The focus of our research has been the A. nidulans calmodulin-dependent protein kinase (ACMPK) which can serve as both a target and a transducer of calcium-dependent signalling. We have shown that ACMPK is encoded by a single copy gene and is essential for viability. The molecular mechanisms by which Cat' and ACMPK control growth are unknown. The ability to measure changes in Ca2+ concentration in vivo during growth, cell division, and in response to external stimuli would aid in the identification of Ca2+ mediated cellular processes. Several laboratories have developed the approach of creating transgenic organisms that express apoaequorin which can be targeted to various intracellular compartments. Treatment such organisms with coelenterazine reconstitutes functional aequorin within the cytosol and subcellular organelles of their cells, thereby producing luminescence whose light emission directly reports the internal Cal' concentration. We have constructed strains of A. nidulans that express apoaequorin with a view toward studying Ca2+ fluxes in vivo as a function of cell division cycle, development and responses to external stimuli. Luminescence requires incubation of the cells with coelenterazine and is calcium-dependent. In both defined and rich media to which no calcim has been added, cytosolic Ca2+ levels are maintained below 50 nM. Characterization of the strains will be presented (Supported by N.I.G.M.S.).
Fungal Genet. Newsl. 46 (Supl):
Full conference title:
Fungal Genetics Conference 20th
- Fungal Genetics Conference 20th (1999)