This is a proposal to develop an improved aequorin method of measuring cytoplasmic calcium levels. Aequorin is a chemiluminescent protein extracted rom a jellyfish. Its luminescence increases rapidly with calcium concentration over the whole range found within living cells and it can be injected without disturbing their function or development. The pattern and level of luminescence which is emitted by an aequorin-loaded cell serves to reveal the changing patterns and levels of free calcium within this cell for days after injection. The aequorin method has several advantages over fluorescent dye techniques currently used for measuring cytoplasmic calcium levels. However, it requires improving for it to reach its full potential. Dr. Jaffe will develop an efficient imaging system and a way for mass loading of aequorin into cells, using fertilized sea urchin eggs, Dictyostelium cells, and other cells. If successful, the improved aequorin method will increase our ability to accurately measure the cytoplasmic calcium levels and will contribute to better understanding of many cell and developmental processes that involve calcium.
