Nous tenons à remercier Atsushi Miyawaki de nous avoir fourni la construction d&#39;expression ratiométrique-pericam-mt. Ce travail a été soutenu par des subventions GM081685 des National Institutes of Health (DB).

1. Pericam-mt transfection et préparation cellulaire. <ol><li> Plate cellules HeLa la nuit avant la transfection sur des lamelles de verre stériles placées dans une plaque de culture de 6 puits standard. </li><li> Préparer l&#39;ADN / Lipofectamine 2000 complexes comme suggéré par le fabricant (Invitrogen). Nous utilisons 4 ug de vecteur d&#39;expression ratiométrique-pericam-mt et 10 pi de Lipofectamine 2000 dilué dans 0,5 ml d&#39;Opti-MEM pour chaque puits. Les cellules doivent être confluentes ~ 70% avant la transfection. </li><li> Remplacer HeLa milieux de culture (Dulbecco Modified Eagles moyenne, 10% de FBS, pénicilline / streptomycine) dans chaque puits avec 1,5 mL de la même support sans antibiotiques. </li><li> Ajouter les complexes ADN / Lipofectamine dans chaque puits à transfecter. Mélanger délicatement avec bascule et incuber pendant 4 heures dans un incubateur de culture cellulaire à 37 o C, 5% de CO 2. </li><li> Remplacez sans antibiotique médias avec les milieux de culture avec des antibiotiques. Laisser les cellules d&#39;exprimer pericam ratiométrique pendant 1-2 jours avant l&#39;imagerie. </li></ol> 2. Installation du microscope et de l&#39;acquisition d&#39;images <ol><li> Placer une lamelle avec des cellules HeLa dans une chambre d&#39;imagerie appropriés avec une solution d&#39;imagerie: 107mm NaCl, KCl 7,2 mm, 1,2 mm de MgCl2, 1 mM CaCl 2, 11.5mm de glucose, 0,1% d&#39;albumine sérique bovine, et 20 mM HEPES 7,2. Notre laboratoire utilise la chambre cellulaire Attofluor chez Invitrogen. Montez la chambre de l&#39;imagerie dans un stade microscope inversé. </li><li> Trouver une zone d&#39;intérêt contenant une ou plusieurs cellules exprimant pericam ratiométrique utilisant un 40x (ou supérieur) objectif à immersion d&#39;huile. Nous avons constaté que pericam ratiométrique est moins résistant au photoblanchiment à 380 nm, et donc nous utilisons cette longueur d&#39;onde pour identifier les cellules appropriées. Pour les images acquises dans la figure 1, un objectif Nikon Superfluor 40X avec une ouverture numérique 1,3 a été utilisé. Objectifs de grossissement supérieur serait aussi approprié (60-100x). Filtres d&#39;excitation utilisées étaient Chroma Technology filtres D380/30x (380 + / - 30 nm) et D495/10 (495 + / - 5 nm), a été 505DCXR séparatrice (505nm longpass) et filtre d&#39;émission a été HQ535/50m (535 + / - 25nm). Notre microscope utilisé pour acquérir les images de la figure 1 se compose d&#39;un Nikon TE2000 microscope inversé équipé d&#39;un Roper Scientific Coolsnap HQ caméra CCD refroidie, Ludl MAC6000 rapide du filtre roue et changeur, et une acquisition de données et MetaFluor logiciel d&#39;analyse. Ce protocole peut être adapté à toute norme à large champ de microscope avec des filtres appropriés et un logiciel capable de capturer et de traitement des images ratiométrique. </li><li> Configurez le logiciel pour l&#39;excitation double en utilisant les filtres 495 et 380 nm. Le calcium se liant à pericam ratiométrique augmente l&#39;absorbance à 495 nm, donc le ratio devrait être mis en place à 495 nm/380 nm. Ratiométrique pericam a sans calcium pic d&#39;excitation à 415 nm 4. Dans ce protocole, nous suggérons d&#39;utiliser un filtre à 380 nm seulement parce qu&#39;elle est omniprésente dans la plupart des laboratoires d&#39;imagerie calcique. Si un filtre 410 nm, est disponible, il est préférable de le filtre 380 nm. </li><li> Acquérir les images séquentiellement par alternativement passionnante à 495 et 380 nm. Acquérir des images de niveaux de calcium de base pour au moins 30 s avant l&#39;application d&#39;un agoniste via un appareil de perfusion. Marquer le temps d&#39;addition pour chaque agoniste. Les temps d&#39;exposition et les intervalles d&#39;acquisition devrait être optimisé pour éviter photoblanchiment tout en permettant par exemple suffisamment resolution.For temporelle, pour la dynamique de calcium semi-rapides suivis en réponse à une stimulation agoniste, les images ont été acquises toutes les deux secondes dans les figures 1b et les taux d&#39;acquisition C. Bien plus efficace Il est également possible 6. À long terme après l&#39;administration d&#39;imagerie staurosporine a été acquis avec un intervalle plus long (30s) entre les acquisitions (figures 1 D et E). </li></ol> 3. Traitement et analyse des images <ol><li> Une fois l&#39;expérience terminée, les images obtenues peuvent être analysées hors ligne. Définir des régions d&#39;intérêt (ROI) dans laquelle vous voulez suivre les changements dans les niveaux de calcium. Utilisez un ROI sélectionnée dans une zone vide du champ de soustraire de fond si nécessaire. Il est à noter que les mitochondries sont très mobiles et dynamiques, et les événements dans une extrapolation mitochondrie unique sur de longues périodes n&#39;est pas toujours possible. Ainsi, compte tenu de la grande mobilité de cet organite dans certains types de cellules, la sélection de ROI doivent être surveillés attentivement. Par ailleurs, comme en témoigne la figure 1, la réponse mitochondries hétérogène à divers stimuli. Il est donc important d&#39;analyser les données hors ligne et de surveiller le placement RO1 sur une base trame par trame. Une description détaillée de la mesure du calcium mitochondrial dans très mobiles des mitochondries a été décrit ailleurs 7. </li><li> Mesure du rapport Obtenu à partir du ROI peut être importé dans Excel ou similaire graphingsoftware. Les données peuvent être présentées comme une trace représentative de l&#39;évolution des taux de calcium mitochondrial dans une seule cellule ou mitochondrie unique, ou en moyenne flsignaux uorescence du ROI plusieurs années. Nous avons également utilisé cette technique pour mesurer la moyenne des niveaux de calcium mitochondrial dans une population de lymphocytes en apoptose induite par Fas ligand 8. </li></ol> 4. Les résultats représentatifs: <img src="/files/ftp_upload/2579/2579fig1.jpg" alt="Figure 1" /> Figure 1. (A) la localisation subcellulaire de ratiométrique-pericam-mt. Cette première image montre des cellules HeLa exprimant direct ratiométrique-pericam-mt. La deuxième image montre coloration fluorescente avec mitochondrie sélectif CMXRos colorant rouge Mitotracker. La fluorescence jaune dans l&#39;image fusionnée démontre la co-localisation des ratiométrique-pericam-mt et la fluorescence Mitotracker. (B) Une série de ratio de pseudo (495:380 nm) des images de cellules HeLa exprimant 4 mt-ratiométrique pericam traitées avec 10 mM d&#39;ATP . (C) Quantification des changements dans les niveaux de calcium mitochondrial dans la région d&#39;intérêt (ROI) a indiqué en (B) traités par 10 mM d&#39;ATP. (D) Série de rapport de pseudo (495:380 nm) des images d&#39;une cellule HeLa exprimant mt -ratiométrique pericam traités avec 0,5 uM de staurosporine. Hétérogénéité de la réponse de calcium dans les mitochondries individuels est évident, ainsi que la fragmentation importante des mitochondries de 60 minutes. Certains ont mitochondries augmente oscillatoires en calcium (tête de flèche blanche), alors que d&#39;autres ne montrent pas d&#39;importants changements dans le niveau de calcium (tête de flèche jaune). (E) de quantification de l&#39;augmentation des niveaux mondiaux de calcium mitochondrial dans une seule cellule HeLa après l&#39;induction de l&#39;apoptose par 0,5 uM de staurosporine.

<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>

Nous présentons ici une méthode très simple pour mesurer le calcium mitochondrial mitochondrial aide ciblée pericam ratiométrique. Comme le montre la figure 1A, en utilisant la norme optiques grand champ sans déconvolution, il est possible de visualiser facilement des mitochondries dans les cellules HeLa individuels avec acceptables signal-bruit. C&#39;est parce que les cellules HeLa, comme la plupart des cellules en culture, s&#39;aplatissent de façon significative lorsque adhérente évitant la nécessité pour la microscopie confocale ou d&#39;autres équipements spécialisés. Nous avons trouvé des résultats similaires dans les cellules Jurkat respectées poly-lysine lamelles revêtement 8. Contrairement aux méthodes d&#39;utilisation de colorants, il est également possible de façon non invasive de surveiller les niveaux de calcium mitochondrial pendant des heures en utilisant des indicateurs de calcium génétiquement codés tels que ratiométrique pericam (figure 1E). Ceci est particulièrement important lors de l&#39;analyse du calcium mitochondrial lors de la mort cellulaire. Par ailleurs, il est également possible de visualiser la fission / fragmentation des mitochondries au cours du processus apoptotique. Comme indiqué dans les figures 1D et E, le traitement des causes staurosporine une lente augmentation de calcium dans les sous-populations de sélectionner des mitochondries qui culmine à 20 minutes. Les taux de calcium redescendre concomitante avec la fragmentation mitochondriale avant de monter à nouveau deux heures après le traitement. Ceci est cohérent avec les ondes lentes en calcium cytosolique induite par le traitement staurosporine mesurée en utilisant Fura-2 9. Ainsi, d&#39;importantes informations cinétiques peuvent être obtenus qui n&#39;est pas possible avec les méthodes employant des mesures statiques. Bien que nous ayons présenté les rapports et les concentrations de calcium n&#39;est pas dans la figure 1, il est possible d&#39;étalonner le capteur in situ afin de calculer les niveaux de calcium absolue 4, 10. Une mise en garde importante à considérer est que pericam ratiométrique est sensible au pH 4, 10. Comme les deux niveaux de pH cytosolique et mitochondrial peut changer de façon spectaculaire lors de l&#39;apoptose 11, c&#39;est une considération importante. Le titrage de pH dans des régions isolées pericam exprimer mitochondries montrent que l&#39;augmentation de [H +] d&#39;émission des augmentations de pericam lorsqu&#39;il est excité à 495 nm, avec peu d&#39;effet sur ​​l&#39;émission stimulée par excitation à 410 nm, une propriété qui a été exploitée pour mesurer simultanément le calcium mitochondrial et pH 12. Ainsi, de façon sélective le contrôle des émissions à 410 nm d&#39;excitation fournit un moyen de surveiller le calcium non ratiometrically mitochondriale avec inquiétude réduite pour le pH. Enfin, le protocole présenté ici ne requiert que l&#39;accès à un microscope standard avec des filtres à épifluorescence largement disponibles, ce qui rend cette technique accessible à la plupart des laboratoires.

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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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>
<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>
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monitoring dynamic changes in mitochondrial calcium levels during apoptosis using a genetically encoded calcium sensor

Changements dynamiques de la concentration de calcium intracellulaire en réponse à divers stimuli régule de nombreux processus cellulaires comme la prolifération, la différenciation et l&#39;apoptose 1. Pendant l&#39;accumulation de calcium apoptose, dans les mitochondries favorise la libération de facteurs pro-apoptotiques de la mitochondrie dans le cytosol 2. Il est donc intéressant de mesurer directement le calcium mitochondrial dans les cellules vivantes in situ lors de l&#39;apoptose. Imagerie à haute résolution fluorescent de cellules chargées avec double excitation ratiométrique et non-ratiométrique colorants synthétiques indicateur de calcium a été prouvé être un outil fiable et polyvalent pour étudier divers aspects de la signalisation du calcium intracellulaire. Mesurer les flux de calcium cytosolique utilisant ces techniques est relativement simple. Toutefois, la mesure des niveaux de calcium dans les cellules intactes intramitochondriales en utilisant des indicateurs tels que le calcium synthétique Rhod-2 et Rhod-FF est plus difficile. Des indicateurs synthétiques destinés aux mitochondries ont émoussé les réponses aux augmentations répétitives en calcium mitochondrial, et perturber la morphologie mitochondriale 3. En outre, les indicateurs synthétiques ont tendance à s&#39;échapper des mitochondries pendant plusieurs heures ce qui les rend impropres à long terme des expériences. Ainsi, les indicateurs de calcium codées génétiquement basé sur la protéine fluorescente verte (GFP) 4 ou 5 aequorine ciblée vers les mitochondries ont grandement facilité la mesure de la dynamique du calcium mitochondrial. Ici, nous décrivons une méthode simple pour mesurer en temps réel des flux de calcium mitochondrial en réponse à différents stimuli. La méthode est basée sur la microscopie par fluorescence de «ratiométrique-pericam &#39;qui est sélectivement ciblé à la mitochondrie. Ratiométrique pericam est un indicateur de calcium repose sur une fusion de circulairement permuté protéine fluorescente jaune et calmoduline 4. Liaison du calcium à pericam ratiométrique provoque un décalage de son pic d&#39;excitation de 415 nm à 494 nm, tandis que le spectre d&#39;émission, qui culmine autour de 515 nm, reste inchangé. Ratiométrique pericam lie un ion calcium unique avec une constante de dissociation in vitro de ~ 1,7 uM 4. Ces propriétés permettent d&#39;pericam ratiométrique la quantification des changements rapides et à long terme de la concentration de calcium mitochondrial. Par ailleurs, nous décrivons l&#39;adaptation de cette méthodologie à un niveau large champ de microscope d&#39;imagerie calcique avec des jeux de filtres couramment disponibles. L&#39;utilisation de deux agonistes distincts, l&#39;agoniste purinergiques ATP et la drogue staurosporine inducteur d&#39;apoptose, nous démontrons que cette méthode est appropriée pour la surveillance des changements dans la concentration de calcium mitochondrial avec une résolution temporelle de secondes à plusieurs heures. Par ailleurs, nous montrent aussi que pericam ratiométrique est également utile pour mesurer la fission mitochondriale / fragmentation lors de l&#39;apoptose. Ainsi, pericam ratiométrique est particulièrement bien adapté pour continu à long terme de mesure de la dynamique mitochondriale de calcium pendant l&#39;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

Ce protocole décrit une méthode pour mesurer en temps réel des flux de calcium mitochondrial par imagerie de fluorescence. La méthode tire parti d&#39;une circulaire permuté YFP à base de capteurs de calcium à double excitation ratiométrique (ratiométrique pericam-mt) sélectivement exprimé dans les mitochondries.

Surveillance des changements dynamiques dans les niveaux de calcium mitochondrial cours de l&#39;apoptose en utilisant un capteur de calcium codées génétiquement

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

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23.9K vues.  University of Texas Medical Branch. Ce protocole décrit une méthode pour mesurer en temps réel des flux de calcium mitochondrial par imagerie de fluorescence. La méthode tire parti d'une circulaire permuté YFP à base de capteurs de calcium à double excitation ratiométrique (ratiométrique pericam-mt) sélectivement exprimé dans les mitochondries.

Vidéo: 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.

Méthodes Single Molecule pour surveiller les changements dans les propriétés élastique bicouche

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

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Biologie

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.

Imagerie calcique des neurones corticaux utilisant Fura-2 heures

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.

Cellules entières d&#39;enregistrement de Calcium Release-Activated calcium (CRAC) Courants dans les lymphocytes T humains

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.

Mesurer les flux de calcium dans les cardiomyocytes rapide

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.

Molecular Probes génétiquement codés pour étude g récepteurs couplés à la protéine

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

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.

Suivi réticulum endoplasmique homéostasie calcique l&#39;aide d&#39;un<em&gt; Gaussia</em&gt; Luciférase 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.

Mesure simultanée de mitochondrial Calcium et mitochondrial potentiel membranaire dans des cellules vivantes par microscopie à fluorescence

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

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.

Surveillance de la libération de calcium ER / SR avec le Ca ciblé<sup&gt; 2+</sup&gt; Sensor CatchER<sup&gt; +</sup

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

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant defense response to microbial elicitors and to wounding caused by chewing insects, eliciting systemic calcium signals in plants. However, the role of calcium in vivo during biotic stress is still unclear. This protocol describes the use of a genetically-encoded calcium sensor to detect calcium signals in plants during feeding by a hemipteran pest. Hemipterans such as aphids pierce a small number of cells with specialized, elongated sucking mouthparts, making them the ideal tool to study calcium dynamics when a plant is faced with a biotic stress, which is distinct from a wounding response. In addition, fluorescent biosensors are revolutionizing the measurement of signaling molecules in vivo in both animals and plants. Expressing a GFP-based calcium biosensor, GCaMP3, in the model plant Arabidopsis thaliana allows for the real-time imaging of plant calcium dynamics during insect feeding, with a high spatial and temporal resolution. A repeatable and robust assay has been developed using the fluorescence microscopy of detached GCaMP3 leaves, allowing for the continuous measurement of cytosolic calcium dynamics before, during, and after insect feeding. This reveals a highly-localized rapid calcium elevation around the aphid feeding site that occurs within a few minutes. The protocol can be adapted to other biotic stresses, such as additional insect species, while the use of Arabidopsis thaliana allows for the rapid generation of mutants to facilitate the molecular analysis of the phenomenon.

Real-time In Vivo Recording of Arabidopsis Calcium Signals During Insect Feeding Using a Fluorescent Biosensor

This protocol outlines a simple method for analyzing calcium signals in plants generated by feeding hemipteran insects, such as aphids. Arabidopsis thaliana transformed with the GFP calcium biosensor GCaMP3 allow for the real-time in vivo imaging of calcium dynamics with a high temporal and spatial resolution.

Enregistrement en temps réel In Vivo des signaux calciques Arabidopsis pendant la tétée insectes à l’aide d’un biocapteur Fluorescent

Calcium ions are predicted to be key signaling entities during biotic interactions, with calcium signaling forming an established part of the plant ...

Forward Genetic Screen Using Transgenic Calcium Reporter Aequorin to Identify Novel Targets in Calcium Signaling

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis of the screen is to generate a mutant population that can be screened with a phenotype of interest. EMS (ethyl methane sulfonate) is a commonly used alkylating agent for inducing random mutation in a classical forward genetic screen to identify multiple genes involved in any given process. Cytosolic calcium (Ca2+) elevation is a key early signaling pathway that is activated upon stress perception. However the identity of receptors, channels, pumps and transporters of Ca2+ is still elusive in many study systems. Aequorin is a cellular calcium reporter protein isolated from Aequorea victoria and stably expressed in Arabidopsis. Exploiting this, we designed a forward genetic screen in which we EMS-mutagenized the aequorin transgenic. The seeds from the mutant plants were collected (M1) and screening for the phenotype of interest was carried out in the segregating (M2) population. Using a 96-well high-throughput Ca2+ measurement protocol, several novel mutants can be identified that have a varying calcium response and are measured in real time. The mutants with the phenotype of interest are rescued and propagated till a homozygous mutant plant population is obtained. This protocol provides a method for forward genetic screens in Ca2+ reporter background and identify novel Ca2+ regulated targets.

A forward genetic screen based on Ca2+ elevation as a read-out leads to identification of genetic components involved in calcium dependent signaling pathways in plants.

Écran génétique avancé utilisant l’aequorin de journaliste transgénique de calcium pour identifier de nouvelles cibles dans la signalisation de calcium

Forward genetic screens have been important tools in the unbiased identification of genetic components involved in several biological pathways. The basis ...