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.

<ol>
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The authors declare that they have no competing financial interests.

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...

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 high scientific interest because calcium is involved in the regulation of gene transcription, cell proliferation, neuronal excitability, cell death and other cell signaling events 1-7. All these cellular calcium signals are functionally connected and contribute to intracellular calcium store dynamics 8-10. 
	A common feature among all calcium signals is the flow of calcium between the extracellular space, the cytosol and organelles, mainly the ER and mitochondria. This causes dynamic changes in calcium concentration within these organelles, which are sensed by different signaling components. In general, the calcium concentration in the ER ranges between 100 to 800 &mu;M, in the cytosol the calcium concentration is close to 100 nM, and in the extracellular space the concentration is around 1-2 mM. Accordingly, there is a high chemical driving force for calcium flow towards the cytosol 2,9,10.
	The most commonly investigated ER-derived calcium signals depend on the stimulation of G-protein coupled receptors (GpcR), which then activate phospholipase C (PLC). PLC in turn produces inositol 1,4,5-trisphosphate (IP3) 1. Upon binding of IP3 to its receptor (IP3-Rec, Figure 1) in the ER-membrane, calcium ions are released from the ER lumen. Historically, IP3-mediated calcium release from the ER was first - even though indirectly - measured in acinar pancreatic cells by Streb et al. in 1983 11. This publication suggested for the first time a signaling cascade involving acetylcholine, phospholipase C, and IP3. This way of calcium release is generally termed IP3-induced calcium release (IiCR) (Figure 1). Kinase-dependent activation of phospholipase C&gamma; by receptor tyrosin kinases links the action of growth factors and neurotrophic factors to ER calcium signaling after IP3 elevation 12. In addition to IiCR, calcium elevation may be mediated by ionotropic calcium entry, for instance via voltage-gated calcium channels (CaV), and subsequent calcium-induced calcium release (CiCR) by ryanodine receptors (RyR). IiCR and CiCR are physiologically linked to store-operated calcium entry (SOCE). SOCE includes the action of STIM (stroma interacting molecule), which is a sensor for ER calcium release. STIM has been shown to stimulate extracellular calcium entry through transient receptor potential channels (Trp) 13, Orai calcium channels 14 and even voltage-gated calcium channels 15 (Figure 1) . Loss of ER calcium is dynamically rescued by the action of the sarco-endoplasmic reticulum calcium ATPase (SERCA), which actively pumps calcium back into the ER. Blocking the SERCA with drugs such as thapsigargin unveils a continuous loss of ER calcium to the cytosolic compartment. This ER calcium "leak" is caused by ER intramembrane pore complexes such as the Sec61 protein complex 16,17 (Figure 1).
	In 1998, Berridge published a model, the "neuron within a neuron model", which suggests a principle physiological role of the ER in integrating neuronal calcium 5. This model considers the existence of a continuous ER membrane system forming an intracellular "image" of the neuronal plasma membrane 5. This binary eukaryotic membrane system was claimed to be a basic prerequisite for temporal and spatial integration of fast and slow calcium signals in neurons. Calcium signals that occur either concomitantly or subsequently in different spines or dendrites of the same neuron are conferred to the cell's soma or nucleus via the ER, where they are summed up 5,18. Then, their sum may have effects on the excitability of the neuron, regulation of gene transcription or integration of signaling cascades. Thus, the ER supports the integration of calcium signals. One prerequisite for this concept is the continuity of the ER in a single cell, which has been claimed by several studies and which has been proven at least for somato-dendritic areas and short distance axonal projections 19-21. Whether there is ER continuity within long axonal projections is a matter of debate.
	Strategies to measure the flow of free calcium over the ER membrane
	Calcium signals are most frequently monitored in the cytosol 22,23. Therefore, it cannot easily be distinguished whether Ca2+ is flowing into the cytosol from extracellular or from intracellular stores 6,24. To overcome this limitation, methodological strategies for direct ER calcium imaging have been developed. In summary, the following strategies are used: (1) ER-targeted genetically engineered protein-indicators 25-27. Protein-based low-affinity Ca2+ indicators use the bioluminescent protein aequorin or GFP in combination with a calcium sensing protein. These genetically engineered Ca2+ indicators (GECIs) can be targeted to the ER with the help of a signal peptide and are actively kept in the ER using a retention and retrieval motif. Common ER Ca2+ indicators base on the Cameleon principle and are the Cameleon YC4.3 26,28; Cameleon split YC7.3ER 29, and Cameleon D1 30. (2) Direct esterase-based dye loading of AM-ester low-affinity Ca2+ indicators 31,32. AM-derivatives of the indicator dyes (Mag-Fura2-AM, Mag-Fluo4-AM or Fluo5N-AM) pass the biological membranes in a lipophilic, calcium-insensitive state. Then, in the cytosol as well as in the ER, endogenous esterases cleave of the AM-ester group and release the Ca2+ indicator, leaving behind a certain amount of active dye in the cytosol and in the ER. Therefore, this approach is useful under conditions of a high calcium concentration in the ER as long as the cytosolic calcium concentration stays well below the detection limit of the low-affinity indicators, particularly during characteristic calcium signals (e.g. nM to low &mu;M). (3) AM-ester loading in combination with plasma membrane permeabilization 32. Any remaining cytosolic Ca2+ indicator is removed by plasma membrane permeabilization with small amounts of a "mild" detergent (e.g. saponin) in an artificial intracellular buffer. Thus, the intracellular membranes may be stimulated, e.g. with IP3 in the intracellular buffer, directly through "pores" in the plasma membrane. (4) Dialysis of the cytosol under whole-cell configuration and simultaneous measurements of Ca2+ in the ER lumen and the cytosol 32,33. A cell is first loaded with a low-affinity Ca2+ indicator (e.g. Mag-Fura2-AM, ratiometric, UV-light). Afterwards, with the help of a patch pipette, any remaining cytosolic low-affinity Ca2+ indicator is dialysed out of the cytosol with a buffer containing a high-affinity Ca2+ indicator (e.g. Fluo-3, visible light). This strategy enables the simultaneous recording of cytosolic and ER derived signals. (5) Targeted-esterase-induced dye loading 8,34. A Carboxylesterase (CES) is targeted to the lumen of the ER and provides a high esterase activity for efficient trapping of the AM-ester form of low-affinity Ca2+ indicators.
	Targeted-esterase induced dye loading (TED)
	To improve targeting of low-affinity Ca2+ indicators to the ER lumen, TED was developed. TED requires the overexpression of an ER targeted mouse carboxylesterase (CES2) (Figure 2), which is achieved via expression constructs. Cells expressing a recombinant CES-construct are incubated with the AM-ester form of a calcium indicator dye (Fluo5N-AM, Figure 2). Then, in the ER, the dye is converted to the Ca2+ sensitive, membrane impermeable Ca2+ indicator complex (Fluo5N/Ca2+) by the high esterase activity, therefore trapping the dye in a high concentration in the ER lumen 8,34. The method is especially useful to investigate ER calcium-release via the IiCR-pathways for instance via metabotropic, purinergic- or glutamate receptors 8,34 and to visualize ER calcium depletion via "leak channels" directly, for instance after blockade of the SERCA 17,34. To our experience the low-affinity Ca2+ indicator Fluo5N-AM is currently the best available indicator to use with the TED dye loading strategy. Fluo5N-AM has a low-affinity for Ca2+ (dissociation constant KD ~90 &mu;M, Figure 2), is almost non-fluorescent in its AM-form, but provides high fluorescence emission upon calcium binding 8,34. The Fluo5N/Ca2+ complex can be excited with a standard light source of ~490 nm, which corresponds to standard dyes such as FITC, Alexa 488, or eGFP. Cytosolic calcium signals scarcely reach a concentration in the low &mu;M range and are therefore barely detected by Fluo5N in the cytosol 35.
	To improve the TED performance, several recombinant vector constructs were developed (Figure 3). Originally, TED vectors based on the coding sequence of CES2 (Refseq accession number NM_145603, CES2c) and best TED performance is observed with stable expression of CES constructs. New TED vectors express a core element of CES2 fused to the red fluorescence protein TagRFP-T2 36. These vectors have the advantage that they can be used to identify transduced cells and to use the red fluorescence as an internal control for the normalization of changes in Fluo5N/Ca2+ fluorescence. The red fluorescence also offers the possibility to visualize the structural distribution of the ER and changes in ER dynamics under stimulation conditions.

<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)

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 &Delta;F/F0.

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,...

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-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.

Direct Imaging of ER Calcium with Targeted-Esterase Induced 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

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19.1K Views.  University of Wuerzburg. 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.