Diese Arbeit wurde durch Zuschüsse der Deutschen Forschungsgemeinschaft (DFG) und dem BL567/3-1 Friedrich-Baur-Stiftung unterstützt. Wir möchten Roger Y. Tsien, Howard Hughes Medical Institute Laboratories an der University of California, San Diego danken für die Bereitstellung von uns mit Tag-RFP-T2. Wir anerkennen dankbar David Baltimore, California Institute of Technology, Pasadena, und Didier Trono, Universität Genf, Genf, für die uns die lentiviralen Plasmide FUGW und psPAX2/pMD2.G.

<ol>
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Die Autoren erklären, dass sie keine finanziellen Interessen haben.

Die Vor-und Nachteile des TED Verfahren wurden ausgiebig in den letzten Publikationen 34,35 diskutiert. Im Vergleich zu anderen oben beschriebenen Verfahren umgeht das Problem der TED zu stören und Perfusion der Zellen. Darüber hinaus müssen zwei Aspekte hervorgehoben werden. Das Prinzip der TED für ER Calcium Imaging erfordert (1.) Die gezielte Expression eines aktiven Carboxylesterase in das ER-Lumen mit Hilfe von TED Vektor-Konstrukte und (2.) Die effiziente und bevorzugte Freisetzung von niedriger Aff...

Um physiologischen Calcium Reaktionen des ER zu beheben, haben wir eine neue Strategie, um das Einfangen von synthetischen Calcium sensitiven Farbstoffen in die Notaufnahme zu verbessern. Das Verfahren ermöglicht die direkte, nicht-störende Echtzeit-Überwachung des freien ER Calcium in Gegenwart von extrazellulärem Calcium. Funktion und Signalisierung von ER Calcium Calcium-Signale werden in verschiedenen Zelltypen, zB Muskelzellen, Neuronen und Gliazellen und ihre Funktionen reichen von der Vermittlung Muskelkontraktion zu einer Beteiligung an der synaptischen Übertragung in Lernen und Gedächtnis 1,2 gefunden. Änderungen im freien Calcium-Konzentration sind von hohem wissenschaftlichen Interesse, da Calcium in der Regulation der Gen-Transkription, Proliferation, neuronale Erregbarkeit, Zelltod und anderen Zelle Signalwege 1-7 beteiligt ist. All diese zellulären Calcium-Signale sind funktional verbunden sind und dazu beitragen, Calcium intrazellulärestore Dynamik 8-10. Ein gemeinsames Merkmal aller Calcium-Signale ist der Fluss von Kalzium zwischen dem extrazellulären Raum, die Cytosol und Organellen, vor allem die ER und Mitochondrien. Dies bewirkt dynamische Änderungen der Calciumkonzentration innerhalb dieser Organellen, die von verschiedenen Signalkomponenten erfasst werden. Im Allgemeinen ist die Calcium-Konzentration in der ER zwischen 100 bis 800 um, in das Cytosol der Calcium-Konzentration in der Nähe von 100 nm und in den extrazellulären Raum die Konzentration etwa 1-2 mM. Dementsprechend gibt es eine hohe chemische Antriebskraft für Calcium Strömung zum Cytosol 2,9,10. Die am häufigsten untersuchten ER abgeleiteten Calcium Signale abhängig von der Stimulation der G-Protein-gekoppelte Rezeptoren (GPCR), die dann aktiviert Phospholipase C (PLC). PLC erzeugt wiederum Inositol 1,4,5-triphosphat (IP 3) 1. Bei der Bindung von IP 3 seiner receptor (IP 3-Rec, Abbildung 1) in der ER-Membran, werden Kalzium-Ionen aus dem ER-Lumen freigesetzt. Historisch gesehen, war IP 3-vermittelte Calcium-Freisetzung aus dem ER ersten - obwohl indirekt - gemessen in acinar Zellen der Bauchspeicheldrüse durch Streb et al 1983 11.. Diese Veröffentlichung vorgeschlagen erstmals eine Signalkaskade mit Acetylcholin, Phospholipase C und IP 3. Diese Art der Calcium-Freisetzung wird allgemein als IP 3-induzierten Calcium-Freisetzung (IICR) (Abbildung 1). Kinase-abhängige Aktivierung von Phospholipase C &amp;ggr; durch Rezeptor-Tyrosinkinasen Verbindungen die Wirkung von Wachstumsfaktoren und neurotrophe Faktoren ER Calciumsignaltransduktion nach IP 3 Erhebung 12. Neben IICR kann Calciumerhöhung durch ionotropen Calcium Eintrag vermittelt werden, zum Beispiel über spannungsabhängigen Calcium-Kanäle (Ca V) und anschließender Calcium-induzierten Calcium-Freisetzung (IKRK) von ryanodine reRezeptoren (RyR). IICR und CICR sind physiologisch Calcium-Shop-Eintrag (SOCE) verknüpft. SOCE umfasst die Wirkung von STIM (Stroma interagierende Molekül), die ein Sensor für ER Calcium-Freisetzung ist. STIM hat sich gezeigt, extrazellulären Calcium Eintrag durch transient receptor potential-Kanäle (Trp) 13, Orai Kalziumkanäle 14 und sogar spannungsabhängigen Calcium-Kanäle 15 (Abbildung 1) zu stimulieren. Loss of ER Calcium wird dynamisch durch die Wirkung der sarco-endoplasmatischen Retikulum Calcium ATPase (SERCA), die aktiv Pumpen Calcium in die ER gerettet. Das Blockieren der SERCA mit Medikamenten wie thapsigargin enthüllt einen kontinuierlichen Verlust von Kalzium ER zum Zytosol. Das ER Calcium &quot;Leck&quot; wird durch ER intramembrane Porenkomplexe wie Sec61 Protein-Komplex 16,17 (Abbildung 1) verursacht. In 1998 veröffentlichte Berridge ein Modell, das &quot;Neuron innerhalb eines Neurons model&quot;, which schlägt ein Prinzip physiologische Rolle des ER bei der Integration von neuronalen Calcium-5. Dieses Modell berücksichtigt die Existenz eines kontinuierlichen ER-Membran-System bildet eine intrazelluläre &quot;Bild&quot; des neuronalen Plasmamembran 5. Diese binäre eukaryotischen Membran-System wurde behauptet, eine Grundvoraussetzung für zeitliche und räumliche Integration von schnellen und langsamen Calcium-Signale in Neuronen. Calcium-Signale auftreten, die entweder gleichzeitig oder anschließend in verschiedenen Stacheln oder Dendriten des gleichen Neurons zu der Zelle soma oder Kern über das ER, wo sie bis 5,18 summiert verliehen. Dann kann ihre Summe haben Auswirkungen auf die Erregbarkeit des Neurons, die Regulierung der Gen-Transkription oder Integration von Signalkaskaden. Somit unterstützt die ER die Integration von Calcium-Signale. Eine Voraussetzung für dieses Konzept ist die Kontinuität des ER in einer einzigen Zelle, die durch mehrere Studien in Anspruch genommen wurde und das hat zumindest für somato-d erwiesenendritic Bereichen und kurze Strecke axonalen Projektionen 19-21. Ob es ER Kontinuität innerhalb langen axonalen Projektionen ist eine Angelegenheit der Debatte. Strategien, um den Fluss der freien Calcium über die ER-Membran zu messen Calcium-Signale werden am häufigsten in das Cytosol 22,23 überwacht. Es kann daher nicht leicht zu unterscheiden, ob Ca2 + in das Cytosol von extrazellulären oder intrazellulären Speichern 6,24 fließenden werden. Um diese Beschränkung zu überwinden, wurden methodische Strategien zur direkten ER Calcium Imaging entwickelt. Zusammenfassend sind die folgenden Strategien: (1) ER-Protein gezielt gentechnisch-Indikatoren 25-27 Protein-basierten Low-Affinität Ca 2 +-Indikatoren verwenden die Biolumineszenz Protein GFP Äquorin oder in Kombination mit einem Calcium-sensitiven Proteins.. Diese gentechnisch veränderten Ca 2 +-Indikatoren (GeCIS) kannder ER mit Hilfe eines Signalpeptids ausgerichtet sein und sich aktiv an der ER mit einem Aufbewahren und Abrufen Motiv gehalten. Gemeinsame ER Ca 2 +-Indikatoren basieren auf der Cameleon-Prinzip und sind die Cameleon YC4.3 26,28; Cameleon Split YC7.3ER 29 und 30 Cameleon D1. (2) Direkter Esterase-basierte Farbstoff Laden von AM-Ester niedriger Affinität Ca 2 + Indikatoren 31,32. AM-Derivate der Indikatorfarbstoffe (Mag-Fura2-AM, Mag-Fluo4-AM oder Fluo5N-AM) gehen die biologischen Membranen in einem lipophilen, Calcium-und Kleinschreibung Zustand. Dann wird in das Cytosol und im ER freizugeben endogene Esterasen gespalten des AM-Estergruppe und die Ca 2 +-Indikator, hinterlässt eine bestimmte Menge des aktiven Farbstoff im Cytosol und im ER. Daher ist dieser Ansatz unter den Bedingungen einer hohen Calcium-Konzentration in der ER nützlich, solange der cytosolischen Calciumkonzentration bleibt weit unter dem deNachweisgrenze der niedriger Affinität Indikatoren, insbesondere während der charakteristischen Calcium-Signale (zB nM bis niedrig uM). (3) AM-Ester Belastung in Kombination mit Plasmamembran Permeabilisierung 32. Ein verbleibender zytosolischen Ca 2 +-Indikator durch Plasmamembran Permeabilisierung mit geringen Mengen eines &quot;mild&quot; Reinigungsmittel (z. B. Saponin) in einem künstlichen intrazelluläre Puffer entfernt. Somit können die intrazellulären Membranen angeregt werden, zB mit IP 3 in der intrazellulären Puffer direkt durch &quot;Poren&quot; in der Plasmamembran. (4) Dialyse des Cytosol unter Whole-Cell-Konfiguration und gleichzeitige Messungen von Ca 2 + in das ER-Lumen und das Cytosol 32,33. Zelle zuerst mit einer niedrigen Affinität Ca 2 +-Indikator (zB Mag-Fura2 geladen AM, ratiometrischen, UV-Licht). Danach mit Hilfe eines Patch-Pipette, verbleibende cytosolic niedriger Affinität Ca 2 +-Indikator aus dem Cytosol mit einem Puffer, der eine hohe Affinität Ca 2 +-Indikator (zB Fluo-3, sichtbares Licht) dialysiert. Diese Strategie ermöglicht die gleichzeitige Aufnahme von zytosolischen und ER abgeleiteten Signale. (5) Gezielte-Esterase-induzierte Farbstoffbeladung 8,34. A Carboxylesterase (CES) mit dem Lumen des ER gezielte und bietet eine hohe Esterase-Aktivität für eine effiziente Abfangen des AM-Ester Form niedriger Affinität Ca 2 +-Indikatoren. Gezielte-Esterase-induzierten Farbstoff Belastung (TED) Zur Verbesserung der Ausrichtung der niedriger Affinität Ca 2 + Indikatoren zur ER-Lumen, wurde TED entwickelt. TED erfordert die Überexpression eines ER gezielte Maus Carboxylesterase (CES2) (Abbildung 2), die über Expressionskonstrukte erreicht wird. Zellen, die ein rekombinantes CES-Konstrukt mit dem AM-Ester Form eines Calcium inkubiertIndikator-Farbstoff (Fluo5N-AM, Abbildung 2). Dann wird in dem ER wird der Farbstoff auf die Ca 2 + umgewandeltes empfindlich, undurchlässigen Membran Ca 2 +-Indikator-Komplex (Fluo5N/Ca 2 +) durch die hohe Esterase daher Einfangen der Farbstoff in einer hohen Konzentration in der ER-Lumen 8 , 34. Die Methode ist besonders nützlich, um ER Calcium-Freisetzung über die IICR-Bahnen zum Beispiel untersuchen, über metabotropic, purinergen-oder Glutamat-Rezeptoren 8,34 und ER Calciumentzug visualisieren über &quot;Leck-Kanäle&quot; direkt, zum Beispiel nach Blockade der SERCA 17 , 34. Um unsere Erfahrungen die Low-Affinität Ca 2 +-Indikator Fluo5N-AM ist derzeit die beste verfügbare Indikator mit dem TED Farbstoffbeladung Strategie zu verwenden. Fluo5N-AM hat eine geringe Affinität für Ca 2 + (Dissoziationskonstante K D ~ 90 pM, Abbildung 2), ist fast nicht fluoreszierenden in seine AM-Form, sondern bietet eine hohe Fluoreszenzemission auf Calcium Bindung 8,34. Die Fluo5N/Ca 2 +-Komplex mit einer Standard-Lichtquelle von ~ 490 nm, die über die normalen Farbstoffen wie FITC, Alexa 488 oder eGFP entspricht angeregt werden. Cytosolic Calcium-Signale kaum erreichen eine Konzentration in den niedrigen pM-Bereich und werden daher kaum von Fluo5N im Cytosol 35 erkannt. Um die TED Leistung zu verbessern, wurden mehrere rekombinanten Vektor-Konstrukte entwickelt (Abbildung 3). Ursprünglich ist TED Vektoren auf der kodierenden Sequenz des CES2 (Refseq Zugangsnummer NM_145603, CES2c) und am besten TED Leistung bezogen mit stabilen Expression CES Konstrukte beobachtet. New TED Vektoren exprimieren ein Kernelement CES2 fusioniert mit dem roten fluoreszierenden Proteins TagRFP-T2 36. Diese Vektoren haben den Vorteil, dass sie verwendet werden, um zu identifizieren transduzierten Zellen und die rote Fluoreszenz als interne Kontrolle für die Normalisierung der Veränderungen in Fluo5N/Ca 2 verwenden werden + Fluoreszenz. Die rote Fluoreszenz bietet auch die Möglichkeit, die strukturellen Verteilung der ER und ER Änderungen in Dynamik unter Stimulationsbedingungen visualisieren.

<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)

Visualisierung Kalziumdynamik ist wichtig, die Rolle von Calcium in der Zellphysiologie zu verstehen. Um Kalzium Dynamik zu untersuchen, wurden synthetische fluoreszierenden Ca 2 + Indikatoren populär geworden. Hier zeigen wir, TED (= gezielte-Esterase-induzierten Farbstoff Laden), eine Methode, um die Freisetzung von Ca 2 + zu verbessern Indikatorfarbstoffe in das ER-Lumen verschiedener Zelltypen. Bisher wurde in TED-Zelllinien, Gliazellen und Neuronen in vitro verwendet. TED basiert auf effizienten, rekombinanten Targeting von einer hohen Aktivität Carboxylesterase dem ER-Lumen mit Vektor-Konstrukte, die ausdrückliche Carboxylesterasen (CES). Die neuesten TED Vektoren enthalten ein Kernelement CES2 fusioniert an ein rot fluoreszierendes Protein, wodurch gleichzeitige zweifarbige Bildgebung. Die Dynamik des freien Calcium in der ER sind in einer Farbe abgebildet, während die entsprechende ER Struktur erscheint in rot. Zu Beginn des Verfahrens werden die Zellen mit einem Lentivirus transduziert. Anschließend werden die infizierten Zellen einere auf Deckgläsern ausgesät, um endlich ermöglichen Live Cell Imaging. Dann lebenden Zellen werden mit dem Acetoxymethylester (AM-Ester) Form niedriger Affinität Ca 2 + inkubiert Indikatoren, zum Beispiel Fluo5N-AM, Mag-Fluo4-AM, oder Mag-Fura2-AM. Die Esterase-Aktivität im ER spaltet hydrophoben Seitenketten vom AM Form des Ca 2 +-Indikator und einem hydrophilen fluoreszierenden Farbstoff / Ca 2 +-Komplex gebildet wird, und eingeschlossen in das ER-Lumen. Nach Farbstoffbeladung, werden die Zellen mit einem invertierten konfokalen Laser-Scanning-Mikroskop analysiert. Die Zellen werden kontinuierlich mit Ringer-ähnliche Lösungen perfundiert und die ER Calcium Dynamik direkt visualisiert Zeitraffer-Bildgebung. Calcium-Freisetzung aus dem ER wird durch eine Abnahme der Fluoreszenz-Intensität in Regionen von Interesse identifiziert, während das Nachfüllen der ER Calcium Speicher erzeugt eine Zunahme der Fluoreszenzintensität. Schließlich wird die Änderung in der Fluoreszenzintensität über die Zeit durch Berechnung der AF / F 0 bestimmt.

Dieses Protokoll stellt eine unterbrechungsfreie Ansatz für die direkte Bildgebung der freien ER Calcium. Low-Affinität synthetischen Ca 2 + Indikatoren gelöst und eingeschlossen in das ER-Lumen mit Hilfe eines ER gezielt rekombinanten Esterase Enzymaktivität. Verbesserte Belastung der Ca 2 +-Indikator Farbstoffe zum ER-Lumen ermöglicht eine direkte und schnelle Darstellung von ER Kalzium-Speicher der Dynamik. Zelltypen für TED Die Durchf...

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

Gezielte-Esterase-induzierten Farbstoff Belastung (TED) unterstützt die Analyse der intrazellulären Kalzium-Speicher der Dynamik von Fluoreszenz-Bildgebung. Das Verfahren stützt sich auf Targeting eines rekombinanten Carboxylesterase zum Endoplasmatischen Retikulum (ER), wo es verbessert die lokale Entlarvung von synthetischen niedriger Affinität Ca<sup&gt; 2 +</sup&gt; Indikatorfarbstoffe in das ER-Lumen.

Direct Imaging von Calcium ER mit Targeted-Esterase Induzierte Dye Loading (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 Aufrufe.  University of Wuerzburg. Gezielte-Esterase-induzierten Farbstoff Belastung (TED) unterstützt die Analyse der intrazellulären Kalzium-Speicher der Dynamik von Fluoreszenz-Bildgebung. Das Verfahren stützt sich auf Targeting eines rekombinanten Carboxylesterase zum Endoplasmatischen Retikulum (ER), wo es verbessert die lokale Entlarvung von synthetischen niedriger Affinität Ca 2 + Indikatorfarbstoffe in das ER-Lumen.

Serie: 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.

Loading Drosophila Nerve Terminals mit Calcium-Indikatoren

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 ...

Forschung

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 Calcium Imaging in Layer 2 / 3 der Mäuse

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.

Calcium Imaging von kortikalen Neuronen mit 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 ...

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.

Montage-, Lade-und Alignment einer analytischen Ultrazentrifuge Messkammerdirektanschluss

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.

Live Cell Imaging mit Calcium siRNA vermittelte Gene Silencing Combined Identifiziert Ca 2 + Leck-Kanäle in der ER-Membran und deren Regulationsmechanismen

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 und Reinigung von virusähnlichen Partikeln zur Vakzinierung

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.

Monitoring ER / SR Calcium Release mit dem gezielten Ca<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

Imaging-Intermediate Filamenten und Mikrotubuli mit 2-dimensionale direkte stochastische optische Rekonstruktion Mikroskopie

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.

Fluoreszenz-Erholung nach Immunofluoreszenz von gelb fluoreszierende Protein Tagged p62 in Aggresome-induzierten Strukturen

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.

Bildgebende Calciumdynamik in Subpopulationen von Maus-Pankreas-Islet-Zellen

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