Morphology of mitochondrial permeability transition: morphometric volumetry in apoptotic cells.

Here we report on the mitochondrial permeability transition (MPT), which refers to the morphology of mitochondria whose inner membrane has lost its selective permeability. In all types of apoptotic cells so far examined, we found outer mitochondrial membranes that had been ruptured. These mitochondria present a swollen matrix covered by an inner membrane herniating into the cytoplasm through the breached outer membrane. Similarly ruptured outer mitochondrial membranes have been reported in studies on mitochondrial fractions induced to undergo MPT, carried out by others. Our observations were made on five types of rat tissue cells and six different cultured cell lines in the early stages of apoptosis. Samples from the cell lines HL-60, HeLa, WEHI-164, and a special batch of PC-12 cells were subjected to various apoptogenic agents and analyzed morphometrically. Nonapoptotic companion cells with unaltered nuclear structure (CUNS) were also analyzed. The mitochondrial volume in microm(3) and the volume fraction of the cytoplasm occupied by mitochondria in cells with typical nuclear signs of apoptosis and also in CUNS were evaluated. The volume of the mitochondria with ruptured membrane represents at least 69% (47-89%) of the total mitochondrial volume of the apoptotic cells. Thus, a considerable fraction of the cellular mitochondrial mass is or was in the state of permeability transition and probably involved in enhancement of the apoptotic program. In all samples, a fraction of the cells with normal nuclei possessed mitochondria with breached outer membranes as described above. In these cells, MPT occurred before the appearance of the typical nuclear phenotype of the apoptotic cells.

[1]  J. Zimmerberg,et al.  Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Grimm,et al.  The permeability transition pore signals apoptosis by directing Bax translocation and multimerization , 2002, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[3]  C Haanen,et al.  A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. , 1995, Journal of immunological methods.

[4]  Guido Kroemer,et al.  Apoptosis: Mitochondrial Membrane Permeabilization – The (W)hole Story? , 2003, Current Biology.

[5]  P. Ho,et al.  Cyclic AMP potentiates bFGF‐induced neurite outgrowth in PC12 cells , 1992, Journal of cellular physiology.

[6]  P. Bernardi,et al.  Physiological effectors modify voltage sensing by the cyclosporin A-sensitive permeability transition pore of mitochondria. , 1993, The Journal of biological chemistry.

[7]  G. Tiegs,et al.  Pre-apoptotic Alterations in Hepatocytes of TNF α-treated Galactosamine-sensitized Mice , 1998, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[8]  R. Jemmerson,et al.  Calcium‐induced Cytochrome c release from CNS mitochondria is associated with the permeability transition and rupture of the outer membrane , 2002, Journal of neurochemistry.

[9]  U. Igbavboa,et al.  EGTA inhibits reverse uniport-dependent Ca2+ release from uncoupled mitochondria. Possible regulation of the Ca2+ uniporter by a Ca2+ binding site on the cytoplasmic side of the inner membrane. , 1988, The Journal of biological chemistry.

[10]  G. Kroemer,et al.  Disruption of the outer mitochondrial membrane as a result of large amplitude swelling: the impact of irreversible permeability transition , 1998, FEBS letters.

[11]  M. Peter,et al.  Two CD95 (APO‐1/Fas) signaling pathways , 1998, The EMBO journal.

[12]  Dean P. Jones,et al.  The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore , 2004, Nature.

[13]  F. Vikhanskaya,et al.  Human papillomavirus type 16 E6‐enhanced susceptibility to apoptosis induced by TNF in A2780 human ovarian cancer cell line , 2002, International journal of cancer.

[14]  H. J. G. GUNDERSEN,et al.  Some new, simple and efficient stereological methods and their use in pathological research and diagnosis , 1988, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica.

[15]  N. Kyprianou,et al.  Activation of programmed cell death in the rat ventral prostate after castration. , 1988, Endocrinology.

[16]  John J Lemasters,et al.  Mitochondrial permeability transition: a common pathway to necrosis and apoptosis. , 2003, Biochemical and biophysical research communications.

[17]  Ruedi Aebersold,et al.  Molecular characterization of mitochondrial apoptosis-inducing factor , 1999, Nature.

[18]  H J Gundersen,et al.  The efficiency of systematic sampling in stereology and its prediction * , 1987, Journal of microscopy.

[19]  M. Zoratti,et al.  The mitochondrial permeability transition. , 1995, Biochimica et biophysica acta.

[20]  J. Kerr,et al.  Cell death by apoptosis during involution of the lactating breast in mice and rats. , 1989, The American journal of anatomy.

[21]  D. Pfeiffer,et al.  Increased permeability of mitochondria during Ca2+ release induced by t-butyl hydroperoxide or oxalacetate. the effect of ruthenium red. , 1982, The Journal of biological chemistry.

[22]  M. Prevost,et al.  Mitochondrial Release of Caspase-2 and -9 during the Apoptotic Process , 1999, The Journal of experimental medicine.

[23]  J. Farber,et al.  Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine. , 2001, Cancer research.

[24]  Gerard I. Evan,et al.  The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant , 2000, Nature Cell Biology.

[25]  D. Pfeiffer,et al.  The relationship between mitochondrial membrane permeability, membrane potential, and the retention of Ca2+ by mitochondria. , 1980, The Journal of biological chemistry.

[26]  B. Kachar,et al.  Structural elements common to mitosis and apoptosis. , 1999, Tissue & cell.

[27]  N. Narula,et al.  Targeting of the c-Abl Tyrosine Kinase to Mitochondria in Endoplasmic Reticulum Stress-Induced Apoptosis , 2001, Molecular and Cellular Biology.

[28]  P. Bradshaw,et al.  Properties of a Cyclosporin-insensitive Permeability Transition Pore in Yeast Mitochondria* , 1997, The Journal of Biological Chemistry.

[29]  A. Rosenzweig,et al.  Comparison of Comet Assay, Electron Microscopy, and Flow Cytometry for Detection of Apoptosis , 2003, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[30]  C. Winterford,et al.  Ethionine‐Induced Atrophy of Rat Pancreas Involves Apoptosis of Acinar Cells , 1993, Pancreas.

[31]  G. Kroemer,et al.  The adenine nucleotide translocator in apoptosis. , 2002, Biochimie.

[32]  S. Pandol,et al.  Mechanisms of cell death after pancreatic duct obstruction in the opossum and the rat. , 1996, Gastroenterology.

[33]  R. Haworth,et al.  The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release. , 1979, Archives of biochemistry and biophysics.

[34]  G. Kroemer,et al.  Mitochondria, the killer organelles and their weapons , 2002, Journal of cellular physiology.

[35]  D. Brenner,et al.  The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. , 1998, Biochimica et biophysica acta.

[36]  B. Herman,et al.  Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species. , 1997, The American journal of physiology.

[37]  Mason R. Mackey,et al.  Bid, Bax, and Lipids Cooperate to Form Supramolecular Openings in the Outer Mitochondrial Membrane , 2002, Cell.

[38]  J. Kerr,et al.  Anatomical methods in cell death. , 1995, Methods in cell biology.

[39]  M. V. Heiden,et al.  Bcl-xL Regulates the Membrane Potential and Volume Homeostasis of Mitochondria , 1997, Cell.

[40]  J. Martinou,et al.  Mitochondria as the central control point of apoptosis. , 2000, Trends in cell biology.

[41]  G. Kroemer,et al.  Quantitation of mitochondrial alterations associated with apoptosis. , 2002, Journal of immunological methods.

[42]  T. Kuwana,et al.  Bcl-2-family proteins and the role of mitochondria in apoptosis. , 2003, Current opinion in cell biology.

[43]  A Khodjakov,et al.  Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells , 2000, Cell Death and Differentiation.

[44]  H. Schmid,et al.  Intramitochondrial phospholipase activity and the effects of Ca2+ plus N-ethylmaleimide on mitochondrial function. , 1979, The Journal of biological chemistry.

[45]  J. Kwong,et al.  Ultrastructural and biochemical observations on the early changes in apoptotic epithelial cells of the rat prostate induced by castration , 1999, Cell and Tissue Research.

[46]  B. Herman,et al.  Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. , 1995, The Biochemical journal.

[47]  P. Bernardi,et al.  Mitochondrial transport of cations: channels, exchangers, and permeability transition. , 1999, Physiological reviews.

[48]  David S. Park,et al.  Caspase-Dependent and -Independent Death of Camptothecin-Treated Embryonic Cortical Neurons , 1999, The Journal of Neuroscience.

[49]  Ingo Schmitz,et al.  Differential Modulation of Apoptosis Sensitivity in CD95 Type I and Type II Cells* , 1999, The Journal of Biological Chemistry.

[50]  J. Martinou,et al.  Bax Is Present as a High Molecular Weight Oligomer/Complex in the Mitochondrial Membrane of Apoptotic Cells* , 2001, The Journal of Biological Chemistry.