Die Autoren möchten FI Ataullakhanov für die Hilfe, für die Bereitstellung von Bildern für Zahlen, N. und P. Gudimchuk Zakharov für die Entwicklung eines Protokolls und die Bereitstellung Reagenzien zu entwickeln und herzustellen wiederverwendbare Strömungskammern, N. Dashkevich, N. und A. Gudimchuk Korbalev danke vorzubereiten Digoxigenin-markierten Mikrotubuli-Samen, A. Potapenko für die Hilfe bei Textbearbeitung und andere Mitglieder der Grishchuk Labor für Tipps und Diskussionen. Diese Arbeit wurde zum Teil unterstützt durch NIH gewähren GM R01-098389 und ein Pilot Zuschuss von Pennsylvania Muscle Institute ELG, der ein Kimmel Scholar ist, durch RFBR gewährt 12-04-00111-eine 13-04-40190-H und 13 -04-40188-H, Russische Akademie der Wissenschaften Präsidium Grants (Mechanismen des Molecular Systems Integration und Molekulare Zellbiologie und Molekulare Programme) zu FI Ataullakhanov, NIH gewähren GM R01 GM033787 JR McIntosh, und Dmitry Zimin Dynastie Stiftung Postdoc-Stipendium an VAV

Die Autoren haben nichts zu offenbaren.

Viele Einzelmolekül-Assays heutzutage routinemäßig speziell behandelt, um unspezifische Proteindeckknack drastisch zu reduzieren. Das Verfahren, das wir hier beschreiben, ist eine Modifikation der in Howard Labor 32 Original-Protokoll entwickelt, und wir finden, dass die Deckgläser Silanisierung ist die Mühe wert, auch mit DIC-basierte Assays Perle, die nicht mit Fluoreszenz. Kammern mit solchen Deckgebaut zeigen viel sauberer Oberflächen, und die in Gegenwart von löslichen Mikrotubuli-Bindungsprotein ...

Mikrotubuli sind hoch konserviert Strukturen des Zytoskeletts, die wichtig für die zelluläre Architektur, Zellbewegung, Zellteilung und intrazellulären Transport 1 sind. Diese dynamischen Polymeren zusammen von Tubulin in Gegenwart von GTP und spontan zwischen Wachstum und Verkürzung 2 wechseln. Mikrotubuli sind sehr dünn (nur 25 nm Durchmesser) daher besondere optische Techniken, um Kontrast verbessern sollte verwendet werden, um Mikrotubuli mit einem Lichtmikroskop zu beobachten. Frühere Arbeiten mit diesen Polymeren untersucht ihr dynamisches Verhalten mit Differentialinterferenzkontrast (DIC) 3. Diese und ähnliche Studien in vitro gezeigt, dass unter typischen experimentellen Bedingungen, die Mikrotubuli unterziehen Katastrophe und Schalter auf der Depolymerisation nur selten, einmal alle 5-15 Minuten (diese Frequenz für 7-15 mM bei 28 bis 32 ° C untersucht löslich Tubulin-Konzentration ) 4. Verschiedene Techniken wurden so Induktions vorgeschlagene Depolymerisation von Mikrotubuli in einer kontrollierten Weise. Mikrotubuli Verkürzung kann durch Waschen entfernt lösliche 5,6 Tubulin, Mikrotubuli Schneiden mit einem Laserstrahl 7 oder 8 unter Verwendung von segmentierten Mikrotubuli, wie hier beschrieben, ausgelöst werden. Frühere Arbeiten mit segmentierten Mikrotubuli sowie stochastisch Schalt Polymere, hat festgestellt, dass die kleinen intrazellulären Ladungen, wie Chromosomen, Vesikel und Perlen-Protein-beschichtet ist, kann an den Enden der Mikrotubuli Verkürzung 9-13 bewegen. Dieses Phänomen wird gedacht, um eine direkte Auswirkungen auf Chromosom Bewegungen in mitotischen Zellen zu haben, und die zugrunde liegenden Mechanismen sind derzeit aktiv untersucht 14-16. Kürzlich, fluoreszenzbasierten Techniken, einschließlich der vollständigen internen Reflexion (TIRF) Mikroskopie, wurden eingesetzt, um die Motilität mit dynamischen Mikrotubuli untersuchen endet 17-24. Der Vorteil dieses Ansatzes ist, dass es ermöglicht die Untersuchung der Wechselwirkungs zwischen Mikrotubuli und Mikrotubuli-bindende Proteine ​​in Echtzeit unter Verwendung von Proteinen mit verschiedenen Fluorophoren markiert sind. Mehrere Protein-Komplexe wurden gefunden, um prozessiv mit Verlängerung und / oder Verkürzung der Mikrotubuli-Enden zu bewegen. Sie umfassen die Mikrotubuli-assoziierten Proteinen Dam1 10,12,18, SKA1 19 und 20 XMAP215 sowie Kinesin-Motoren Kif18A 21,22, MCAK 23 und CENP-E 24. Diese Proteine ​​zeigen prozessive tip-Tracking, die sich grundlegend von der der klassischen Spitze-Tracking-Proteine ​​wie EB1 25 ist. Obwohl EB1-Moleküle und die assoziierten Partner erscheinen stabil mit dynamischen Mikrotubuli-Enden, die einzelnen Moleküle bleiben an der Spitze der Mikrotubuli für nur ~ 0,8 s gebunden ist, schnell den Austausch mit dem löslichen Pool 26 zugeordnet bleiben. Im Gegensatz dazu prozessive Spitze-trackers, wie Dam1, reisen mit Mikrotubuli-Enden für viele Mikrometer und ihre Assoziation mit Mikrotubuli-Tipps können für m dauernkeine Sekunden. Die Spitze Assoziation Zeit sowie die resultierende Geschwindigkeit der Verfolgung, hängt stark von der Anzahl der Moleküle, die die Spitze-Tracking-Komplex 27 zu bilden. Größere Protein-Ensembles sind in der Regel viel besser Spitze-trackers. Zum Beispiel können solche komplexen Baugruppen der isolierten Hefe Kinetochore Mikrotubuli gekoppelt bleiben Enden für 28 Stunden. Einige Mikrotubuli-bindende Proteine, wie zB Ndc80 Kinetochorenprotein Komplex gefunden werden nicht mit Mikrotubuli Bahnenden an einer Einzelmolekülebene, noch Ndc80 ist sehr effizient bei der Kopplung der Bewegung der Wulst Ladung 19,29-31. So, um den Mechanismus der Spitze-Tracking von verschiedenen Protein-Komplexe, sowie deren biologische Funktionen zu verstehen, ist es wichtig, Spitze-Tracking in Abhängigkeit von der Anzahl der Moleküle in der Spitze-Tracking-Komplex zu untersuchen, sowie um zu bestimmen, die Fähigkeit dieser Komplexe Tarif Motilität auf der Oberfläche der Perle Ladung aufweisen. (1A) vorzubereiten und durchzuführen Experimenten. Zunächst werden die im Handel erhältlichen Glasobjektträger modifiziert, um kurze Polyethylenschlauch (Protokoll 1) befestigen. Die wiederverwendbare Mikroskopie Flusskammer wird dann aus wie eine Folie die chemisch oder Plasma-gereinigt und silanisierten Deckglas (Protokoll 2) 32-34 montiert und. Die daraus resultierende Raumvolumen ist nur 20-25 ul (oder so klein wie 15 ul, siehe Anmerkung 3 in Protokoll Nr. 1), einschließlich des Volumens der Einlassschlauch. Kommerziell erhältlich Strömungskammern können auch verwendet werden, aber ihr Volumen ist in der Regel größer, was zu einer unnötigen Verschwendung von Proteinen. Wenn eine größere Kammer verwendet wird, sollte das Volumen aller Lösungen in folgenden Protokolle proportional skaliert werden. Mikrotubuli Samen werden dann mit langsam hydrolysierbaren GTP-Analog, GMPCPP (Guanosin-5&#39;-[(α, β)-methyleno] Triphosphat) (Protokoll 3 hergestellt, zum Beispiel, siehe auch Hyman &lt;em&gt; et al. 35). Die Samen werden auf einem gereinigten Deck immobilisiert und die Oberfläche wird anschließend blockiert, um die unspezifische Absorption von anderen Proteinen verhindern, 32 (Protokoll 4 beschreibt Samen Immobilisierung mit Digoxigenin). Die segmentierten Mikrotubuli können dann mit Protokoll Nr. 5 hergestellt werden. Der Hauptgrund für diesen Ansatz ist, dass dynamische Mikrotubuli-Polymere, die in Gegenwart von GTP zu bilden, kann vorübergehend durch Addieren der kurzen &quot;Kappen&quot; von stabilen Tubulin-Segmente, die GMPCPP enthalten stabilisiert werden. Diese Kappen enthalten Rhodamin-markierten Tubulin, so können sie einfach mit einem 530-550 nm-Laser-oder Quecksilberbogenlampe (Protokoll Nr. 6) 36 Beleuchten des Sichtfeldes entfernt werden. Die Fluoreszenzintensität der Spitze-Tracking-Signal kann dann verwendet werden, um die Anzahl der Moleküle, die mit der Demontage der Mikrotubuli-Enden reisen zu schätzen, unter Berücksichtigung der Unebenheiten des Mikroskops Feldbeleuchtung (Protokoll 7) werden. Ein ähnlicher Ansatz kann verwendet werden,Wechselwirkungen zwischen Depolymerisation von Mikrotubuli und Perlen-Protein-beschichteten, hergestellt wie in 27 (Protokoll 8) beschrieben zu studieren. Einige Proteine ​​werden leicht an den Wänden des segmentierten Mikrotubuli binden, aber Laserpinzette können auch verwendet werden, um den Wulst in der Nähe der Mikrotubuli Wand halten, wodurch die Förderung ihrer verbindlich.

<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 1: Microscopy and other equipment.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss 
			Nikon</td>
			<td>Axio Imager 2 
			Eclipse Ti</td>
			<td>other microscope models capable of DIC and epi-fluorescence-imaging can be used</td>
		</tr>
		<tr>
			<td>Objective</td>
			<td>Zeiss 
			Nikon</td>
			<td>420490-9900-000 
			CFI Apo 100x Oil 1.49</td>
			<td>100X, DIC, 1.3-1.49 NA</td>
		</tr>
		<tr>
			<td>Objective heater</td>
			<td>Bioptechs</td>
			<td>150803, 150819-19</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent filter cube</td>
			<td>Chroma</td>
			<td>49004 or 49008 
			41017 or 49020</td>
			<td>optimized for Rhodamine fluorescence&#160; 
			optimized for GFP fluorescence</td>
		</tr>
		<tr>
			<td>Acquisition software</td>
			<td>freeware MicroManager 
			Molecular Devices</td>
			<td>not applicable 
			MetaMorph 7.5</td>
			<td>http://valelab.ucsf.edu/~MM/MMwiki/ 
			other software can be used to acquire images and for a particle tracking</td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon3, DU-897E-cs0-#BV</td>
			<td>Highly sensitive EMCCD camera</td>
		</tr>
		<tr>
			<td>Trapping laser</td>
			<td>IPG Photonics</td>
			<td>YLR-10-1064-LP</td>
			<td>1064 nm laser, 10 W</td>
		</tr>
		<tr>
			<td>Fluorescence excitation lasers</td>
			<td>Coherent, Inc. 
			Coherent, Inc.</td>
			<td>Sapphire 488 LP 
			Sapphire 552 LP</td>
			<td>excitation of green fluorophores 
			excitation of red fluorophores</td>
		</tr>
		<tr>
			<td>Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flow chambers</td>
			<td>Warner Instruments</td>
			<td>RC-20 or RC-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Perfusion pump</td>
			<td>Cole Palmer 
			Harvard Apparatus</td>
			<td>Masterflex 77120-52 
			Pico Plus</td>
			<td>Both pumps provide the required rate of liquid flow but a peristaltic pump may pulse at very slow speed. The flow with a syringe pump is more consistent for a wide range of rates but this pump has inertia.&#160;</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 2: Microscopy chamber preparation.</td>
		</tr>
		<tr>
			<td>Modified microscope slides for reusable chambers</td>
			<td>Precision Glassblowing of Colorado</td>
			<td>Custom order www.precisionglassblowing.com</td>
			<td>Sonic slots in slides using schematics in Figure 1</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427410</td>
			<td>I.D. 0.58 mm, O.D. 0.965 mm; use these tubes to connect assembled chamber to the pump and waste container</td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>Intramedic</td>
			<td>427400</td>
			<td>I.D. 0.28 mm, O.D. 0.61 mm; use these tubes to make the reusable chamber</td>
		</tr>
		<tr>
			<td>Regular microscope slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>Other similar slides can be used</td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>VWR</td>
			<td>48393-150, 48366-067</td>
			<td>Other similar coverslips can be used</td>
		</tr>
		<tr>
			<td>Silicon sealant</td>
			<td>World Precision Instruments</td>
			<td>KIT, SILICON SEALANT 5 MIN CURE</td>
			<td></td>
		</tr>
		<tr>
			<td>Epoxy glue</td>
			<td>Loctite</td>
			<td>83082</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyanoacrylate adhesive</td>
			<td>Scotch 3M</td>
			<td>AD114</td>
			<td>Or cyanoacrylate adhesive from other manufacturers</td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 3: Coverslips cleaning and coating.</td>
		</tr>
		<tr>
			<td>Molecular Sieves, Grade 564</td>
			<td>Macron</td>
			<td>4490-04</td>
			<td width="200px"></td>
		</tr>
		<tr>
			<td>Coverglass Staining Jar</td>
			<td>Ted Pella, Inc.</td>
			<td>21036</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslip Ceramic Holder</td>
			<td>Thomas Scientific</td>
			<td>8542e40</td>
			<td></td>
		</tr>
		<tr>
			<td>PlusOne Repel Silane</td>
			<td>GE Healthcare Biosciences</td>
			<td>17-1332-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Sigma-Aldrich</td>
			<td>P2443</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-digoxigenin AB</td>
			<td>Roche applied science</td>
			<td>11093274910</td>
			<td></td>
		</tr>
	</tbody>
</table>
<table>
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr>
			<td></td>
<td></td>
<td></td>
<td>Table 4: Preparation of seeds and segmented microtubules.</td>
		</tr>
		<tr>
			<td>Tubulin</td>
			<td>purified from cow brains 
			Cytoskeleton, Inc</td>
			<td>&#160; 
			T238P</td>
			<td>For purification protocols see 49&#8211;51 
			Unlabeled porcine tubulin</td>
		</tr>
		<tr>
			<td>Labeled tubulin</td>
			<td>Cytoskeleton, Inc 
			Invitrogen 
			Invitrogen</td>
			<td>TL590M 
			C1171 (Rhodamine) 
			A-2952 (Digoxigenin)</td>
			<td>Rhodamine-labeled porcine tubulin 
			Tubulin can be labeled with any amine-reactive dye as in reference52.</td>
		</tr>
		<tr>
			<td>GMPCPP</td>
			<td>Jena Biosciences</td>
			<td>NU-405</td>
			<td>Aliquot and store at -70 &#176;C</td>
		</tr>
		<tr>
			<td>VALAP</td>
			<td>vaseline, lanolin, and paraffin at 1:1:2 by mass</td>
			<td></td>
			<td>see ref. 9</td>
		</tr>
	</tbody>
</table>

preparation of segmented microtubules to study motions driven by the disassembling microtubule ends

Mikrotubuli-Depolymerisation Kraft zu liefern, um verschiedene Proteinkomplexe und Perlen-Protein-beschichteten in vitro zu transportieren. Die zugrunde liegenden Mechanismen sind vermutlich eine wichtige Rolle bei der Mikrotubuli-abhängige Chromosomenbewegungen während der Zellteilung spielen, aber die entsprechenden Proteine ​​und ihre genauen Aufgaben sind schlecht definiert. Somit gibt es einen wachsenden Bedarf an Assays, mit denen eine solche Beweglichkeit in vitro-Studie unter Verwendung von gereinigten Komponenten und definierten biochemischen Milieu entwickeln. Mikrotubuli sind jedoch inhärent instabil Polymere, ihre Umschaltung zwischen Wachstum und Verkürzung stochastisch und schwierig zu steuern. Die Protokolle beschreiben wir hier die Vorteile der segmentierten Mikrotubuli, die mit der Stabilisierung photoablatable Kappen hergestellt werden. Depolymerisation von solchen segmentierten Mikrotubuli mit hoher zeitlicher und räumlicher Auflösung ausgelöst werden, wodurch die Unterstützung der Motilität Studien an den Mikrotubuli-Enden der Demontage. Diese Technik kann verwendet werden, um ca.RRY eine quantitative Analyse der Anzahl der Moleküle in der fluoreszenzmarkierte Proteinkomplexe, die mit dynamischen Mikrotubuli prozessiv Enden zu bewegen. Ein Signal-zu-Rausch-Verhältnis in diesem und anderen quantitativen Fluoreszenz-Assays zu optimieren, sollte Deck behandelt, um unspezifische Absorption von löslichen fluoreszierend markierten Proteinen zu reduzieren. Detaillierte Protokolle vorgesehen sind, um der Unebenheit der Fluoreszenzbeleuchtung zu nehmen und zu bestimmen, die die Intensität eines einzelnen Fluorophors mit äquidistanten Gauß Form. Schließlich beschreiben wir die Verwendung von segmentierten Mikrotubuli Mikrotubuli-abhängigen Bewegungen der Protein-beschichteten Mikroperlen zu untersuchen, die einen Einblick in die Fähigkeit der verschiedenen Motor-und nichtmotorisiertem Proteine ​​zu koppeln Mikrotubuli-Depolymerisation prozessive Fracht Bewegung.

Protein-Tracking mit Depolymerisation von Mikrotubuli-Enden. Hefe Kinetochor Komponente Dam1 ist mit Abstand der beste Tipp-Tracker der Depolymerisation von Mikrotubuli-Enden 14. Dieses 10-Untereinheit Komplex mit GFP-markierten leicht ausgedrückt und aus Bakterienzellen 18,38 gereinigt, so dass wir empfehlen, es als eine positive Kontrolle für den Tipp-Tracking-Assay. Ein fluoreszierendes Protein, das mit dem Ende einer Depolymerisation der Mikrotubuli-Tracks als hell fluoreszie...

Watch this Scientific Journal Video about Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends at JoVE.com

Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Mikrotubuli sind Polymere von Natur aus instabil, und deren Umschaltung zwischen Wachstum und Verkürzung stochastisch und schwierig zu steuern. Hier beschreiben wir Protokolle mit segmentierten Mikrotubuli mit photoablatable stabilisierende Kappen. Depolymerisation von Mikrotubuli segmentiert mit hoher zeitlicher und räumlicher Auflösung ausgelöst werden, wodurch die Unterstützung Analyse der Bewegungen mit den Mikrotubuli-Enden der Demontage.

Vorbereitung der segmentierte Mikrotubuli auf Anträge von der Demontage Microtubule Ends Driven Studieren

Microtubule depolymerization can provide force to transport different protein complexes and protein-coated beads in vitro. The underlying mechanisms are ...

preparation-segmented-microtubules-to-study-motions-driven

Research

JoVE Journal

Biology

14.3K Aufrufe.  Russian Academy of Sciences.  Mikrotubuli sind Polymere von Natur aus instabil, und deren Umschaltung zwischen Wachstum und Verkürzung stochastisch und schwierig zu steuern. Hier beschreiben wir Protokolle mit segmentierten Mikrotubuli mit photoablatable stabilisierende Kappen. Depolymerisation von Mikrotubuli segmentiert mit hoher zeitlicher und räumlicher Auflösung ausgelöst werden, wodurch die Unterstützung Analyse der Bewegungen mit den Mikrotubuli-Enden der Demontage. 

Serie: Preparation of Segmented Microtubules to Study Motions Driven by the Disassembling Microtubule Ends

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testen visueller Sensibilität für die Geschwindigkeit und Richtung der Bewegung in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Forschung

Biologie

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Computer-Generated-Tiermodell Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite morphogenesis at the functional and molecular levels. To aid in these analyses, we have developed a strategy for the isolation of a subclass of PNS neurons called dendritic arborization (da) neurons that have been widely used for studying dendrite morphogenesis1,2. These neurons are very difficult to isolate as a pure population, due in part to their extremely low occurrence and their difficult-to-reach location below the tough chitinous larval cuticle. Our newly developed method overcomes these challenges, and is based on a fast and specific cell enrichment using antibody-coated magnetic beads. For our magnetic bead sorting studies, we have used age-matched third instar larvae expressing a mouse CD8 tagged GFP fusion protein (UAS-mCD8-GFP)3 under the control of either the class IV dendritic arborization (da) neuron-specific pickpocket (ppk)-GAL4 driver4 or the control of the pan-da neuron-specific GAL421-7 driver5. Although this protocol has been optimized for isolating PNS cells which are attached to the inner wall of the larval cuticle, by varying a few parameters, the same protocol could be used to isolate many different cell types attached to the cuticle at larval or pupal stages of development (e.g. epithelia, muscle, oenocytes etc.), or other cell types from larval organs depending upon the GAL4-specific driver expression pattern. The RNA isolated by this method is of high quality and can be readily used for downstream genomic analyses such as microarray gene expression profiling studies. This approach offers a powerful new tool to perform studies on isolated Drosophila dendritic arborization (da) neurons thereby providing novel insights into the molecular mechanisms underlying dendrite morphogenesis.

Isolation and Purification of Drosophila Peripheral Neurons by Magnetic Bead Sorting

In this video-article we present a method for the isolation and purification of Drosophila peripheral neurons using a fast magnetic bead assisted cell sorting strategy. RNA obtained from the isolated cells can be readily used for downstream applications including microarray analyses.

Isolierung und Reinigung von Drosophila Peripheren Neuronen durch Magnetic Bead Sorting

The Drosophila peripheral nervous system (PNS) is a powerful model for investigating the complex processes of neuronal development and dendrite ...

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

Many organisms localize mRNAs to specific subcellular destinations to spatially and temporally control gene expression. Recent studies have demonstrated that the majority of the transcriptome is localized to a nonrandom position in cells and embryos. One approach to identify localized mRNAs is to biochemically purify a cellular structure of interest and to identify all associated transcripts. Using recently developed high-throughput sequencing technologies it is now straightforward to identify all RNAs associated with a subcellular structure. To facilitate transcript identification it is necessary to work with an organism with a fully sequenced genome. One attractive system for the biochemical purification of subcellular structures are egg extracts produced from the frog Xenopus laevis. However, X. laevis currently does not have a fully sequenced genome, which hampers transcript identification. In this article we describe a method to produce egg extracts from a related frog, X. tropicalis, that has a fully sequenced genome. We provide details for microtubule polymerization, purification and transcript isolation. While this article describes a specific method for identification of microtubule-associated transcripts, we believe that it will be easily applied to other subcellular structures and will provide a powerful method for identification of localized RNAs.

Production of Xenopus tropicalis Egg Extracts to Identify Microtubule-associated RNAs

We describe the collection of unfertilized Xenopus tropicalis eggs and production of a meiosis II-arrested egg extract. This egg extract can be used to purify microtubules and microtubule-associated RNAs.

Produktion von<em&gt; Xenopus tropicalis</em&gt; Egg Extrakte Mikrotubuli-assoziierten RNAs identifizieren

Many  organisms localize mRNAs to specific subcellular destinations to spatially and  temporally control gene expression. Recent studies have demonstrated ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

Vorbereitung des Mgm101 Rekombinationsprotein von MBP-based Tagging-Strategie

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Using Caco-2 Cells to Study Lipid Transport by the Intestine

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely accepted as the in vivo model of dietary fat absorption, the surgical techniques involved are challenging and expensive. Genetic manipulation of the animal model is also costly and time consuming. The alternative in vitro model is arguably more affordable, timesaving, and less challenging. Importantly, the in vitro model allows investigators to examine the enterocytes as an isolated system, reducing the complexity inherent in the whole organism model. This paper describes how human colon carcinoma cells (Caco-2) can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs and vitamins. It explains the proper maintenance of Caco-2 cells and the preparation of their lipid mixture; and it further discusses the valuable option of using the permeable membrane system. Since differentiated Caco-2 cells are polarized, the main advantage of using the permeable membrane system is that it separates the apical from the basolateral compartment. Consequently, the lipid mixture can be added to the apical compartment while the lipoproteins can be collected from the basolateral compartment. In addition, the effectiveness of the lentivirus expression system in upregulating gene expression in Caco-2 cells is discussed. Lastly, this paper describes how to confirm the successful isolation of intestinal lipoproteins by transmission electron microscopy (TEM).

Caco-2 cells can serve as an in vitro model to study the enterocyte transport of lipids, and lipid-soluble drugs/vitamins. The permeable membrane system separates the apical from the basolateral compartment, while the lentivirus expression system offers an effective gene overexpression method. The isolation of lipoproteins is confirmed by TEM.

Verwenden von Caco-2-Zellen, die Lipidtransport Studie der Darm

Studies of dietary fat absorption are generally conducted by using an animal model equipped with a lymph cannula. Although this animal model is widely ...

Measurement of Microtubule Dynamics by Spinning Disk Microscopy in Monopolar Mitotic Spindles

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a genetically encoded marker for the positive ends of microtubules (EB3 labelled with tdTomato fluorescent protein) and high-speed, high-resolution, live-cell imaging using spinning disk confocal microscopy. Cell cycle synchronization and increased density of microtubules are achieved by inhibiting centrosomal separation in mitotic cells, and analysis of growth is performed using open-source U-Track software. The use of a bright and red-shifted fluorescent protein, in combination with the lower laser power and reduced exposure time required for spinning disk microscopy reduce phototoxicity and the probability of light-induced artifacts. This allows for imaging a larger number of cells in the same preparation while maintaining the cells in a growth medium under standard culture conditions. Because the analysis is performed in a supervised automatic fashion, the results are statistically robust and reproducible.

Here we present a robust and detailed method of microtubule dynamics analysis in cells synchronized in prometaphase using live-cell spinning disk confocal microscopy and MATLAB-based image processing.

Messung der Mikrotubuli-Dynamik durch Spinnen der Disk-Mikroskopie in Monopolar-Mitotischen Spindeln

We describe a modification of an established method to determine microtubule dynamics in living cells. The protocol is based on the expression of a ...

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, to elucidate the function and assembly of flagellar parts, and to examine and understand patterns of movement. Here we demonstrate a combination of three of these methodologies. Motility in soft agar is the oldest, offering a strong selection for isolating gain-of-function suppressor mutations in motility-impaired strains, where motility is restored through a second mutation. The cell-tethering technique, first employed to demonstrate the rotary nature of the flagellar motor, can be used to assess the impact of signaling effectors on the motor speed and its ability to switch rotational direction. The &#8220;border-crossing&#8221; assay is more recent, where swimming bacteria can be primed to transition into moving collectively as a swarm. In combination, these protocols represent a systematic and powerful approach to identifying components of the motility machinery, and to characterizing their role in different facets of swimming and swarming. They can be easily adapted to study motility in other bacterial species.

Investigating Flagella-Driven Motility in Escherichia coli by Applying Three Established Techniques in a Series

Many bacteria use flagella-driven motility to navigate their environment and colonize favorable surroundings both individually and as a collective. Demonstrated here is the use of three established methods that exploit motility as a selection tool to identify components/pathways contributing to swimming and swarming motility.

Untersuchung der Flagellen-getriebenen Motilität in Escherichia coli durch Anwendung von drei etablierten Techniken in einer Serie

Motility is crucial to the survival and success of many bacterial species. Many methodologies exist to exploit motility to understand signaling pathways, ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Eine Schritt-für-Schritt-Anleitung zur Mücken-Elektroantennographie

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

Ein verbesserter Chemotaxis-Assay zur schnellen Identifizierung von rhizobakteriellen Chemoattraktantien in Wurzelexsudaten

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...