Our group studies the structure and function of mitochondria using state of the art microscopic techniques, principally Electron Tomography. Electron Tomography is a technique which calculates the three-dimensional structure from a series of electron micrographs of cells or cellular components tilted over a range of angles. Our study of mitochondria has led, along with the work of several other research groups, to a new paradigm of mitochondrial structure in which the inner membrane of mitochondria is divided into two components. The inner boundary membrane is the component that lies along the outer membrane separated from it by approximately 6-7 nanometers. At numerous sites we find 30 nm diameter tubular extensions of the inner membrane projecting into the matrix toward the center of the mitochondrion forming cristae, the second component of the inner membrane. Numerous tubular cristae often merge forming large disklike cristae. These results have raised a number of questions about mitochondrial structure and function that we are currently addressing. In all of these projects we work in collaboration with the National Center for Microscopy and Imaging Research at UCSD directed by Dr. Mark Ellisman.

(1) What are the changes in mitochondria structure during apoptosis? Mitochondria play a key role in initiating the apoptosis program with the release of cytochrome c into the cytosol. We are using Electron tomography to study the structural changes in mitochondrial membranes in order to determine whether cytochrome c is released through specific pores or through rupture of the outer membrane following swelling of the matrix. Through correlated light and electron microscopy/tomography using cells labeled with specific fluorescent markers to indicate the location of cytochrome c and the presence of a membrane potential we have discovered a remarkable transition in the structure of the inner membrane to one in which the inner membrane forms large vesicles containing the mitochondrial matrix. This change appears to be unrelated to the release of cytochrome c but may be an indication of mitochondrial fragmentation. In this context we are also studying the possible role of the mitochondrial permeability transition in cytochrome c release.

(2) What effects does hypoxia and reperfusion have on the structure of mitochondria? Prolonged hypoxia results in irreversible changes in cell physiology that can lead to cell death, and this damage is exacerbated upon re-exposure to oxygen, reoxygenation. This experimental condition mimics ischemia/reperfusion injury that occurs during heart attacks and strokes. We are working on a cell culture model for hypoxia and reoxygenation in order to study the effects on mitchondrial structure and function by correlated light and electron microscopy/tomography. This is a collaboration with Dr. Roberta Gottlieb at The Scripps Research Institute.

(3) What determines the architecture of the mitochondrial inner membrane? We believe that the changes in mitochondrial structure observed during apoptosis may be related to the release of a protein, OPA1, that is a dynamin-related GTPase believed to play an important role in regulating inner membrane topology. We will test this hypothesis by correlated light and electron microscopy in cells transfected with a fluorescent OPA1 protein. In a second approach we are developing a thermodynamic model based upon the energetics of membrane bending in an attempt to explain both the shape of crista junctions and their uniform sizes.