The biological focus is to study the roles of cytosolic free Ca2+ ([Ca2+]i) changes in helping trigger the entry of quiescent cells into the cell cycle and in controlling their progression through mitosis. Emphasis will be placed on resolving differences in [Ca2+]i between heterogeneous neighboring cells and between different regions of large mitotic and motile cells. Methodology developed during the previous grant period offers the unprecedented sensitivity and spatial resolution needed. New analogs of the tetracarboxylate Ca2+ indicator quin2 offer about thirty-fold increased brightness of fluorescence, with change in excitation and sometimes emission wavelengths in response to Ca2+. Wavelength shifts mean that [Ca2+]i changes can now be detected by ratios of fluorescence at two wavelengths, without interference from variations in dye content or cell size, in individual 6 um cells studied by fluorescence microscopy or by flow cytrometry. Spatial resolution to image [Ca2+]i merely requires a video processing system whose feasibility has been demonstrated by collaboration with a better-equipped laboratory. This system will be used to examine the time course of [Ca2+]i in individual lymphocytes treated with mitogenic and some non-mitogenic lectins, antibodies, and lymphokines. Lymphocyte surface markers will be assessed by the usual antibodies to selectively stain cells or adhere them to the chamber floor. Flow cytometry will provide an independent view of the population statistics. The video system will also be used to study [Ca2+]i and pHi in fibroblasts stimulated with growth factors or transformed by a temperature-sensitive oncogene. Present studies showing [Ca2+]i rises correlated with several major events in mitosis of sea urchin zygotes will be extended by the imaging capability to see if and where the rises are localized in the cell. Cell lines such as PtK1 or CHO will be checked to see if they too have [Ca2+]i fluctuations. The importance of [Ca2+]i transients during mitosis should be tested by using photoreactive Ca2+ chelators to generate spatially and temporally defined rises or falls in [Ca2+]i. Meanwhile, chemical efforts will be devoted to yet further improvements of [Ca2+]i indicators by increasing their wavelengths of operation. Further work on photoreactive Ca2+ chelators is aimed at increasing the quantum efficiency, speed, and wavelengths of photolysis. Prototype Na+ selective indicators need optimization of fluorescent properties and addition of carboxylate groups to make them physiologically useful.
