Nos gustaría dar las gracias a Andrea Kremsreiter por su excelente asistencia técnica. El trabajo en nuestro laboratorio es apoyado por el NIH GM096088 y el Vilas Confianza de la Universidad de Wisconsin - Madison.

1. Métodos luminometría Levadura Utilice cepa BYYT de Saccharomyces cerevisiae. Es un yvc1-tok1-derivado de BY4741 (Mata, ura3D0, his3D1, leu2D0, lys2D0, yvc1 :: HIS3, tok1 :: kanMX4). Transformar las células con los plásmidos pEVP1/Aeq Aequorina que expresan-Leu seleccionable y la rata TRPV4-expresión de plásmido-ura seleccionable p416GPDV4, como se describe en Batiza et al. 26 y Loukin et al. 24 Use métodos estándar de la construcción del plásmido y la transformación 27 . La cepa de levadura y plásmidos están disponibles bajo petición. <ol><li> Cultura 2 ml de células de levadura durante toda la noche a fase de post-logarítmica en un agitador 30 ° C en Leu-URA-&quot;DCD&quot; medio de abandono 28 (una variante sulfato-empobrecido del medio de CMD-Leu-ura 29), suplementado con sorbitol 1M . Inocular 200 l de estos cultivos en 1,8 ml de medio fresco suplementado con 2 mM de la Luciferin coelenterazina. Crecer en la oscuridad a temperatura ambiente durante otras 24 horas sin agitación. </li><li> Medir la osmolaridad de la cultura (típicamente a 1400 mOsM) y otras soluciones (a continuación) utilizando un osmómetro de presión de vapor. </li><li> Alícuota de 20 l de cultivo fresco en un tubo luminómetro 12 mm para un solo tubo luminómetro. </li><li> Para el choque hipotónico, añadir 200 l de una solución, que contiene 25 mM de NaEGTA (pH 7,2) o nombres (pH 7,2) y NaCl 500-100 mM, (osmolaridad total de 1,200-400 mOsM) dependiendo del grado de descarga deseada. Monitorear continuamente la luminiscencia antes y por lo menos 120 segundos después de la downshock osmótica, sólo interrumpido por la breve operación de dilución. Registro de salida luminómetro en un ordenador de escritorio como unidades relativas de luminiscencia (RUL). </li></ol> Aunque no para el estudio de TRPV4 transgénico, hemos utilizado una variante del protocolo anterior en un sistema automatizado para examinar las respuestas de un gran número de cepas de levadura. Estudio of las respuestas a la hipo-30 o hiperosmóticos 31 estímulos del deletome levadura (la colección de 4906 de la levadura deletants de un solo gen) utilizando un luminómetro de microplacas y analizadas con el software correspondiente ha sido un éxito. 2. y 3. Métodos de ovocitos Electrofisiología Electrofisiología utiliza métodos básicos 32. PCR amplificar el marco de lectura abierta de tipo salvaje o mutante TRPV4 utilizando alta fidelidad PfuUtra polimerasa e integrado en pGH19 33. Esto implica una inserción precisa de la ORF entre los 5 &#39;y 3&#39; UTRs de el gen β-globina de Xenopus y aguas abajo de un promotor de la ARN-polimerasa de T7. Linealizar el constructo de aguas abajo de la UTR 3 &#39;y utilizado como plantillas en una reacción in vitro de la polimerasa de ARN de T7 en. Utilizar técnicas de biología molecular estándar 34. Utilice ovocitos V-VI de la etapa de X. laevis. La cría de animales, ovariecto parcialmi, la eliminación del sobre defolliculation y vitelina siga los procedimientos estándar 32,33,35. Inyectar 2-40 ng de ARNc por oocito solución usando un microinyector automática Drummond Nanoinject II. Inyectar ~ 30 ovocitos por cada conjunto de experimentos. Después de la inyección de ARNc de TRPV4-, incubar los ovocitos en el medio ND96 con gentamicina (100 mg / ml) y 1 M rojo de rutenio 14. 2. Dos Electrodo abrazadera de voltaje <ol><li> Tire de borosilicato pipetas de registro de vidrio de precisión desechables Micropipetas 100 l individualmente con un extractor de pipeta. </li><li> Tire tanto voltaje de medición y electrodos de pipeta corrientes inyección se tiran a tener una abertura punta de ~ 1 m de diámetro. </li><li> Electrodos de reposición con 2 M KCl resultante en 0,1-0,2 resistencia mΩ. Montarlos en Headstages HS2A, que junto con una abrazadera de baño virtual con conexión a tierra VG-2Ax100, están conectados a una interfaz GeneClamp 500 amplificador a través de un digitalizador Digidata 1440. Adquirir<html> datos utilizando el software pClamp10. </li><li> Coloque el ovocito para ser probado en un baño de 1 ml en una cámara de plástico fabricada montada en el escenario de un microscopio de disección con 20 aumentos. La solución del baño es virtual a tierra por un puente de agar 3 M KCl y un cable de plata clorada colocado cerca del ovocito y conectado a una etapa de tierra virtual VG-2A. </li><li> Construir la cámara para la perfusión continua con tubos de plástico como de entrada y salida de solución. Conectar el tubo de entrada está conectado a un sistema de perfusión fabricada, que es un múltiple de seis conchas 50 ml de jeringa, cada uno lleno con una solución diferente. Tipos de alimentación por gravedad de cualquier solución particular se controla para ~ 2-3 ml / min utilizando &quot;Keck&quot; abrazaderas de rampa en los tubos. </li><li> Montar los dos electrodos con sus Headstages sobre micromanipuladores. Realice las penetraciones de electrodo del ovocito por micromanipulación. </li></ol> 3. Patch Clamp Examen de directo mecanosensibilidad &quot;&gt; Los métodos básicos de patch clamp incluyendo la preparación de la pipeta, la formación GΩ-sello, y formación de en-célula o modos extirpados son aquellos en los Hamill et al. 36 y Conn 32. <ol><li> Llenar pipetas de vidrio de borosilicato con una abertura ~ 1 m de diámetro en la punta (número de burbujas 5-6) con 98 mM de KCl, 1 mM de MgCl 2, y 10 mM de K +-HEPES, pH 7,2. </li><li> Coloque la pipeta de pinzamiento zonal a través de un tubo de plástico a una jeringa de 5 ml y por medio de una junta en T, también a un manómetro. Utilice un ordenador para manejar tanto el electrodo y las salidas de manómetro. Adquirir datos a 10 kHz, y luego jugar de nuevo a través de un filtro de ocho polos Bessel a 1 kHz para el análisis. </li><li> Diferentes parches pueden ser extirpados de la misma ovocito repetidamente. Esta práctica hace que el análisis de la actividad molecular de TRPV4 más eficiente y confiable. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Como se dijo en la introducción, los métodos comúnmente utilizados para estudiar las funciones TRPV4 veces resultaron en inconsistencias y controversias. Los dos conjuntos de métodos descritos aquí ofrecen algunas ventajas y pueden complementar los métodos existentes. Mientras describimos sólo los estudios sobre TRPV4, los métodos pueden ser extendidos para estudiar otros canales iónicos también. 1. Métodos luminometría Levadura Levadura en ciernes tien...

TRP (potencial de receptor transitorio) comprenden siete subfamilias de canales de cationes que sirven funciones sensoriales 1,2. En los mamíferos, TRPV, la subfamilia vanilloid de TRP, tiene seis variedades. TRPV4 (tipo 4) 3,4 responde al calor, ciertos productos químicos, hinchazón osmótica, o esfuerzo de corte. El gen TRPV4 se aisló repetidamente por el candidato-gen y / o clonación de expresión 5-8. El último método siguió la respuesta del producto génico a la hipo-osmolaridad. TRPV4 se expresa en casi todos los órganos y funciones en el desarrollo, fisiología, o patología de muchos tipos de células dispares 3,4. Llama la atención son las mutaciones TRPV4 autosómicos dominantes humana&gt; 50, provocando neuropatías periféricas y / o displasias esqueléticas (anormalidades en el desarrollo del esqueleto) 9-11. Oscilan Las displasias esqueléticas de leves tipo brachyomia 3 (tipo-3 enanismo), displasia espondilometafisaria tipo Kozlowski, a Severe displasias, algunas causando la muerte neonatal o embrionario. Aunque parecen posibles toda clase de mecanismos, ninguno explica la diversidad, la complejidad, variabilidad y superposiciones ocasionales de estas enfermedades 4. Al igual que otros canales TRP 1, TRPV4 es un tetrámero. En rata o TRPV4 humana, cada subunidad está constituida por 871 residuos. Su elemento central es el de seis hélices transmembrana (S1-S6), lo que probablemente se disponen de una manera similar a canales de K + dependientes de voltaje. Hay, S1 a S4 formar un dominio periférica y la S5 y S6 forman el dominio de poro de permeación. Entre S5 y S6 de TRPV4 es una hélice de poro corto seguido por la secuencia de LDLFKLTIGMGDL, cuatro de los cuales presumiblemente convergen para formar el filtro de cationes. El segmento citoplasmático N-terminal de 470 residuos contiene 6 repeticiones de anquirina, conocidos por unirse a proteínas o ligandos pequeños. El segmento citoplásmico 152-residuo C-terminal incluye una secuencia de unión a calmodulina-entre otros sitios posibles que se unen OTsus elementos 3. TRPV4 es un canal catiónico que excluye esencialmente aniones 1. Aunque su función fisiológica es para transducir estímulos a Ca 2 + afluencia, que también es permeable a otros cationes con una secuencia de permeabilidad Eisenman IV, favoreciendo divalente a un Ca P: P Na ~ 7 12. Conductancia de un canal rectifica a ~ 90 pS hacia afuera y hacia adentro ~ 40 pS 6,13,14. Heteróloga expresada actual (abajo) se puede activar por hipo-osmótica hinchazón, tensión de corte o calor 15. También es activado por los ácidos grasos poliinsaturados 16,17 y el éster de forbol sintética 4αPDD 18. En la actualidad, el agonista más potente es GSK1016790A 19 y antagonista es GSK2193874, eficaz en 10 -9 a 10 -8 M 20, ambas descubiertas por el alto rendimiento, la pequeña pantalla-molecular. Dos áreas clave de la investigación TRPV4 permanecen confusos: (1) A pesar de que TRPV4 es estudiado en gran medida por su mechanosensitivity, su base molecular es controversial. Un modelo describe hipo-osmolaridad de alguna manera activa la fosfolipasa A2 (PLA2) para producir el ácido graso poliinsaturado (PUFA) de ácido araquidónico (AA), que se convierte en ácido epoxieicosatrienoicos (EET) por una epoxigenasa, y la unión de EET activa TRPV4 16, 17. Sin embargo, sí TRPV4 se ha demostrado que responden directamente a estiramiento de la membrana 14 (a continuación), que proporciona una explicación más simple. (2) Las patologías mutantes TRPV4 son desconcertantes. En la base, es necesario saber si las enfermedades se deben a la pérdida, la reducción o el aumento de las actividades de canal. Incluso en este caso, la literatura está lejos de ser clara. Aunque se reportaron múltiples alelos esquelético-displasia tener actividades más altas, (ganancia de función, GOF) 4,21, varios se informó que han reducido las actividades (pérdida de la función, LOF) 10,22. Un estudio sistemático de 14 alelos que los ha encontradotodas las mutaciones se GOF (continuación) 23. La afirmación de que algunos son LOF parece contradecir el fenotipo de TRPV4-/ - ratón knockout, que son viables o fértiles, con sólo pequeños defectos, a pesar de una pérdida completa de la función TRPV4. Una parte de estas controversias tiene origen metodológico. Los laboratorios usan diferentes métodos, o variantes de un método, y emplean diferentes normas de juzgamiento. Por lo general, TRPV4 se expresa transitoriamente en células de mamífero en cultivo (HEK, CHO, HeLa) y el surgimiento de interior [Ca 2 +] sobre agonista o estimulación hipotónica está registrada con el colorante fluorescente sensible a Ca + 2, Fura-2. El exceso de confianza en este ensayo fluorométrico ha sido criticada 1. El nivel de expresión en diferentes poblaciones, necesita ser controlado y documentado la distribución en el mismo, así como la concentración de colorante eficaz,. Más fiable es los exámenes electrofisiológicos directos. Incluso esto, como commonly practica, también no está exenta de problemas. Debido a que los niveles de expresión en células cultivadas individuales son difíciles de controlar, las corrientes de células enteras tienen grandes variaciones. Además, puesto que las corrientes son pequeñas, las estadísticas fiables tendrán que depender de muestras de gran tamaño, a menudo no es práctico. Rara vez se han realizado los exámenes Patch-clamp. Algunas de estas grabaciones muestran racimos de estallidos de actividad que componen la evaluación estadística desafiante 16,17. Para comprender mejor el mechanosensitivity molecular, se han desarrollado dos sistemas adicionales para examinar TRPV4. (1) Para aislar TRPV4 lejos de sus socios de mamíferos habituales, hemos expresado TRPV4 rata en ciernes de levadura 24. La expresión funcional de TRPV4 en este contexto evolutivamente distantes mostró que aún podría responder a la fuerza osmótica sin sus socios habituales. Debido a que la levadura no hace PUFAs como AA o EET, y su genoma no tiene genes PLA2 o epoxigenasa, esta expression también muestra que no se requieren para TRPV4 para detectar la fuerza. Tener TRPV4 en los eucariotas biológicamente más susceptibles moleculares también permite la manipulación eficiente de avanzar-o revertir-genéticos 25. (2) Para los análisis biofísicos en profundidad de TRPV4, expresamos TRPV4 en ovocitos de Xenopus. A diferencia de las células cultivadas, que producen sub-nA a nA (10 -10 a 10 -9 A) corrientes, un oocito expresa corrientes en μA de (10 -6 a 10 -4 A). Señal mucho más grande sobre el ruido permite una mejor cuantificación y la comparación con más confianza. TRPV4, así expresado, también se puede examinar como moléculas individuales utilizando un patch clamp. Un único ovocito permite el muestreo de parches repetido, de nuevo haciendo una cuantificación más fiable. Estos estudios mostraron que la propia canal TRPV4 directamente puede ser activado por fuerza de estiramiento de la membrana 14. Los análisis también mostraron que 14 alelos representativos esquelético-displasia son todas las mutaciones con ganancia de función. Además, el degree de este constitutiva Ca 2 + fugas es paralelo a la gravedad de las enfermedades esqueléticas 23. Debido a su novedad y utilidad, los procedimientos detallados de estos dos métodos se ensamblan aquí para permitir repeticiones en futuras investigaciones sobre TRPV4 o canales similares.

<table border="0" cellpadding="0" cellspacing="0" width="595">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Vapor Pressure Osmometer</td>
			<td style="width:93px;">Wescor</td>
			<td style="width:119px;">Wescor 5500</td>
			<td style="width:167px;">Logan, UT, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sirius Single Tube Luminometer </td>
			<td style="width:93px;">Titertek-Berhold</td>
			<td style="width:119px;">Sirius </td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microplate luminometer</td>
			<td style="width:93px;">Titertek-Berthold </td>
			<td style="width:119px;">Mithras LB940</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Luminometry software</td>
			<td style="width:93px;">Titertek-Berthold</td>
			<td style="width:119px;">MikroWin2000</td>
			<td>Huntsville, LA, USA</td>
		</tr>
		<tr height="210">
			<td height="210" style="height:210px;width:216px;">Patch clamp and software</td>
			<td style="width:93px;">Axon Instrument</td>
			<td style="width:119px;">HS2A headstage, VG-2Ax100 virtual grounded bath clamp, GeneClamp 500 amplifier, digidata 1440 digitizer, pClamp10 software </td>
			<td>Union City, CA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">manometer</td>
			<td style="width:93px;">Bio-Tec</td>
			<td style="width:119px;"> </td>
			<td>Winooski, VT, USA</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Pipet puller</td>
			<td style="width:93px;">Sutter Instrument Co</td>
			<td style="width:119px;">Model P-97</td>
			<td>Novata, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Microinjector</td>
			<td style="width:93px;">Drummond Scientific</td>
			<td style="width:119px;">Nanoinject II</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Material:</td>
			<td style="width:93px;"> </td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">luciferin coelenterazine</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;"> </td>
			<td>Carlsbad, CA, USA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">T7 RNA polymerase reaction</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">mMessage mMachine</td>
			<td>Austin, TX, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">gentamicin</td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ruthenium red </td>
			<td style="width:93px;">Sigma</td>
			<td style="width:119px;"> </td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">micropipets </td>
			<td style="width:93px;">Drummond</td>
			<td style="width:119px;">100 microliter micropipets</td>
			<td>Broomall, PA, USA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">high-fidelity PfuUtra polymerase </td>
			<td style="width:93px;">Stratagene</td>
			<td style="width:119px;"> </td>
			<td>La Jolla, CA, USA</td>
		</tr>
	</tbody>
</table>

yeast luminometric and xenopus oocyte electrophysiological examinations of the molecular mechanosensitivity of trpv4

TRPV4 (Transient Receptor Potenciales familia vanilloid, tipo 4) es ampliamente expresado en tejidos de vertebrados y se activa por varios estímulos, incluyendo por fuerzas mecánicas. Ciertas mutaciones causan TRPV4 ósea hereditaria compleja o patologías neuronales en humanos. De tipo salvaje o mutante TRPV4 los transgenes se expresan comúnmente en células de mamífero cultivadas y se examinaron por Fura-2 fluorometría y por los electrodos. En cuanto al mecanismo de mechanosensitivity y las bases moleculares de las enfermedades, la literatura actual es confuso y controvertido. Para complementar métodos existentes, se describen dos métodos adicionales para examinar las propiedades moleculares de TRPV4. (1) Rata TRPV4 y un transgén aecuorina se transforman en levadura en ciernes. Un choque hipo osmtic de la población transformante produce una señal luminométrica debido a la combinación de aecuorina con Ca 2 +, lanzado a través del canal TRPV4. Aquí TRPV4 está aislado de sus proteínas asociadas mamíferos habitualesy revela su propio mechanosensitivity. (2) ARNc de TRPV4 se inyectó en oocitos de Xenopus. Después de un período de incubación adecuado, la corriente TRPV4 macroscópica se examinó con una pinza de voltaje de dos electrodos. El aumento de la intensidad después de la retirada de osmoticum inerte del baño de los ovocitos es indicativa de mechanosensitivity. Los microamperios (10 -6 a 10 -4 C) Las corrientes de ovocitos son mucho más grandes que las subnano a nanoamperio (10 -10 a 10 -9 C) Las corrientes de células cultivadas, produciendo cuantificaciones más claras y las evaluaciones de más confianza. Corrientes microscópicas que reflejan las actividades de proteínas de los canales individuales también pueden ser directamente registrados bajo una grapa de parche, en el de las células o el modo extirpado. Lo mismo oocito ofrece varias muestras de parches, lo que permite la replicación de mejores datos. Succiones aplicadas a los parches pueden activar TRPV4 evaluar directamente mechanosensitivity. Estos métodos también deben ser útiles en el estudio de otros tipos de canales TRP.

En un experimento típico luminometría, una gama de 400-1,200 mOsM choques hipotónicas abajo se logra mediante la adición de 200 l de soluciones de choque de diferente osmolaridad baja a 20 l de cultivo a 1.400 mOsM. La actividad TRPV4 es evidente cuando el RLU de la experimental se comparó con la de dos controles negativos: células de levadura transformadas con el plásmido p416GPDV4 vacía o una que contiene el gen TRPV4 con una mutación en el filtro de iones (Figura 1) 24. <...

Watch this Scientific Journal Video about Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4 at JoVE.com

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

Para complementar los métodos comúnmente utilizados para el estudio de TRPV4, dos métodos se describen: Su mechanosensitivity puede ser estudiado por Ca 2 +-luminometría aecuorina en la levadura transgénica a partir del desafío hipo-osmótica. También puede ser examinado en TRPV4-ARN inyectado ovocitos de Xenopus por células enteras de fijación de voltaje de dos electrodos o parche abrazadera en en-célula o el modo extirpado.

Luminométricos levadura y<em&gt; Xenopus</em&gt; ovocitos electrofisiológicos Exámenes del mecanosensibilidad Molecular de TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

yeast-luminometric-xenopus-oocyte-electrophysiological-examinations

Research

JoVE Journal

Biology

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

10.0K vistas.  University of Wisconsin – Madison.  Para complementar los métodos comúnmente utilizados para el estudio de TRPV4, dos métodos se describen: Su mechanosensitivity puede ser estudiado por Ca 2 +-luminometría aecuorina en la levadura transgénica a partir del desafío hipo-osmótica. También puede ser examinado en TRPV4-ARN inyectado ovocitos de Xenopus por células enteras de fijación de voltaje de dos electrodos o parche abrazadera en en-célula o el modo extirpado. 

Video: Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

Una técnica rápida para la visualización en vivo de las células de levadura inmovilizada

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

Investigación

Biología

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Parche de grabación Clamp de canales iónicos expresados ​​en ovocitos de Xenopus

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

Visualizar la localización del ARN en Xenopus Los ovocitos

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Un ensayo de microscopía de fluorescencia para Mitophagy Vigilancia de la levadura Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

La microinyección de ovocitos de ratón, Maduración y Evaluación de ploidía

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Pantallas competitiva Genómica de Bibliotecas de levadura con código de barras

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

Purificación por cromatografía de ribosomas de levadura de gran actividad

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Green Monster Process for the Generation of Yeast Strains Carrying Multiple Gene Deletions

Phenotypes for a gene deletion are often revealed only when the mutation is tested in a particular genetic background or environmental condition1,2. There are examples where many genes need to be deleted to unmask hidden gene functions3,4. Despite the potential for important discoveries, genetic interactions involving three or more genes are largely unexplored. Exhaustive searches of multi-mutant interactions would be impractical due to the sheer number of possible combinations of deletions. However, studies of selected sets of genes, such as sets of paralogs with a greater a priori chance of sharing a common function, would be informative.
In the yeast Saccharomyces cerevisiae, gene knockout is accomplished by replacing a gene with a selectable marker via homologous recombination. Because the number of markers is limited, methods have been developed for removing and reusing the same marker5,6,7,8,9,10. However, sequentially engineering multiple mutations using these methods is time-consuming because the time required scales linearly with the number of deletions to be generated.
Here we describe the Green Monster method for routinely engineering multiple deletions in yeast11. In this method, a green fluorescent protein (GFP) reporter integrated into deletions is used to quantitatively label strains according to the number of deletions contained in each strain (Figure 1). Repeated rounds of assortment of GFP-marked deletions via yeast mating and meiosis coupled with flow-cytometric enrichment of strains carrying more of these deletions lead to the accumulation of deletions in strains (Figure 2). Performing multiple processes in parallel, with each process incorporating one or more deletions per round, reduces the time required for strain construction.
The first step is to prepare haploid single-mutants termed 'ProMonsters,' each of which carries a GFP reporter in a deleted locus and one of the 'toolkit' loci&#x2014;either Green Monster GMToolkit-a or GMToolkit-&alpha; at the can1&Delta; locus (Figure 3). Using strains from the yeast deletion collection12, GFP-marked deletions can be conveniently generated by replacing the common KanMX4 cassette existing in these strains with a universal GFP-URA3 fragment. Each GMToolkit contains: either the a- or &alpha;-mating-type-specific haploid selection marker1 and exactly one of the two markers that, when both GMToolkits are present, collectively allow for selection of diploids.
The second step is to carry out the sexual cycling through which deletion loci can be combined within a single cell by the random assortment and/or meiotic recombination that accompanies each cycle of mating and sporulation.

The Green Monster method enables the rapid assembly of multiple deletions marked with a reporter gene encoding green fluorescent protein. This method is based on driving yeast strains through repeated cycles of sexual assortment of deletions and fluorescence-based enrichment of cells carrying more deletions.

El Proceso de Monstruo Verde para la generación de cepas de levadura que llevan deleciones múltiples genes

Phenotypes  for a gene deletion are often revealed only when the mutation is tested in a  particular genetic background or environmental condition1,2. ...

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.
Here, we employ the human cardiac sodium channel (hNaV1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.
The properties of fast activating ion channels, such as hNaV1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.
In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest1. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open Vaseline gap approach is used to obtain low noise recordings of ionic and gating currents from voltage-dependent ion channels expressed in Xenopus oocytes with high resolution of fast channel kinetics. With minor modification, voltage clamp fluorometry can be coupled to the cut-open oocyte protocol.

La<em&gt; Xenopus</em&gt; Ovocitos-Cut abierta vaselina Gap Técnica Voltage-clamp Con Fluorometría

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

Difusión de superficies y inmunotinción de cromosomas de la levadura

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...