Biology Reference
In-Depth Information
3. Peroxisome
Cameleon D3cpv was furnished with a modified peroxisome localization
sequence (D3cpv-KVK-SKL) to monitor calcium concentrations in this organelle
in HeLa cells in response to agonists or depolarization (
Drago
et al.
, 2008
).
4. Golgi
The Citrine cameleon YC3.3 has been expressed in the Golgi using an 81 residue
N-terminal sequence from human galactosyl transferase type II (
Griesbeck
et al.
,
2001
); it was saturated, o
ering no useful information but that the Golgi has a very
high resting calcium concentration.
V
5. Plasma Membrane
Sub-plasmalemmal calcium concentrations may di
er from those in bulk cyto-
plasm. Localized calcium concentrations around secretory vesicles were shown to
be higher than those in cytoplasm by using a phogrin chimera to target YC2 to
secretory vesicle membrane (
Emmanouilidou
et al.
, 1999
). A number of targeting
strategies have proved successful in localizing sensors to the plasma membrane.
The cpVenus cameleon YC3.60 has been targeted using a Ki-Ras chimera (
Nagai
et al.
, 2004
). The TN-L15 sensor localized to the plasma membrane as GAP43,
Ras, or synaptobrevin chimeras (
Heim and Griesbeck, 2004
). Localization
can also be achieved with a myristoyl/palmitoyl N-terminal tag (
Zacharias
et al.
, 2002
), an approach that was used with the cameleon D series (
Palmer
et al.
, 2006
). A chimera of GCaMP2 and synaptotagmin (SyGGCamp2) has been
used to monitor synaptic calcium signals, in this case
in vivo
in zebrafish (
Dreosti
et al.
, 2009
).
V
B. Tissue-Specific Expression
The other major advantage of genetically encoded calcium sensors is tissue-
specific expression in intact organisms.
1. YC2.1
The first transgenic tissue-specific expression of genetically encoded calcium
sensors was demonstrated in plants. YC2.1 was expressed in
Arabidopsis
guard
cells of the leaf, first using a CaMV promoter (
Allen
et al.
, 1999
) and then a guard
cell-specific
det
promoter (
Allen
et al.
, 2000
), demonstrating that aspects of the
calcium-signaling response in guard cells were under di
erential genetic control.
YC3.1 was used in transgenic
Aradidopsis
plants to visualize calcium signals in the
pollen grain (
Iwano
et al.
, 2004
).
V
