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First published online 28 February 2006
doi: 10.1242/jcs.02819

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Functional genomics in Dictyostelium: MidA, a new conserved protein, is required for mitochondrial function and development

Instituto de Investigaciones Biomédicas Alberto Sols. C.S.I.C./U.A.M., Calle Arturo Duperier 4, 28029 Madrid, Spain

View larger version (72K): [in a new window]   Fig. 1. MidA disruption and expression. (A) The sequence of Dictyostelium MidA protein was aligned with the human protein PRO1853 (GenBank accession number: NP_653337) and the most similar prokaryote protein from the alpha-proteobacteria Magnetospirillum magnetotacticum (GenBank accession number: ZP_00207869) using the ClustalW program. Black background corresponds to identical residues in three species, dark gray to identical residues in two species and light gray to similar residues. (B) The coding region of midA gene is depicted as two open boxes. The line between the open boxes represents an intron. KO1 and KO2 are two mutant strains generated by disruption of the midA coding region by homologous recombination at the indicated locations. Horizontal arrows show the oligonucleotides (1-4) used for checking the disruption of midA genomic locus in these strains. (C) Disruption of the midA gene was assessed by PCR. DNA from wild-type, KO1 (right panel) and KO2 (left panel) strains were subjected to PCR using the indicated oligonucleotides. A pair of oligonucleotides from an unrelated locus was used as internal control of the PCR reaction. The expected bands were absent in the KO strains due to the insertion of the BS-cassette. The upper bands in the KO samples (labeled as BS) corresponded to the inserted cassette. (D) RNA isolated at different times of development was hybridized with a radioactive probe derived from the coding sequence of the midA gene (upper panel). Lower panel shows the absence of midA mRNA in RNA samples from the KO strains isolated from vegetative stage.

View larger version (41K): [in a new window]   Fig. 2. Phenotype of midA- at the vegetative stage. (A) Appropriate dilutions of wild-type and mutant Dictyostelium cells were plated in association with Klebsiella aerogenes in SM plates to determine differences in the size of the clearing zone. The plates were incubated at 22°C for the indicated times. Bar, 1 cm. (B) Growth rate in axenic medium, cell diameter and the amount of protein per cell were compared between the wild type and midA- cells. Significance of differences is indicated by the P value shown inside the panels. (C) Proportion of cells (%) whose diameter is in the within the indicated sizes (µm). Most of the mutant cells had a diameter of 8-10 µm, whereas the majority of wild-type cells had a diameter of 10-14 µm. At least 200 cells were analyzed for each strain. (D) Axenically growing cells were exposed for 30 minutes to fluorescent beads (phagocytosis) or soluble fluorescent-dextran (macropinocytosis). The fluorimetry values were expressed as arbitrary units. The mean of three independent experiments is shown and the significance of differences indicated by the P value. (E) Representative flow-cytometry assay to determine the amount of fluorescent beads taken up by wild-type and midA- cells. Both the total number of labeled cells and the proportion of cells with multiple beads were reduced in the mutant. For comparison, the vertical line is given as a reference.

View larger version (107K): [in a new window]   Fig. 3. Phenotype of midA- during development. (A) Wild-type and mutant cells were deposited in nitrocellulose filter for development under overhead light at 22°C. The underlying cellulose pad was soaked with water and representative photographs are shown at the indicated times. Under these conditions (without buffer) the mutant cells were unable to form fruiting bodies. Bars, 0.5 mm. (B) Cells were deposited on filters as described before, but in this case the underlying pad was soaked with PDF buffer to induce direct culmination. Under these conditions midA- is able to culminate after a period of migration. Notice the presence of slime-sheath trails left behind at the base of the fruiting bodies. Bars, 0.5 mm. (C) Spores taken from 15-day-old fruiting bodies and photographed with a phase-contrast microscope. Mutant cells are rounder and less refringent than wild-type cells. Bar, 10 µm.

View larger version (62K): [in a new window]   Fig. 4. MidA is a mitochondrial protein. (A) Scheme of the construct that was used for the expression of MidA fused to GFP. Coding sequences are depicted as open boxes and the intron by a thin line. The expression of the fused genes was driven by the actin 15 promoter. (B) Colony size after transfection of the construct A15::midA-GFP into midA- cells. The indicated strains were mixed with Klebsiella aerogenes and plated on SM plates. Photographs were taken after 5 days at 22°C. Bar, 1 cm. (C) Cells expressing midA fused to GFP were incubated with the mitochondrial marker MitoTracker Red, fixed and observed in a confocal microscope. Series of 2-µm confocal images from the same cell show GPF location in green and the mitochondrial marker in red. Colocalization can be seen in the merged images. Bar, 10 µm.

View larger version (24K): [in a new window]   Fig. 5. Mitochondrial dysfunction in midA-/- cells. Mitochondria were stained with Mito Tracker Red (A) or Mito Tracker Green (B) and fluorimetric quantification of the respective fluorescence from similar numbers of wild type and mutant cells was carried out. (C) The amount of ATP was measured in a bioluminescence assay. (D) Cellular oxygen consumption from wild-type and midA- cells was measured in a Clark-type electrode. Significance of differences is indicated by the P value shown inside the panels; n.s., nonsignificant differences.

View larger version (41K): [in a new window]   Fig. 6. Changes in the accumulation of glycogen, glutamate and ammonia. (A) Natural-abundance proton-decoupled 13C-NMR spectra (125.13 MHz, 25°C, pH 7.2) of perchloric acid extracts from wild-type (WT) and mutant (KO) cells grown to late exponential phase. Resonances from carbons C1 (100.6 ppm) and C4 (77.8 ppm) of glycogen (Gly 1:4) and carbons C3 (27.7 ppm), C4 (34.2 ppm) and C5 (181.9 ppm) of glutamate (Glu) are shown in representative spectra of wild-type (left) and mutant cells (right). Arrows highlight the disappearance of glycogen and glutamate resonances. (B) Similar amount of wild-type and mutant cells were washed and deposited on nitrocellulose filters over cellulose pads. After 5 hours of starvation at 22°C, cells were taken off the filter for protein quantification, and the ammonia released to the underneath cellulose pad was determined by a colorimetric assay; ammonia concentration (mM) normalized to the number of cells (left panel) and total protein content (right panel). (C) Wild-type and mutant cells were deposited on filters for development with one or two PDF-soaked cellulose pads. Photographs were taken at the indicated times. When one cellulose pad was used under the filter, the wild-type cells culminated normally, whereas the mutant remained at the slug stage. This delay in culmination was rescued in midA- when two cellulose pads were used underneath the filter to dilute the secreted ammonia. Bar, 0.5 mm.