DISRUPTION OF MITOCHONDRIAL FUNCTION DURING APOPTOSIS

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. During apoptosis, pro-apoptotic members of the Bcl-2 family induce mitochondrial outer membrane permeabilization and cytochrome c release resulting in caspase activation. Among the first targets of the activated caspases are the permeabilized mitochondria themselves, leading to disruption of electron transport, loss of mitochondrial transmembrane potential (??m), decline in ATP levels, production of reactive oxygen species (ROS) and loss of mitochondrial structural integrity. In 2003, we identified NDUFS1, the 75 kDa subunit of respiratory complex I, as a major caspase substrate in the mitochondria. Cells expressing a cleavage site mutant of p75 (D255A) sustained ??m and ATP levels during apoptosis and produced reduced ROS in response to apoptotic stimuli. While cytochrome c release and DNA fragmentation were unaffected by the uncleavable p75 mutant, mitochondrial morphology was maintained in the dying cells, and loss of plasma membrane integrity was delayed. Therefore, caspase cleavage of NDUFS1 promotes mitochondrial changes in apoptosis. This work has been submitted for publication. In 2003, we started studies with new labeling techniques being developed at NCMIR. ReAsH is particularly useful as it can be employed for both fluorescence and electron microscopy. Upon intense illumination in fixed samples, ReAsH generates singlet oxygen that in turn can oxidize diaminobenzidine into a highly localized polymer, and this can be stained with osmium tetroxide (Gaietta, et al., 2002). We have started to use ReAsH to localize the cytochrome c-4C in the mitochondria and determine the extent to which it may be sequestered in inner membrane cisternae. Further, using isolated mitochondria from cytochrome c-4C-expressing cells, we are inducing permeabilization of the mitochondrial outer membrane with a recombinant truncated form of the pro-apoptotic Bcl-2 family member Bid, as we have done previously (Kuwana, et al., 2002). This causes a rapid mitochondrial outer membrane permeability (MOMP) that we should be able to visualize, at least in terms of how the cytochrome c is released (i.e., whether any is at least temporarily trapped in the process. Similarly, we are also examining cytochrome c-4C release in cells undergoing apoptosis to compare the ultrastucture of the mitochondria in such cells to that of isolated mitochondria undergoing MOMP. Once these approaches are perfected, we will then fully reconstruct mitochodria from semi-thick sections to produce three-dimensional reconstructions of mitochondria before and after MOMP, complete with localized cytochrome c-4C. We will work with Guy Perkins and Tom Deerinck to best determine how to use electron tomography with our labeled samples with the advantages first described by Perkins et al. (1997b). After the kinetics of apoptotic effector protein release from mitochondria are determined, we will need to quickly fix our cells to capture the events of the release of these proteins during MOMP. A three-dimensional analysis of labeled mitochondria is needed to pinpoint the regions of release. We need the high resolution capabilities of electron tomography to correlate points of release with structural elements characteristic of mitochondria, such as contact sites and crista junctions (Perkins et al., 1997a). Initially, we will conduct tomographic three-dimensional reconstruction using the JEOL 4000 microscope (operated at 400 kV) with 0.5 ?m or thinner sections according to empirical optimization. After the new JEOL 300 kV energy-filtering microscope is operational, we will use it because it will allow for much thicker sections (several ?m thick) at the same resolution as the older 400 kV microscope.