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Abstract

Introduction

Protocol

Representative Results

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Acknowledgements

Materials

References

Biology

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

Published: May 7th, 2013

DOI:

10.3791/50317

1Institute for Clinical Neurobiology, University of Wuerzburg, 2Department of Synapses - Circuits - Plasticity, Max Planck Institute of Neurobiology, Martinsried, 3Walter Brendel Centre of Experimental Medicine, Ludwig-Maximilians University of Munich

Targeted-esterase induced dye loading (TED) supports the analysis of intracellular calcium store dynamics by fluorescence imaging. The method bases on targeting of a recombinant Carboxylesterase to the endoplasmic reticulum (ER), where it improves the local unmasking of synthetic low-affinity Ca2+ indicator dyes in the ER lumen.

Visualization of calcium dynamics is important to understand the role of calcium in cell physiology. To examine calcium dynamics, synthetic fluorescent Ca2+ indictors have become popular. Here we demonstrate TED (= targeted-esterase induced dye loading), a method to improve the release of Ca2+ indicator dyes in the ER lumen of different cell types. To date, TED was used in cell lines, glial cells, and neurons in vitro. TED bases on efficient, recombinant targeting of a high carboxylesterase activity to the ER lumen using vector-constructs that express Carboxylesterases (CES). The latest TED vectors contain a core element of CES2 fused to a red fluorescent protein, thus enabling simultaneous two-color imaging. The dynamics of free calcium in the ER are imaged in one color, while the corresponding ER structure appears in red. At the beginning of the procedure, cells are transduced with a lentivirus. Subsequently, the infected cells are seeded on coverslips to finally enable live cell imaging. Then, living cells are incubated with the acetoxymethyl ester (AM-ester) form of low-affinity Ca2+ indicators, for instance Fluo5N-AM, Mag-Fluo4-AM, or Mag-Fura2-AM. The esterase activity in the ER cleaves off hydrophobic side chains from the AM form of the Ca2+ indicator and a hydrophilic fluorescent dye/Ca2+ complex is formed and trapped in the ER lumen. After dye loading, the cells are analyzed at an inverted confocal laser scanning microscope. Cells are continuously perfused with Ringer-like solutions and the ER calcium dynamics are directly visualized by time-lapse imaging. Calcium release from the ER is identified by a decrease in fluorescence intensity in regions of interest, whereas the refilling of the ER calcium store produces an increase in fluorescence intensity. Finally, the change in fluorescent intensity over time is determined by calculation of ΔF/F0.

In order to resolve physiological calcium responses of the ER, we developed a new strategy to improve the trapping of synthetic calcium sensitive dyes into the ER. The method enables the direct, non-disruptive real-time monitoring of free ER calcium in presence of extracellular calcium.

Function and signaling of ER Calcium

Calcium signals are found in different cell types, e.g. muscle-cells, neurons and glial cells and their functions range from mediating muscle contraction to an involvement in synaptic transmission in learning and memory 1,2. Changes in free calcium concentration are of h....

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This protocol introduces TED applied to cell lines, hippocampal neurons and cortical glial cells. TED performance is best when TED vectors are stably expressed, for instance by lentiviral vectors. A schematic overview of the TED method is shown in Figure 4.

1. Preparation of Solutions

The following solutions should be prepared before starting and can be stored as indicated. We generally use sterilized glass and plastic material and autoclaved water.

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This protocol provides a non-disruptive approach for direct imaging of free ER calcium. Low-affinity synthetic Ca2+ indicators are released and trapped in the ER lumen with the help of an ER targeted, recombinant esterase enzyme activity. Improved loading of the Ca2+ indicator dyes to the ER lumen enables a direct and fast imaging of ER calcium store dynamics.

Cell types for TED

The feasibility of the method has been shown in the cell lines BHK21,.......

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The advantages and disadvantages of the TED method have been discussed extensively in recent publications 34,35. Compared to other methods described above, TED circumvents the problem of disrupting and perfusing the cells. Furthermore, two aspects need to be emphasized. The principle of TED for ER calcium imaging requires (1.) the targeted expression of an active carboxylesterase in the ER lumen with the help of TED vector constructs and (2.) the efficient and preferential release of low-affinity synthe.......

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This work has been supported by grants of the Deutsche Forschungsgemeinschaft (DFG) BL567/3-1 and the Friedrich-Baur-Stiftung. We would like to thank Roger Y. Tsien, Howard Hughes Medical Institute Laboratories at the University of California, San Diego for providing us with Tag-RFP-T2. We thankfully acknowledge David Baltimore, California Institute of Technology, Pasadena, and Didier Trono, University of Geneva, Geneva, for providing us the lentiviral plasmids FUGW, and psPAX2/pMD2.G.

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

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