Biomedical Engineering Reference
In-Depth Information
system and the QF system (Lai and Lee
2006
; Potter et al.
2010
). All of those
technical refi nements allow for a very precisely controllable expression of the trans-
gene of interest, which can, of course, be a Ca
2+
sensor protein.
7.3
Genetically Encoded Ca
2+
Indicators
Genetically encoded Ca
2+
indicators (GECIs) [also called fl uorescent calcium
indicator proteins (FCIPs)] are widely used to image activity in defi ned neuronal
populations. GECIs consist of a calcium-binding domain such as calmodulin or
troponin C, which is fused to GFP-derived fl uorescent proteins (Mank et al.
2008
;
Miyawaki et al.
1997
; Nakai et al.
2001
). Two principal kinds of Ca
2+
sensor pro-
teins have been described, single-chromophore sensors and two-chromophore sen-
sors. In single-fl uorescent-protein indicators, the fl uorescence intensity of a
modifi ed fl uorescence protein is modulated by calcium-binding-dependent changes
in the chromophore environment. These indicators show very low fl uorescence at
the unbound state but increase their emission intensities drastically after binding
calcium. Among them, the GCaMP (Nakai et al.
2001
) is the most widely used one
across multiple model organisms from nematodes, fruit fl ies, zebrafi sh to mice
(Hasan et al.
2004
; Higashijima et al.
2003
; Kerr et al.
2000
; Wang et al.
2003
).
GCaMP consists of an enhanced GFP that has been circularly permuted (cpEGFP)
such that new N- and C-termini have been introduced. To the new termini, a
calmodulin sequence and the calmodulin-binding M13 fragment of myosin light
chain kinase have been fused (Nakai et al.
2001
). When Ca
2+
binds to calmodulin,
conformational changes due to the Ca
2+
-calmodulin-M13 interaction induce a sub-
sequent conformational change in cpEGFP such that the emission intensity changes
when the chromophore is excited. Site-directed mutagenesis has improved the
GCaMP indicators signifi cantly (Muto et al.
2011
; Tian et al.
2009
). In
Drosophila
,
the version GCaMP3 is widely used to monitor neural Ca
2+
activity in intact fl ies
(Chiappe et al.
2010
; Seelig et al.
2010
), and it shows high signal-to-noise ratio,
high sensitivity with respect to transient neural activity, and fast kinetics in vivo
(Tian et al.
2009
).
In two-chromophore indicators, calcium binding modulates the effi ciency of
Förster resonance energy transfer (FRET) between a pair of fl uorescent proteins,
usually an enhanced cyan (eCFP) and an enhanced yellow (eYFP) fl uorescent pro-
tein (Miyawaki et al.
1999
; Miyawaki et al.
1997
), between which a calcium-binding
domain is sandwiched. If the eCFP (donor chromophore) is excited at ~440 nm
wavelength, the emission light energy is transferred to the eYFP (acceptor chromo-
phore) in the form of FRET, which decreases the emission intensity from the donor
chromophore and increases the emission intensity from the acceptor chromophore.
As a result the ratio of emissions between these chromophores refl ects the extent by
which intracellular calcium concentration changes. Such ratiometric, FRET-based
Ca
2+
indicators have potential advantages over single-fl uorescent-protein GECIs,
including higher baseline brightness and relative insensitivity to motion artifacts
