Targeting of calcium-sensing protein cameleon from cytoplasm to nucleus

Abstract

Ca²⁺ concentrations in the cell have been measured in past by Ca²⁺ specific indicators (chelators) and recombinant aequorins. The chelators are easily imaged but are difficult to target precisely to specific intracellular locations. Aequorin requiring the incorporation of coelenterazine, is irreversibly consumed by Ca²⁺ and very difficult to image. To combine the brightness of fluorescent indicators with the targetability of a biosynthetic indicator, new fluorescent indicators for Ca²⁺ that are genetically encoded without cofactors have been constructed. Such Ca²⁺ sensing indicators are called cameleons as they change color with changing Ca²⁺ concentrations. Gene for cytoplasmic cameleon having fused cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) was available. For measuring Ca²⁺ in nucleus, appropriate constructs were made in the cloning vector. Gene for cytoplasmic cameleon was hooked to nuclear localization signal of 45 bp from transcription factor C2 cDNA of tobacco. Constructs were mobilized into transformation vector. Transient gene expression assays undertaken in onion peel cells showed that cameleon was expressed in the nucleus with the modified construct.