Dynamic and quantitative Ca²⁺ measurements using improved cameleons

Abstract

Cytosolic and organellar free Ca ^{concentrations show the most dramatic spatial and temporal fluctuations of any intracellular messenger. Ca signals are most often measured by using synthetic fluorescent chelators (}1) or the photoprotein aequorin (2). The chelators are easily imaged, but they are hard to target to specific intracellular locations, and they gradually leak out of cells, particularly at warmer temperatures. Loading of chelators via their acetoxymethyl esters is fairly noninvasive and easy in isolated cells from vertebrate organisms but is harder or impossible in thicker tissues or cells from other phyla. Genetically expressed aequorin is easily targeted, but it requires the incorporation of the cofactor coelenterazine to emit light and is very difficult to image because its luminescence produces much less photons than fluorescence. Because aequorin is irreversibly destroyed by Ca ^{ion, its light output depends on the entire past history of Ca exposure. To combine the advantages of molecular biological targeting and fluorescence readout, we previously designed fluorescent protein indicators for Ca, “cameleons” (}3). Cameleons are chimeric proteins consisting of a blue or cyan mutant of green fluorescent protein (GFP), calmodulin (CaM), a glycylglycine linker (4), the CaM-binding domain of myosin light chain kinase (M13) (5), and a green or yellow version of GFP. Ca ^{binding to the CaM causes intramolecular CaM binding to M13. The resulting change from an extended to a more compact conformation increases the efficiency of fluorescence resonance energy transfer between the shorter to the longer wavelength mutant GFP. To obtain adequate expression and brightness of the mutant GFPs in mammalian cells, enhanced genes with mammalian codon usage and mutations for improved folding at 37°C were developed (}6). Also the blue mutant proved to be the dimmest and most bleachable of the GFPs. It also required ultraviolet excitation, which is potentially injurious, excites the most cellular autofluorescence, and could interfere with the use of caged compounds. Therefore, we substituted enhanced cyan and yellow fluorescent proteins (ECFP and EYFP) for the original blue and green mutants, respectively (3), to make “yellow cameleons” (YCs). Their affinity for Ca ^{was tunable by mutations in the Ca binding loops of CaM (}3, 7). Cameleons were targeted to specific intracellular sites by fusing them to appropriate organellar targeting signals or localized host proteins to observe local Ca ^{dynamics. For example, we engineered two low-affinity indicators, YC3 and YC4, to reside in the lumen of endoplasmic reticulum, where the [Ca] ranged from 60 to 400 μM at rest, and 1 to 50 μM after agonist-induced mobilization of Ca (}3).

The original YCs had problems such as sensitivity of EYFP to quenching by acidification at pHs close to physiological (8). Such quenching perturbs the signals of YCs, mimicking a decrease in [Ca^{]. Furthermore, the incorporation of CaM into the indicator naturally raised concerns that it may interfere with endogenous CaM-stimulated pathways or that it may be perturbed by endogenous CaM or CaM-binding proteins. We have now developed improved YCs that are more resistant to acidification and examined the effects of varying protein expression levels.}

Acknowledgments

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