It is widely assumed that Ca is a regulator of mitosis. Changes in the intracellular free ?Ca! could modulate a variety of processes that control the formation and/or function of the mitotic apparatus. Recent studies show that free ?Ca! changes during mitosis; however, there are reported differences concerning the timing, kinetics, and location of these changes. The problem will first be clarified by comparing the timing and kinetics of Ca transients in a single cell type (Tradescantia stamen hair) using two different Ca reporting techniques: differential absorbance measurements from arsenazo-III-loaded cells, and light emission from aequorin-injected cells. Second, the timing and kinetics of Ca transients will be compared in cell types of different origin, including Tradescantia stamen hair, Haemanthus endosperm, newt lung epithelium, and cultured kangaroo rat (PtK1) cells, to see if there are biological variations. Third, the role of G-binding proteins and components of the phosphatidylinositol cycle in regulating the metaphase/anaphase transition will be examined. Fourth, the roles of Ca and calmodulin in regulating the rate of chromosome movement will be studied, by microinjecting Ca, calmodulin, GTP-gamma-S, inositol triphosphate, or Ca chelators directly into the living cells. Fifth and finally, the possible occurrence of ultrastructural changes in the mitotic apparatus during anaphase that might be related to chromosome movement will be addressed, using rapid freezing and freeze-substitution techniques to elucidate the structure and periodicity of microtubule connections to other microtubules or to the membrane and to see if changes can be related to chromosome movement. Cell division is a fundamental aspect of life on earth. In eukaryotic cells, the orderly distribution of genetic material in the form of mitotic chromosomes to daughter cells at each division event is absolutely necessary for normal maintenance of individual organisms and populations at all stages of life. The mechanism whereby this orderly distribution occurs is a complex phenomenon involving structural and regulatory aspects. In the proposed studies, Dr. Hepler will extend his previous work on mitosis, as well as definitively address a current technical controversy in the field.
