Ultrastructural localization of cytochrome c in apoptosis demonstrates mitochondrial heterogeneity

Abstract

Release of apoptogenic factors into the cytosol including cytochrome c is triggering the execution phase of apoptosis through activation of cytoplasmic effector caspases. How loss of function of the electron transport chain can be reconciled with an adequate energy supply necessary for executing the apoptotic program was studied in granulosa cell (GC) sheets cultured up to 72 h without gonadotrophic support. Cytochrome c was localized ultrastructurally by oxidation of diaminobenzidine tetrahydrochloride both in living and fixed cells. In uncultured GC sheets all cells show staining over their entire mitochondrial population. In 72 h cultured sheets in the absence of FSH pre-apoptotic GC's display two subsets of mitochondria: normal sized stained mitochondria and small orthodox mitochondria without demonstrable cytochrome function. Apoptotic cells contain several mitochondria with preservation of respiratory function besides unstained orthodox mitochondria. The cytochrome c containing mitochondria typically display dilated intracristal spaces, a mitochondrial conformation related to increased ATP production. Cytochrome c release was confirmed by Western blotting. In 72 h cultures supplemented with FSH, GC's displayed staining over their entire mitochondrial population. In cultures lacking FSH, but partially protected from apoptosis through caspase inhibition, the cytochrome c release was not inhibited. Thus in the present studied model dysfunction of only a subset of mitochondria is instrumental to initiate the apoptotic program while a functional electron transport chain is maintained until the degradation phase in a subset of respiring mitochondria. Cell Death and Differentiation (2000) 7, 331 ± 337.

High Glucose Disrupts Mitochondrial Morphology in Retinal Endothelial Cells

“…13 We report that an increase in mitochondrial heterogeneity coincides with the progression of RRECs through apoptosis. In other systems, it has been reported that increased mitochondrial heterogeneity may reflect permeability transition pore opening and the release of cytochrome c. 32,33 Our findings coincide with recent studies of mitochondria biology that have focused on the role of mitochondrial morphology as a regulator of mitochondrial functioning. Most mammalian cell types have long, tubular networks of mitochondria, which is critical to normal mitochondria functioning.…”

Externalized Glycolytic Enzymes Are Novel, Conserved, and Early Biomarkers of Apoptosis

“…Importantly, some of the proteins identified here as enriched among apoptotic vesicles (for example, the actin-associated proteins cofilin and Arp2/3 (37), numerous heat shock proteins, and other chaperones associated with stress (38 -40)) fulfill expectations derived from other studies. Similarly, the mitochondrial protein cytochrome c is known to be released from mitochondria during apoptotic cell death (41,42). It is significant that not all of the vesicle-enriched molecules are membrane-exposed; in particular, we did not detect surfaceexposed cytochrome c (data not shown).…”

Mitochondrial Membrane Permeabilization and Superoxide Production during Apoptosis

“…It is known that a large fraction of cyt-c does not participate in electron transport (27) and hence that cyt-c is present in excess in the mitochondrial intermembrane space. Electron microscopy studies have indicated that the release of cyt-c during apoptosis is a complete process, leading to a total depletion of cyt-c in affected mitochondria (28). There is also evidence that cyt-c is released specifically out of apoptotic cells prior to a loss of plasma membrane integrity (18,29).…”