Creation of ρ0 cells by delivering Eco RI into mitochondria
As the first step to be tested, we needed to know if conditional targeting of restriction endonucleases such as Eco RI into Dictyostelium mitochondria can eliminate mtDNA completely to give mtDNA-null cells (ρ0-cells). In the Wt-mtDNA there are 5 Eco RI sites (Figure 1a). To deliver Eco RI into mitochondria, the Eco RI gene was fused with the presequence that encodes the signal peptide (MTS) of cytochrome c oxidase subunit IV (pCoxIV), and then the fusion gene was inserted into a Dictyostelium TRE-Pmin (Tet-mediated promoter)-plasmid (pCE38) shown in Additional data file 1b. Subsequently, Dictyostelium Tet-Off cells (MB35 cells) were transformed with the pMB38/pCoxIV-Eco RI (pCE38) vector, and the resulting double-transformants were clonally selected in the presence of 20 μg/ml of tetracycline (Tet), 10 μg/ml of blasticidin S and 50 μg/ml of G418. In a total of about 600 clones examined, the pCox-Eco RI expression after removal of tetracycline was found to vary widely, and most of clones exhibited only poor induction of pCox-Eco RI under the tetracycline-minus (-Tet) conditions (data not shown). The inefficient expression in the Tet-Off system is possibly due to unfit integration of the transactivator plasmid (pMB35) [5] and response plasmid (pCE38). A wide range of the expression levels was also reported in essentially the same Tet-Off system using the TRE-Pmin/luciferase [5]. Fortunately, we could obtain one blasticidin S-resistant clone (referred to as LpCEco cells) in which the mRNA of Eco RI was expressed at a high level within 24 hours after removal of Tet. We initially needed about 600 clones to generate a doubly-transformed cell line. Recently, however, 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, as noted in methods.
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
We have previously reported that ρΔ cells with a reduced amount (25%) of mtDNA, which were produced from parental Ax-2 cells by exposing them to ethidium bromide (EtBr), exhibit a series of developmental defects, such as delay of differentiation and abnormal cell patterning after starvation [8]. Therefore the developmental phenotypes of ρ0 cells (LpCEco cells grown for 48 hours under the -Tet condition) were examined and compared with those of parental MB35 cells and ρΔ cells that were prepared by a relatively short period (8 hours) of growth under the -Tet condition. With respect to the membrane potential of mitochondria, mitochondrial staining with MitoTracker Orange was found to be rather stronger in the ρΔ cells than in MB35 cells (Figure 2a), as observed in ρΔ cells produced by EtBr-exposure [8]. By contrast, the membrane potential-dependent staining with MitoTracker Orange was completely abolished in ρ0 cells (Figure 2a). Electronmicroscopic observations showed that mitochondria in ρΔ cells exert marked structural transformation, and that ρ0 cells contain quite abnormal mitochondria in which cristae are highly disorganized (data not shown). In this connection, progressive external opthalmoplegia and Kearns-Sayre syndrome, which are caused by deletion of mtDNA [9, 10], are known to exert marked morphological changes in the mitochondria of muscle tissues [11]. When the ρ0 cells and parental MB35 cells were starved and incubated on 1.5% non-nutrient agar, the former showed no sign of cell aggregation even after 60 hours of incubation at 22°C (Figure 2b). Therefore, it is most likely that at least 25% of the mtDNA might be required for maintenance of cell growth. The ρΔ cells also exhibit a series of fascinating behaviors after starvation: they show a marked delay of differentiation including cell aggregation and abnormal cell-type proportioning [8], thus suggesting strongly the importance of mtDNA in a variety of cellular functions. One conclusion to be drawn is that neither growth nor differentiation occurred in the ρ0 cells, presumably because of ATP depletion. Interestingly, the mitochondrial membrane potential does not necessarily decrease linearly coupled with reduced levels of mtDNA, and is abruptly eliminated below a certain level of mtDNA (Figure 2a). This phenomenon is called the threshold effect [12]. Taken together the data presented here strongly suggest that the threshold value is around 20–25% in Dictyosyelium mitochondria, and that the mtDNA level less than 20% may cause growth arrest and also a failure of cells to differentiate after starvation.
Strategy for creating rps4-null cells by a combination of homologous recombination and targeted Sfo I introduction
Impairment of mitochondrial function has been associated with a wide range of severe human disorders, which have been regrouped under the name of mitochondrial diseases [13]. Frequently, mtDNA with pathogenic mutations coexist with Wt-mtDNA (mtDNA heteroplasmy). MtDNA mutations manifest their phenotypes only when the proportion of mutated mtDNAs is high (threshold effect) [12]. To reduce this proportion, an effective therapy for these disorders has been tried by use of mitochondrially targeted restriction endonucleases to eliminate specifically mutated mtDNAs [6, 7, 14, 15]. The new method presented in this work was devised around the inverse concept. In Wt-mtDNA, there is a single Sfo I site in the upstream of rps4 gene. This raised the possibility that the conditioned expression of Sfo I in mitochondria of the rps4-heteroplasmic cells might allow us to create rps4-null cells, as schematically shown in Figure 3. To test this possibility, we first needed to prepare a transformant in which the fusion protein MTS-SfoI is expressed in response to removal of tetracycline from the PS medium, as in the case of LpCEco cells described in the preceding section. For this purpose, MB35 cells were transformed with the pMB38/pCoxIV-Sfo I (pCS38) vector (Additional data file 1c), and the resulting double-transformants were clonally selected in the presence of 20 μg/ml of tetracycline (Tet), 10 μg/ml of blasticidin S and 50 μg/ml of G418. In a total of about 800 clones examined, 24 likely clones were initially obtained as ones that expressed the MTS-Sfo I after removal of Tet from growth medium, but most of them were found to exhibit a poor response to Tet. Fortunately, however, we could gain one nice transformant (referred to as LpCSfo cells) that stably expressed the MTS-Sfo I coupled with removal of Tet. The method adopted here is based on introduction of Sfo I into mitochondria and its exact expression under the -Tet condition. Therefore, the use of MB35 cells in which the Tet-Off system is strictly regulated is quite critical for efficient selection of transformants such as LpCSfo cells.
Heteroplasmic LpCSfoHR cells, in which both of Wt-mtDNA and Mut-mtDNA lacking the Sfo I site and a 5'-half of rps4 coding region are intermingled, were prepared by means of homologous recombination using the vector construct as described in Additional data file 3. When LpCSfoHR cells (+Tet) grown with Tet and parental MB35 cells were starved, the large polycistronic dia3 transcripts (about 6.5 kb) including the rps4 or its deleted gene were transiently expressed after 2 hours of starvation (Figure 4b). In LpCSfoHR (-Tet) cells grown for 48 hours in the absence of Tet, however, the dia3 transcript containing the intact rps4 gene was never expressed after starvation (Figure 4b), though the smaller dia3 transcript with the 5'-deleted rps4 gene was expressed 2 hours after starvation (Figure 4c). It is quite difficult to detect the size-difference between the two-types of dia3 transcripts, because the deleted region of rps4 gene is not so large compared to the whole dia3 gene cluster. As shown in Figure 4d, a 5 kb fragment that should be obtained by digestion of the mutant dia3 gene cluster with Nde I and Sfo I was scarcely observed in LpCSfoHR (+Tet) cells grown for 2 weeks in the presence of Tet. This seemed to indicate that a majority of Mut-mtDNA was lost during a prolonged time of growth under the Tet-plus condition. This is consistent with our previous findings [2] that the heteroplasmic state is not stable, and therefore that Wt-mtDNA becomes dominant gradually during culture, possibly because of preferential duplication of Wt-mtDNA compared to Mut-mtDNA. Importantly, however, a small proportion of Mut-mtDNA was found to be enough to create rps4-null cells containing only the Mut-mtDNA, because LpCSfoHR (+Tet) cells were converted to LpCSfoHR (-Tet) cells within 48 hours of growth under the Tet-minus condition (Figure 4d). When MB35, LpCSfo and LpCSfoHR cells grown for 48 hours under the -Tet conditions were stained with DAPI, LpCSfo cells were confirmed to be completely devoid of mitochondrial staining, though dot-like stains of nuclei were retained well (Figure 4e). By contrast, since MB35 cells and LpCSfoHR (-Tet) cells have Wt-mtDNA and Mut-mtDNA respectively, their mitochondria were stained well with DAPI (Figure 4e). In LpCSG cells produced by fusing the hsEGFP sequence with MTS-Sfo I, the fluorescence was specifically observed in mitochondria. Unexpectedly, however, mitochondrial DAPI-staining was still retained in the LpCSG cells (Figure 4e). This seemed to indicate that the enzyme activity of the relatively small Sfo I molecule might be reduced or abolished when fused with hsEGFP, probably because of a steric hindrance. The lack of of wild-type rps4 gene in LpCSfoHR cells incubated for 48 hrs under the -Tet condition was confirmed by PCR analysis (Figure 4f). Again, transformed cells with Mut-mtDNA in which only the rps4 gene had been mutated were able to grow even in the presence of introduced Sfo I. Conversely, wild-type mtDNA (WT-mtDNA) was selectively digested by Sfo I, and the ratio of Mut-mtDNA/WT-mtDNA in the heteroplasmic cells was gradually increased. Thus, cells with little or no Mut-mtDNA became a ρ0-state and eventually died because of ATP depletion.
Developmental phenotypes of rps4-null cells
LpCSfo cells stopped growing in PS medium within 48 hours after their transfer to Tet-minus medium, because they were converted to a ρ0 state. However, LpCSfoHR (-Tet) cells (rps4-null cells) were able to grow normally with almost the same doubling-time as parental MB35 cells, indicating that the intact rps4 gene is not required for growth (data not shown). When MB35 cells and LpCSfoHR cells (+Tet) cells were starved and incubated on 1.5% non-nutrient agar at 22°C, they formed aggregation streams after 6 hours and mounds after 12 hours (Figure 5b). This was followed by formation of migrating slugs after 16 hours of incubation. By contrast, starving rps4-null cells exhibited a great delay of differentiation: no sign of cell aggregation was noticed even after 16 hours of incubation (Figure 5b), and aggregation-streams were formed only after 60 hours of incubation (data not shown).
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.
