Biology Reference
In-Depth Information
not much has changed with respect to these particular techniques since Volume 40 of
the Methods in Cell Biology series was published.
II. Expression of Apoaequorin, GFP-Apoaequorin, and Other
Apoaequorin-Based Spectral Variants in Cells, Tissues, and
Whole Organisms
Microinjected holoaequorin has been used since the late 1960s for monitoring
changes in [Ca
2
þ
]
i
in di
erent cells and tissues. The earliest reports describe the use
of holoaequorin to detect Ca
2
þ
transients in muscle and nerve cells (
Baker et al.,
1971; Ridgway and Ashley, 1967
) as well as during activation in medaka eggs
(
Ridgway et al., 1977
). This approach is only practical, however, for introducing
aequorin into giant cells and large embryos, which are easy to microinject. The
more recent development, from the mid 1980s to early 1990s, of genetic engineering
techniques to introduce and express apoaequorin (the protein moiety of aequorin)
cDNA in cells, tissues, and whole organisms (
Inouye et al., 1989; Knight et al.,
1991a,b; Nakajima-Shimada et al., 1991; Prasher et al., 1985; Saran et al., 1994
), as
well as to target apoaequorin to specific organelles within cells (
Brini et al., 1993;
Rizzuto et al., 1992
), has paved the way for aequorin to be used as the Ca
2
þ
reporter of choice in many more biological systems today, from cells in culture to
complex multicellular organisms.
GFP-aequorin was developed approximately 10 years ago in order to improve
the stability and light emission properties of aequorin for single-cell imaging
(
Baubet et al., 2000
). Based on the naturally occurring phenomenon of BRET,
GFP-aequorin emits a red-shifted light emission (
V
l
¼
509 nm) relative to that of
470 nm) in the presence of elevated free Ca
2
þ
ion concentra-
tions. GFP-aequorin has a number of advantages over aequorin for monitoring
changes in cellular Ca
2
þ
concentrations, including increased stability and total
light output. Furthermore, the expression level and distribution of the GFP reflects
the expression level and distribution of apoaequorin; thus, the expression of
apoaequorin can be directly visualized in living cells or tissues. Although the
l
¼
aequorin alone (
incubated with f-coelenterazine starting at the 64-cell stage to reconstitute aequorin. Each panel
represents 120 s of accumulated light and consecutive panels are stepped at 60-s intervals. Scale bar is
200
m
m. (C) D. melanogaster (P[GAL4] OK107 line) stably expressing GFP-aequorin in the mushroom
bodies. Exposed fly brains were incubated for
>
1 h at room temperature with native coelenterazine,
prior to imaging. The first panel shows the whole brain and the localization of GFP in the mushroom
bodies. The following panels show consecutive bioluminescent images, each panel representing 15 s of
accumulated light, following treatment with 70 mM KCl to induce K
þ
-depolarization. Scale bar is
100
m
m. Reproduced with permission, from
Martin et al. (2007)
. (D) Nine-day old seedlings of
Arabidopsis thaliana (ecotype RLD1) that constitutively express apoaequorin were incubated in the
dark overnight in coelenterazine solution. These images show the total Ca
2
þ
-dependent biolumines-
cence recorded from seedlings exposed to air or to di
V
erent concentrations of ozone for 1 h. Scale bar is
5 mm.
#
John Wiley and Sons Ltd. Reproduced with permission, from
Evans et al. (2005)
.
