraethylester basierten Na + -spezifische ionenspezifischen Elektrode, gekennzeichnet durch Messerli et. al. 22. Ionenspezifischen Elektroden wurden ebenfalls verwendet, um die Ionenkonzentrationen in Flüssigkeit durch Malpighischen im Ramsay Assay bei Erwachsenen 9,23 und 16 Larven Drosophila melanogaster sezerniert zu messen, der Neuseeland-Alpine Weta (Hemideina maori) 24 und in Moskitos 17. Hier beschreiben wir im Detail die Verwendung des so Ramsaysage Flüssigkeitssekretion Raten in Malpighischen von Drosophila melanogaster, sowie die Verwendung von ionenspezifischen Elektroden, die Konzentrationen von K + und Na + innerhalb von sekretierten Flüssigkeit zu bestimmen und somit die Berechnung der transepithelialen Ionenflüsse zu messen. Eine Übersicht des Tests ist in Figur 1 vorgesehen. <img alt="Abbildung 1" src="/files/ftp_upload/53144/53144fig1.jpg" /> Abbildung 1. Schematische Darstellung des Vasa Malpighi und der Ramsay-Assay unter Verwendung von ionenspezifischen Elektroden, die an Ionenkonzentrationen zu messen. Diese Abbildung zeigt das Setup für die Ramsay Assay. (A) Jede Fliege hat vier Röhrchen, ein Paar von vorderen Tubuli und ein Paar von hinteren Tubuli, die schwimmen in der Bauchhöhle von Hämolymphe umgeben. In jedem Paar, verbinden die beiden Röhrchen in den Harnleiter, die dann leert den Urin an der Kreuzung des Mitteldarms und hindgut. Die Röhrchen sind blind digen. Urin wird durch die (in rot dargestellt) Flüssigkeit absondernden Hauptsegment erzeugt und strömt in Richtung des Harnleiters und hinaus in den Darm. Nach der Präparation wird die Tubuli Paar aus dem Darm durch Durchtrennung des Harnleiters dissoziiert. (B) Das Paar von Röhrchen wird dann in einen Tröpfchenbadesalzlösung innerhalb einer Vertiefung des Assays Schale überführt. Eines der beiden Röhrchen, hier als die &quot;Anker Tubulus&quot; ist um einen Metallstift gewickelt und ist inert. Die andere Röhrchen ist die sezernierenden Röhrchen. Das Anfangssegment (die nicht Flüssigkeit sezerniert) und Hauptsegment der sezernierenden Röhrchen bleiben innerhalb des Tröpfchens des Badens Kochsalzlösung. Ionen und Wasser Umzug von der Badekochsalzlösung und in die Tubuluslumen des Hauptsegments und dann in Richtung des Harnleiters zu bewegen, wie es in vivo auftreten. Das untere Segment (blau) ist außerhalb der Badesalzlösung und daher inert. Da der Harnleiter geschnitten wird, tritt der sekretierten Flüssigkeit als ein Tröpfchen von dem geschnittenen Ende des Ureters. Ter sezerniert Fluidtröpfchen vergrößert Laufe der Zeit als Sekret setzt, und sein Durchmesser wird unter Verwendung eines okularen Mikrometers gemessen. Eine Schicht von Mineralöl verhindert die Verdunstung des sekretierten Flüssigkeit. Die Referenz- und ionenspezifische Elektroden messen die Ionenkonzentration der Flüssigkeit ausgeschieden. <a href="https://www.jove.com/files/ftp_upload/53144/53144fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieser Figur zu sehen.</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 Nierenepithelzellen Ionentransport erlaubt Organismen ionische und osmotische Homöostase angesichts der unterschiedlichen äußeren Bedingungen aufrechtzuerhalten. Die Drosophila melanogaster Malpighischen (Nieren) Tubulus bietet eine einmalige Gelegenheit, um die molekularen Mechanismen der epithelialen Ionentransport zu untersuchen, aufgrund der leistungsfähigen Genetik dieses Organismus und der Zugänglichkeit ihrer Nierentubuli auf physiologische Studie. Hier beschreiben wir die Verwendung des Ramsay Assayfluid Sekretionsraten von isolierten fly Nierentubuli zu messen, mit der Verwendung von ionenspezifischen Elektroden, die Natrium- und Kaliumkonzentrationen in der sekretierten Flüssigkeit zu messen. Dieser Assay ermöglicht Studium der transepithelialen Flüssigkeit und Ionenflüsse von ~ 20 Tubuli zu einer Zeit, ohne die Notwendigkeit, das sezernierte Fluid zu einer separaten Vorrichtung zu übertragen, um die Ionenkonzentration zu messen. Genetisch unterschiedlichen Tubuli können analysiert werden, um die Rolle spezifischer Gene, die in Transportprozessen zu beurteilen. Zusätzlich wird die bathing Salzlösung kann geändert werden, um die Wirkungen der chemischen Eigenschaften oder Arzneimittel oder Hormone zugesetzt untersuchen. Zusammenfassend ermöglicht diese Technik die molekulare Charakterisierung der Grundmechanismen von epithelialen Ionentransport in Drosophila Tubulus sowie Regelung dieser Transportmechanismen.

Die 7 und 8 zeigen, dass die Verwendung des Ramsay Assay mit ionenspezifischen Elektroden gegenüber K + und Na + -Konzentration zu messen, kann die genetische und pharmakologisch verschieden K + und Na + Flüsse, Informationen, die nicht durch die Messflüssigkeit Sekretionsraten allein erfasst wird unterscheiden. Fig 7 zeigt, dass Fluid-Sekretion in Tubuli von Fliegen, die eine homozygote Nullmutation in der NKCC verringert wird durch eine A...

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

Dieses Protokoll beschreibt die Verwendung des Ramsay Assay Flüssigkeitssekretion Raten von isolierten Malpighischen (Nieren) Tubuli aus Drosophila melanogaster zu messen. Darüber hinaus ist die Verwendung von ionenspezifischen Elektroden, die Natrium- und Kaliumkonzentrationen in der sekretierten Flüssigkeit zu messen, mit Berechnung der transepithelialen Ionenfluss, wird beschrieben.

Die Nutzung der Ramsay Assay zu Flüssigkeitssekretion und Ionenfluss Preise in der Maßnahme<em&gt; Drosophila melanogaster</em&gt; Malpighischen Tubulus

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 Aufrufe.  University of Texas Southwestern Medical Center.  Dieses Protokoll beschreibt die Verwendung des Ramsay Assay Flüssigkeitssekretion Raten von isolierten Malpighischen (Nieren) Tubuli aus Drosophila melanogaster zu messen. Darüber hinaus ist die Verwendung von ionenspezifischen Elektroden, die Natrium- und Kaliumkonzentrationen in der sekretierten Flüssigkeit zu messen, mit Berechnung der transepithelialen Ionenfluss, wird beschrieben. 

Serie: 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.

Mikroinjektion Techniken zur Untersuchung Mitose in der 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 ...

Forschung

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 Assays (FRAP &amp; FLIP) zu Chromatin Protein Dynamics in Living embryonalen Stammzellen Measure

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.

Insulin-Injektion und Hämolymphe Extraction die Insulinsensitivität in Adult Measure 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.

Messung der Lebensdauer in<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.

Live-Imaging von apoptotischen Ausverkauf während<em&gt; Drosophila</em&gt; Embryogenese

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.

Imaging Durch die Puppenhülle von<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.

Quantitative Immunfluoreszenz-Assay, um die Variation der Protein-Ebene an Zentrosomen Messen

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.

Optische Quantifizierung der intrazellulären pH in Drosophila Melanogaster Malpighian Röhrchen Epithelien mit einem fluoreszierenden genetisch codierte pH Indikator

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.

Mündliche bakterielle Infektion und 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 ...

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.

Visualisierung und Verfolgung endogenes mRNAs in Live Drosophila melanogaster Eierkammern

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