Ce travail a été soutenu par des subventions de la Deutsche Forschungsgemeinschaft (DFG) BL567/3-1 et la Friedrich-Baur-Stiftung. Nous tenons à remercier Roger Y. Tsien, Howard Hughes Medical Laboratories Institute à l&#39;Université de Californie, San Diego pour nous fournir avec Tag-DP-T2. Nous reconnaissons heureusement David Baltimore, California Institute of Technology, Pasadena, et Didier Trono, Université de Genève, Genève, pour nous avoir fourni le lentivirus plasmides FUGW et psPAX2/pMD2.G.

<ol>
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Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Les avantages et les inconvénients de la méthode TED ont été largement débattues dans les publications récentes 34,35. Comparé à d&#39;autres méthodes décrites ci-dessus, TED contourne le problème de la perturbation et la perfusion des cellules. En outre, deux aspects méritent d&#39;être soulignés. Le principe de TED pour ER imagerie de calcium nécessite (1.) L&#39;expression ciblée d&#39;une carboxylestérase actif dans la lumière du RE à l&#39;aide de vecteurs construits TED et (2.) La lib...

Afin de résoudre réponses calciques physiologiques de l&#39;ER, nous avons développé une nouvelle stratégie pour améliorer le piégeage des colorants sensibles de calcium synthétiques dans l&#39;ER. La méthode permet à l&#39;, non perturbatrice surveillance en temps réel directement sans calcium ER en présence de calcium extracellulaire. Fonction et signalisation de calcium ER signaux de calcium se trouvent dans différents types cellulaires, cellules musculaires par exemple, les neurones et les cellules gliales et leur gamme de fonctions de médiation contraction musculaire à une implication dans la transmission synaptique dans l&#39;apprentissage et de la mémoire 1,2. Les variations de la concentration en calcium libre sont de grand intérêt scientifique parce que le calcium est impliqué dans la régulation de la transcription génique, la prolifération cellulaire, l&#39;excitabilité neuronale, la mort cellulaire et autre signalisation cellulaire événements 1-7. Tous ces signaux calciques cellulaires sont fonctionnellement liés et contribuent à intracellulaires de calciummagasin 8-10 Dynamics. Une caractéristique commune à tous les signaux de calcium est le flux de calcium entre l&#39;espace extracellulaire, le cytosol et organites, principalement l&#39;ER et les mitochondries. Cela provoque des changements dynamiques de la concentration de calcium dans ces organites, qui sont détectées par différents éléments de signalisation. En général, la concentration en calcium dans les intervalles entre ER 100 à 800 pM, dans le cytosol de la concentration de calcium est proche de 100 nm, et dans l&#39;espace extracellulaire de la concentration est de l&#39;ordre de 1 à 2 mM. Par conséquent, il est une force motrice chimique élevée pour le flux de calcium vers le cytosol 2,9,10. Les signaux calciques ER dérivés les plus couramment étudiées dépendent de la stimulation des récepteurs couplés aux protéines G (RCPG), qui a ensuite activent la phospholipase C (PLC). PLC à son tour produit l&#39;inositol 1,4,5-triphosphate (IP 3) 1. Lors de la liaison de IP3 à son receptor (IP 3-Rec, figure 1) dans le ER-membrane, les ions calcium sont libérés à partir de la lumière du RE. Historiquement, IP libération de calcium 3 médiation de l&#39;ER est le premier - même si indirectement - mesurée dans les cellules pancréatiques acineuses par Streb et al en 1983. 11. Cette publication a suggéré pour la première fois une cascade de signalisation impliquant l&#39;acétylcholine, la phospholipase C et IP 3. Cette façon de libération de calcium est généralement appelé IP libération de calcium 3 induit (IICR) (Figure 1). Activation de kinases dépendant de la phospholipase Cy par récepteurs tyrosines kinases liens à l&#39;action des facteurs de croissance et des facteurs neurotrophiques pour ER signalisation IP après 3 Altitude 12 calcium. En plus de IICR, l&#39;élévation du calcium peut être médiée par l&#39;entrée du calcium ionotropique, par exemple via des canaux calciques voltage-dépendants (Ca V), et après la libération de calcium induite par le calcium (CICR) par ryanodine reprécepteurs (RyR). IICR et CICR sont physiologiquement liés à l&#39;entrée du calcium magasin fonctionnant (SOCE). SOCE comprend l&#39;action de STIM (molécule interagissant stroma), qui est un capteur pour ER libération de calcium. STIM a été montré pour stimuler l&#39;entrée du calcium extracellulaire par les voies de passage des récepteurs potentiels (TRP) 13, les canaux calciques Orai 14 et même des canaux calciques voltage-dépendants 15 (Figure 1). Perte de calcium ER est dynamiquement sauvé par l&#39;action du réticulum sarco-endoplasmique ATPase de calcium (SERCA), qui active les pompes de retour de calcium dans le RE. Blocage de la SERCA avec des médicaments tels que thapsigargine dévoile une perte continue de calcium ER dans le compartiment cytosolique. Ce ER calcium «fuite» est causée par des complexes de pores intramembranaires ER tels que le complexe de protéine Sec61 16,17 (figure 1). En 1998, Berridge a publié un modèle, le «neurone dans un modèle neuronal&quot;, which suggère un rôle physiologique de principe de l&#39;ER dans l&#39;intégration de calcium neuronal 5. Ce modèle estime l&#39;existence d&#39;un système de membrane du RE continue formant une &quot;image&quot; intracellulaire de la membrane plasmique des neurones 5. Ce système de membrane eucaryote binaire a été prétendu être une condition sine qua non pour l&#39;intégration spatiale et temporelle des signaux calciques lents et rapides dans les neurones. signaux calciques qui se produisent soit concomitamment ou ultérieurement dans différents épines ou dendrites du neurone même sont conférés au soma ou le noyau via l&#39;ER, où ils se résument 5,18 de la cellule. Ensuite, leur somme peut avoir des effets sur l&#39;excitabilité des neurones, la régulation de la transcription des gènes ou l&#39;intégration des cascades de signalisation. Ainsi, l&#39;ER soutient l&#39;intégration de signaux calciques. Une condition préalable à ce concept est la continuité de l&#39;ER dans une seule cellule, qui a été revendiquée par plusieurs études et qui a été prouvé au moins pour somato-dzones endritic et à courte distance des projections axonales 19-21. Qu&#39;il y ait continuité ER dans les projections axonales longues est un sujet de débat. Les stratégies visant à mesurer le flux de calcium libre sur la membrane du RE signaux calciques sont le plus souvent suivies dans le cytosol 22,23. Par conséquent, il ne peut pas facilement être distingué s&#39;il Ca 2 + se déverse dans le cytosol de extracellulaire ou des réserves intracellulaires 6,24. Pour contourner cette limitation, les stratégies méthodologiques pour l&#39;imagerie directe de calcium ER ont été développés. En résumé, les stratégies suivantes sont utilisées: (1) ER-ciblées génétiquement protéines indicateurs 25-27 faible affinité Ca 2 + indicateurs à base de protéines utilisent la protéine bioluminescente aequorin ou GFP en combinaison avec une protéine de détection de calcium.. Ces Ca 2 + indicateurs génétiquement modifiés (GECIS) peutêtre ciblée à l&#39;urgence à l&#39;aide d&#39;un peptide signal et sont activement conservés à l&#39;urgence en utilisant un motif de rétention et de récupération. Common ER Ca 2 + base des indicateurs sur le principe de Cameleon Cameleon et sont la YC4.3 26,28; Cameleon scission YC7.3ER 29 et Cameleon D1 30. (2) Direct chargement de colorant à base d&#39;estérase de AM-ester de faible affinité Ca 2 + 31,32 indicateurs. Des colorants indicateurs (Mag-Fura2-AM, Mag-Fluo4-AM ou Fluo5N-AM) dérivés AM passer l&#39; membranes biologiques dans un état lipophile, calcium insensible. Puis, dans le cytosol, ainsi que dans l&#39;urgence, estérases endogènes cliver du groupe AM-ester et relâchez le Ca 2 + indicateur, laissant derrière lui une certaine quantité de colorant actif dans le cytosol et dans l&#39;urgence. Par conséquent, cette approche est utile dans des conditions d&#39;une forte concentration de calcium dans le RE aussi longtemps que la concentration de calcium cytosolique reste bien en dessous de la deLimite de protection des indicateurs de faible affinité, en particulier pendant signaux calciques caractéristiques (par exemple nM à faible M). (3) chargement AM-ester en association avec la membrane plasmique perméabilisation 32. Cytosolique de préférence Ca + indicateur restante est éliminée par deux plasma perméabilisation de la membrane avec de petites quantités d&#39;un détergent &quot;doux&quot; (par exemple saponine) dans un tampon intracellulaire artificielle. Ainsi, les membranes intracellulaires peuvent être stimulées, par exemple avec l&#39;IP 3 dans le tampon intracellulaire, directement par &quot;pores&quot; dans la membrane plasmique. (4) Dialyse du cytosol sous configuration cellule entière et des mesures simultanées de Ca 2 + dans la lumière du RE et le cytosol 32,33. Une cellule est d&#39;abord chargé avec une faible affinité Ca 2 + indicateur (par exemple Mag-Fura2- AM, quotientométrique, UV). Ensuite, à l&#39;aide d&#39;une pipette de patch, tout reste CYTosolic faible affinité Ca + indicateur 2 est dialysée sur le cytosol avec un tampon contenant un Ca2 + indicateur de haute affinité (par exemple Fluo-3, de la lumière visible). Cette stratégie permet l&#39;enregistrement simultané de signaux dérivés cytosolique et ER. (5) Targeted-estérase colorant induit chargement 8,34. Un carboxylestérase (CES) est destiné à la lumière de l&#39;ER et fournit une haute activité de l&#39;estérase de piégeage efficace de la forme AM-ester de faible affinité Ca 2 + indicateurs. Targeted-estérase induite colorant chargement (TED) Pour améliorer le ciblage de faible affinité Ca 2 + indicateurs à la lumière du RE, TED a été développé. TED nécessite la surexpression d&#39;une carboxylestérase ER ciblée de la souris (CES2) (figure 2), ce qui est réalisé par l&#39;intermédiaire d&#39;assemblage d&#39;expression. Les cellules exprimant un CES-construction recombinante sont incubés avec la forme AM-ester d&#39;un calcium dansdicateur colorant (Fluo5N-AM, figure 2). Puis, dans l&#39;urgence, le colorant est converti en Ca 2 + sensibles, membrane imperméable Ca 2 + complexe indicateur (Fluo5N/Ca 2 +) par la forte activité de l&#39;estérase, piégeant ainsi le colorant à une concentration élevée dans la lumière du RE 8 , 34. La méthode est particulièrement utile pour étudier ER calcium-release via les IICR-voies, par exemple via des récepteurs métabotropiques, purinergiques ou glutamate 8,34 et de visualiser ER épuisement de calcium via &quot;canaux de fuite&quot; directement, par exemple après le blocage de la SERCA 17 , 34. Pour notre expérience, la faible affinité Ca 2 + indicateur Fluo5N-AM est actuellement le meilleur indicateur disponible pour une utilisation avec la stratégie de chargement colorant TED. Fluo5N-AM a une faible affinité pour le Ca 2 + (constante de dissociation K D ~ 90 um, figure 2), est presque non-fluorescent dans sa AM-forme, mais fournit une grande émission de fluorescence sur Calciucontraignant m 8,34. Le complexe Fluo5N/Ca + 2 peut être excité par une source de lumière standard de ~ 490 nm, ce qui correspond à des colorants standards tels que le FITC, Alexa 488, ou EGFP. Signaux calciques cytosoliques guère atteindre une concentration dans la gamme des basses pM et sont donc à peine détectés par Fluo5N dans le cytosol 35. Pour améliorer les performances de TED, plusieurs constructions de vecteurs recombinants ont été développés (Figure 3). A l&#39;origine, les vecteurs TED basé sur la séquence codante du CES2 (RefSeq numéro d&#39;accession NM_145603, CES2c) et meilleure performance TED est observée avec une expression stable de constructions CES. Nouveaux vecteurs TED expriment un élément central de CES2 fusionnée à la protéine de fluorescence rouge TagRFP-T2 36. Ces vecteurs ont l&#39;avantage qu&#39;ils peuvent être utilisés pour identifier les cellules transduites et d&#39;utiliser la fluorescence rouge comme un contrôle interne pour la normalisation des changements dans Fluo5N/Ca 2 + fluorescence. La fluorescence rouge offre également la possibilité de visualiser la répartition structurelle de l&#39;ER et des changements dans la dynamique ER dans des conditions de stimulation.

<table border="1">
	 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalogue Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>(S)-3,5-DHPG (Dihydroxyphenylglycine)</td>
 <td>Tocris</td>
 <td>0805</td>
 <td></td>
 </tr>
 <tr>
 <td>5';-ATP-Na2, ATP disodium salt hydrate</td>
 <td>Sigma</td>
 <td>A26209-1G</td>
 <td></td>
 </tr>
 <tr>
 <td>B-27 supplement,50x</td>
 <td>Invitrogen</td>
 <td>17504-044</td>
 <td></td>
 </tr>
 <tr>
 <td>Carbamoylcholine chloride (Charbachol)</td>
 <td>Sigma</td>
 <td>C4382-1g</td>
 <td></td>
 </tr>
 <tr>
 <td>Cyclopiazonic acid (CPA)</td>
 <td>Ascent scientific</td>
 <td>Asc-300</td>
 <td></td>
 </tr>
 <tr>
 <td>Dimethylsulfoxide (DMSO)</td>
 <td>Sigma</td>
 <td>D2650</td>
 <td></td>
 </tr>
 <tr>
 <td>DMEM with Glutamax</td>
 <td>Invitrogen-Gibco</td>
 <td>31966-047</td>
 <td></td>
 </tr>
 <tr>
 <td>Dulbecco's PBS without Ca/Mg 1x</td>
 <td>PAA</td>
 <td>H15-002</td>
 <td></td>
 </tr>
 <tr>
 <td>Fetal calf serum</td>
 <td>Invitrogen-Gibco</td>
 <td>10270-106</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluo-5N-AM, 10 x 50 &mu;g</td>
 <td>Invitrogen</td>
 <td>F14204</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamate</td>
 <td>Ascent scientific</td>
 <td>Asc-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Glutamax</td>
 <td>Invitrogen</td>
 <td>35050-038</td>
 <td></td>
 </tr>
 <tr>
 <td>Ham's F12 nutrients mixture</td>
 <td>Invitrogen-Gibco</td>
 <td>21765-029</td>
 <td></td>
 </tr>
 <tr>
 <td>Hank's BSS(1x) without Ca,without Mg, with Phenol Red, HBSS</td>
 <td>PAA Laboratories</td>
 <td>H15-010</td>
 <td></td>
 </tr>
 <tr>
 <td>Horse serum</td>
 <td>SHD3250YK</td>
 <td>Linearis</td>
 <td></td>
 </tr>
 <tr>
 <td>Ionomycin Ca2+ salt</td>
 <td>Ascent scientific</td>
 <td>Asc-116</td>
 <td></td>
 </tr>
 <tr>
 <td>murine EGF </td>
 <td>PreproTech</td>
 <td>315-09</td>
 <td></td>
 </tr>
 <tr>
 <td>N2 supplement 100x</td>
 <td>Invitrogen</td>
 <td>17502-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Neurobasal medium</td>
 <td>Invitrogen-Gibco</td>
 <td>21103-049</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127, low UV absorbance,2g</td>
 <td>Invitrogen</td>
 <td>P6867</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-DL-ornithine hydrobromide PORN</td>
 <td>Sigma</td>
 <td>P8638</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-D-lysine hydrobromide</td>
 <td>Sigma</td>
 <td>P7280</td>
 <td></td>
 </tr>
 <tr>
 <td>Poly-L-Lysin</td>
 <td>Sigma</td>
 <td>P2636</td>
 <td></td>
 </tr>
 <tr>
 <td>Thapsigargin</td>
 <td>Calbiochem</td>
 <td>586005-1mg</td>
 <td></td>
 </tr>
 <tr>
 <td>TryLE Express</td>
 <td>Invitrogen</td>
 <td>12605-028</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsin</td>
 <td>Worthington</td>
 <td>LS-003707</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypsininhibitor from Glycine MAX (soybean)</td>
 <td>Sigma</td>
 <td>T6522</td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Materials</td>
 </tr>
 <tr>
 <td>Confocal system: inverted laser scanning microscope: FV1000 with IX81</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: laser combiner FV10 and diode lasers (405, 473, 559, 635)</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Confocal system: scan head FV10-SPD with spectraldetector</td>
 <td>Olympus</td>
 <td></td>
 <td>user-specific configuration</td>
 </tr>
 <tr>
 <td>Heater controller TC-344B</td>
 <td>Warner Instruments</td>
 <td>64-0101</td>
 <td>to control in line solution heater</td>
 </tr>
 <tr>
 <td>NIH ImageJ software</td>
 <td>WS Rasband, ImageJ, US National Institutes of Health, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997--2006</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UAPON20XW340 NA 0,7 WD 0,35</td>
 <td>Olympus</td>
 <td>N2709100</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLFLN40XO</td>
 <td>Olympus</td>
 <td>N1478700</td>
 <td></td>
 </tr>
 <tr>
 <td>Objective: UPLSAPO60XO/1.35, WD=0.15</td>
 <td>Olympus</td>
 <td>N1480700</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, inverse</td>
 <td>custom-made</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber, RC-49FS</td>
 <td>Warner Instruments</td>
 <td>W4 64-1709 </td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion tube: PT-49</td>
 <td>Warner Instruments</td>
 <td>64-1730</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Peristaltic pump MiniPlus 3 (4 channels)</td>
 <td>Gilson</td>
 <td>n.a. </td>
 <td>perfusion pump</td>
 </tr>
 <tr>
 <td>Single inline solution heater SH-27B</td>
 <td>Warner Instruments</td>
 <td>64-0102</td>
 <td>controlled by heater controller</td>
 </tr>
 <tr>
 <td>Suction tubes: ST3R</td>
 <td>Warner Instruments</td>
 <td>64-1407</td>
 <td>for perfusion</td>
 </tr>
 <tr>
 <td>Heating insert: Tempocontrol 37-2 digital 2-channel</td>
 <td>Pecon</td>
 <td>n.a. </td>
 <td></td>
 </tr>
	 </tbody>
 </table>

direct imaging of er calcium with targeted-esterase induced dye loading (ted)

Visualisation de la dynamique de calcium est important de comprendre le rôle du calcium dans la physiologie cellulaire. Pour étudier la dynamique de calcium, Ca 2 + fluorescents indicateurs de synthèse sont devenus populaires. Ici, nous démontrons TED (= ciblée estérase induite colorant chargement), une méthode pour améliorer la libération de Ca 2 + colorants indicateurs dans la lumière du RE de différents types de cellules. À ce jour, TED a été utilisé dans des lignées cellulaires, les cellules gliales et les neurones in vitro. Bases TED sur l&#39;efficacité, le ciblage d&#39;une activité à haute carboxylesterase à la lumière du RE en utilisant vecteurs constructions recombinant qui carboxylestérases express (CES). Les derniers vecteurs TED contiennent un élément central de CES2 fusionné à une protéine fluorescente rouge, permettant ainsi à l&#39;imagerie à deux couleurs simultanément. La dynamique du calcium libre dans l&#39;ER sont imagés dans une couleur, tandis que la structure ER correspondant apparaît en rouge. Au début de la procédure, les cellules sont transduites avec un lentivirus. Ensuite, les cellules infectées par unre ensemencées sur des lamelles pour enfin permettre l&#39;imagerie des cellules vivantes. Ensuite, les cellules vivantes sont mises en incubation avec l&#39;ester d&#39;acétoxyméthyle (AM-ester) sous forme de faible affinité de Ca 2 + indicateurs, par exemple Fluo5N-AM, Mag-Fluo4-AM, ou en alliage d&#39;aluminium-Fura2-AM. L&#39;activité de l&#39;estérase dans les clive ER hors chaînes latérales hydrophobes à partir de la forme AM du Ca 2 + et un indicateur fluorescent + complexe colorant / Ca 2 hydrophile est formé et pris au piège dans la lumière du RE. Après chargement colorant, les cellules sont analysées au microscope à balayage laser confocal inversé. Les cellules sont continuellement perfusées avec des solutions de Ringer-like et la dynamique de calcium ER sont directement visualisés par l&#39;imagerie time-lapse. libération de calcium à partir de l&#39;ER est identifiée par une diminution de l&#39;intensité de fluorescence dans les régions d&#39;intérêt, tandis que le remplissage de la réserve de calcium ER produit une augmentation de l&#39;intensité de fluorescence. Enfin, la variation de l&#39;intensité de fluorescence au cours du temps est déterminée par le calcul de AF / F 0.

Ce protocole prévoit une approche non perturbatrice pour l&#39;imagerie directe de libre calcium ER. Faible affinité synthétique Ca 2 + indicateurs sont libérés et piégés dans la lumière du RE à l&#39;aide d&#39;une ER ciblée, l&#39;activité de l&#39;enzyme estérase recombinante. Amélioration de chargement des Ca 2 + colorants indicateurs à la lumière du RE permet une imagerie directe et rapide des urgences dynamique de magasins de calcium. Les types de cell...

Watch this Scientific Journal Video about Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED at JoVE.com

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Targeted-estérase induite colorant chargement (TED) appuie l&#39;analyse de la dynamique intracellulaire des magasins de calcium par imagerie de fluorescence. La méthode se fonde sur le ciblage d&#39;une carboxylestérase recombinant pour le réticulum endoplasmique (RE), où il améliore le démasquage locale de synthétique de faible affinité Ca<sup&gt; 2 +</sup&gt; Colorants indicateurs dans la lumière du RE.

Imagerie directe du calcium ER avec Targeted-estérase colorant induit Chargement (TED)

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent ...

direct-imaging-er-calcium-with-targeted-esterase-induced-dye-loading

Research

JoVE Journal

Biology

Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading (TED)

19.1K vues.  University of Wuerzburg. Targeted-estérase induite colorant chargement (TED) appuie l'analyse de la dynamique intracellulaire des magasins de calcium par imagerie de fluorescence. La méthode se fonde sur le ciblage d'une carboxylestérase recombinant pour le réticulum endoplasmique (RE), où il améliore le démasquage locale de synthétique de faible affinité Ca 2 + Colorants indicateurs dans la lumière du RE.

Vidéo: Direct Imaging of ER Calcium with Targeted-Esterase Induced Dye Loading TED

Loading Drosophila Nerve Terminals with Calcium Indicators

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as the messenger essential for the synaptic plasticity that supports learning and memory. To further elucidate the molecular underpinnings of Ca2+-dependent synaptic mechanisms, a model system is required that is both genetically malleable and physiologically accessible. Drosophila melanogaster provides such a model. In this system, genetically-encoded fluorescent indicators are available to detect Ca2+ changes in nerve terminals. However, these indicators have limited sensitivity to Ca2+ and often show a non-linear response. Synthetic fluorescent indicators are better suited for measuring the rapid Ca2+ changes associated with nerve activity. Here we demonstrate a technique for loading dextran-conjugated synthetic Ca2+ indicators into live nerve terminals in Drosophila larvae. Particular emphasis is placed on those aspects of the protocol most critical to the technique's success, such as how to avoid static electricity discharges along the isolated nerves, maintaining the health of the preparation during extended loading periods, and ensuring axon survival by providing Ca2+ to promote sealing of severed axon endings. Low affinity dextran-conjugated Ca2+-indicators, such as fluo-4 and rhod, are available which show a high signal-to-noise ratio while minimally disrupting presynaptic Ca2+ dynamics. Dextran-conjugation helps prevent Ca2+ indicators being sequestered into organelles such as mitochondria. The loading technique can be applied equally to larvae, embryos and adults.

Calcium is a ubiquitous messenger in the nervous system, essential for triggering neurotransmitter release and changes in synaptic strength. Here we demonstrate a technique for loading Ca2+-indicators into Drosophila nerve terminals. We also demonstrate fabrication of the required apparatus and emphasize points critical for the technique's success.

Terminaux de chargement nerveuse chez la drosophile avec des indicateurs de calcium

Calcium plays many roles in the nervous system but none more impressive than as the trigger for neurotransmitter release, and none more profound than as ...

Recherche

Biologie

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

In Vivo 2-Photon Imaging calcium dans la couche 2 / 3 de souris

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

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 ...

Assembly, Loading, and Alignment of an Analytical Ultracentrifuge Sample Cell

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed centrifugation. When properly planned and executed, an AUC sedimentation velocity or sedimentation equilibrium experiment can reveal a great deal about a protein in regards to size and shape, sample purity, sedimentation coefficient, oligomerization states and protein-protein interactions.
This technique, however, requires a rigorous level of technical attention. Sample cells hold a sectored center piece sandwiched between two window assemblies. They are sealed with a torque pressure of around 120-140 in/lbs. Reference buffer and sample are loaded into the centerpiece sectors and then after sealing, the cells are precisely aligned into a titanium rotor so that the optical detection systems scan both sample and reference buffer in the same radial path midline through each centerpiece sector while rotating at speeds of up to 60, 000 rpm and under very high vacuum
Not only is proper sample cell assembly critical, sample cell components are very expensive and must be properly cared for to ensure they are in optimum working condition in order to avoid leaks and breakage during experiments. Handle windows carefully, for even the slightest crack or scratch can lead to breakage in the centrifuge. The contact between centerpiece and windows must be as tight as possible; i.e. no Newton s rings should be visible after torque pressure is applied. Dust, lint, scratches and oils on either the windows or the centerpiece all compromise this contact and can very easily lead to leaking of solutions from one sector to another or leaking out of the centerpiece all together. Not only are precious samples lost, leaking of solutions during an experiment will cause an imbalance of pressure in the cell that often leads to broken windows and centerpieces. In addition, plug gaskets and housing plugs must be securely in place to avoid solutions being pulled out of the centerpiece sector through the loading holes by the high vacuum in the centrifuge chamber. Window liners and gaskets must be free of breaks and cracks that could cause movement resulting in broken windows.
This video will demonstrate our procedures of sample cell assembly, torque, loading and rotor alignment to help minimize component damage, solution leaking and breakage during the perfect AUC experiment.

The analytical ultracentrifuge (AUC) sample cell holds sample and reference buffer and during experiments and is exposed to high vacuum and rotor speeds up to 60,000 rpm. This video will demonstrate the rigorous attention to detail necessary for assembly, loading and alignment of this very important component of an AUC experiment.

Assemblée, de chargement, et l&#39;alignement d&#39;une cellule échantillon ultracentrifugation analytique

The analytical ultracentrifuge (AUC) is a powerful biophysical tool that allows us to record macromolecular sedimentation profiles during high speed ...

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 complex in the ER membrane provides an aqueous path for newly-synthesized polypeptides into the lumen of the ER. Recent work from various laboratories suggested that this heterotrimeric complex may also form transient Ca2+ leak channels2-8. The key observation for this notion was that release of nascent polypeptides from the ribosome and Sec61 complex by puromycin leads to transient release of Ca2+ from the ER. Furthermore, it had been observed in vitro that the ER luminal protein BiP is involved in preventing ion permeability at the level of the Sec61 complex9,10. We have established an experimental system that allows us to directly address the role of the Sec61 complex as potential Ca2+ leak channel and to characterize its putative regulatory mechanisms11-13. This system combines siRNA mediated gene silencing and live cell Ca2+ imaging13. Cells are treated with siRNAs that are directed against the coding and untranslated region (UTR), respectively, of the SEC61A1 gene or a negative control siRNA. In complementation analysis, the cells are co-transfected with an IRES-GFP vector that allows the siRNA-resistant expression of the wildtype SEC61A1 gene. Then the cells are loaded with the ratiometric Ca2+-indicator FURA-2 to monitor simultaneously changes in the cytosolic Ca2+ concentration in a number of cells via a fluorescence microscope. The continuous measurement of cytosolic Ca2+ also allows the evaluation of the impact of various agents, such as puromycin, small molecule inhibitors, and thapsigargin on Ca2+ leakage. This experimental system gives us the unique opportunities to i) evaluate the contribution of different ER membrane proteins to passive Ca2+ efflux from the ER in various cell types, ii) characterize the proteins and mechanisms that limit this passive Ca2+ efflux, and iii) study the effects of disease linked mutations in the relevant components.

Live Cell Calcium Imaging Combined with siRNA Mediated Gene Silencing Identifies Ca2+ Leak Channels in the ER Membrane and their Regulatory Mechanisms

The endoplasmic reticulum plays a key role in protein biogenesis and in calcium homeostasis. We have established an experimental system that allows us to address the role of Ca2+ leak channels and to characterize their putative regulatory mechanisms. This system involves siRNA mediated gene silencing and live cell Ca2+ imaging.

Direct imagerie calcique cellulaire Combiné avec Gene Silencing siARN Mediated Identifie Ca 2 + Canaux de fuite dans la membrane du RE et de leurs mécanismes de régulation

In mammalian cells, the endoplasmic reticulum (ER) plays a key role in protein biogenesis as well as in calcium signalling1. The heterotrimeric Sec61 ...

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

Expression et purification de particules de virus-like pour la vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

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 ...

Imaging Intermediate Filaments and Microtubules with 2-dimensional Direct Stochastic Optical Reconstruction Microscopy

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, polarity, and motility. The organization of the actin and microtubule networks has been extensively studied but that of IFs is not yet fully characterized. IFs have an average diameter of 10 nm and form a network extending throughout the cell cytoplasm. They are physically associated with actin and microtubules through molecular motors and cytoskeletal linkers. This tight association is at the heart of the regulatory mechanisms that ensure the coordinated regulation of the three cytoskeletal networks required for most cell functions. It is therefore crucial to visualize IFs alone and also together with each of the other cytoskeletal networks. However, IF networks are extremely dense in most cell types, especially in glial cells, which makes its resolution very difficult to achieve with standard fluorescence microscopy (lateral resolution of ~250 nm). Direct STochastic Optical Reconstruction Microscopy (dSTORM) is a technique allowing a gain in lateral resolution of one order of magnitude. Here, we show that lateral dSTORM resolution is sufficient to resolve the dense organization of the IF networks and, in particular, of IF bundles surrounding microtubules. Such tight association is likely to participate in the coordinated regulation of these two networks and may, explain how vimentin IFs template and stabilize microtubule organization as well as could influence microtubule dependent vesicular trafficking. More generally, we show how the observation of two cytoskeletal components with dual-color dSTORM technique brings new insight into their mutual interaction.

The overall goal of this methodology is to give the optimal experimental conditions from sample preparation to image acquisition and reconstruction in order to perform 2D dual color dSTORM images of microtubules and intermediate filaments in fixed cells

Imagerie des Filaments intermédiaires et Microtubules avec directes 2 dimensions de Reconstruction stochastique optiques de microscopie

The cytoskeleton, composed of actin microfilaments, microtubules, and intermediate filaments (IF), plays a key role in the control of cell shape, ...

Fluorescence Recovery after Photobleaching of Yellow Fluorescent Protein Tagged p62 in Aggresome-like Induced Structures

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical FRAP experiment, steady-state fluorescence is observed by repeated imaging with low-intensity laser light. Subsequently, the fluorescent molecules are rapidly and irreversibly impaired via brief exposure to high-intensity laser light. Information about protein mobility is obtained by monitoring the recovery of fluorescence. We used FRAP to determine the mobility of p62 in aggresome-like induced structures (ALIS) in murine macrophages after stimulation with lipopolysaccharide (LPS). Because many existing FRAP protocols are either incomplete or complex, our goal was to provide a comprehensive, practical, and straightforward step-by-step protocol for FRAP experiments with live cells. Here, we describe RAW264.7 macrophage transfection with yellow fluorescent protein-p62 (YFP-p62), induction of ALIS by exposing the cells to LPS, and a step-by-step method for collecting prebleach and postbleach FRAP images and data analysis. Finally, we discuss important factors to consider when conducting a FRAP experiment.

We describe a comprehensive and practical protocol for fluorescence recovery after photobleaching experiments with live cells. Although the protocol was used to measure the mobility of yellow fluorescent protein-tagged p62 in aggresome-like induced structures, it can be applied to a variety of microscopy systems and fluorescent proteins.

Récupération de fluorescence après Photoblanchiment du jaune Fluorescent Protein tag p62 en Structures induites ressemblant à Aggresome

Fluorescence recovery after photobleaching (FRAP)&#160;is a microscopy technique that can be used to quantify protein mobility in live cells. In a typical ...

Imaging Calcium Dynamics in Subpopulations of Mouse Pancreatic Islet Cells

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells that trigger secretion of three main hormones: insulin (from &#946;-cells), glucagon (&#945;-cells) and somatostatin (&#948;-cells). &#946;-Cells, which make up the majority of islet cells and are electrically coupled to each other, respond to the glucose stimulus as one single entity. The excitability of the minor subpopulations, &#945;-cells and &#948;-cells (making up around 20% (30%) and 4% (10%) of the total rodent1 (human2) islet cell numbers, respectively) is less predictable and is therefore of special interest.
Calcium sensors are delivered into the peripheral layer of cells within the isolated islet. The islet or a group of islets is then immobilized and imaged using a fluorescence microscope. The choice of the imaging mode is between higher throughput (wide-field) and better spatial resolution (confocal). Conventionally, laser scanning confocal microscopy is used for imaging tissue, as it provides the best separation of the signal between the neighboring cells. A wide-field system can be utilized too, if the contaminating signal from the dominating population of &#946;-cells is minimized.
Once calcium dynamics in response to specific stimuli have been recorded, data are expressed in numerical form as fluorescence intensity vs. time, normalized to the initial fluorescence and baseline-corrected, to remove the effects linked to bleaching of the fluorophore. Changes in the spike frequency or partial area under the curve (pAUC) are computed vs. time, to quantify the observed effects. pAUC is more sensitive and quite robust whereas spiking frequency provides more information on the mechanism of calcium increase.
Minor cell subpopulations can be identified using functional responses to marker compounds, such as adrenaline and ghrelin, that induce changes in cytosolic calcium in a specific populations of islet cells.

Here, we present a protocol for imaging and quantifying calcium dynamics in heterogeneous cell populations, such as pancreatic islet cells. Fluorescent reporters are delivered into the peripheral layer of cells within the islet, which is then immobilized and imaged, and per-cell analysis of the dynamics of fluorescence intensity is performed.

Imagerie de la dynamique du calcium dans les sous-populations de cellules d'islet pancréatique de souris

Pancreatic islet hormones regulate blood glucose homeostasis. Changes in blood glucose induce oscillations of cytosolic calcium in pancreatic islet cells ...