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First published online 13 May 2003
doi: 10.1242/jcs.00476

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Unexpected roles of a Dictyostelium homologue of eukaryotic EF-2 in growth and differentiation

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

View larger version (48K): [in a new window]   Fig. 1. Expression patterns of the Dd-ef2h mRNA and the Dd-EF2H protein during early development in Ax-2 cells and various transformed cells. Cells were harvested at the exponential growth phase, washed twice in BSS and shaken for the indicated times (hours) at 22°C. Total RNAs were prepared as described (Nellen et al., 1987). Northern hybridization was performed using the RI (Amersham), as previously described (Hirose et al., 2000). As a probe for detection of the Dd-ef2h mRNA, a PCR-product (3 kb) obtained by amplification of the cDNA clone SLE406 with the full-length of Dd-ef2h using M13-20 and M13R primers was used. Western blotting was performed as described in Materials and Methods. The expression patterns in ef-2AS cells underexpressing the Dd-ef2h mRNA (A), Dd-ef2 null cells produced by homologous recombination (B), and ef-2OE cells overexpressing the Dd-ef2h mRNA (C) are presented in comparison with those in parental Ax-2 cells. In the lower panel in B, the amount of actin in each lane (stained with CBB) is shown.

View larger version (27K): [in a new window]   Fig. 2. Changes of the Dd-EF2H protein during progression of cell cycle and starvation of synchronized cells. (A) Growth-phase Ax-2 cells synchronized by the temperature-shift method (Maeda, 1986) were withdrawn at the indicated cell-cycle phases. Exponentially growing Ax-2 cells (1-2x106 cells/ml) at 22.0°C, with a doubling time of about 7.5 hours, were shifted to 9.5°C, shaken for 14.5 hours and then reshifted to 22.0°C. Under these conditions, the cell number doubled within about 2 hours after a lag phase of about 1 hour. Tt cells, t hours after the shift-up from 9.5°C to 22.0°C, were harvested for western blot analysis. In another experiment, T7 cells, 7 hours after the shift-up, were harvested just before the PS-point, starved by washing twice in 20 mM Na/K-phosphate buffer (pH 6.5), and shaken at 1x107 cells/ml for 2 hours at 150 rpm. This yielded T7+2 cells, i.e. newly differentiating cells from the PS-point. T1+2 and T3+2 cells were also prepared by starving T1 and T3 cells for 2 hours in the buffer, as starved but not differentiated cells. The cell pellets were dissolved in 9 volumes of SDS-sample buffer, and the samples derived from the same number of cells (5x104 cells) were applied to SDS-PAGE (10% gel), followed by transfer to PVDF membranes and western blotting. In another experiment, synchronized T1, T3 and T7 cells were starved in BSS for 2 hours to obtain T1+2, T3+2 and T7+2 cells, respectively. Their western blot analyses were carried out as described above. (B) A schematic representation of the cell-cycle of an Ax-2 cell. The checkpoint (PS-point) of growth/differentiation is interposed just after T7.

View larger version (28K): [in a new window]   Fig. 3. Growth kinetics of ef-2OE, ef-2AS, ef2-null and parental Ax-2 cells. The four kinds of cells were separately shaken in growth medium without G418- or blasticidin S-addition at 22.0°C at 150 rpm. The number of cells at the exponential growth phase was determined using a hemocytometer. Similar results were obtained by cell counts in three independent experiments (, Ax-2; , ef-2OE; , ef-2AS; , ef2-null).

View larger version (173K): [in a new window]   Fig. 4. Development of ef2-null (B,D,F) and parental Ax-2 cells (A,C,E) under submerged conditions. ef2-null and Ax-2 cells were harvested during the exponential growth phase, washed twice in BSS and plated in a 24-well titer plate at a density of 5x105 cells/ml (1 ml of cell suspension/ well). This was followed by incubation at 22°C for 6.0 hours (A,B), for 7.5 hours (C,D) and 10.0 hours (E,F). At 6.0 hours of incubation, Ax-2 cells acquire aggregation competence (A), while ef2-null cells show no sign of cell aggregation (B). At 7.5 hours, aggregation streams are formed in Ax-2 cells (C), but not in ef2-null cells (D). At 10.0 hours, Ax-2 cells form tight aggregation streams (E), but ef2-null cells still remain as aggregation-competent cells (F). Similar results were obtained by observations in at least three independent experiments. Bars, 200 µm.

View larger version (189K): [in a new window]   Fig. 5. Development of ef-2OE (B,D,F) and parental Ax-2 cells (A,C,E) under submerged conditions. ef-2OE and Ax-2 cells were processed as described in the legend of Fig. 4. At 5.0 hours of incubation, Ax-2 cells remain as round-shaped single cells (A), but ef-2OE cells are elongated in shape with aggregation competence (B). At 6.0 hours, Ax-2 cells acquire aggregation competence (C), while ef-2OE cells exhibit enhanced differentiation to form aggregation streams (D). At 7.5 hours, ef-2OE cells form a more advanced stage of aggregation streams (F), compared with Ax-2 cells (E). Similar results were obtained by observations in three independent experiments. Bars, 200 µm.

View larger version (58K): [in a new window]   Fig. 6. Localization of Dd-EF2H in ef-2AS, ef2-null and parental Ax-2 cells. (A-H) Cells were harvested at the exponential growth phase, fixed in absolute methanol and double-stained with the anti-Dd-EF2H antibody or non-immune serum, and then with DAPI, as noted in Materials and Methods. In Ax-2 cells (A-C), the cytoplasm was immuno-stained by the anti-Dd-EF2H antibody (B). Surprisingly, in ef-2AS cells (D-F), immunostaining of cytoplasmic granules is retained. Higher magnification of ef2-null cells double-stained with the anti-Dd-EF2H antibody and DAPI (G,H) indicates that the distribution of cytopasmic granules stained with the antibody is exactly the same as that of mitochondria stained with DAPI. (I-K) ef2-null cells were double-stained with the anti-Dd-EF2H antibody (I) and MitoTracker Orange (K), as described in Materials and Methods. It is clear that both the stains are completely merged (J). Bars, 10 µm.

View larger version (87K): [in a new window]   Fig. 7. Western blot analyses of proteins extracted from a different number of cells and subcellular fractions, using the anti-Dd-EF2H antibody. (A,B) Cell lysates prepared from the indicated number of cells were applied to lanes of 10% SDS-PAGE, followed by western blotting. (C) Ax-2 cells were fractionated to cytosolic (Cyt) and mitochondria-rich (Mit) fractions. Vegetative cells were harvested at the mid-late exponential growth phase and pelleted by centrifugation (400 g, 1 minute). The pellet was suspended in ice-cold 50 mM Tris-HCl buffer (pH 7.5) containing 2.5 M sucrose and homogenized using a glass homogenizer. The homogenate was centrifuged for 10 minutes at 900 g to remove undisrupted cells and nuclei, and the resulting supernatant was centrifuged for 20 minutes at 2000 g. The pellet thus obtained was used as a Mit fraction. The supernatant was again centrifuged for 1 hour at 16,000 g, and the supernatant was put to use as a Cyt fraction. All of the processes were carried out around 4°C. In the Mit fraction, a band of 71 kDa protein can be detected in addition to contaminated 101 kDa Dd-EF2H. Based on the band patterns observed, it is most likely that the 58 kDa and 36 kDa proteins in B are hydrolysis products of 101 kDa Dd-EF2H, while the 41 kDa protein may be a hydrolysis product of the mitochondrial 71 kDa protein.