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Heim, Nicola (2005): Genetically Encoded Calcium Indicators Based on Troponin C and Fluorescent Proteins. Dissertation, LMU München: Faculty of Biology
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Abstract

Genetically encoded calcium probes allow the visualization and quantification of intracellular calcium dynamics with great specificity and sensitivity. Until now, all genetically encoded calcium indicators have shared a common design that consists of mutants of the green fluorescent protein (GFP) as fluorophores and calmodulin as the calcium binding moiety, in several configurations. However, most of these calmodulin-based probe types show deficiencies such as reduced dynamic ranges when expressed within transgenic organisms and a lack of calcium sensitivity in certain subcellular targetings. A likely reason for this reduced sensitivity is that calmodulin is an ubiquitous signal protein in cell metabolism and thus stringently regulated. Thus, we chose to develop novel types of calcium probes based on the muscle calcium sensor troponin C, a protein that is not a constituent of non-muscle cells and therefore less likely to interact with cytosolic activities. By going through a series of cloning optimization steps, a set of new ratiometric calcium indicators was created using domains of skeletal and cardiac muscle troponin C variants as calcium binding moieties. These constructs showed in vitro FRET ratio changes of up to 140 %, had calcium dissociation constants ranging from 470 nM to 29 µM, and were functional in intracellular targetings in which previous indicators had failed. The new indicators expressed homogenously with no signs of aggregation in HEK293 cells as well as in rat hippocampal neurons, and large and dynamic ratio changes could be quantified after drug stimulation in cell culture. Membrane labeling experiments with the indicator construct TN-L15 were successful in HEK293 cells and hippocampal neurons. When targeted to the plasma membrane, the indicator readily responded to agonist-induced increases in cytosolic calcium and kept its full dynamic range. In the last part of this work, transgenic mouse lines were created expressing one of the new calcium indicators in the cytosol of neurons. Imaging experiments in live tissue cultures and brain slices revealed responses to rises in calcium that were superior to previously published indicator performance in mouse lines expressing other calcium probes. The novel troponin C-based probes of intracellular calcium developed in this work have the potential for monitoring calcium dynamics in applications in which previous calmodulin-containing calcium indicators failed, possibly because they interact less with the cellular biochemical machinery and are thus more compatible with transgenic expression in tissue and whole organisms.