Establishment of a new method for precisely determining the functions of individual mitochondrial genes, using Dictyostelium cells
© Chida et al; licensee BioMed Central Ltd. 2008
Received: 09 November 2007
Accepted: 21 March 2008
Published: 21 March 2008
Disruption of mitochondrial genes may become a powerful tool for elucidating precisely the functions of individual mitochondrial genes. However, it is generally difficult to manipulate genetically mitochondrial genes, because 1) a mitochondrion is surrounded by inner and outer membranes, and 2) there are a large number of mtDNA copies in a single cell. This is the reason why we tried to establish a novel method for disrupting a certain mitochondrial gene (rps4), using Dictyostelium cells.
Here, we have developed a new method for specifically disrupting a mitochondrial gene (rps4 ; ribosomal protein subunit S4), by a combination of homologous recombination and delivery of an appropriate restriction endonuclease (Sfo I) into mitochondria. First, mitochondrially targeted Sfo I whose expression is under control of the tetracycline (Tet)-regulated gene expression system was introduced into cells heteroplasmic with respect to the rps4 gene. Then, the heteroplasmic cells were produced by homologous recombination by use of the construct in which the unique Sfo I site and the 5'-half of the rps4 coding region were deleted not to be digested by Sfo I, and therefore their mitochondria have both the wild-type mtDNA and the mutant mtDNA with the disrupted rps4 gene. In response to removal of Tet from growth medium, Sfo I was selectively delivered into mitochondria and digested only the wild-type mtDNA but not the mutated rps4. Thus one can gain rps4-null cells with only the mutated mtDNA, under the Tet-minus condition.
The mitochondrial gene-disruption method presented here must be widely useful for precisely determining the functions of individual mitochondrial genes. This is the first report to demonstrate complete and specific mitochondrial gene disruption.
Mitochondrial DNA (mtDNA) is a maternally inherited circular genome present in hundreds to thousands of copies in most cells. A single mitochondrion contains hundreds of proteins that together are responsible for the various biological functions of the organelle. Most of these proteins are encoded in the nucleus, synthesized in the cytosol, and then directed to the mitochondria by specific targeting sequences (mitochondria targeting sequence; MTS) to be inserted in the mitochondrion by a complex import machinery largely conserved across species. Mitochondrial functions are generally presented as the central pathway for energy metabolism including ATP synthesis through respiration, but several other aspects of mitochondrial function have been noted, such as key roles in apoptosis, free radical production, thermogenesis and calcium signaling. In addition, increasing evidence indicates that mitochondria possess novel and critical functions as the regulatory machinery of the growth/differentiation transition (GDT), cell movement, cell-type choice, and pattern formation, as exemplified well in Dictyostelium, a wonderful model organism in the field of cell and developmental biology . For example, the expression of the mitochondrial ribosomal protein (rps4) that is encoded by mtDNA is required for differentiation from the GDT point in the cell cycle .
The mtDNA of Dictyostelium discoideum is a 55.6-kb circular double-stranded DNA molecule which encodes 2 rRNAs, 10 subunits of the NADH dehydrogenase complex (NAD1, 2, 3, 4, 4L, 5, 6, 7, 9, and 11), apocytochorome b (cyt b), 3 subunits of cytochrome c oxidase (Cox 1, 2, and 3), 15 ribosomal proteins, and 5 other open reading frames (ORFs), excluding intronic ORFs (essential peptides of enzymes for oxidative phosphorylation, 3 rRNAs and 18 tRNAs) [3, 4]. The mitochondrial gene cluster (dia3) including rps4 is specifically expressed in response to starvation around the GDT point and plays a critical role in the initiation of differentiation in D. discoideum Ax-2 cells. The rps4 gene is present as a single copy in mtDNA, but the copy number is multiple because numerous mitochondria are contained in a cell. In spite of this situation, we attempted homologous recombination to determine the function of rps4, inactivating the subpopulation of the rps4 gene . The homologous recombination was beautifully accomplished with high efficiency and specificity, and resulting G418-resistant transformants exhibited mtDNA heteroplasmy with both the wild-type rps4 and its inactivated gene. The partial inactivation of the rps4 gene greatly impaired differentiation, including cell aggregation. Provided that it is possible to completely inactivate rps4 expression, one can expect that the rps4-null cells would never differentiate from the GDT point in response to starvation. This is the principal reason why we tried to establish an efficient system for specifically disrupting a mitochondrial gene such as rps4 in the present work.
In the present work, we first aimed to preparing transformants in which a certain portion of the rps4 gene was disrupted by homologous recombination. For this purpose, a single Sfo I site located in the upstream region of rps4 and a 5' half of the rps4-coding region were deleted by Bam HI and Sma I digestion so that the deleted rps4 (mutant rps4; Mut-mtDNA) gene is no longer able to be digested by Sfo I. The vector constructs for homologous recombination were introduced into a transformant in which MTS-Sfo I is expressed under control of the tetracycline (Tet)-regulated gene expression system to produce heteroplasmic cells with respect to the rps4 gene. After removal of Tet from the growth medium, MTS-Sfo I was expressed and imported into the mitochondria where it digested the Sfo I site of wild-type mtDNA (Wt-mtDNA) to eliminate Wt-mtDNA. Since the Mut-mtDNA was not digested by Sfo I, the ratio of Mut-mtDNA/Wt-mtDNA increased strikingly during the growth of the heteroplasmic cells under the Tet-minus condition. This resulted in the creation of Wt-mtDNA-null cells, that is rps4-null cells. The method presented here gives us a powerful tool for targeted mitochondrial-gene disruption and therefore for elucidation of the precise function of each mitochondrial gene.
Results and Discussion
Creation of ρ0 cells by delivering Eco RI into mitochondria
Dose-response analysis of MTS-Eco RI mRNA expression levels was done by use of LpCEco cells and parental MB35 cells, both of which were exposed to various Tet-concentrations ranging from 0 μg/ml to 30 μg/ml during 24 hours of incubation in growth (PS) medium. As shown in Figure 1b, the MTS-Eco RI was expressed only in LpCEco cells incubated under the -Tet condition. Figure 1c shows a time course of the MTS-Eco RI mRNA expression in LpCEco cells and parental MB35 cells after complete removal of Tet from PS-medium: the mRNA began to be expressed 12 hours after removal of Tet in LpCEco cells and reached maximal levels by 24 hours of incubation. Incidentally, LpCEco cells incubated under the -Tet condition completely lost the ability to grow in PS medium within 48 hours of incubation.
To know if mtDNA in LpCEco cells is actually eliminated 48 hours after removal of Tet, Southern blot analysis was carried out using the nuclear DNA-specific probe (dd-trap1) or mtDNA-specific probe (rps4). From densitometric measurements of the autoradiograms obtained, the mtDNA in LpCEco cells was found to be almost completely eliminated, but no difference in the amount of the nuclear DNA was detected between LpCEco cells and parental MB35 cells (Figure 1d). This indicates that LpCEco cells are efficiently converted to the ρ0-state within 48 hours after removal of Tet. Similar observations have been reported in human 293T and cybrid NARP cells [6, 7], indicating that restriction-enzyme mediated destruction of mtDNA is an all-or-none process.
When parental MB35 cells were stained with DAPI, their nuclei and mitochondria were stained as relatively large dots and small granules, irrespective of the presence or absence of Tet (Figure 1e). Essentially the same staining pattern was attained in LpCEco cells (referred to as LpCEco (+Tet) cells) grown for 48 hours in the presence of Tet (+Tet). In LpCEco cells (referred to as LpCEco (-Tet) cells) grown for 48 hours under the -Tet condition, however, DAPI-staining of mitochondria was never observed, though the staining of nuclei was retained well (Figure 1e). This was also the case in LpCEG and LpCGE cells that express fusion proteins consisting of MTS-Eco RI and hsEGFP (Additional data file 2). When LpCEG and LpCGE cells were examined under fluorescence microscopy, the fluorescence of hsEGFP was confirmed to locate in the mitochondria of some cells grown without Tet (Figure 1e). Therefore, both of Eco RI-hsEGFP and hsEGFP-Eco RI fusion proteins were concluded to be properly targeted to mitochondria in a Tet-regulated manner. During incubation of LpCEG and LpCGE cells under the -Tet condition, there were transiently observed a wide range of variation in intramitocondrial hsEGFP expression, and more or half of cells in the field of Figure 1e seemed to have little or no fuorecsence. Thus, although there were a significant number of cells in which the expression of hsEGFP is low, there are five Eco RI sites in mtDNA, and also Eco RI has very high activity to cleave mtDNA. The cleaved mtDNA has no duplication ability and is eventually digested in mitochondria, thus resulting in generation of ρ0-state cells. We detected no signs of apoptosis, such as the formation of DNA ladders (Figure 1d) or the appearance of nuclear fragmentation and condensation (Figure 1e), in the mtDNA-null cells (ρ0 cells). These results indicate that the restriction enzyme imported into mitochondria is tightly retained within the mitochondrial matrix compartment without attacking the nuclear genome.
Depletion of mtDNA induces mitochondrial dysfunction
Strategy for creating rps4-null cells by a combination of homologous recombination and targeted Sfo I introduction
Developmental phenotypes of rps4-null cells
When the membrane potential of mitochondria was monitored by staining of cells with MitoTracker Orange, the staining of mitochondria was almost completely abolished in LpCSfo cells grown without Tet, as the case of LpCEco (-Tet) cells (Figure 5a). Here it is of importance to note that mitochondria of LpCSfoHR (-Tet) cells (rps4-null cells) were stained well with the dye (Figure 5a), suggesting that the expression of rps4 is not necessarily required for the occurrence of mitochondrial membrane potential.
To our knowledge, this is the first report to demonstrate complete and specific disruption of a certain mitochondrial gene by a combination of homologous recombination and introduction of mitochondrially targeted endonuclease. In the present study, we constructed vectors containing the sequence for Eco RI or Sfo I downstream of and fused in frame to the mitochondrial targeting sequence (MTS) under control of the Tet-response element and introduced them into Dictyostelium cells. This system permits the reversible expression of individual mitochondrial genes in a Tet-regulated manner, to create ρ0 cells or rps4 null cells. The usefulness of mitochondrially targeted restriction endonucleases in modulating mtDNA is limited by the presence of an appropriate restriction site. However, this approach may modulate mtDNA heteroplasmy in the context of multiple cleavage sites if expression of the restriction endonuclease could be tightly regulated. In conclusion, we believe that the new method presented here can provide a powerful tool for precisely determining the functions of individual mitiochondrial genes as well as for genetic therapy of mitochondrial diseases.
Culture conditions and transformation of cells
Vegetative cells of Dictyostelium discoideum Ax-2 (clone 8A) were axenically grown in growth (PS)-medium, as previously described . The PCR product containing both the mitochondrial targeting sequence (MTS) and endonuclease gene (Sfo I or Eco RI) was inserted into pMB38 bearing the blasticidin S resistant cassete, which is the response plasmid of tetracycline-regulated gene expression system (Tet-Off system) for Dictyostelium cells  (Additional data file 1b, 1c, Additional data file 3 and Additional data file 4). The transactivator plasmid of Tet-Off system (MB35) was initially introduced into Ax-2 cells, and the transformed cells (MB35 cells) were selected and cloned with 30 μg/ml G418. The pCE38 or pCS38 vector construct, both of which have TRE- Pmin (tetracycline response element), MTS, endonuclease (Eco RI or Sfo I) and balasticidin S cassette, was introduced into MB35 cells to obtain LpCEco cells or LpCSfo cells. The transformed cells thus obtained were maintained in PS medium containing 20 μg/ml of tetracycline (Tet), 10 μg/ml of blasticidin S and 50 μg/ml of G418. Recently, we have succeeded in obtaining the doubly-transformed cells (LpCEco cells or LpCSfo cells) much more efficiently by the use of MB35 cells that are strictly regulated by the Tet-Off system. That is, the transformed cells were cloned in 96-well tissue culture plates (Greiner bio-one, Inc., Maybachstr., Frickenhausen, Germany) and incubated in the presence of tetracyclin (+Tet) or in the absence of tetracyclin (-Tet). Subsequently, 20–30 clones that had been able to grow only under the +Tet-condition were isolated, followed by Northern blottings to know if they are LpCEco cells or LpCSfo cells that express the restriction enzyme (Eco RI or Sfo I).
Isolation of genomic DNAs and Southern blot analysis
Genomic DNAs were extracted according to the method described previously . Genomic Southern hybridization was performed using the Southern method with a slight modification.
Isolation of total RNAs and Northern hybridization
Total RNAs were prepared from vegetatively growing cells or cells starved for various periods in 1.5% (wt/vol) non-nutrient agar, using TRIzol Reagent (Life Technologies, Inc., Gaithersburg, MD, USA) as described by the manufacturer. Northern hybridization was performed as described previously .
Staining of cells with a mitochondrion-selective dye, MitoTracker Orange
The vegetatively growing cells were incubated in PS medium containing 0.5 μM MitoTracker Orange CMTMRos (Molecular Probes, Eugene, OR, USA) for 30 minutes at room temperature. After staining with the dye, stained cells were washed twice with BSS. One drop of the cell suspension was put on a cleaned coverslip and cells were fixed with 3.7% (vol/vol) formaldehyde in 20 mM Na/K-phosphate buffer (pH 7.2) for 20 minutes. Subsequently, the cells were washed with PBS (10 mM Na/K-phosphate buffer (pH 7.0), 0.9% (wt/vol) NaCl), as previously described . The stained cells were observed under a fluorescence microscope.
List of abbreviations
mitochondrial targeting signal
differentiation-associated gene 3
ribosomal protein subunit S4 gene
mitochondrial large ribosomal RNA
tumor necrosis factor receptor-associated protein
neuronal muscle weakness, ataxia, and retinitis pigmentosa
We thank P. R. Fisher for his critical reading of the manuscript and insightful comments. We are also grateful to Dr. Donald G. Comb for kindly providing p19SfoIRM vector constructs. This work was supported by Grant-in-Aid (No. 16370030 and 16657020) from JSPS. This work was also funded by the Mitsubishi Foundation.
- Maeda Y: Regulation of growth and differentiation in Dictyostelium. Int Rev Cytol. 2005, 244: 287-332. 10.1016/S0074-7696(05)44007-3.View ArticlePubMedGoogle Scholar
- Inazu Y, Chae SC, Maeda Y: Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase-shift of Dictyostelium cells from growth to differentiation. Dev Genet. 1999, 25: 339-352. 10.1002/(SICI)1520-6408(1999)25:4<339::AID-DVG8>3.0.CO;2-3.View ArticlePubMedGoogle Scholar
- Gray MW, Lang BF, Cedergren R, Golding GB, Lemieux C, Sankoff D, Turmel M, Brossard N, Delage E, Littlejohn TG, Plante I, Rioux P, Saint-Louis D, Zhu Y, Burger G: Genome structure and gene content in protest mitochondrial DNAs. Nucleic Acid Res. 1998, 26: 865-878. 10.1093/nar/26.4.865.PubMed CentralView ArticlePubMedGoogle Scholar
- Ogawa S, Yoshino R, Angata K, Iwamoto M, Pi M, Kuroe K, Matsuo K, Morio T, Urushihara H, Yanagisawa K, Tanaka Y: The mitochondrial DNA of Dictyostelium discoideum: complete sequence, gene content, and genome organization. Mol Gen Genet. 2000, 263: 514-519.PubMedGoogle Scholar
- Blaauw M, Linskens MH, van Haastert PJ: Efficient control of gene expression by a tetracycline-dependent transactivator in single Dictyostelium discoideum cells. Gene. 2000, 252: 71-82. 10.1016/S0378-1119(00)00227-4.View ArticlePubMedGoogle Scholar
- Tanaka M, Borgeld HJ, Zhang J, Muramatsu S, Gono JS, Yoneda M, Maruyama W, Naoi M, Ibi T, Sahashi K: Gene therapy for mitochondrial disease by delivering restriction endonuclease Sma I into mitochondria. J Biomed Sci. 2002, 9: 534-541.PubMedGoogle Scholar
- D'Souza GG, Weissig V: Approaches to mitochondrial gene therapy. Curr Gene Ther. 2004, 4: 169-180.View ArticleGoogle Scholar
- Chida J, Yamaguchi H, Amagai A, Maeda Y: The necessity of mitochondrial genome DNA for normal development of Dictyostelium cells. J Cell Sci. 2004, 117: 3141-3152. 10.1242/jcs.01140.View ArticlePubMedGoogle Scholar
- Moraes CT, Dimauro S, Zeviani M, Lombes A, Shanske S, Miranda AF, Nakase H, Bonilla E, Werneck LC, Serividel S, Nonaka I, Koga Y, Spiro AJ, Bromwell KW, Schmidt B, Schotland DL, Zupanc M, DeVivo DC, Scon EA, Rowland LP: Mitochondrial DNA deletions in progressive external ophthalmoplegia and Kearns-Sayre syndrome. N Engl J Med. 1989, 320: 1293-1299.View ArticlePubMedGoogle Scholar
- Saiwaki T, Shiga K, Fukuyama R, Tsutsumi Y, Fushiki S: A unique junctional palimdromic sequence in mitochondrial DNA from a patient with progressive external ophthalmoplegia. Mol Pathol. 2000, 53: 333-335. 10.1136/mp.53.6.333.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwartzkopff B, Frenzel H, Breithardt G, Deckert M, Losse B, Toyoka KV, Borggrefe M, Hort W: Ultrastructural findings in endomyocardial biopsy of patients with Kearns-Sayre syndrome. J Am Coll Cardiol. 1988, 12: 1522-1528.View ArticlePubMedGoogle Scholar
- Rossignol R, Faustin B, Rocher C, Malgat M, Mazat JP, Letellier T: Mitochondrial threshold effects. Biochem J. 2003, 340: 751-762. 10.1042/BJ20021594.View ArticleGoogle Scholar
- Fischel-Ghodsian N: Mitochondrial genetics and hearing loss: the missing link between genotype and phenotype. Proc Soc Exp Biol Med. 1998, 218: 1-6.View ArticlePubMedGoogle Scholar
- Srivastava S, Moraes CT: Manipulating mitochondrial DNA heteroplasmy by a mitochondrially targeted restriction endonuclease. Hum Mol. 2001, 10: 3093-3099. 10.1093/hmg/10.26.3093.View ArticleGoogle Scholar
- Bayona-Bafaluy MP, Blits B, Battersby BJ, Shoubridge EA, Moraes CT: Rapid directional shift of mitochondrial DNA heteroplasmy in animal tissues by a mitochondrially targeted restriction endonuclease. Proc Natl Acad Sci USA. 2005, 102: 14392-14397. 10.1073/pnas.0502896102.PubMed CentralView ArticlePubMedGoogle Scholar
- Nellen W, Datta S, Reymond C, Sivertsen A, Mann S, Crowley T, Firtel RA: Molecular biology in Dictyostelium: tools and applications. Methods in Cell Biology. Edited by: Spudich JA. 1989, New York: Academic Press, 28: 67-100.Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.