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the concentration of the free Ca2+ is highly INNOVATION heterogeneous within cells  it ranges from micromolar to nanomolar concentrations in the endoplasmic reticulum (ER) and resting Looking forward to seeing calcium cytoplasm, respectively1. Moreover, the changes in the cytoplasmic and organellar Ca2+ concentrations that are induced by cell Rüdiger Rudolf, Marco Mongillo, Rosario Rizzuto and Tullio Pozzan stimulation occur with a defined spatial and/or temporal pattern, which is pivotal in From molecules, single cells and tissues to For the past five decades, the study of the determining the final functional outcome2.As whole organisms, our insights into Ca2+ dynamics of intracellular Ca2+ concentra- a result of this biological complexity, it is 2+ signalling and the corresponding physiological tion, [Ca ]i, has been a main focus of life therefore not surprising that our understand- phenomena are growing exponentially. Here, sciences research. More important than ing of Ca2+ signalling has been largely depen- we describe the improvements that have for other signalling molecules, the study of dent on the development of methodologies been made in the development of the probes the role of Ca2+ in cell physiopathology that can be used to monitor Ca2+ concentra- and instrumentation that are used for Ca2+ requires the ability to monitor the dynam- tion in living cells. imaging and the expanding applications of ics of its concentration in living cells with Here, we describe the development of Ca2+ imaging in basic and applied research. both spatial and temporal accuracy. In fact, the various Ca2+ probes and the scientific

Box 1 | Ca2+ sensors a R–Rmin Sf2 Ratiometric dyes 2+ × × [Ca ] = Kd The excitation (or emission) spectrum (see figure, part a) of ratiometric dyes, such as fura-2 and Rmax–R Sb2 indo-1, changes according to the free Ca2+ concentration, [Ca2+]. The Ca2+ concentration is High Ca2+ measured as the ratio between two intensity values that are taken at two wavelengths, Low Ca2+ λ1 and λ2. Ratiometric dyes correct for unequal dye loading, bleaching and focal-plane shift, for example, as the ratio does not depend on the absolute intensity of the two signals. This can be illustrated by a simple example: if there are two cells, A and B, that have the same intracellular Ca2+ 2+ concentration, [Ca ]i, but different concentrations of the dye, the ratio between the two 2+ wavelengths will immediately reveal that their [Ca ]i is identical, whereas a complex calibration procedure would be necessary to obtain the same information with an indicator that only changes its intensity as a function of Ca2+ concentration. Fluorescence intensity (AU) Fluorescence Non-ratiometric dyes Ca2+ concentration is determined solely by a relative increase in the fluorescence intensity (see figure, λ (nm) part b) of non-ratiometric dyes, such as fluo dyes and rhod dyes, on elevation of the free Ca2+ λ1 λ2 concentration. The single excitation allows for simpler instrumentation or simultaneous observation b F–Fmin 2+ × of other parameters. These dyes mainly work in the visible range. [Ca ] = Kd Fmax–F

It should be noted that dyes are available with a range of Kd values and spectral properties that make them suitable for use at common laser lines, and some of the single-wavelength indicators give High Ca2+ extremely strong fluorescence changes on Ca2+ binding. It is also important to note that relative, Low Ca2+ approximate, and not absolute, Ca2+ concentration values are usually all that is measured. Measuring the Fmin/Fmax or Rmin/Rmax values in cells or tissues requires the exposure of the dye within intact cells to a known Ca2+ concentration by harsh biochemical methods, such as high doses of ionophores, that are often incompatible with cell survival. This is an even bigger problem with probes targeted to organelles. AU, arbitrary units; F, fluorescence intensity; Fmax, F at saturating 2+ 2+ Ca concentration; Fmin, F at zero Ca concentration; Kd, dissociation constant; R, ratio between two wavelengths; Rmax, R at saturating Ca2+ concentration; Rmin, R at zero Ca2+ concentration; intensity (AU) Fluorescence Sb2, value at saturating Ca2+ concentration for wavelength two; Sf2, value at zero Ca2+ concentration for wavelength two. λ (nm)

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a Dye loading Measuring Ca2+ concentration It was only in the late 1970s that Roger Lipophilic Ca2+ probes (also known as indicators, Tsien synthesized the first, rationally AM ester 2+ Microinjection reporters or sensors) are molecules that can designed, fluorescent Ca probes for intracel- not only form selective and reversible com- lular use6. These fluorescent polycarboxylate plexes with Ca2+ , but, uniquely, the dyes are based on a very simple design, as they physicochemical characteristics of the free are derivatives of the best known selective and bound form are sufficiently different to Ca2+-chelator available, EGTA. In BAPTA, the enable their relative concentrations to be prototype fluorescent polycarboxylate dye, measured. Most commonly, the differences in the two methylene groups of EGTA have been Trapped compound the absorbance and/or emission of light are replaced by two benzene rings to enable it to used (BOX 1), but other properties, such as the function as a chromophore. The conforma- b Calibration and use nuclear magnetic spectrum4, have also been tional change caused by Ca2+ binding to the High used to this end. So, similar to a pH indicator, carboxyl groups is transmitted to the chro- the concentration of free Ca2+ is not mea- mophore and results in changes in the excita- sured directly, but instead the indicator mon- tion and/or emission properties of the dye. itors the amounts of free and complexed Despite being largely different in terms of probe. The concentration of free Ca2+ is then their spectral properties and Ca2+-binding calculated on the basis of the effective disso- affinities, all of the polycarboxylate dyes now 2+ ciation constant (Kd) of the probe for Ca in available are derived from this original design. the specific environment (BOX 1). Another BAPTA itself could not be used as an intracel- basic concept is that all Ca2+ indicators are lular indicator because it absorbs light in the Ca2+ buffers and so, unavoidably, the mea- far ultraviolet (UV) spectrum, but its deriva- Fluorescence intensity/ratio Fluorescence surement of the Ca2+ concentration with tive quin2 became immediately popular as an indicators leads to an increase in the Ca2+ intracellular indicator, despite being far from Low buffering capacity. ideal in terms of its selectivity and fluores- 7 2+ Ca2+ concentration cence intensity . The polycarboxylate Ca Evolution of synthetic Ca2+ indicators indicators became such popular probes owing 2+ Figure 1 | Procedure of a typical Ca In the 1960s and 1970s, the spectral proper- to the synthesis of their acetoxymethyl esters8, measurement experiment. a | The polycarboxylate Ca2+ probe is introduced into ties of the organic coloured compounds which allowed the trapping of these indica- the cells, either by incubation of the lipophilic murexide, the azo dyes and chlortetracycline, tors in living cells (FIG. 1). Rather than the acetoxymethyl (AM) esters (intracellular cleavage were serendipitously found to change on time-consuming and complex microinjection by cellular esterases releases the original binding of Ca2+ (REF. 5). Whereas murexide technique (FIG. 1), the ester-loading technique hydrophilic compound) or forced introduction of has been mainly used to monitor the ability is simple, requires no specific expertise and is the hydrophilic compound (for example, by of isolated organelles (such as mitochondria highly effective in virtually all cell types. The microinjection or through a patch pipette). Other methods (for example, using liposomes or or the sarcoplasmic reticulum) to take up flexibility of the chemical design (which 2+ electroporation) have rarely been used, whereas Ca from the medium, the azo dyes have enabled the synthesis of many new and better genetically encoded probes can be introduced been extensively used to investigate Ca2+ sig- dyes within a few years), the efficacy and by standard transfection, transgene techniques nalling in skeletal muscle and giant axons. simplicity of the loading technique and the or microinjection of the purified protein. The main limitations of azo dyes are the minimal side effects have made these dyes 2+ b | To quantify Ca concentration, the complex and variable Ca2+–dye stoichiome- invaluable for many researchers. dissociation constant (K ) of the probe should d tries, the small signal-to-noise ratio and the ideally be determined both in situ and in the need for the dyes to be introduced into cells Protein-based Ca2+ indicators specific cell used. However, the Kd from the literature is most often used. To transform the by technically demanding methods, such as In the first century AD, the Roman scientist fluorescent data into Ca2+ concentration values, microinjection or intracellular perfusion, and historian Plinius Secundus described it is necessary to measure the fluorescence at that are mainly applicable to large and medusae, which contract on contact and very low and at saturating Ca2+ concentrations robust cells. simultaneously emit a bright green light; with the instrumentation and the experimental By contrast, chlortetracycline shows “urticae noctu vagantus locumque mutant … setting used in the experiment ( ). For BOX 1 2+ further details, see the Introduction to Ca2+ large increases in fluorescence on Ca cum admoveri sibi manum sentit, colorem 2+ 9 measurement with fluorescent indicators web binding. Changes in the Ca concentration mutat et contrahitur” (“in the night medusae site (see Online Links). that are monitored by chlortetracycline have often been referred to as representing 2+ changes in membrane Ca concentration; “In AD 79 the volcano concepts that form the basis of this sophis- however, chlortetracycline might measure ticated measurement toolkit, which has no the Ca2+ concentration close to membranes, Mount Vesuvius entombed equivalent counterpart in other biological but not within membranes. Chlortetracycline bioluminescence research fields. These advances are not only pushing is also sensitive to several other parameters, research in Ca2+ signalling forward, but are including Mg2+ concentration, membrane under metres of ash, also stimulating neighbouring fields (for potential and pH, and the advances in the together with Plinius who example, the development of new probes understanding of Ca2+ signalling resulting for measuring protein phosphorylation or from the use of chlortetracycline have been died in Pompeii during the cyclic nucleotides3). limited. eruption.”

580 | JULY 2003 | VOLUME 4 www.nature.com/reviews/molcellbio PERSPECTIVES float and change their position … when it Box 2 | Protein-based Ca2+ sensors realizes that a hand is approaching, it changes its colour and contracts”). In AD 79 A Photoproteins the volcano Mount Vesuvius entombed bio- luminescence research under metres of ash, together with Plinius who died in Pompeii during the eruption. However, in the 1960s the phenomenon of bioluminescence was reinvestigated in the medusa Aequorea Ca2+ Ca2+ victoria. These studies identified two pro- Ca2+ teins — aequorin (AEQ)10 and green fluo- Ca2+ rescent protein (GFP)11, both of which have impacted greatly on the life sciences and are central to this article. B GFP-based probes Aequorin. Although several cnidarian a chemiluminescent proteins, which include CFP mnemiopsin and obelin, emit photons on CFP YFP 2+ Ca 2+ YFP Ca2+ binding12, there is no doubt that the CaM M13 Ca 10 Ca2+ Ca2+ AEQ photoprotein (BOX 2) has dominated Ca2+ the Ca2+-signalling field. For almost 30 years, the protein had to be painstakingly extracted from the jellyfish, carefully purified to pre- b 2+ vent contact with Ca (as this would cause YFP 2+ Ca 2+ the immediate emission of chemilumines- Ca2+ Ca CaM Ca2+ cence, thereby rendering the protein unsuit- YFP able for measuring purposes) and eventually Ca2+ microinjected. However, after the cloning of AEQ complementary DNA13, the possibility of recombinant expression of the AEQ pro- c tein gave a new impetus to the use of this YFP YFP probe. However, the most important incen- 2+ tive to re-examine the use of AEQ was the CaCa2+ Ca2+ introduction of a new concept in the field of Ca2+ Ca2+ Ca2+ indicators — that of specifically tar- M13 geted probes14. With the exception of rhod-2 CaM (the positively charged Ca2+ indicator that is largely retained within the mitochondr- Photoproteins 2+ ial matrix15), the targeting of chemical- A Ca -induced conformational change of the apoprotein (depicted as a rod-shaped compound dyes had been based largely on structure) leads to peroxidation of the coenzyme (the reduced form of the coenzyme coelenterazine is shown in black and the oxidized form, coelenteramide, is shown in dark unanticipated, cell-specific conditions, and blue), which results in the release of blue light (see figure, part A). Red circles represent the was often unsatisfactory. By contrast, AEQ peroxide molecules. could be selectively targeted to most subcel- lular compartments by the insertion of spe- GFP-based probes cific signal sequences16. The use of chimeric In the case of cameleon, a Ca2+-induced interaction between calmodulin (CaM) and the AEQs allowed substantial advances in our CaM-binding peptide M13 increases fluorescence resonance energy transfer (FRET) leading to understanding of Ca2+ signalling, such as the a decrease in the fluorescence of cyan fluorescent protein (CFP) and an increase in the interplay between the ER and mitochon- fluorescence of yellow fluorescent protein (YFP) (see figure, part Ba). A similar FRET-based 2+ dria17, the presence of sub-plasmalemmal probe, FIP-CB, was constructed that measured the concentration of free Ca as a decrease of FRET (not shown). For the camgaroo probe, the Ca2+-induced conformational change in CaM Ca2+ microdomains18 and the role of the leads to an increase in the fluorescence of YFP (see figure, part Bb). The Ca2+-induced Golgi as an important Ca2+ store19, but was interaction between CaM and the binding peptide M13 of pericam leads to changes in the plagued by their small inherent signal. So, fluorescence characteristics of circularly permuted (cp)YFP (see figure, part Bc). A similar although the amount of photons that are probe with circularly permuted enhanced green fluorescent protein (cpEGFP) has also been emitted from a cell population is more than developed (not shown). 2+ adequate for the measurement of the Ca Sensitivity to pH is a bigger problem with GFP-based indicators than with synthetic dyes. concentration, the amount of photons that With the synthetic dyes, pH has an effect on fluorescence and Ca2+ affinity only below about are emitted by a single cell (with the excep- pH 6.5, whereas with most GFP-based probes, even pH changes around neutral can lead to tion of very large cells such as oocytes or marked changes in fluorescence. Finally, it is worth pointing out that the loss of signal (owing muscle fibres) is very low, and substantial to photobleaching and/or photoisomerization) with protein dyes is more significant than with signal accumulation is required to overcome synthetic dyes, and that some GFPs have absorption spectra that are not suitable for standard the background noise, at the expense of both confocal microscope laser lines.

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no FRET FRET strategy — the change of fluorescence reso- nance energy transfer (FRET) (FIG. 2) between two different coloured mutants of GFP that 2+ Non-radiative energy transfer is caused by the interaction between Ca - activated CaM and the target peptide 23,24.In cameleon23, the probe designed by Tsien and

R co-workers, the CaM-binding peptide M13 0 and CaM are fused together. The resulting protein construct is flanked at the carboxyl and amino termini by blue and green mutants of GFP,respectively, and the addition of Ca2+ leads to an increase in FRET23 (BOX 2). The probe developed by Persechini and col-

leagues, FIP-CBSM (REF.24), comprised a blue and a green mutant of GFP linked by the M13 peptide. After an increase in Ca2+ concentra- tion, endogenous or transfected CaM binds to M13, causing a drop in FRET24. Several varia- tions of these probes have been produced by Fluorescence Fluorescence replacing the blue and green GFP mutants

Fluorescence emission Fluorescence with cyan and yellow mutants, by introducing mutations in the CaM domain and by replacing Wavelength (nm) Wavelength (nm) M13 with a different CaM-binding pep- tide23,27,29,30. Many of the recombinant expres- τ τ 1 2 sion strategies initially used for AEQ have also been used with the cameleons, and several variants with targeting sequences and Ca2+ sensitivity that are suitable for many subcellu- lar compartments are now available23,31-33.The Fluorescence Fluorescence success of the cameleons, however, is limited by two principal experimental problems. The Fluorescence decay Fluorescence first is the relatively small change in signal on 2+ Time (ns) Time (ns) Ca binding, and the second is the large size Figure 2 | Fluorescence resonance energy transfer. Fluorescence resonance energy transfer (FRET) and molecular complexity that might, in is the physicochemical phenomenon that is characterized by the transfer of energy from an excited some cases, significantly impair its targeting donor chromophore to an acceptor chromophore, without associated radiation release. FRET occurs efficiency, for example to the mitochondrial when the donor emission and acceptor excitation spectra overlap considerably (not shown) and the two matrix. However, the low diffusion rate owing dipoles are very close to each other (2–7 nm). FRET is proportional to the 6th power of the distance to its high molecular mass, might turn into an between the chromophores and, therefore, even minor conformational changes can induce considerable advantage when high spatial accuracy is FRET changes. At the Förster radius, R , 50% of the energy is transmitted to the acceptor. The 0 needed, for example, to measure and localize occurrence of FRET results in the change of several fluorescence parameters — two used in the 2+ measurement of Ca2+ concentration are changes in the at constant excitation and in Ca ‘puffs’ or ‘sparks’. the fluorescence decay time. The former has been used the most because of the simplicity of its use, To overcome these limitations, efforts to the relatively limited cost and the high velocity of image acquisition, which mainly depends on the speed understand the structure-to-photochemistry of the imaging system and the dissociation time constant of Ca2+ from the probe. Fluorescence decay or relationship of the GFPs34,35 has led to new, lifetime imaging microscopy (FLIM), however, requires a dedicated apparatus and is slow, but it offers the rationally designed, Ca2+ probes that are advantage of being relatively insensitive to other environmental parameters, such as pH changes and τ τ based on a single GFP.For this generation of unequal loading of the probe. 1 and 2 indicate the time constants of fluorescence decay in the absence or the presence of FRET, respectively. new , GFP has been engineered so that its chromophore and surrounding hydrogen network are affected by even sub- space and time resolution. As a result, only the camgaroos25 and the pericams26, as well tle changes in its protein structure25.The very few researchers have attempted to as some variations25-29 (BOX 2). All of these mechanism for the fluorescence change of image recombinant AEQ and, at best, the probes use calmodulin (CaM) as a molecu- most of these probes is that the binding of time resolution in a small mammalian cell lar switch, which changes its conformation Ca2+ ions to CaM that is fused to GFP alters 20,21 2+ has been a few seconds . on the binding of Ca . In turn, this confor- the pKa (the pH at which 50% of the probe mational change alters the fluorescence is protonated) of the chromophore. In GFP-based indicators. Soon after it was shown properties of the GFP-based moieties, and other words, the binding of Ca2+ mimics that heterologously expressed GFP maintains this is then used to calculate the concentra- alkalization or acidification and so results its strong fluorescence22, recombinant Ca2+ tion of Ca2+ (BOX 2). in the increase or decrease of the chro- probes based on GFP were developed23,24.At The first two GFP-based fluorescent mophore fluorescence25. To achieve such present, there are three main types of this sen- Ca2+ indicators were developed in 1997 sensitivity, two types of modification have sor that are in use; the so-called cameleons23, (REFS 23, 24). Both probes are based on a similar been introduced. First, it was discovered

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Table 1 | The ‘ideal’ Ca2+ probe and the status of indicators available at present Probe Fluorescence Spectral shift Ca2+ Toxicity Targetability Variable pH sensitivity Molecular specificity Ca2+ affinity mass ‘Ideal’ probe Strong Yes High None Yes Yes None Variable Synthetic Variable* Some yes, but Good‡ In the short Poor (except for Yes Only below Variable§ polycarboxylates only in the UV term, Rhod-2) about pH 6.5 range relatively low GFP-based Variable Some yes, and Good ND Yes Yes Generally high Limited probe in the visible variability range (44–83 kDa) *The new generation of polycarboxylates are quite variable in terms of the fluorescence intensities of the Ca2+-free and Ca2+-bound forms. Some are strongly fluorescent without Ca2+, but the changes on Ca2+ binding are relatively small (for example, Oregon Green), whereas others are very dim without Ca2+ and the fluorescence increases many times on Ca2+ binding (for example, fluo-3). ‡Although the Ca2+ selectivity over Mg2+ and monovalent cations of most polycarboxylate indicators is very good, they bind with a significantly higher affinity than other divalent and trivalent cations. §Although the molecular mass of all polycarboxylate indicators is about 1 kDa, they can be conjugated to dextrans of different molecular weights. GFP, green fluorescent protein; ND, not determined; UV, ultraviolet. that there is a unique site in the GFP pri- systems. In contrast to FRET-based probes, Towards an ideal Ca2+ probe mary sequence (tyrosine (Tyr) 145) where ratiometric pericams do not change their As described previously, the past three relatively long peptides can be inserted emission spectra on Ca2+ binding, instead decades have seen a tremendous development without impairing the ability of the protein they change their excitation spectra26 .The of fluorescent Ca2+ indicators that can be used to fold and form the chromophore25.In normalization for shifts in the microscopic for a wide range of in vitro and in vivo appli- camgaroos25 (BOX 2), the insertion of CaM focal plane and protein distribution inferred by cations. Despite these advances, however, we between position 145 and 146 of yellow flu- the ratiometric nature of the measurement are still far from having the ideal Ca2+ probe orescent protein (YFP), a yellow mutant of allows simultaneous imaging of Ca2+ in two (TABLE 1). Here we speculate about future GFP,resulted in a protein in which binding spatially resolved cellular compartments in directions in the development of both syn- of Ca2+ mimics alkalization25. However, this cells that co-express two differently targeted thetic polycarboxylate dyes and protein-based makes all insertional mutants of GFP variants of the probe26,36.However,ratiometric probes. intrinsically pH sensitive. Second, Tyr145 pericams have to be excited at two different How could the qualities of polycarboxy- can be at the centre of a more spectacular wavelengths and are therefore inadequate for lates be ameliorated? One problem that is modification, circular permutation25.In very fast imaging experiments, and for most often neglected is that the hydrolysis products this variant, the original amino and car- confocal microscopes. of acetoxymethyl esters, in particular boxyl termini of YFP are linked by a spacer, and the new amino and carboxyl termini ab c Before During allow different fusion strategies. Pericam Histamine stimulation stimulation stimulation 26 High Ca2+ reporters (BOX 2) were designed on this Before 0 s 26 s basis by sandwiching circularly permuted

YFP between CaM and M13 (REF.26). Random CFP or rational mutations introduced into the sequence generated three variants of peri- 54 s 72 s cams26, one of which, known as ratiometric pericam, has an excitation spectrum that During YFP shifts on Ca2+ binding, thereby allowing ratiometric measurement26 (BOX 1). For the 78 s 104 s advantages of ratiometric measurement, see BOX 1. Similar to camgaroos, pericams are also highly pH sensitive. YFP/CFP 2+ The ‘readout’ of all single-GFP-based Low Ca biosensors, with the notable exception of Figure 3 | Examples of Ca2+-concentration signalling in living cells. a | Pseudo-colour-coded ratio- ratiometric pericam26, is a fluorescence inten- images obtained by conventional epifluorescence micrographs of a HeLa cell transfected with ratiometric sity change26. Such imaging is easily attainable pericam. Ca2+ concentrations are colour coded with a basal Ca2+ concentration in blue and a high Ca2+ 2+ with most confocal and conventional experi- concentration in yellow and red. Note the strong increase in mitochondrial Ca concentration on histamine-induced release from the endoplasmic reticulum (see also Online Video 1). b | The pseudo- mental systems, but this fluorescence intensity colour-coded ratio-images (as in FIG. 3a) of indo-1-loaded acute brain slices were obtained by confocal change might represent an important limita- microscopy and show the Ca2+ waves in astrocytes that were induced by stimulation of the CA1 nerve tion in the ability to quantify the signal, for fibres that contact the CA3 region of the hippocampus (known as the ‘Schaffer collateral pathway’) with an example owing to movement of the sample or extracellular electrode, which results in the depolarization of the nerve terminals and the release of of the cell (for example owing to contraction), neurotransmitter. The panels show sequential frames of Online Video 2. Note that the waves are sweeping photobleaching or a change in the microscope from one process to another, traversing the whole cell. The numbers indicate the time after stimulation. c | Two-photon confocal micrographs of a muscle fibre from tibialis anterior muscle transfected with focus. One possible solution to correct for any cytoplasmic cameleon in live mouse. The upper four panels show the yellow (YFP) and cyan fluorescence such problem is the co-transfection with a channel micrographs (CFP) from the cameleon probe that are responsible for the pseudo-colour-coded 2+ Ca -insensitive GFP mutant, although this ratio-images (lower 2 panels) as in FIG. 3a. Note the increase in the intracellular Ca2+ concentration in the might not be practical in many experimental muscle on stimulation of the sciatic nerve.

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Experimental system Cell/tissue Cell Tissue

Measurement Photon counting Conventional microscope Confocal laser scanning Multi-photon microscope* hardware board with FW or MC* microscope*

Principle of Ca2+ measurement Photon counting XR INT MR XR INT MR INT MR

Targetable‡ Aequorin Ratiometric Camgaroo Cameleon Ratiometric Camgaroo Cameleon Camgaroo? Cameleon pericam Inverse pericam Inverse Inverse pericam pericam pericam? Flash Flash Flash pericam pericam pericam? Rhod dyes Rhod dyes Rhod dyes?

Non-targetable Fura dyes Fluo dyes Indo dyes Fluo dyes Indo dyes Fluo dyes Indo dyes

Figure 4 | Description of major decisions for the planning of a Ca2+-concentration imaging experiment. The flow chart summarizes the main decisions to be made in a typical Ca2+-concentration imaging experiment (left column, green) and the corresponding hardware that is available at present for each of these steps (right columns, blue and yellow). At certain points, according to the experimental approach, there are several choices of hardware, which are indicated by forked lines. *The use of a monochromator increases the time resolution considerably when compared with filter wheel-equipped microscopes. While the costs associated with confocal laser scanning microscopy are considerably higher than those associated with conventional microscopy, the costs associated with multi- photon microscopy are even greater. ‡Note that targeted chemical probes are only available in the form of rhod2 for mitochondria. Low-affinity dyes could be used under certain instances for Ca2+-concentration measurements in the endoplasmic reticulum. FW, filter wheel; INT, intensity measurement; MC, monochromator; MR, emission ratioing; XR, excitation ratioing. formaldehyde, are potentially highly toxic. optics and expensive UV lasers for confocal However, CaM is also known to be important Although in short experiments these by- microscopy), not to mention background flu- in many physiological processes, and overex- products can be surprisingly innocuous to orescence, lower penetration of the exciting pression of these probes might therefore result cells, in longer experiments acetoxymethyl beam and the toxicity of UV illumination. in a substantial alteration of some of the many hydrolysis per se might have important delete- It could be argued that the introduction of reactions that depend on CaM in cells. So, it rious effects37. Although in the early days the protein-based indicators has reduced the might not be entirely surprising that trans- Tsien considered the possibility of designing efforts of scientists and companies to develop genic models expressing GFP-based probes alternative esters, the efficacy of ace- new, optimized dyes. However, this should have so far been described only in lower toxymethyl esters has deterred other investi- only be true in part. Although these probes organisms, such as Caenorhabditis elegans38 gators from designing alternatives. The use of have undoubtedly solved the problem of selec- and Drosophila39-42, but not in mammals. new esters could also be exploited as an tive targeting, there is still room for substantial Indeed, the construction of new protein-based approach to improve targeting of the polycar- improvement, especially of the GFP-based Ca2+ biosensors would require a protein com- boxylate indicators. Indeed, acetoxymethyl probes, which are still lacking a thorough ponent as sensitive and selective as CaM that esters are cleaved by enzymes that are methodological analysis of their advantages undergoes a marked Ca2+-dependent confor- expressed at different levels in most cellular and disadvantages. This might explain why the mational change, but which is characterized compartments. But if esters could be designed use of these probes, until now, has been rela- by negligible interference from endogenous that were selectively cleaved by enzymes that tively limited. In particular, a crucial feature components of the cell. Finally, biosensors that are localized or recombinantly expressed in of the available GFP-based probes is that they exploit CaM inevitably suffer from the rela- organelles, a simple and effective way to target all contain CaM as the Ca2+-sensing compo- tively slow kinetics of the Ca2+-induced con- the probes will have been found. 20 years after nent. This protein has been chosen for its formational change. Therefore, whether these their synthesis, it is surprising that the only high Ca2+ affinity and the marked conforma- tools can be used to monitor fluctuations in ratiometric probes available are fura-2 and tional change that is induced by Ca2+ binding. Ca2+ concentration of the order of millisec- indo-1 (and their low-affinity derivatives). onds (a technically feasible advance with mod- This highly desirable property (that is, the ern imaging systems), is still under evaluation. spectral shift and so the ratiometric property) “…the past three decades So, when the need for speed is compelling, flu- would be a great advantage for the use of orescent Ca2+ dyes still rule. A further problem indicators that absorb light in the visible have seen a tremendous is the necessity to overcome the bleaching and range (loading with two different probes, as development of fluorescent photoisomerization of GFP on illumination43. described above for two GFPs has been con- 2+ It is not easy to imagine a solution to these sidered as an alternative, but has inherent Ca indicators … despite problems, but the identification of new fluo- technical problems). Both fura-2 and indo-1 these advances, however, we rescent proteins in organisms other than need to be excited in the UV spectrum and Aequorea could provide alternatives. An insur- this, in turn, poses technical problems for the are still far from having the mountable problem of GFP-based Ca2+ use of microscopes (for example, special ideal Ca2+ probe.” probes is their large size. Unless fluorescent

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Rüdiger Rudolfand Tullio Pozzan are at the 20. Rutter, G. A. et al. Subcellular imaging of proteins much smaller than GFP are found, it 2+ Department of Biomedical Sciences, CNR Institute intramitochondrial Ca with recombinant targeted will be hard to significantly reduce the size of aequorin: significance for the regulation of pyruvate probes that are based on this protein. Methods of Neurosciences, University of Padova, dehydrogenase activity. Proc. Natl Acad. Sci. USA 93, via G. Columbo 3, 35121 Padova, Italy. 5489–5494 (1996). for selective in situ labelling of endogenous 21. Villalobos, C., Nunez, L., Chamero, P., Alonso, M. T. & proteins have been introduced by Tsien’s labo- Marco Mongillo and Tullio Pozzan are at the Garcia-Sancho, J. Mitochondrial [Ca2+] oscillations driven Venetian Institute for Molecular Medicine, by local high [Ca2+] domains generated by spontaneous ratory that take advantage of small peptide via Orus 2, 35129 Padova, Italy. electric activity. J. Biol. Chem. 276, 40293–40297 (2001). tags and organic fluorescent probes44. It would 22. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & not be surprising if, eventually, such method- Rosario Rizzuto is at the Department of Prasher, D. C. Green fluorescent protein as a marker for Experimental and Diagnostic Medicine, Section of gene expression. Science 263, 802–805 (1994). 23. Miyawaki, A. et al. Fluorescent indicators for Ca2+ based ologies could be applied to the generation of General Pathology, University of Ferrara, 2+ on green fluorescent proteins and calmodulin. Nature a new family of Ca indicators that would via Borsari 46, 44100 Ferrara, Italy. 388, 882–887 (1997). couple the selectivity of localization of pro- 24. Romoser, V. A., Hinkle, P. M. & Persechini, A. Correspondence to T.P. Detection in living cells of Ca2+-dependent changes in the tein-based probes to the small size of the poly- e-mail: [email protected] fluorescence emission of an indicator composed of two carboxylate dyes. doi:10.1038/nrm1153 green fluorescent protein variants linked by a calmodulin- 2+ binding sequence. A new class of fluorescent indicators. So far, most research in Ca signalling has J. Biol. Chem. 272, 13270–13274 (1997). been carried out in cultured cells (FIG. 3a) or 1. Pozzan, T., Rizzuto, R., Volpe, P. & Meldolesi, J. 25. Baird, G. S., Zacharias, D. A. & Tsien, R. Y. Circular Molecular and cellular physiology of intracellular calcium permutation and receptor insertion within green (FIG. 3b) acute tissue slices .However,modern stores. Physiol. Rev. 74, 595–636 (1994). fluorescent proteins. Proc. Natl Acad. Sci. USA 96, biology is advancing towards the analysis of 2. Berridge, M. J., Lipp, P. & Bootman, M. D. The versatility 11241–11246 (1999). and universality of calcium signalling. Nature Rev. Mol. highly complex systems and possibly whole liv- 26. Nagai, T., Sawano, A., Park, E. S. & Miyawaki, A. Cell Biol. 1, 11–21 (2000). Circularly permuted green fluorescent proteins ing organisms (FIG. 3c). The challenge to advance 3. Zhang, J., Campbell, R. E., Ting, A. Y. & Tsien, R. Y. engineered to sense Ca2+. Proc. Natl Acad. Sci. USA 98, Creating new fluorescent probes for cell biology. Nature the measurement of Ca2+-concentration is 3197–3202 (2001). Rev. Mol. Cell Biol. 3, 906–918 (2002). 27. Miyawaki, A., Griesbeck, O., Heim, R. & Tsien, R. Y. therefore not only to develop new and better 4. Smith, G. A., Hesketh, R. T., Metcalfe, J. C., Feeney, J. & Dynamic and quantitative Ca2+ measurements using probes, but also instruments that can take Morris, P. G. Intracellular calcium measurements by 19F improved cameleons. Proc. Natl Acad. Sci. USA 96, NMR of fluorine-labeled chelators. Proc. Natl Acad. Sci. 2135–2140 (1999). advantage of the new probes and that, at the USA 80, 7178–7182 (1983). 28. Nakai, J., Ohkura, M. & Imoto, K. A high signal-to-noise same time, are flexible and potent enough to 5. Thomas, M. V. in Techniques in Calcium Research Ca2+ probe composed of a single green fluorescent (eds Treherne, J. E. & Rubery, P. H.) (Academic, London, protein. Nature Biotechnol. 19, 137–141 (2001). allow the investigation of the whole living New York, Paris, 1982). 29. Truong, K. et al. FRET-based in vivo Ca2+ imaging by a organism. This is likely to be one of the rea- 6. Tsien, R. Y. New calcium indicators and buffers new calmodulin–GFP fusion molecule. Nature Struct. with high selectivity against magnesium and Biol. 8, 1069–1073 (2001). sons why the genetically encoded probes are protons: design, synthesis, and properties of 30. Persechini, A., Lynch, J. A. & Romoser, V. A. Novel rapidly becoming popular, given that by judi- prototype structures. Biochemistry 19, 2396–2404 fluorescent indicator proteins for monitoring free (1980). intracellular Ca2+. Cell Calcium 22, 209–216 (1997). cious genetic manipulation the new organism 7. Tsien, R. Y., Pozzan, T. & Rink, T. J. T-cell mitogens 31. Emmanouilidou, E. et al. Imaging Ca2+ concentration 2+ will already be intrinsically equipped with the cause early changes in cytoplasmic free Ca and changes at the secretory vesicle surface with a membrane potential in lymphocytes. Nature 295, 68–71 recombinant targeted cameleon. Curr. Biol. 9, 915–918 probe ready for in vivo measurement. Two- (1982). (1999). photon confocal microscopes, although still 8. Tsien, R. Y. A non-disruptive technique for loading 32. Jaconi, M. et al. Inositol 1,4,5-trisphosphate directs Ca2+ calcium buffers and indicators into cells. Nature 290, flow between mitochondria and the very expensive and often cumbersome to use, 527–528 (1981). endoplasmic/sarcoplasmic reticulum: a role in regulating at the moment seem to be the ideal instru- 9. Plinius, G. S. Naturalis Historia Liber IX, §146 (77). cardiac autonomic Ca2+ spiking. Mol. Biol. Cell 11, 10. Shimomura, O., Johnson, F. H. & Saiga, Y. 1845–1858 (2000). ments for this type of in vivo analysis (FIGS 4 Extraction, purification and properties of aequorin, 33. Isshiki, M., Ying, Y. S., Fujita, T. & Anderson, R. G. and 3c). In the meantime, however, important a bioluminescent protein from the luminous A molecular sensor detects signal transduction from hydromedusan, Aequorea. J. Cell. Comp. Physiol. 59, caveolae in living cells. J. Biol. Chem. 277, 43389–43398 improvements in the more classical 223–239 (1962). (2002). approaches could be the development of 11. Morise, H., Shimomura, O., Johnson, F. H. & Winant, J. 34. Ormo, M. et al. Crystal structure of the Aequorea victoria Intermolecular energy transfer in the bioluminescent green fluorescent protein. Science 273, 1392–1395 multicolour excitation for the simultaneous system of Aequorea. Biochemistry 13, 2656–2662 (1996). observation of different processes, online (1974). 35. Yang, F., Moss, L. G. & Phillips, G. N. Jr. The molecular 12. Tsuji, F. I., Ohmiya, Y., Fagan, T. F., Toh, H. & Inouye, S. structure of green fluorescent protein. Nature Biotechnol. emission spectrum analysis for an increased 2+ Molecular evolution of the Ca -binding photoproteins of 14, 1246–1251 (1996). sensitivity to FRET and video-rate acquisition the Hydrozoa. Photochem. Photobiol. 62, 657–661 36. Robert, V. et al. Beat-to-beat oscillations of mitochondrial in . As to the latter fea- (1995). [Ca2+] in cardiac cells. EMBO J. 20, 4998–5007 (2001). 13. Prasher, D., McCann, R. O. & Cormier, M. J. 37. Murgia, M. et al. Cytosolic free calcium concentration ture, the need for better time resolution of Cloning and expression of the cDNA coding in the mitogenic stimulation of T lymphocytes by confocal imaging has been limited by the rela- for aequorin, a bioluminescent calcium-binding protein. anti-CD3 monoclonal antibodies. Cell Calcium 16, Biochem. Biophys. Res. Commun. 126, 1259–1268 167–180 (1994). tive low speed of laser scanning. Line scan has (1985). 38. Kerr, R. et al. Optical imaging of calcium transients in been the only practical alternative, but at the 14. Rizzuto, R., Simpson, A. W., Brini, M. & Pozzan, T. Rapid neurons and pharyngeal muscle of C. elegans. Neuron changes of mitochondrial Ca2+ revealed by specifically 26, 583–594 (2000). expense of poor spatial resolution. The new targeted recombinant aequorin. Nature 358, 325–327 39. Diegelmann, S., Fiala, A., Leibold, C., Spall, T. & Buchner, E. confocal microscopes with a Nipkow disc (1992); erratum in 360, 768 (1992). Transgenic flies expressing the fluorescence calcium 15. Minta, A., Kao, J. P. & Tsien, R. Y. Fluorescent indicators sensor cameleon 2.1 under UAS control. Genesis 34, already ensure time resolution for full images for cytosolic calcium based on rhodamine and 95–98 (2002). in the order of 10 ms per image. Furthermore, fluorescein chromophores. J. Biol. Chem. 264, 40. Fiala, A. et al. Genetically expressed cameleon in 8171–8178 (1989). Drosophila melanogaster is used to visualize olfactory taking into consideration the complex spatio- 16. Rizzuto, R., Brini, M. & Pozzan, T. Targeting information in projection neurons. Curr. Biol. 12, temporal compartmentalization of Ca2+, the recombinant aequorin to specific intracellular organelles. 1877–1884 (2002). Methods Cell Biol. 40, 339–358 (1994). 41. Reiff, D. F., Thiel, P. R. & Schuster, C. M. Differential field will greatly profit from the close interac- 17. Rizzuto, R. et al. Close contacts with the endoplasmic regulation of active zone density during long-term tion with biocomputing scientists for the reticulum as determinants of mitochondrial Ca2+ strengthening of Drosophila neuromuscular junctions. responses. Science 280, 1763–1766 (1998). J. Neurosci. 22, 9399–9409 (2002). development of more powerful image analy- 18. Marsault, R., Murgia, M., Pozzan, T. & Rizzuto, R. 42. Yu, D., Baird, G. S., Tsien, R. Y. & Davis, R. L. Detection sis software. In summary, methodologies, Domains of high Ca2+ beneath the plasma of calcium transients in Drosophila mushroom body membrane of living A7r5 cells. EMBO J. 16, 1575–1581 neurons with camgaroo reporters. J. Neurosci. 23, 64–72 hardware and measurement probes are evolv- (1997). (2003). ing in a synergistic manner and will undoubt- 19. Pinton, P., Pozzan, T. & Rizzuto, R. The Golgi apparatus 43. Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R. is an inositol 1,4,5-trisphosphate-sensitive Ca2+ store, & Piston, D. W. Use of the green fluorescent protein and edly affect our future insights into the biology with functional properties distinct from those of the its mutants in quantitative fluorescence microscopy. of the fascinating field of Ca2+ signalling. endoplasmic reticulum. EMBO J. 17, 5298–5308 (1998). Biophys. J. 73, 2782–2790 (1997).

NATURE REVIEWS | MOLECULAR CELL BIOLOGY VOLUME 4 | JULY 2003 | 585 PERSPECTIVES

44. Griffin, B. A., Adams, S. R. & Tsien, R. Y. Specific Venetian Institute of Molecular Medicine: covalent labeling of recombinant protein molecules inside http://www.vimm.it Box 1 | Inositides live cells. Science 281, 269–272 (1998). Introduction to Ca2+ measurement with fluorescent indicators: The word ‘inositide’ has had various Acknowledgements http://www.probes.com/handbook/sections/2001.html The original work by the authors was supported by grants from Atsushi Miyawaki’s laboratory: meanings over the years that are covered by the Italian Ministry of University, the Italian Research Council http://www.riken.go.jp/engn/ this article. In the present context, inositide is (CNR), Italian Telethon, the Italian Association for Cancer Research r-world/research/lab/nokagaku/tip/faculty/ used as a blanket term for any compound (AIRC), ASI, Human Science Frontier and from the European Roger Y. Tsien’s laboratory: Union (EU) to T.P. and R. Rizzuto. R. Rudolf is supported by an EU http://www.tsienlab.ucsd.edu/ containing inositol as a part of its structure. Marie Curie fellowship. We are grateful to G. Carmignoto and The two principal forms of inositide are L. Filippin for supplying artwork and to P. Magalhães for help with MICROSCOPES AND CAMERAS image processing. Biorad: inositol lipids and inositol phosphates. http://microscopy.bio-rad.com/products/products.htm Leica: http://www.leica-microsystems.com/website/lms.nsf Online links Perkin Elmer: http://lifesciences.perkinelmer.com principal milestones that led to the publica- Sutter Instruments: http://www.sutter.com DATABASES T.I.L.L. Photonics: http://www.till-photonics.de/ tion of this original paper and, for reasons The following terms in this article are linked online to: Zeiss: http://www.zeiss.de/ discussed below, it focuses more on the inosi- Swiss-Prot: http://www.expasy.ch/ 2+ AEQ | CaM | GFP FLUORESCENT PROBES AND FILTERS tides (BOX 1) than on Ca . An extensive his- Chroma: http://www.chroma.com tory has been written by Bob Michell2, and FURTHER INFORMATION Molecular Probes: http://www.probes.com Tullio Pozzan’s laboratory: Omega Opticals: http://www.omegafilters.com briefer personal accounts have been written http://www.bio.unipd.it/~pozzan/home.html Access to this interactive links box is free online. by Lowell Hokin3 and Michael Berridge4.

The ‘PI effect’ The original demonstration of receptor- TIMELINE stimulated lipid turnover in 1953 (which is another anniversary to celebrate this year!) was a classic piece of serendipity that was 20 years of Ins(1,4,5)P3, imaginatively exploited5. Lowell and Mabel Hokin (FIG. 1a,b) were investigating what they and 40 years before believed to be an increase in the incorpora- tion of 32P into RNA, which was caused by the stimulation of pancreatic slices with acetyl- Robin F. Irvine choline. However, as they purified the RNA (they carried the radiolabelled samples with This year marks the 20th birthday of the Streb et al.1 were the first to show that inositol- them from Sheffield, UK, to Halifax, Canada, discovery of inositol-1,4,5-trisphosphate as 1,4,5-trisphosphate (Ins(1,4,5)P3) can mobilize before doing the purification), they noticed a second messenger. The background to Ca2+ from stores in the endoplasmic reticu- that the radioactivity was lost. So, they rein- this discovery is a complex story that goes lum (ER), and that it could therefore function vestigated the discarded ‘junk’,and found back more than 50 years and involves a as a second messenger that is generated by that most of the radioactivity was in the large cast of characters, both chemical and receptor activation and that regulates intracel- phospholipid fraction. Separating individual human. lular Ca2+. This account can only highlight the phospholipids for analysis was difficult in

Timeline | Milestones on the road to the discovery of Ins(1,4,5)P3

Quin-2, a calcium-sensing dye, was loaded into cell populations and used to Many reports confirmed 2+ 24 A link was Mitochondria were measure intracellular Ca Ins(1,4,5)P3’s function , First measurement suggested identified as mostly a (REF. 38). Introduction of and a more complex between inositol inositide metabolism was PtdIns(4,5)P2 of changes in target, rather than a lithium amplification assay The Ins(1,4,5)P3 ‘Diphosphoinositide’ was intracellular Ca2+ lipid turnover and source, of mobilizable to measure inositol revealed by the presence receptor was 14 15–18 10 30 53 40–42 was first isolated . discovered . (REF. 35). calcium entry . intracellular calcium . phosphate generation . of Ins(1,3,4)P3 (REF. 44). identified .

1949 1953 1960-61 1964 1966 1969 1975 1979 1980 1981 1982 1983 1984 1988 1989

First report of PtdIns(4,5)P2-PLC and First suggestion First direct PtdIns(4,5)P2 was First suggestion that Ins(1,4,5)P3 PI3K was 55,56 2+ 47 receptor-stimulated phosphatase were discovered . of PtdIns(4,5)P2’s evidence for a reaffirmed as a is a Ca -mobilizing second identified . 23 phospholipid A PLC reaction was identified as involvement in link between principal substrate messenger , and Ins(1,4,5)P3 metabolism5. the primary event in stimulated receptor action9. inositides and for receptor- was shown to mobilize Ca2+ inositol lipid turnover51. Ca2+ (REF. 11). activated PI-PLC from non-mitochondrial stores in (REF. 21). permeabilized pancreatic acinar cells1.

Ins(1,3,4)P3, inositol-1,3,4-trisphosphate; Ins(1,4,5)P3, inositol-1,4,5-trisphosphate; PI, phosphoinositide; PI3K, phosphoinositide 3-kinase; PLC, phospholipase C; PtdIns(4,5)P2, phosphatidylinositol-4,5-bisphosphate.

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