Gostaríamos de agradecer a Atsushi Miyawaki para fornecer-nos com a construção de expressão raciométrica-pericam-mt. Este trabalho foi financiado por bolsa GM081685 do National Institutes of Health (DB).

1. Pericam-mt Transfection e Preparação Cell. <ol><li> Células HeLa placa na noite anterior transfecção em lamínulas estéreis colocados em uma placa de cultura padrão de 6 poços. </li><li> Prepare DNA / 2000 Lipofectamine complexos, como sugerido pelo fabricante (Invitrogen). Nós usamos 4 mg de raciométrica-pericam-mt vetor de expressão e de 10 mL de 2000 Lipofectamine diluído em 0,5 mL de Opti-MEM para cada poço. As células devem ser confluentes ~ 70% antes de transfecção. </li><li> Substituir HeLa cultura da mídia (modificado Dulbecco águias médio, 10% de SFB, penicilina / estreptomicina) em cada poço com 1,5 mL do mesmo meio sem antibióticos. </li><li> Adicionar os complexos DNA / Lipofectamine a cada poço a ser transfectadas. Misture delicadamente com balanço e incubar por quatro horas em uma cela cultura incubadora a 37 o C, 5% CO 2. </li><li> Substituir antibióticos de mídia livre com meios de cultura com antibióticos. Permitem que as células para expressar pericam raciométrica por 1-2 dias antes da imagem. </li></ol> 2. Setup microscópio e Aquisição de Imagem <ol><li> Coloque uma lamela com células HeLa em uma câmara de imagem apropriado com solução de imagem: 107mm NaCl, KCl 7.2mm, 1.2mm MgCl 2, 1 mM CaCl 2, 11,5 mm de glicose, 0,1% de albumina bovina sérica e HEPES 20mM 7.2. Nosso laboratório utiliza a câmara de célula Attofluor da Invitrogen. Monte a câmara de imagem em um estágio do microscópio invertido. </li><li> Encontrar uma área de interesse contendo uma ou mais células que expressam pericam raciométrica usando um 40x (ou superior) objetiva de imersão em óleo. Descobrimos que pericam proporcional está menos resistentes à fotodegradação a 380 nm e, assim, usar esse comprimento de onda para identificar células adequado. Para as imagens obtidas na Figura 1, a Nikon objetivo Superfluor 40X com uma abertura numérica 1,3 foi utilizado. Objetivos maior ampliação também seria apropriado (60-100x). Filtros de excitação utilizadas foram Chroma Tecnologia filtros D380/30x (380 + / - 30 nm) e D495/10 (495 + / - 5 nm), refletores foi 505DCXR (505nm longpass), e emissão de filtro foi HQ535/50m (535 + / - 25nm). Nosso microscópio usado para adquirir as imagens na Figura 1 consiste de um microscópio invertido Nikon TE2000 equipado com um Roper Scientific Coolsnap HQ resfriado câmera CCD, Ludl MAC6000 rápida roda de filtros e changer, e um MetaFluor aquisição de dados e software de análise. Este protocolo pode ser adaptado a qualquer microscópio de campo amplo padrão com os filtros adequados e software capaz de capturar e processar imagens raciométrica. </li><li> Configurar o software para a excitação dupla usando o 495 e 380 nm filtros. Cálcio ligação a pericam raciométrica aumenta a absorvância a 495 nm, assim, a relação deve ser configurado para ser 495 nm/380 nm. Raciométrica pericam tem um pico de cálcio livre de excitação em 415 nm 4. Neste protocolo, sugerimos usar um filtro de 380 nm só porque ele é onipresente na maioria dos laboratórios de imagem de cálcio. Se um filtro de 410 nm está disponível, é preferível o filtro nm 380. </li><li> Adquirir imagens sequencialmente por alternativamente emocionante em 495 e 380 nm. Adquirir imagens dos níveis de cálcio de base por pelo menos 30 s antes da aplicação do agonista através de um aparelho de perfusão. Marcar o tempo de adição para cada agonista. Tempos de exposição e os intervalos de aquisição deve ser otimizado para evitar fotobranqueamento enquanto ainda permitindo exemplo resolution.For suficiente temporal, para a dinâmica de semi-rápidas de cálcio monitorados em resposta à estimulação agonista, imagens foram adquiridas a cada dois segundos nas Figuras 1B e taxas C. Muito mais rápido aquisição Também é possível 6. Longo prazo de imagens após a administração estaurosporina foi adquirido com um intervalo mais longo (30s) entre as aquisições (Figura 1 D e E). </li></ol> 3. Processamento de Imagem e Análise <ol><li> Uma vez que a experiência é completa, imagens obtidas podem ser analisadas offline. Definir as regiões de interesse (ROI) em que você deseja monitorar mudanças nos níveis de cálcio. Use um ROI selecionada dentro de uma área vazia do campo para subtrair fundo, se necessário. Note-se que as mitocôndrias são bastante móveis e dinâmicos, e eventos extrapolando em uma única mitocôndria durante longos períodos nem sempre é possível. Assim, considerando a alta motilidade dessa organela em alguns tipos de células, a seleção de ROI devem ser cuidadosamente monitorizados. Além disso, como evidenciado na Figura 1, a resposta mitocôndrias heterogeneamente a vários estímulos. Portanto, é importante para analisar os dados offline e monitorar RO1 colocação em uma base quadro-a-quadro. A descrição detalhada de medição de cálcio mitocondrial em alta motilidade mitocôndrias tem sido descrito em outros lugares 7. </li><li> Medições obtidas a partir do ROI pode ser importado para o Excel ou graphingsoftware similar. Os dados podem ser apresentados como um traço que representa as alterações nos níveis de cálcio mitocondrial em uma única célula ou mitocôndria única, ou uma média de fluorescence sinais de ROI diversas. Temos também utilizado esta técnica para medir os níveis de cálcio média mitocondrial em uma população de linfócitos em apoptose induzida pelo Fas ligante 8. </li></ol> 4. Resultados representativos: <img src="/files/ftp_upload/2579/2579fig1.jpg" alt="Figura 1" /> Figura 1. (A) localização subcelular de raciométrica-pericam-mt. Esta primeira imagem mostra células HeLa viver expressando raciométrica-pericam-mt. A segunda imagem mostra a coloração fluorescente com mitocôndria seletivo corante CMXRos MitoTracker Vermelho. A fluorescência amarelo na imagem fundiu demonstra co-localização de raciométrica-pericam-mt e fluorescência MitoTracker. (B) Uma série de pseudo-relação (495:380 nm) imagens de 4 células HeLa expressar mt-raciométrica pericam tratados com 10 mM ATP . (C) Quantificação das mudanças dos níveis de cálcio mitocondrial na região de interesse (ROI) indicada em (B) tratados com 10 mM ATP. Series (D) de pseudo-relação (495:380 nm) imagens de uma célula HeLa expressar mt -raciométrica pericam tratados com 0,5 mM estaurosporina. Heterogeneidade na resposta de cálcio na mitocôndria indivíduo é evidente, bem como a fragmentação significativa das mitocôndrias por 60 minutos. Alguns têm mitocôndrias aumenta oscilatório em cálcio (cabeça de seta branca), enquanto outros não demonstram alterações significativas no nível de cálcio (cabeça de seta amarela). (E) Quantificação dos aumentos globais nos níveis de cálcio mitocondrial em uma célula HeLa única após a indução de apoptose com 0,5 mM 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>

Não há conflitos de interesse declarados.

Aqui apresentamos um método muito simples para a medição de cálcio mitocondrial utilizando-alvo mitocondrial pericam raciométrica. Conforme mostrado na Figura 1A, usando o padrão Widefield óptica sem deconvolução é possível visualizar facilmente mitocôndrias nas células HeLa individuais com relação sinal-ruído aceitável. Isso ocorre porque as células HeLa, como a maioria das células em cultura, achatar significativamente quando aderente evitando a necessidade de microscopia confocal ou outros equipamentos especializados. Temos encontraram resultados semelhantes em células Jurkat adere a poli-lisina lamínulas revestido 8. Em contraste com metodologias usando corantes, também é possível de forma não invasiva monitorar os níveis de cálcio mitocondrial durante horas utilizando indicadores de cálcio geneticamente codificado como pericam raciométrica (Figura 1E). Isto é especialmente importante quando se analisa o cálcio mitocondrial durante a morte celular. Além disso, também é possível visualizar a cisão / fragmentação da mitocôndria durante o processo de apoptose. Como mostrado nas Figuras 1D e E, o tratamento estaurosporina causa um aumento lento em cálcio em subpopulações seleto de mitocôndrias, que picos em 20 minutos. Níveis de cálcio descer novamente concomitante com a fragmentação mitocondrial antes de subir novamente 2 horas após o tratamento. Isto é consistente com as ondas lentas em cálcio citosólico induzido pelo tratamento estaurosporina medida usando Fura-2 9. Assim, as informações cinética importantes podem ser obtidos que não é possível com métodos que empregam medições estáticas. Embora tenhamos apresentado índices e concentrações de cálcio não na Figura 1, é possível calibrar o sensor in situ para calcular os níveis de cálcio absoluta 4, 10. Uma ressalva importante a considerar é que pericam raciométrica é sensível ao pH 4, 10. Como ambos os níveis de pH citosólico e mitocondrial podem mudar dramaticamente durante a apoptose 11, esta é uma consideração importante. Titulação de pH em pericam mitocôndrias isoladas expressar demonstrar que o aumento da [H +] de emissão aumentos de pericam quando animado em 495 nm, com pouco efeito sobre a emissão estimulada pela excitação a 410 nm, uma propriedade que tem sido explorada para medir simultaneamente tanto cálcio mitocondrial e pH 12. Assim, seletivamente monitoramento de emissões com 410 nm de excitação fornece um meio de não-ratiometrically cálcio mitocondrial acompanhar com preocupação reduzida para pH. Finalmente, o protocolo aqui apresentado requer apenas o acesso a um microscópio de epifluorescência com filtros padrão amplamente disponível, tornando essa técnica acessível para a maioria dos laboratórios.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<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>
<tr>
<td>Imaging Software</td>
<td>&nbsp;</td>
<td>Molecular Devices, Inc</td>
<td>Metafluor</td>
<td>&nbsp;</td>
<tr>
<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>
<tr>
<td>MitoTracker Red CMXRos</td>
<td>&nbsp;</td>
<td>Invitrogen Corporation</td>
<td>M7512</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

Mudanças dinâmicas na concentração de cálcio intracelular em resposta a vários estímulos regula muitos processos celulares como a proliferação, diferenciação e apoptose 1. Durante a apoptose acúmulo de cálcio, na mitocôndria promove a liberação de fatores pró-apoptóticos da mitocôndria para o citosol 2. É, portanto, de interesse para medir diretamente cálcio mitocondrial em células vivas in situ durante a apoptose. Alta resolução de imagem fluorescente de células carregadas de excitação dupla raciométrica e não raciométrica corantes indicador sintético de cálcio tem sido provado ser uma ferramenta confiável e versátil para estudar vários aspectos da sinalização do cálcio intracelular. Medir os fluxos de cálcio citosólico utilizando estas técnicas é relativamente simples. No entanto, medindo os níveis de cálcio nas células intactas intramitocondrial utilizando indicadores de cálcio sintéticos como Rhod-2 e Rhod-FF é mais desafiador. Indicadores sintéticos direcionados para as mitocôndrias têm embotado respostas repetitivas a um aumento no cálcio mitocondrial, e interromper a morfologia mitocondrial 3. Além disso, indicadores sintéticos tendem a vazar para fora da mitocôndria durante várias horas o que os torna inadequados para experimentos de longa duração. Assim, os indicadores de cálcio geneticamente codificado com base em proteína fluorescente verde (GFP) 4 ou 5 aequorin direcionados para as mitocôndrias têm muito facilitada medição da dinâmica do cálcio mitocondrial. Aqui, descrevemos um método simples para medição em tempo real dos fluxos de cálcio mitocondrial em resposta a estímulos diferentes. O método é baseado em microscopia de fluorescência de &#39;raciométrica-pericam &quot;, que é seletivamente direcionados para as mitocôndrias. Pericam raciométrica é um indicador de cálcio baseado em uma fusão de circularmente permutados proteína fluorescente amarela e calmodulina 4. A ligação do cálcio para pericam raciométrica provoca uma mudança de seu pico de excitação de 415 nm a 494 nm, enquanto que o espectro de emissão, que picos em torno de 515 nm, permanece inalterado. Raciométrica pericam liga um íon de cálcio com uma única constante de dissociação in vitro de ~ 1,7 mM 4. Estas propriedades do pericam raciométrica permitem a quantificação de mudanças rápidas e de longo prazo na concentração de cálcio mitocondrial. Além disso, descrevemos a adaptação desta metodologia a um padrão de imagem do microscópio de campo amplo de cálcio com comumente disponíveis conjuntos de filtros. Usando dois agonistas distintos, o agonista purinérgico ATP e indução de apoptose estaurosporina de drogas, demonstram que este método é apropriado para monitorar mudanças na concentração de cálcio mitocondrial com uma resolução temporal de segundos a horas. Além disso, também demonstram que pericam raciométrica também é útil para medir a fissão mitocondrial / fragmentação durante a apoptose. Assim, pericam raciométrica é particularmente adequado para a medição contínua de longo prazo da dinâmica do cálcio mitocondrial durante a apoptose.

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 descreve um método para medição em tempo real dos fluxos de cálcio mitocondrial por imagem fluorescente. O método se aproveita de uma circular permutated sensor de cálcio YFP baseado excitação dupla raciométrica (raciométrica pericam-mt) seletivamente expressos na mitocôndria.

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

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 visualizações.  University of Texas Medical Branch. Este protocolo descreve um método para medição em tempo real dos fluxos de cálcio mitocondrial por imagem fluorescente. O método se aproveita de uma circular permutated sensor de cálcio YFP baseado excitação dupla raciométrica (raciométrica pericam-mt) seletivamente expressos na mitocôndria.

Vídeo: 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 única molécula para acompanhar as alterações nas propriedades elásticas bicamada

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

Biologia

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.

Imagem de cálcio dos neurônios corticais usando Fura-02:00

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.

Todo-Cell Gravação de Cálcio O cálcio Release-Ativado (CRAC) Correntes em linfócitos humanos T

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.

Fluxos de medição de cálcio rápido no Cardiomyocytes

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.

Geneticamente codificados Molecular Probes para estudar G receptores acoplados à proteína

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.

Avaliação do cálcio Faíscas intactas 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.

Monitoramento retículo endoplasmático homeostase do cálcio Usando um<em&gt; Gaussia</em&gt; Luciferase 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.

Medição simultânea de mitocondrial de cálcio e mitocondrial potencial de membrana em células vivas por Microscopia 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.

PH intracelular de quantificação de óptica em Drosophila melanogaster túbulo de Malpighi epitélios com um indicador de pH fluorescente geneticamente codificado

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.

Monitorização da libertação de cálcio ER / SR com o Ca<sup&gt; 2+</sup&gt; Captador<sup-benzóico.</sup

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

Mudanças Dinâmicas de monitoramento dos níveis de cálcio mitocondrial durante a apoptose através de um sensor de cálcio geneticamente codificado

Resumo

Explore mais vídeos

Capítulos neste vídeo