9308024 Silver Cell division is a central feature in the life of every living cell. It is known from the work of this and other laboratories that intracellular free calcium is an essential regulator of specific biochemical and physiological aspects of mitosis. This laboratory is identifying patterns of calcium transients during the cell cycle in normal sand dollar blastomeres, using calcium- dependent aequorin luminescence, multi-spectral video microscopy and high performance computational analysis of discrete calcium signals in intracellular microdomains. Prior results show that calcium is released into the cytoplasm in discrete, short-lived, and highly localized pulses within local regions, or microdomains. Computational analyses, including three-dimensional Fourier analysis, show that spatial and temporal patterns of calcium release vary in characteristic fashions throughout the cell cycle. The goal is to decipher the spatial and temporal patterns of calcium release associated with specific steps in mitosis known to be dependent upon calcium-sensitive biochemical pathways (e.g. nuclear envelope breakdown, anaphase onset, and cytokinesis). In this way a library of descriptors of calcium release patterns associated with specific mitotic events will be assembled. In addition, biochemical studies of how the cell mediates the localized release of calcium within microdomains of the cytoplasm are underway. The present focus of the biochemical studies is the role of phospholipid metabolism in regulating calcium release, particularly phospholipases and other enzymes associated with the arachidonate pathway whose lipid-derived products (e.g. inositol trisphosphate, leukotrienes, arachidonic acid) are known to mobilize calcium from intracellular stores. Monoclonal antibodies to phospholipases A2, C and D, and antibodies to thromboxane synthase and cyclooxygenase label endomembranes associated with the cytoskeleton and mitotic apparatus. Which of these enzyme s are associated with which endomembrane system, which lipid-derived products can serve to release calcium from endomembranes in vitro, and which may be acting in vivo to mediate calcium release associated with mitotic events will be determined. %%% It is known from the work of this and other laboratories that intracellular free calcium is an essential regulator of biochemical and physiological aspects of mitosis, the process by which one cell divides to become two. The long-term goal of this technically demanding research is to understand the regulation of intracellular calcium on a physiological time scale and in three dimensions within an egg cell undergoing division. In vitro experiments on the biochemical role of lipid-derived mediators of calcium release will complement in vivo experiments using sophisticated video imagery to develop a three-dimensional picture of when and where within the cell calcium is released. The results of this research will be a major contribution to our understanding of the regulation of mitosis. ***
