(51) MITOCHONDRIAL DESIGN: IMPLICATIONS FOR THE STRUCTURAL BASIS OF THE PERMEABILITY TRANSITION

C.A. Mannella (1,2), K. Buttle (1), D.R. Pfeiffer (3), B.K. Rath (1) and M. Marko (1)
(1) Wadsworth Center, New York State Dept of Health, Albany NY
(2) Dept of Biomedical Sciences, School of Public Health, Univ at Albany
(3) Dept Medical Biochemistry, Ohio State Univ.

Electron microscopic tomography is providing important new information about the 3-D structure of mitochondria from different cell types, including the connectivity between internal compartments and interactions of the outer membrane with the inner membrane and with endoplasmic reticulum. While the shape and organization of the cristae may vary considerably with cell type and osmotic conditions, there appears to be a common design principle: the cristae connect to the surface of the inner membrane (and to each other) through narrow tubular regions. The nature and apparent universality of this structural feature suggest (a) that formation of cristae is a controlled process and not merely a passive infolding of the inner membrane, and (b) that this design feature plays a fundamental (if as yet undefined) role in mitochondrial function. Possibilities include restriction of diffusion of solutes (ions, ATP, cytochrome c) between internal compartments, and control (together with contact sites) of the "flow" of inner membrane between the cristal and peripheral surfaces (e.g., during osmotic adjustments). That the "permeability transition" might be associated with irreversible loss of this fundamental structural feature is being explored. Specifically, we are determining whether the tubular cristae regions are retained/reformed during swelling and subsequent recontraction of mitochondria.

Until now, 3-D reconstructions have been done on tissues or isolated organelles that have been chemically fixed, stained and embedded in plastic. Using an intermediate-voltage electron microscope equipped with an automated (CCD-based) imaging system and cryo-accessories, we have been able to tomographically reconstruct unfixed, unstained mitochondria quick-frozen in vitreous ice ("frozen-hydrated"). While the resolution is lower (because fewer images can be collected from the beam-sensitive "naked" organelles), these tomograms provide direct visualization of native mitochondrial structure, e.g., confirming the presence of the tubular cristae connecting regions. The ability to directly image frozen-hydrated mitochondria in the electron microscope makes feasible a wide range of experiments to explore the dynamics of 3-D structural changes associated with such factors as energy state and induction of the permeability transition.

This work is supported by NSF grant MCB-9506113 and uses the facilities of the Wadsworth Center's Biological Microscopy and Image Reconstruction Resource, funded by NIH grant RR01219.