Nos gustaría dar las gracias a Atsushi Miyawaki por darnos la construcción de expresión radiométrica-pericam-mt. Este trabajo fue apoyado por la beca GM081685 de los Institutos Nacionales de Salud (DB).

1. Pericam-mt Transfección y preparación de células. <ol><li> Placa de células HeLa la noche antes de la transfección en cubreobjetos de vidrio estériles colocados en una placa de cultivo estándar de 6 pocillos. </li><li> Preparar ADN / Lipofectamine 2000 complejos como sugerido por el fabricante (Invitrogen). Usamos 4 mg del vector de expresión radiométrica-pericam-mt y 10 l de Lipofectamine 2000 diluido en 0,5 ml de Opti-MEM para cada pozo. Las células deben ser confluentes ~ 70% antes de la transfección. </li><li> Vuelva a colocar HeLa medios de cultivo (Dulbecco modificado águilas medio, 10% de SFB, penicilina / estreptomicina) en cada pocillo con 1,5 ml del mismo medio sin antibióticos. </li><li> Añadir los complejos ADN / Lipofectamine a cada pocillo para ser transfectadas. Mezclar suavemente con la oscilación y se incuba durante 4 horas en un cultivo celular incubadora a 37 º C, 5% de CO 2. </li><li> Reemplazar a los antibióticos sin los medios de comunicación con los medios de cultivo con antibióticos. Permiten a las células para expresar pericam radiométrica por 1-2 días antes de la imagen. </li></ol> 2. Microscopio de instalación y de adquisición de imágenes <ol><li> Coloque un cubreobjetos con células HeLa en una cámara de imágenes apropiadas con una solución de imagen: 107 mm NaCl, KCl 7,2 mm, 1,2 mm de MgCl2, 1 mM CaCl 2, la glucosa 11,5 mm, 0,1% de albúmina de suero bovino, 20 mM HEPES y 7,2. Nuestro laboratorio utiliza la cámara del celular Attofluor de Invitrogen. Monte la cámara de imágenes en una etapa microscopio invertido. </li><li> Encontrar un área de interés que contienen una o más células que expresan pericam radiométrica con un 40x (o superior) objetivo de inmersión en aceite. Hemos encontrado que pericam radiométrica es menos resistente a photobleaching a 380 nm, y por lo tanto utilizamos esta longitud de onda para identificar las células adecuadas. Para las imágenes adquiridas en la Figura 1, un objetivo Nikon Superfluor 40X con una apertura numérica 1.3 se utilizó. Objetivos más altos de aumento también podría ser adecuado (60-100x). Filtros de excitación utilizada se Chroma Technology filtros D380/30x (380 + / - 30 nm) y D495/10 (495 + / - 5 nm), divisor se 505DCXR (505nm paso largo), y la emisión de filtro se HQ535/50m (535 + / - 25 nm). Nuestro microscopio usado para adquirir las imágenes en la Figura 1 se compone de una Nikon TE2000 microscopio invertido equipado con un Roper Científico Coolsnap HQ enfriado cámara CCD, Ludl MAC6000 rápida rueda de filtros y un cambiador, y una adquisición de datos MetaFluor y software de análisis. Este protocolo se puede adaptar a cualquier punto de vista amplio campo del microscopio con los filtros apropiados y software capaz de capturar y procesar imágenes radiométrica. </li><li> Configurar el software para la excitación de doble uso de los 495 nm y 380 filtros. Unión del calcio a pericam radiométrica aumenta la absorbencia a 495 nm, con lo que la relación debe ser configurado para ser 495 nm/380 nm. Radiométrica pericam tiene un libre de calcio pico de excitación a 415 nm 4. En este protocolo le sugerimos que utilice un filtro de 380 nm sólo porque es omnipresente en la mayoría de los laboratorios de imagen de calcio. Si un filtro de 410 nm está disponible, es preferible que el filtro de 380 nm. </li><li> Adquirir imágenes secuencialmente como alternativa interesante a 495 y 380 nm. Adquirir imágenes de los niveles de calcio de referencia por lo menos 30 s antes de la aplicación de agonistas a través de un aparato de perfusión. Además de marcar el tiempo para cada agonista. Los tiempos de exposición y los intervalos de adquisición debe ser optimizado para evitar photobleaching al tiempo que permite suficiente ejemplo resolution.For temporal, para la dinámica de calcio semi-rápido seguimiento en respuesta a la estimulación agonista, las imágenes fueron adquiridas cada dos segundos en las figuras 1B y C. Mucho más rápido las tasas de adquisición También es posible 6. A largo plazo después de la administración de imágenes estaurosporina fue adquirida con un intervalo más largo (30 años) entre las adquisiciones (Figuras 1 D y E). </li></ol> 3. Procesamiento de imágenes y análisis <ol><li> Una vez que el experimento está completo, las imágenes obtenidas pueden ser analizados fuera de línea. Definir regiones de interés (ROI) en la que desea monitorear los cambios en los niveles de calcio. Utilice un retorno de la inversión seleccionados dentro de un área vacía del campo para restar el fondo si es necesario. Cabe señalar que las mitocondrias son muy móviles y dinámicas, y la extrapolación de los acontecimientos en una sola mitocondria durante largos períodos de tiempo no siempre es posible. Por lo tanto, teniendo en cuenta la movilidad de alta de este orgánulo en algunos tipos de células, la selección de retorno de la inversión deben ser monitoreados cuidadosamente. Además, como se evidencia en la Figura 1, la respuesta de las mitocondrias heterogénea a diversos estímulos. Por tanto, es importante analizar los datos en línea y monitorear RO1 colocación en un fotograma a fotograma. Una descripción detallada de la medición de calcio mitocondrial en la mitocondria muy móviles ha sido descrito en otra parte 7. </li><li> Obtuvieron mediciones de la relación de retorno de la inversión se pueden importar en Excel o similar graphingsoftware. Los datos pueden ser presentados como una huella que representan los cambios en los niveles de calcio mitocondrial en una sola celda o mitocondria sola, o un promedio de flseñales de fluorescencia de retorno de la inversión de varios. También hemos utilizado esta técnica para medir los niveles promedio de calcio mitocondrial en una población de linfocitos en la apoptosis inducida por Fas ligando 8. </li></ol> 4. Los resultados representativos: <img src="/files/ftp_upload/2579/2579fig1.jpg" alt="Figura 1" /> Figura 1. (A) la localización subcelular de radiométrica-pericam-mt. Esta primera imagen muestra en vivo de células HeLa que expresan radiométrica-pericam-mt. La segunda imagen muestra la tinción fluorescente selectiva mitocondria-dye MitoTracker CMXRos Roja. La fluorescencia de color amarillo en la imagen fusionada demuestra co-localización de radiométrica-pericam-mt y fluorescencia MitoTracker. (B) una serie de relación pseudocolor (495:380 nm) imágenes de 4 células HeLa que expresan mt-radiométrica pericam tratados con 10 mM ATP . (C) Cuantificación de los cambios en los niveles de calcio mitocondrial en la región de interés (ROI) se indica en (B) tratados con 10 mM ATP. (D) de la serie de la relación de pseudocolor (495:380 nm) imágenes de una célula HeLa expresar mt -radiométrica pericam tratados con estaurosporina M 0.5. Heterogeneidad en la respuesta de calcio en las mitocondrias individuales es evidente, así como una fragmentación significativa de las mitocondrias en 60 minutos. Algunos mitocondrias aumenta oscilatorio en calcio (cabeza de flecha blanca), mientras que otros no muestran cambios significativos en el nivel de calcio (cabeza de flecha amarilla). (E) Cuantificación de los incrementos en los niveles globales de calcio mitocondrial en una sola célula HeLa después de la inducción de la apoptosis con 0,5 M estaurosporina.

<ol>
	<li>Berridge, M. J., Lipp, P., Bootman, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+versatility+and+universality+of+calcium+signalling.">The versatility and universality of calcium signalling.</a> Nat Rev Mol Cell Biol. 1, 11-21 (2000).</li><li>Hajnoczky, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+calcium+signalling+and+cell+death:+approaches+for+assessing+the+role+of+mitochondrial+Ca2++uptake+in+apoptosis.">Mitochondrial calcium signalling and cell death: approaches for assessing the role of mitochondrial Ca2+ uptake in apoptosis.</a> Cell Calcium. 40, 553-560 (2006).</li><li>Nagai, T., Sawano, A., Park, E. S., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circularly+permuted+green+fluorescent+proteins+engineered+to+sense+Ca2.">Circularly permuted green fluorescent proteins engineered to sense Ca2.</a> Proc Natl Acad Sci U S A. 98, 3197-3202 (2001).</li><li>Rizzuto, R., Simpson, A. W., Brini, M., Pozzan, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+changes+of+mitochondrial+Ca2++revealed+by+specifically+targeted+recombinant+aequorin.">Rapid changes of mitochondrial Ca2+ revealed by specifically targeted recombinant aequorin.</a> Nature. 358, 325-327 (1992).</li><li>Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+indicators+for+Ca2++based+on+green+fluorescent+proteins+and+calmodulin.">Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin.</a> Nature. 388, 882-887 (1997).</li><li>Shimozono, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+of+subcellular+Ca2++concentrations+using+a+dual-excitation+ratiometric+indicator+based+on+green+fluorescent+protein.">Confocal imaging of subcellular Ca2+ concentrations using a dual-excitation ratiometric indicator based on green fluorescent protein.</a> Sci STKE. , pl4-pl4 (2002).</li><li>Wozniak, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+of+biphasic+calcium+release+from+the+endoplasmic+reticulum+for+Fas-mediated+apoptosis.">Requirement of biphasic calcium release from the endoplasmic reticulum for Fas-mediated apoptosis.</a> J Cell Biol. 175, 709-714 (2006).</li><li>Boehning, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytochrome+c+binds+to+inositol+(1,4,5)+trisphosphate+receptors,+amplifying+calcium-dependent+apoptosis.">Cytochrome c binds to inositol (1,4,5) trisphosphate receptors, amplifying calcium-dependent apoptosis.</a> Nat Cell Biol. 5, 1051-1061 (2003).</li><li>Csordas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+interorganelle+contacts+and+local+calcium+dynamics+at+the+ER-mitochondrial+interface.">Imaging interorganelle contacts and local calcium dynamics at the ER-mitochondrial interface.</a> Mol Cell. 39, 121-132 (2010).</li><li>Matsuyama, S., Llopis, J., Deveraux, Q. L., Tsien, R. Y., Reed, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+intramitochondrial+and+cytosolic+pH:+early+events+that+modulate+caspase+activation+during+apoptosis.">Changes in intramitochondrial and cytosolic pH: early events that modulate caspase activation during apoptosis.</a> Nat Cell Biol. 2, 318-325 (2000).</li><li>Jiang, D., Zhao, L., Clapham, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+RNAi+screen+identifies+Letm1+as+a+mitochondrial+Ca2+/H++antiporter.">Genome-wide RNAi screen identifies Letm1 as a mitochondrial Ca2+/H+ antiporter.</a> Science. 326, 144-147 (2009).</li></ol>

Aquí presentamos un método muy sencillo para medir el calcio mitocondrial utilizando mitocondrial orientada pericam radiométrica. Como se muestra en la Figura 1, la utilización de la óptica de campo amplio con deconvolución no se puede ver fácilmente cada mitocondrias en las células HeLa con aceptable relación señal-ruido. Esto se debe a las células HeLa, como la mayoría de las células en cultivo, se aplanan de manera significativa cuando adherente obviando la necesidad de microscopía confocal u otro equipo especializado. Hemos encontrado resultados similares en las células Jurkat conforme con poli-lisina cubreobjetos recubiertos 8. A diferencia de los métodos de uso de tintes, también es posible controlar de forma no invasiva los niveles de calcio mitocondrial durante horas utilizando genéticamente codificados indicadores de calcio, tales como radiométrica pericam (Figura 1). Esto es especialmente importante cuando se analiza el calcio mitocondrial en la muerte celular. Por otra parte, también es posible visualizar la fisión / fragmentación de las mitocondrias durante el proceso de apoptosis. Como se muestra en las figuras 1D y E, el tratamiento estaurosporina causa un incremento lento de calcio en las subpoblaciones de selección de las mitocondrias que culmina a 20 minutos. Los niveles de calcio volver a bajar concomitante con la fragmentación mitocondrial antes de subir otra vez 2 horas después del tratamiento. Esto es consistente con las ondas lentas en el calcio citosólico inducida por el tratamiento estaurosporina mide utilizando Fura-2 9. Así, la información cinética importantes se pueden obtener lo que no es posible con los métodos que emplean las mediciones estáticas. A pesar de que han presentado razones y no las concentraciones de calcio en la figura 1, es posible calibrar el sensor in situ para el cálculo de los niveles absolutos de calcio 4, 10. Una advertencia importante a considerar es que pericam radiométrica es sensible a un pH de 4, 10. Dado que tanto los niveles de pH citosólico y mitocondrial puede cambiar drásticamente durante la apoptosis 11, esta es una consideración importante. La titulación de pH en las mitocondrias aisladas pericam expresar demostrar que el aumento de [H +] aumenta la emisión de pericam cuando se excita a 495 nm, con poco efecto sobre la emisión estimulada por la excitación a 410 nm, una propiedad que ha sido explotada para medir simultáneamente el calcio mitocondrial y pH 12. Por lo tanto, de manera selectiva el control de emisiones con 410 nm de excitación proporciona un medio para controlar el calcio no ratiometrically mitocondrial con preocupación reducción de pH. Finalmente, el protocolo que aquí se presenta sólo requiere acceso a un microscopio de epifluorescencia con filtros estándar ampliamente disponible, con lo que esta técnica accesible a la mayoría de los laboratorios.

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<td>Inverted wide-field fluorescent calcium imaging microscope</td>
<td>&nbsp;</td>
<td>Nikon</td>
<td>MEA53310</td>
<td>Any wide-field inverted microscope equipped with 380 nm/495 nm excitation filters and appropriate longpass and emission filter can be easily adapted to use this protocol.</td>
</tr>
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<td>Imaging Software</td>
<td>&nbsp;</td>
<td>Molecular Devices, Inc</td>
<td>Metafluor</td>
<td>&nbsp;</td>
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<td>Attofluor cell chamber</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>A-7816</td>
<td>Any imaging chamber for an inverted microscope which allows perfusion would suffice.</td>
</tr>
<tr>
<td>Lipofectamine 2000</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>11668-019</td>
<td>Most transfection reagents/protocols would be appropriate for these studies </td>
</tr>
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<td>MitoTracker Red CMXRos</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>M7512</td>
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monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

Los cambios dinámicos en la concentración de calcio intracelular en respuesta a diversos estímulos regula muchos procesos celulares como la proliferación, diferenciación y apoptosis 1. Durante la acumulación de calcio en la apoptosis, en las mitocondrias promueve la liberación de factores pro-apoptóticos de la mitocondria en el citosol 2. Por lo tanto, de interés para medir directamente el calcio mitocondrial en las células vivas in situ durante la apoptosis. De alta resolución de imágenes fluorescentes de las células de carga con doble excitación radiométrica y no radiométrico, colorantes sintéticos de calcio indicador se ha demostrado ser una herramienta fiable y versátil para estudiar diversos aspectos de la señalización del calcio intracelular. Medición de los flujos de calcio citosólico uso de estas técnicas es relativamente sencillo. Sin embargo, la medición de los niveles de calcio en las células intramitocondrial intacta a través de indicadores sintéticos de calcio, tales como Rhod-2 y Rhod FF-es más difícil. Indicadores sintéticos dirigidos a la mitocondria han mitigado las respuestas a los aumentos de repetitivo en el calcio mitocondrial, y alterar la morfología mitocondrial 3. Adicionalmente, los indicadores sintéticos tienden a salirse de la mitocondria durante varias horas que los hace inadecuados para experimentos a largo plazo. Por lo tanto, genéticamente codificados indicadores de calcio basado en la proteína verde fluorescente (GFP) 4 o 5 aequorin dirigidos a la mitocondria han facilitado en gran medida de la dinámica del calcio mitocondrial. Aquí se describe un método sencillo para medición en tiempo real de los flujos de calcio mitocondrial en respuesta a diferentes estímulos. El método se basa en la microscopía de fluorescencia de &#39;radiométrica-pericam &quot;, que es selectivamente a las mitocondrias. Radiométrica pericam es un indicador de calcio basado en una fusión de circularmente permutado proteína amarilla fluorescente y calmodulina 4. Unión del calcio a pericam radiométrica provoca un desplazamiento de su punto máximo de excitación de 415 nm a 494 nm, mientras que el espectro de emisión, que alcanza su máximo alrededor de 515 nm, se mantiene sin cambios. Radiométrica pericam une un ion de calcio solo con una constante de disociación in vitro de ~ 1,7 M 4. Estas propiedades de pericam radiométrica permite la cuantificación de los cambios rápidos y de largo plazo en la concentración de calcio mitocondrial. Además, se describe la adaptación de esta metodología a un nivel de campo amplio microscopio imágenes de calcio con los conjuntos de filtros comúnmente disponibles. El uso de dos agonistas diferentes, los agonistas purinérgicos ATP y de inducción de apoptosis estaurosporina de drogas, demuestran que este método es apropiado para observar los cambios en la concentración de calcio mitocondrial, con una resolución temporal de segundos a horas. Por otra parte, también demuestran que pericam radiométrica es también útil para la medición de la fisión mitocondrial / fragmentación durante la apoptosis. Por lo tanto, pericam radiométrica está particularmente bien adaptado para la continua a largo plazo la medición de la dinámica del calcio mitocondrial de la apoptosis.

Watch this Scientific Journal Video about Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor at JoVE.com

Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

Este protocolo describe un método para la medición en tiempo real de los flujos de calcio mitocondrial de imagen fluorescente. El método se aprovecha de una circular permutaciones YFP basada en sensor de calcio de doble excitación radiométrica (radiométrica pericam-mt) expresa selectivamente en las mitocondrias.

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

Dynamic changes in intracellular calcium concentration in response to various stimuli regulates many cellular processes such as proliferation, ...

monitoring-dynamic-changes-mitochondrial-calcium-levels-during

Research

JoVE Journal

Biology

23.9K vistas.  University of Texas Medical Branch. Este protocolo describe un método para la medición en tiempo real de los flujos de calcio mitocondrial de imagen fluorescente. El método se aprovecha de una circular permutaciones YFP basada en sensor de calcio de doble excitación radiométrica (radiométrica pericam-mt) expresa selectivamente en las mitocondrias.

Video: Monitoring Dynamic Changes In Mitochondrial Calcium Levels During Apoptosis Using A Genetically Encoded Calcium Sensor

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

Calcium Imaging of Cortical Neurons using Fura-2 AM

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium indicator dyes, which are BAPTA-based organic molecules that change their spectral properties in response to the binding of Ca2+ ions. Calcium indicator dyes fall into two categories, ratio-metric dyes like Fura-2 and Indo-1 and single-wavelength dyes like Fluo-4. Ratio-metric dyes change either their excitation or their emission spectra in response to calcium, allowing the concentration of intracellular calcium to be determined from the ratio of fluorescence emission or excitation at distinct wavelengths.  The main advantage of using ratio-metric dyes over single wavelength probes is that the ratio signal is independent of the dye concentration, illumination intensity, and optical path length allowing the concentration of intracellular calcium to be determined independently of these artifacts. One of the most common calcium indicators is Fura-2, which has an emission peak at 505 nM and changes its excitation peak from 340 nm to 380 nm in response to calcium binding.  Here we describe the use of Fura-2 to measure intracellular calcium elevations in neurons and other excitable cells.

Calcium signals play a key role in many cellular processes including gene expression, survival and differentiation. Here we demonstrate how to perform calcium imaging using Fura-2 AM. Calcium imaging is a valuable tool to study the regulation of intracellular calcium in real time and its regulation of signaling cascades.

Imagen de calcio de las neuronas corticales con Fura-2 a.m.

Calcium imaging is a common technique that is useful for measuring calcium signals in cultured cells. Calcium imaging techniques take advantage of calcium ...

Whole-Cell Recording of Calcium Release-Activated Calcium (CRAC) Currents in Human T Lymphocytes

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated Calcium (CRAC) channels. CRAC channels play important role in regulation of T cell proliferation and gene expression. Abnormal CRAC channel function in T cells has been linked to severe combined immunodeficiency and autoimmune diseases 1, 2 . Studying CRAC channel function in human T cells may uncover new molecular mechanisms regulating normal immune responses and unravel the causes of related human diseases. Electrophysiological recordings of membrane currents provide the most accurate assessment of functional channel properties and their regulation. Electrophysiological assessment of CRAC channel currents in Jurkat T cells, a human leukemia T cell line, was first performed more than 20 years ago 3, however, CRAC current measurements in normal human T cells remains a challenging task. The difficulties in recording CRAC channel currents in normal T cells are compounded by the fact that blood-derived T lymphocytes are much smaller in size than Jurkat T cells and, therefore, the endogenous whole-cell CRAC currents are very low in amplitude. Here, we give a step-by-step procedure that we routinely use to record the Ca2+ or Na+ currents via CRAC channels in resting human T cells isolated from the peripheral blood of healthy volunteers. The method described here was adopted from the procedures used for recording the CRAC currents in Jurkat T cells and activated human T cells 4-8.

Whole-Cell Recording of Calcium Release-Activated Calcium CRAC Currents in Human T Lymphocytes

We provide a step-by-step protocol for whole-cell patch clamp recording of Calcium Release-Activated Calcium (CRAC) currents in peripheral blood mononuclear cell-derived human T lymphocytes.

De células enteras de grabación de calcio activados por calcio de lanzamiento (CRAC) Las corrientes en los linfocitos T humanos

In T lymphocytes, depletion of Ca2+ from the intracellular Ca2+ store leads to activation of plasmalemmal Ca2+ channels, called Calcium Release-Activated ...

Measuring Fast Calcium Fluxes in Cardiomyocytes

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to extracellular agents is predominantly regulated by the phospholipase C&beta;- G&alpha;q pathway localized on the plasma membrane which is stimulated by agents such as acetylcholine 3,4. We have recently found that plasma membrane protein domains called caveolae5,6 can entrap activated G&alpha;q 7. This entrapment has the effect of stabilizing the activated state of G&alpha;q and resulting in prolonged Ca2+ signals in cardiomyocytes and other cell types8. We uncovered this surprising result by measuring dynamic calcium responses on a fast scale in living cardiomyocytes. Briefly, cells are loaded with a fluorescent Ca2+ indicator. In our studies, we used Ca2+ Green (Invitrogen, Inc.) which exhibits an increase in fluorescence emission intensity upon binding of calcium ions. The fluorescence intensity is then recorded for using a line-scan mode of a laser scanning confocal microscope. This method allows rapid acquisition of the time course of fluorescence intensity in pixels along a selected line, producing several hundreds of time traces on the microsecond time scale. These very fast traces are transferred into excel and then into Sigmaplot for analysis, and are compared to traces obtained for electronic noise, free dye, and other controls. To dissect Ca2+ responses of different flux rates, we performed a histogram analysis that binned pixel intensities with time. Binning allows us to group over 500 traces of scans and visualize the compiled results spatially and temporally on a single plot. Thus, the slow Ca2+ waves that are difficult to discern when the scans are overlaid due to different peak placement and noise, can be readily seen in the binned histograms. Very fast fluxes in the time scale of the measurement show a narrow distribution of intensities in the very short time bins whereas longer Ca2+ waves show binned data with a broad distribution over longer time bins. These different time distributions allow us to dissect the timing of Ca2+fluxes in the cells, and to determine their impact on various cellular events.

We present a method to isolate rapid (microsecond) calcium events from slower fluxes in living cells using laser scanning confocal microscopy. The method measures fluorescence intensity fluctuations of calcium indicators by recording line scans of several hundred pixels in a cell. Histogram analysis allows us to isolate the time scales of different calcium fluxes.

La medición de flujos rápidos de calcio en los cardiomiocitos

Cardiomyocytes have multiple Ca2+ fluxes of varying duration that work together to optimize function 1,2. Changes in Ca2+ activity in response to ...

Genetically-encoded Molecular Probes to Study G Protein-coupled Receptors

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce informative probes or labels into expressed receptors that do not perturb receptor function. We used amber codon suppression technology to genetically-encode the unnatural amino acid, p-azido-L-phenylalanine (azF) at various targeted positions in GPCRs heterologously expressed in mammalian cells. The versatility of the azido group is illustrated here in different applications to study GPCRs in their native cellular environment or under detergent solubilized conditions. First, we demonstrate a cell-based targeted photocrosslinking technology to identify the residues in the ligand-binding pocket of GPCR where a tritium-labeled small-molecule ligand is crosslinked to a genetically-encoded azido amino acid. We then demonstrate site-specific modification of GPCRs by the bioorthogonal Staudinger-Bertozzi ligation reaction that targets the azido group using phosphine derivatives. We discuss a general strategy for targeted peptide-epitope tagging of expressed membrane proteins in-culture and its detection using a whole-cell-based ELISA approach. Finally, we show that azF-GPCRs can be selectively tagged with fluorescent probes. The methodologies discussed are general, in that they can in principle be applied to any amino acid position in any expressed GPCR to interrogate active signaling complexes.

We genetically-encode the unnatural amino acid, p-azido-L-phenylalanine at various targeted positions in GPCRs and show the versatility of the azido group in different applications. These include a targeted photocrosslinking technology to identify residues in the ligand-binding pocket of a GPCR, and site-specific bioorthogonal modification of GPCRs with a peptide-epitope tag or fluorescent probe.

Codificados genéticamente-sondas moleculares para Estudiar G receptores acoplados a proteínas

To facilitate structural and dynamic studies of G protein-coupled receptor (GPCR) signaling complexes, new approaches are required to introduce ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Evaluación de calcio en Sparks intactas las fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic levels. Tight control over ER calcium is imperative for protein folding, modification and trafficking. Perturbations to ER calcium can result in the activation of the unfolded protein response, a three-prong ER stress response mechanism, and contribute to pathogenesis in a variety of diseases. The ability to monitor ER calcium alterations during disease onset and progression is important in principle, yet challenging in practice. Currently available methods for monitoring ER calcium, such as calcium-dependent fluorescent dyes and proteins, have provided insight into ER calcium dynamics in cells, however these tools are not well suited for in vivo studies. Our lab has demonstrated that a modification to the carboxy-terminus of Gaussia luciferase confers secretion of the reporter in response to ER calcium depletion. The methods for using a luciferase based, secreted ER calcium monitoring protein (SERCaMP) for in vitro and in vivo applications are described herein. This video highlights hepatic injections, pharmacological manipulation of GLuc-SERCaMP, blood collection and processing, and assay parameters for longitudinal monitoring of ER calcium.

Monitoring Endoplasmic Reticulum Calcium Homeostasis Using a Gaussia Luciferase SERCaMP

Endoplasmic reticulum calcium homeostasis is disrupted in diverse pathologies. A secreted ER calcium monitoring protein (SERCaMP) reporter can be used to detect disruptions in the ER calcium store. This protocol describes the use of a Gaussia luciferase SERCaMP to examine ER calcium homeostasis in vitro and in vivo.

Monitoreo de la homeostasis del calcio del retículo endoplasmático Usando una<em&gt; Gaussia</em&gt; Luciferasa SERCaMP

The endoplasmic reticulum (ER) contains the highest level of intracellular calcium, with concentrations approximately 5,000-fold greater than cytoplasmic ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

Medición simultánea de calcio mitocondrial y mitocondrial potencial de membrana en células vivas por microscopía fluorescente

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH (pHi) is influenced by many ion transporters and thus monitoring pHi is a useful tool for assessing transporter activity. Modern Genetically Encoded pH-Indicators (GEpHIs) provide optical quantification of pHi in intact cells on a cellular and subcellular scale. This protocol describes real-time quantification of cellular pHi regulation in Malpighian Tubules (MTs) of Drosophila melanogaster through ex vivo live-imaging of pHerry, a pseudo-ratiometric GEpHI with a pKa well-suited to track pH changes in the cytosol. Extracted adult fly MTs are composed of morphologically and functionally distinct sections of single-cell layer epithelia, and can serve as an accessible and genetically tractable model for investigation of epithelial transport. GEpHIs offer several advantages over conventional pH-sensitive fluorescent dyes and ion-selective electrodes. GEpHIs can label distinct cell populations provided appropriate promoter elements are available. This labeling is particularly useful in ex vivo, in vivo, and in situ preparations, which are inherently heterogeneous. GEpHIs also permit quantification of pHi in intact tissues over time without need for repeated dye treatment or tissue externalization. The primary drawback of current GEpHIs is the tendency to aggregate in cytosolic inclusions in response to tissue damage and construct over-expression. These shortcomings, their solutions, and the inherent advantages of GEpHIs are demonstrated in this protocol through assessment of basolateral proton (H+) transport in functionally distinct principal and stellate cells of extracted fly MTs. The techniques and analysis described are readily adaptable to a wide variety of vertebrate and invertebrate preparations, and the sophistication of the assay can be scaled from teaching labs to intricate determination of ion flux via specific transporters.

Optical Quantification of Intracellular pH in Drosophila melanogaster Malpighian Tubule Epithelia with a Fluorescent Genetically-encoded pH Indicator

Cellular ion transport can often be assessed by monitoring intracellular pH (pHi). Genetically Encoded pH-Indicators (GEpHIs)&#160;provide optical quantification of intracellular pH in intact cells. This protocol details the quantification of intracellular pH through cellular ex vivo live-imaging of Malpighian tubules of Drosophila melanogaster with pHerry, a pseudo-ratiometric genetically encoded pH-indicator.

Óptica pH cuantificación de intracelular en epitelios de túbulos de Malpighi de Drosophila melanogaster con fluorescentes genéticamente codificado pH indicador

Epithelial ion transport is vital to systemic ion homeostasis as well as maintenance of essential cellular electrochemical gradients. Intracellular pH ...

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of intracellular calcium release are the major intracellular calcium storage organelles, the endoplasmic reticulum (ER) and the more specialized sarcoplasmic reticulum (SR) in muscle cells. The dynamic release of calcium from these organelles is mediated by the ryanodine receptor (RyR) and the inositol 1,4,5-triphosphate receptor (IP3R) with refilling occurring through the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump. A genetically encoded calcium sensor (GECI) called CatchER was created to monitor the rapid calcium release from the ER/SR. Here, the detailed protocols for the transfection and expression of the improved, ER/SR-targeted GECI CatchER+ in HEK293 and C2C12 cells and its application in monitoring IP3R, RyR, and SERCA pump-mediated calcium transients in HEK293 cells using fluorescence microscopy is outlined. The receptor agonist or inhibitor of choice is dispersed in the chamber solution and the intensity changes are recorded in real time. With this method, a decrease in ER calcium is seen with RyR activation with 4-chloro-m-cresol (4-cmc), the indirect activation of IP3R with adenosine triphosphate (ATP), and inhibition of the SERCA pump with cyclopiazonic acid (CPA). We also discuss protocols for determining the in situ Kd and quantifying basal [Ca2+] in C2C12 cells. In summary, these protocols, used in conjunction with CatchER+, can elicit receptor mediated calcium release from the ER with future application in studying ER/SR calcium related pathologies.

Monitoring ER/SR Calcium Release with the Targeted Ca2+ Sensor CatchER+

We present protocols for the application of our targeted genetically-encoded calcium indicator (GECI) CatchER+ for monitoring rapid calcium transients in the endoplasmic/sarcoplasmic reticulum (ER/SR) of HEK293 and skeletal muscle C2C12 cells using real-time fluorescence microscopy. A protocol for the in situ Kd measurement and calibration is also discussed.

Seguimiento de la liberación de calcio ER / SR con el Ca<sup&gt; 2+</sup&gt; Sensor de Captura<supUnesdoc.unesco.org unesdoc.unesco.org</sup

Intracellular calcium (Ca2+) transients evoked by extracellular stimuli initiate a multitude of biological processes in living organisms. At the center of ...

Seguimiento de los cambios dinámicos en niveles de calcio mitocondrial de la apoptosis mediante un sensor de calcio genéticamente codificados

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