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Kendall 1998
Kendall 1998
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.
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
Coelenteramide remains noncovalently bound to the
protein, emitting blue light (lmax 5 470 nm) as it
+Ca2+ relaxes to the ground state (Fig. 1)7,8. The quantum
yield of this reaction (FCL, defined in Box 2) is
Ca3–Apo-aequorin–coelenteramide*
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
+GFP be calibrated as rate constants over a range of [Ca21]
In vivo In vitro (effectively 100 nM – 10 mM9), which corrects for the
Green light Blue light cooperativity between the three Ca21-binding sites
λ max = 509 nm λ max = 469 nm {photons emitted are proportional to [Ca21]2.5–[Ca21]3
(Ref. 10)} and consumption of the photoprotein
Figure 2 during the reaction11.
The pathway responsible for the bioluminescence of A. victoria. Ca21 Coelenterazine is chemically modified during the
binds to aequorin, which catalyses the oxidation of coelenterazine light-emitting reaction, and so active aequorin must be
to coelenteramide. The excited state (*) of coelenteramide bound regenerated before the cycle can continue. The Ca21
to apo-aequorin emits blue light in vitro. However, in vivo, radiation- must therefore be removed from the protein and fresh
less energy is transferred from the excited state to green fluorescent coelenterazine supplied. Although the mechanism by
protein, which emits green light. which the jellyfish achieves this has yet to be estab-
lished, the apoprotein can easily be reactivated in vitro
when these sites are occupied by Ca21, aequorin to form the photoprotein by the addition of a Ca21
undergoes a conformational change that converts it chelator and coelenterazine in the presence of a
into an oxygenase (luciferase) and provides the appro- reducing agent and molecular oxygen7.
priate environment for the oxidation of coelenterazine.
Coelenterazine is converted into a highly unstable Green fluorescent protein
dioxetanone intermediate, which loses carbon dioxide A. victoria bioluminescence is blue–green owing to
to form the excited phenolate anion of coelenteramide. the presence of GFP, which derives its light-emitting
Aequorin is the most widely studied of the seven Ca21-binding The successful biotechnological application of light-
photoproteins described to date2. Sequence homologies suggest that emitting proteins is dependent on efficient methods
aequorin has evolved from Ca21-binding proteins such as calmodulin to detect and monitor light. These must be capable of
(CaM)60. Recombinant-DNA technology and the sensitivity of the light- detecting light signals over a wide range of intensities
emitting reaction18 have permitted the generation of mutated and spectral responses. There are a number of dif-
recombinant aequorins, that have illuminated the structure–function ferent methods for detecting light2, for example lumi-
relationship of the photoprotein. nometers and image-intensified charge-coupled
The position of the EF hands in aequorin are highly conserved com- devices (CCDs). Luminometers usually house photo-
pared with CaM, with the exception that site II has been lost from the multiplier tubes, which can operate over six to seven
photoprotein60. Substitution of the key glycine residue (position 6) in orders of magnitude of detection and have a wide
the EF-hand sites of apo-aequorin showed that sites I and II are spectral response. In order to capture the spatial and
required for the bioluminescence of the protein9,40,61,62. This led to temporal organization of events, particularly at the
speculation that the area between these two sites (corresponding to subcellular level, a process called ‘imaging’ has been
site II in CaM) may have evolved for coelenterazine binding or catalytic- developed. The basic principle of imaging relies on
site formation, controlled by Ca21-binding to the two EF-hand sites converting light signals to electrical signals, which
that surround it. Aequorin contains a relatively high content of aromatic can be amplified whilst retaining spatial resolution.
amino acid residues compared with other Ca21-binding proteins. Sub- The practical choice for imaging is a CCD linked to a
stitution of tryptophan with phenylalanine between EF-hand sites I and microscope. One problem encountered with all light-
II identified two residues that may play key roles in the catalytic activ- detection systems is that their efficiency is poor and
ity of the protein; residue 58 in oxygen binding62 and residue 86 in that .90% of light is lost. The limits of detection will,
generating the excited state in aequorin bioluminescence63. obviously, also depend on the amount of light gen-
The most interesting discovery to have emerged from the structure– erated by the recombinant protein. This is called the
function analyses is that the C-terminal proline residue (position 189) quantum yield (FCL) and is equivalent to the number
is essential for the normal bioluminescent activity of the protein, poss- of photons emitted divided by the number of molecules
ibly in stabilizing the coelenterazine. Removal or substitution of this reacting; the ΦCL for aequorin is between 0.12 and
residue has deleterious effects on the specific activity, stability and 0.20 (Ref. 2), whereas that for green fluorescent
Ca21-independent light emission of the recombinant protein64,65. protein is 0.72–0.85 (Ref. 17).
energy from aequorin. This process of radiationless ent spatial and temporal patterns of changing cytosolic
energy transfer occurs as the excited Ca21–apo- [Ca21], both in cell populations and at the single-cell
aequorin–coelenteramide complex decays to its ground level. This probably reflects the need for the specifi-
state, exciting the GFP, which, in turn, emits green cation of signals conveyed by a wide array of agonists
light (Fig. 2). GFP is a 238 amino acid protein12, that must operate in many different cell types. Regu-
arranged as an 11-stranded barrel of b sheets sur- lation of Ca21 signals involves the storage of Ca21 within
rounding a central a-helical heptapeptide that forms intracellular organelles, stimulus-activated release and
the fluorescent centre (Fig. 1)12,13,15. The fluorophore regulated influx of Ca21 from outside the cell19.
is formed by the cyclization and oxidation of three Aequorin was first used to investigate intracellular
amino acids, serine–dehydrotyrosine–glycine, at pos- calcium homeostasis in contracting muscle fibres from
itions 65–6716. GFP has a major excitation peak at giant barnacles20. The photoprotein was introduced
395 nm (with a minor peak at 475 nm), an emission into these cells by microinjection, a technique that has
maximum at 508 nm and a quantum yield of been applied to progressively smaller cells, for example,
0.72–0.8517. As the light-emitting properties of GFP oocytes, myocytes and hepatocytes9. A major goal in
are independent of substrates and cofactors, the protein biological research has, however, been the development
has rapidly become a popular choice for monitoring a of readily available, user-friendly, noninvasive tech-
vast array of biological processes, ranging from gene niques to monitor events inside living cells or organ-
expression to organelle organization. isms. The cloning of the gene for aequorin6 has allowed
recombinant expression of the photoprotein, circum-
Biotechnological applications of aequorin and venting the need for microinjection. This led to genetic
GFP transformation of bacteria, yeast, plant and animal cells
Light-emitting reactions have assumed an important with the gene encoding apo-aequorin. A cDNA
role in many biotechnological applications because of encoding apo-aequorin expressed in Escherichia coli pro-
their sensitivity and specificity, ease of detection, and duced a functional recombinant protein when bac-
noninvasive nature18. The protein components, and the terial extracts were combined with coelenterazine21; as
very mechanism responsible for the bioluminescence coelenterazine can readily permeate cell membranes,
of the jellyfish itself, are now being exploited in an this facilitated activation of the recombinant protein in
exciting new era for A. victoria. situ. The first measurements of cytosolic Ca21 transients
were made with recombinant aequorin from stimuli as
Aequorin diverse as wind (plants), complement attack (bacteria)
Many diverse cellular functions are regulated by and mating pheromones (yeast)22–24. These early experi-
changes in intracellular [Ca21] in response to a ments have been elaborated by imaging light signals
number of different classes of extracellular stimuli. (Box 2) and equating them with cytosolic Ca21 signals
Extensive use of fluorescent Ca21-chelating dyes (e.g. in transgenic multicellular organisms expressing recom-
fura-2) has revealed that agonists produce many differ- binant aequorin25 and in an organotypic context by
restricting expression of the transgene to specific cell These increases resulted in the activation of intramito-
types26. chondrial enzymes such as pyruvate dehydrogenase31.
The most exciting development to have arisen from Changes in intranuclear [Ca21] were also shown to dif-
the cloning of the apo-aequorin cDNA has been the fer from those occurring in the cytosol32. These differ-
development of methods to measure [Ca21] in discrete ent signals were dependent on whether the Ca21
subcellular domains, such as the lumen of different originated from an intracellular or extracellular source
organelles. This has been achieved both with minimal (Fig. 4) and may play a role in regulating nuclear
targeting sequences and by fusion with resident events33. Targeted aequorin has also revealed that the
organellar proteins that already include the information area directly beneath the plasma membrane has a [Ca21]
necessary for efficient localization (Table 1)27. Chimeras that is significantly higher than the bulk cytosol34. Such
of apo-aequorin and organellar proteins or targeting sub-plasma-membrane microdomains may be involved
sequences are simple to generate by standard cloning in controlling intercellular communication via gap
techniques, as is their introduction into cells by tran- junctions35.
sient or stable transfection (Fig. 3; Table 1)28. The tar- Despite these pioneering experiments, measuring
geting strategy offers clear advantages over alternative [Ca21] in discrete subcellular locations with targeted
methods, for example, fluorescent Ca21-chelator dyes, aequorin has not been without problems. This is exem-
which display no selective localization and are more plified by the difficulties encountered when measuring
sensitive to changes in their environment than [Ca21] in the endoplasmic reticulum (ER). Ca21 in the
aequorin29. ER controls an array of cellular functions including
The first successful investigation of organellar Ca21 protein biosynthesis36 and Ca21 influx through the
homeostasis using an aequorin chimera was made in plasma membrane, as well as providing an agonist-
mitochondria (Table 1). Rizzuto et al.30 showed that sensitive store of rapidly mobilizable Ca21 (Ref. 19).
the stimulation of receptors coupled to the generation Different strategies have been adopted to find a suitable
of the second messenger inositol-1,4,5-trisphosphate way to employ recombinant aequorin as an ER
produced increases in intramitochondrial [Ca21], Ca21 probe. Addition of the –KDEL sequence to the
exceeding the accompanying rise in cytosolic [Ca21]. C terminus of the protein aids ER targeting (Fig. 3),
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.
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). Figure 4
Comparison of nuclear and cytosolic Ca21 signals measured with
appropriately targeted aequorins. (a) HeLa cells expressing nuclear-
but results in Ca21-independent light emission and targeted or cytosolic aequorin were challenged by exposure to ATP.
problems in calibrating the light signal37. Although Initial increases in nuclear and cytosolic [Ca21] were similar, followed
alternative targeting strategies, which do not involve by a greater increase in the cytosolic signal than the nuclear signal,
modification of the C terminus have been used38,39, the with the nuclear signal returning to baseline more rapidly. Subse-
sensitivity of aequorin for Ca21 was too high to quent stimulation with histamine (Hist) changed neither nuclear nor
measure steady-state ER [Ca21] values, even when cytosolic [Ca21] but restoration of extracellular Ca21 induced an
using an aequorin engineered to have a kd 20 times increase in only cytosolic [Ca21]. (b) Treatment with cyclopiazonic
lower than the wild type (Box 1)38,40. acid induced an increase in cytosolic [Ca21] that clearly preceded
A range of coelenterazine analogues (h-, n- and e-) the increase in nuclear [Ca21].
have been synthesized and used to generate semisyn-
thetic aequorins with different light-emitting proper-
ties and Ca21 sensitivities41. Despite the theoretical both proteins and are highly suitable for use in
attraction of using semisynthetic aequorins to measure immunoassays46.
a wider range of [Ca21], they actually have limited
application42,43, as they are less stable and have signifi- Green fluorescent protein
cantly lower quantum yields than native aequorin41. Mutational analyses of GFP, particularly in the fluoro-
A further application has been the use of recombi- phore region, have generated a variety of GFP iso-
nant aequorin as a quantitative label in different ana- forms with different characteristics to those of the
lytical assay systems, thereby enabling high detection native protein. The first functional mutation to have a
sensitivity in the presence of excess Ca21. The proper- significant biotechnological impact was S65T. This
ties of aequorin make it an ideal label in a number of generated not only a brighter and more stable GFP but
different assay systems: it can be readily conjugated (e.g. also altered the protein’s excitation spectrum charac-
to antibodies and to substrates); it generates a high teristics (Table 2)47. The increase in brightness and
signal-to-noise ratio with a broad range of detection improved folding have been of paramount importance
sensitivity; and it generates a signal rapidly18. Fusion of to the success of GFP. Combined with modifications
apo-aequorin cDNA to the coding sequence of pro- such as species-specific codon optimization48, GFP has
tein-A signal peptide and its subsequent expression in now assumed a role as an ideal reporter of protein
bacteria leads to secretion of the apoprotein, which can dynamics and gene expression both in multicellular
be purified and subjected to a variety of chemical organisms and at the subcellular level. Over the past few
modifications for use in different assay systems44,45. years, there has been an explosion of interest in the
Alternatively, fusion proteins have been generated (e.g. employment of GFP as a protein tag, a reporter of gene
antibody–apo-aequorin) that retain the functions of expression and to investigate organelle structure17,49,50.
Table 2. Wild-type GFP and GFP variants with altered spectral characteristics
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
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 posed to allow FRET53. A similar strategy showed that
fluorophore, have generated a new range of GFPs with chimeras of red- and blue-shifted variants emitted a
altered excitation and emission spectra. Substitution of fluorescence peak at 505 nm when excited at 385 nm
the central tyrosine residue at position 66 shifts the owing to FRET that was lost when the two proteins
excitation and emission spectra to shorter wavelengths, were separated55. Fusion proteins capable of FRET
to generate a family of proteins called ‘blue-shifted’ from BGFP to RGFP have been linked by the calmodu-
variants51. Mutations of other amino acid residues lin (CaM)-binding domain from smooth-muscle light-
have generated cyan-, red- and yellow-shifted variants chain kinase (Fig. 5a). When this protein (termed
of GFP with altered spectral characteristics FIP–CBSM) is excited at 380 nm, the fluorescence
(Table 2)14,52–54. Radiationless transfer of energy from emission peaks at 505 nm. However, on binding of acti-
aequorin to GFP in A. victoria relies on the emission vated CaM [(Ca21)4–CaM] to the linker, the two GFP
spectrum of aequorin overlapping with the excitation moieties move apart, diminishing the FRET and result-
spectrum of GFP. The mutations in GFP have gener- ing in a decrease in emission at 505 nm (RGFP) and
ated fluorescent proteins with excitation and emission an increase at 440 nm (BGFP) (Fig. 5a). Because FRET
spectra that overlap and it therefore became feasible to is a nondestructive process, continual photon emission
exploit radiationless energy transfer, in this case fluor- can occur as long as a source of excitation is provided
escence-resonance-energy transfer (FRET), to moni- through appropriate filter sets. Expression of FIP–CBSM
tor molecular interactions. For example, Y66H (blue- in living cells means, therefore, that emission ratios can
shifted variant, or BGFP) was joined to S65C be used to image spatial and temporal changes in fluor-
(red-shifted variant, or RGFP) by fusing their cDNAs escence (Box 2). Changes in cytosolic [Ca21] (in the
with an amino acid linker sequence incorporating a range of 50 nM–1 mM) paralleled the changes in fluor-
protease site. Excitation of the chimera at 368 nm escence emission caused by the binding of
resulted in the emission of green light at 510 nm. When (Ca21)4–CaM to FIP–CBSM (Fig. 6)56. Further modi-
the chimera was cleaved with trypsin, there was an fications of FIP–CBSM have been made by engineering
increase in the emission of blue light and a concomi- modified CaM onto its C-terminus. This modification
tant decrease in the emission of green light, illustrating rendered FIP–CBSM sensitive to [Ca21] (kd 5 100 nM),
that the variants were no longer appropriately juxta- the affinity of which could be decreased by altering the
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
Figure 6
at 380 nm, there is a decrease in the emission of light at 510 nm and a concomitant
Real-time detection of calmodulin activation in living cells using
increase in emission of light at 440 nm. Fusion of an engineered CaM to the C terminus
FIP–CBSM (chimera of red- and blue-shifted green fluorescent pro-
of this chimera resulted in an indicator that responded directly to [Ca21]57 (not shown).
teins linked by the calmodulin-binding domain56). Cells were micro-
(b) ‘Cameleons’ are chimeras of GFP variants linked by CaM and the CaM-binding
injected with solutions containing 88 mM FIP–CBSM (a) or 88 mM
peptide M1358. Increases in [Ca21] are quantified by an increase in the ratio of
FIP–CBSM and 88 mM calmodulin (b). At the indicated times, 3 mM
440–510 nm emission (green cameleon) or 480–535 nm emission (yellow cameleon).
BAPTA [1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9-tetraacetic acid],
4 mM TRH (thyrotropin hormone-releasing hormone), 5 mM CaCl2 and
amino acid composition of the linker (CaM-binding) 500 nM ionomycin were added to the bathing solution. The average
sequence, to a kd of 280 nM. These modifications have emission intensity at 510 nm (F) of 12–27 individual cells following
generated ideal indicators that can be targeted to dif- excitation at 380 nm is expressed as F/F0, with F0 defined as the
ferent subcellular locations (Fig. 7) and used to moni- average of the first ten values in the time course. (c) A representa-
tor changes in cytosolic [Ca21], which has a dynamic tive cell [human embryonic kidney-cell line (HEK-293)] stably trans-
range in nonexcitable cells of between about 100 nM fected with a humanized version of FIP–CBSM. At the indicated times,
and 1 mM57. 200 mM ATP, 5 mM EGTA [ethylene glycol-bis(b-aminoethylether)-
GFP-based FRET technology has opened an excit- N,N,N9,N9-tetraacetic acid], 35 mM BAPTA-AM (acetoxy methyl ester
ing new era in intracellular signalling with a new gen- of BAPTA) 5 mM CaCl2 and 2 mM ionomycin were added to the bathing
eration of GFP-based fluorescent indicators called solution, which was replaced with a nominally Ca21-free solution at
‘cameleons’, with different spectral characteristics and the indicated time (A. Persechini, unpublished).
affinities for Ca21. These indicators differ in basic con-
cept to that outlined above, in that an increase in FRET suitable for reporting a resting [Ca21] in the region of
occurs in response to an increase in [Ca21] [as measured 60–400 mM, which falls to 1–50 mM following agonist
by the ratio of the intensity of light emitted at 440 nm to stimulation58. This generation of indicators has the
510 nm (green cameleon) or 480 nm to 535 nm (yellow potential to overcome the limitations encountered with
cameleon)]. This has been achieved by generating a recombinant aequorin, such as Ca21-dependent con-
fusion of two GFP molecules linked by both CaM and sumption of the protein, low quantum yield and the
the CaM-binding peptide, M13 (Fig. 5b). When Ca21 requirement for coelenterazine.
binds to CaM, it interacts with M13, bringing the two The future for GFP remains exciting. Its mutational
GFP moieties into close proximity and allowing FRET analysis will no doubt continue, with the development
to occur. Further mutations, engineered into the Ca21- of brighter mutants with more suitable excitation and
binding sites of the CaM moiety of these chimeras, emission spectra. Indeed, a recent publication describes
have resulted in an array of cameleons with different a GFP that is photoactivated by blue light (488 nm) in
affinities for Ca21 (10 nM–10 mM). Cameleons have anaerobic bacteria to generate a GFP that absorbs green
been targeted to the ER (using a similar strategy to that light (525 nm) and emits red light (lmax 600 nm)59. In
used with recombinant aequorin37), in which a com- addition, it is likely that FRET between GFP variants
bination of their Ca21 affinity, the nondestructive will generate other indicators for visualizing key events
nature of FRET and a high quantum yield makes them at the subcellular level.
41 Shimomura, O., Musicki, B., Kishi, Y. and Inouye, S. (1993) Cell 56 Romoser, V. A., Hinkle, P. M. and Persechini, A. (1997) J. Biol.
Calcium 14, 373–378 Chem. 272, 13270–13274
42 Knight, M. R., Read, N. D., Campbell, A. K. and Trewavas, A. J. 57 Persechini, A., Lynch, J. A. and Romoser, V. A. (1997) Cell Calcium
(1993) J. Cell Biol. 121, 83–90 22, 209–216
43 Montero, M., Barrero, M. J. and Alvarez, J. (1997) FASEB J. 11, 58 Miyawaki, A. et al. (1997) Nature 388, 882–887
881–885 59 Elowitz, M. B., Surette, M. G., Wolf, P-E., Stock, J. and Leibler, S.
44 Jackson, R. J., Fujihashi, K., Kiyono, H. and McGhee, J. R. (1996) (1997) Curr. Biol. 7, 809–812
J. Immunol. Methods 190, 189–197 60 Tsuji, F. I., Ohmiya, Y., Fagan, T. F., Toh, H. and Inouye, S. (1995)
45 Lizano, S., Ramanathan, S., Feltus, A., Witowski, A. and Daunert, S. Photochem. Photobiol. 62, 657–661
(1997) Methods Enzymol. 279, 296–303 61 Persechini, A., Moncrief, N. D. and Kretsinger, R. H. (1989) Trends
46 Casadei, J., Powell, M. J. and Kenten, J. H. (1990) Proc. Natl. Acad. Neurosci. 12, 462–467
Sci. U. S. A. 87, 2047–2051 62 Tsuji, F. I., Inouye, S., Goto, T. and Sakaki, Y. (1986) Proc. Natl.
47 Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995) Nature 373, Acad. Sci. U. S. A. 83, 8107–8111
663–664 63 Ohmiya, Y., Ohashi, M. and Tsuji, F. I. (1992) FEBS Lett. 301,
48 Haas, J., Park, E-C. and Seed, B. (1996) Curr. Biol. 6, 197–201
315–324 64 Nomura, M., Inouye, S., Ohmiya, Y. and Tsuji, F. I. (1991) FEBS
49 Misteli, T. and Spector, D. L. (1997) Nat. Biotechnol. 15, 961–964 Lett. 295, 63–66
50 Lippincott-Schwartz, J. and Smith, C. L. (1997) Curr. Opin. 65 Watkins, N. J. and Campbell, A. K. (1993) Biochem. J. 293, 181–185
Neurobiol. 5, 631–639 66 Badminton, M. N., Kendall, J. M., Sala-Newby, G. and Campbell, A. K.
51 Heim, R., Prasher, D. C. and Tsien, R. Y. (1994) Proc. Natl. Acad. (1995) Exp. Cell Res. 216, 236–243
Sci. U. S. A. 91, 12501–12504 67 Brini, M., Murgia, M., Pasti, L., Picard, D., Pozzan, T. and Rizzuto, R.
52 Delagrave, S., Hawtin, R. E., Silva, C. M., Yang, M. M. and (1993) EMBO J. 12, 4813–4819
Youvan, D. C. (1995) Biotechnology 13, 151–154 68 Xiong, Z-H. and Ruben, L. (1996) Mol. Biochem. Parisitol. 83, 57–67
53 Heim, R. and Tsien, R. Y. (1996) Curr. Biol. 6, 178–182 69 Kendall, J. M., Dormer, R. L. and Campbell, A. K. (1992) Biochem.
54 Cormack, B. P., Valdivia, R. H. and Falkow, S. (1996) Gene 173, Biophys. Res. Commun. 189, 1008–1016
33–36 70 Brini, M. et al. (1997) Mol. Biol. Cell 8, 129–143
55 Mitra, R. D., Silva, C. M. and Youvan, D. C. (1996) Gene 173, 71 Daguzan, C., Nicolas, M-T., Mazars, C., Leclerc, C. and Moreau, M.
13–17 (1995) Int. J. Dev. Biol. 39, 653–657
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-
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)