Agradecemos a los Dres. Erez Eliyahu, Daniel Melamed, Ophry Pines y Doron Rapaport ayuda y comentarios durante la creación de este protocolo. Este trabajo está financiado por la ISF (número de concesión 1193-1109).

1. Las mitocondrias Purificación Pesar el pellet de células. Aproximadamente 0,6 g se obtienen a partir de células 100 ml. <ol><li> Crecer 100-150 ml de células de levadura a OD 600 = 1-1,5 a 30 ° C en un medio de crecimiento no fermentable, como medio de crecimiento a base de galactosa, con el fin de enriquecer para las mitocondrias. </li><li> Centrifugar las células a 3000 xg durante 5 minutos a temperatura ambiente y deseche el sobrenadante. </li><li> Lavar el precipitado con agua destilada dos veces y centrifugar de nuevo. Eliminar el sobrenadante. </li><li> Resuspender el precipitado en 1 ml de tampón por TDT 0,5 g de células. Es importante para el tratamiento de las células con un agente reductor con el fin de romper los enlaces disulfuro dentro de la pared celular, mejorando de este modo la hidrólisis. </li><li> Se incuban las células durante 10 min a 30 ° C con agitación suave. Mientras tanto, sopesar 6 mg zimoliasa por 1 g de células y suspender en tampón de Zymolyase. </li><li> Centrifugar las células a 3000 × g durante 5 min atemperatura t habitación y descartar el sobrenadante. </li><li> Resuspender el precipitado en 1 ml de Zymolyase tampón por 0,15 g de células. No vórtice. </li><li> Mida OD 600 de 10 l alícuota de las células en 990 l de agua. </li><li> Añadir Zymolyase (paso 1,5) a las células para hidrolizar polímeros de glucosa en las β-1 ,3-glucano vínculos y generar esferoplastos. Desde este paso, esferoplastos se deben mantener en una solución isotónica a fin de evitar la lisis. </li><li> Se incuban las células durante 15 min a 30 ° C (para la actividad óptima de zimoliasa) con agitación suave. Para verificar la hidrólisis de la pared celular y la generación de esferoplastos, mezclar 10 l de las células con 990 l de agua. Se espera Esferoplastos para lisar debido a la diferencia osmótica y el OD 600 deben ser por lo menos 10 veces menor que el valor determinado en el paso 1.9. Si no, continuar la incubación con zimoliasa durante otros 15 minutos. </li><li> Centrifugar las células a 3000 xg durante 5 minutos a temperatura ambiente. Dis cuidadosamentetarjeta el sobrenadante, el pellet como podría ser inestable. </li><li> Lave esferoplastos con Zymolyase Buffer y descartar el sobrenadante. </li><li> Resuspender esferoplastos con 100 ml de medio de recuperación. Transferencia de esferoplastos a un matraz Erlenmeyer y se incuba durante 1 hora a 30 ° C con agitación. Esta etapa de recuperación es necesario ya que durante la traducción en el tratamiento Zymolyase está detenido y la localización del mRNA se interrumpe (Figura 1B). </li><li> Añadir 0,1 mg / ml de CHX y esferoplastos de transferencia a un tubo cónico de 50 ml previamente enfriado. Además CHX es importante para congelar asociación &#39;con los ribosomas ARNm. Por lo tanto, se mantiene ribosomas dependientes de ARNm asociación con las mitocondrias. </li><li> Esferoplastos se centrifuga a 3000 × g durante 5 min a 4 ° C y descartar el sobrenadante. </li><li> Lavar dos veces con frío Manitol Buffer. </li><li> Resuspender esferoplastos con 4 ml de frío Manitol Buffer y transferirlos a un homogeneizador Dounce de 15 ml de capacidad equipado con cierre bienmaja. Romper suavemente esferoplastos con 15 golpes y transferir el lisado a un tubo de 13 ml. </li><li> Centrifugar el lisado a 1.500 xg durante 6 min a 4 º C para sedimentar los núcleos y las células sin romper. </li><li> Transferir con cuidado el sobrenadante a un nuevo tubo. Ponga a un lado 1 ml (25%) de la muestra como una muestra no fraccionada (&quot;total&quot; de la muestra). Transferencia de 50 ml de esta muestra a un tubo nuevo y añadir 15 ml de 4X LSB para el análisis de Western blot. El precipitado de ARN del resto de la muestra &quot;Total&quot; (Ver Protocolo 3, RNA precipitación). </li><li> Centrifugar el sobrenadante a 10.000 xg durante 10 min a 4 ° C para sedimentar las mitocondrias. </li><li> Transferir el sobrenadante (~ 3 ml) a un nuevo tubo y se mantiene en hielo. Esta es la fracción &quot;citosólica&quot;. Transferencia de 50 ml de esta muestra a un tubo nuevo y añadir 15 ml de 4x LSB para el análisis de Western blot. El precipitado de ARN del resto de la muestra &quot;citosólica&quot; (Ver Prot.Ocol 3, ARN precipitación). </li><li> Lavar el precipitado con 3 ml de manitol tampón y se centrifuga de nuevo a 10.000 xg durante 10 min a 4 ° C. </li><li> Resuspender el precipitado con 3 ml Manitol Buffer. Este ejemplo contiene las mitocondrias, y se refiere como fracción &quot;mitocondrias&quot;. Transferencia de 50 ml de esta muestra a un tubo nuevo y añadir 15 ml de 4x LSB para el análisis de Western blot. </li></ol> 2. La extracción de RNA <ol><li> Añadir a cada muestra un volumen de M guanidinio-HCl 8 y dos volúmenes de etanol 100%. Agitar e incubar durante al menos 2 horas a -20 ° C. </li><li> Centrifugar las muestras a 10.000 xg durante 20 min a 4 ° C. Desechar el sobrenadante. Ten cuidado ya que la pastilla puede ser inestable. </li><li> Lavar el precipitado con 70% de EtOH. </li><li> Resuspender el precipitado con 400 ml de agua libre de RNasa y transferir la muestra a un nuevo tubo Eppendorf. </li><li> Precipitar el ARN de nuevo mediante la adición de 0,1 volumen de 3 M acetato de sodioe pH 5,2 y dos volúmenes de EtOH al 100%. Agitar e incubar durante al menos 2 horas a -20 ° C. </li><li> Centrifugar las muestras a 20.000 xg durante 20 min a 4 ° C. Descartar el sobrenadante y lavar con EtOH al 70%. </li><li> Secar el pellet y resuspender con RNasa libre de agua. Las muestras de ARN Almacenar a -80 ° C. Las muestras pueden ser utilizados para el análisis de Northern 19 o el análisis de microarrays 18 (Véase el Protocolo 3). </li></ol> 3. Preparación del ARN para el análisis de microarrays <ol><li> Resuspender el ARNm de la etapa 2.2 con 650 ml de agua libre de RNasa. </li><li> Para eliminar cualquier ADN residual o proteínas, añadir el mismo volumen (650 ml) de fenol ácido: cloroformo (5:1, pH 4.7) y agitar vigorosamente. Centrifugar a máxima velocidad durante 2 min. </li><li> Transferir 500 ml de la fase superior (la fase acuosa) a un nuevo tubo. Evite tomar la interfase, ya que contiene el ADN. También tenga cuidado de tomar fenol, que puede inhibir la reacción de transcripción inversa. &lt;/ Li&gt; <li> La muestra de ARN contiene una cantidad significativa de heparina, que es un potente inhibidor de la transcriptasa inversa. Por lo tanto, para la RT-PCR o microarrays de etiquetado que tiene que ser eliminado. Retire la heparina por precipitación con LiCl: añadir LiCl a una concentración final de 2 M y se incuban las muestras durante la noche a -20 ° C. </li><li> Descongele las muestras a 4 ° C y se centrifuga a 20.000 xg durante 20 min a 4 ° C. </li><li> Desechar con cuidado el sobrenadante y lavar el precipitado con etanol al 80%. Centrifugar de nuevo como se describe en el paso 3.5. </li><li> Desechar el sobrenadante, secar al aire y resuspender el precipitado en 150 ml de agua libre de RNasa. </li><li> Para eliminar cualquier residuo de LiCl, precipitar de nuevo con 0,1 volumen de acetato sódico 3 M pH 5,3 y 3 volúmenes de etanol 100%. Incubar a -20 ° C durante al menos 2 h. </li><li> Centrifugar a máxima velocidad durante 20 minutos a 4 ° C. Lave cuidadosamente la bolita transparente con etanol al 80% y secar al aire. </li><li> Resuspender elpellet con 25 ml de agua libre de ARNasa y mantener las muestras a -80 ° C. </li><li> Siga los pasos para el etiquetado y la hibridación de ARN descrito en 18,20. </li></ol>

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Los avances tecnológicos en las imágenes habían dado las herramientas de alta resolución para estudiar la localización del mRNA. Hoy en día, se puede medir el movimiento de incluso una sola molécula de ARNm en la escala de tiempo mseg 23-26. Sin embargo, los enfoques bioquímicos tradicionales, como el descrito anteriormente, también son informativos y son preferibles en algunos casos. Aislamiento bioquímica permite la purificación de un gran repertorio de los ARNm y proteínas, y por tanto es prefe...

Las células eucariotas están organizados en compartimentos distintos que tienen funciones específicas. Para cumplir su función, cada compartimiento contiene un conjunto único de las proteínas que son esenciales para su actividad. Un posible mecanismo a través del cual estas proteínas se acercan a su compartimento es por 1,2 localizada traducción. En este proceso, la proteína se sintetiza en su destino por los ribosomas y mRNAs que se encuentran allí. Entre las ventajas probables de traducción adaptada aumentan la eficiencia de la focalización de proteínas, disminución de la necesidad de chaperones de la proteína, y permitiendo a los mecanismos de regulación específicos del sitio. Además, los ARNm y ribosomas localizados pueden ser un depósito aislado de la maquinaria de traducción en los casos de estrés celular, cuando se inhibe la traducción general. Las mitocondrias se convirtió en los últimos años un modelo central para estudiar traducción localizada. La mayoría de las proteínas mitocondriales están codificadas en el núcleo, traducido en el citosol,e importado en el orgánulo. Varias líneas de evidencia indican que muchas de estas proteínas son producidas a través de un proceso de traducción local. Inicialmente, los estudios de microscopía electrónica y de fraccionamiento bioquímico detectan los ribosomas asociados con las mitocondrias 3-5. Estos estudios en los que luego corroboraron in vivo por el trabajo en los ARNm específicos, que se encontraron se importen solamente de una manera cotraduccional 6,7. Los estudios del genoma completo de los ARNm asociación con mitocondrias revelaron que una fracción significativa de los ARNm se localiza en las proximidades mitocondrias 8-10. Algunos de estos ARNm se caracterizaron además por fluorescencia in vivo métodos, como el pescado o MTag 9,11. Una interpretación sencillo de esta asociación es que estos mRNAs sirven como moldes para la traducción localizada. Los mecanismos por los que estos ARNm se acercan a las mitocondrias son desconocidos. Dominios no codificantes (más significantly 3 &#39;UTR), se mostró a participar en el ARNm asociación a la mitocondria 12. Estos dominios son probablemente para servir como un sitio de unión a las proteínas de unión al ARN que median su transporte. Los estudios en levadura revelaron que un miembro de la familia de proteínas PUM (Puf3) apoya asociación ARNm con mitocondrias 8,13. Un papel plausible para Puf3, que se basa en las funciones de otros miembros de la familia, es para inhibir la traducción mientras que el ARNm está en ruta 14. Por lo tanto, los ARNm pueden ser transportados en un estado no traducida, por las proteínas de unión de ARN que interactúan con regiones no codificantes. Por otra parte, un gran cuerpo de trabajo sugiere que el transporte se produce mientras se sintetiza la proteína. En particular, los inhibidores de la traducción, se mostró a afectar ARNm asociación 8,13. Además, características traducidas tales como el AUG, secuencia de direccionamiento mitocondrial (MTS) o regiones ORF se muestran para ayudar en la localización 8,11,15. Proteína Hsp70 familiar chaperone y receptor de la proteína en la membrana externa de la mitocondria también se muestran para apoyar asociación ARNm, lo que implica que más características codificada en proteínas son importantes para la localización del mRNA 16. Esto es consistente con un modelo en el que la proteína ribosoma-emergente sirve como elemento de reconocimiento para la orientación complejo proteína ARNm-ribosoma a la mitocondria 17. Traducción localizada cerca de la mitocondria fue estudiada por varios métodos, incluyendo la microscopía electrónica (para visualizar los ribosomas) 3, FISH 9, ARN verde (para detectar ARNm específicos) 11, y el fraccionamiento bioquímico (para detectar tanto el ARN y ribosomas) 10,18. Mientras que los métodos antiguos detectar la localización in vivo y pueden permitir la visualización de la dinámica de transporte, este último permite la detección de muchos ARNm diferentes en un solo experimento. Además, para los dominios de codificación de fraccionamiento bioquímico o no codificantes no necesitan ser colcados, por lo tanto, sus funciones específicas se puede evaluar. Fraccionamiento bioquímico ha sido utilizado con éxito durante muchos años, para el aislamiento de muchos diferentes compartimentos celulares. Sus directores y limitaciones están bien establecidos, y uno puede fácilmente modificar los protocolos existentes para diferentes propósitos. La instrumentación necesaria es estándar en muchos laboratorios, por lo que suele ser el primer método de elección para el estudio de la localización intracelular. Se describe un protocolo que se ha optimizado para el aislamiento de ARNm, mientras que los ribosomas están asociados con las mitocondrias. Por tanto, este protocolo es óptimo para los factores implicados en la traducción localizada cerca de las mitocondrias estudiando.

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			<td height="21" style="height:21px;width:216px;">Yeast Extract</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">BactoPeptone</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Galactose</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;">Do not autoclave Galactose</td>
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			<td height="60" style="height:60px;">Growth medium</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;">For mitochondrial enrichment, you should use any non- fermentable carbon source, such as Galactose-based growth medium sterilized 1% Yeast Extract, 2% BactoPeptone, 2% Galactose</td>
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			<td height="21" style="height:21px;">0.1 M TrisHCl pH9.4</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">30 mM TrisHCl pH7.6</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">&#160;10 mM DTT Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.1 M TrisHCl pH 9.4, 10 mM Dithiothreitol (DTT). Make fresh every time</td>
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			<td height="21" style="height:21px;width:216px;">1.2 M Sorbitol</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="25" style="height:25px;width:216px;">4 mM KH2PO4</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="25" style="height:25px;width:216px;">16 mM K2HPO4</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;width:216px;">0.2&#956;m filter</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="46" style="height:46px;">Zymolyase Buffer&#160;</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;">1.2 M Sorbitol, 4 mM KH2PO4 , 16 mM K2HPO4. Filter this&#160; buffer (0.2&#956;m) and keep at room temperature for future use</td>
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			<td height="21" style="height:21px;">&#160;Zymolyase 20T&#160;</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;">20,000 U/gr</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Recovery medium</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">Galactose-based growth medium supplemented with 1 M Sorbitol</td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.1 mg/mL Cycloheximide (CHX)</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">0.6 M Mannitol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">5 mM MgAc</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100 mM KCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5 mg/mL Heparin</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="63" style="height:63px;width:216px;">1 mM phenylmethanesulfonylfluoride (PMSF)</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Mannitol Buffer</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">0.6 M Mannitol, 30 mM TrisHCl pH7.6, 5 mM MgAc, 100 mM KCl. Add freshly: 0.5 mg/mL Heparin, 0.1 mg/mL CHX and 1mM (PMSF). Filter this buffer and use it ice-cold</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:216px;">8M Guanidinium-HCl</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;100% and 70% Ethanol (EtOH)</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">&#160;3 M Sodium Acetate pH5.2</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">&#160;10 M LiCl stock solution</td>
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			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 mM Tris HCl pH 6.8</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SDS</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">&#160;Glycerol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
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			<td height="21" style="height:21px;">&#946;-mercaptoethanol</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bromophenolblue</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#160;4xLSB</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;">250 mM Tris HCl pH 6.8, 8% SDS, 40% Glycerol, 20% &#946;-mercaptoethanol and 0.02% Bromophenol blue</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dounce homogenizer of 15-ml capacity equipped with tight- fitting pestle</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td style="width:563px;"></td>
		</tr>
	</tbody>
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isolation of mrnas associated with yeast mitochondria to study mechanisms of localized translation

La mayoría de las proteínas mitocondriales están codificadas en el núcleo y deben ser importados en el orgánulo. Import puede ocurrir mientras que la proteína se sintetiza cerca de las mitocondrias. El apoyo a esta posibilidad se deriva de estudios recientes, en los que se muestran muchos mRNAs que codifican proteínas mitocondriales estar localizado en las proximidades mitocondrias. Junto a las manifestaciones anteriores de asociación ribosomas &#39;con la membrana externa, estos resultados sugieren un proceso de traducción localizada. Dicha traducción adaptada puede mejorar la eficiencia importan, suministran sitios únicos de regulación y reducir al mínimo los casos de expresión ectópica. Diversos métodos han sido utilizados para caracterizar los factores y elementos que median la traducción localizada. Estándar entre ellos es de fraccionamiento subcelular por centrifugación diferencial. Este protocolo tiene la ventaja de aislamiento de ARNm, ribosomas y proteínas en un solo procedimiento. Estos pueden ser caracterizados por diversas molecular y bimétodos ochemical. Además, transcriptómica y proteómica métodos se pueden aplicar al material resultante, por ende facilitaría ideas de todo el genoma. La utilización de la levadura como organismo modelo para tales estudios tiene las ventajas de velocidad, los costos y simplicidad. Además, las herramientas genéticas avanzadas y supresión cepas disponibles facilita la verificación de los factores de candidatos.

Este protocolo permite la separación de una fracción que contiene mitocondrias-a partir de componentes citosólicos. La mejor manera de probar su éxito es para llevar a cabo análisis de Northern y análisis de Western (Figura 1) a las muestras de las diferentes etapas de aislamiento. Tres parámetros para la calidad de aislamiento se derivan de estos análisis. En primer lugar, si el ARN o proteínas en las muestras están intactos - éstos serán detectados como bandas distintas en los análisis. E...

Watch this Scientific Journal Video about Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation at JoVE.com

Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

Muchos ARNm que codifican proteínas mitocondriales se asocian con la membrana externa mitocondrias. Se describe un procedimiento de fraccionamiento subcelular destinada a aislamiento de las mitocondrias de levadura con sus ARNm y ribosomas asociados. Este protocolo se puede aplicar a las células cultivadas bajo diversas condiciones con el fin de revelar los mecanismos de la localización del mRNA y la traducción localizada cerca de las mitocondrias.

Aislamiento de ARNm asociados a la mitocondria de levadura para estudiar los mecanismos de traducción localizada

Most of mitochondrial proteins are encoded in the nucleus and need to be imported into the organelle. Import may occur while the protein is synthesized ...

isolation-mrnas-associated-with-yeast-mitochondria-to-study

Research

JoVE Journal

Biology

12.8K vistas.  Technion - Israel Institute of Technology.  Muchos ARNm que codifican proteínas mitocondriales se asocian con la membrana externa mitocondrias. Se describe un procedimiento de fraccionamiento subcelular destinada a aislamiento de las mitocondrias de levadura con sus ARNm y ribosomas asociados. Este protocolo se puede aplicar a las células cultivadas bajo diversas condiciones con el fin de revelar los mecanismos de la localización del mRNA y la traducción localizada cerca de las mitocondrias. 

Video: Isolation of mRNAs Associated with Yeast Mitochondria to Study Mechanisms of Localized Translation

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

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

Purificación de las mitocondrias de las células de levadura

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

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 ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

La visualización de los ARNm retículo endoplásmico localizados en células de mamíferos

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial dysfunction often accompanies and contributes to human disease. Majority of the approaches that have been developed to evaluate mitochondrial function and dysfunction are based on in vitro or ex vivo measurements. Results from these experiments have limited ability in determining mitochondrial function in vivo. Here, we describe a novel approach that utilizes confocal scanning microscopy for the imaging of intact tissues in live aminals, which allows the evaluation of single mitochondrial function in a real-time manner in vivo. First, we generate transgenic mice expressing the mitochondrial targeted superoxide indicator, circularly permuted yellow fluorescent protein (mt-cpYFP). Anesthetized mt-cpYFP mouse is fixed on a custom-made stage adaptor and time-lapse images are taken from the exposed skeletal muscles of the hindlimb. The mouse is subsequently sacrificed and the heart is set up for Langendorff perfusion with physiological solutions at 37 &deg;C. The perfused heart is positioned in a special chamber on the confocal microscope stage and gentle pressure is applied to immobilize the heart and suppress heart beat induced motion artifact. Superoxide flashes are detected by real-time 2D confocal imaging at a frequency of one frame per second. The perfusion solution can be modified to contain different respiration substrates or other fluorescent indicators. The perfusion can also be adjusted to produce disease models such as ischemia and reperfusion. This technique is a unique approach for determining the function of single mitochondrion in intact tissues and in vivo.

Confocal Imaging of Single Mitochondrial Superoxide Flashes in Intact Heart or In Vivo

Confocal scanning microscopy is applied for imaging single mitochondrial events in perfused heart or skeletal muscles in live animal. Real-time monitoring of single mitochondrial processes such as superoxide flashes and membrane potential fluctuations enables the evaluation of mitochondrial function in a physiologically relevant context and during pathological perturbations.

Imagen confocal de Soltero mitocondriales superóxido Parpadea en corazón intacto o<em&gt; En Vivo</em

Mitochondrion is a critical intracellular organelle responsible for energy production and intracellular signaling in eukaryotic systems. Mitochondrial ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Aislamiento y análisis funcional de las mitocondrias de las células cultivadas y ratón de Tejidos

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally scales with the size of the cell, notably during embryogenesis when dramatic reductions in both cell and nuclear sizes occur. Mechanisms of nuclear size regulation are largely unknown and may be relevant to cancer where altered nuclear size is a key diagnostic and prognostic parameter. In vivo approaches to identifying nuclear size regulators are complicated by the essential and complex nature of nuclear function. The in vitro approach described here to study nuclear size control takes advantage of the normal reductions in nuclear size that occur during Xenopus laevis development. First, nuclei are assembled in X. laevis egg extract. Then, these nuclei are isolated and resuspended in cytoplasm from late stage embryos. After a 30 - 90 min incubation period, nuclear surface area decreases by 20 - 60%, providing a useful assay to identify cytoplasmic components present in late stage embryos that contribute to developmental nuclear size scaling. A major advantage of this approach is the relative facility with which the egg and embryo extracts can be biochemically manipulated, allowing for the identification of novel proteins and activities that regulate nuclear size. As with any in vitro approach, validation of results in an in vivo system is important, and microinjection of X. laevis embryos is particularly appropriate for these studies.

A Cell-Free Assay Using Xenopus laevis Embryo Extracts to Study Mechanisms of Nuclear Size Regulation

Mechanisms of cellular and intra-cellular scaling remain elusive. The use of Xenopus embryo extracts has become increasingly common to elucidate mechanisms of organelle size regulation. This method describes embryo extract preparation and a novel nuclear scaling assay through which mechanisms of nuclear size regulation can be identified.

Un ensayo libre de células Utilizando<em&gt; Xenopus laevis</em&gt; Embrión Extrae para estudiar los mecanismos de regulación del tamaño nuclear

A fundamental question in cell biology is how cell and organelle sizes are regulated. It has long been recognized that the size of the nucleus generally ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Análisis de la transferencia dependiente de la unión Gap de miARN con 3D-FRAP Microscopía

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

The Use of the Patch-Clamp Technique to Study the Thermogenic Capacity of Mitochondria

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat metabolic syndrome. Thermogenic tissues such as brown and beige fats develop highly specialized mitochondria for heat production. Mitochondria of other tissues, which primarily produce ATP, also convert up to 25% of the total mitochondrial energy production into heat and can, therefore, have a considerable impact on the physiology of the whole body. Mitochondrial thermogenesis is not only essential for maintaining the body temperature, but also prevents diet-induced obesity and reduces the production of reactive oxygen species (ROS) to protect cells from oxidative damage. Since mitochondrial thermogenesis is a key regulator of cellular metabolism, a mechanistic understanding of this fundamental process will help in the development of therapeutic strategies to combat many pathologies associated with mitochondrial dysfunction. Importantly, the precise molecular mechanisms that control acute activation of thermogenesis in mitochondria are poorly defined. This lack of information is largely due to a dearth of methods for the direct measurement of uncoupling proteins. The recent development of patch-clamp methodology applied to mitochondria enabled, for the first time, the direct study of the phenomenon at the origin of mitochondrial thermogenesis, H+ leak through the IMM, and the first biophysical characterization of mitochondrial transporters responsible for it, the uncoupling protein 1 (UCP1), specific of brown and beige fats, and the ADP/ATP transporter (AAC) for all other tissues. This unique approach will provide new insights into the mechanisms that control H+ leak and mitochondrial thermogenesis and how they can be targeted to combat metabolic syndrome. This paper describes the patch-clamp methodology applied to mitochondria to study their thermogenic capacity by directly measuring H+ currents through the IMM.

This method article details the main steps in measuring H+ leak across the inner mitochondrial membrane with the patch-clamp technique, a new approach to study the thermogenic capacity of mitochondria.

El uso de la técnica Patch-Clamp para estudiar la capacidad termogénica de las mitocondrias

Mitochondrial thermogenesis (also known as mitochondrial uncoupling) is one of the most promising targets for increasing energy expenditure to combat ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Aplicación del movimiento pasivo de la cabeza para generar aceleraciones definidas en las cabezas de roedores

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...

Aislamiento de ARNm asociados a la mitocondria de levadura para estudiar los mecanismos de traducción localizada

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