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  • Correlated images reveal details of apoptosis

Nov 2007
Electron and confocal microscopy reveal mitochondrial changes

Kevin Robinson

Recent research has revealed information on structural changes in mitochondria that happen during apoptosis. A better understanding of apoptosis could lead to new treatments for cancer and genetic disorders and possibly even to treatments that prevent cell death during heart attacks or strokes.

Researchers at San Diego State University, at St. Jude Children’s Research Hospital in Memphis, Tenn., and at the University of California, San Diego, collaborated on an approach to studying the process of etoposide-induced apoptosis in HeLa cells. Etoposide interferes with the cells’ ability to repair breaks in their DNA, and when the breaks become too numerous, a signaling system activates, initiating apoptosis so that the cell will die in an orderly manner.

In normally functioning cells, cytochrome c is part of the electron transfer process that generates ATP. However, when it is released from the mitochondria, it activates a series of caspase enzymes that break down other proteins and thus leads to cell death.

The researchers, led by Terrence G. Frey, used a line of HeLa cells permanently transfected to produce the protein cytochrome c with a short extension of its amino acid sequence that could be labeled with a fluorescent dye.

They used a biarsenical fluorescein derivative called FlAsH to label the cytochrome c. They also used the fluorescent probe tetramethylrhodamine ethyl ester (TMRE) to monitor mitochondrial membrane potential.

The basic experiment consisted of treating cells with etoposide to initiate apoptosis, labeling the cells with FlAsH and TMRE and then using various techniques, including confocal microscopy and transmission electron microscopy, to assess the progression of cell death.

Frey said that one benefit of HeLa cells is that they will grow while attached to a surface or substrate. The scientists grew the cells in petri dishes with a glass coverslip that had a grid etched on it. “Since the HeLa cells are grown on a coverslip with a labeled grid, we can fix, embed and section for transmission electron microscopy the identical cells that we have observed by confocal light microscopy.”

The grid allowed the researchers also, using confocal fluorescence microscopy, to monitor the individual cells as they progressed through apoptosis.

By correlating confocal fluorescence microscopy and electron microscope tomography, researchers identified three stages and five mitochondrial morphologies that cells progress through during apoptosis induced with etoposide. The morphologies are, from left to right and top to bottom: normal, normal-vesicular, vesicular, vesicular-swollen and swollen. Images courtesy of Terrence G. Frey.

They used a Leica confocal inverted microscope, exciting FlAsH with its 488-nm argon laser line attenuated to 35 percent. To excite TMRE, they used a HeNe laser at 543 nm that they attenuated to 34 percent. They also used two transmission electron microscopes: one made by Jeol Ltd. of Tokyo that has an accelerating voltage of 400 kV to image thicker sections of cells (up to 0.5 mm thick) and a microscope made by FEI Co. of Hillsboro, Ore., that has an accelerating voltage of 120 kV and a cooled slow-scan CCD camera made by Tietz Video and Image Processing Systems GmbH of Gauting, Germany, to image thinner sections. They tilted sections of cells in the electron microscope to calculate a 3-D structure of the mitochondria.

They discovered that apoptosis induced with etoposide has three basic stages. In the first, the mitochondria have normal membrane potential, and no cytochrome c has been released. In the second, the mitochondria have normal membrane potential; however, FlAsH fluorescence indicates that cytochrome c has been released. In some stage two cells, the mitochondrial structures display abnormal vesicular matrix compartments. Stage three cells have lost membrane potential, and the mitochondria are swollen.

During apoptosis induced by etoposide, the substructures in the mitochondria progress from normal (left) to normal with some vesicular components (center) to vesicular (right).

he researchers also described five mitochondrial morphologies leading to cell death: normal, normal-vesicular, vesicular, vesicular-swollen and swollen. They found that the change from normal to swollen progresses from one end of a mitochondrion to the next.

The investigators repeated the experiment, this time including the caspase inhibitor zVAD-fmk with the etoposide. The caspase inhibitor led to a much more normal mitochondrial structure even in cells that had released cytochrome c. Frey explained that this was a key finding. “The structural changes we have observed in the study ultimately appear not to be a cause of the release of proteins such as cytochrome c from mitochondria but are an effect of the release of proteins and activation of caspases.”

In the September issue of Nature Cell Biology, the researchers hypothesize that vesicles formed after cytochrome c release actually result from the transformation of the mitochondrial inner membrane. The vesicles appear to form as the membrane’s crista junctions elongate and fuse. In fact, the elongation and fusion of the crista junctions caused by etoposide-induced apoptosis are similar to morphology found in mitochondria of a laboratory model of autosomal dominant optic atrophy, a hereditary disorder characterized by progressive loss of vision and caused by a mutation of the OPA1 gene.

In addition, they reported that the vesicular mitochondrial morphology is identical to morphology found in the mouse model for Bethlehem myopathy, a genetic muscular disease.

From here, Frey said that they plan to examine other methods for inducing apoptosis, including generating reactive oxygen species.

“We are studying the alternate mechanisms and find that they proceed by different pathways than that initiated by etoposide.”

The researchers also are examining whether the mitochondria’s change from normal to vesicular to swollen is a more generalized manifestation of the mechanism of mitochondrial fission observed in all cells.

“We are currently studying the reverse process — mitochondrial fusion — in order to determine whether its mechanism is the reverse of the fission process.”

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