This proposal continues the application of ion microscopy, a mass spectrometry based subcellular imaging technique, for answering a wide variety of biological problems related to calcium. The central theme of the proposed research depends on the use of a stable isotope 44Ca, as a calcium transport tracer. Ion microscopy is capable of imaging the transported calcium at mass 44 (44Ca) and the endogenous calcium at mass 40 (40CA) independently. This unique capability will enable us to investigate a number of fundamental cell biology and biomedical problems. The cellular calcium constantly undergoes a rapid exchange with the extracellular calcium (calcium cycling), which we believe is fundamental to the signaling of intracellular calcium stores with a stimulus. We, therefore, propose to investigate calcium cycling under a variety of circumstances. First of all, the intracellular stores of calcium and the cycling of stored calcium will be investigated in mitotic mammalian cells, where several dynamic structures assemble and disassemble during the course of cell division. Knowledge of intracellular calcium stores and their rates of calcium cycling on the various stages of mitosis will provide new insights into the regulatory role of calcium in cell division. Secondly, the Golgi apparatus, structure recently identified as a calcium sequestration site, will be investigated for its regulatory role in cellular calcium homeostasis. A non-toxic drug, brefeldin A, will be used for disassembling the Golgi apparatus, and calcium cycling will be measured while the Golgi apparatus is disabled for exocytosis. In addition, we are continuing two biomedical studies in collaboration with Cornell researchers in which the use of 44Ca and ion microscopy have already provided answers to the questions previously unattainable. This is a rare opportunity since these researchers have pioneered in their respective areas and the ion microscope is utilized to compliment their research. We will continue to investigate the complex role of calcium in the secretory response of mast cells during the allergic reaction in collaboration with Dr. C. Fewtrell. In collaboration with Drs. R. Wasserman and C. Fullmer we are continuing the investigation of intestinal calcium absorption by using stable 44Ca. Strict cryogenic frozen freeze-dry methods of sample preparation will be used in all studies. A successful completion of this program would add a new understanding to the fundamental knowledge of """"""""cellular calcium homeostasis"""""""" during interphase and mitosis, and also provide critical information towards understanding the complex role of calcium in biomedical problems.
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