Cell cultures and developmental conditions

Vegetative cells of Dictyostelium discoideum Ax-2 were grown axenically in PS-medium (1% Special Peptone (Oxoid: Lot. No. 333 56412), 0.7% Yeast extract (Oxoid), 1.5% D-glucose, 0.11% KH₂PO₄, 0.05% Na₂HPO₄ · 12H₂O, 40 ng/ml of vitamin B₁₂, 80 ng/ml of folic acid, pH 6.4). rps4-underexpressing transformants (rps4^AS cells) produced by antisense gene inactivation and rps4-overexpressing transformants (rps4^OE cells) isolated previously by Inazu et al. (1999) were grown axenically in shaking cultures in PS-medium containing 50 μg/ml of G418. rps4^HR cells, in which the rps4 gene was disrupted in about half the mitochondrial genomes by means of homologous recombination (Inazu et al., 1999), were also used for comparison. To allow cells to differentiate, cells were harvested at the exponential growth phase, washed twice in BSS (Bonner's salt solution; 10 mM NaCl, 10 mM KCl, 2.3 mM CaCl₂; Bonner, 1947) as starvation medium, and either shaken at a density of 1.5×10⁷cells/ml or incubated in a 24-well titer plate (Falcon, #3047) at a density of 5×10⁵ cells/well at 22°C.

Transformation of cells

pDNeo2 (Witke et al., 1987) was used the original vector for preparation of antisence-mediated gene inactivation. This vector was cut by digestion with BamHI and SalI and then ligated overnight with the rps4 gene that had been amplified by PCR using pBluescript II KS(+) with the mitochondrial DNA of D. discoideum as the template and then digested with BamHI and XhoI. The ligates were inserted into XL1-blue competent cells. To produce cells underexpressing the rps4 mRNA, Ax-2 cells were transformed with the antisense construct by electroporation, as described by Nellen et al. (Nellen et al., 1987). Transformed cells were selected in 10 ml of PS-medium containing 10 μg/ml G418 in Petri dishes (9 cm diameter). Two days after the appearance of colonies of transformed cells, the colonies were cultured by shaking in PS-medium containing 20–50 μg/ml G418 for 2–3 days and then cloned in 96-well titer plates (Iwaki, Chiba, Japan).

Preparation of the anti-RPS4 antibody and western blot analysis

Chemically synthesized oligopeptide (EEPKLTAIKYPFTLQPEK; from the 368th to 385th amino acid of RPS4) with an additional cysteine residue at the C-terminus was conjugated with KLH (keyhole limpet hemocyanin) as a carrier protein by Research Genetics, Inc. (Huntsville, Alabama, USA). The KLH-conjugated oligopeptide was injected 4×1 ml s.c. into the foot pads of rabbits with complete Freund's adjuvant. The total amount of the antigen was 5 mg per animal. 5 weeks later, a total amount of 1 mg KLH-conjugated oligopeptide per animal with adjuv. was supplied s.c.. Samples of blood (about 50 ml) were collected 10 days after the final injection, and aliquoted serum containing the polyclonal anti-RPS4 antibody was stored at −80°C. The IgG fraction of the serum was absorbed by homogenates prepared from vegetatively growing Ax-2 cells that were almost devoid of RPS4 protein, and the absorbed antibody (referred to as the anti-RPS4 antibody) was used for western analysis and immunostaining.

Cells were harvested from shaken cultures and lysed in SDS sample buffer (2% SDS, 10% glycerol, 41.7 mM dithiothreitol, 0.01% bromophenol blue, and 62.5 mM Tris-HCl (pH 6.8)). Proteins were size fractionated on 10% SDS gels and blotted onto polyvinylidene difluoride (PVDF) membranes (Millipore). After blotting, the membranes were gently shaken in TBS-T (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% Triton X-100) containing 5% BSA or 5% skim milk, overnight at 4°C. Subsequently, the membranes were incubated in the primary antibody solution (1/2,500 anti-RPS4 antibody in TBS-T with 5% BSA or 5% skin milk and 0.15% Tween 20), overnight at 4°C. After washing in TBS-T for 20 min, the membranes were incubated in the secondary antibody solution (1/30,000 HRP-conjugated anti-rabbit IgG, goat (Amersham Pharmacia Bio-technology) in TBS-T with 5% BSA for 1 hr, according to the suggestions made by ICN Biochemicals. The chemical enhanced chemiluminescence (ECL kit; Amersham Biosciences) was used for detection of the RPS4 protein.

Staining of cells with a mitochondrion-selective dye, MitoTracker Orange

Ax-2 cells were starved for 4 hr and then incubated in BSS containing 0.5 μM MitoTracker Orange CMTMRos (Molecular Probes) for 15 min. The stained cells were washed twice with BSS, and observed under a scanning confocal fluorescence microscope.

Double staining of cells with the anti-RPS4 antibody and DAPI

After 0–4 hr of starvation, Ax-2 cells and rps4 -overexpressing cells (rps4^OE cells) that had been allowed to adhere to coverslips were fixed with 100% methanol for 10 min. To prevent cell shrinkage during fixation, samples were pre-fixed with 50% and then 100% methanol (1 min for each). The fixed samples were incubated for 16 hr at 22°C in the primary antibody (50-times diluted anti-RPS4 antibody) solution. Following threefold washing (10 min for each) in phosphate-buffered saline (PBS; 0.14 M NaCl, 3 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄, pH 7.2), the samples were incubated with the secondary antibody solution (FITC-conjugated anti-rabbit IgG antibody, goat; 2,000 times-diluted with 1% BSA) for additional 3 hr at 22°C. After repeated washings with PBS, the samples were mounted in PBS containing 20% glycerol. They were visualized by a fluorescence microscope. In the case of double staining with the anti-RPS4 antibody and DAPI (4′,6′-diamidino-2-phenylindole-dihydrochroride), cells adhering to coverslips were fixed with 2.0% formaldehyde solution containing 0.1% glutaralde-hyde for 20 min, followed by treatment with 0.1% Triton X-100 solution to make cells permeable. The fixed samples were immunostained with the anti-RPS4 antibody as described above, and then stained with 0.5 μg/ml of DAPI for 1–4 hr at room temperature.

Subcellular fractionation

Vegetatively growing Ax-2 cells (t₀-cells) and t₄-cells starved for 4 hr were fractionated by a slight modification of the method of Ikeda and Takeuchi (1971). Vegetative and starving Ax-2 cells were separately harvested from shaken cultures, washed twice with BSS, and suspended in 5 mM Tris-HCl (pH 7.5) containing 0.25 M sucrose. The cells were homogenized and centrifuged at 400×g for 10 min to remove undisrupted cells. The supernatant was centrifuged twice at 900×g for 10 min to obtain the membrane fraction that mainly contained the cell membrane and nuclei as the pellet. The resulting supernatant was then centrifuged twice at 2,000×g for 20 min to the pelleted particulate fraction including mitochondria. The supernatant was centrifuged at 18,000×g for 60 min, and the resulting supernatant was collected as the cytosolic faction. The pellet fractions were washed with 5 mM Tris-HCl (pH, 7.5) containing 0.25 M sucrose, and then were dissolved in the SDS sample buffer for western blot analysis. The cytosol fraction was two-times diluted with 2×SDS sample buffer, followed by western blotting.

Uniqueness of Dictyostelium RPS4 protein

An alignment of the deduced amino acid sequence of Dictyostelium discoideum mitochondrial RPS4 (Dd-mtRPS4) with sequences of RPS4 homologues from other species show a moderate degree of homology (25–35%) (Fig. 1). Although the homology of Dd-mtRPS4 to human mt-RPS4 is not so high as a whole, the N-terminus of Dd-mtRPS4 which is supposed to be an RNA-binding region is well conserved. More significantly for this work, several putative nuclear localization signals are present in Dd-mtRPS4, but not in mt-RPS4 protein of other organisms (Fig. 1). Thus the Dd-mtRPS4 protein, if present in the cytosol, would be predicted to be transferred to the nucleus with 95% or more probability according to the PSORTII Search.

Fig. 1

Amino acid alignment of Dictyostelium discoideum mitochondrial RPS4 (D.d-mt), Clostridium RPS4 (Clostridium), Reclinomonas RPS4 (Reclinomonas), Oryza mitochondrial RPS4 (Oryza) and Arabidopsis mitochondrial RPS4 (Arabidopsis). Identical amino acids are blocked in black and similar amino acids are indicated with gray shading. In D.d-mt, part 4-type and bipartite-type nuclear localization signals predicted by PSORTII are underlined. The nuclear localization signals are specifically present in D.d-mt; such signals have not been reported in mt-RPS4 protein of other organisms, yet.

Expression pattern of RPS4 protein during early development

Western blottings using the anti-RPS4 antibody absorbed beforehand by homogenates of vegetative growth phase cells that were supposed to be almost devoid of RPS4 protein gave a single band at the predicted position of 30 kDa RPS4, as shown in Fig. 2. As was expected, the RPS4 protein was scarcely noticed in vegetatively growing Ax-2 cells, and began to increase transiently in response to starvation, reaching the maximum level after 4–6 hr (Fig. 3). In rps4^AS cells expressing the antisense rps4 RNA, the expression level of RPS4 protein was considerably reduced as a whole (Fig. 3A). In contrast, rps4^OE cells overexpressing the rps4 mRNA in extra-mitochondrial space exhibited an augmented expression of RPS4 even during the vegetative growth phase (Fig. 3B). Like the rps4^AS cells, rps4^HRcells in which about a half of the mitochondrial copies of the rps4 gene were disrupted by homologous recombination showed a reduced level of RPS4 expression (Fig. 3)C>. The time course of RPS4 expression in Ax-2 cells was found to vary fairly from experiment to experiment, the expression peak being 6 hr, 4 hr, and 2 hr in Fig. 3A, B, and C, respectively. In this connection, it has been recently revealed that the expression of rps4 mRNA is moderately augmented depending on increased cell density during the vegetative growth phase: the rps4 mRNA is scarcely expressed at low cell densities less than 1×10⁶ cells/ml in growth medium, whereas the expression becomes detectable at 2–3×10⁶cells/ml and increases gradually coupled with increased cell densities in growth medium. This seems to indicate that the rps4 mRNA expression is under control of prestarvation factor(s) (PSFs) which accumulates as a function of cell density in growth medium, as the case for certain proteins that were previously believed to be induced by starvation. Thus Ax-2 cells acquire differentiation-competence during the vegetative growth phase in a PSF-dependent manner, and cells exposed to more PSF exhibit more rapid differentiation and morphogenesis when starved. Considering from these facts, it is most likely that the variation of RPS4-expression time course, as observed in Ax-2 cells, might be due to slight differences in cell density at the time-point of cell's starvation.

Fig. 2

Western blot analysis of the RPS4 protein in Ax-2 cells, using the absorbed anti-RPS4 antibody. Cells were harvested at the exponential growth phase (v), washed twice in BSS and shaken for the indicated times (hr) at 22°C. Western blottings were performed as described in Materials and Methods. It is clear that the antibody detects monospecifically the 30 kDa RPS4 protein, particularly in t_4-cells starved for 4 hr.

Fig. 3

Expression patterns of the RPS4 protein during the early development of Ax-2 cells and several transformed cells. Cells were harvested at the exponential growth phase (v), washed twice in BSS and shaken for the indicated times (hr) at 22°C, followed by western blot analysis using the anti-RPS4 antibody. The expression patterns in Ax-2 cells, (A) rps4^AS cells expressing the antisense rps4 RNA, (B) rps4^OEcells overexpressing the rps4 mRNA in the extra-mitochondrial cytoplasm and (C) rps4^HR cells in which the rps4 gene was disrupted by homologous recombination in about half mitochondrial genomes in the cell. The relative amount of RPS4 protein at t₄- and t₆-cells of Ax-2 varies from experiment to experiment.

Relation of the RPS4 protein to the car1 expression

Our previous findings suggested that in the regulatory cascade controlling early development, rps4 expression at the onset of differentiation might be located upstream of the expression of cAMP receptor 1 (car1), because car1 expression is markedly reduced in the rps4-inactivated cells like rps4^HR cells (Inazu et al., 1999). To investigate this further, the expression patterns of rps4 in car1-null cells (JB4 cells) and parental Ax-3 cells were compared by western blot analysis. The result has demonstrated that the RPS4 protein exists in the car1-null cells, though its amount seems to be slightly reduced as compared to that in Ax-3 cells, particularly at the early stage of starvation (data not shown). Therefore, car1 expression might have some effect on the synthesis of RPS4, but the cAMP receptor1 (CAR1) is not necessarily required for RPS4 formation.

Antisense rps4 RNA expressed in the extra-mitochondrial cytoplasm greatly impairs the progression of cell differentiation

When cells of the transformed rps^AS strain and its parent Ax-2 cells were separately starved and incubated in BSS under submerged conditions at 5×10⁵ cells/cm², most of rps^AS cells showed no sign of cell aggregation and remained as round-shaped single cells at 8 hr of incubation (Fig. 4A), while Ax-2 cells were elongated in shape, acquiring aggregation-competence, and some of them formed tiny aggregates (Fig. 4B). After a more prolonged time of incubation (12–16 hr), Ax-2 cells formed aggregation streams and then tight mounds (Fig. 4C, E). In contrast, some of rps^AS cells form aggregation streams, but many cells were still rounded in shape and remained as nonaggregated single cells (Fig. 4D, F).

As expected, starving rps^AS cells also exhibited marked delay of development on agar. At 10 hr of incubation, while most rps^AS cells showed no sign of cell aggregation, Ax-2 cells had already aggregated to form tight mounds. During further incubation, Ax-2 cells formed a tip on each aggregate, migrated as a slug, and eventually constructed a soro-carp after 24 hr of incubation. In contrast, a small population of rps^AS cells participated in sorocarp formation, but a considerable number of cells still remained as nonaggregated single cells.

Fig. 4

Development of rps4^AS cells (B, D, F) and parental Ax-2 cells (A, C, E) under submerged conditions. rps4^AS cells and parental 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 5×10⁵ cells/ml (1 ml of cell suspension/well). This was followed by incubation at 22°C. At 8 hr of incubation, (A) Ax-2 cells acquire aggregation-competence and form small cell clumps (arrows), while (B) rps4^AS cells show no sign of cell aggregation and remain as round-shaped single cells. At 12–16 hr, (C, E) Ax-2 cells form aggregation streams and tight aggregates, but (D, F) many of rps4^AS cells still remain as round-shaped single cells, though a small number of cells participate in formation of aggregation streams. Bar, 200 μm.

Since rps^AS cells grew normally by binary fission in growth medium, with almost the same doubling time as parental Ax-2 cells, it was concluded that the observed effect of rps4-inactivation by the antisense RNA was limited to the process of cell differentiation. Here it is of interest to note that the developmental phenotype of rps4^AS cells is quite similar to that of rps4^HR cells in which about a half of the mitochondrial rps4 genes have been disrupted by homologous recombination. During a prolonged time of axenic culture in growth medium, the phenotype of transforments such as rps4^HR cells has been shown to change moderately even in the presence of G418, eventually resulting in a return into the parental Ax-2-like phenotype. In the phenotypic revertants, we previously demonstrated that the dia3 RNA including the rps4 mRNA was recovered at almost the same level as that in Ax-2 cells (Inazu et al., 1999). In the rps4^AS cells, however, the developmental phenotype was retained stably during a prolonged time of successive axenic culture in growth medium.

When starved rps4^AS cells and Ax-2 cells (vitally stained with MitoTracker) were mixed in various number-ratios and incubated in BSS under submerged conditions at a density of 5×10⁵ cells/cm², no synergism was observed between the two. In a mixed culture of rps4^AS and Ax-2 (10:1), they completely sorted out after aggregation: a few number of small aggregates, consisting of Ax-2 cells were formed in a non-aggregated sheet composed of rps4^AS cells. The inability of Ax-2 cells to compensate for the development of rps4^AS cells indicates that the phenotype of rps4^AS cells is cell-autonomous.

Is RPS4 protein synthesized in the cytoplasm capable of moving actively to the nucleus?

Immunostaining of Ax-2 cells by the anti-PRS4 antibody has revealed that RPS4 is only slightly detected in granular structures (presumably mitochondria) of vegetatively growing cells (Fig. 5B). In cells (t₄-cells) starved for 4 hr, their cytoplasmic granules were strongly stained, as shown in Fig. 5D. The granules were confirmed to be mitochondria by double-stainings of the cells with DAPI or MitoTracker Orange. Here it is of interest to note that the extra-mitochondrial region of t₄-cells is only slightly stained, and that only a weak nuclear staining is sometimes recognized in some of t₄-cells. In this connection, the presence of a small amount of RPS4 in the cytosolic fraction of t₄-cells was detected by subcellular fractionation of cell homogenates and subsequent immuno-blottings using the anti-RPS4 antibody. Although these observations seemed to suggest that a trace of RPS4 protein might be released from mitochondria to the cytosol and then to the nucleus, this possibility remains to be tested using a more sensitive detection method, because the staining, if present in the cytosol and nucleus, is quite weak.

Fig. 5

Localization of RPS4 in Ax-2 cells just before starvation (B) and after 4 hr of starvation (D). Cells were harvested at the exponential growth phase, washed twice in BSS and fixed either immediately or after 4 hr of starvation at 22°C, followed by staining with the anti-RPS4 antibody or non-immune serum. At the vegetative growth phase (B), granular structures like mitochondria are weakly stained. In starving t_4-cells (D), the staining of mitochondria becomes highly marked. (E–G) Starving t₄-cells were double-stained with the anti-RPS4 antibody (E) and MitoTrtacker Orange (G), as described in Materials and Methods. Although both the stains are mostly colocalized, the extra-mitochondrial cytoplasm is very weekly stained only with the anti-RPS4 antibody (F, arrows). Bar; (A–D) 30 μm, (E–G) 15 μm.

When rps4^OE cells overexpressing the RPS4 protein were immunostained with the anti-RPS4 antibody and DAPI, they were strongly stained all over at the vegetative growth phase (Fig. 6A). In the t₄-cells, however, their nuclei in addition to the cytoplasm were strongly stained (Fig. 6D), thus at least indicating that the RPS4 protein synthesized in the cytoplasm may be preferentially transferred into the nucleus.

Fig. 6

Localization of RPS4 in rps4-overexpressing (rps4^OE cells) just before starvation (A) and after 4 hr of starvation (D). Cells were harvested at the exponential growth phase, washed twice in BSS and fixed either immediately or after 4 hr of starvation at 22°C, followed by double-staining with the anti-RPS4 antibody and DAPI. At the vegetative growth phase (A), the cells were strongly stained all over (A). In the t_4-cells, however, it is clear that their nuclei are strongly stained (D, arrows). Bar; (A–C) 20 μm, (D-F) 30 μm.