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First published online June 28, 2004
doi: 10.1242/10.1242/jcs.01140

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The necessity of mitochondrial genome DNA for normal development of Dictyostelium cells

Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aoba, Sendai 980-8578, Japan

View larger version (20K): [in a new window]   Fig. 1. Effect of EtBr on growth of Dictyostelium discoideum Ax-2 cells. Various concentrations of EtBr were added to exponentially growing cells (2x105 cells/ml) in axenic growth medium (PS medium), followed by cell counts under a haemocytometer. () control (non-EtBr-treated cells); () cells treated with 10 µg/ml of EtBr, () 20 µg/ml of EtBr, () 30 µg/ml of EtBr, () 40 µg/ml of EtBr and () 50 µg/ml of EtBr. Similar results were obtained by cell counts in three independent experiments.

View larger version (82K): [in a new window]   Fig. 2. Stainings of Ax-2 cells treated with 30 µg/ml of EtBr for 40 hours and nontreated cells with DAPI (A,C) and MitoTracker Orange (B,D). In nontreated Ax-2 cells, DAPI stains are noticed in nuclei (A, arrowheads) and mitochondria as granular structures (A, arrows). In EtBr-treated cells, however, the DAPI-staining of mitochondria is almost vanished, although the staining of nuclei is retained (C). However, rather stronger staining by MitoTracker Orange is observed in a limited number of mitochondria (D, arrows) contained in EtBr-treated cells compared with nontreated cells (B) at the vegetative growth phase. Bars, 10 µm.

View larger version (98K): [in a new window]   Fig. 3. Southern blot analysis of total DNAs extracted from Ax-2 cells and cells treated with 30 µg/ml of EtBr for 40 hours. The DNAs were digested with the indicated restriction enzymes and electrophoresed. After transfer of the size-fractionated DNA fragments to nylon membranes, they were hybridized with the 32P-labeled (A) nuclear DNA-specific probe Dd-trap1 or (B) mtDNA-specific probe rps4, followed by autoradiography.

View larger version (182K): [in a new window]   Fig. 4. Electron micrographs showing marked structural transformation of mitochondria in cells. (A,C) Vegetatively growing Ax-2 cells have normal-shaped mitochondria, whereas (B,D) cells have markedly transformed mitochondria having a sort of vacuoles (arrows), engulfing the nearby cytoplasm. Mt, mitochondria; N, nucleus. Bars, 1 µm.

View larger version (177K): [in a new window]   Fig. 5. Development of starved Ax-2 cells and cells on agar. Ax-2 cells and cells were washed twice in BSS and plated on 1.5% non-nutrient agar at a density of 5x106 cells/cm2. This was followed by incubation for the indicated times at 22°C. (A) Nontreated Ax-2 cells formed aggregation streams after 6 hours and mounds after 12 hours of incubation. Subsequently a tip was formed at the apex of each mound, elongated and constructed a migrating slug after 16 hours of incubation. This was followed by formation of a fruiting body at about 26 hours of incubation. By contrast, cells exhibited delayed and somewhat abnormal morphogenesis; large aggregation streams were formed after 16 hours of incubation, followed by their subdivision to smaller cell masses (A). (B) Gross morphology of final structures: fruiting bodies derived from nontreated cells and irregular-shaped slugs derived from cells. The cells stopped their development at the slug stage and never formed fruiting bodies. Bars, 1 mm.

View larger version (121K): [in a new window]   Fig. 6. Development of starved Ax-2 cells and cells under submerged conditions. Ax-2 cells and cells were washed twice in BSS and plated in a 24-well plate (1 ml cell suspension/well) at 5x105 cells/cm2. This was followed by incubation for the indicated times at 22°C. Almost all the cells showed no sign of cell aggregation and remained as round-shaped single cells even after 12 hours of incubation, while nontreated Ax-2 cells acquired aggregation-competence and began to aggregate at 7 hours of incubation, followed by formation of tight mounds during 12-24 hours of incubation. By contrast, cells formed large aggregation streams at 24 hours, which were then subdivided into smaller mounds during another 4 hours of incubation. Bars, 200 µm.

View larger version (92K): [in a new window]   Fig. 7. Expression patterns of the rps4, car1 and csA mRNAs during early development in Ax-2 cells and cells. Cells were harvested at the growth phase (V), washed twice in BSS and shaken for the indicated times (hours) at 22°C. Total RNAs were prepared according to the method of Nellen et al. (Nellen et al., 1987). Northern hybridization was performed using the RI (Amersham), as previously described (Hirose et al., 2000). As probes for detection of the three kinds of mRNAs, PCR products obtained by amplification of the respective cDNA clones, using M13-20 and M13R primers, was used. The lower panel shows for each lane the amount of ribosomal 17S and 26S RNA stained with EtBr.

View larger version (71K): [in a new window]   Fig. 8. Expression patterns of prestalk- and prespore-specific genes in slugs derived from transformed cells (ecmAO-gal cells and D19-gal cells) and their cells. ecmAO-gal cells are shown in (A) and (C), while D19-gal cells are shown in (B) and (D). Nontreated transformed cells and their EtBr-treated cells ( cells) were separately washed twice in BSS and incubated for 16 or 28 hours on filters supported by 1.5% agar to obtain respective slugs. This was followed by histochemical staining of slug with X-gal to visualize the activity of ß-galactosidase. In slugs derived from nontreated cells, the prestalk-specific ecmAO is expressed in the anterior prestalk region (A), whereas the prespore-specific D19 is expressed in the posterior prespore region (B). In slugs derived from cells, however, the boundary between the prestalk and prespore regions becomes unclear, presumably because of incomplete sorting between the two cell types (C,D). Images of respective DAPI-stained cells at the time-point of starvation are shown in (A'-D'). The DAPI staining of cytoplasmic granules (mitochondria) is observed in nontreated Ax-2 cells (A',B'), but not in cells (C',D'). Bars, 0.4 mm (A-D); 10 µm (A'-D').

View larger version (53K): [in a new window]   Fig. 9. Impaired prespore differentiation in slugs derived from cells. Non-treated Ax-2 cells and cells were separately washed twice in BSS and incubated for 16 or 28 hours on 1.5% agar to obtain respective slugs. This was followed by immunostaining of dissociated slug cells with FITC-conjugated anti-D. mucoroides spore IgG, as described in Materials and Methods. (A) Slug cells derived from nontreated Ax-2 cells. Prespore cells have many strongly stained granules (PSVs) in the cytoplasm. (B) In slug cells derived from cells, however, it is clear that the number-ratio of prespore cells with PSVs is considerably decreased. In addition, both the number of PSVs in each prespore cell and the strength of fluorescent FITC in each PSV are lower (arrows) compared with those in slug cells derived from nontreated Ax-2 cells. Bars, 20 µm.

View larger version (178K): [in a new window]   Fig. 10. Electron micrographs showing (A) mature PSVs in the prespore cells of a normal slug formed without EtBr-treatment, and (B) marked mitochondrial transformation and a scarce number of PSVs in the posterior region of a slug ( slug) formed from cells. Markedly transformed mitochondria (tMt) as observed in cells are still retained in slugs (B). Mt, mitochondria; PSV, prespore-specific vacuole. Bars, 1 µm.

View larger version (12K): [in a new window]   Fig. 11. A phototaxis-deficient phenotype of migrating slugs derived from cells. The light source is shown by the large arrows. Whereas slugs derived from non-EtBr-treated Ax-2 cells migrated almost directly towards the light source, slugs ( slug) derived from cells were highly disoriented. Phototactic behaviors of totally about 100 of respective slugs were traced, and the representative results of the slug no. 1-8 are shown in this figure. Bar, 1 cm.

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