Development of Methods to Analyze Intraorganelle Calcium

9307746 Gill The objectives of this research are to develop imaging methods to characterize the calcium pools within cultured cells and ascertain the biological significance of calcium within calcium-signaling organelles. The project aims to address an area of weakness in our understanding of cellular signalling events, that is, the nature, distribution, and function of the pools of calcium inside cells which contribute to generation of intracellular calcium signals. High resolution imaging techniques will be applied to study the location, control, and biological significance of calcium regulatory organelles functioning within established smooth muscle cell lines. The project will develop and utilize novel single cell and subcellular imaging technologies to spatially resolve those organelles that function as calcium signaling pools in cells. The specific goals are to answer the following questions: (1) What are the levels of calcium within calcium pools? The objectives are to visualize calcium pools directly, measure intraluminal calcium levels, and correlate distribution of calcium pools with known calcium regulatory proteins and specific organelle markers. (2) How is calcium inside calcium pools controlled? The question will be addressed by investigating the function and interrelationship of the mechanisms that pump calcium into pools, release calcium from pools in response to intracellular mediators, and transfer calcium between distinct intracellular pools activated by GTP-binding proteins. By developing and applying high resolution z-axis controlled fluorescence imaging to the analysis of calcium pools, the studies provide a novel approach to understanding the distribution of calcium signalling pools, and address the hypothesis that calcium levels within organelles are of fundamental biological importance to cellular function. %%% An early, measurable event in many cellular responses to external signals is an increase in internal calcium. A variet y of cellular functions are regulated by calcium, so it is critical that cells be able to regulate the internal levels and subcellular distribution of this ion. An active area of research in recent years has been development of techniques to measure the levels of free calcium in the cytoplasm of cells. The goals of this research will complement the available techniques for measurement of cytoplasmic calcium by developing and using novel techniques to study the location, control, and biological function of calcium sequestered within regulatory organelles (calcium pools). High-resolution, single cell and subcellular imaging will be used to study calcium- containing organelles in cultured smooth muscle cell lines. Experiments will be designed to answer the questions: What are the levels of calcium within particular calcium pools, and how is calcium inside these pools controlled? The results of this research will provide new techniques and information to fill an important gap in our understanding of cellular signalling events. ***