Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level

Monitoring calcium fluxes in real time could help to understand the develop ment, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associ ated with the green fluorescent protein and a green bioluminescent signal i s emitted upon Ca2+ stimulation. We decided to use this chemiluminescence r esonance energy transfer between the two molecules. Calcium-sensitive biolu minescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemilumi nescent and fluorescent activities of these fusion proteins have been asses sed in mammalian cells. Cytosolic Ca2+ increases were imaged at the single- cell level with a cooled intensified charge-coupled device camera. This bif unctional reporter gene should allow the investigation of calcium activitie s in neuronal networks and in specific subcellular compartments in transgen ic animals.