NCMIR In The News
December 12, 2008
NCMIR's Electron Tomography Resource is Helping to Reveal the Role of Mitochondria in Cell Death
Summary
Mitochondria, often described as the powerhouses of cells, also play a key role in carrying out programmed cell death, or apoptosis. Increasingly, they are being seen as a key determinant of cellular health and a host of metabolic and neurodegenerative diseases and are even targets of viral infection. By applying NCMIR's expertise in light microscopy and electron tomography, three teams of researchers are enhancing our understanding of how mitochondria contribute to neurodegeneration, mediate apoptosis, and serve as viral replication complexes. Each of the three groups recently published reports detailing how aspects of a mitochondrion's three-dimensional structure are influenced by a variety of stresses.
In one study, Dr. Ella Bossy-Wetzel and colleagues (University of Central Florida) used NCMIR's electron tomography resources to study the events of nitric oxide-induced mitochondrial fission in order to characterize the mechanism underlying mitochondrial injury during neurodegeneration (Yuan et al. 2007).
A second collaborative study by Dr. Paul Ahlquist (Inst. Molecular Virology, Univ. Wisconsin, Madison) and collaborators, describes how his group leveraged NCMIR's biochemical, electron tomography, and modeling approaches to characterize aspects of how a virus infects mitochondria, providing the first 3-D analysis of RNA replication complexes of a (+)RNA virus (Kopek et al. 2007).
In a third publication by Dr. Terrence Frey's laboratory (Dept. Biology, San Diego State University) and collaborators from St Jude Children's Research Hospital in Memphis, Tennessee, researchers used fluorescence microscopy with three-dimensional electron microscope tomography to characterize the time-course of morphological changes occurring in the mitochondria of single cells undergoing apoptosis (Sun et al. 2007). This study is the first to show the sequence of structural changes of mitochondria during programmed cell death and identifies the precise mechanisms responsible for the release of mitochondrial proteins such as cytochrome c, an essential step in apoptosis.
In most cell culture models of apoptosis, when mitochondria release cytochrome c they subsequently undergo a loss of membrane potential within minutes. To examine the ultrastructure of mitochondria during defined stages of cell death, The Sun et al. team used correlated light and electron microscopy to observe cell growth and death. By capturing a composite time-course overview of the mitochondrion's structural and functional changes during apoptosis, they observed that the inner portion of the mitochondrial membrane was remodeled into several separate compartments with a concomitant release of proteins. Leveraging NCMIR's tetracysteine and electron tomography technologies, Sun and colleagues monitored apoptosis initiated in cells engineered with the tetracysteine domain tagged cytochrome c fusion protein, Cyt. c-4Cys. This offered a means to visually monitor cytochrome c release during cell death. To follow changes in the mitochondrial membrane potential, they visually monitored the uptake of a fluorescent marker, tetramethylrhodamine ethyl ester (TMRE), across the membrane. Fluorescence microscopy allowed them to concurrently monitor cytochrome c release Cyt. c-4Cys and the membrane's potential during apoptosis. Using electron tomography, the researchers observed extensive rearrangement of the inner mitochondrial membrane during cell death (Figure 1). Time-course monitoring of apoptosis revealed cristae remodeling from an organized arrangement of membranes into a vesicular patchwork. This structural change co-occurred with the release of several mitochondrial proteins into the cytoplasm of the cell. The researchers observed, via fluorescence microscopy, that cytochrome c was released prior to and independent of cristae remodeling. By inhibiting caspases, a set of specialized proteases critical to apoptosis, mitochondria remodeling was inhibited but permitted the normal release of cytochrome c. Likewise, by inhibiting the normal changes in the permeability of the inner mitochondrial membrane, the researchers prevented release of cytochrome c and inhibited the formation of vesicular mitochondria that normally occurs during cell death. This finding suggests that the formation of vesicular mitochondria is not required for efficient release of cytochrome c but may be related to the fragmentation of mitochondria occurring during apoptosis
This study demonstrated that using three-dimensional transmission electron microscopy to study mitochondrial ultrastructure is helping to enhance a new paradigm of mitochondrial structure originally determined by electron tomography, as noted in the 5-Oct-2007 edition of Science's Editor's Choice: "Death Throes in Living Color". Science 318(5):19.
Publications:
Kopek, B., Perkins, G., Ellisman, M.H. and Ahlquist, P. (2007) Three-dimensional Structure and Organization of a Positive-strand RNA Virus RNA Replication Complex. PLoS Biology, 5:e220.
Sun MG, Williams J, Munoz-Pinedo C, Perkins GA, Brown JM, Ellisman MH, Green DR, Frey TG. Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis. Nat Cell Biol. 2007; 9:1057-65.
Yuan H, Gerencser AA, Liot G, Lipton SA, Ellisman M, Perkins GA, Bossy-Wetzel E. Mitochondrial fission is an upstream and required event for bax foci formation in response to nitric oxide in cortical neurons. Cell Death Differ. 2007;14:462-71.
