Biology Reference
In-Depth Information
1. Endoplasmic Reticulum
ER calcium concentrations have been measured using low molecular mass
calcium sensors and with aequorin (
Solovyova and Verkhratsky, 2002
), but it
seems fair to say that the cameleon-based sensors (YC-3er and YC-4er) have
given the best estimates of ER calcium concentration and turnover (
Foyouzi-
Youssefi
et al.
, 2000; Graves and Hinkle, 2003a,b; Varadi and Rutter, 2002a; Yu
and Hinkle, 2000
). In summary, cameleon-based indicators have presented a
picture of the ER as an organelle with resting calcium concentrations in the
range 250-600
m
M and a very active calcium turnover that depends very heavily
on the activity of the SERCA ATPase (
Demaurex and Frieden, 2003
). Transgenic
YC3.3er has been engineered to give tissue-specific expression in mouse pancreatic
beta cells (
Hara
et al.
, 2004
). The interpretation of calcium changes in the ER
measured by cameleon indicators is tempered by the finding that pH changes
within the organelle may interfere with estimates of dynamic calcium concentra-
tion (
Varadi and Rutter, 2004
). Improved sensors for ER calcium are now avail-
able (
Palmer Amy
et al.
, 2004; Zou
et al.
, 2007
).
2. Mitochondria
Mitochondrial targeting of recombinant aequorin was achieved using the
N-terminal presequence of subunit VIII of cytochrome oxidase (
Rizzuto
et al.
,
1992
). The same targeting strategy was used to locate ratiometric pericam within
mitochondria (
Robert
et al.
, 2001
) and to show that the pericam tracked beat to
beat calcium changes in cardiomyocytes, just as did aequorin. Cameleon probes
targeted to mitochondria were e
ective only at very low expression levels
(
Arnaudeau
et al.
, 2001
). In a comparison of mitochondrially targeted cameleon
(mtYC2), camgaroo-2, and Ratiometric Pericam (
Nagai
et al.
, 2001
) in HeLa
cells, it was found that Ratiometric Pericam was the most reliable and faithful
of the sensors (
Filippin
et al.
, 2003
). Mislocalization and poor expression of the
mitochondrially targeted YC2 sensor could be improved by inserting a tandem
repeat of the subunit VIII presequence as the targeting sequence (2mtYC2)
(
Filippin
et al.
, 2005
). 2mtYC2 was used successfully to demonstrate calcium
handling by skeletal muscle mitochondria during contraction (
Rudolf
et al.
,
2004
). Insertion of a tandem targeting repeat was an ine
V
ective strategy
for the preferred citrine or Venus variants (
Filippin
et al.
, 2005
), but in contrast,
the D2cpv, D3cpv, and D4cpv cameleons (
Palmer
et al.
, 2006
) functioned
well as mitochondrial calcium sensors when targeted with the cytochrome
oxidase tandem repeat (
Palmer
et al.
, 2006
). These constructs are now the
recommended genetically encoded mitochondrial calcium sensors. An recent
overview of calcium sensor approaches in mitochondria is available (
Pozzan
and Rudolf, 2009
).
V
