Damos las gracias a Jordan Davis, Yanyan Lin, Eric Schadler y Jimiao Zheng por su ayuda técnica con este estudio. Este trabajo fue apoyado por NSF CARRERA conceder MCB-1054962 de ALA

1. Preparación grandes de Apple Juice Placas de agar <ol><li> Para hacer 4 platos, añadir 375 ml de H2O, 11,25 g de agar con mosca y una barra de agitación en un matraz de 1000 ml. Se trata de Mix A. Autoclave Mix A con la tapa frasco sin apretar tope en un ciclo de 30 min-esterilización para productos líquidos. </li><li> Añadir 125 ml de jugo de manzana, 12,5 g azúcar de mesa y una barra de agitación a un vaso de 500 ml. Esta es la mezcla B. El calor de la mezcla B en una plataforma calentada mientras se agitaba y mantener la temperatura a aproximadamente 70 ° C hasta que se terminó el tratamiento en autoclave de la mezcla A. </li><li> Tras la finalización de Mix A autoclave, transferir Mix B en Mix A. Mantenga la agitación de la mezcla combinada con suavidad y deje que se enfríe durante 15 minutos. Evitar el exceso de enfriamiento y la solidificación de agar dado lugar antes de la adición de conservante. </li><li> Hacer 10% (peso / volumen) metil solución de 4-hidroxibenzoato en etanol. Esto actuará como un conservante. Añadir 3,75 ml de la solución a la mezcla de jugo de manzana-agar anteriormente. Mantener la agitación durante anotheR 5 min. </li><li> Vierta 125 ml manzana mezcla de jugo-agar con conservante en cada 300 cm2 de plástico o placa de espuma de poliestireno. Deje que se enfríe a temperatura ambiente y se solidifique. Evite la formación de burbujas de aire mientras se vierte. Nota: Las placas de manzana jugo de agar están listos para usar inmediatamente. Cubra las placas utilizadas con los abrigos claros y almacenar a 4 ° C durante un máximo de una semana. </li></ol> 2. Drosophila embriones Recolección y Tratamiento Pre-reticulación <ol><li> Cargue una jaula de población con aproximadamente 40.000 a 50.000 moscas de la que se recogen los embriones. </li><li> Haga dos placas que contienen cada uno 125 ml de harina de maíz-melaza estándar medio 12 y espolvorear aproximadamente 4 g de levadura de panadería seca en cada plato. Coloque estas placas en la jaula de la población en una incubadora a 25 ° durante 48 horas para engordar a las moscas. </li><li> Caliente 2 manzana jugo de placas de agar a 25 ° C. Espolvorear aproximadamente 1 g de levadura de panadería seca en cada unoplato. Coloque las 2 placas en la jaula de la población para comenzar a recoger 0-1 embriones-hr de edad. </li><li> Saque las placas al final de la colección 1 hr. Reemplace con 2 nuevas placas si se necesita otra ronda de recolección. </li><li> Agregar suficiente agua para cubrir las placas y volver a suspender los embriones suavemente con un pincel fino. Vierta los embriones se volvieron a suspender por un tamiz de dos capas hechas de malla de alambre de acero inoxidable. Nota: La capa superior está hecha de tamaño de poro medio (850 m) de malla que recoge gran parte de la mosca vez que deja pasar los embriones. La capa inferior está hecha de malla fina (75 micras), que recoge los embriones vez que deja pasar la levadura y el agua. </li><li> Utilice el pincel fino para transferir los embriones recogidos en una canasta de la colecta hecha con un tubo de 50 ml y malla de nylon fino. Enjuague los embriones brevemente con agua y luego seque la malla en toallas de papel para secarse. </li><li> Sumerja los embriones en el 50% de lejía con agitación suave durante 3 min. Enjuague a fondoLY con agua para eliminar cualquier lejía restante. Seque la malla en toallas de papel para secarse. </li><li> Sumerja los embriones dechorionated en isopropanol con agitación suave durante 15 segundos o hasta que la descomposición de agregados múltiples del embrión (esto no debe durar más de 30 segundos). Seque la malla en toallas de papel para secarse a fondo. </li><li> Sumerja los embriones en heptano durante 5 minutos mientras se utiliza una pipeta para transmitir heptano sobre los embriones para mantenerlos resuspendido. Seque la malla en toallas de papel para secarse. </li><li> Enjuague los embriones con corrientes de Tampón fosfato salino, pH 7,4 (PBS) que contenía 0,1% de Triton X-100 (PBST) durante 3 min. </li><li> Desmontar la canasta de la colecta y transferir los embriones junto con la malla a un tubo de 15 ml que contiene 10 ml de PBST. Invertir el tubo suavemente para lavar los embriones de la malla. </li><li> Retire la malla del tubo. Poner el tubo en posición vertical y dejar que los embriones se hunden hasta el fondo del tubo por gravedad. Nota: Aproximadamente 100-300l de embriones se espera para una colección de 1 hr de una jaula. </li><li> Retire el PBST y continuar realizando reticulación inmediatamente. </li></ol> 3. In vivo La reticulación de embriones obtenidos <ol><li> Preparar 0,2% de formaldehído en PBST inmediatamente antes de su uso. Añadir 10 ml de fijador formaldehído para los embriones y se incuba a 25 ° C con agitación suave en un agitador rotatorio durante 10 min. Desechar fijador no utilizada por el final del día. </li><li> Se detiene la reacción de reticulación <ol><li> Al final de la reticulación, dejar que los embriones se hunden hasta el fondo del tubo. </li><li> Eliminar la solución de formaldehído. Inmediatamente después, añadir 10 ml de 0,25 M de glicina en PBST para inactivar la reacción de reticulación. Incubar a 25 ° C con agitación suave en un agitador rotatorio durante 5 min. </li><li> Deje que los embriones se hunden hasta el fondo del tubo. Eliminar la solución de enfriamiento. Lavar los embriones con 10 ml de PBST tres veces. </li><li> Retire completamente el PSolución de lavado BST utilizando una pipeta. Embriones Almacenar a -80 ° C hasta su uso. </li></ol></li></ol> 4. Proteína preparación de la muestra y la inmunoprecipitación <ol><li> Homogeneizar 500 l embriones reticulados usando un homogeneizador Dounce en 2 ml de tampón de lisis (urea 0,5 M, 0,01% de SDS, 2% de Triton-X 100, fluoruro de fenilmetanosulfonilo 2 mM de [PMSF] y cóctel inhibidor de la proteasa en PBS). Nota: Realice este y los siguientes pasos a temperatura ambiente a menos que se especifique lo contrario. </li><li> Transferir el lisado a 1,5 ml de tubos de centrífuga y mueva suavemente sobre una plataforma giratoria durante 15 minutos para asegurar la lisis completa. Centrifugar el lisado a 16.000 xg durante 5 min para sedimentar los restos celulares. Guardar el sobrenadante en un tubo limpio 15 ml. </li><li> Añadir 40 l perlas de agarosa anti-HA suspensión al sobrenadante y se incuba en una plataforma giratoria durante 2,5 h. </li><li> Se precipitan las perlas mediante centrifugación a 2500 xg durante 5 min. Eliminar el sobrenadante, pero dejan aproximadamente 250 l en el tubo. Resuspender las perlas en el sobrenadante restante y transferir a una columna de centrifugación de 1,5 ml con un filtro de poro de 10 micras. </li><li> Se centrifuga la columna de centrifugación a 12.000 xg durante 10 seg y desechar el flujo a través. </li><li> Lavar las perlas mediante la adición de 200 l de tampón de lavado (0,1 M de glicina, 1 M de NaCl, 1% de Igepal CA-630, 0,1% de Tween-20, en PBS). Centrifugar la columna a 12.000 xg durante 10 seg y deseche el lavado. Repetir el lavado dos veces más. </li><li> Añadir 40 l de tampón de elución (2 mg / ml de HA-péptido en PBS) a las perlas y se incuba a 37 ° C durante 15 min con agitación suave cada 5 min. Centrifugar la columna a 12.000 xg durante 10 segundos y recoger el eluido. </li><li> Confirmar elución del complejo por análisis de transferencia Western usando anticuerpo anti-HA 13. </li></ol>

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Los autores no tienen nada que revelar.

El formaldehído se ha usado comúnmente como un reactivo de reticulación para la identificación de proteína-proteína y las interacciones proteína-ácido nucleico. Su buena solubilidad y permeabilidad de la membrana celular, junto con la compatibilidad con los procedimientos de espectrometría de masas aguas abajo, crea formaldehído un agente candidato ideal para aplicaciones de reticulación intracelulares 3,15-17. En particular, se utiliza con éxito para identificar los ARNm asociados con Vasa, una h...

El aislamiento de las asambleas de proteínas y complejos de múltiples subunidades de ADN o ARN-proteína se realiza para identificar los complejos de proteínas, genómica loci reconocida por proteínas reguladoras se unen al ADN o ARN objetivos de las proteínas de unión de ARN. Diferentes métodos permiten la identificación de todo el genoma de ADN sitios reconocidos por factores de transcripción o proteínas de la cromatina (ChIP-seq) 1 y los objetivos de ARN asociados con una proteína de unión al ARN dado (CLIP-seq) 2. Las bibliotecas de ADNc de ARN derivados u objetivos de ADN son entonces profundamente secuenciados. Estos métodos utilizan química o reticulación para estabilizar los complejos seguido por inmunoprecipitación (IP) con un anticuerpo contra un componente de proteína del complejo estudiado inducida por UV. Durante el desarrollo de un organismo, muchos complejos de proteínas forman transitoriamente. Por lo tanto, es crucial para analizar la composición y función de estos complejos in vivo para comprender los mecanismos moleculares que controlan development. Tal análisis in vivo sería superior a enfoque in vitro, ya que es prácticamente imposible de reproducir concentraciones nativas de los componentes que interactúan y entorno bioquímico celular in vitro. Aquí se demuestra un enfoque in vivo que utilizamos con éxito para aislar grandes complejos de proteínas a partir de embriones de Drosophila. En este método, los complejos de proteínas en los embriones vivos se reticulan con una baja concentración de formaldehído y, posteriormente, los complejos de proteínas de interés se aíslan por IP con un anticuerpo contra un componente conocido de los complejos seguido de purificación en gel de los complejos y el análisis de espectrometría de masas para identificar los componentes complejos desconocidos. Dado que el formaldehído es capaz de permear la membrana de la célula y tiene un rango de reticulación de 2.3 a 2.7 Å 3, los complejos de proteínas se pueden reticular in vivo y los componentes complejos son propensos a estar cerca el uno al otro. En este artículodescribir este método usando el aislamiento de entramado (Tud) complejo de la proteína como un ejemplo. Tud es una proteína de la línea germinal que es esencial para el desarrollo de la línea germinal 4-7. Esta proteína contiene 11 dominios TUD conocidos para interactuar con argininas metiladas o lisinas de otros polipéptidos 8-10. Anteriormente, hemos generado una línea de Drosophila transgénica que expresa HA-etiquetados funcional Tud 5 y por lo tanto, el anticuerpo específico anti-HA se utiliza para tirar hacia abajo compleja Tud después de la reticulación. Además de reticulaciones proteína-proteína, el formaldehído puede generar enlaces cruzados de proteína-ácido nucleico y se utiliza en los experimentos de ChIP-ss. Además, en Drosophila, in vivo de reticulación con formaldehído ha permitido la identificación de una diana de ARN de la proteína helicasa Vasa ARN 11. Aunque en este artículo se describe un método para la reticulación in vivo y purficación de complejos de proteínas a partir de embriones de Drosophila, este método se puede adaptar para otros organismos y tejidos.

<table border="0" cellpadding="0" cellspacing="0" width="858">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Drosophila agar</td>
			<td style="width:235px;">Lab Scientific (http://www.labscientific.com/)</td>
			<td style="width:119px;">FLY-8020-1</td>
			<td style="width:289px;">Do not autoclave the water and agar mix for prolonged period of time as it will cause the apple juice plates to become fragile</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Methyl 4-hydroxybenzoate</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">H5501</td>
			<td style="width:289px;">Other name: TEGOSEPT</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Population cage</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">59-104</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Fine nylon mesh</td>
			<td style="width:235px;">Flystuff (http://www.flystuff.com/)</td>
			<td style="width:119px;">57-102</td>
			<td style="width:289px;">When making the collection basket, cut the mesh slightly larger than the opening of the falcon tube cap to ensure a tight seal</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">D8938-1SET</td>
			<td style="width:289px;">Chill the homogenizer on ice and prerinse with cold lysis buffer before use to prevent protein degradation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Protease inhibitor cocktail&#160;</td>
			<td style="width:235px;">Roche (http://www.rocheusa.com/portal/usa)</td>
			<td style="width:119px;">4693132001</td>
			<td style="width:289px;">PBS could be used to prepare concentrated protease inhibitor stock solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-HA agarose beads</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">The kit also includes HA-peptide and spin columns</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">HA-peptide</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Prepare to 2 mg/ml with PBS&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Spin Column</td>
			<td style="width:235px;">MBL international (http://www.mblintl.com/)</td>
			<td style="width:119px;">561-8</td>
			<td style="width:289px;">Spin columns are included as part of the kit</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isopropanol</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP26324</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP151-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Heptane</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">H350-4</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">PBS</td>
			<td style="width:235px;">Invitrogen (https://www.lifetechnologies.com/us/en/home.html)</td>
			<td style="width:119px;">AM9625</td>
			<td style="width:289px;">Dilute from 10 X to 1 X with nanopure water before use</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Formaldehyde</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP531-500</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycine</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0724</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SDS</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0301</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Urea</td>
			<td style="width:235px;">BioRad (www.bio-rad.com/&#8206;)</td>
			<td style="width:119px;">161-0730</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Phenylmethanesulfonyl fluoride</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">P7626-1G</td>
			<td style="width:289px;">Prepare 200 mM stock solution in isopropanol then dilute to working concentration of 2 mM in lysis buffer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">IGEPAL CA-630</td>
			<td style="width:235px;">Sigma (http://www.sigmaaldrich.com/united-states.html)</td>
			<td style="width:119px;">I8896-50ML</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tween 20</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">BP337-100</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15-ml tubes</td>
			<td style="width:235px;">USA Scientific (http://www.usascientific.com/)</td>
			<td style="width:119px;">1475-0511</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bleach</td>
			<td style="width:235px;">Clorox brand</td>
			<td style="width:119px;"></td>
			<td style="width:289px;">Dilute with equal volume of nanopure water to make 50% bleach</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50-ml tubes</td>
			<td style="width:235px;">BD Biosciences (http://www.bdbiosciences.com/home.jsp)</td>
			<td style="width:119px;">352098</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Top layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1AK</td>
			<td style="width:289px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Bottom layer sieve</td>
			<td style="width:235px;">Fisher Scientific (www.fishersci.com/&#8206;)</td>
			<td style="width:119px;">04-884-1BA</td>
			<td style="width:289px;"></td>
		</tr>
	</tbody>
</table>

an in vivo crosslinking approach to isolate protein complexes from drosophila embryos

Muchos procesos celulares están controlados por complejos de proteínas con múltiples subunidades. Frecuentemente, estos complejos se forman transitoriamente y requieren entorno nativo de montar. Por lo tanto, para identificar estos complejos de proteínas funcionales, es importante para estabilizar ellos in vivo antes de la lisis celular y la posterior purificación. Aquí se describe un método para aislar grandes complejos de proteínas de buena fe a partir de embriones de Drosophila. Este método se basa en la permeabilización del embrión y la estabilización de los complejos dentro de los embriones de reticulación en vivo usando una baja concentración de formaldehído, que puede atravesar fácilmente la membrana celular. Posteriormente, el complejo proteína de interés se inmunopurificó seguido por purificación en gel y se analizaron por espectrometría de masas. Se ilustra este método mediante la purificación de un complejo de proteínas de Tudor, que es esencial para el desarrollo de la línea germinal. Tudor es una proteína grande, que contiene varios dominios Tudor - pequeñamódulos que interactúan con argininas metiladas o lisinas de las proteínas diana. Este método puede adaptarse para el aislamiento de complejos de proteínas nativas de diferentes organismos y tejidos.

La eficacia de la reticulación y la purificación con éxito del complejo de la proteína reticulada Tud se analizaron por SDS-PAGE en un 3% - 7% de gel de paso (ilustrado en la Figura 1), seguido de Western blot (Figura 2). El propósito de utilizar el 3% - gel de paso 7% se basa en la separación efectiva de reticulado complejo de proteínas Tud de la proteína Tud no reticulada restante y la concentración del complejo. Bajo nuestras condiciones in v...

Watch this Scientific Journal Video about An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos at JoVE.com

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Complejos de proteínas con múltiples componentes juegan un papel crucial durante la función y el desarrollo celular. Aquí se describe un método usado para aislar los complejos de proteínas nativas a partir de embriones de Drosophila después de la reticulación en vivo seguido de la purificación de los complejos reticulados para el análisis de estructura-función subsiguiente.

Un In vivo Enfoque de reticulación para aislar Complejos de Proteína Desde Drosophila Los embriones

Many cellular processes are controlled by multisubunit protein complexes. Frequently these complexes form transiently and require native environment to ...

an-vivo-crosslinking-approach-to-isolate-protein-complexes-from

Research

JoVE Journal

Biology

An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

16.6K vistas.  Murray State University.  Complejos de proteínas con múltiples componentes juegan un papel crucial durante la función y el desarrollo celular. Aquí se describe un método usado para aislar los complejos de proteínas nativas a partir de embriones de Drosophila después de la reticulación en vivo seguido de la purificación de los complejos reticulados para el análisis de estructura-función subsiguiente. 

Video: An in vivo Crosslinking Approach to Isolate Protein Complexes From Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Preparación de cultivos neuronales a partir de embriones de Drosophila Midgastrula Etapa

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

Investigación

Biología

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

Microfabricated Post-Array-detectores (mPADs): un enfoque para aislar las fuerzas mecánicas

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparación de embriones para Microscopía Electrónica de la Drosophila Tubo cardíaco embrionario

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

El aislamiento de lábil Multi-complejos de proteínas por In vivo Control Celular entrecruzamiento y Inmuno-cromatografía de afinidad magnética

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

En vivo-La centrifugación de embriones de Drosophila

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Imágenes de interacciones proteína-proteína In vivo</em

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

Imagen de la posición vertical Drosophila Los embriones

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

Cultivos de células primarias de Drosophila Gástrula embriones

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

Celular imagen Cambiar forma de vida Drosophila Los embriones

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Aislamiento y purificación de Kinesin desde Drosophila Los embriones

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...