Ca2+ as a Second Messenger: New Reporters for Calcium (Cameleons and Camgaroos)
The intracellular Ca2+ ion concentration has been found to be associated with a wide variety of cellular processes (Carafoli, 2003). These include diverse events such as secretion, fertilization, cleavage, nuclear envelope breakdown, and apoptosis. Several diseases, including types of muscular dystrophy, diabetes, and leukemia, involve proteins that directly respond to or control Ca2+. Indeed, it may be more difficult to find cellular processes that do not involve Ca2+ than ones that do. From decades of research we have learned that the process of Ca2+ signaling consists, in general terms, of molecules for Ca2+ signal production, spatial and temporal shaping, sensors, and targets that elicit changes in biological function. Hence, this has prompted the development of sensitive signaling techniques to measure and image submicromolar levels of [Ca2+] and decode the dynamic Ca2+ messages throughout the propagation of the signal.
Ca2+ transients have traditionally been measured using synthetic fluorescent chelators (such as Fura-2 and Quin2) or recombinant aequorin (Grynkiewicz et al., 1985: Montero et al., 1995). Synthetic molecules provide a bright fluorescent signal, but these dyes are not easy to load and gradually leak out of cells at physiological temperatures. Cellular targeting is also not specific and some chemical indicators have been shown not to accumulate well in certain organelles. Aequorin is targeted easily but it requires incorporation of the cofactor coelenterazine, is irreversibly consumed by Ca2+, and is very difficult to image due to low bioluminescence. By comparison, green fluorescent protein (GFP) and calmodulin (CaM)-based "cameleon" probes have been developed and retain several of the benefits of the aforementioned indicators, yet also provide significant improvements for in vivo imaging (Miyawaki, 2003; Zhang et al., 2002; Truong and Ikura, 2001).
The use of cameleon indicators is gradually becoming more common within the Ca2+ signaling community and the literature is rich with examples of various applications. For instance, fusion of cameleons to specific signal sequences has successfully sorted them to nuclei, endoplasmic reticulum, caveolae, and secretory granule membranes (Isshiki et al., 2002; Demaurex and Frieden, 2003; Emmanouilidou et al., 1999). In addition to their use in detecting rapid stimulusinduced [Ca2+] transients, genetic studies in which cameleons were stably expressed in Arabidopsis stomatal guard cells (Allen et al., 1999), nematode pharyngeal muscle (Kerr et al., 2000), or larval thermoresponsive neurons of Drosophila (Liu et al., 2003) show that the sensors are also applicable to long-term monitoring of Ca2+ concentration. This has been demonstrated further for murine cells where the circadian rhythm of cytosolic but not nuclear Ca2+ in hypothalmic suprachiasmatic neurons was demonstrated (Ikeda et al., 2003).
It is hoped that this article provides some explicit and practical information relevant to the laboratory use of cameleon fluorescence resonance energy transfer (FRET) indicators. Our aim is that it will benefit and be of interest to colleagues both unfamiliar or experienced in using fluorescent Ca2+ indicators.
II. MATERIALS AND INSTRUMENTATION
A. Expression and Purification of Cameleons
Enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) expression constructus (Clontech Cat. No. 6075-1 and 6004-1, respectively); calmodulin cDNA (M. Ikura); pRSETB prokaryotic expression vector (Invitrogen Cat. No. V351-20); Luria broth (LB) media; isopropyl-β-D-thiogalactopyranoside (IPTG, Fermentas Cat. No. R0391); complete protease inhibitor cocktail tablets (Roche Cat. No. 1697498); Ni-NTA agarose (Qiagen Cat. No. 1018240); Escherichia coli BL21 (DE3) strain (Stratagene Cat. No. 200133); sonicator; EGTA buffer: 100 mM KCl, 50 mM HEPES (pH 7.4), and 10 mM EGTA; CaCl2 buffer: 100 mM KCl, 50 mM HEPES (pH 7.4), 10 mM EGTA, and 10 mM CaCl2.
B. In Vitro Fluorescence Quantitation
Shimadzu spectrofluorometer RF5301; 10-mm pathlength quartz cuvette.
C. In Vitro Imaging of Cameleons
pcDNA3 eukaryotic transient expression vector (Invitrogen Cat. No. V790-20); uncoated; γ-irradiated, 35-mm tissue culture dishes with glass bottom No. 0 (MatTek Cat. No. P35G-0-10-C); Dulbecco's modified Eagle medium (DMEM) supplemented with 10% dialyzed fetal bovine serum (FBS, Invitrogen Cat. No. 26400044), Hanks' balanced salts solution (HBSS) with Ca2+ (Invitrogen Cat. No. 14170120); 37°C CO2 incubator; HeLa cells or appropriate eukaryotic strain; Lipofectamine (Invitrogen Cat. No. 18324012) and PLUS (Invitrogen Cat. No. 11514015) reagents; histamine, ionomycin, ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl ester) (BAPTA-AM) (Sigma Cat. No. H7125, I0634, E0396, and A1076, respectively); Olympus IX70 inverted epifluorescence microscope; Olympus Xenon lamp; MicroMax 1300YHS CCD camera and Sutter Lambda 10-2 filter changers controlled by Metafluor 4.5r2 software (Universal Imaging); ECFP-EYFP FRET filter set (Omega Optical); 440AF21 excitation filter (ECFP excitation), 455DRLP dichroic mirror, 480AF30 emission filter (ECFP emission), and 535AF26 emission filter (EYFP emission); neutral density (ND) filter set (Omega Optical); UApo 40xOil Iris/340 objective (Olympus); U-MNIBA bandpass mirror cube unit (Olympus).
A. Engineering Cameleon Constructs
The history of cameleon engineering is reflected in its nomenclature (Table I). Optimization of Ca2+ affinities, pH dependency, maturation time, and other parameters is by no means complete. However, we present construction of a general cameleon designed in our laboratory, YC6.1, to serve as a reference point for future work (Truong et al., 2001). Molecular biology techniques for manipulating recombinant DNA are not given as they can be obtained from common reference books.
In these constructs, ECFP and EYFP function as a donor-acceptor pair for nonradiative, intramolecular FRET. During FRET, excitation of the donor (cyan) leads to emission from the acceptor (yellow), provided that the molecules are close enough (within 80 Å) and in a parallel orientation. In this way, on binding Ca2+ the CaM wraps around its adjacent CKKp target peptide and ECFP and EYFP are brought closer to each other and FRET increases (Fig. 1).
B. Overexpression and Purification of Cameleons
If necessary, it is possible to perform the characterization using a mammalian cell lysate. For harvesting, cells should be transfected in several 100-mmdiameter culture dishes, washed thoroughly to remove traces of phenol red and serum, and lysed in a hypotonic lysis buffer [50 mM HEPES (pH 7.4) 100 mM KCl, 5mM MgCl2, and 0.5% Triton X-100]. Following removal of cellular debris by centrifugation, dialyze the supernatant in 2 liter of buffer [50 mM HEPES (pH 7.4) and 100mM KCl]. Finally, the sample can be used for characterization as described.
C. In Vitro Cameleon Fluorescence Spectroscopy
The Ca2+-binding curve is used to assess the effective range of [Ca2+] measurement. Ca2+/EDTA and Ca2+/EGTA buffers are used as standards because even trace Ca2+ contaminants can significantly distort [Ca2+]free values at low [Ca2+] (Bers et al., 1994; Miyawaki et al., 1997).
D. Live Cell Cameleon Fluorescence Imaging
This section describes a Ca2+-imaging experiment using HeLa cells; however, with minor modifications the method can be applied to other cellular and physiological contexts.
IV. OTHER APPROACHES
Several other Ca2+ probes are also being developed, including the so-called "camgaroos" and "pericams." Camgaroos take an alternative approach to designing fluorescent Ca2+ sensors based on CaM and GFP family members (Baird et al., 1999). While ECFP and EYFP in cameleons are appended to the amino and carboxyl termini of CaM and Ca2+ binding is detected by FRET, camgaroo indicator proteins take advantage of the robust structure and profound fluorescence sensitivity of GFP to altered pKa values and chromophore orientation. Circular permutations and insertion of whole CaM in place of Tyr-145 within EEYFP thereby render this indicator responsive to Ca2+ binding. As a result, both excitation and emission spectra of camgaroo simply increase in amplitude by up to seven-fold upon saturation with Ca2+, without any significant shift in peak wavelength. This Ca2+-dependent fluorescence enhancement is substantially larger than other published genetically encoded fluorescent indicators. However, camgaroos are limited by pH sensitivity inherent in the current mechanism of modulating fluorescence via changes in pKa of the chromophore and have to date only been preliminarily subjected to systematic mutational improvement.
Pericams, in which EYFP is circularly fused to CaM and the M13 myosin light chain kinase peptide, improve approximately 10-fold upon the low affinity of camgaroo for Ca2+ (Kd = 7 µM) and are thereby better capable of sensing low physiological changes in intracellular [Ca2+] (Nagai et al., 2001). Taken together, these strategies offer alternatives complementary to cameleons for creating genetically encoded, physiological Ca2+ indicators.
V. OTHER CONSIDERATIONS AND PITFALLS
A. Ca2+ Ion Sensitivity
Although cameleon probes vary in their Ca2+ affinities, most are suitable for monitoring [Ca2+] between 0.5 and 100 µM. This poses a problem for examining the relatively high [Ca2+] found in the endoplasmic reticulum of resting cells (approximately 500 µM). However, CaM mutagenesis studies have shown that substitution of a conserved glutamic acid residue at the 12th position of each Ca2+-binding loop abolishes its Ca2+-binding ability (Zhu et al., 1998). The effect of combinations of these mutations on cameleon Ca2+ range is currently being examined further in our laboratory.
GFP variants have been developed in which chromophore oxidative maturation (and thereby become fluorescent) occurs more quickly and efficiently at 37°C. It would be advantageous to utilize efficiently folding versions, such as the recently developed "Venus" form of EYFP (F46L/F64L/M153T/V163A/ S175G). These EYFP mutations confer an eight-fold increase of fluorescence intensity when expressed in mammalian cells (Nagai et al., 2002; Rekas et al., 2002). This will enable assay of cells 24 h postrecovery transfection, if so desired.
C. pH Sensitivity
The hydrogen bond network within the 13 barrel of the chromophore is sensitive to external pH. Hence, in order to analyze Ca2+ levels in acidic organelles (such as secretory vesicles), the FRET donor/acceptor pair must be engineered so that it is pH resistant. Two mutations within EEYFP (V68L and Q69K) have been shown to decrease its pKa to 6.1 (Miyawaki et al., 1999). The pH sensitivity was improved further via a Q69M or "citrine" mutation (pKa 5.7; Griesbeck et al., 2001). Another approach would be to change the donor/acceptor pair to the pH-insensitive sapphirered cameleon probe (SapRC2). Although this construct has a tendency to aggregate and form homotetramers, the recent engineering of a monomeric mRFP1 offers an interesting alternative (Campbell et al., 2002).
GFP family proteins have been observed to form obligate dimers and may thereby generate falsepositive FRET signals. However, this aggregation problem need not preclude their use in biological systems, even when present in higher local concentrations. Nonoligomerizing mutants of EYFP have been suggested from its crystal structure (Wachter et al., 1998; Rekas et al., 2002), but these have yet to be validated experimentally. Alternatively, using a monomeric version of the evolutionary distinct RFP (mRFP1) in tandem with a EYFP donor would also serve to eliminate this issue (Campbell et al., 2002).
E. Influences on Biological Systems
It is very important to consider potential competition of the FRET indicator for native CaM or CaMdependent enzymes. Previous comparisons of the effect of recombinant CaM and cameleon chimeras on prototypical CaM-dependent enzymes have revealed that the primary effect of cameleons is on buffering [Ca2+] and not interfering with CaM-mediated signaling (Miyawaki et al., 1999). This could be due to the CaM component of the cameleon being inhibited by the adjoining CKKp efficiently occupying its substratebinding site. Also, as the YC6.1 CKKp is embedded within the cameleon polypeptide, it is not likely to interact with endogenous CaM proteins.
F. Interpretation of Live Cell FRET Data
There are two commonly used simple and practical approaches. The first, measurement of donor emission quenching and acceptor emission enhancement by using three filter sets and then mathematical processing to determine emission/FRET ratios, is described in Section III. The alternative approach is by detection of donor dequenching following acceptor bleaching. We find bleaching with a minimum of 200ms, compared to 1 ms for control excitation, is sufficient.
G. Additional Ways to Do Ratio Imaging
FRET requires rapid intensity measurements at different wavelengths. Switching time (in the millisecond range) may be an important parameter for some applications. New imaging systems have become available in the marketplace, notably TILLvisION (T.I.L.L. Photonics) and AquaCosmos (Hamamatsu), which allow for excellent time resolution for fluorescenceintensity ratio imaging.
H. FRET Using Red Fluorescence Protein
For YC6.1 applications, the ECFP-EYFP FRET filter set is sufficient. However, if you are using RFP for FRET, the following dichoric mirrors and filters from Omega Optical (or equivalents) will be needed: 450- 520-590TBDR for the dichoric mirror; 440DF21 for ECFP excitation; 510DF23 for EYFP excitation; 575DF26 for RFP excitation; 480AF30 for ECFP emission; 535AF26 for EYFP emission; and 600ALP for RFP emission. This filter set allows you to excite or acquire emission from ECFP, EYFP, and RFP individually, albeit with a tradeoff in efficiency.
I. Imaging Systems
Using a confocal microscope is the best way to increase spatial resolution for FRET experiments. Confocal YC6.1 measurements can be performed with single-photon excitation using the 458-nm line of an argon laser, but much more efficient excitation of ECFP is attained with the 442-nm line of a HeCd laser. Twophoton excitation microscopy, in addition to providing optical sections of a specimen as with confocal microscopy, offers certain advantages. Its applicability to cameleons has been demonstrated using video-rate scanning instrumentation (Fan et al., 1999). This latter imaging approach may not be readily available to most laboratories, however.
J. Preparation of Ca2+/EGTA Buffers
The accuracy of the Ca2+-binding curve depends on accurate preparation of the Ca2+/EGTA and Ca2+/HEEDTA systems below 10-5M free Ca2+ and unbuffered Ca2+ above (Bers et al., 1994). The purity of EGTA, temperature, and pH are all practical issues.
We are grateful to Atsushi Miyawaki for his help in setting up a FRET microscope system in our laboratory, as well as for much advice on the use of GFP variants. This work was supported by grants from the Cancer Research Society Inc. and the Institute for Cancer Research of the Canadian Institutes of Health Research (CIHR). K.P.H. is a recipient of a NCIC Research Fellowship, K.T. holds a CIHR scholarship, and M.I. is a CIHR senior investigator.
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