The goal of this work is to understand (1) the mechanisms of intracellular calcium regulation in motile cells, (2) the relationship between calcium changes and other intracellular signalling mechanisms, and (3) the role of these signalling mechanisms in regulating cell motility. The proposed work will examine two types of phagocytic cells: the human neutrophil and the mouse peritoneal macrophage. For both types of cells, there is extensive biochemical characterization of the signal generating mechanisms and the mechanisms for regulation of the cytoskeleton. There is also substantial information on the motile behavior of these cells in response to various stimuli. Despite intensive study, the mechanisms for controlling motility in vivo remain unclear, although roles for (Ca2+)i, pHi, kinase C, and other signals have been proposed and investigated. Optical microscopy techniques will be used to measure both the generation of intracellular signals and the motile response in single cells. (Ca2+)i will be measured with high temporal and spatial resolution. These measurements will be made simultaneous with measurements of pHi and cell morphology to provide detailed correlations between the generation of signals and the motion of cells. Methods to measure kinase C activation in single cells will be developed. The interplay between three signals ((Ca2+)i, pHi, and kinase C) will be examined. Both chemotaxis and phagocytosis exhibit desensitization/resensitization phenomena. The mechanism for this will be studied based on analysis of the properties of these three intracellular signals and their inter-relationships. Finally, the role of these signals in the morphological and cytoskeletal changes will be examined. Both chemotaxis and phagocytosis are processes that require study at the single cell level to understand how biochemical mechanisms are orchestrated to produce specific cell behavior. The microscopic methods described in this proposal provide a means to study intracellular signals and cell responses at the single cell level.
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