Mitochondrial "swirls" induced by oxygen stress and in the Drosophila mutant hyperswirl

Abstract

Mitochondrial dysfunction and reactive oxygen species have been implicated in the aging process as well as a wide range of hereditary and age-related diseases. Identifying primary events that result from acute oxidative stress may provide targets for therapeutic interventions that preclude aging. By using electron microscopy, we have discovered a striking initial pattern of degeneration of the mitochondria in Drosophila flight muscle under hyperoxia (100% O2). Within individual mitochondria, the cristae become locally rearranged in a pattern that we have termed a "swirl." Serial sections through individual mitochondria reveal the reorganization of the cristae in three dimensions. The cristae involved in a swirl are deficient in respiratory enzyme cytochrome c oxidase activity, within an otherwise cytochrome c oxidase-positive mitochondrion. In addition, under hyperoxia cytochrome c undergoes a conformational change, manifested by display of an otherwise hidden epitope. The conformational change is correlated with widespread apoptotic cell death in the flight muscle, as revealed by in situ terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. In normal flies, mitochondrial swirls accumulate slowly with age. To investigate the molecular mechanisms involved in oxygen toxicity, we conducted a genetic screen for mutants that display altered survival under hyperoxia, and we identified both sensitive and resistant mutants. We describe a mutant, hyperswirl, which displays an overabundance of swirls with associated respiratory and flight defects and a greatly reduced lifespan. Such mutants can identify genes that are needed to maintain mitochondrial homeostasis throughout the lifespan.

Fig. 1.

Mitochondrial swirls accumulate under hyperoxia. (A) Electron micrograph of flight muscle of Drosophila (white¹¹¹⁸ strain) maintained for 7 days under normoxic conditions. Mitochondria (M) are aligned between the myofibrils. (B) Electron micrograph of flight muscle of Drosophila (white¹¹¹⁸ strain) after 7 days under hyperoxia (100% O₂). Mitochondrial swirls are seen in most mitochondria. (C) A normal mitochondrion at higher magnification. (D) A typical swirl at higher magnification. (Scale bars indicate 1 μm.)

Fig. 2.

Frequency of swirl formation under hyperoxia. Sections of flight muscle were examined for swirl formation after 4 and 7 days under hyperoxia. Age-matched controls were maintained under normoxia. Data are averages of 10 flies ± SD, with >80 mitochondria scored from each individual fly.

Fig. 3.

Exposure to hyperoxia induces defects in complex IV of the electron transport chain. (A) Electron micrograph of flight muscle (white¹¹¹⁸ strain) showing staining for COX activity. After 6 days under normoxia, mitochondria stain uniformly. (B) After 6 days under hyperoxia, cristae within a swirl are COX deficient (COX^–), whereas cristae in the remainder of the mitochondrion are COX⁺. (C) Electron micrograph of flight muscle from control flies maintained under normoxia and stained with mAb 2G8, which specifically recognizes the apoptogenic cytochrome c epitope. No immunoreactivity is seen. (D) After exposure to hyperoxia for 4 days, the preapoptotic epitope for mAb 2G8 is exposed. (Scale bars indicate 1 μm.)

Fig. 4.

Apoptosis induced in the flight muscle by hyperoxia. (A) Flight muscle from a control fly, maintained under normoxia for 4 days, exhibits no TUNEL-positive nuclei, whereas flies exposed to hyperoxia for 4 days (B), and assayed by TUNEL staining 2 days later, have many apoptotic nuclei (green). (Scale bars indicate 100 μm.)

Fig. 5.

Serial sections through a mitochondrion containing a swirl. (A–T) Electron micrographs of flight muscle of Drosophila (white¹¹¹⁸ strain), after 4 days under hyperoxia. Section thickness is 0.08 μm. Higher magnification of boxed region in H shows projection of swirl to the outer membrane. (Scale bars indicate 1 μm.)

Fig. 6.

The long-lived mth mutant displays resistance to oxygen stress whereas hys is hypersensitive. Survival curves (± SE) under hyperoxia of mth mutant males compared with its parental line, white¹¹¹⁸ and the WT strain Canton-S (C-S). The mth displays increased resistance to hyperoxia (log-rank test; P < 0.0001). The hys is extremely sensitive to hyperoxia (log-rank test; P < 0.0001).

Fig. 7.

The recessive mutant hys is short-lived and rapidly accumulates swirls. (A) Survival curves (±SE) of white¹¹¹⁸ (w), hys heterozygotes (hys+/–), and homozygotes (hys–/–). Flies were maintained at 25°C, transferred every 2–4 days, and scored for survival. The hys homozygotes are very short-lived even under normoxia (log-rank test; P < 0.0001). (B) Electron micrograph of flight muscle of hys maintained under normoxic conditions for 2 days at 25°C. (C) Flight muscle of hys maintained under normoxic conditions for 7 days at 25°C. (Scale bars indicate 1 μm.)

Fig. 8.

The hys mutant displays respiratory and flight defects. (A) CO₂ production was measured for white¹¹¹⁸, the WT strain Canton-S (C-S), and the mutant hys after 7 days under normoxia. The mutant hys displays a 31% decrease in metabolic rate (P < 0.001). (B) Flight ability was measured in a simple flight assay, as described (40). In brief, control (C-S) flies were dropped into a 500-ml graduated cylinder with its internal walls coated with paraffin oil. Normal flies quickly initiate horizontal flight, striking the wall close to the entry level, whereas poor fliers land at lower levels or at the bottom of the cylinder. (C) The hys mutants (2 days old) perform poorly.