Biology Reference
In-Depth Information
The first recombinant fluorescent calcium sensors were described by Tsien and
Persecchini in 1997 (
Miyawaki
et al.
, 1997; Persechini
et al.
, 1997; Romoser
et al.
,
1997
). They were based on a concatenation of a recombinant calcium-binding
domain with GFP-derived fluorescent protein pairs. This approach has bred a
family of these cameleon indicators, so called because they are based on a long
tongue-like interaction between calmodulin (CaM) and a binding peptide and
change color (
Miyawaki
et al.
, 1997
). Later, when it was realized that the GFP
beta-can structure lent itself to circular permutation without loss of function
(
Baird
et al.
, 1999
), insertion of a calcium-binding domain within the GFP
(
Baird
et al.
, 1999
) or concatenated to new N- or C-terminals (
Nakai
et al.
, 2001
)
led to a second family of calcium sensors based on the fluorescence of a single
GFP-derived molecule, the camgaroos, pericams and their relatives.
The last 10 years have been marked by a rapid evolution in the laboratory of
these two families and their relatives, both figuratively and literally, as random
mutagenesis and clonal selection in bacteria has on occasion been used to opti-
mize the properties of the sensors (
Griesbeck
et al.
, 2001
). This rapid diversifica-
tion has generated not only continuing improvements in the performance of the
sensors, but also a plethora of choice. Reviews have been written to track progress
in the field (
Barth, 2007; Demaurex and Frieden, 2003; Garaschuk
et al.
, 2006;
Griesbeck, 2004; Hires
et al.
, 2008; Kotliko
, 2007; Mank and Griesbeck, 2008;
Miyawaki, 2003a,b, 2005; Pozzan and Rudolf, 2009; Solovyova and Verkhratsky,
2002; Zacharias
et al.
, 2000
). Most of the new variants have first been tested by
their makers in living cells as proof of principle rather than to answer substantial
questions in biology. I shall first set out the evolution of this growing tribe of
genetically encoded calcium sensing probes, dealing with the two broad families
in turn and then describe their application and utility in various biological
settings.
V
II. Genetically Encoded Sensors
A. The Cameleon Family
1. Origins
The family founders were described in three papers that followed rapidly in
succession in 1997. Their conception was aided by previous work in which GFP
had been altered by directed mutagenesis to produce di
erent colored variants
with altered excitation and emission spectra (
Heim
et al.
, 1995
). As an aside, these
di
V
erently colored variants are sometimes referred to collectively as GFPs, though
they are not green. Persechini's group described a construct (FIP-CB
sm
) in which a
red-shifted excitation variant of GFP (RSGFP;
Delagrave
et al.
, 1995
, hereafter
GFP) and blue fluorescent protein (BFP) are linked by a sequence that includes 17
amino acids from the calmodulin-binding domain of avian myosin light chain
kinase (MLCK). This novel protein indirectly senses calcium concentrations inside
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