(65) INVESTIGATION BY ELECTRON TOMOGRAPHY OF ISOLATED MITOCHONDRIA FROM A CELL FREE APOPTOTIC SYSTEM

C.W. Renken (2), G.A. Perkins (1), R. Kluck (3), L. Tarsa (1), C. McCarty (1), D. Neumeyer (3), M.H. Ellisman (1), and T.G. Frey (2)
(1) National Center for Microscopy and Imaging Research at San Diego, University of California, San Diego, La Jolla, CA 92093-0608
(2) Biology Department, San Diego State University, San Diego, CA 92182-4614
(3) La Jolla Institute for Allergy and Immunology, La Jolla, CA 92093

The cell free apoptotic system using Xenopus oocyte extract has been used to order the molecular events of B-galactosidase stimulated apoptosis. In this system release of cytochrome c from mitochondria is an early event, preceding caspase-3 activation and an observed transition in the permeability of the mitochondrial inner membrane (MPT). We have looked at three different types of isolated mitochondria from this system by electron tomography: 1) Control mitochondria, prior to treatment with B-galactosidase, 2) apoptotic mitochondria, after the detected caspapse-3 activity, and 3) mitochondria from oocytes treated with Bcl-2 after treatment with B-galactOsidase. All mitochondria from this system are in the condensed state and no structural difference between the three mitochondria types were observed in projection. No swelling of mitochondria was seen. Tomographic reconstruction to 5nm resolution shows no evidence of any mechanical disruption of the outer membrane. Our evidence suggests that the mechanism of cytochrome c release from mitochondria is limited to morphological changes of less than 50 angstroms.