Les auteurs tiennent à remercier FI Ataullakhanov pour aider à concevoir et fabriquer des chambres d&#39;écoulement réutilisables, N. Dachkevitch, N. et A. Gudimchuk Korbalev pour fournir des images de figures, N. et P. Gudimchuk Zakharov pour l&#39;élaboration d&#39;un protocole et de fournir des réactifs à préparer les graines des microtubules marquées à la digoxigénine, A. Potapenko de l&#39;aide pour l&#39;édition de texte et d&#39;autres membres de Grishchuk laboratoire pour obtenir des conseils et des discussions. Ce travail a été financé en partie par des subventions du NIH GM R01-098 389 et une subvention pilote de Pennsylvanie Muscle Institut ELG, qui est un Kimmel Scholar, par RFBR accorde 12-04-00111-a, le 13-04-40190-H et 13 -04-40188-H, Académie russe des Sciences subventions Présidium (mécanismes de l&#39;intégration des systèmes moléculaires et les programmes de biologie cellulaire et moléculaire et moléculaires) à FI Ataullakhanov, NIH GM R01 GM033787 à JR McIntosh, et une bourse de recherche postdoctorale de la Fondation Dmitry dynastie Zimin à VAV

Les auteurs n&#39;ont rien à révéler.

De nombreux essais de molécules simples de nos jours utilisent couramment des lamelles spécialement traitées pour réduire considérablement non spécifique de protéines de collage. La procédure que nous décrivons ici est une modification du protocole original développé en laboratoire Howard 32, et nous constatons que silaniser les lamelles vaut bien l&#39;effort même avec les dosages de perles à base de DIC, qui n&#39;utilisent pas de fluorescence. Chambers assemblés avec ces lamelles présentent ...

Les microtubules sont des structures hautement conservées du cytosquelette qui sont importants pour l&#39;architecture cellulaire, la motilité cellulaire, la division cellulaire, le transport intracellulaire et 1. Ces polymères dynamiques assembler à partir de tubuline en présence de GTP, et ils commutent spontanément entre la croissance et le raccourcissement 2. Les microtubules sont très mince (seulement 25 nm de diamètre) donc des techniques optiques spéciales pour renforcer le contraste doit être utilisé pour observer les microtubules avec un microscope optique. Les travaux antérieurs de ces polymères a examiné leur comportement dynamique utilisant contraste interférentiel différentiel (DIC) 3. Et d&#39;autres semblables études in vitro ont montré que dans des conditions expérimentales typiques, les microtubules subissent catastrophe et passer à la dépolymérisation que rarement, une fois tous les 5-15 min (cette fréquence est de 7-15 mM concentration de tubuline soluble examiné à 28-32 ° C ) 4. Différentes techniques ont ainsi été proposés à inductione dépolymérisation des microtubules d&#39;une manière contrôlée. Raccourcissement des microtubules peut être déclenché par élimination par lavage de la tubuline soluble 5,6, coupant microtubules avec un faisceau laser à 7, ou en utilisant des microtubules segmentées 8, comme décrit ici. Des travaux antérieurs utilisant microtubules segmentés, ainsi que les polymères de commutation stochastique, a trouvé que les petites cargaisons intracellulaires, comme les chromosomes, vésicules, et perles de revêtus de protéine, peuvent se déplacer à l&#39;extrémité des microtubules de raccourcissement 9-13. Ce phénomène est supposé avoir une incidence directe sur les mouvements des chromosomes dans les cellules en mitose, et les mécanismes sous-jacents sont actuellement sous enquête 14-16. Récemment, des techniques fluorescentes base, y compris la fluorescence totale interne de réflexion (FRBR) microscopie, ont été utilisées pour étudier la motilité de microtubules dynamique extrémités 17-24. L&#39;avantage de cette approche est qu&#39;elle permet l&#39;examen de l&#39;interactions entre les microtubules et les protéines des microtubules contraignant en temps réel à l&#39;aide de protéines marquées avec des fluorophores différents. Plusieurs complexes de protéines ont été trouvés pour déplacer processively avec allongement et / ou raccourcissement des extrémités des microtubules. Ils comprennent les protéines associées aux microtubules Dam1 10,12,18, SKA1 19, et XMAP215 20, ainsi que les moteurs de kinésine Kif18A 21,22, MCAK 23 et CENP-E 24. Ces protéines présentent pointe de suivi processive, qui est fondamentalement différente de celle des protéines de pointe de suivi classiques comme EB1 25. Bien que les molécules EB1 et les partenaires associés semblent rester stable associée à des microtubules dynamique se termine, les molécules individuelles restent liés à la pointe des microtubules pour seulement ~ 0,8 sec, échanger rapidement avec la piscine soluble 26. En revanche, la pointe des trackers processives, comme Dam1, voyagent avec des microtubules se termine pour beaucoup de microns, et leur association avec des conseils de microtubules peuvent durer mdes secondes. La pointe de temps de l&#39;association, ainsi que le taux de suivi résultant, dépend fortement du nombre de molécules qui forment la pointe de suivi complexe 27. Plus grands ensembles de protéines sont généralement beaucoup mieux la pointe des trackers. Par exemple, ces ensembles complexes que les kinétochores de levure isolées peuvent rester couplé à des microtubules extrémités pour heures 28. Certaines protéines de microtubules liaison, par exemple Ndc80 complexe protéique kinétochore, ont été trouvés dans l&#39;impossibilité de suivre avec microtubules se termine à un niveau de la molécule unique, encore Ndc80 est très efficace dans le couplage de la motion de perle cargaison 19,29-31. Ainsi, afin de comprendre le mécanisme de la pointe de poursuite par différents complexes protéiques, ainsi que leurs rôles biologiques, il est important d&#39;examiner la pointe de suivi en fonction du nombre de molécules dans le complexe de la pointe de poursuite, ainsi que pour déterminer la capacité de ces complexes à exposer la motilité collective sur la surface de chargement des billes. (figure 1A). Tout d&#39;abord, les lames de verre sont disponibles dans le commerce modifiés pour attacher court tube en polyéthylène (protocole 1). La chambre d&#39;écoulement de microscopie réutilisable est ensuite assemblée à partir d&#39;un tel coulisseau et le plasma nettoyé et traité au silane lamelle (protocole 2) 32 à 34 chimiquement ou. Le volume de la chambre résultant est seulement 20-25 pi (ou aussi petite que 15 pi, voir la note 3 dans le Protocole 1), y compris le volume de la tubulure d&#39;admission. Disponibles dans le commerce chambres d&#39;écoulement peuvent également être utilisés, mais leur volume est généralement plus grande, ce qui conduit à la perte inutile de protéines. Si une chambre plus grande est utilisé, le volume de toutes les solutions dans les protocoles ci-dessous doit être réduite proportionnellement. graines de microtubules sont ensuite préparées, par exemple en utilisant lentement hydrolysable du GTP analogique, GMPCPP (guanosine-5&#39;-[(α, β)-methyleno] triphosphate) (protocole 3, voir aussi Hyman &lt;em&gt; et al. 35). Les graines sont immobilisés sur une lamelle couvre-objet et la surface nettoyée est ensuite bloquée pour empêcher l&#39;absorption non spécifique d&#39;autres protéines 32 (protocole 4 décrit l&#39;immobilisation de graines à l&#39;aide de la digoxigénine). Les microtubules segmentés peuvent être préparés en utilisant le protocole 5. La raison principale de cette approche est que les polymères de microtubules dynamiques, qui se forment dans la présence de GTP, peuvent être temporairement stabilisée par l&#39;ajout des «caps» de courtes segments de tubuline stables, qui contiennent GMPCPP. Ces capsules contiennent également de la tubuline marqué à la rhodamine, de sorte qu&#39;ils peuvent être éliminés simplement en illuminant le champ de vision avec un laser de 530 à 550 nm ou une lampe à arc au mercure (Protocole 6) 36. L&#39;intensité de fluorescence du signal de suivi de pointe peut alors être utilisée pour estimer le nombre de molécules qui se déplacent avec les extrémités de désassemblage des microtubules, en tenant compte de l&#39;irrégularité de l&#39;éclairement de champ de microscope (Protocole 7). Une approche similaire peut être utiliséepour étudier les interactions entre les microtubules de dépolymérisation et de perles de protéine revêtu, préparé comme décrit dans 27 (protocole n ° 8). Certaines protéines se lient facilement aux parois des microtubules segmentées, mais pinces laser peuvent également être utilisés pour la tenue du bourrelet à proximité de la paroi des microtubules, ce qui favorise sa fixation.

<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

Dépolymérisation des microtubules peut fournir une force pour le transport de différents complexes de protéines et de perles revêtus de protéine in vitro. Les mécanismes sous-jacents sont supposés jouer un rôle essentiel dans les mouvements des chromosomes microtubules dépend cours de la division cellulaire, mais les protéines concernées et leurs rôles exacts sont mal définis. Ainsi, il ya un besoin croissant de développer des tests qui permettent d&#39;examiner cette motilité in vitro en utilisant des composants purifiés et milieu biochimique défini. Les microtubules, cependant, sont par nature instables des polymères; leur commutation entre la croissance et le raccourcissement est stochastique et difficile à contrôler. Les protocoles que nous décrivons ici profiter des microtubules segmentées qui sont faites avec le photoablatable stabilisation bouchons. La dépolymérisation des microtubules tels segmentées peut être déclenchée avec une résolution spatiale et temporelle élevée, aidant de ce fait les études de motilité au niveau des extrémités de désassemblage des microtubules. Cette technique peut être utilisée pour carry à une analyse quantitative du nombre de molécules dans les complexes de protéines marquées par fluorescence, qui se déplacent avec processively dynamique des microtubules se termine. Afin d&#39;optimiser un rapport signal sur bruit dans cet et d&#39;autres dosages quantitatifs fluorescentes, des lamelles doivent être traités pour réduire l&#39;absorption non spécifique de protéines solubles marqués par fluorescence. Des protocoles détaillés sont prévus pour prendre en compte les irrégularités de l&#39;illumination fluorescente, et déterminer l&#39;intensité d&#39;un fluorophore unique en utilisant l&#39;ajustement gaussien équidistants. Enfin, nous décrivons l&#39;utilisation des microtubules segmentés pour étudier les mouvements des microtubules dépendant des microbilles de revêtus de protéine, apportant des éclairages sur la capacité des différentes protéines motrices et non motrices à deux dépolymérisation des microtubules au mouvement de la cargaison processive.

Suivi de la dépolymérisation des microtubules protéines se termine. Levure kinetochore composant Dam1 est de loin le meilleur conseil-tracker de la dépolymérisation des microtubules se termine 14. Ce complexe de 10 sous-unité marquée par la GFP peut être facilement exprimée et purifiée à partir de cellules bactériennes 18,38, nous vous recommandons donc l&#39;utiliser comme un contrôle positif pour le dosage de la pointe de suivi. Une protéine fluorescente qui permet ...

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

Les microtubules sont des polymères intrinsèquement instables, et leur commutation entre la croissance et de raccourcissement est stochastique et difficile à contrôler. Nous décrivons ici les protocoles à l&#39;aide de microtubules segmentés avec des bouchons de stabilisation photoablatable. Dépolymérisation des microtubules segmentées peut être déclenchée avec une résolution temporelle et spatiale, aidant ainsi l&#39;analyse de mouvements avec les extrémités des microtubules démontage.

Préparation des microtubules sectoriels pour étudier les motions Poussé par le démontage microtubules Ends

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 vues.  Russian Academy of Sciences.  Les microtubules sont des polymères intrinsèquement instables, et leur commutation entre la croissance et de raccourcissement est stochastique et difficile à contrôler. Nous décrivons ici les protocoles à l'aide de microtubules segmentés avec des bouchons de stabilisation photoablatable. Dépolymérisation des microtubules segmentées peut être déclenchée avec une résolution temporelle et spatiale, aidant ainsi l'analyse de mouvements avec les extrémi...

Vidéo: 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.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

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

Recherche

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 Stimuli modèle animal

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.

Isolement et purification de Drosophile Neurones périphériques en triant bille magnétique

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.

Production d&#39;<em&gt; Xenopus tropicalis</em&gt; Œuf Extraits pour identifier les ARN associée aux microtubules

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.

Préparation de la recombinaison de protéines Mgm101 par la stratégie de marquage MBP basée

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.

Utilisation de cellules Caco-2 à étudier Lipid Transport par l&#39;intestin

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.

Mesure de la dynamique des microtubules par la microscopie du disque filature dans des spindles mitotiques monopolaires

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.

Étudier la motilité induite par flagelle chez Escherichia coli en appliquant trois techniques établies dans une série

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.

Un guide étape par étape de l’électroanténnographie des moustiques

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.

Un test de chimiotaxie amélioré pour l’identification rapide des chimioattractifs rhizobactériens dans les exsudats racinaires

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