EXPRESSION OF APO-AEQUORIN DURING EMBRYONIC-DEVELOPMENT - HOW MUCH ISNEEDED FOR CALCIUM IMAGING

Aequorin is a bioluminescent calcium indicator consisting of a 21 kDa protein (apo-aequorin) that is covalently linked to a lipophilic cofac tor (coelenterazine). The aequorin gene can be expressed in a variety of cell lines and tissues, allowing non-invasive calcium imaging of sp ecific cell types. In the present paper, we describe the possibilities and limitations of calcium imaging with genetically introduced apo-ae quorin during embryonic development. By injecting aequorin into sea ur chin, Drosophila and zebrafish eggs, we found that higher aequorin con centrations are needed in smaller eggs. Our results suggest that for m easuring resting levels of free cytosolic calcium, one needs aequorin concentrations of at least 40 mu M in sea urchin eggs, 2 mu M in Droso phila eggs, and only 0.11 mu M in zebrafish eggs. A simple assay was u sed to determine the absolute concentrations of expressed apoaequorin and the percentage of aequorin formation in vivo. The use of this assa y is illustrated by expression of the aequorin gene in Drosophila oocy tes. These oocytes form up to 1 mu M apo-aequorin. In our hands, only 0.3% of this apo-aequorin combined with coelenterazine entering from t he medium to form aequorin, which was not enough for calcium imaging o f the oocytes, but did allow in vivo imaging of the ovaries. From thes e studies, we conclude that coelenterazine entry into the cell is the rate limiting step in aequorin formation. Based on the rate of coelent erazine uptake in Drosophila, we estimate that complete conversion of 1 mu M apo-aequorin would take 50 days in zebrafish eggs, 19 days in D rosophila eggs, 7 days in sea urchin eggs or 18 h in a 10 mu m tissue culture cell. Our results suggest that work based on genetically intro duced apo-aequorin will be most successful when large amounts of small cells can be incubated in coelenterazine. During embryonic developmen t this would involve introducing coelenterazine into the circulatory s ystem of late stage embryos. Calcium imaging in early stage embryos ma y be best done by injecting aequorin, which circumvents the slow proce ss of coelenterazine entry.