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Genetically encoded optical sensors of neuronal activity and cellular function Giovanna Guerrero and Ehud Y Isacoff* Fluorescent proteins (FPs) have been engineered to produce an optical report in response to cellular signals. FP fluorescence can be made directly sensitive to the chemical environment, via specific mutations of or around the chromophore. Alternatively, FPs can be made indirectly sensitive to cellular signals by their fusion to ‘detector’ proteins that respond to specific cellular signals with structural rearrangements that act on the FP to alter fluorescence. These optical sensors of membrane voltage, neurotransmitter release, and intracellular messengers, including powerful new sensors of Ca2+, cyclic nucleotides and nitric oxide, are likely to provide new insights into the workings of cellular signals and of information processing in neural circuits. Addresses *Department of Molecular and Cell Biology, Physical Biosciences Division, Lawrence Berkeley National Laboratory, University of California, Berkeley, 271 LSA, MC#3200, Berkeley, CA 94720-3200, USA; e-mail: [email protected] Current Opinion in Neurobiology 2001, 11:601–607 0959-4388/01/$ — see front matter © 2001 Elsevier Science Ltd. All rights reserved. Abbreviations BFP blue fluorescent protein CFP cyan fluorescent protein cGMP guanine 3′, 5′-cyclic monophosphate CREB cAMP response element binding FP fluorescent protein FRET fluorescence resonance energy transfer GFP green fluorescent protein PKA protein kinase YFP yellow fluorescent protein

Introduction Two proteins, green fluorescent protein (GFP) and Aequorin, of the jellyfish Aequora victoria [1–3] and a set of fluorescent proteins (FPs) from coral [4] have emerged as important tools for biology. FPs have been used to track when and where proteins are produced and targeted [5], and to understand how signaling proteins are re-localized in response to cellular signaling [6–9]. Here we review another biological application, the broad strategy of converting FPs into optical sensors of cellular signaling by engineering them to change fluorescence in response to physiological signals in the manner of the earlier generation of chemical indicator dyes. Aequora victoria produces a natural optical report of a cellular signal when a rise in intracellular Ca2+ evokes a flash of blue-green fluorescence. The fluorescence is triggered when Ca2+ binds to a co-factor associated with Aequorin, converting the energy of chemical bonds into emission of blue light [10]. Close proximity between Aequorin and

another protein, GFP, results in fluorescence resonance energy transfer (FRET) of the excited state from the ‘donor’ Aequorin to the ‘acceptor’ GFP, releasing energy as green fluorescence [11]. The first experimental protein-based optical sensor simply used Aequorin to detect Ca2+ inside cells, similar to what had been done for many years with chemical dyes like fura and flo-3 [12–14]. Aequorin, however, is limited in that fluorescence emission is accompanied by dissociation and breakdown of the Ca2+-binding co-factor, and reloading with fresh co-factor is slow and inefficient, leading to fading of the signal with repeated use. In contrast to Aequorin (emission peak = 469 nm), the longer wavelength GFP (emission peak = 509 nm) is stable during prolonged excitation, but does not naturally change fluorescence in response to cellular signals. The advantages of longer wavelength and increased stability have encouraged efforts to engineer GFP to change fluorescence in response to cellular signals. Two general approaches have been used: mutating GFP to make it directly sensitive to physiological changes in its chemical environment; and fusing an FP to a ‘detector’ protein that endows it with the new functionality of responding to the cellular signals that activate the detector protein. The latter strategy is premised on the idea that a cellular signal that leads to an activation rearrangement of the detector protein can either induce a structural change in the attached FP, thus altering fluorescence, or else can change FRET between an attached donor–acceptor pair of FPs.

Structure and photophysics of GFP To explore how GFP fluorescence can be modulated, we first examine its structure and photophysics [3]. This chromophore is cyclized autocatalytically from amino acids 65–67 of a helix located inside a 42Å × 24Å 11-stranded β barrel (Figure 1a) [15]. The photophysics of GFP depend on the structure of the residues that make the chromophore (S/T65–Y66–G67), on the electronic resonance and proton–transfer interactions between the chromophore and the barrel, and on protons in solution (Figure 1b) [15–18]. The chromophore exists mainly in three protonation states (Figure 1b), each with distinct fluorescence properties: a neutral state (A) that requires high-energy excitation (short wavelength peak), three forms of a deprotonated anionic state (I, B and D) that require lower-energy excitation (longer wavelength peaks), and a zwitterionic state (Z) that requires lower energy excitation (Figure 2) [18–23]. These states inter-convert as the chromophore excites and de-excites, and is protonated and de-protonated. The inter-conversions result in a flickering between fluorescent (A, B and I) and non-fluorescent states (D and Z), with a wide range of dwell times in the

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Figure 1 GFP and its chromophore. (a) The structure of GFP. Amino acids that influence color, temperature stability, sensitivity to pH and halides, are highlighted, as is a site of detector protein insertion near the chromophore. The chromophore is highlighted in green. Mutations that change color to YFP and CFP are highlighted in yellow and cyan, respectively. Two versions of YFP are shown, the second has reduced pH sensitivity as a result of the mutation Q69K. Residues that contribute to pH sensitivity are in red, with those that contribute to native pH sensitivity [29,44,52] indicated by amino acid identity and position (e.g. Q69) and mutations that enhance pH sensitivity indicated by identity of substitution (e.g. H148D). For ecliptic and ratiometric GFPs, it is unclear which of the mutations enhance pH sensitivity. H148 endows YFP with a native Cl– sensitivity that is enhanced by the H148Q mutation, both indicated in purple. Residue Y145, in pink, has been found to allow insertions of detector proteins into GFP that make the fluorescence sensitive to detector rearrangements. Mutations that promote folding at 37ºC are in black [3]. Multiple mutations in a single construct are separated by slashes, mutations of different constructs that individually endow specific properties are separated by commas. (b) Protonation states of the chromophore.

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The chromophore, produced by cyclization and dehydration of residues 65–67, is shown in three protonation states: neutral, zwitterionic

states that de-excite either directly (I*→I; B*→B) or indirectly (A* de-exciting to I via I*), and dark states (D* and Z*) that de-excite without fluorescence (Figure 2) [21–23]. The time spent in these states varies from the subnanosecond to the second time-scale [16,22,24,25]. If structural changes were induced in GFP by a signal, the rates of proton exchange and the change in the balance of occupancy of its different fluorescent states might be altered. This could change fluorescence in color (e.g. an increased de-excitation via path A1 in Figure 2 would produce a higher energy emission and thus bluer fluorescence) and brightness (e.g. accumulation of molecules into the dark states D* or Z* in Figure 2 would reduce fluorescence). Additional changes in fluorescence could occur if a structural rearrangement of GFP changed the way that the chromophore packs against residues of the barrel, such as πstacking of phenylalanine or tyrosine at position 203 with chromophore residue Y66 [17]. This could affect fluorescence color in a proton-independent manner via a change in resonance, or in rates of inter-conversion of B* ↔ I* [17,23]. Brightness may also change due to alterations in the rate of transition from B* to the D* dark state [22].

Direct sensitivity of GFP to bulk ionic environment The dependence of GFP fluorescence on protonation makes it directly sensitive to pH. Several groups have capitalized on this and made mutants of GFP with

and anionic — where the negative charge can resonate between the tyrosine hydroxyl group and the imidazole ketone.

heightened sensitivity to pH [3,26–29]. The yellow fluorescing mutant of GFP, yellow fluorescent protein (YFP) has also been made sensitive to halides [30,31] by introducing the mutation H148Q, which places a glutamine residue near the chromophore (see Figure 1a). Glutamine favors deprotonation of the chromophore. Cl– association from the bulk solution, which might be aided by a larger cavity size in H148Q, favors protonation and reduces fluorescence [31]. Kuner and Augustine [32••] took advantage of the quenching of regular YFP by Cl– to make a ratiometric sensor in which FRET from a cyan fluorescent protein (CFP) donor to a YFP acceptor is reduced when Cl– quenches YFP. GFPs that act as sensors of pH and Cl– can be turned into sensors of cell signals. For example, by fusing pH-sensitive GFPs to the lumenal domain of the synaptic vesicle membrane protein vesicle associated membrane protein (VAMP; Figure 3), Miesenböck et al. [28] made ‘synaptopHluorins’. These report synaptic neurotransmitter secretion by detecting fusion between synaptic vesicles and the plasma membrane, an event which abruptly exposes the GFP in the interior of the vesicle (where pH is ~5) to the outside of the cell (where pH is neutral). One of the synapto-pHluorins increases in brightness (‘ecliptic’) and the other shifts to longer wavelength excitation (‘ratiometric’) in the higher pH, so fusion is reported as either an increase in fluorescence intensity or as a ratiometric change in excitation spectrum.

Genetically encoded optical sensors of neuronal activity and cellular function Guerrero and Isacoff

Converting the activity of a signaling protein into a change in the intensity of protein fluorescence

The first of these approaches was used to make the membrane voltage sensor ‘FlaSh’. Insertion of GFP near a gating domain of the Shaker voltage-gated potassium channel generated a sensor in which voltage-driven rearrangements of the channel alter the brightness of GFP [33]. To maximize transmission of the channel’s structural rearrangement to GFP, a portion of the the unstructured C terminus of GFP, which may act as a floppy linker to the channel, was removed. In an alternative approach, the detector was inserted into GFP. GFP was found to fold and fluoresce when circularly permutated — linking its native N and C terminals together and opening up sites elsewhere to become new termini [34,35] — suggesting that it may tolerate the insertion of a detector at the new terminal sites. Baird et al. [35] made the Ca2+ sensor ‘Camgaroo’ by inserting calmodulin into one of these sites (Y145). The resulting GFP, with normal N- and C-termini, produces changes of up to 40% in its emission intensity, upon increases of Ca2+. Nagai et al. [36••] used a circularly permuted YFP and attached the detectors calmodulin and M13 to the new N and C termini to make ‘Pericam’ Ca2+ sensors. Changes in Pericam fluorescence intensity in response to Ca2+ are as large as 180%. Motions of the ion channel gate in FlaSh, of calmodulin in Camgaroo, and association between calmodulin and M13 in Pericams, ‘pull’ in some way on the barrel of GFP leading to a change in fluorescence.

Converting the activity of a signaling protein into a change in FRET between a pair of FPs Another alternative sensor design strategy has been to convert the structural change of a detector protein into a change in the FRET interaction between pairs of GFPs of different color. The first FRET sensors used color-shifted mutants of GFP that paired a blue fluorescent protein (BFP) ‘donor’ with a GFP ‘acceptor,’ or a longer wavelength pair consisting of a CFP donor and YFP acceptor [37]. More recently, a Sapphire GFP donor has been paired with a DsRed acceptor from coral [38••]. If the maturation and oligomerization problems of DsRed could be solved, the new FRET pair would provide an alternative to CFP/YFP that has the dual advantages of lower auto-fluorescence (due to the longer wavelength) and a very low pH-sensitivity.

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A second approach to GFP sensors has been to fuse GFP to a ‘detector’ protein that responds to a cellular signal with a structural rearrangement that, in turn, forces a rearrangement of GFP and changes its fluorescence properties. Because the amino (N) and carboxyl (C) termini of GFP are close to one another at one end of the barrel (Figure 1), GFP can be inserted into a detector protein without seriously disrupting its function. Alternatively, it can be inserted between two interacting detector proteins without preventing association (Figure 3).

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Model of de-excitation and inter-conversions between states (modified with permission from Weber et al. [21] and Schwille et al. [22]). The y-axis represents relative energy levels. Some proton transfers are fast in the excited state (*), and some are fast in the resting state (thicker arrows represent faster rates). The anionic states I and B (see Figure 1b) are the main fluorescent states. The neutral A* state tends to de-excite via inter-conversion, deprotonating to I* and then deexciting to I (from which it quickly re-protonates and returns to A). Because of this, it emits a photon of about the same energy as does B*, which usually de-excites directly to B. As a result, in a population of molecules divided between stable resting states A and B, high energy light will excite molecules in the A state and lower energy light will excite molecules in the B state, but both molecules will emit green light. B* also inter-converts to the zwitterionic Z* and anionic D* nonfluorescent (dark) states, which de-excite via non-radiative pathways.

Two general strategies exist for using FRET interactions between FPs in the detection of cellular signals. The first is to link together the donor–acceptor pairs via a detector protein that undergoes a structural change (Figure 3). In this way the ‘Cameleon’ FRET sensors were made for Ca2+, employing calmodulin and M13 as two detector components that associate when calmodulin binds Ca+ [37,38••]. Subsequently, other sensors have been made for nitric oxide using metallothionein as the detector [39], for cAMP, using cAMP response element binding (CREB) protein as a detector of cAMP dependent phosophorylation by protein kinase A (PKA) [40••], and for cyclic cGMP, using cGMP dependent protein kinase as the detector [41••]. The second design strategy is to fuse the donor and acceptor FPs to separate proteins whose interaction changes in response to the signal (Figure 3). In this way a FRET sensor was made for cAMP, by fusing one FP to the catalytic subunit of PKA and the other to its regulatory domain, so that binding of cAMP leads to dissociation of the subunits and a drop in FRET [42••]. A second FRET sensor in this class monitors G-protein coupled receptor activation by labeling the Gα and Gβ subunits with the different color GFPs and observing a drop in FRET upon receptor-driven dissociation of these subunits [43••].

Improving and tuning FP sensors The intrinsic pH sensitivity and the temperature lability of GFP have posed problems for sensors. To prevent changes in pH from triggering optical reports or interfering with a

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Figure 3 Four classes of FP-based reporters of cellular signals. The reporters make use of the sensitivity of FPs (a) to the bulk ionic environment, (b,c) to protein interactions between the chromophore and amino acids of the FP barrel, and (d) to FRET interactions with other FPs. (a) FPs sensitized by mutations to the ionic environment (such as pH or Cl–) can be targeted to specific subcellular compartments by fusing them to targeting proteins, as shown here for synaptopHluorin, a fusion of ecliptic or ratiometric GFP to the lumenal end of the synaptic vesicle protein VAMP. These sensors can measure the pH inside the acidic vesicle, and they detect vesicle fusion and transmitter release by the effect on GFP fluorescence of an abrupt transition from the inside of the vesicle to the near neutral extracellular solution. (b) Insertion fluorescence sensitivity. FlaSh is an insertion of GFP into each of the four identical subunits of the voltagedependent Shaker K+ channel, ~20 residues away from the S6 gating structure at the internal mouth of the pore. The construct is depicted in a diagonal cross-section through two subunits. The S6 FlaSh linker threads away from the internal mouth of the channel between linkers on neighboring subunits that connect the tetrameric T1 cytoplasmic assembly domain with S1. Voltage-driven rearrangements of the channel evoke changes in the brightness of GFP, yielding a sensor of membrane potential. (c) Insertion of detectors into GFP also confers fluorescence sensitivity to detector activity. GFP tolerates insertion of a detector protein into the GFP barrel at site Y145. The closeness of Y145 to the chromophore (Figure 1a) heightens fluorescence sensitivity to rearrangements in the detector. Insertion of the Ca2+ binding protein calmodulin (shown as two Ca2+ binding lobes connected by a coil) at GFP

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site Y145 yields the Ca2+ sensor Camgaroo (top). GFP can also be circularly permuted (bottom) by linking the native N and C termini and opening up the structure elsewhere, such as Y145, to make new N and C termini near the chromophore. The Pericam Ca2+ sensors attach calmodulin and M13 (shown as a gray rectangle) to the new N and C termini. A Ca2+-induced rearrangement of calmodulin in Camgaroo, or binding of calmodulin to M13 in Pericams acts on the GFP barrel to change fluorescence brightness or color. (d) FRET

signal-induced optical report, mutants of GFP have been made which have a reduced pH sensitivity, such as the Q69M mutant of YFP [44] and Sapphire GFP [38••] (see Update). Moreover, the coral FP DsRed has been found to be naturally insensitive to pH [38••]. To overcome temperature lability, several mutations have been identified that increase the efficiency of folding at 37°C [3], and although some circularly permuted GFP sensors do not fold efficiently at 37°C, variants have been constructed which do fold well [36••,45]. The use of the coral FPs may also help improve some reporters. The structure and photophysics of DsRed reveal remarkable similarity to GFP (Figure 4), including a β-can fold, a chromophore composed of tyrosine and glycine, and several key chromophore-interacting residues in the barrel [46–48]. The homology suggests that DsRed may not only be suitable for FRET, but, like GFP, may also provide an optical readout of structural distortion induced by activation of an

between donor–acceptor pairs of FPs can be used to report altered detector structure or association of two detector moeities upon detector activation. In the Ca2+ sensor Cameleon binding of calmodulin to M13 in high Ca2+ increases FRET between a CFP donor and YFP acceptor (top). Disassociation of the PKA catalytic and regulatory subunits upon cAMP binding decreases FRET between CFP on one subunit and YFP on the other, yielding a sensor of cAMP (bottom).

attached detector protein. Unfortunately, while attractive for its long wavelength (emission peak = 583 nm), photostability and natural pH insensitivity, DsRed tetramerizes [45,48] and aggregates into large clumps [38••]. However, recent alterations to DsRed are promising, since one case of fusion to a detector protein and one DsRed mutation have already been found to reduce aggregation [38••] (Clontech Living Colors: DsRed2, 2001 — see http://www.clontech.com/products/ literature/pdf/brochures/DsRed2.pdf). Moreover, the tetramerization interface of DsRed is known from the crystal structure, paving the way for mutations that will disrupt interaction and lead to stable monomers. One of the attractions of FP sensors is that both detectors and FPs can be tuned rationally. Mutations of the Ca2+ binding sites of the calmodulin detector of a Cameleon have been used to make lower affinity Ca2+ sensors [37]. Mutations known to alter the voltage dependence and gating kinetics of the ion channel detector of FlaSh have also

Genetically encoded optical sensors of neuronal activity and cellular function Guerrero and Isacoff

been used to make voltage sensors with an altered dynamic range and rates of response (G Guerrero et al., unpublished data). In addition, the crystal structure of GFP has been used to rationally guide mutagenesis of GFP barrel residues that interact with the chromophore, thereby altering fluorescence properties [17].

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FP sensors in biological systems We are still in the early days of FP sensor engineering and so far they have been used in just a handful of applications. One of the most successful FP sensors to date has been Cameleon. It was one of the first genetically encoded fluorescent reporters of cellular function and has the advantage of providing ratiometric measurements based on FRET. Cameleon has been expressed in different cell types through the use of cell-specific promoters, and targeted to specific cellular compartments by the addition of targeting peptides [49]. In this way it has been used to observe and quantify Ca2+ oscillations in the endoplasmic reticulum [37,50,51], nucleus [37], plant stomatal guard cells [52], mammalian cardiac myocytes [53], and secretory granule membranes [54]. Ca2+ oscillations have also been analyzed in neurons and muscle of Caenorhabditis elegans [55]. A shortcoming of Cameleon is that its rates of binding and unbinding Ca2+ (particularly its unbinding rate) are slow. As a result of the slow kinetics, optical responses diminish significantly in size at signal rates greater than 3 Hz [56]. A second shortcoming of the original Cameleon — the sensitivity of YFP to pH — can now be circumvented by the replacement of CFP-YFP with Sapphire GFP-DsRed, which are much less sensitive to pH. This new type of Cameleon has been used in dissociated hippocampal neurons [38••]. Intracellular signals other than Ca2+ have also been observed in neurons through the use of FP sensors. cGMP has been measured in Purkinje neurons [41], and Cl– has been visualized in hippocampal neurons [32••].

Conclusions We have described the design principles of a new class of optical reporters of cellular signaling. By basing these sensors on proteins, it should be possible to harness the sensitivity and specificity of biological systems, to rationally tune the sensors, and to expand detection of almost any cellular signaling event. These optical reporters should particularly benefit studies in preparations where electrophysiological or microinjection approaches are technically challenging, and where cell loading of dyes by bath or localized superfusion give the problem of decreasing signal to noise ratios. These sensors can be targeted to specific cell types, via the use of promoters, and to precise subcellular compartments, by fusion to targeted peptides. This should enable the exclusion of signals from nonneuronal cells and the selective visualization of the activity of molecular signals in subcellular elements, such as nerve terminals or dendrites, of particular neuronal populations, thus adding enormously to the capability of efforts to image neural activity.

Current Opinion in Neurobiology

Structures of GFP and DsRed monomers ‘cut-out’ to show internal chromophore. The chromophores of both structures are depicted in black. N and C termini are labeled in gray. Although the two proteins show only 22% sequence identity, the topology of DsRed is very similar to that of GFP. Both are 11-stranded β-cans with a central α-helix, on which lies an autocatalytically created chromophore. The conjugated π-system of the chromophore is extended in DsRed, which probably accounts for its longer absorbance (max = 558 nm) and emission (max = 583 nm). Unlike GFP, which exists mostly as a monomer, DsRed is found as a tetramer in solution. This occurs through two conserved protein interfaces along the β-can, a typical hydrophobic cluster of residues and a polar dimer interface that might be involved in hetero-oligomerization with other DsRed-like proteins.

Update A description of ‘Citrine’, a new pH-insensitive YFP, has recently appeared [57••]. YFP’s pKa is lowered to 5.7 with the introduction of mutation Q69M. This mutation also confers insensitivity to chloride and a greater photostability (Citrine photobleaches at half the rate of YFP), but maintains quantum yield (0.76 for Citrine versus 0.71 for YFP). Replacement of YFP by Citrine improves the brightness and folding of Camgaroo at 37°C and shifts the pH sensitivity of Cameleon.

Acknowledgements We are grateful to Camin Dean, Arnd Pralle and Alois Sonnleitner for their helpful comments on the manuscript.

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Llopis J, McCaffery JM, Miyawaki A, Farquhar MG, Tsien RY: Measurement of cytosolic, mitochondrial, and Golgi pH in single living cells with green fluorescent proteins. Proc Natl Acad Sci USA 1998, 95:6803-6808.

28. Miesenböck G, De Angelis DA, Rothman JE: Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 1998, 394:192-195. 29. Elsliger MA, Wachter RM, Hanson GT, Kallio K, Remington SJ: Structural and spectral response of green fluorescent protein variants to changes in pH. Biochemistry 1999, 38:5296-5301. 30. Jayaraman S, Haggie P, Wachter RM, Remington SJ, Verkman AS: Mechanism and cellular applications of a green fluorescent protein-based halide sensor. J Biol Chem 2000, 275:6047-6050. 31. Wachter RM, Yarbrough D, Kallio K, Remington SJ: Crystallographic and energetic analysis of binding of selected anions to the yellow variants of green fluorescent protein. J Mol Biol 2000, 301:157-171. 32. Kuner T, Augustine GJ: A genetically encoded ratiometric indicator •• for chloride: capturing chloride transients in cultured hippocampal neurons. Neuron 2000, 27:447-459. First report of a novel FRET-based halide reporter that consists of a CFP and a YFP fused in tandem, separated by a flexible linker. The naturally halidesensitive YFP is quenched by Cl–, decreasing the YFP/CFP ratio of emission (although in a somewhat slow manner). This new halide reporter has noticeable advantages (being ratiometric, genetically-encoded, and non-toxic) over existing chloride indicators. 33. Siegel MS, Isacoff EY: A genetically encoded optical probe of membrane voltage. Neuron 1997, 19:735-741. 34. Topell S, Hennecke J, Glockshuber R: Circularly permuted variants of the green fluorescent protein. FEBS Lett 1999, 457:283-289. 35. Baird GS, Zacharias DA, Tsien RY: Circular permutation and receptor insertion within green fluorescent proteins. Proc Natl Acad Sci USA 1999, 96:11241-11246. 36. Nagai T, Sawano A, Park ES, Miyawaki A: Circularly permuted green •• fluorescent proteins engineered to sense Ca2+. Proc Natl Acad Sci USA 2001, 98:3197-3202. The proximity of Y145 to the chromophore of GFP suggests that opening GFP at this site to make new N- and C-termini would make the chromophore more accessible to outside protons and more susceptible to structural rearrangements in detectors attached to the termini. Here, attachment of calmodulin and M13 to the new termini yields a change in fluorescence intensity, in response to Ca2+, of as much as 180% in one construct, and another construct, a ratiometric reporter, gives a ratio change of 10-fold. 37.

Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY: Fluorescent indicators for Ca2+ based on green fluorescent proteins and calmodulin. Nature 1997, 388:882-887.

38. Mizuno H, Sawano A, Eli P, Hama H, Miyawaki A: Red fluorescent •• protein from Discosoma as a fusion tag and a partner for fluorescence resonance energy transfer. Biochemistry 2001, 40:2502-2510. Cameleons were made with pH-resistant DsRed as the FRET acceptor, and either CFP, YFP, or Sapphire GFP as the FRET donor. DsRed maturation problems were circumvented by incubation of cells at 37ºC. Out of all the donors tested, Sapphire GFP was the most resistant to pH, and had the longest Stoke’s shift (distance between excitation and emission maxima), its pairing with DsRed thus exhibited minimal cross-excitation. Red-shifted Cameleons show promise particularly when pH oscillations are a concern. 39. Pearce LL, Gandley RE, Han W, Wasserloos K, Stitt M, Kanai AJ, McLaughlin MK, Pitt BR, Levitan ES: Role of metallothionein in nitric oxide signaling as revealed by a green fluorescent fusion protein. Proc Natl Acad Sci USA 2000, 97:477-482. 40. Nagai Y, Miyazaki M, Aoki R, Zama T, Inouye S, Hirose K, Iino M, •• Hagiwara M: A fluorescent indicator for visualizing cAMP-induced phosphorylation in vivo. Nat Biotechnol 2000, 18:313-316. This recently engineered reporter uses BFP and GFP separated by the kinase-inducible domain of CREB. Phosphorylation of CREB by PKA decreases FRET between the donor and acceptor FPs. Substitution of the FRET pair used in this paper, with less light-sensitive FPs might make this a more viable option for studying cAMP fluctuations in neurons.

Genetically encoded optical sensors of neuronal activity and cellular function Guerrero and Isacoff

41. Honda A, Adams SR, Sawyer CL, Lev-Ram VV, Tsien RY, •• Dostmann WR: Spatiotemporal dynamics of guanosine 3′′, 5′′-cyclic monophosphate revealed by a genetically encoded, fluorescent indicator. Proc Natl Acad Sci USA 2001, 98:2437-2442. Once again, the paradigm of flanking a detector domain (cGMP-dependent kinase) with FRET donor and acceptor FPs (CFP and YFP) to produce an optical sensor of a cytosolic signal (cGMP) is successful, producing a decrease in FRET upon increased cGMP levels.

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48. Yarbrough D, Wachter RM, Kallio K, Matz MV, Remington SJ: Refined crystal structure of DsRed, a red fluorescent protein from coral, at 2.0-Å resolution. Proc Natl Acad Sci USA 2001, 98:462-467. 49. Pinton P, Ferrari D, Magalhaes P, Schulze-Osthoff K, Di Virgilio F, Pozzan T, Rizzuto R: Reduced loading of intracellular Ca2+ stores and downregulation of capacitative Ca2+ influx in Bcl-2overexpressing cells. J Cell Biol 2000, 148:857-862.

42. Zaccolo M, De Giorgi F, Cho CY, Feng L, Knapp T, Negulescu PA, •• Taylor SS, Tsien RY, Pozzan T: A genetically encoded, fluorescent indicator for cyclic AMP in living cells. Nat Cell Biol 2000, 2:25-29. Another FRET-based cAMP reporter, this one employs PKA as the detector moeity. In contrast to the previously reviewed study [41••], the researchers here label separate peptides (the catalytic and regulatory subunits of PKA) with BFP and GFP. Thus, this indicator uses changes in protein association rather than changes in conformation as the signal to be followed by FRET. In addition to using the easily bleached BFP, this reporter design has the caveat of being able to interact with endogenous PKA substrates, possibly changing the physiology of overexpressing cells.

50. Fan GY, Fujisaki H, Miyawaki A, Tsay RK, Tsien RY, Ellisman MH: Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 1999, 76:2412-2420.

43. Janetopoulos C, Jin T, Devreotes P: Receptor-mediated activation of •• heterotrimeric G-proteins in living cells. Science 2001, 291:2408-2411. The authors tagged Gα and Gβ of Dictyostelium discoideum with CFP and YFP and visualized G-protein coupled receptor activation as a decrease in FRET upon disassociation of the G-protein heterotrimer. In terms of general applicability, one problem with the labeling of individual G-protein subunits is that there are numerous Gαs, Gβs, and Gγs, and many more different combinations of G-protein trimers. As different receptors use different sets of G-proteins to transduce ligand binding, one set of G-protein reporters might not be able to report on the activation of all G-protein coupled receptors.

52. Allen GJ, Kwak JM, Chu SP, Llopis J, Tsien RY, Harper JF, Schroeder JI: Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells. Plant J 1999, 19:735-747.

44. Miyawaki A, Griesbeck O, Heim R, Tsien RY: Dynamic and quantitative Ca2+ measurements using improved cameleons. Proc Natl Acad Sci USA 1999, 96:2135-2140. 45. Baird GS, Zacharias DA, Tsien RY: Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 2000, 97:11984-11989. 46. Gross LA, Baird GS, Hoffman RC, Baldridge KK, Tsien RY: The structure of the chromophore within DsRed, a red fluorescent protein from coral. Proc Natl Acad Sci USA 2000, 97:11990-11995. 47.

Heikal AA, Hess ST, Baird GS, Tsien RY, Webb WW: Molecular spectroscopy and dynamics of intrinsically fluorescent proteins: coral red (dsRed) and yellow (Citrine). Proc Natl Acad Sci USA 2000, 97:11996-12001.

50. Yu R, Hinkle PM: Rapid turnover of calcium in the endoplasmic reticulum during signaling. J Biol Chem 2000, 275:23648-23653. 51. Foyouzi-Youssefi R, Arnaudeau S, Borner C, Kelley WL, Tschopp J, Lew DP, Demaurex N, Krause K-H: Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum. Proc Natl Acad Sci USA 2000, 97:5723-5728.

53. Fan GY, Fujisaki H, Miyawaki A, Tsay RK, Tsien RY, Ellisman MH: Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 1999, 76:2412-2420. 54. Emmanoulidou E, Teschemacher AG, Pouli AE, Nicholls LI, Seward EP, Rutter GA: Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon. Curr Biol 1999, 9:915-918. 55. Kerr R, Lev-Ram V, Baird G, Vincent P, Tsien RY, Schafer WR: Optical imaging of calcium transients in neurons and pharyngeal muscle of C. elegans. Neuron 2000, 26:583-594. 56. Brejc K, Sixma TK, Kitts PA, Kain SR, Tsien RY, Ormo M, Remington SJ: Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein. Proc Natl Acad Sci USA 1997, 94:2306-2311. 57. ••

Griesbeck O, Baird GS, Campbell RE, Zacharias DA, Tsien RY: Reducing the environmental sensitivity of yellow fluorescent protein. J Biol Chem 2001, 276:29188-29194. Description of 'Citrine', a new YFP variant.