Several lines of evidence including vibrating calcium specific electrode measurements and calcium-imaging data support the hypothesis that calcium gradients are involved in setting up cell polarity. Of particular interest is the relationship between calcium gradients in sea urchin eggs which develop during pronuclear migration/fusion and the subsequent axis of cleavage. We also propose to study the role of calcium gradients in orienting motile cells such as macrophages and T lymphocytes. We propose to test this hypothesis directly, through the development of new optical tools including new long-wavelength, ratioable calcium indicators, probes for following the movements of the centrosome and methods for making localized gradients of calcium ions in cells. Included in the proposal is a proposed solution to the well recognized problems of dye loading and compartmentation seen with fura-2 and other ion indicators.
