Ka
Kaleidoscop
pe 6.1, Charrlotte Hurstt, “Aequoriin: glow in the dark p
protein”
Aequoorin: glow
w in the da
ark prote in
CHARLO
OTTE HURS
ST
Introduction
Co
olour is an indispensable mode of communica
ation within nature. It iss used to co
onvey all
kinds off information
n between both
b
individ
duals and sp
pecies as a whole.
w
For eexample, ma
any frogs
use brig
ght colours to advertise
e that they are poison
nous to disssuade poten
ntial predato
ors. Male
peacockks also use distinctive
d
brightly colou red and pattterned tails to attract m
mates. Howev
ver, these
colours are only effe
ective during
g daylight ho
ours. There are many an
nimals that o
occupy envirronments
where light levels are very low,, such as du
uring the nig
ght or in the
e depths off the ocean to which
colours are of no usse. For these
e species, pro
oducing ligh
ht instead off displaying different colours can
be used as a meanss of commun
nication. Thiss illuminating ability is known
k
as bio
oluminescen
nce.
Allthough “glo
ow-in-the-da
ark” is not a trait norm
mally associiated with n
nature, it is a more
common
n occurrence than man
ny would b
believe. Num
merous biolu
uminescent organisms exist on
Earth, fro
a
glow wo
orms to various fungi and bacteria,, to name but a few.
om Anglerfish, fireflies and
While ttheir natural habitat iss far-removved from la
aboratories and scienttific researcch, these
biolumin
nescent orga
anisms have their part to
o play in the
e world of sc
cientific reseearch. Many will have
heard o
of the bioluminescent jellyfish, Aeq
quorea victo
oria, as is it where thee Green Flu
uorescent
Protein (GFP), a tool widely used in molecu
ular biology, originates. Much
M
like a GFP-tagged
d protein,
Aequoreea victoria’s naturally grreenish hue is also due
e to GFP fluorescence. H
However, GF
FP is not
the only useful protein
p
isola
ated from this specie
es: within the
t
jellyfish
h, GFP fluo
orescence
is stimullated by the photon emissions from
m another pro
otein named
d aequorin (M
Morise et all., 1974).
Discoverry of aequorrin
Ovver 50 yea
ars ago, a scientist n
named Osamu Shimom
mura was p
presented with
w
the
opportunity to stud
dy biolumine
escence in jjellyfish. Aftter arriving in Princeton
n, he was presented
p
ht when mixxed with sea
awater (Shim
momura et al.,
a 1962).
with a “squeezate” that would give off ligh
ueezate wass made fro
om the biolluminescent Aequorea victoria jelllyfish found
d around
This squ
the nea
arby Friday Harbour, Washington
n. After ma
any setback
ks and tho
ousands of jellyfish,
23
Kaleidoscope 6.1, Charlotte Hurst, “Aequorin: glow in the dark protein”
the proteins responsible for Aequorea’s bioluminescence were purified and identified (Shimomura,
1995). Shimomura named the proteins “aequorin”, after the jellyfish genus, and “Green Fluorescent
Protein” (GFP). Further research revealed the link between aequorin and GFP, which causes
the greenish hue given off by aequorea in nature, but the secrets to aequorin’s bioluminescence
still remained elusive. As the squeezate had produced luminescence in the presence of seawater,
Shimomura quickly discovered that it was calcium ions (Ca2+) present in the seawater that held
the key to unlock aequorin’s luminescence. Further research showed that the aequorin protein
is made of two subunits, apoaequorin and coelenterazine which are required for luminescence.
Binding of Ca2+ to apoaequorin causes the coelenterazine to undergo a conformational change
and emit low levels of blueish light. This is absorbed by GFP and the resulting fluorescence gives
Aequorea victoria its characteristic greenish hue (Morise et al., 1975, Shimomura & Johnson, 1975).
Since its discovery, the properties of aequorin have made it a useful tool in molecular biology
research. The creation of plants and animal cells genetically modified to express apoaequorin
has allowed the study of intracellular Ca2+ levels. As apoaequorin alone cannot bioluminesce,
coelenterazine must be provided to the cells to produce bioluminescence (Knight et al., 1991,
1993).
Uses of aequorin
Calcium is important for all living organisms, not just for those like Aequorea that use it
to bioluminesce (Campbell 1988, White & Broadley, 2003). It is important as a nutrient, and as
part of information networks within cells. Much research has been carried out using aequorin
to investigate Ca2+ in cells (Campbell, 1983, Williamson & Ashley 1982). Aequorin was first
stably transformed into plant cells (Knight et al., 1991), and later into animal cells, to uncover
more about the information networks in cells. The discovery of aequorin as a Ca2+-responsive
protein, and the creation of transgenic plants expressing apoaequorin created a powerful tool
that allowed the possibility to dissect these cellular information networks. Highly sensitive
imaging equipment is used to capture the luminescence given off by aequorin upon it binding
Ca2+ in cells, as the luminescence signal is generally very low (Knight et al., 1993).
In plants, information about the external environment such as light intensity, water and
nutrient availability, temperature or bacterial pathogens is passed into cells by specialised
receptors built into the cell membrane. This information must be encoded and relayed to the
nucleus of cells so that the organism can respond. This environmental information can be
encoded many ways, but notably by waves of Ca2+ ions (McAinsh & Pittman, 2009).
Information about different environmental conditions is stored in the duration and intensity
of Ca2+ influx into cells. These have been called “calcium signatures” as each is unique to the
environmental condition that caused it (Whalley & Knight, 2012). As Ca2+ is released
throughout the cell in response to a stimulus, this causes transgenic plants expressing
aequorin to produce a luminescent signal, which can be captured by imaging equipment
24
Kaleidoscope 6.1, Charlotte Hurst, “Aequorin: glow in the dark protein”
and used to analyse Ca2+ signatures (Knight et al., 1993). Ca2+ influx affects many cellular
processes such as protein phosphorylation, which ultimately alters gene expression within cells
allowing them to respond and adapt to changing environmental conditions (Whalley & Knight,
2012).
Transgenic plants and animal cell lines have since been created to express the aequorin
protein at specific locations within the cell (Mehlmer et al., 2012, Davies & Terhaz 2009). Research
is now centred on decoding these calcium signatures within cells to find out more about
the cellular language of calcium signalling.
Bibliography
Campbell, A. K. (1988). “Calcium as an intracellular regulator.” In Calcium in Human Biology, edited
by B. E. C. Nordin, pp. 261-316. London: Springer-Verlag, 1988.
Campbell, A.K. (1983). “Intracellular Calcium: Its Universal Role As Regulator” p. 556. Chichester:
Wiley, 1983.
Davies S.A. & Terhzaz S. “Organellar calcium signalling mechanisms in Drosophila epithelial
function” J. Exp. Biol. 212 (2009): 387-400.
Knight M.R., Campbell A.K., Smith S.M. & Trewavas A.J. “Transgenic plant aequorin reports the
effects of touch and cold-shock and elicitors on cytoplasmic calcium” Nature 352 (1991):
524 – 526.
Knight M.R., Read N.D., Campbell A.K., & Trewavas A.J. “Imaging calcium dynamics in living plants
using semi-synthetic recombinant aequorins.” JCB 121(1993): 83-90.
McAinsh M.R. & Pittman J.K. “Shaping the calcium signature” New Phytologist 181 (2008): 275-294.
Mehlmer N., Parvin N., Hurst C.H., Knight M.R., Teige M. & Vothknecht U.C. “A toolset of aequorin
expression vectors for in planta studies of subcellular calcium concentrations in Arabidopsis
thaliana” J Exp Bot 63 (2012): 1751-1761.
Morise, H., Shimomura, O., Johnson, F. H. & Winant, J. “Intermolecular energy transfer in the
bioluminescent system of Aequorea” Biochemistry 13 (1974): 2656-2662.
Shimomura O. “A short story of aequorin.” Biol Bull 189 (1995): 1–5.
Shimomura O, Johnson FH, Saiga Y. “Extraction, purification and properties of aequorin, a
bioluminescent protein from the luminous hydromedusan, Aequorea.” J Cell Comp Physiol
59 (1962): 223–239.
Shimomura O. & Johnson F.H. “Chemical Nature of Bioluminescence Systems in Coelenterates”
Proc. Nat. Acad. Sci. USA 72 (1975): 1546-1549.
Whalley H.J. & Knight M.R. “Calcium signatures are decoded by plants to give specific gene
responses” New Phytologist 197 (2013): 690–693.
White P.J. & Brodley M.R. “Calcium in Plants.” Annals of Botany 92 (2003): 487-511.
Williamson RE & Ashley CC. “Free Ca2+ and cytoplasmic streaming in alga Chara” Nature 296
(1982): 647-651.
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Kaleidoscope 6.1, Charlotte Hurst, “Aequorin: glow in the dark protein”
Charlotte Hurst,
Division of Plant Sciences,
Dundee University.
[email protected]
Charlotte completed both her undergraduate degree and MSc in biological sciences at Durham
University. She first encountered aequorin while working as a summer project student with Professor
Marc Knight. She is now studying for a PhD in plant sciences at Dundee University and the James
Hutton Institute.
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