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Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis

Nature Cell Biology volume 9, pages 1057–1065 (2007)Cite this article

Abstract

In addition to their role in cellular bioenergetics, mitochondria also initiate common forms of programmed cell death (apoptosis) through the release of proteins such as cytochrome c from the intermembrane and intracristal spaces1,2. The release of these proteins is studied in populations of cells by western blotting mitochondrial and cytoplasmic fractions of cellular extracts, and in single cells by fluorescence microscopy using fluorescent indicators and fusion proteins3,4. However, studying the changes in ultrastructure associated with release of proteins requires the higher resolution provided by transmission electron microscopy5,6,7. Here, we have used fluorescence microscopy to characterize the state of apoptosis in HeLa cells treated with etoposide followed by electron microscopy and three-dimensional electron microscope tomography of the identical cells to study the sequence of structural changes. We have identified a remodelling of the inner mitochondrial membrane into many separate vesicular matrix compartments that accompanies release of proteins; however, this remodelling is not required for efficient release of cytochrome c. Swelling occurs only late in apoptosis after release of cytochrome c and loss of the mitochondrial membrane potential.

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Figure 1: Cyt. c-4CYS staining with FlAsH colocalizes with TMRE staining of mitochondria that maintain a ΔΨm.
Figure 2: Mitochondrial inner membrane conformation changes to the vesicular form during cytochrome c release, and the matrix swells during or after the loss of ΔΨm.
Figure 3: Five mitochondrial morphologies are identified.
Figure 4: Electron microscope tomography of examples of the five mitochondrial morphologies.
Figure 5: Mitochondria progress from normal to vesicular during release of cytochrome c and then swell during or after the loss of ΔΨm.

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Acknowledgements

This project was supported by a Blasker Science and Technology Grant from the San Diego Foundation to T.G.F., by the National Institutes of Health (NIH) National Center for Research Resources (Grant No. P41 RR004050; G.A.P. and M.H.E.), by NIH Roadmap Grant GM72033 (Roger Y. Tsien and M.H.E.) and by NIH Grants AI40646, AI52735 and CA69381 (D.R.G.). C.M.-P. was supported by the Secretaria de Estado de Universidades Investigacion and the Fondo de Investigaciones Sanitarias of Spain. We thank Y. Jones (National Center for Microscopy and Imaging Research, University of California, San Diego) for assistance with high pressure freezing and freeze substitution, and J. Nulton for suggesting the mechanism of transformation mitochondria to the vesicular form.

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Authors and Affiliations

  1. Department of Biology, San Diego State University, San Diego, 92182-4614, CA, USA

    Mei G. Sun, James Williams & Terrence G. Frey

  2. Department of Immunology, St Jude Children's Research Hospital, Memphis, 38105–2794, TN, USA

    Cristina Munoz-Pinedo & Douglas R. Green

  3. Center for Research in Biological Systems, National Center for Microscopy and Imaging Research, University of California, San Diego, La Jolla, 92093-0608, CA, USA

    Guy A. Perkins, Joshua M. Brown & Mark H. Ellisman

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Correspondence to Terrence G. Frey.

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Sun, M., Williams, J., Munoz-Pinedo, C. et al. Correlated three-dimensional light and electron microscopy reveals transformation of mitochondria during apoptosis. Nat Cell Biol 9, 1057–1065 (2007). https://doi.org/10.1038/ncb1630

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