REVIEWS

Aequorea victoria bioluminescence moves

into an exciting new era
Jonathan M. Kendall and Michael N. Badminton

Bioluminescence has revolutionized research into many cellular and molecular-biological processes, ranging from intracellu-
lar signalling to gene transcription. This article focuses on the chemistry and biotechnological exploitation of the two proteins
involved in bioluminescence of the jellyfish Aequorea victoria – aequorin and green fluorescent protein. Engineered recombi-
nant aequorin has led to a novel technological approach to monitoring calcium signals in organelles and subcellular domains.
A new generation of intracellular calcium indicators has been produced in which engineered variants of green fluorescent
protein are used to probe their ionic environment using intramolecular fluorescence-resonance-energy transfer.

ioluminescence is a natural phenomenon in (most commonly by molecular oxygen) of a substrate,

B which visible light is generated by an organism as

a result of a chemical reaction. These reactions
can be reconstructed outside the organisms from which
they originate, thereby enabling exploitation of this
natural process. Bioluminescent organisms occur in a
variety of habitats, particularly the deep sea, where light
is employed for functions including defence, repro-
duction and feeding. The chemical reactions underly-
ing bioluminescence have fascinated scientists for many
years, the terms ‘luciferase’ and ‘luciferin’ first being
used over a century ago to describe light-generating
components of the bioluminescent mollusc Pholas
dactylus1,2. The specificity and sensitivity of biolumi-
nescence have led to detailed analyses of the compo-
nents and reactions involved in generating light.
There are two protein components involved in the
bioluminescent system of the jellyfish Aequorea victoria:
aequorin and the green fluorescent protein (GFP;
Fig. 1). Both proteins have been cloned, and the
recombinant proteins have assumed important roles in
an array of biotechnological applications, ranging from
monitoring protein dynamics and gene transcription to
sensitive labels in analytical assays. This review focuses
on how both the proteins and the mechanism
employed in the bioluminescence of A. victoria have
been exploited to investigate subcellular calcium
signalling in living cells.
The bioluminescence of A. victoria
Bioluminescent reactions require three major com-
ponents: a luciferin, a luciferase and molecular oxygen.
However, as many as three other components may also
be required in some reactions, including cations (e.g.
Ca21 and Mg21), cofactors [e.g. ATP, NAD(P)H or
flavin mononucleotide] and fluorophores (e.g. GFP). A
luciferase is an enzyme that catalyses the oxidation
J. M. Kendall (kendall@cardiff.ac.uk) is at the Department of
Medical Biochemistry, University of Wales College of Medicine,
Heath Park, Cardiff, UK CF4 4XN, and M. N. Badminton
(mikeb@rascal.med.harvard.edu) is at the Cardiovascular Division,
Enders Building Room 1306, Children’s Hospital, Harvard Medical
School, 300 Longwood Ave., Boston, MA 02115, USA.
the luciferin, to produce an unstable, electronically
excited intermediate. Light is emitted when this unsta-
ble intermediate decays to its ground state, generating
the product, an oxyluciferin. As this compound differs
chemically from the luciferin, the reaction only pro-
duces light once. Photoproteins differ from luciferases
per se in that they are stabilized oxygenated intermedi-
ate complexes of the luciferase and luciferin. Decay of
these photoprotein intermediates and emission of
light can only occur when the luciferase provides the
appropriate environment2.
Fluorescent reactions, on the other hand, differ from
bioluminescent reactions in that they derive energy
from electromagnetic radiation of a specific wave-
length. This results in an excited-state molecule with a
short half-life, which rapidly decays to its ground state,
emitting light of a longer wavelength. The fluorescent
molecule remains chemically unchanged during the
reaction and can therefore emit light continuously in
the presence of an appropriate source of energy3.
A. victoria is a hydromedusan jellyfish found mainly
at Friday Harbor, WA, USA. Its bioluminescence arises
from a photoprotein found in specialized photocytes in
the umbrella of the organism. The photoprotein,
aequorin, contains its luciferin (coelenterazine) bound
to the protein with oxygen. Binding of calcium ions
(Ca21) to the protein triggers the oxidation of co-
elenterazine (Fig. 1) and the consequent emission of
blue light; for this reason, the complex is often called
the blue fluorescent protein (BFP). Light emitted by
A. victoria is, however, blue–green, because the excited-
state BFP undergoes radiationless energy transfer to
GFP to produce excited-state GFP, which emits green
light as it relaxes to its ground state (Fig. 2).
Aequorin
Aequorin is the most widely studied of the Ca21-
binding photoproteins (Box 1) and is composed of an
apoprotein (apo-aequorin; 189 amino acid residues;
molecular weight approximately 22 kDa) and a
luciferin (coelenterazine, an imidazopyrazine com-
pound; molecular weight 423 Da)2,4–6. The protein
contains three EF-hand Ca21-binding sites (Box 1) and,

216 Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0167 – 7799/98/$19.00. PII: S0167-7799(98)01184-6 TIBTECH MAY 1998 (VOL 16)
 REVIEWS

a b

Figure 1
(a) Primary structure of apo-aequorin. The sequence is arranged to illustrate the Ca21-binding sites (I, II, III). Oxidation of coelenterazine to coelenteramide
is triggered when Ca21 binds to the protein. The excited state of coelenteramide bound to the protein is the light emitter in the reaction. (Reproduced with
permission from Ref. 62.) (b) Crystal structure of an S65T mutant of the green fluorescent protein. The fluorophore (shown as ball-and-stick model) is located
in a central a-helical heptapeptide surrounded by an 11-stranded barrel of b sheets14 (S. J. Remington, pers. commun.).

Aequorin
+Ca2+
Ca3–Apo-aequorin–coelenteramide*
+GFP
In vivo In vitro
Green light Blue light
λ max = 509 nm λ max = 469 nm
Figure 2
The pathway responsible for the bioluminescence of A. victoria. Ca21
binds to aequorin, which catalyses the oxidation of coelenterazine
to coelenteramide. The excited state (*) of coelenteramide bound
to apo-aequorin emits blue light in vitro. However, in vivo, radiation-
less energy is transferred from the excited state to green fluorescent
protein, which emits green light.
when these sites are occupied by Ca21, aequorin
undergoes a conformational change that converts it
into an oxygenase (luciferase) and provides the appro-
priate environment for the oxidation of coelenterazine.
Coelenterazine is converted into a highly unstable
dioxetanone intermediate, which loses carbon dioxide
to form the excited phenolate anion of coelenteramide.
Coelenteramide remains noncovalently bound to the
protein, emitting blue light (lmax 5 470 nm) as it
relaxes to the ground state (Fig. 1)7,8. The quantum
yield of this reaction (FCL, defined in Box 2) is
approximately 0.15–0.20 (Ref. 2).
As light emission from aequorin is sensitive to Ca21,
the protein provides an excellent method for the detec-
tion and determination of Ca21 concentration, denoted
[Ca21]. However, light emission from aequorin must
be calibrated as rate constants over a range of [Ca21]
(effectively 100 nM – 10 mM9), which corrects for the
cooperativity between the three Ca21-binding sites
{photons emitted are proportional to [Ca21]2.5–[Ca21]3
(Ref. 10)} and consumption of the photoprotein
during the reaction11.
Coelenterazine is chemically modified during the
light-emitting reaction, and so active aequorin must be
regenerated before the cycle can continue. The Ca21
must therefore be removed from the protein and fresh
coelenterazine supplied. Although the mechanism by
which the jellyfish achieves this has yet to be estab-
lished, the apoprotein can easily be reactivated in vitro
to form the photoprotein by the addition of a Ca21
chelator and coelenterazine in the presence of a
reducing agent and molecular oxygen7.
Green fluorescent protein
A. victoria bioluminescence is blue–green owing to
the presence of GFP, which derives its light-emitting

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Box 1. Ca21-binding photoproteins
Aequorin is the most widely studied of the seven Ca21-binding
photoproteins described to date2. Sequence homologies suggest that
aequorin has evolved from Ca21-binding proteins such as calmodulin
(CaM)60. Recombinant-DNA technology and the sensitivity of the light-
emitting reaction18 have permitted the generation of mutated
recombinant aequorins, that have illuminated the structure–function
relationship of the photoprotein.
The position of the EF hands in aequorin are highly conserved com-
pared with CaM, with the exception that site II has been lost from the
photoprotein60. Substitution of the key glycine residue (position 6) in
the EF-hand sites of apo-aequorin showed that sites I and II are
required for the bioluminescence of the protein9,40,61,62. This led to
speculation that the area between these two sites (corresponding to
site II in CaM) may have evolved for coelenterazine binding or catalytic-
site formation, controlled by Ca21-binding to the two EF-hand sites
that surround it. Aequorin contains a relatively high content of aromatic
amino acid residues compared with other Ca21-binding proteins. Sub-
stitution of tryptophan with phenylalanine between EF-hand sites I and
II identified two residues that may play key roles in the catalytic activ-
ity of the protein; residue 58 in oxygen binding62 and residue 86 in
generating the excited state in aequorin bioluminescence63.
The most interesting discovery to have emerged from the structure–
function analyses is that the C-terminal proline residue (position 189)
is essential for the normal bioluminescent activity of the protein, poss-
ibly in stabilizing the coelenterazine. Removal or substitution of this
residue has deleterious effects on the specific activity, stability and
Ca21-independent light emission of the recombinant protein64,65.
energy from aequorin. This process of radiationless
energy transfer occurs as the excited Ca21–apo-
aequorin–coelenteramide complex decays to its ground
state, exciting the GFP, which, in turn, emits green
light (Fig. 2). GFP is a 238 amino acid protein12,
arranged as an 11-stranded barrel of b sheets sur-
rounding a central a-helical heptapeptide that forms
the fluorescent centre (Fig. 1)12,13,15. The fluorophore
is formed by the cyclization and oxidation of three
amino acids, serine–dehydrotyrosine–glycine, at pos-
itions 65–6716. GFP has a major excitation peak at
395 nm (with a minor peak at 475 nm), an emission
maximum at 508 nm and a quantum yield of
0.72–0.8517. As the light-emitting properties of GFP
are independent of substrates and cofactors, the protein
has rapidly become a popular choice for monitoring a
vast array of biological processes, ranging from gene
expression to organelle organization.
Biotechnological applications of aequorin and
GFP
Light-emitting reactions have assumed an important
role in many biotechnological applications because of
their sensitivity and specificity, ease of detection, and
noninvasive nature18. The protein components, and the
very mechanism responsible for the bioluminescence
of the jellyfish itself, are now being exploited in an
exciting new era for A. victoria.
Aequorin
Many diverse cellular functions are regulated by
changes in intracellular [Ca21] in response to a
number of different classes of extracellular stimuli.
Extensive use of fluorescent Ca21-chelating dyes (e.g.
fura-2) has revealed that agonists produce many differ-
218
Box 2. Measurement of light
The successful biotechnological application of light-
emitting proteins is dependent on efficient methods
to detect and monitor light. These must be capable of
detecting light signals over a wide range of intensities
and spectral responses. There are a number of dif-
ferent methods for detecting light2, for example lumi-
nometers and image-intensified charge-coupled
devices (CCDs). Luminometers usually house photo-
multiplier tubes, which can operate over six to seven
orders of magnitude of detection and have a wide
spectral response. In order to capture the spatial and
temporal organization of events, particularly at the
subcellular level, a process called ‘imaging’ has been
developed. The basic principle of imaging relies on
converting light signals to electrical signals, which
can be amplified whilst retaining spatial resolution.
The practical choice for imaging is a CCD linked to a
microscope. One problem encountered with all light-
detection systems is that their efficiency is poor and
that .90% of light is lost. The limits of detection will,
obviously, also depend on the amount of light gen-
erated by the recombinant protein. This is called the
quantum yield (FCL) and is equivalent to the number
of photons emitted divided by the number of molecules
reacting; the ΦCL for aequorin is between 0.12 and
0.20 (Ref. 2), whereas that for green fluorescent
protein is 0.72–0.85 (Ref. 17).
ent spatial and temporal patterns of changing cytosolic
[Ca21], both in cell populations and at the single-cell
level. This probably reflects the need for the specifi-
cation of signals conveyed by a wide array of agonists
that must operate in many different cell types. Regu-
lation of Ca21 signals involves the storage of Ca21 within
intracellular organelles, stimulus-activated release and
regulated influx of Ca21 from outside the cell19.
Aequorin was first used to investigate intracellular
calcium homeostasis in contracting muscle fibres from
giant barnacles20. The photoprotein was introduced
into these cells by microinjection, a technique that has
been applied to progressively smaller cells, for example,
oocytes, myocytes and hepatocytes9. A major goal in
biological research has, however, been the development
of readily available, user-friendly, noninvasive tech-
niques to monitor events inside living cells or organ-
isms. The cloning of the gene for aequorin6 has allowed
recombinant expression of the photoprotein, circum-
venting the need for microinjection. This led to genetic
transformation of bacteria, yeast, plant and animal cells
with the gene encoding apo-aequorin. A cDNA
encoding apo-aequorin expressed in Escherichia coli pro-
duced a functional recombinant protein when bac-
terial extracts were combined with coelenterazine21; as
coelenterazine can readily permeate cell membranes,
this facilitated activation of the recombinant protein in
situ. The first measurements of cytosolic Ca21 transients
were made with recombinant aequorin from stimuli as
diverse as wind (plants), complement attack (bacteria)
and mating pheromones (yeast)22–24. These early experi-
ments have been elaborated by imaging light signals
(Box 2) and equating them with cytosolic Ca21 signals
in transgenic multicellular organisms expressing recom-
binant aequorin25 and in an organotypic context by
TIBTECH MAY 1998 (VOL 16)
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restricting expression of the transgene to specific cell
types26.
The most exciting development to have arisen from
the cloning of the apo-aequorin cDNA has been the
development of methods to measure [Ca21] in discrete
subcellular domains, such as the lumen of different
organelles. This has been achieved both with minimal
targeting sequences and by fusion with resident
organellar proteins that already include the information
necessary for efficient localization (Table 1)27. Chimeras
of apo-aequorin and organellar proteins or targeting
sequences are simple to generate by standard cloning
techniques, as is their introduction into cells by tran-
sient or stable transfection (Fig. 3; Table 1)28. The tar-
geting strategy offers clear advantages over alternative
methods, for example, fluorescent Ca21-chelator dyes,
which display no selective localization and are more
sensitive to changes in their environment than
aequorin29.
The first successful investigation of organellar Ca21
homeostasis using an aequorin chimera was made in
mitochondria (Table 1). Rizzuto et al.30 showed that
the stimulation of receptors coupled to the generation
of the second messenger inositol-1,4,5-trisphosphate
produced increases in intramitochondrial [Ca21],
exceeding the accompanying rise in cytosolic [Ca21].
These increases resulted in the activation of intramito-
chondrial enzymes such as pyruvate dehydrogenase31.
Changes in intranuclear [Ca21] were also shown to dif-
fer from those occurring in the cytosol32. These differ-
ent signals were dependent on whether the Ca21
originated from an intracellular or extracellular source
(Fig. 4) and may play a role in regulating nuclear
events33. Targeted aequorin has also revealed that the
area directly beneath the plasma membrane has a [Ca21]
that is significantly higher than the bulk cytosol34. Such
sub-plasma-membrane microdomains may be involved
in controlling intercellular communication via gap
junctions35.
Despite these pioneering experiments, measuring
[Ca21] in discrete subcellular locations with targeted
aequorin has not been without problems. This is exem-
plified by the difficulties encountered when measuring
[Ca21] in the endoplasmic reticulum (ER). Ca21 in the
ER controls an array of cellular functions including
protein biosynthesis36 and Ca21 influx through the
plasma membrane, as well as providing an agonist-
sensitive store of rapidly mobilizable Ca21 (Ref. 19).
Different strategies have been adopted to find a suitable
way to employ recombinant aequorin as an ER
Ca21 probe. Addition of the –KDEL sequence to the
C terminus of the protein aids ER targeting (Fig. 3),

Table 1. Strategies used to target aequorin to subcellular domains

Subcellular domain Cell type Target strategy Notes Ref.

Cytosola HeLa Luciferase chimera. Increase size prevents free 66

diffusion into nucleus.
Mitochondria HeLa Leader sequence of cytochrome-c IP3 increased mitochondrial [Ca2+] 30
CHO oxidase, subunit VIII. to more than cytosolic [Ca2+].
Leader sequence of cytochrome c High expression by injecting nuclei – 31
oxidase, subunit VIII. single cell analyses.
Nucleus HeLa Part of glutocorticoid receptor Cytosolic and nuclear Ca2+ signals 67
(117 residues). identical.
HeLa Nucleoplasmin chimera. Different cytosolic and nuclear 32
Ca2+ signals, dependent on Ca2+
source.
Trypanosoma brucei L25 signal motif (6 residues). 68
Endoplasmic reticulum HeLa Calreticulin N-terminal signal motif First estimates of endoplasmic 69
(17 residues) and C-terminal KDEL reticulum [Ca2+] (1–5 mM).
motif.
HEK-293 MHC-II invariant-chain chimera. IP3-sensitive microdomains identified. 39
HeLa Immunoglobulin heavy-chain chimera. Low-Ca2+-affinity aequorin measured 38
mM levels of Sr2+.
HeLa Immunoglobulin heavy-chain chimera. Semisynthetic low-affinity n-aequorin 43
measured 600 mM levels of Ca2+.
Sarcoplasmic reticulum Cardiac myotubes Calsequestrin chimera. Low-Ca2+-affinity aequorin-measured 70
mM levels of Sr2+.
Golgi HEK-293 Galactosyltransferase chimera. No [Ca2+] estimates made. 39
Plasma membrane Xenopus oocytes 5-HT1A-receptor chimera. No [Ca2+] estimates made. 71
Ar57 SNAP-25 chimera. Localized high [Ca2+] under plasma 34
membrane.
COS7 Connexin chimera. Localized high [Ca2+] under plasma 34
membrane.

aNumerous studies have used aequorin without targeting modifications to estimate cytosolic [Ca2+]. This strategy does not exclude free diffusion of
the protein (Mr 22 kDa) into other domains, such as the nucleus.
Abbreviations: Ar57, rat aortic smooth muscle cells; [Ca2+], calcium-ion concentration; CHO, Chinese hamster ovary cells; COS7, monkey kidney
fibroblasts; 5-HT1A, 5-hydroxytryptamine sub-type 1A; HeLa, human epithelial cells; HEK-293, human embryonic kidney cells; IP3, inositol-1,4,5-
trisphosphate; SNAP-25, soluble N-ethylmaleimide-sensitive fusion-protein-attachment protein.

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REVIEWS
Figure 3
Immunofluorescent staining of a COS7 cell transiently expressing apo-aequorin tar-
geted to the endoplasmic reticulum (ER) observed by light microscopy through
488 nm excitation and 515 nm emission filters. Cells were fixed, stained with mouse
anti-apo-aequorin antibody and visualized with FITC-labelled antimouse antibody. The
staining pattern shows a highly localized distribution, with a heavy perinuclear stain
and a fine network extending to the periphery of the cell. This pattern confirms
efficient ER targeting. (Reproduced with permission from Ref. 37).
but results in Ca21-independent light emission and
problems in calibrating the light signal37. Although
alternative targeting strategies, which do not involve
modification of the C terminus have been used38,39, the
sensitivity of aequorin for Ca21 was too high to
measure steady-state ER [Ca21] values, even when
using an aequorin engineered to have a kd 20 times
lower than the wild type (Box 1)38,40.
A range of coelenterazine analogues (h-, n- and e-)
have been synthesized and used to generate semisyn-
thetic aequorins with different light-emitting proper-
ties and Ca21 sensitivities41. Despite the theoretical
attraction of using semisynthetic aequorins to measure
a wider range of [Ca21], they actually have limited
application42,43, as they are less stable and have signifi-
cantly lower quantum yields than native aequorin41.
A further application has been the use of recombi-
nant aequorin as a quantitative label in different ana-
lytical assay systems, thereby enabling high detection
sensitivity in the presence of excess Ca21. The proper-
ties of aequorin make it an ideal label in a number of
different assay systems: it can be readily conjugated (e.g.
to antibodies and to substrates); it generates a high
signal-to-noise ratio with a broad range of detection
sensitivity; and it generates a signal rapidly18. Fusion of
apo-aequorin cDNA to the coding sequence of pro-
tein-A signal peptide and its subsequent expression in
bacteria leads to secretion of the apoprotein, which can
be purified and subjected to a variety of chemical
modifications for use in different assay systems44,45.
Alternatively, fusion proteins have been generated (e.g.
antibody–apo-aequorin) that retain the functions of
220
Figure 4
Comparison of nuclear and cytosolic Ca21 signals measured with
appropriately targeted aequorins. (a) HeLa cells expressing nuclear-
targeted or cytosolic aequorin were challenged by exposure to ATP.
Initial increases in nuclear and cytosolic [Ca21] were similar, followed
by a greater increase in the cytosolic signal than the nuclear signal,
with the nuclear signal returning to baseline more rapidly. Subse-
quent stimulation with histamine (Hist) changed neither nuclear nor
cytosolic [Ca21] but restoration of extracellular Ca21 induced an
increase in only cytosolic [Ca21]. (b) Treatment with cyclopiazonic
acid induced an increase in cytosolic [Ca21] that clearly preceded
the increase in nuclear [Ca21].
both proteins and are highly suitable for use in
immunoassays46.
Green fluorescent protein
Mutational analyses of GFP, particularly in the fluoro-
phore region, have generated a variety of GFP iso-
forms with different characteristics to those of the
native protein. The first functional mutation to have a
significant biotechnological impact was S65T. This
generated not only a brighter and more stable GFP but
also altered the protein’s excitation spectrum charac-
teristics (Table 2)47. The increase in brightness and
improved folding have been of paramount importance
to the success of GFP. Combined with modifications
such as species-specific codon optimization48, GFP has
now assumed a role as an ideal reporter of protein
dynamics and gene expression both in multicellular
organisms and at the subcellular level. Over the past few
years, there has been an explosion of interest in the
employment of GFP as a protein tag, a reporter of gene
expression and to investigate organelle structure17,49,50.
TIBTECH MAY 1998 (VOL 16)
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Table 2. Wild-type GFP and GFP variants with altered spectral characteristics

GFP variants Excitation Emission Relative Mutationsa Comments Ref.

wavelength wavelength fluorescence
(nm) (nm) intensity (compared
with wild-type GFP)

Wild-type GFP 395 510 1 64F–S–Y–G–V–69Q

470b

Red-shifted variants
S65T mutant 489 511 4–6 64F–T–Y–G–V–69Q Fourfold enhancement in
oxidation of fluorophore
S65C mutant 479 507 n/a 64F–C–Y–G–V–69Q Used for FRET 53
Enhanced GFPc 488 507 35 64L–T–Y–G–V–69Q Based on GFPmut1. 54
Available from Clontech.
Red-shifted 490 509 n/a 64M–G–Y–G–V–69L Used for FRET 55

Blue-shifted variants
Blue-shifted 382 448 n/a 64F–S–H–G–V–69Q Denoted P4 53
Blue-shifted 381 445 n/a 64F–S–H–G–V–69Q Denoted P4-3 53
+Y145F
BFP5 385 450 64M–S–H–G–I–69Q Used for FRET 55
Enhanced blue- 380 440 1 64L–T–H–G–V–69Q Available from Clontech
shiftedc +Y145F

Other variants
Enhanced cyanc 433 475 n/a 64L–T–W–G–V–69Q Mutations F64L and S72A
501b +N146I, M153T, improve folding at 378C
V163A, N212L but do not change spectral
characteristics
Enhanced 502 512 n/a 64F–G–W–G–V–69Q

yellow c +S72A, T203Y

aAmino acid sequence at positions 64 to 69; bold denotes mutated amino acid, other mutations as noted.
bMinorexcitation or emission peak.
cGFP uses mammalian codon usage, which results in 190 silent mutations.

Abbreviations: BFP, blue fluorescent protein; FRET, fluorescence-resonance-energy transfer; GFP, green fluorescent protein; n/a, not available.

Other mutational analyses, in and surrounding the
fluorophore, have generated a new range of GFPs with
altered excitation and emission spectra. Substitution of
the central tyrosine residue at position 66 shifts the
excitation and emission spectra to shorter wavelengths,
to generate a family of proteins called ‘blue-shifted’
variants51. Mutations of other amino acid residues
have generated cyan-, red- and yellow-shifted variants
of GFP with altered spectral characteristics
(Table 2)14,52–54. Radiationless transfer of energy from
aequorin to GFP in A. victoria relies on the emission
spectrum of aequorin overlapping with the excitation
spectrum of GFP. The mutations in GFP have gener-
ated fluorescent proteins with excitation and emission
spectra that overlap and it therefore became feasible to
exploit radiationless energy transfer, in this case fluor-
escence-resonance-energy transfer (FRET), to moni-
tor molecular interactions. For example, Y66H (blue-
shifted variant, or BGFP) was joined to S65C
(red-shifted variant, or RGFP) by fusing their cDNAs
with an amino acid linker sequence incorporating a
protease site. Excitation of the chimera at 368 nm
resulted in the emission of green light at 510 nm. When
the chimera was cleaved with trypsin, there was an
increase in the emission of blue light and a concomi-
tant decrease in the emission of green light, illustrating
that the variants were no longer appropriately juxta-
posed to allow FRET53. A similar strategy showed that
chimeras of red- and blue-shifted variants emitted a
fluorescence peak at 505 nm when excited at 385 nm
owing to FRET that was lost when the two proteins
were separated55. Fusion proteins capable of FRET
from BGFP to RGFP have been linked by the calmodu-
lin (CaM)-binding domain from smooth-muscle light-
chain kinase (Fig. 5a). When this protein (termed
FIP–CBSM) is excited at 380 nm, the fluorescence
emission peaks at 505 nm. However, on binding of acti-
vated CaM [(Ca21)4–CaM] to the linker, the two GFP
moieties move apart, diminishing the FRET and result-
ing in a decrease in emission at 505 nm (RGFP) and
an increase at 440 nm (BGFP) (Fig. 5a). Because FRET
is a nondestructive process, continual photon emission
can occur as long as a source of excitation is provided
through appropriate filter sets. Expression of FIP–CBSM
in living cells means, therefore, that emission ratios can
be used to image spatial and temporal changes in fluor-
escence (Box 2). Changes in cytosolic [Ca21] (in the
range of 50 nM–1 mM) paralleled the changes in fluor-
escence emission caused by the binding of
(Ca21)4–CaM to FIP–CBSM (Fig. 6)56. Further modi-
fications of FIP–CBSM have been made by engineering
modified CaM onto its C-terminus. This modification
rendered FIP–CBSM sensitive to [Ca21] (kd 5 100 nM),
the affinity of which could be decreased by altering the

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a 380 nm 510 nm 380 nm 440 nm
b 370 nm 440 nm 370 nm 510 nm
440 nm 480 nm 440 nm 535 nm
BGFP or CGFP CaM
RGFP, EGFP or YGFP CaM-binding site
Figure 5
Scheme illustrating fluorescence-resonance-energy transfer between covalently linked
green fluorescent protein (GFP) variants. (a) Chimera of blue-shifted (BGFP) and red-
shifted (RGFP) GFP variants joined by the calmodulin (CaM)-binding domain of smooth-
muscle myosin-light-chain kinase. When (Ca21)4–CaM binds to this chimera and is excited
at 380 nm, there is a decrease in the emission of light at 510 nm and a concomitant
increase in emission of light at 440 nm. Fusion of an engineered CaM to the C terminus
of this chimera resulted in an indicator that responded directly to [Ca21]57 (not shown).
(b) ‘Cameleons’ are chimeras of GFP variants linked by CaM and the CaM-binding
peptide M1358. Increases in [Ca21] are quantified by an increase in the ratio of
440–510 nm emission (green cameleon) or 480–535 nm emission (yellow cameleon).
amino acid composition of the linker (CaM-binding)
sequence, to a kd of 280 nM. These modifications have
generated ideal indicators that can be targeted to dif-
ferent subcellular locations (Fig. 7) and used to moni-
tor changes in cytosolic [Ca21], which has a dynamic
range in nonexcitable cells of between about 100 nM
and 1 mM57.
GFP-based FRET technology has opened an excit-
ing new era in intracellular signalling with a new gen-
eration of GFP-based fluorescent indicators called
‘cameleons’, with different spectral characteristics and
affinities for Ca21. These indicators differ in basic con-
cept to that outlined above, in that an increase in FRET
occurs in response to an increase in [Ca21] [as measured
by the ratio of the intensity of light emitted at 440 nm to
510 nm (green cameleon) or 480 nm to 535 nm (yellow
cameleon)]. This has been achieved by generating a
fusion of two GFP molecules linked by both CaM and
the CaM-binding peptide, M13 (Fig. 5b). When Ca21
binds to CaM, it interacts with M13, bringing the two
GFP moieties into close proximity and allowing FRET
to occur. Further mutations, engineered into the Ca21-
binding sites of the CaM moiety of these chimeras,
have resulted in an array of cameleons with different
affinities for Ca21 (10 nM–10 mM). Cameleons have
been targeted to the ER (using a similar strategy to that
used with recombinant aequorin37), in which a com-
bination of their Ca21 affinity, the nondestructive
nature of FRET and a high quantum yield makes them
222
Figure 6
Real-time detection of calmodulin activation in living cells using
FIP–CBSM (chimera of red- and blue-shifted green fluorescent pro-
teins linked by the calmodulin-binding domain56). Cells were micro-
injected with solutions containing 88 mM FIP–CBSM (a) or 88 mM
FIP–CBSM and 88 mM calmodulin (b). At the indicated times, 3 mM
BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid],
4 mM TRH (thyrotropin hormone-releasing hormone), 5 mM CaCl2 and
500 nM ionomycin were added to the bathing solution. The average
emission intensity at 510 nm (F) of 12–27 individual cells following
excitation at 380 nm is expressed as F/F0, with F0 defined as the
average of the first ten values in the time course. (c) A representa-
tive cell [human embryonic kidney-cell line (HEK-293)] stably trans-
fected with a humanized version of FIP–CBSM. At the indicated times,
200 mM ATP, 5 mM EGTA [ethylene glycol-bis(b-aminoethylether)-
N,N,N9,N9-tetraacetic acid], 35 mM BAPTA-AM (acetoxy methyl ester
of BAPTA) 5 mM CaCl2 and 2 mM ionomycin were added to the bathing
solution, which was replaced with a nominally Ca21-free solution at
the indicated time (A. Persechini, unpublished).
suitable for reporting a resting [Ca21] in the region of
60–400 mM, which falls to 1–50 mM following agonist
stimulation58. This generation of indicators has the
potential to overcome the limitations encountered with
recombinant aequorin, such as Ca21-dependent con-
sumption of the protein, low quantum yield and the
requirement for coelenterazine.
The future for GFP remains exciting. Its mutational
analysis will no doubt continue, with the development
of brighter mutants with more suitable excitation and
emission spectra. Indeed, a recent publication describes
a GFP that is photoactivated by blue light (488 nm) in
anaerobic bacteria to generate a GFP that absorbs green
light (525 nm) and emits red light (lmax 600 nm)59. In
addition, it is likely that FRET between GFP variants
will generate other indicators for visualizing key events
at the subcellular level.
TIBTECH MAY 1998 (VOL 16)

Figure 7
Fluorescence of a plasma-membrane-targeted FIP–CBSM (chimera
of red- and blue-shifted green fluorescent proteins linked by the
calmodulin-binding domain56) in live cells. Confocal microscopy sec-
tion of HEK-293 (human embryonic kidney-cell line) cells transiently
expressing the FIP–CBSM chimera targeted to the plasma membrane
by the addition of a myristoylation consensus sequence. The
fluorescence of the red-shifted moiety of the chimera was detected
at 510 nm following excitation at 488 nm. Illumination of the
plasma membrane illustrates that green fluorescent protein, or vari-
ants of it, can be used to visualize organelle structure and protein
distribution in living cells (C. Strubing, unpublished).
Conclusion
The cloning of the gene for aequorin led to exploi-
tation of the recombinant protein in many biotechnologi-
cal applications, particularly in monitoring subcellular
Ca21 homeostasis in living cells. The other protein
involved in the bioluminescence of A. victoria, GFP, con-
tains its own chromophore and is brighter than aequorin,
making it more amenable to imaging events in single
cells. Mutations of GFP have resulted in brighter recom-
binant proteins with different colours. These GFPs have
been engineered and, learning from the mechanism
employed by the jellyfish to emit light, rendered sensi-
tive to changes in their local environment. Combined
with the targeting technology applied to recombinant
aequorin, GFP has become a powerful new tool in cell
biology to visualize key events such as mobilization of
intracellular Ca21 and protein dynamics in living cells.
Acknowledgments
We thank A. K. Campbell and D. E. Clapham for
their support, and C. H. George for help in preparation
of the figures. J. M. Kendall is funded by the Medical
Research Council; M. N. Badminton is the recipient of
a Wellcome Trust Advanced Training Fellowship.
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Development of biocompatible synthetic

extracellular matrices for tissue engineering
Byung-Soo Kim and David J. Mooney

Tissue engineering may provide an alternative to organ and tissue transplantation, both of which suffer from a limitation of
supply. Cell transplantation using biodegradable synthetic extracellular matrices offers the possibility of creating completely
natural new tissues and so replacing lost or malfunctioning organs or tissues. Synthetic extracellular matrices fabricated from
biocompatible, biodegradable polymers play an important role in the formation of functional new tissue from transplanted
cells. They provide a temporary scaffolding to guide new tissue growth and organization, and may provide specific signals
intended to retain tissue-specific gene expression.

he loss or failure of an organ or tissue is one of with approximately 50 000 patients registered on trans-

T the most severe human health problems. Treating

patients with these health problems consumes
approximately half of the total annual health-care costs
in the USA1. Tissue or organ transplantation is a stan-
dard therapy to treat these patients, but this is severely
limited by a donor shortage. For example, fewer than
7500 organs were available for transplantation in 1996,
B-S. Kim and D. J. Mooney (mooneyd@umich.edu) are at the
Department of Chemical Engineering, University of Michigan, Ann
Arbor, MI 48109, USA. D. J. Mooney is also at the Department of
Biologic and Materials Sciences, University of Michigan, Ann Arbor,
MI 48109, USA.
plant waiting lists in the USA alone (United Network
for Organ Sharing, 1997). Other available therapies to
treat these patients include surgical reconstruction (e.g.
heart), drug therapy (e.g. insulin for a malfunctioning
pancreas), synthetic prostheses (e.g. polymeric vascular
prostheses) and mechanical devices (e.g. kidney dialysers).
Although these therapies are not limited by supply, they
do not replace all functions of a lost organ or tissue and
often fail in the long term1.
Tissue engineering has emerged as a promising
approach to treat the loss or malfunction of a tissue or
organ without the limitations of current therapies. This
approach has a foundation in the biological observation

224 Copyright © 1998, Elsevier Science Ltd. All rights reserved. 0167 – 7799/98/$19.00. PII: S0167-7799(98)01191-3 TIBTECH MAY 1998 (VOL 16)