Premo Cameleon Calcium Sensor (P36207, P36208) is a ratiometric calcium-sensitive fluorescent protein that is delivered by BacMam baculovirus-mediated transduction (BacMam Gene Delivery and Expression Technology—Note 11.1) to a variety of mammalian cell types (Figure 19.5.1). This content and delivery system provides an effective and robust technique for measuring Ca2+ mobilization in transduced cells using microplate assays or fluorescence microscopy.
The Premo Cameleon Calcium Sensor is based on the YC3.60 version of the fluorescent protein–based sensor (cameleon) family developed by Tsien, Miyawaki and coworkers, which is reported to have a Ca2+ dissociation constant of 250 nM. The sensor comprises two fluorescent proteins (enhanced cyan-fluorescent protein or ECFP and Venus variant of yellow-fluorescent protein or YFP), linked by the calmodulin-binding peptide M13 and calmodulin. Upon binding four calcium ions, calmodulin undergoes a conformational change by wrapping itself around the M13 peptide, which changes the efficiency of the fluorescence resonance energy transfer (FRET) between the CFP donor and the YFP acceptor fluorophores (Figure 19.5.2). Following this conformational change, there is an increase in YFP emission (525–560 nm) and a simultaneous decrease in CFP emission (460–500 nm) (Figure 19.5.3), making Cameleon an effective reporter of calcium mobilization. This Ca2+-dependent emission ratio response reduces assay variations due to compound or cellular autofluorescence, nonuniform cell plating, differences in expression levels between cells, instability of instrument illumination and changes in illumination pathlength.
The Premo Cameleon Calcium Sensor readily and accurately detects intracellular calcium flux from different receptors. An example of the robustness and reproducibility and accuracy of the system is demonstrated using the endogenous histamine receptor in conjunction with histamine, pyrilamine, and thioperamide in HeLa cells (Figure 19.5.4). The no-wash, no-dye format and ratiometric readout eliminates wash steps that can dislodge cells, reduces data variability and increases data integrity. Expression levels will be maintained for several days, enabling iterative assays to be run; for instance, when examining agonist/antagonist relationships on the same cells. Premo Cameleon Calcium Sensor is provided as a ready-to-use baculovirus stock suspension containing the Cameleon DNA, which is efficiently delivered to target cells, including primary and stem cells, prior to cell plating. If required, immunolocalization of Premo Cameleon Calcium Sensor in fixed specimens can be accomplished using our anti–green-fluorescent protein (anti-GFP) antibodies (Anti–Epitope Tag and Anti-Reporter Antibodies—Section 7.5). Both stable cell lines and human primary cells can be prepared frozen and "assay-ready" and can be subsequently plated as little as four hours prior to screening. Cell-based assays or imaging experiments can be conducted in complete medium without any intervening wash steps.
Figure 19.5.1 Schematic representation of BacMam-mediated transduction and expression of the Premo Cameleon Calcium Sensor (P36207, P36208). The combination of fluorescent protein biosensors and BacMam delivery technology yields easy-to-use, genetically encoded sensors for cell-based assays. A BacMam virus carrying the Cameleon fusion gene (blue-gray-yellow) transduces a cell and traffics to the nucleus where only the Cameleon gene is transcribed; baculovirus promoters are not recognized by the mammalian transcriptional machinery, hence no virus replication occurs. Following transcription, the Cameleon mRNA is expressed in the cytosol, or in a specific compartment, depending on the presence of targeting tags.
Figure 19.5.3 Fluorescence emission spectra of Premo Cameleon Calcium Sensor (P36207, P36208). The dashed line indicates the spectra in the absence of Ca2+; the solid line shows the fluorescence resonance energy transfer (FRET)–based change upon Ca2+ binding.
Figure 19.5.4 Agonist and antagonist dose response curves. HeLa cells were plated in a 96-well plate at a density of 15,000 cells/well, transduced with Premo Cameleon Calcium Sensor (P36207, P36208), and incubated overnight at 37°C. The following day, a histamine dose response was performed (A). A separate plate was used to evaluate an antagonist dose response with pyrilamine (closed squares) and thioperamide (closed triangles) in the presence of an EC80 concentration of histamine (B). Pyrilamine is a known H1 receptor antagonist that couples through Gq proteins and the second messenger Ca2+. Thioperamide is a known H3 receptor antagonist that couples through Gi proteins and the second messenger cAMP.
Bioluminescence is defined as the production of light by biological organisms. Because light is produced by a chemical reaction of specific photoproteins within the organism and does not require illumination, bioluminescence-based assays can be extremely sensitive and free of background. However, the intensity of light produced by bioluminescent cells is often very low, necessitating the use of image enhancement to obtain sufficient signals.
Properties and Applications of Aequorin
We offer recombinant aequorin as well as a variety of synthetic coelenterazine analogs for quantitative Ca2+ measurements with aequorin, a photoprotein originally isolated from luminescent jellyfish and other marine organisms. The aequorin complex comprises a 22,000-dalton apoaequorin protein, molecular oxygen and the luminophore coelenterazine (Figure 19.5.5). When three Ca2+ ions bind to this complex, coelenterazine is oxidized to coelenteramide, with a concomitant release of carbon dioxide and blue light (Figure 19.5.6, Figure 19.5.7). The approximately third-power dependence of aequorin's bioluminescence on Ca2+ concentration gives it a broad detection range, allowing the measurement of Ca2+ concentrations from ~0.1 µM to >100 µM.
Unlike fluorescent Ca2+ indicators, Ca2+-bound aequorin can be detected without illuminating the sample, thereby eliminating interference from autofluorescence and allowing simultaneous labeling with caged probes (Photoactivatable Reagents, Including Photoreactive Crosslinkers and Caged Probes—Section 5.3). Moreover, aequorin that has been microinjected into eggs usually reports higher wave amplitudes (3–30 µM) than do fluorescent ion indicators. Aequorin is not exported or secreted, nor is it compartmentalized or sequestered within cells; thus, aequorin measurements can be used to detect Ca2+ changes that occur over relatively long periods. In several experimental systems, aequorin's luminescence was detectable many hours to days after cell loading. Aequorin also does not disrupt cell functions or embryo development (Figure 19.5.8).
Figure 19.5.5 Ribbon representation of the aequorin/coelenterazine complex showing the secondary structural elements in the protein. Coelenterazine and the side chain of Tyr 184 are shown as stick representations. Reproduced with permission from .
Figure 19.5.6 The Ca2+-induced luminescence emission spectrum of native aequorin incorporating the coelenterazine luminophore (C2944).
Figure 19.5.7 Ca2+-dependent generation of luminescence by the aequorin complex, which contains apoaequorin (APO) and coelenterazine (C2944).
Figure 19.5.8 Images of Ca2+ waves in gastrulating zebrafish embryos detected by microinjected f aequorin (recombinant aequorin reconstituted with the coelenterazine f luminophore C6779). The images are pseudocolored to represent Ca2+-dependent luminescent flux in (photons/pixel/second × 10-2) according to the color scales shown at the left of each of the three time-lapse image sequences (a,b,c). Time in seconds is indicated in the lower left-hand corner of each frame. The sequences depict three different spatial wave types that are represented schematically at the end of each sequence. PM indicates the dorsal midline pacemaker; its position in the luminescence images is marked by a red asterisk. The image was contributed by Edwin Gilland, Marine Biological Laboratory, Woods Hole, MA, and reproduced with permission from Proc Natl Acad Sci U S A (1999) 96:157.
Conventional purification of aequorin from the jellyfish Aequorea victoria requires laborious extraction procedures and sometimes yields preparations that are substantially heterogeneous or that are toxic to the organisms under study. Two tons of jellyfish typically yield ~125 mg of the purified photoprotein. In contrast, recombinant AquaLite aequorin (A6785) is produced by purifying apoaequorin from genetically engineered Escherichia coli, followed by reconstitution of the aequorin complex in vitro with pure coelenterazine. This method of preparation yields a pure, nontoxic, fully charged aequorin complex that is suitable for measuring intracellular Ca2+ by microinjection or other loading techniques, as well as for calibrating aequorin-based assays. Pressure injection is a commonly cited loading method, despite the fact that only large cells can be loaded in this way. Pressure injection has been employed to study the effects of caffeine on mouse diaphragm muscle fibers and the role of Ca2+ in the fertilization of sea urchin eggs. Alternatively, human platelets have been transiently permeabilized to the aequorin complex with DMSO, and monkey kidney cells have been loaded by hypoosmotic shock. A method based on the osmotic lysis of pinocytic vesicles—a technique that can be conveniently implemented using our Influx pinocytic cell-loading reagent (I14402, Chelators, Calibration Buffers, Ionophores and Cell-Loading Reagents—Section 19.8)—has been successfully used for cellular loading of aequorin and the related photoprotein obelin.
Because of its Ca2+-dependent luminescence, the aequorin complex has been extensively used as an intracellular Ca2+ indicator. Aequorea victoria aequorin has been used to:
- Analyze the secretion response of single adrenal chromaffin cells to nicotinic cholinergic agonists
- Calibrate micropipets with injection volumes of as little as 3 picoliters
- Clarify the role of Ca2+ release in heart muscle damage
- Demonstrate the massive release of Ca2+ during fertilization
- Study the regulation of the sarcoplasmic reticulum Ca2+ pump expression in developing chick myoblasts
Coelenterazine and Its Synthetic Analogs
We offer coelenterazine and several synthetic coelenterazine analogs for reconstituting aequorin in cells that have been transfected with apoaequorin cDNA (Coelenterazines and their properties—Table 19.4). Cell permeation of coelenterazine, which has been demonstrated in organisms as diverse as Escherichia coli, yeast, Dictyostelium cells, fish eggs, mammalian cells and plants, is the rate-limiting step in the reconstitution process. Coelenterazine is also required for generating the bioluminescent aequorin complex when using chimeric aequorin constructs. Furthermore, coelenterazine and its analogs are substrates for the bioluminescent Renilla luciferase.
In addition to native coelenterazine (C2944), we have synthesized three derivatives of coelenterazine that confer different Ca2+ affinities and spectral properties on the aequorin complex (Coelenterazines and their properties—Table 19.4). Like native coelenterazine, these derivatives can be used to reconstitute the aequorin complex both in vivo and in vitro. However, intracellular reconstitution of aequorin from coelenterazine analogs can be relatively slow. Aequorins containing the cp, f or h form of coelenterazine (C14260, C6779, C6780) exhibit relative intensities that are reported to be 10–20 times that of apoaequorin reconstituted with native coelenterazine. Coelenterazine cp has been used in an automated high-throughput screening assay for G-protein–coupled receptors. Coelenterazine is readily solubilized in aqueous solutions containing 50 mM hydroxypropyl-β-cyclodextrin.
|C2944||423.47||FF,D,LL,AA||MeOH||429||7500||see Notes||pH 7||1, 2, 3|
|C6779||425.46||FF,D,LL,AA||MeOH||437||8700||see Notes||MeOH||1, 2|
|C6780||407.47||FF,D,LL,AA||MeOH||437||9500||see Notes||MeOH||1, 2|
|C14260||415.49||FF,D,LL,AA||MeOH||430||7000||see Notes||MeOH||1, 2|