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. 2008 Feb 29;4(2):e1000022.
doi: 10.1371/journal.pgen.1000022.

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals

Takayuki Kanazawa  1 , Mauro D Zappaterra, Ayako Hasegawa, Ashley P Wright, Erin D Newman-Smith, Karolyn F Buttle, Kent McDonald, Carmen A Mannella, Alexander M van der Bliek
Affiliations

The C. elegans Opa1 homologue EAT-3 is essential for resistance to free radicals

Takayuki Kanazawa et al. PLoS Genet. .

Abstract

The C. elegans eat-3 gene encodes a mitochondrial dynamin family member homologous to Opa1 in humans and Mgm1 in yeast. We find that mutations in the C. elegans eat-3 locus cause mitochondria to fragment in agreement with the mutant phenotypes observed in yeast and mammalian cells. Electron microscopy shows that the matrices of fragmented mitochondria in eat-3 mutants are divided by inner membrane septae, suggestive of a specific defect in fusion of the mitochondrial inner membrane. In addition, we find that C. elegans eat-3 mutant animals are smaller, grow slower, and have smaller broodsizes than C. elegans mutants with defects in other mitochondrial fission and fusion proteins. Although mammalian Opa1 is antiapoptotic, mutations in the canonical C. elegans cell death genes ced-3 and ced-4 do not suppress the slow growth and small broodsize phenotypes of eat-3 mutants. Instead, the phenotypes of eat-3 mutants are consistent with defects in oxidative phosphorylation. Moreover, eat-3 mutants are hypersensitive to paraquat, which promotes damage by free radicals, and they are sensitive to loss of the mitochondrial superoxide dismutase sod-2. We conclude that free radicals contribute to the pathology of C. elegans eat-3 mutants.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Mutations in C. elegans eat-3 and dyn-1 Mutants.
(A) Dynamin family members in C. elegans. DYN-1 is required for scission of vesicles from the plasma membrane. DRP-1 is required for scission of mitochondrial outer membranes. FZO-1 is required for fusion of mitochondrial outer membranes. EAT-3 is required for fusion of mitochondrial inner membranes. The GTPase, Middle, and GTPase Effector (GED) domains are shared between dynamin family members. In addition, DYN-1 has a pleckstrin homology (PH) domain and a proline rich domain (PRD), FZO-1 has two transmembrane segments that anchor the protein in the mitochondrial outer membrane, and EAT-3 has a mitochondrial leader sequence (mls) that targets the protein to the mitochondrial intermembrane space. Some key alleles are shown on the right. (B) Sequence alignment of the GTPase domains of C. elegans EAT-3 (D2031.5), human Opa1, human Dyn1, and C. elegans DYN-1. The GTP binding consensus sequences (G1-4) are indicated with white circles. The primary mutations in eat-3(ad426) and dyn-1(ky51) alleles are shown in the red circles. Secondary mutations in the intragenic revertants are shown in the blue circles. The dyn-1(ky51) revertants cq2, cq3, and cq4 are shown below the sequences and the eat-3(ad426) revertants cq6, cq7, cq8, cq9, and cq10 are shown above the sequences. (C) The positions of the GTP binding motifs (open circles labeled G1-4), the positions of primary mutations (red circles), and the positions of secondary mutations (blue circles) superimposed on the structure of the rat Dyn1 GTPase domain . The arrows point to the primary mutation suppressed by each secondary mutation.
Figure 2
Figure 2. Fragmented Mitochondria Caused by RNAi or Mutations in eat-3.
(A) Regular tubular array of mitochondria in muscle cells of a wildtype worm. (B) Fragmented mitochondria in muscle cells subjected to eat-3 RNAi generated with a Pmyo-3:: antisense-eat-3 construct. (C) Fragmented mitochondria in muscle cells of worms with a Pmyo-3:: eat-3(T322A) construct. (D) Fragmented mitochondria in muscle cells eat-3(ad426) worms. (E) Fragmented mitochondria in the muscle cells of eat-3(tm1107) worms. (F) Tubular mitochondria in the muscle cells of eat-3(ad426) worms that were rescued by a construct containing wildtype eat-3 cDNA under control of the eat-3 gene promoter (Ex[eat-3(wt)]). (G) Mitochondria in the fzo-1(tm1133) deletion strain. (H-J) Mitochondria in the eat-3(ad426) revertants cq6, cq8, and cq10. Mitochondria were detected with matrix GFP driven by the muscle specific myo-3 promoter. The bar indicates 5 µm.
Figure 3
Figure 3. Aberrant Internal Structures in eat-3(ad426) Mitochondria.
(A) Longitudinal section of a body wall muscle in a wildtype worm. This section shows one long mitochondrion (m) and myofibrils (f). The insert is an enlargement of a muscle cell mitochondrion. (B) Longitudinal section of a body wall muscle in an eat-3(ad426) worm. (C) Section of an intestinal cell in a wildtype worm. The mitochondria (m) are randomly oriented and therefore sectioned at many different angles. (D) Section of an intestinal cell in an eat-3(ad426) worm. The pairs of arrows indicate matrix compartments with different cristae morphologies enclosed by a single outer membrane. (E) Enlarged portion of an intestinal cell showing mitochondria (m) with more inner membrane aberrations. Aberrant structures indicated with numbers: 1. Inner membrane septae. 2. Short tubular cristae. 3. Long invaginations of inner membrane. 4. Novel compartments within the mitochondrial matrix enclosed by a double membrane. 5. Electron dense inclusions in the mitochondrial matrix. The inserts show further enlargements of three different places with double membranes. (F) Section of an intestinal cell in an eat-3(ad426) worm rescued by microinjection of a wildtype eat-3 transgene. Bars are 0.5 µm.
Figure 4
Figure 4. Tomographic Reconstruction of an eat-3(ad426) Mitochondrion.
(A,B) Front and rear views of an eat-3 mitochondrion reconstructed by electron tomography. Outer membrane is shown in red. Inner membrane is shown in green. Bubbles on the side of the mitochondrion, which are also shown in green, are matrix compartments separated inner membrane septae. Invaginations of the inner membrane, which are likely to be short tubular cristae, are shown in light green. Internalized membrane-bound compartments are shown in magenta. Large clusters of electron dense material without membrane are shown in blue. (C–F) Tomographic sections used to make the three-dimensional renderings in (A) and (B). The bar is 0.5 µm.
Figure 5
Figure 5. Slow Growth and Disrupted Gonads Caused by eat-3 RNAi.
(A) Growth curve comparing wildtype worms to the progeny of worms injected with eat-3 RNAi. The plots show the average lengths obtained with 5 wildtype worms (closed circles) and 20 eat-3 RNAi progeny (closed squares) with standard deviations. (B) Chromosomal eat-3 mutants are also smaller than wild type, but this defect can be restored by a mutation in drp-1. The lengths of eat-3(ad426), eat-(ad426); drp-1(cq5) and eat-3(tm1107) are compared with the lengths of wild type (N2) animals four days after hatching. The lengths are averages of 12, 13, 5, and 16 animals respectively with standard deviations. (C) Close-up of a gonad arm from a wild type worm. Mitochondria were stained with rhodamine 123 (red) and nuclear DNA was stained with Hoechst (green). The nuclei form an orderly pattern near the surface of the gonad. These nuclei are always surrounded by mitochondria. (D) Gonad of a worm injected with eat-3 RNAi. The gonads are dissected two days after injection with dsRNA. The mitochondria appear more dispersed than in wildtype but are not notably less abundant. Instead, there is a paucity of nuclei consistent with the reduced brood size of eat-3 RNAi animals. The bar is 5 µm.
Figure 6
Figure 6. Suppression of eat-3(ad426) Phenotypes.
(A) Mitochondria in a wildtype (N2) muscle cell with their normal tubular morphology. (B) Fragmented mitochondria in a muscle cells of the eat-3(ad426) mutant. (C) Mitochondria with highly connected outer membranes (green) but not connected matrix compartments (red) in a muscle cell of the eat-3(ad426); drp-1(cq5) double mutant. (D) Similarly connected mitochondrial outer membranes in a muscle cell of the drp-1(cq5) mutant after removal of the eat-3(ad426) mutation by backcrossing. The mitochondria in muscle cells were detected with the transmembrane segment of C. elegans Tom70 fused to YFP (shown in green) and the matrices are labeled with a mitochondrial leader sequence fused to CFP (red). Nuclei are marked with n. The bar indicates 5 µm. (E) Histograms showing rescue of the eat-3(ad426) broodsize defect by drp-1 and fzo-1 RNAi, but not rescue of the eat-3(tm1107) deletion allele by drp-1 or fzo-1 RNAi. Wildtype (N2) and mutant animals were grown on bacteria with the feeding RNAi plasmid pILL4440 without insert, with fzo-1 cDNA or with drp-1 cDNA. The bars on the right show that the brood size of the eat-3(ad426) mutant is also rescued by a chromosomal drp-1 mutation (drp-1(cq5)). This rescue depends on residual eat-3 function in the ad426 allele, because it is eliminated by eat-3 RNAi. The brood sizes were defined as the numbers of viable larvae that developed to the L4 stage. Error bars indicate SE. An unpaired Student's t test was used for statistical analysis. The single asterisk indicates P<0.0001 and the double asterisk indicates P<0.01 (n = 24 for eat-3(ad426) alone, n = 14 for the same with fzo-1 RNAi and n = 7 for drp-1 RNAi).
Figure 7
Figure 7. Lack of Evidence for Increased Cell Death in eat-3 Mutant Animals.
(A) The broodsizes of eat-3(ad426) animals alone were not appreciably different from the broodsizes of the ced-3(n717); eat-3(ad426) or ced-4(n1894); eat-3(ad426) double mutants. Feeding RNAi for the other caspases (csp-1, csp-2 and csp-3) also had no effect (data not shown). (B) Numbers of dying cells were counted in comma stage embryos of wildtype (N2), eat-3(ad426), eat-3(tm1107), ced-3(n717) and ced-4(n1894) animals. The numbers of dying cells were not significantly increased in the eat-3 mutants when compared with wildtype. The numbers of dying cells in ced-3(n717) and ced-4(n1894) embryos were included to show that cell death is effectively blocked in these mutants. The bars indicate SE.
Figure 8
Figure 8. Paraquat Sensitivity of the eat-3(ad426) Mutant and Its Reversal by drp-1(cq5).
(A) The histogram shows the percentages of worms that survive from the L1 larval stage to adulthood when grown on plates with increasing concentrations of paraquat. Fifty L1 larvae with the indicated genotypes were transferred to five fresh plates for each data point and monitored for five days. The entire experiment was done in triplicate, except for drp-1(cq5); eat-3(ad426), which was done in duplicate. The bars show averages and SD for the experiments. An unpaired Student's t test was used for statistical analysis. The single asterisk indicates P<0.0001 and the double asterisk indicates P<0.0005 at 0.4 mM paraquat (n = 3). The N2 (wildtype) and eat-3(ad426) strains were tested two more times to more accurately determine the IC50 (see text). (B) A comparison between eat-3(ad426), eat-3(tm1107) and eat-3(ad426); drp-1(cq11) at 0.4 mM paraquat. The histogram shows averages of three experiments with their standard deviations. The increased sensitivity of eat-3(tm1107) animals to paraquat confirms that the effect is due to loss of eat-3 function, because it was observed with two independent alleles (ad426 and tm1107). In this same manner, reversal of paraquat sensitivity by the cq11 allele in eat-3(ad426); drp-1(cq11) animals confirm that the reversal is due to loss of drp-1 function, because it was also observed with two independent alleles (cq5 and cq11). The absolute numbers for N2 and eat-3(ad426) in panels A and B show minor differences because of variations between experiments, but the trends are the same.
Figure 9
Figure 9. Increased Sensitivity to the Mitochondrial Matrix Superoxide Dismutase sod-2.
(A) Effects of sod-1, sod-2, sod-3, and sod-5 RNAi on the broodsize of eat-3(ad426) animals. The histogram shows the numbers of hatched worms on plates with eat-3(ad426) animals with or without sod feeding RNAi as percentages of the numbers of hatched worms on plates with wildtype (N2) worms with or without feeding RNAi. (B) Effects of eat-3 RNAi on the broodsizes of sod-1, sod-2, sod-3 and sod-5 mutants. Wildtype (N2) worms are compared with the sod mutants, sod-1(tm776), sod-2(gk257), sod-3(gk235), sod-4(gk101), and sod-5(tm1146). The histogram shows the numbers of hatched worms on plates with eat-3 feeding RNAi as percentages of the numbers of hatched worms on plates without feeding RNAi (vector alone). Both (A) and (B) show the average results for three independent experiments. For each point in each experiment five L4 larvae were transferred to individual bacterial plates with or without feeding RNAi. Eggs that hatched on those plates were counted as viable progeny. In each experiment the average values of five plates were determined. The error bars show SD for variation between the three experiments. An unpaired Student's t test was used for statistical analysis. The asterisk indicates P<0.005 in (A) and P<0.05 in (B) (n = 3). (C) Expression levels of Fe/Mn-SOD proteins relative to the expression levels in wildtype (N2) animals. Expression was determined by probing Western blots with an antibody that detects SOD-2 and SOD-3 proteins. Densitometric scans of SOD were normalized to tubulin levels for each lane and then expressed as a percentage of wildtype (N2) levels. The mutant strains used here have fzo-1(tm1133), eat-3(ad426), eat-3(ad426); drp-1(cq5) and eat-3(ad426cq8) alleles. (D) Expression levels in mutant animals grown with or without feeding RNAi bacteria, as indicated below the histogram. The mutant alleles used here are eat-3(ad426), sod-2(gk257) and sod-3(gk235).
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