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TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK

In the last 3 months, we developed a method for quantitative analysis of the reactive oxygen species content in mitochondria as a function of position within the yeast cell. We performed ratiometric imaging of mitochondria-targeted GFP to dihydroethidium, which stain superoxide. Since mitochondria are the primary endogenous source of superoxide, the majority of the DHE staining co-localizes with mitochondria-targed GFP. For image acquisition, we obtain an optical z-series through the depth of the cell. For quantitation, we perform digital deconvolution of the z-series, generate a rendered 3D reconstruction of structures labeled with both fluorophores and measure the fluorescence from DHE and Cit1p-GFP in regions of interest. We now have quantitative data that the amount of mitochondrial ROS per unit organelle is significantly lower in bud tip compared to other mitochondria within the cell. Since mitochondria that are anchored in the bud tip are destined for inheritance to daughter cells, we obtained the first evidence that the mitochondrial inheritance machinery has the capacity to distinguish high-from low-functioning mitochondria and to promote inheritance of the fittest mitochondria. We also purchased equipment from these studies. since the funds awarded for equipment is less than that requested, we re-evaluated our equipment needs, and decided that the funds would be best used to replace antiquated equipment that we use daily. We purchased 3 incubators, polyacrylamide gel electrophoresis boxes, and a plate reader for automated quantitation of cell growth rates. We also updated our >7 year old microscope image acquisition softare, and are in the process of evaluating fluorescence filter sets. The new equipment has had a remarkable effect on morale and productivity in the laboratory.

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