We began using the IVEM facility when I was on sabbatical during the Spring semester of 1995. My initial goals were to develope the IVEM as a tool for the structural study of cytochrome c oxidase in two-dimensional crystals isolated from beef heart mitochondria. This is a project on which I had been working for a number of years, and I planned to use the IVEM and the slow-scan CCD camera to collect data on crystals containing multiple layers of cytochrome oxidase monomers. Although the CCD camera was not ready for use in data collection, the IVEM and the Tv camera proved very suitable for scanning and collecting images of crystals frozen in thin layers of vitreous ice (maintained at - 1700C using the Gatan cryospecimen holder) on film. I managed to collect images of cytochrome oxidase crystals in both the fully oxidized and fully reduced state in order to see if reduction of the four metal centers was accompanied by a detectable conformational change. Mter careful analysis of the 4-5 best images of each sample (oxidized and reduced), I was not able to identify significant differences. During my sabbatical I became interested in use of the """"""""rEM in electron tomography of cellular and subcellular structures. Given my interest in mitochondria and energy transduction, I decided to exploit the facility and the expertise of the personnel to study mitochondria ultrastructure with the goal of first understanding the membrane topologies of various types of mitochondria and then the possible compartmentalization of mitochondrial function as seen by the distribution of different membrane protein components. This goal is being carried out primarily by a postdoc, Dr. Guy Perkins, supported by my Grant-in-Md from the California Affiliate of the American Heart Association and by a graduate student, Mr. Christian Renken, who is finishing his M.Sc. in Physics at SDSU and applying to our Joint Doctoral Program (with UCSD) in Cell and Molecular Biology. They have completed the first three-dimensional reconstructions of mitochondria from different areas of nerve cells, and we are attempting to correlate their different ultrastructures with different cellular location and function. We will also study mitochondria structure in heart tissue and in cultured heart cells in order to compare their ultrastructures in various functional states (e.g. normal vs. ischaemic) as well as attempt to map various classes of mitochondrial membrane proteins by specific labeling.
Showing the most recent 10 out of 384 publications
