Los autores desean agradecer a FI Ataullakhanov para ayudar a diseñar y fabricar cámaras de flujo reutilizables, N. Dashkevich, N. y A. Gudimchuk Korbalev para proporcionar imágenes de figuras, N. y P. Gudimchuk Zakharov para el desarrollo de un protocolo y proporcionar reactivos para preparar semillas microtúbulos marcadas con digoxigenina, A. Potapenko para obtener ayuda con la edición de texto y otros miembros del laboratorio Grishchuk para consejos y discusiones. Esta obra fue financiada en parte por subvención NIH R01 GM-098389 y una subvención piloto de Pennsylvania Muscle Instituto de ELG, que es un Kimmel Scholar, por RFBR ofrece 12-04-00111-A, 13-04-40190-H y 13 -04-40188-H, Academia Rusa de Ciencias del Presidium de Subvenciones (Mecanismos de Sistemas Molecular Integración y programas de Biología Celular Molecular y Moleculares) a FI Ataullakhanov, NIH subvención GM R01 GM033787 a JR McIntosh, y una beca postdoctoral de la Fundación Dinastía Zimin Dmitry al VAV

Los autores no tienen nada que revelar.

Muchos ensayos de una sola molécula hoy en día utilizan habitualmente cubreobjetos especialmente tratadas para reducir drásticamente la adherencia no específica de proteínas. El procedimiento se describe aquí es una modificación del protocolo original desarrollado en Howard laboratorio 32, y nos encontramos con que silanizar los cubreobjetos es bien vale la pena el esfuerzo, incluso con ensayos de cuentas basados ​​en la DIC, que no utilizan la fluorescencia. Cámaras ensambladas con tales cubreobj...

Los microtúbulos son altamente conservadas estructuras citoesqueléticas que son importantes para la arquitectura celular, la motilidad celular, la división celular, y el transporte intracelular 1. Estos polímeros dinámicos se ensamblan a partir de tubulina en presencia de GTP, y cambian de forma espontánea entre el crecimiento y acortamiento 2. Los microtúbulos son muy delgado (sólo 25 nm de diámetro), por lo tanto las técnicas ópticas especiales para incrementar el contraste debe ser utilizado para observar los microtúbulos con un microscopio óptico. El trabajo previo con estos polímeros examinó su comportamiento dinámico mediante contraste de interferencia diferencial (DIC) 3. Este y otros estudios in vitro revelaron que en condiciones típicas experimentales, los microtúbulos se someten a la catástrofe y cambie a la despolimerización sólo en raras ocasiones, una vez cada 5-15 minutos (esta frecuencia es de 7-15 concentración de tubulina soluble mM examinada en 28-32 ° C ) 4. Así, se han propuesto diferentes técnicas para la induccióne microtúbulos despolimerización de una manera controlada. Acortamiento de microtúbulos puede ser activado mediante el lavado de distancia tubulina soluble en 5,6, corte microtúbulos con un haz de láser 7, o el uso de los microtúbulos segmentados 8, tal como se describe aquí. El trabajo previo utilizando los microtúbulos segmentados, así como polímeros de conmutación estocásticamente, se ha encontrado que pequeñas cargas intracelulares, tales como cromosomas, vesículas, y perlas de proteína recubiertos, se pueden mover en los extremos de los microtúbulos acortamiento 9-13. Este fenómeno se cree que tiene una implicación directa de los movimientos de los cromosomas en las células mitóticas, y los mecanismos subyacentes están actualmente bajo investigación activa 14-16. Recientemente, las técnicas a base de fluorescentes, incluyendo la reflexión interna total de fluorescencia (TIRF) microscopía, se han empleado para estudiar la motilidad con microtúbulos dinámicos extremos 17-24. La ventaja de este enfoque es que permite el examen de la interaccións entre los microtúbulos y proteínas de unión a microtúbulos en tiempo real utilizando proteínas marcadas con diferentes fluoróforos. Se encontraron varios complejos de proteínas para mover processively con el alargamiento y / o acortamiento de los extremos de los microtúbulos. Ellos incluyen las proteínas asociadas a los microtúbulos Dam1 10,12,18, Ska1 19, y XMAP215 20, así como motores de quinesina Kif18A 21,22, MCAK 23 y CENP-E 24. Estas proteínas exhiben processive punta-de seguimiento, que es fundamentalmente diferente de la de las proteínas de punta de seguimiento de clásicos como EB1 25. Aunque las moléculas EB1 y los socios parecen permanecer de manera estable asociada a los microtúbulos dinámica termina, las moléculas individuales permanecen atados a la punta de los microtúbulos por sólo ~ 0,8 seg, intercambiar rápidamente con la piscina soluble 26. Por el contrario, procesivas punta-trackers, como Dam1, viajan con los microtúbulos termina para muchos micras, y su asociación con las puntas de los microtúbulos pueden durar mningún segundo. La punta tiempo asociación, así como el que resulte de seguimiento, depende en gran medida del número de moléculas que forman la punta de rastreo complejo 27. Grandes conjuntos de proteínas suelen ser mucho mejores de punta-trackers. Por ejemplo, dichos ensamblajes complejos como los cinetocoros de levadura aisladas pueden permanecer acoplado a microtúbulos termina por hora 28. Algunas proteínas de unión a microtúbulos, por ejemplo Ndc80 complejo de proteínas cinetocoro, se han encontrado para ser incapaces de realizar un seguimiento con microtúbulos termina en un solo nivel de molécula, sin embargo Ndc80 es muy eficiente en el acoplamiento del movimiento del talón de carga 19,29-31. Por lo tanto, para entender el mecanismo de la punta de seguimiento por diferentes complejos de proteínas, así como sus funciones biológicas, es importante examinar la punta de seguimiento como una función del número de moléculas en el complejo punta de seguimiento, así como para determinar la capacidad de estos complejos para exhibir la motilidad colectiva en la superficie de la carga de talón. (Figura 1A). En primer lugar, los portaobjetos de vidrio disponibles comercialmente son modificados para unir tubos de polietileno corto (Protocolo 1). La cámara de flujo microscopía reutilizable se ensambla a partir de una diapositiva tal y el plasma-limpiado y cubreobjetos silanizada (protocolo 2) 32-34 químicamente o. El volumen de la cámara resultante es sólo 20-25 l (o tan pequeño como 15 l, véase la nota 3 en el Protocolo 1), incluyendo el volumen de la tubería de entrada. Cámaras de flujo disponibles comercialmente también se pueden usar, pero su volumen es generalmente más grande, que conduce a la pérdida innecesaria de proteínas. Si se emplea una cámara más grande, el volumen de todas las soluciones en los protocolos a continuación debe ser escalado proporcionalmente. Semillas de microtúbulos continuación, se preparan, por ejemplo, usando GTP analógico lentamente hidrolizable, GMPCPP (guanosina-5&#39;-[(α, β)-methyleno] trifosfato) (Protocolo 3, ver también Hyman &lt;em&gt; et al. 35). Las semillas se inmovilizan sobre un cubreobjetos de limpiado y la superficie es posteriormente bloqueando los comandos para prevenir la absorción no específica de otras proteínas 32 ​​(protocolo de 4 describe semillas inmovilización utilizando digoxigenina). Los microtúbulos segmentados pueden ser preparados usando el Protocolo 5. La razón principal de este enfoque es que los polímeros de microtúbulos dinámicos, que se forman en presencia de GTP, se pueden estabilizar temporalmente mediante la adición de los cortos &quot;topes&quot; de segmentos de tubulina estables, que contienen GMPCPP. Estas tapas también contienen tubulina marcada con rodamina, para que puedan ser retirados simplemente iluminando el campo de visión con un láser de 530-550 nm o lámpara de arco de mercurio (Protocolo 6) 36. La intensidad de fluorescencia de la señal de la punta de seguimiento se puede usar entonces para estimar el número de moléculas que se desplazan con los extremos de los microtúbulos desmontaje, teniendo en cuenta la falta de uniformidad de la iluminación de campo del microscopio (Protocolo 7). Un enfoque similar se puede utilizarpara estudiar las interacciones entre los microtúbulos depolymerizing y perlas de proteína-revestido, preparado como se describe en el 27 (Protocolo de 8). Algunas proteínas se unen fácilmente a las paredes de los microtúbulos segmentados, pero pinzas láser también pueden utilizarse para sostener el cordón cerca de la pared de microtúbulos, promoviendo así su unión.

<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

Despolimerización de microtúbulos puede proporcionar la fuerza para el transporte de diferentes complejos de proteínas y los granos proteicos recubierto in vitro. Los mecanismos subyacentes se cree que desempeñan un papel vital en los movimientos de los cromosomas microtúbulos dependientes durante la división celular, pero las proteínas correspondientes y sus funciones exactas están mal definidos. Por lo tanto, hay una necesidad creciente de desarrollar ensayos con el que estudiar tales motilidad in vitro utilizando componentes purificados y medio bioquímica definida. Los microtúbulos, sin embargo, son inherentemente inestables polímeros; su conmutación entre el crecimiento y acortamiento es estocástico y difícil de controlar. Los protocolos que describimos aquí se aprovechan de los microtúbulos segmentados que se hacen con el fotoescindible estabilizar tapas. La despolimerización de microtúbulos tales segmentados se puede activar con alta resolución temporal y espacial, ayudando así a los estudios de la motilidad en los extremos de los microtúbulos desmontaje. Esta técnica se puede utilizar para CArry a cabo un análisis cuantitativo del número de moléculas en los complejos de proteínas marcadas con fluorescencia, que se mueven processively con los extremos de los microtúbulos dinámica. Para optimizar una relación señal-ruido en este y otros ensayos fluorescentes cuantitativos, cubreobjetos deben ser tratados para reducir la absorción no específica de proteínas marcadas fluorescentemente-solubles. Protocolos detallados se proporcionan a tener en cuenta la falta de uniformidad de la iluminación fluorescente, y determinar la intensidad de un solo fluoróforo utilizando ajuste gaussiano equidistantes. Por último, se describe el uso de los microtúbulos segmentados para estudiar los movimientos de los microtúbulos dependientes de las microperlas de proteína-revestido, proporcionando conocimientos sobre la capacidad de las diferentes proteínas motoras y no motoras para acoplar la despolimerización de microtúbulos al movimiento de carga processive.

Proteína seguimiento con depolymerizing microtúbulos termina. Cinetocoro levadura componente Dam1 es, con mucho, el mejor consejo-tracker de los microtúbulos depolymerizing termina 14. Este complejo de 10 subunidad marcada con GFP se puede expresar fácilmente y se purifica a partir de células bacterianas 18,38, por lo que recomendamos su uso como un control positivo para el ensayo de la punta de seguimiento. Una proteína fluorescente que rastrea con el extremo despolimerizaci...

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

Los microtúbulos son polímeros inherentemente inestables, y su conmutación entre el crecimiento y acortamiento es estocástico y difícil de controlar. Aquí se describen los protocolos que utilizan microtúbulos segmentados con tapones estabilizadores fotoescindible. La despolimerización de los microtúbulos segmentados puede ser activado con una alta resolución temporal y espacial, ayudando de este modo el análisis de los movimientos con los extremos de los microtúbulos desmontaje.

Preparación de segmentados microtúbulos para estudiar las mociones impulsadas por las que desmontan microtúbulos 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 vistas.  Russian Academy of Sciences.  Los microtúbulos son polímeros inherentemente inestables, y su conmutación entre el crecimiento y acortamiento es estocástico y difícil de controlar. Aquí se describen los protocolos que utilizan microtúbulos segmentados con tapones estabilizadores fotoescindible. La despolimerización de los microtúbulos segmentados puede ser activado con una alta resolución temporal y espacial, ayudando de este modo el análisis de los movimientos con los extremos de los micro...

Video: 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.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

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

Investigación

Biología

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.

Los estímulos generados por ordenador Modelo 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.

Aislamiento y purificación de Drosophila Clasificación de las neuronas periféricas por bolas magnéticas

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.

Producción de<em&gt; Xenopus tropicalis</em&gt; Huevos Extrae para identificar los ARN asociada a los microtúbulos

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.

Preparación de la proteína de recombinación Mgm101 por Estrategia de etiquetas MBP basado

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.

El uso de células Caco-2 para el Estudio de lípidos Transporte por el Intestino

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

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

-Alta resolución de imagen de lapso de tiempo y análisis automatizado de los microtúbulos en la dinámica de estar umbilical humana Las células endoteliales de vena

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

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.

Medición de microtúbulos dinámicos mediante microscopía de disco giratorio en husillos mitoticos Monopolar

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.

Investigación De La Motilidad Impulsada Por Flagelos En Escherichia coli mediante la Aplicación De Tres Técnicas Establecidas En Una 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.

Una guía paso a paso para la electroantenografía de mosquitos

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 ...