Name: detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy
Uploaded: 2017-04-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

The authors of this paper would like to thank Prof. Jiayan Xie for technical assistance and equipment usage. This work was financially supported by the National Natural Science Foundation of China (41406137).

1. Preparación de células <ol><li> La recuperación celular <ol><li> Transferencia de las 293 células criopreservadas de riñón embrionario humano (HEK293) a partir de un tanque de nitrógeno líquido a un baño de agua C 37 ° y agitar continuamente durante 1 - 2 min. </li><li> Añadir 10 ml de medio de crecimiento completo equilibrado (medio de Eagle modificado por Dulbecco (DMEM), 10% de solución de penicilina-estreptomicina suero bovino (FBS) y 1% fetal) a una placa de cultivo celular de 10 c...

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El protocolo presentado aquí proporciona un método eficiente y fácilmente interpretable para detectar la localización subcelular y la internalización de la proteína de fusión GPCR expresado en células HEK293. La técnica se puede adaptar fácilmente para muchos genes diferentes y tipos de células, tales como para la localización celular y la internalización del receptor corazonin de Bombyx mori en células HEK293 y BMN 16. La aplicación exitosa de...

Las células poseen la maquinaria tráfico de carga para el transporte de materiales -tales como ligandos, microorganismos, nutrientes y proteínas de transmembrana en la célula de información, la energía y otros fines 1, 2 extracelular. Es crítico para la homeostasis celular, la función del tejido, y la supervivencia celular en general. La expresión basada en células de proteínas de fusión fluorescentes es una forma poderosa para estudiar la localización y la internalización de los receptores transmembrana, tales como G receptores acoplados a proteínas (GPCR), en las vías de señalización 3. La expresión de proteínas de fusión etiquetadas con proteína verde fluorescente (GFP) de medusa Aequorea victoria, combinada con técnicas de detección de fluorescencia directa, ha ganado amplia aceptación en proteínas de metas de investigación 4, 5, 6. detecti basada en GFPOn tiene la ventaja de permitir la obtención de imágenes en tiempo real de las células vivas y evitar artefactos de fijación 7. Una serie de vectores de clonación versátiles se ha construido para facilitar la expresión de fusiones de proteínas a GFP en diversas células 8, 9. El vector pEGFP-N1 es un vector plásmido ampliamente utilizado y comercializado que codifica una proteína verde fluorescente (EGFP), que ha sido optimizado de GFP de tipo salvaje para más brillante de fluorescencia y mayor expresión en células de mamífero. Para observar la señal fluorescente de EGFP expresa en células de mamífero, la microscopía de fluorescencia debe llevarse a cabo con la excitación a 488 nm y la emisión a 507 nm, preferiblemente con microscopía confocal. Control de la localización subcelular y la internalización mediada por ligando de los receptores es una técnica común para investigar la transducción de la señal y la función de los GPCR 10 </shasta&gt; 11. En la mayoría de los casos, la internalización conduce a la desensibilización de los GPCRs (la atenuación de la respuesta de estímulo a pesar de la presencia de agonistas) 12, 13. Los receptores internalizados pueden ser incorporados en el lisosoma y degradado, o pueden ser reciclados de nuevo a la superficie celular, dependiendo de la naturaleza de los receptores y las líneas celulares utilizadas en los experimentos. Los receptores de la hormona liberadora de gonadotropina (GnRHRs) pertenecen a la familia GPCR y tienen una estructura típica de proteínas GPCR, con un terminal amino extracelular, un terminal -COOH intracelular, y siete transmembrana (TM) Dominios 14. La transfección del plásmido recombinante Sj GnRHR-EGFP (con el gen GnRHR de Sepiella japonica) y la detección la microscopía confocal de GnRHR Sj EGFP-etiquetados (expresado en células HEK293) se informó Previouastuto, y la fluorescencia de GFP se estableció como un reportero para los ensayos de localización de la proteína transmembrana 15. Ahora, la internalización de Sj GnRHR, activado por S. japonica hormona liberadora de gonadotropina (GnRH Sj), se demostró en modales tiempo- y dependientes de la dosis por microscopía confocal.

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			<td height="21" style="height:21px;width:300px;">HEK293 cell line&#160;</td>
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			<td style="width:203px;"></td>
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			<td height="21" style="height:21px;width:300px;">DMEM/High glucose</td>
			<td style="width:236px;">Hyclone</td>
			<td style="width:119px;">SH30022.01</td>
			<td>High glucose 4.0 mM L-glutamine</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Foetal Bovine Serum (FBS)</td>
			<td style="width:236px;">PuFei</td>
			<td style="width:119px;">1101-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:236px;">Genom</td>
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			<td height="21" style="height:21px;width:300px;">0.25% Trypsin &#38; 0.02% EDTA</td>
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			<td height="21" style="height:21px;width:300px;">Opti-MEM (1X)</td>
			<td style="width:236px;">GIBCO, by Life Technologies</td>
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			<td>reduced serum media</td>
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			<td height="24" style="height:25px;width:300px;">Lipofectamine 2000 Transfection Reagent</td>
			<td style="width:236px;">Invitrogen</td>
			<td style="width:119px;">11668027</td>
			<td>Reagent 1</td>
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		<tr height="24">
			<td height="24" style="height:25px;width:300px;">X-tremeGENE HP DNA Transfection Reagent</td>
			<td style="width:236px;">Roche</td>
			<td style="width:119px;">6366236001</td>
			<td>Reagent 2</td>
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		<tr height="22">
			<td height="22" style="height:22px;width:300px;">DAPI Staining Solution</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1006</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">DiI</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">C1036</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Antifade Mounting Medium</td>
			<td style="width:236px;">Beyotime</td>
			<td style="width:119px;">P0126</td>
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			<td height="42" style="height:42px;width:300px;">Paraformaldehyde</td>
			<td style="width:236px;">Sinopharm Chemical Reagent Co.(SCRC)</td>
			<td style="width:119px;">80096618</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">100 mm cell culture</td>
			<td style="width:236px;">CORNING</td>
			<td style="width:119px;">430167</td>
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			<td height="21" style="height:21px;width:300px;">6-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">12-well plate&#160;</td>
			<td style="width:236px;">ExCell Bio</td>
			<td style="width:119px;">CS016-0093</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Cover Glass</td>
			<td style="width:236px;">NEST</td>
			<td style="width:119px;">801007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Microscope Slides</td>
			<td style="width:236px;">Citoglas</td>
			<td style="width:119px;">10127105P-G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">Thermo Scientific Forma CO2 Incubator</td>
			<td style="width:236px;">Thermo</td>
			<td style="width:119px;">3111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:300px;">TCS SP5II laser scanning confocal microscope</td>
			<td style="width:236px;">Leica&#160;</td>
			<td style="width:119px;">5100001311</td>
			<td></td>
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detection of ligand-activated g protein-coupled receptor internalization by confocal microscopy

Confocal laser scanning microscopy (CLSM) is an optical imaging technique for high-contrast imaging. It is a powerful approach to visualize fluorescent fusion proteins, such as green fluorescent protein (GFP), to determine their expression, localization, and function. The subcellular localization of target proteins is important for identification, characterization, and functional analyses. Internalization is one of the predominant mechanisms controlling G protein-coupled receptor (GPCR) signaling to ensure the appropriate cellular responses to stimuli. Here, we describe an experimental method to detect the subcellular localization and internalization of GPCR in HEK293 cells with confocal microscopy. In addition, this experiment provides some details about cell culture and transfection. This protocol is compatible with a variety of widely available fluorescent markers and is applicable to the visualization of the subcellular localization of a majority of proteins, as well as of the internalization of GPCR. This technique should enable researchers to efficiently manipulate GPCR gene expression in mammalian cell lines and should facilitate studies on GPCR subcellular localization and internalization.

La Figura 1 muestra un ejemplo de un sistema de microscopio confocal. La Figura 2 presenta la expresión de pEGFP-N1 en células HEK293. Se detectó la señal de GFP en el citoplasma y núcleo. La Figura 3 muestra la localización subcelular de GnRHR de S. japonica en células HEK293, consistentes con nuestros resultados publicados previamente 15. La proteína de fusión con la etiqueta de EGFP en el extr...

Watch this Scientific Journal Video about Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy at JoVE.com

Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy (Video) | JoVE

Este protocolo describe la detección de microscopía confocal de receptor acoplado a proteína G internalización (GPCR) en células de mamífero. Incluye la cultura básica celular, transfección, y el procedimiento de microscopía confocal y proporciona un método eficiente y fácilmente interpretable para detectar la localización subcelular y la internalización de GPCR de fusión expresada.

La detección de activados por ligando G receptor acoplado a proteína internalización por microscopía confocal

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Detection of Ligand-activated G Protein-coupled Receptor Internalization by Confocal Microscopy

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