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Content archived on 2024-05-29

Labelling of fusion proteins in living mice with synthetic probes

Final Activity Report Summary - IN VIVO LABELING (Labelling of fusion proteins in living mice with synthetic probes)

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 introduced in this project 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 cultured primary muscle cells of mice expressing a nucleus-localised SNAP-tag fusion. Ca2+ concentrations inside nuclei of resting cells were measured by SEER (shifted excitation and emission ratio-ing) of confocal microscopic images of fluorescence.

After permeabilising, 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.