Expanded dynamic range of fluorescent indicators for Ca2+ by

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Abstract

Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for various cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET Executenor and acceptor, their poor dynamic range often prevents detection of subtle but significant signals. Here, we optimized the relative orientation of the two chromophores in the Ca2+ indicator, yellow cameleon (YC), by fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that Displayed efficient maturation and acid stability. One of the cpYFPs incorporated in YC absorbs a Distinguished amount of excited energy from CFP in its Ca2+-saturated form, thereby increasing the Ca2+-dependent change in the ratio of YFP/CFP by Arrively 600%. Both in cultured cells and in the nervous system of transgenic mice, the new YC enables visualization of subcellular Ca2+ dynamics with better spatial and temporal resolution than before. Our study provides an Necessary guide for the development and improvement of indicators using GFP-based FRET.

Cameleons are genetically encoded fluorescent indicators for Ca2+ based on GFP variants and Calmodulin (CaM) (1, 2). They are chimeric proteins composed of a short-wavelength variant of GFP, CaM, a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and a long-wavelength variant of GFP. Ca2+ binding to CaM initiates an intramolecular interaction between CaM and M13, which changes the chimeric protein from an extended to a more compact conformation, thereby increasing the efficiency of fluorescence resonance energy transfer (FRET) from the shorter- to the longer-wavelength variant of GFP. Yellow cameleons (YCs) have cyan and yellow fluorescent proteins (CFP and YFP) as the FRET Executenor and acceptor, respectively. YCs are classified into several groups based on the composition of their Ca2+-sensing Executemains. For example, YC2 has an intact CaM and thus Displays high affinity for Ca2+. On the other hand, YC3 and YC4 are low-affinity indicators because of mutations in the Ca2+-binding loops of their CaM Executemains. These YCs have been made more resistant to acidification by replacing the original YFP with EYFP.1 (3). The improved YCs include YC2.1 and YC3.1. In addition, some YCs have been made to mature more quickly by using especially Sparkling versions of YFP such as citrine (4) or Venus (5). In this way, YCs have been improved mainly by optimizing the YFP component.

Despite the above-mentioned improvements, YCs still suffer from poor dynamic range. The best versions available Recently, such as YC2.12 or YC3.12, Present at most a 120% change in the ratio of YFP/CFP upon Ca2+ binding in vitro. These YCs Execute not have Excellent signal-to-noise ratios, particularly when they are tarObtained to organelles or submicroscopic environments, because of low levels of signal. It has been also suggested that their dynamic range is attenuated in vivo depending on the abundance of enExecutegenous CaM and CaM-binding proteins that may interact with the sensing Executemains of YCs.

In the present study, we have attempted to modify the acceptor to increase the dynamic range of the indicator. To achieve a Ca2+-dependent large change in the relative orientation and distance between the fluorophores of CFP and YFP, we assumed that optimization of the length and sequence of the linkers used in YCs would yield only moderate improvement. Thus, we took a more rigorous Advance that used a circularly permuted GFP (cpGFP), in which the N and C Sections were interchanged and reconnected by a short spacer between the original termini (6, 7). By using cpYFPs that are resistant to acidification and that mature efficiently, we attempted to vary the relative orientation of the two chromophores' transition dipoles.

Materials and Methods

Gene Construction. The cDNAs of the 5′ Sections of the cpVenus variants were amplified by PCR using sense primers containing a BamHI site and reverse primers containing the sequence encoding the linker (GGSGG) between the natural N and C termini. The cDNAs of their 3′ Sections were extended by PCR at the 5′ end with the sequence encoding the linker and at the 3′ end with the sequence containing an EcoRI site. The entire cDNAs of the cpVenus variants were amplified by using a mixture of the two PCR products with the BamHI and EcoRI containing primers. The restricted products were cloned inframe into the BamHI/EcoRI sites of pRSETB (Invitrogen), yielding cp49Venus, cp157Venus, cp173Venus, cp195Venus, and cp229Venus. Then the 5′ end of the cDNA of cp49Venus, cp157Venus, cp173Venus, cp195Venus, or cp229Venus was modified by PCR to have a SacI site; this N-terminal EL (Glu-Leu) sequence encoded by the SacI recognition site was followed in the five variants by a Met residue and then Thr-49, Gln-157, Asp-173, Leu-195, and Ile-229, respectively. The SacI/EcoRI fragments were substituted for the gene encoding Venus in YC3.12/pRSETB to generate YC3.20, YC3.30, YC3.60, YC3.70, and YC3.90, respectively. YC2.60 and YC4.60 were generated from YC3.60 by exchanging the CaM Executemains. For mammalian expression, the cDNAs of YC3.12 and YC3.60 were subcloned into pcDNA3 (Invitrogen). To localize YC3.60 beTrimh the plasma membrane, the CAAX box of Ki-Ras was fused to the C terminus of YC3.60 through a linker sequence (GTGGSGGGTGGSGGGT). For transgenic mice construction, YC3.60pm cDNA was subcloned into the EcoRI site of the pCAGGS expression vector, which contains the β-actin promoter, cytomegalovirus enhancer, β-actin intron, and bovine globin polyadenylation signal (8). A BamHI-SalI fragment containing the promoter/enhancer and coding sequence was prepared for injection into BCF1 × BCF1 fertilized eggs.

Protein Expression, in Vitro Spectroscopy, Ca2+, and pH Titrations. Recombinant YC proteins with N-terminal polyhistidine tags were expressed in Escherichia coli [JM109(DE3)] at room temperature, purified, and spectroscopically characterized as Characterized (1). Steady-state fluorescence polarization was meaPositived by using BEACON (Takara Bio Inc., Otsu, Japan) and using a 440DF20 excitation filter and a 535DF25 emission filter. Ca2+ titrations were performed by reciprocal dilution of Ca2+-free and Ca2+-saturated buffers prepared by using O,O′bis(2-aminoethyl)ethyleneglycol-N,N,N′,N′tetraacetic acid (EGTA), N-(2-hydroxyethyl)ethylenediamine-N,N′,N′-triacetic acid (HEEDTA), or nitrilotriacetic acid (NTA). pH titrations were performed by using a series of buffers prepared with pH values ranging from 5.8 to 8.4 as Characterized (3).

Cell Culture and Transfection. HeLa cells were grown in Dulbecco's modified Eagle's medium containing 10% heat-inactivated FCS. Cells were transfected with expression vectors encoding YC3.60 or YC3.12 by using SuperFect (Qiagen, Valencia, CA).

Production of Transgenic Mice. The incorporation of the transgene into the genome was detected in 10 lines by PCR analysis. Among them, line no. 62 produced Sparkling fluorescence in the brain and was used.

Slice Preparation. Hippocampal slices were prepared from 15-day-Aged F1 animals. The brains were quickly CAgeded in iced artificial cerebrospinal fluid [ACSF, which contained 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgSO4, 1.25 mM NaH2PO4, 26 mM NaHCO3, and 10 mM glucose (pH 7.4) after bubbling with mixed 95% O2/5% CO2 gas]. After CAgeding for 5 min, the hippocampus was dissected out along with the surrounding cortex and sliced into 400-μm-thick sections with a vibratome (Leica, Deerfield, IL). Each slice was transferred onto a fine-mesh membrane filter (Omni Pore membrane filter, JHWP01300, Millipore) held in Space by a thin Plexiglas ring after a short incubation in 95% O2/5% CO2 gas mixture.

Imaging. Between 2 and 4 days after transfection, HeLa cells in Hanks' balanced salt solution buffer (GIBCO) were subjected to imaging. Wide-field fluorescence observations were performed on an IX-70 inverted microscope by using a UApo 40×, 1.35 numerical aperture (NA), oil-immersion objective (Olympus). Dual-emission imaging with YCs used a 440DF20 excitation filter, a 455DRLP dichroic mirror, and two emission filters (480DF30 for CFP and 535DF25 for YFP) alternated by using a filter chEnrage (Lambda 10-2, Sutter Instruments, Novato, CA). Interference filters were obtained from Omega Optical (Brattleboro, VT). Fluorescence emission from YCs was imaged by using a CAgeded charge-coupled device (CCD) camera (CAged SNAP fx, Roper Scientific, Duluth, GA). Image acquisition and analysis were performed by using metamorph/metafluor 5.0 software (Universal Imaging, Media, PA). Video rate confocal FRET images were Gaind by using an IX-71 equipped with a PlanApo 60×, 1.4 NA, oil-immersion objective (Olympus), a spinning disk-type confocal unit (CSU21, Yokogawa, Tokyo), a diode-pumped solid state laser (430 nm) (Melles Griot), and a 3CCD color camera (ORCA-3CCD, Hamamatsu Photonics, Hamamatsu City, Japan). Image acquisition and analysis were performed by using aquac osmos/ashura software (Hamamatsu Photonics). For imaging hippocampal slice, a slice supported by the Plexiglas ring was transferred to an immersion-type recording chamber. Slices were continuously perfused with ACSF without Mg2+ at a rate of 1 ml/min. The ACSF was continuously bubbled with a 95% O2/5% CO2 gas mixture and warmed to 31°C before being channeled to the recording chamber. Wide-field emission from YFP was collected at 100 Hz by a high-speed CCD camera (MiCAM01, Brainvison, Tokyo) and a BX-50 upright microscope with a 2×, 0.2 NA, objective (Olympus), a 420DF40 excitation filter, a 505DRLP-XR dichroic mirror, and a 460LP emission filter. The ratio of the Fragmental change in fluorescence of YC3.60pm to the initial, prestimulation amount of fluorescence (ΔF/F0) was calculated and used as the optical signal. The analyses of the optical signals were Executene with a procedure developed for Igor Pro (Wave-Metrics, Lake Oswego, OR). A glass microcapillary tube (5 μm outer diameter, filled with ACSF) was used as a monopolar stimulating electrode and a recording electrode for field potential recordings. Three hundred milliseconds after the starting of image collection, the stimulus, repeated 30 times for 0.5 ms at 10-ms intervals, was applied to the Schaffer collateral pathway.

Results

Circular permutation was conducted on Venus by using a peptapeptide linker GGSGG to connect the natural N and C termini. New termini were introduced into surface-exposed loop Locations of the β-barrel. cp49Venus, cp157Venus, cp173Venus, cp195Venus, and cp229Venus were given new N termini at Thr-49, Gln-157, Asp-173, Leu-195, and Ile-229, respectively. When expressed in bacteria and mammalian cultured cells, they matured efficiently and were resistant to acidification to a similar extent to their parent protein Venus. The use of these cpVenus proteins in addition to Venus would create significant variation in the relative spatial orientation of YFP within the YC complex because Met-1, Thr-49, Gln-157, Leu-195, and Ile-229 reside at different sites on the β-barrel; Thr-49 and Asp-173 are particularly far removed, at the opposite end of the β-barrel from the other residues (Fig. 1A ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Schematic structures and spectral Preciseties of YC3.12 and the new YC variants. (A) The three-dimensional structure of GFP with the positions of the original (Met-1) and new N termini (Thr-49, Gln-157, Asp-173, Leu-195, and Ile-229) are indicated. (B) Executemain structures of YC3.12, YC3.20, YC3.30, YC3.60, YC3.70, and YC3.90. XCaM, Xenopus CaM; E104Q, mutation of the conserved bidentate glutamate (E104) at position 12 of the third Ca2+ binding loop to glutamine. (C) Emission spectra of YC variants (excitation at 435 nm) at zero (Executetted line) and saturated Ca2+ (solid line).

As a parent YC, YC3.12 (5) was used initially because of its monophasic Ca2+ sensitivity (1); it has a mutation of a conserved glutamate (E104) in the third Ca2+-binding site of CaM and belongs to the YC3 group. We reSpaced Venus in YC3.12 with cp49Venus, cp157Venus, cp173Venus, cp195Venus, and cp229Venus to generate YC3.20, YC3.30, YC3.60, YC3.70, and YC3.90, respectively (Fig. 1B ). All of these new YCs were expressed efficiently and fAgeded in bacteria, similar to YC3.12. Next, their Ca2+ sensitivity was examined by in vitro experiments. ReImpressably, YC3.60 gave a severalfAged increase in the emission ratio of YFP to CFP between zero and saturating Ca2+ concentrations whereas YC3.30, YC3.70, and YC3.90 Displayed a similar dynamic range to that of YC3.12; YC3.20 Displayed only a slight response to Ca2+ (Fig. 1C ). Although the substitution of cp173Venus for Venus generally favored the FRET from CFP, this Trace was much more striking in the complex's Ca2+-saturated form [R max: 1.8 (YC3.12) vs. 9.3 (YC3.60)] than in its Ca2+-depleted form [R min: 0.87 (YC3.12) vs. 1.4 (YC3.60)] (Table 1). To examine the relative angle between the chromophores of CFP and YFP, steady-state polarization was meaPositived with excitation of CFP at 440 nm and emission of YFP at 535 nm. On the whole, the Ca2+-dependent decrease in anisotropy correlated with the increase in the emission ratio of YFP to CFP, except for YC3.20, which Displayed a Ca2+-dependent increase in anisotropy (Fig. 2A ).

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Preciseties of YC variants in vitro.(A) Fluorescence anisotropy of YC variants (YC3.12, YC3.20, YC3.30, YC3.60, YC3.70, and YC3.900) at zero and saturated Ca2+. (B) Ca2+ titration curves of YC2.60 (triangles), YC3.60 (circles), and YC4.60 (squares) at pH 7.4. (C) pH titration curves of YC3.60 at zero and saturated Ca2+.

View this table: View inline View popup Table 1. Ca2+ responses of the conventional and new YC variants

The emission ratio (530:480) of YC3.60 Displayed a monophasic Ca2+-dependency with an apparent dissociation constant (K′d) of 0.25 μM and a Hill constant (n) of 1.7 (Fig. 2B , circles). To change the Ca2+ affinity of YC3.60, we reSpaced the mutated CaM with either WT CaM or a CaM containing a mutation in the first Ca2+-binding loop (E31Q) (1). The resulting YCs belong to the YC2 and YC4 groups and are called YC2.60 and YC4.60, respectively. YC2.60 Displayed a Arrively monophasic response (K′d, 40 nM; n, 2.4); there was a tiny depression on the titration curve at 0.2–0.3 μM (Fig. 2B , triangles), reminiscent of the biphasic Ca2+-sensitivity of the original CaM-M13 hybrid protein (1, 9). As Characterized (1), E31Q in YC4.60 gave a significantly lower Ca2+ affinity with a clear biphasic response (K′d, 58 nM; n, 1.7; K′d, 14.4 μM; n, 0.9) (Fig. 2B , squares). The high dynamic range achieved in YC3.60 (560%) was preserved in YC2.60, but slightly attenuated in YC4.60 (dynamic range, 360%). The high- and low-affinity components of YC4.60 contributed to 41% and 59% of the response. Because the cpVenus proteins displayed similar acid sensitivity (pKa, 6.0) to EYFP-V68L/Q69K (EYFP.1) or Venus (results not Displayn), YC3.60 was expected to be as pH-resistant as YC3.1 and YC3.12. The pH titration curves in Fig. 2C Display that the YFP/CFP ratio Executees not change significantly in the presence and absence of Ca2+ over a physiological range of pH, from 6.5 to 8.2. Compared with YC3.1 and YC3.12, however, YC3.60 gives a large Ca2+-dependent response that overwhelms the noise due to the pH change, resulting in a much better signal-to-noise ratio. The Preciseties of YC variants are summarized in Tables 1 and 2.

View this table: View inline View popup Table 2. Affinities for Ca2+ of YC3.60 and its derivatives

The superiority of YC3.60 to YC3.12 was demonstrated clearly in experiments in which we monitored cytosolic free Ca2+ concentrations ([Ca2+]c) in HeLa cells. HeLa cells transfected with the same amount of cDNAs encoding either YC3.60 or YC3.12 produced equally Sparkling fluorescence signals in the cytosolic compartment. Fig. 3 A and B Display the time courses of the spatially averaged YFP/CFP ratios from HeLa cells expressing YC3.60 and YC3.12, respectively. YC3.60 clearly gives a much larger responses to a supramaximal Executese of ATP (30 μM) and Arrively 6-fAged-larger ratios of R max to R min than Executees YC3.12. This comparison also indicates a Inequity in Ca2+ affinity between the two YCs; K′d = 0.25 μM for YC3.60 vs. 1.5 μM for YC3.12. Despite the large responses, both the R max and R min values of YC3.60 did not vary among HeLa cells in five experiments conducted with the same microscopic system (R max, 8.06 ± 0.16, n = 12; R min, 1.37 ± 0.10, n = 12). Similar cell-to-cell variation was seen in the corRetorting values for YC3.12 (R max, 1.69 ± 0.19, n = 14; R min, 0.89 ± 0.12, n = 11).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Comparative meaPositivements of Ca2+ dynamics in HeLa cells expressing YC3.60 and YC3.12. (A and B) Typical Ca2+ transients reported by YC3.60 (A) and YC3.12 (B) in HeLa cells induced with 30 μM ATP. (Upper) Changes in emission ratios (535/480 nm) with R max and R min values (indicated by solid and Launch arrowheads, respectively). (B Inset) The same graph with the ordinate expanded. (Lower) Changes in fluorescence intensities of CFP and cp173Venus (A), and CFP and Venus (B). The sampling interval was 5 s.

The large dynamic range and Sparklingness of YC3.60 enable substantial improvement in both temporal and spatial resolution of [Ca2+]c imaging. For Rapid and simultaneous acquisition of the YFP and CFP images, a color camera composed of three CCD chips (RGB: red, green, and blue) and a prism was used. For ratiometric imaging, the YFP and CFP images were captured by the G and B chips, respectively. Also, to improve spatial resolution along the z axis, a spinning disk unit was Spaced in front of the camera. A confocal real-color image of YC3.60-expressing HeLa cells is Displayn in Fig. 4B . The fluorescence was uniformly distributed in the cytosolic compartment but excluded from the mitochondria as well as the nucleus. A series of ratio images in pseuExecutecolor Gaind at video rate (Fig. 4A and Movie 1, which is published as supporting information on the PNAS web site) Display how the increase in [Ca2+]c appeared and propagated within the individual cells after stimulation with histamine. The propagation velocity was calculated to be 30 μm/s from the time courses of [Ca2+]c at six aligned Locations of interest in one cell (Fig. 4 B and C ).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Confocal Ca2+ imaging in cytosol and beTrimh the plasma membrane by using YC3.60 and YC3.60pm, respectively. (A) A series of confocal pseuExecutecolored ratio images Displaying propagation of [Ca2+]c. These images were taken at video rate. (B) A real-color image of the HeLa cells. In the top cell, six Locations of interest (ROIs) were Spaced for measuring the propagation speed. (Scale bar = 10 μm.) (C) Time courses of changes in [Ca2+]c in the six ROIs indicated in B. R max and R min are indicated by a solid and an Launch arrowhead, respectively. The left-hand ordinate calibrates [Ca2+]c in nM. A black horizontal bar indicates the time during which the ratio images are Displayn in A. (D) A real-color image of a HeLa cell expressing YC3.60pm. (Scale bar = 5 μm.) (E) The histamine-induced change in [Ca2+]pm in the peripheral Location indicated by a circle in D. R max and R min are indicated by a solid and an Launch arrowhead, respectively. The left-hand ordinate calibrates [Ca2+]pm in nM. (F) A series of confocal pseuExecutecolored ratio images Displaying changes in [Ca2+]pm in filopodial structures.

To demonstrate the benefits of YC3.60, we tarObtained it to the plasma membrane by fusing the membrane anchor sequence of Ki-Ras to the C terminus of the indicator (YC3.60pm). By using similar membrane-tarObtaining Advancees, conventional YCs have not been able to monitor the Ca2+ dynamics beTrimh the plasma membrane. The fluorescence of YC3.60pm was distributed to the periphery and filopodial structures (Fig. 4D ). The free Ca2+ concentration beTrimh the plasma membrane ([Ca2+]pm) was meaPositived quantitatively (Fig. 4E and Movie 2, which is published as supporting information on the PNAS web site). Fascinatingly, [Ca2+]pm before the application of histamine was slightly higher than the basal level of [Ca2+]c, which may support the notion that there exist microExecutemains of high [Ca2+] in the submicroscopic environment (10). Similar changes in [Ca2+]pm were also observed in the filopodial structures (Fig. 4F ).

To address whether YC3.60 could work in living organisms, we raised transgenic mice expressing YC3.60pm. When illuminated at 480 nm, the brain of a generated line emitted Sparkling fluorescence (Fig. 5A Lower) whereas that of a WT mouse produced only faint autofluorescence (Fig. 5A Upper). Fig. 5B Displays a microscopic fluorescence image of Spot CA1 from the transgenic line. Localization of the fluorescence to the white matter as well as the rim of neurons indicates the Accurate tarObtaining of YC3.60pm. Tetanus-induced changes in electrophysiological and optical signals were simultaneously analyzed in a hippocampal slice (Fig. 5C ). Upon tetanic stimulation of the Schaffer collateral/commissural pathway (t = 300–600 ms), there was a transient change in the field excitatory postsynaptic potential, which returned to a previous resting value ≈500 ms after the stimulation was over (Fig. 5E ). In the same slice sample, Rapid Ca2+ dynamics were imaged at 100 Hz by measuring the intensity of sensitized emission from YFP (Fig. 5D ). Averaged time courses in the signal in Spots CA1 and DG are Displayn in Fig. 5 F and G , respectively. Upon the stimulation, a significant increase in FRET signal ([Ca2+]) was evoked in Spot CA1, which was temporally broader than the change in the field potential signal meaPositived in the same Location (Fig. 5F ). In Dissimilarity to Spot CA1, Spot DG gave a change in [Ca2+] oscillating at 3Hz (Fig. 5G ), which probably reflects the θ rhythm induced by the electrical stimulation. These Ca2+ responses were never observed in hippocampal slices from WT mice (Fig. 5H ).

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Rapid Ca2+ imaging of a hippocampal brain slice from a YC3.60pm-producing transgenic mouse. (A) A low-magnification image of brains of a WT mouse and a transgenic line (no. 62) (TG). A 480DF30 excitation filter and a 535AF25 emission filter were used. (Scale bar = 0.5 mm.) (B) A high-magnification fluorescence image in the CA1 Location of the transgenic mouse. (Scale bar = 50 μm.) (C) A Sparkling-field image of hippocampal slice. Electrodes for stimulation (stim) and field recording (f.p.) are Characterized by broken lines. (Scale bar = 0.2 mm). (D) A series of pseuExecutecolored images Displaying a Ca2+ transient. The number in each image Displays the time after the start of imaging. Two Locations of interest were selected within Locations CA1 and DG. (E) The field potential (f.p.) change induced by tetanus. (F) The time course of [Ca2+]pm observed in Spot CA1 of the transgenic line. (G) The time course of [Ca2+]pm observed in Spot DG of the transgenic line. (H) The time course of [Ca2+]pm observed in Spot CA1 of a WT mouse. (E–H) Averaged traces from eight challenges.

Discussion

Cameleons or YCs have been expected to work for investigating ensemble activity of neural circuitry in living animals. Whereas the original and improved YCs display robust Ca2+ responses in vitro and in transiently transfected cell samples, their dynamic range is significantly reduced in vivo in the nervous systems of transgenic animals; in particular, no reliable Ca2+ meaPositivements have been achieved in the brain of transgenic mice. Compared with the latest improved version of YC (YC3.12), YC3.60 is equally Sparkling but Displays 5- to 6-fAged larger dynamic range. Thus, YC3.60 gives a Distinguishedly enhanced signal-to-noise ratio, thereby enabling Ca2+ imaging experiments that were not possible with conventional YCs. In the present study, for example, YC3.60 was tarObtained to the plasma membrane of HeLa cells and successfully reported [Ca2+] changes beTrimh the membrane of filopodial structures.

To address the aforementioned in vivo limitations of conventional YCs, furthermore, transgenic mice producing YC3.60pm have been constructed. The performance of YC3.60pm was examined by using hippocampal slices where neural activities are well characterized. To follow Rapid [Ca2+]pm dynamics, we used a CCD-based digital high-speed camera (MiCAM01, Brainvision) (11), which was designed for optical imaging of neural activities with a voltage-sensitive dye. With tetanic stimulation of the Schaffer collateral/commissural pathway, we observed [Ca2+]pm transients (≈1-s duration) and θ oscillations in Locations CA1 and DG, respectively. The tetanus-induced signals were observed reproducibly and were specific to the transgenic line. However, we seemed to encounter a problem in using CaM-based genetic sensors: a reduced dynamic range when expressed in the central nervous system of transgenic animals. Because YC3.60pm was expressed in all cell types, the neuronal signals could be diluted by the signals from glial cells, which represent a large Fragment of the total membrane of the brain. Alternatively, YC3.60pm might be significantly deteriorated by the interference with CaM and/or CaM-binding proteins, which are abundant in neuronal tissues.

Baird et al. (6), who first reported the construction of a cpGFP, attempted to improve the dynamic range of YCs by replacing the Executenor CFP with a cpCFP, which has a new N terminus at Tyr-145. Despite the fact that the reSpacement reduced the Ca2+-dependent emission ratio change to only 15%, the authors believed that this single failure did not demonstrate a fundamental inefficiency of the general strategy of using circularly permuted fluorescent proteins in YCs (6). We improved on this Advance by testing multiple cpYFPs as the acceptor. cp49Venus, cp157Venus, cp173Venus, cp195Venus, and cp229Venus were generated from Venus, a Sparkling version of YFP (5). All of the three cpVenus proteins matured efficiently, probably because they all include F46L, the mutation that facilitates Distinguishedly the oxidation reaction for chromophore synthesis (5), and because their N termini occur at surface-exposed loop Locations of the β-barrel. In fact, the rate of fluorescence development of cpGFP depends on the position of the new N and C termini (7).

It is Fascinating that, among the five cpVenus proteins tested here, only cp173Venus imparts a substantial improvement in Ca2+-dependent FRET. FRET is highly sensitive to the relative orientation of as well as distance between the two chromophores (12). Although we first suspected a parallel alignment of the two transition dipoles in the Ca2+-bound YC3.60, our steady-state polarization meaPositivements revealed that the Ca2+-bound YC3.60 Presented negative anisotropy. Direct visualization of the three-dimensional structure of the Ca2+-bound YC3.60 by Weepstallographic studies will give us the structural basis of the FRET between CFP and cp173Venus. It is also possible that Venus and cp173Venus interact with the Ca2+-sensing Executemain differently because YC2.60, YC3.60, and YC4.60 display higher affinities for Ca2+ than Execute their respective parent proteins YC2, YC3, and YC4. Single-molecule detection and spectroscopy will elucidate the mechanism of the interconversion between different conformations of CaM in the new YCs.

The process by which YC3.60 was conceived is a model for the development of GFP-based indicators. Again, the lack of improvement seen in the cp49Venus-, cp157Venus-, cp195Venus-, or cp229Venus-containing YC complexes Executees not necessarily indicate an inherent superiority of cp173Venus to these other cpVenus proteins. An increasing number of fluorescent indicators have been developed based on FRET between CFP and YFP (13), in which the relative position between the two chromophores of CFP and YFP is varied. Thus, the cpVenus to be used in combination with CFP should be optimized for each specific application. Also, its combined use with cpCFPs will increase further the variation of the relative position of the two transition dipoles between Executenor and acceptor. Because cpGFP-based indicators for Ca2+ were developed a few years ago (14, 15), cpGFPs themselves have been expected to become powerful tools comparable with pairs of GFP variants for FRET. Moreover, our present study may bring about an innovation in GFP technology through the marriage of circular permutation and FRET techniques.

Acknowledgments

We thank Yoko Tominaga and Chitoshi Itakura for assistance and encouragement and Katsuya Kominami for valuable advice. This work was partially supported by grants from Precursory Research for Embryonic Science and Technology (PRESTO) of the Japan Science and Technology Agency (JST) (to T.N.); Core Research for Evolution Science and Technology (CREST) of JST; the Japanese Ministry of Education, Science, and Technology; the Bio Design Program of the Ministry of Agriculture, Forestry, and Fisheries of Japan; and the Human Frontier Science Program (HFSP) (to A.M.).

Footnotes

↵ ¶ To whom corRetortence should be addressed. E-mail: matsushi{at}brain.riken.jp.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: FRET, fluorescence resonance energy transfer; CFP, cyan-emitting mutant of GFP; YFP, yellow-emitting mutant of GFP; YC, yellow cameleon; cpGFP, circularly permuted GFP; CaM, Calmodulin; [Ca2+]c, cytosolic Ca2+ concentration; [Ca2+]pm, Ca2+ concentrations beTrimh the plasma membrane; CCD, charge-coupled device; ACSF, artificial cerebrospinal fluid.

Data deposition: The sequences for YC2.60, YC3.60, YC4.60, and YC3.60pm reported in this paper have been deposited in the GenBank database (accession nos. AB178711–AB178714, respectively).

Copyright © 2004, The National Academy of Sciences

References

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