This work was supported by NIH grant GM085232 and an Irma T. Hirschl/ Monique Weill-Caulier Scholar Award (to A.A.).

1. Preparación de lípidos <ol><li> Alícuota de los lípidos cantidad deseados en un tubo de vidrio transparente. Tu Ves. coli extracto lipídico polar, pero la mayoría de composiciones lipídicas pueden ser utilizados. Si los lípidos están en forma de polvo, ellos resuspender en cloroformo a una concentración de 20 mg / ml. Secar los lípidos a TA bajo N2 gas hasta que todo el disolvente se haya evaporado. </li><li> Resuspender los lípidos en pentano y secar de nuevo un flujo constante de gas N 2 para eliminar los restos sobrantes de cloroformo. Seco hasta que todo el disolvente se haya evaporado. Si bien el secado, gire suavemente el tubo de manera que el lípido se distribuye en una película uniforme que cubre el tercio inferior del tubo en lugar de una formación de una masa densa en la parte inferior. </li><li> Añadir el tampón de reconstitución que contiene detergente (300 mM KCl, 50 mM de ácido cítrico, 25 mM de K 2 HPO 4, pH 4,5, 35 mM CHAPS) para disolver los lípidos. Utilice otros detergentes suaves para este paso si la proteína es poco tolerante a la CHAPS. </li><li> Resuspend los lípidos a la claridad utilizando un aparato de ultrasonidos baño de agua. Sumergir el tubo en el centro del baño de agua sonicador la máxima potencia a corto (30 seg-1 min) con impulsos cortos (30 seg-1 min) pausas. La solución debería quedar claro. NOTA: El grado final de la claridad lograrse depende de la composición lipídica específico usado. Alguna variación de lote a lote en este paso, incluso entre los lípidos nominalmente idénticos, también es posible. Bruma residual es debido a la dispersión de la luz por las grandes partículas en la suspensión, que se pueden eliminar por centrifugación. </li><li> Incubar los lípidos se resuspendieron a temperatura ambiente durante 20-60 min. </li></ol> 2. Proteoliposoma Formación NOTA: Varias estrategias se puede emplear para insertar la proteína solubilizada con detergente en liposomas. Para CLC-EC1 diálisis funciona bien y por lo tanto es el método de elección 6,9,10. <ol><li> Añadir la proteína purificada a la deseada proteína-lípido (P / L) relación (expresada en microgramos o<html> f proteína / mg de lípidos). La selección de la relación P / L depende de la finalidad del experimento. <ol><li> Si el objetivo es evaluar la actividad de la proteína de interés, reconstituir a alta P / L&#39;s para maximizar la señal, ya que cada liposoma contiene múltiples copias de la proteína. Para cuantificar la actividad y / o la velocidad de transporte unitario de la proteína, reconstituir en un régimen de dilución de Poisson a baja P / L&#39;s, de modo que cada vesícula contiene en promedio proteína 1. Vea el paso 7 y Discusión para un análisis más profundo de cuándo utilizar cada régimen. </li></ol></li><li> Para determinar los valores de P / L óptimas para los diferentes regímenes, realizar una valoración completa de la actividad como una función de la concentración de proteína 8,11-13. Sin embargo, para cualquier proteína para la que el peso molecular (MW, expresado en kDa) de la unidad activa se conocen los siguientes valores P / L se pueden utilizar como guesstimates iniciales: pg &quot;/&gt; <img alt="Ecuación 2" fo:content-width="1.5in" src="/files/ftp_upload/52369/52369eq2.jpg" /></li><li> Cargue la proteína y mezcla de lípidos en un tubo de diálisis pre-humedecida o un casete. Coloque el dispositivo de diálisis en un vaso de precipitados que contiene 500-1,000x el volumen del tampón de reconstitución de lípidos (es decir, 1 L de tampón de 1 ml de la resuspensión de lípidos) bajo agitación suave. <ol><li> Cambio búfer 3-4 veces a intervalos de&gt; 3 horas. En cada cambio de solución, lavar la casete / tubo y el vaso de precipitados con agua destilada y llenar el vaso de precipitados con tampón de reconstitución fresco. Incluya 2.1 O / N intervalos para el cambio de solución. El número exacto y la duración de los cambios solución depende de la exacta de lípidos y detergentes utilizados. </li></ol></li><li> Después de la última etapa, retire los liposomas de la vasija de diálisis, alícuota en tubos y flash les congele en N2 líquido o congelación a -80 ° C. </li></ol> 3. Registro de Configuración &lt;p class = &quot;jove_content&quot;&gt; NOTA: La grabación de configuración (Figura 2A) se compone de dos cámaras (cilindros de fondo plano, ~ volumen 3-4 ml), un Cl - (ver más abajo) de electrodo, un medidor de pH con un análogo o salida digital eléctrica, un digitalizador, y un ordenador con un software de adquisición correspondiente. <ol><li> Conectar el Cl - electrodo al medidor de pH, la salida del medidor de pH para el digitalizador que está conectado a la computadora. Coloque el electrodo de referencia en una cámara y el cable de recodificación en la otra (Figura 2B). NOTA: La polaridad de los cables es correcta si? V Cal (ver Paso 6.4 y 7.2) es positiva. </li><li> Coloque las cámaras en una placa de agitación con una barra de agitación en la cámara de grabación (Figura 1B). Asegúrese de que la barra de agitación no toque el electrodo. </li><li> Conectar las cámaras utilizando un puente de agar (Figura 2B) (100 mM KCl y 2% de agarosa). Guarde los puentes de agar en 100 mM KCl. </li&gt; </ol> 4. Preparación de la Cl - Electrodo <ol><li> Tome un electrodo de edad y -eventualmente- que no funciona pH. Romper el recubrimiento de vidrio para revelar completamente los cables de plata. </li><li> Retire cuidadosamente el recubrimiento de los cables de plata usando una hoja de bisturí. Limpie los cables usando copiosas cantidades de H 2 O y EtOH. </li><li> Colocar en una solución saturada de FeCl 3 (o lejía) hasta que los cables están cubiertos con una capa oscura uniforme de AgCl. </li></ol> 5. Preparación de vesículas unilamelares <ol><li> La noche antes de los experimentos, se hinchan 1 g de Sephadex G-50 bolas en 15 ml de tampón externo (1 mM KCl, 150 mM de K 2 SO 4, ácido cítrico 25 mM, pH 4,5) con agitación suave (no revuelva) para al menos 3 horas a RT antes de su uso. Cada experimento requiere ~ 3 ml de perlas hinchadas. Mantenga las perlas hinchadas a 4 ° C durante unos días como máximo. </li><li> Mientras que los liposomas están descongelando a temperatura ambiente, se vierte en cada columna ~ 3 ml de hinchadas G-50 perlas. Deje que se sequen por gravedad a RT; esto por lo general toma 1-2 min. </li><li> Preparar vesículas unilamelares por extrusión de los proteoliposomas 11 veces a través de un Mini-Extrusora utilizando un punto de corte 0,4 m de teflón. </li><li> Colocar la columna en un tubo de fondo redondo de plástico. Haga girar las columnas para eliminar el exceso de solución. Centrifugar durante 20-30 segundos a 1400 xg en una centrífuga clínica. </li><li> Desechar el flujo a través, la columna de lugar en un tubo de vidrio de 13 x 100 mm y añadir 100 l de las vesículas extruidos a la columna. Girar la columna durante 1 min a 500 xg en una centrífuga clínica. </li><li> Recoger ~ 200 l de flujo a través que se añadirán a la cámara de grabación en el paso 6.5. </li></ol> 6. Eflujo Medición <ol><li> Coloque 2 ml de KCl 100 mM en la cámara (tierra) de referencia y 1,8 ml del tampón externo (1 mM KCl, 150 mM K 2 SO 4, ácido cítrico 25 mM, pH 4,5) a la cámara de grabación. El desequilibrio leve osmótico entre el interiory soluciones externas no afecta </li><li> Inicie el programa de adquisición. Deje que la línea de base se equilibre, esto puede tardar unos minutos. </li><li> Una vez que la señal llega a una línea de base estable (los liposomas están listos y las columnas seco), iniciar la grabación (Figura 3A). </li><li> Añadir 15 l de una solución 10 mM KCl para calibrar el sistema (Figura 2A). </li><li> Añadir liposomas. Un pequeño salto podría ser visible debido a la eliminación incompleta de Cl externa - a partir de los proteoliposomas (Figura 3A). Espere hasta que se estabilice la línea de base. </li><li> Añadir valinomicina (1 l de 1 mg / ml en EtOH) para iniciar el eflujo (Figura 3A). </li><li> Deje que el flujo de salida siga su curso temporal hasta que mesetas (Figura 3A). </li><li> Añadir 40 l de 1,5 M β-octilglucósido (β-OG) preparado en el tampón externo para disolver todos los liposomas (figura 3A). Termine la grabación. </li></ol> 7. Datos Análisis <ol><li> Exportar el archivo de rastreo de en un formato ASCII o texto e importarlo al programa de análisis de la elección. </li><li> Mida la tensión en los siguientes puntos (Figura 3A): Al comienzo de la grabación (V 0); Después de la adición del pulso de calibración (V cal); Después de la adición de los liposomas (V lipo); Al final de el flujo de salida (V fin); Después de la adición de detergente (V tot); Los voltajes de línea de base de manera que V 0 = 0. </li><li> Use la ecuación de Nernst-Plank para determinar el valor experimental de <img alt="Ecuación 3" fo:content-width=".3in" src="/files/ftp_upload/52369/52369eq3.jpg" /> midiendo <img alt="Ecuación 4" fo:content-width="3in" src="/files/ftp_upload/52369/52369eq4.jpg" /> Cuando Vol Cal es el volumen del impulso de calibración en l, 1,800 es el volumen de cámara en el comienzo del experimento se expresa en1; l, [Cl] cal / en son el Cl - concentraciones del impulso de calibración y de la memoria intermedia externa en mM y cal? V es el salto en mV medido después del pulso de calibración. NOTA: Esta determinación empírica de α sirve para tres propósitos: asegura que el sistema responde correctamente, ofrece una medida de la consistencia de la respuesta del instrumento entre los experimentos, así como para comprobar que no se han cometido errores en la fabricación solución. </li><li> Calcular las concentraciones de Cl después de cada paso de la siguiente manera: <img alt="Ecuación 5" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq5.jpg" /> <img alt="Ecuación 6" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq6.jpg" /> <img alt="Ecuación 7" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq7.jpg" /> El factor de 3/400 proviene de la dilución del impulso de calibración 15 l en el volumen final de 2.000 l. </li><li> Calculcomieron los cambios en [Cl] en cada paso, ΔCl lipo / def / tot. </li><li> Convertir V (t) en Cl (t) con <img alt="Ecuación 8" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq8.jpg" /> Determinar directamente los valores [CL] en los puntos críticos y compararlos con los valores calculados como un control interno. </li><li> Normalizar la traza para que la lipo Cl rel = 0 y Cl tot rel = 1 <img alt="Ecuación 9" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq9.jpg" /></li><li> Montar el supuesto tiempo de flujo a la siguiente ecuación: <img alt="Ecuación 10" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq10.jpg" /> Donde L es la tasa de Cl - fuga que se determina experimentalmente mediante la medición de Cl - flujo de salida a partir de liposomas libres de proteínas preparados en las mismas condiciones y τ es la constante de tiempo del proceso. </li><li> Calcular v, la velocidad inicial: Cuando ΔCl aleta se expresa en milimolar y V es el volumen de la cámara en l. </li><li> Calcular la tasa de transporte / conductancia <img alt="Ecuación 12" fo:content-width="3.5in" src="/files/ftp_upload/52369/52369eq12.jpg" /> Donde p es el P / L, MW es el peso molecular del complejo activo expresada en kDa </li></ol>

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The authors declare no competing financial interests.

Hemos descrito un protocolo detallado para medir la Cl - transporte mediado por canales o transportadores reconstituidas en liposomas aniones selectiva purificados. El ejemplo utilizado fue el procariota H + / Cl - Intercambiador CLC-ec1. Sin embargo, la metodología se puede adaptar fácilmente para estudiar canales cerrados por ligandos 12,13,15, 11,12 tensión, o deportivo selectividad diferente aniónico 15,16 sustituyendo el Ag: electrodo AgCl con uno adecuado ...

En las últimas dos décadas la capacidad para sobreexpresar y purificar las proteínas de transporte de membrana ha aumentado dramáticamente: canales iónicos, transportadores primario y secundario están ahora purificaron rutinariamente a partir de sistemas de expresión heterólogos, así como de fuentes naturales. Constantemente se están desarrollando nuevos enfoques para controlar la expresión, mejorar y facilitar la extracción y mejorar la estabilidad de estas proteínas 1-5. Estos avances tecnológicos han sido fundamentales en el desencadenamiento de la explosión de la información estructural a nivel atómico en las proteínas de membrana que, a su vez, mejorado nuestra comprensión de las bases estructurales de su función. En contraste, nuestra capacidad para investigar las propiedades funcionales de las proteínas purificadas no aumentó en la misma proporción, de modo que en algunos casos alta resolución información estructural está acompañada por datos funcionales cualitativos, lo que limita nuestra capacidad para probar cuantitativamente predicciones basadas en la estructura. Por lo tanto, la development de ensayos funcionales cuantitativos y generalizables es un paso clave hacia la elucidación de las bases mecanicistas de la función de las proteínas de membrana. Aquí se describe un ensayo de flujo de salida que se puede utilizar para determinar cuantitativamente propiedades funcionales clave de purificado y reconstituido Cl - canales y transportadores. Los principios subyacentes del ensayo se pueden generalizar a una variedad de sistemas de transporte, así como a las proteínas no-iónicos transporte. Los liposomas se reconstituyen con purificados Cl - canal / transportadores en la presencia de un gran Cl - gradiente (Figura 1A, B). Cl - flujo de salida se inicia mediante la adición de un ionóforo para permitir flujo de contra-ión, en nuestro caso el K + valinomicina ionóforo, que la tensión de derivación establecido por el Cl - y establecer el gradiente de potencial inicial membrana para el potencial de equilibrio de K + 6,7. Sin tque ionóforo no significativa Cl neta - flujo de salida se produce, ya que se evita mediante la generación de un potencial transmembrana. Los datos se describe cuantitativamente por dos parámetros medibles (Figura 1C): τ, la constante de tiempo de eflujo de Cl -, y f 0, la fracción de liposomas que no contienen una proteína activa. De τ f 0 y el unitaria Cl - velocidad de transporte, la fracción de proteínas activas y la masa molecular del complejo activo se pueden derivar 8. La técnica se ilustra aquí usando proteoliposomas reconstituidos con CLC-EC1, un bien caracterizado de tipo CLC H + / Cl - intercambiador de la estructura y función conocida. Este ensayo se generaliza fácilmente a los canales o transportadores con diferente selectividad iónica o cuya actividad depende de la presencia de voltaje y / o ligandos. Además, este ensayo se puede usar para determinar si las moléculas pequeñas afectan directamente a la proteína reconstituida,para cuantificar los efectos de estos compuestos y cómo composición de la membrana o de los reactivos modificadores de lípidos afectan a la función de los canales y transportadores reconstituidas.

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			<td height="21" style="height:21px;width:372px;">Name of Reagent/ Equipment</td>
			<td style="width:337px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;">Liposomicator, Avanti Polar Lipids Inc.</td>
			<td style="width:337px;">&#160;Avanti Polar Lipids Inc.</td>
			<td>610200</td>
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			<td height="21" style="height:21px;">IEC Centra CL2 Benchtop</td>
			<td style="width:337px;">Thermo Scientific</td>
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			<td height="21" style="height:21px;">Orion Research Model 701A Digital pH-mV meter</td>
			<td style="width:337px;"></td>
			<td style="width:119px;"></td>
			<td>These can be found on Ebay.</td>
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			<td height="21" style="height:21px;">&#160;Non-functional pH probe</td>
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			<td style="width:119px;"></td>
			<td>Any pH meter probe with silver wires will work. The glass/plastic coating needs to be removed and the wires cleaned.</td>
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			<td height="21" style="height:21px;">&#160; DI-710 Data Logger</td>
			<td style="width:337px;">DATAQ instruments</td>
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			<td height="21" style="height:21px;">WinDAQ acquisition software</td>
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			<td height="21" style="height:21px;">Pierce Disposable Plastic Columns, Gravity-flow, 2ml</td>
			<td style="width:337px;">Pierce (Thermo Scientific)</td>
			<td>29922</td>
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			<td height="21" style="height:21px;">KIMAX Culture Tubes, Disposable, Borosilicate Glass</td>
			<td style="width:337px;">Kimble Chase</td>
			<td>73500-13100</td>
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			<td height="21" style="height:21px;">Extruder Set With Holder/Heating Block</td>
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			<td align="right" style="width:119px;">610000</td>
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			<td height="21" style="height:21px;">Computer</td>
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a proteoliposome-based efflux assay to determine single-molecule properties of cl- channels and transporters

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as from eukaryotes. This increase has been largely driven by the successful push to obtain structural information on membrane proteins. However, the ability to functionally interrogate these proteins has not advanced at the same rate and is often limited to qualitative assays of limited quantitative value, thereby limiting the mechanistic insights that they can provide. An assay to quantitatively investigate the transport activity of reconstituted Cl- channels or transporters is described. The assay is based on the measure of the efflux rate of Cl- from proteoliposomes following the addition of the K+ ionophore valinomycin to shunt the membrane potential. An ion sensitive electrode is used to follow the time-course of ion efflux from proteoliposomes reconstituted with the desired protein. The method is highly suited for mechanistic studies, as it allows for the quantitative determination of key properties of the reconstituted protein, such as its unitary transport rate, the fraction of active protein and the molecular mass of the functional unit. The assay can also be utilized to determine the effect of small molecule compounds that directly inhibit/activate the reconstituted protein, as well as to test the modulatory effects of the membrane composition or lipid-modifying reagents. Where possible, direct comparison between results obtained using this method were found to be in good agreement with those obtained using electrophysiological approaches. The technique is illustrated using CLC-ec1, a CLC-type H+/Cl- exchanger, as a model system. The efflux assay can be utilized to study any Cl- conducting channel/transporter and, with minimal changes, can be adapted to study any ion-transporting protein.

Se describe un protocolo detallado y robusto para medir Cl - transporte mediado por purificada CLC-ec1, un procariota tipo CLC H + / Cl - intercambiador, reconstituido en liposomas. Una representación esquemática del experimento se muestra en la Figura 3 proteoliposomas reconstituidos con purificada CLC-ec1 y contiene alta Cl interna -. Se sumergen en un baño de solución que contiene bajo Cl -. Bajo estas condiciones netas Cl - flujo ...

Watch this Scientific Journal Video about A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters at JoVE.com

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Un Proteoliposoma Basada Eflujo Ensayo para determinar una sola molécula Propiedades de Cl<sup&gt; -</sup&gt; Canales y Transportadores

The last 15 years have been characterized by an explosion in the ability to overexpress and purify membrane proteins from prokaryotic organisms as well as ...

a-proteoliposome-based-efflux-assay-to-determine-single-molecule

Research

JoVE Journal

Biology

A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

9.7K vistas.  Weill Cornell Medical College. Proteoliposomes are used to study purified channels and transporters reconstituted in a well-defined biochemical environment. An experimental procedure to measure efflux mediated by these proteins is illustrated. The steps to prepare proteoliposomes, perform the recordings, and analyze data to quantitatively determine the functional properties of the reconstituted protein are described.

Video: A Proteoliposome-Based Efflux Assay to Determine Single-molecule Properties of Cl- Channels and Transporters

Single Molecule Methods for Monitoring Changes in Bilayer Elastic Properties

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein interactions and more general lipid bilayer-protein interactions. These interactions are particularly important in pharmacological research, as many current pharmaceuticals on the market can alter the lipid bilayer material properties, which can lead to altered membrane protein function.   The formation of gramicidin channels are dependent on conformational changes in gramicidin subunits which are in turn dependent on the properties of the lipid.  Hence the gramicidin channel  current is a reporter of altered properties of the bilayer due to certain compounds.  

Membrane protein function is regulated by the cell membrane lipid composition. This video-article details how to form a patch using bilayer patch electrodes, as well as how to use gramicidin channels as reporters of altered membrane properties.

Métodos de una sola molécula para vigilar los cambios en las propiedades elásticas bicapa

Membrane protein function is regulated by the cell membrane lipid composition.  This regulation is due to a combination of specific lipid-protein ...

Investigación

Biología

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

Visualización de una sola molécula de ADN de replicación con microscopía de fluorescencia

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

La observación directa de ADN Enzimas Replicar El uso de un ADN de una sola molécula de estiramiento de ensayo

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

Imagen de una sola molécula de transporte nuclear

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

A Fluorescent Screening Assay for Identifying Modulators of GIRK Channels

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in the brain, heart, skeletal muscle and endocrine tissue1,2. GIRK channels become activated following the binding of ligands (neurotransmitters, hormones, drugs, etc.) to their plasma membrane-bound, G protein-coupled receptors (GPCRs). This binding causes the stimulation of G proteins (Gi and Go) which subsequently bind to and activate the GIRK channel. Once opened the GIRK channel allows the movement of K+ out of the cell causing the resting membrane potential to become more negative. As a consequence, GIRK channel activation in neurons decreases spontaneous action potential formation and inhibits the release of excitatory neurotransmitters. In the heart, activation of the GIRK channel inhibits pacemaker activity thereby slowing the heart rate.
GIRK channels represent novel targets for the development of new therapeutic agents for the treatment neuropathic pain, drug addiction, cardiac arrhythmias and other disorders3. However, the pharmacology of these channels remains largely unexplored. Although a number of drugs including anti-arrhythmic agents, antipsychotic drugs and antidepressants block the GIRK channel, this inhibition is not selective and occurs at relatively high drug concentrations3.
Here, we describe a real-time screening assay for identifying new modulators of GIRK channels. In this assay, neuronal AtT20 cells, expressing GIRK channels, are loaded with membrane potential-sensitive fluorescent dyes such as bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC4(3)] or HLB 021-152 (Figure 1). The dye molecules become strongly fluorescent following uptake into the cells (Figure 1). Treatment of the cells with GPCR ligands stimulates the GIRK channels to open. The resulting K+ efflux out of the cell causes the membrane potential to become more negative and the fluorescent signal to decrease (Figure 1). Thus, drugs that modulate K+ efflux through the GIRK channel can be assayed using a fluorescent plate reader. Unlike other ion channel screening assays, such atomic absorption spectrometry4 or radiotracer analysis5, the GIRK channel fluorescent assay provides a fast, real-time and inexpensive screening procedure.

A real-time screening procedure for identifying drugs that interact with G protein-gated inward rectifier K+ (GIRK) channels is described. The assay utilizes membrane potential-sensitive fluorescent dyes to measure GIRK channel activity. This technique is adaptable for use on a number of cell lines.

Un ensayo de cribado para la identificación de moduladores fluorescente de canales GIRK

G protein-gated inward rectifier K+ (GIRK) channels function as cellular mediators of a wide range of hormones and neurotransmitters and are expressed in ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

Combinando Manipulación sola molécula y de imagen para el estudio de las interacciones proteína-ADN

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

El estudio de ADN Looping por FRET sola molécula

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Medición y Análisis de la producción de ácido extracelular para Determinar glicolítica Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

The Lactate Dehydrogenase Sequestration Assay &#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a simple protocol to monitor this process is described. Moreover, typical results and experimental validation of the method under autophagy-inducing conditions in various types of cultured mammalian cells are provided. During bulk autophagy, autophagosomes sequester cytosol, and thereby also soluble cytosolic proteins, alongside other autophagic cargo. LDH is a stable and highly abundant, soluble cytosolic enzyme that is non-selectively sequestered into autophagosomes. The amount of LDH sequestration therefore reflects the amount of bulk autophagic sequestration. To efficiently and accurately determine LDH sequestration in cells, we employ an electrodisruption-based fractionation protocol that effectively separates sedimentable from cytosolic LDH, followed by measurement of enzymatic activity in sedimentable fractions versus whole-cell samples. Autophagic sequestration is determined by subtracting the proportion of sedimentable LDH in untreated cells from that in treated cells. The advantage of the LDH sequestration assay is that it gives a quantitative measure of the autophagic sequestration of endogenous cargo, as opposed to other methods that either involve ectopic expression of sequestration probes or semi-quantitative protease protection analyses of autophagy markers or receptors.

The Lactate Dehydrogenase Sequestration Assay &amp;#8212; A Simple and Reliable Method to Determine Bulk Autophagic Sequestration Activity in Mammalian Cells

Here a simple and well-validated protocol for measuring bulk autophagic sequestration activity in mammalian cells is described. The method is based on quantifying the proportion of lactate dehydrogenase (LDH) in sedimentable cell fractions compared to total cellular LDH levels.

El ensayo de secuestro del lactato deshidrogenasa, Es un método Simple y confiable para determinar la actividad de autofagia secuestro a granel en células de mamíferos

Bulk autophagy is characterized by the sequestration of large portions of cytoplasm into double/multi-membrane structures termed autophagosomes. Here a ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Ensayo cinético de molécula única de alto rendimiento paralelo para la escoja de ADN específica del sitio

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...