Expanded dynamic range of fluorescent indicators for Ca2 by circularly permuted yellow fluorescent proteins Takeharu Nagai*���, Shuichi Yamada���, Takashi Tominaga��, Michinori Ichikawa��, and Atsushi Miyawaki*�� *Laboratory for Cell Function and Dynamics, Advanced Technology Development Group, and ��Laboratory for Brain-Operative Devices, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan ���Structure and Function of Biomolecules, Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Agency (JST), 4-1-8 Hon-cho, Kawaguchi, Saitama 351-0198, Japan and ���Laboratory of Signal Transduction, Institute for Virus Research, Kyoto University, Kawaharacho 53, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan Edited by Roger Y. Tsien, University of California at San Diego, La Jolla, CA, and approved May 19, 2004 (received for review January 29, 2004) Fluorescence resonance energy transfer (FRET) technology has been used to develop genetically encoded fluorescent indicators for vari- ous cellular functions. Although most indicators have cyan- and yellow-emitting fluorescent proteins (CFP and YFP) as FRET donor 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)thatshowedefficientmaturationandacidstability.One of the cpYFPs incorporated in YC absorbs a great amount of excited energy from CFP in its Ca2 -saturated form, thereby increasing the Ca2 -dependent change in the ratio of YFP CFP by nearly 600%. Both inculturedcellsandinthenervoussystemoftransgenicmice,thenew YC enables visualization of subcellular Ca2 dynamics with better spatial and temporal resolution than before. Our study provides an important guide for the development and improvement of indicators using GFP-based FRET. CCa2 ameleons are genetically encoded fluorescent indicators for 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 short- er- to the longer-wavelength variant of GFP. Yellow cameleons (YCs) have cyan and yellow fluorescent proteins (CFP and YFP) as the FRET donor and acceptor, respectively. YCs are classified into several groups based on the composition of their Ca2 - sensing domains. For example, YC2 has an intact CaM and thus shows 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 domains. 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 bright 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 currently, such as YC2.12 or YC3.12, exhibit at most a 120% change in the ratio of YFP CFP upon Ca2 binding in vitro. These YCs do not have good signal-to-noise ratios, particularly when they are targeted 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 endogenous CaM and CaM-binding proteins that may interact with the sensing domains 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 approach that used a circularly permuted GFP (cpGFP), in which the N and C portions 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 portions 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 portions 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 in- frame 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 en- coding 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 domains. For mammalian expression, the cDNAs of YC3.12 and YC3.60 were subcloned into pcDNA3 (Invitrogen). To localize YC3.60 beneath the plasma membrane, the CAAX box of Ki-Ras was This paper was submitted directly (Track II) to the PNAS office. Freely available online through the PNAS open access option. Abbreviations:FRET,fluorescenceresonanceenergytransfer CFP,cyan-emittingmutantof 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 beneath 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). ��To whom correspondence should be addressed. E-mail: matsushi@brain.riken.jp. �� 2004 by The National Academy of Sciences of the USA 10554���10559 PNAS July 20, 2004 vol. 101 no. 29 www.pnas.org cgi doi 10.1073 pnas.0400417101

fused to the C terminus of YC3.60 through a linker sequence (GTGGSGGGTGGSGGGT). For transgenic mice construc- tion, YC3.60pm cDNA was subcloned into the EcoRI site of the pCAGGS expression vector, which contains the -actin pro- moter, cytomegalovirus enhancer, -actin intron, and bovine globin polyadenylation signal (8). A BamHI-SalI fragment con- taining the promoter enhancer and coding sequence was pre- pared 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 tem- perature, purified, and spectroscopically characterized as de- scribed (1). Steady-state fluorescence polarization was measured 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 described (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 bright fluorescence in the brain and was used. Slice Preparation. Hippocampal slices were prepared from 15- day-old F1 animals. The brains were quickly cooled 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 cooling for 5 min, the hippocampus was dissected out along with the sur- rounding cortex and sliced into 400- m-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 place 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 changer (Lambda 10-2, Sutter Instruments, Novato, CA). Interference filters were obtained from Omega Optical (Brattle- boro, VT). Fluorescence emission from YCs was imaged by using a cooled charge-coupled device (CCD) camera (Cool 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 acquired 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 AQUACOSMOS 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 cham- ber. 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 di- chroic mirror, and a 460LP emission filter. The ratio of the fractional change in fluorescence of YC3.60pm to the initial, prestimulation amount of fluorescence ( F F0) was calculated and used as the optical signal. The analyses of the optical signals were done with a procedure developed for Igor Pro (Wave- Metrics, Lake Oswego, OR). A glass microcapillary tube (5 m outer diameter, filled with ACSF) was used as a monopolar stimulating electrode and a recording electrode for field poten- tial 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 Fig. 1. Schematic structures and spectral properties 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) Domain 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 (dotted line) and saturated Ca2 (solid line). Nagai et al. PNAS July 20, 2004 vol. 101 no. 29 10555 BIOCHEMISTRY