Indo-1 derivatives for local calcium sensing.
ACS Chem. Biol.. 2009-02-04; 4(3): 179-190
DOI: 10.1021/cb800258g
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Bannwarth M(1), Correa IR, Sztretye M, Pouvreau S, Fellay C, Aebischer A, Royer L, Rois E, Johnsson K.
Author information:
(1)Institute of Chemical Sciences and Engineering, Ecole Polytechnique Fédérale
de Lausanne (EPFL), CH-1015, Lausanne, Switzerland.
The role of calcium in signal transduction relies on the precise spatial and
temporal control of its concentration. The existing means to detect fluctuations
in Ca2+ concentrations with adequate temporal and spatial resolution are
limited. We introduce here a method to measure Ca2+ concentrations in defined
locations in living cells that is based on linking the Ca2+-sensitive dye Indo-1
to SNAP-tag fusion proteins. Fluorescence spectroscopy of SNAP-Indo-1 conjugates
in vitro showed that the conjugates retained the Ca2+-sensing ability of Indo-1.
In a proof-of-principle experiment, local Ca2+ sensing was demonstrated in
single cells dissociated from muscle of adult mice expressing a
nucleus-localized SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting
cells were measured by shifted excitation and emission ratioing of confocal
microscopic images of fluorescence. After permeabilizing the plasma membrane,
changes in the bathing solution induced corresponding changes in nuclear [Ca2+]
that were readily detected and used for a preliminary calibration of the
technique. This work thus demonstrates the synthesis and application of
SNAP-tag-based Ca2+ indicators that combine the spatial specificity of
genetically encoded calcium indicators with the advantageous spectroscopic
properties of synthetic indicators.