| Abstract |
Green fluorescent protein (GFP)-based fluorescent indicators for Ca(2+) offer significant promise for monitoring Ca(2+) in previously unexplored organisms, tissues, and submicroscopic environments because they are genetically encoded, function without cofactors, can be targeted to any intracellular location, and are bright enough for single-cell imaging. These probes use simple GFP variants, circularly permuted GFP (cpGFP), in which the amino and carboxyl portions have been interchanged and reconnected by short spacers between the original termini, or pairs of GFP variants that permit fluorescence resonance energy transfer (FRET). Yellow cameleons (YCs) use FRET between cyan- and yellow-emitting variants of Aequorea GFP (cyan fluorescent protein [CFP] and yellow fluorescent protein [YFP], respectively). YCs are composed of a linear combination of CFP, calmodulin (CaM), a glycylglycine linker, the CaM-binding peptide of myosin light-chain kinase (M13), and YFP. Binding of Ca(2+) to the CaM moiety of the YC initiates an intramolecular interaction between the CaM and the M13 domains, causing the chimeric protein to shift from an extended conformation to a more compact one, thereby increasing the efficiency of FRET from CFP to YFP. This technique is amenable to emission ratioing, which is more quantitative than single-wavelength monitoring. YC3.60 was engineered to enhance its performance as a fluorescent Ca(2+) indicator and here we describe the use of this cameleon to image rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)]i) within HeLa cells. FRET imaging is performed using a laser-scanning confocal microscope.
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