Biology Reference
In-Depth Information
organelles, for the most part in single cells
in vitro
. Their success as quantitative
calcium sensors in tissues
in vitro
and
in vivo
is qualified, but they have proved
valuable in imaging the pattern of calcium signals within tissues in whole animals.
Some branches of the calcium sensor evolutionary tree continue to evolve rapidly
and the steady progress in optimizing sensor parameters leads to the certain hope
that these drawbacks will eventually be overcome by further genetic engineering.
I. Introduction
Small, fluorescent, calcium-sensing molecules have been enormously useful
in mapping intracellular calcium signals in time and space, as chapters in this
volume attest. Despite their widespread adoption and utility, they su
V
er some
disadvantages.
All low molecular mass fluorescent cytoplasmic calcium sensors are highly
charged molecules, so cross the cell's plasma membrane very poorly. They are
placed into the cytoplasm by microinjection using fine-tipped micropipette or a
patch clamp pipette in whole cell mode. This limits their utility. Cell-permeant
fluorescent calcium sensors can be made by masking the charged carboxylic groups
by forming acetoxymethyl (AM) esters. Once inside the cell, the ester bonds are
cleaved, trapping the sensor in the cell. It is straightforward to bathe cells in culture
with the aposensor at low concentration and these AM esters have been very
widely used. One major drawback of the method is that the calcium sensor finds
itself not only in the cytoplasm, but also in intracellular compartments such as the
endoplasmic reticulum (ER) (
Silver
et al.
, 1992
). Calcium concentrations are
higher in the ER than in the cytoplasm, so this leads to a significant unwanted
fluorescence signal from sensor in the ER that makes interpretation of the true
cytoplasmic concentration changes di
Y
cult. It is also very challenging to use low-
molecular-mass fluorescent calcium sensors in whole animals.
For these reasons, genetically encoded calcium sensors that can be expressed
inside cells by transfection or transgenesis are desirable. One such sensor is
aequorin, a calcium-sensing protein found in the jellyfish
Aequoria victoria
. Origi-
nally, aequorin was isolated as a protein from jellyfish and placed inside cells by
microinjection (
Baker, 1978; Gilkey
et al.
, 1978
). More recently, a construct
encoding recombinant aequorin has been used to express the aequorin apoprotein
in cells directly (see Chapter 10). Aequorin is a luminescent molecule and at the
concentrations used inside cells emits relatively few photons compared to fluores-
cent molecules at appropriate excitation intensities (
Varadi and Rutter, 2002b
).
However, proteins that are fluorescent at the visible wavelengths best suited to
fluorescence imaging are relatively rare. As it happens,
A. victoria
also expresses
a fluorescent protein, green fluorescent protein (GFP), and it is the work that
has produced the variously colored versions of GFP that has improved our
knowledge of this fluorophore and led to recombinant fluorescent calcium sensors
(
Tsien, 2010
).
