This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.ABSTRACT:During the early stages of apoptosis, the pro-apoptotic protein Bax moves from the cytosol to the mitochondrial outer membrane (OM) where it oligomerizes and initiates a process that results in release of cytochrome c and other proteins from the intermembrane space (IMS) into the cytosol (Wei et al., 2000). This process is initiated by the truncated form of the protein Bid (tBid) and is inhibited by anti-apoptotic proteins like Bcl-2. There are several hypotheses in play regarding the mechanism of release of cytochrome c, including (1) the formation of large pores in the OM by Bax and possibly other proteins, such as the channel VDAC; (2) the formation of structural defects in the OM, perhaps involving lipids like ceremides; and (3) inner membrane expansion, causing OM lysis, perhaps at regions weakened by Bax (e.g., Korsmeyer et al., 2000; Shimizu et al., 2000; Mootha et al., 2001; Degterev et al., 2001; Waterhouse et al., 2002). Strong support for hypothesis (1) has been provided by electrophysiological studies indicating the appearance of a new channel activity in mitochondrial outer membranes concurrent with release of cytochrome c during early stages of apoptosis induced by IL-3 deprivation in murine FL5.12 cells (Pavlov et al., 2001). That the new channel (called MAC for Mitochondrial Apoptosis-induced Channel) might be the cytochrome c release pathway is suggested by two observations. First, its maximum conductance amplitudes are consistent with permeability to proteins having diameters of 5 nm or larger (the diameter of cytochrome c is around 3 nm). Second, cytochrome c and other basic proteins cause a transient blockade of the channel conductance, consistent with penetration and possible translocation (although the latter has not been directly demonstrated). We will undertake cryo-electron tomographic studies of mitochondria in mammalian cells at progressive stages of apoptosis (over 24 hours). For in-situ studies, the cells will be deprived of IL-3 and high-pressure frozen (in our Bal-Tec HPM 010) at times (4, 8 and 16 hours) characterized in terms of MAC activity and cytochrome c release in parallel experiments. Freezing will be done in 200- m-diameter cellulose capillary tubes, which will then be sectioned by either cryo-ultramicrotomy or FIB-milling. However, while these techniques are under development, first experiments will be done with mitochondria isolated from the murine cell line at the specified times following induction of apoptosis. The mitochondria would be embedded in ca. 400-nm-thick films of vitreous ice by plunge-freezing suspensions on EM grids (coated with a holey carbon film) using the FEI Vitrobot. Cryo-electron tomography (using the JEOL 4000 with GIF) will be undertaken initially on randomly selected sets of 6-8 mitochondria at each time point. Control reconstructions will be done on equal numbers of randomly selected mitochondria from untreated cells at the same time points. Double-axis tilt series would be needed for these studies to reduce artifacts and information loss due to the missing wedge in single-axis data (Penczek et al, 1995). We estimate 3-4 nm resolution should be attainable in our best tomograms. At this resolution, the largest MAC pores expected on the basis of conductance amplitude (approximately 8-10 nm) should be detectable in the tomograms, especially if MAC forms clusters. Immuno-labelling of mitochondria with anti-Bax antibodies suggests that Bax does cluster on the OM. Likewise, unusual surface topology, including defects such as linear cracks or tears, on the order of 10-20 nm or larger, should be visible in OM profiles. Finally, unusual inner membrane morphologies (Scorrano et al., 2002) or herniation of the OM by local bulging of the IM (Mootha et al, 2001) would be noted. In parallel experiments, we will employ immuno-labeling of intact mitochondria with anti-Bax and anti-cytochrome c antibodies (commercially available). Decoration of OM regions by the distinctive IgG molecules (in the absence of colloidal gold-labeled secondary antibody) may help to localize the structural features we hope to characterize.It should be noted that an earlier study failed to detect unusual structures in the mitochondrial outer membrane during apoptosis (Kuwana et al., 2002). That study employed conventional TEM and SEM preparative procedures, which may have limited resolution and/or obscured fine detail. It should be noted in this regard that pores 5-200 nm in diameter have been detected in flattened Bax-treated liposomes by atomic force microscopy (Epand et al. 2002). While there is no guarantee that we will detect the structural basis for cytochrome c release from mitochondria in these studies, we will  at the very least  establish a lower limit for the physical size of the pore or other membrane structural feature underlying this very important phenomenon. References. 1. Degterev, A., Boyce, M., and Yuan, J. (2001). The channel of death. J Cell Biol 155:695-697.2. Epand, R. F., Martinou, J. C., Montessuit, S., Epand, R. M., and Yip, C. M. (2002). Direct evidence for membrane pore formation by the apoptotic protein Bax. Biochem Biophys Res Comm 298:744-749.3. Korsmeyer, S. J., Wei, M. C., Saito, M., Weiler, S., Oh, K. J., and Schlesinger, P. H. (2000). Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 7:1166-1173.4. Kuwana, T., Mackey, M., Perkins, G., Ellisman, M., Latterich, M., Schneiter, R., Green, D. R., and Newmeyer, D. D. (2002). Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111:331-342.5. Mootha, V. K., Wei, M. C., Buttle, K., Scorrano, L., Panoutsakopoulou, V., Mannella, C. A., and Korsmeyer, S. J. (2001). A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J 20:661-671.6. Pavlov, E. V., Priault, M., Pietkiewicz, D., Cheng, E. H. Y., Antonsson, B., Manon, S., Korsmeyer, S. J., Mannella, C. A., and Kinnally, K. W. (2001). A novel, high conductance channel of mitochondria linked to apoptosis in mammalian cells and Bax expression in yeast. J Cell Biol 155:719-724.7. Penczek, P., Marko, M., Buttle, K., and Frank, J. (1995). Double-tilt electron tomography. Ultramicroscopy 60:393-410.8. Scorrano, L., Ashiya, M., Buttle, K., Oakes, S. A., Mannella, C. A., and Korsmeyer, S. J. (2002). A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis. Devel Cell 2:55-67.9. Shimizu, S., Konishi, A., Kodama, T., and Tsujimoto, Y. (2000). BH4 domain of antiapoptotic Bcl-2 family members closes voltage-dependent anion channel and inhibits apoptotic mitochondrial changes and cell death. Proc Natl Acad Sci USA 97:3100-3105.10. Waterhouse, N. J., Ricci, J.-E., and Green, D. R. (2002). All of a sudden it's over: mitochondrial outer-membrane permeabilization in apoptosis. Biochimie 84:113-121.In previous work at the RVBC, mitochondria in four cultured neuronal cells were laser-ablated. The cells were processed for same-cell correlative LM/EM, and serially-sectioned. Tomographic reconstructions were made, using the HVEM, from semi-thick sections of ablated and control mitochondria. Work on this project will continue shortly.

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