From the Institute of Biological Sciences and
§ Center for Tsukuba Advanced Research Alliance, University
of Tsukuba, Ibaraki 305-8572, Japan
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ABSTRACT |
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Two cell lines were used for determination of
whether interaction occurred between different types of
respiration-deficient mitochondria. One was a respiration-deficient
In yeast and plant cells, the occurrence of mitochondrial
interaction has been suggested by the presence of recombinant
mitochondrial DNA (mtDNA)1
molecules from two parental cells (1, 2). In mammalian species,
however, it has been shown that the mtDNA population is very
homoplasmic in a given individual (3, 4) probably because of inhibition
of the coexistence of mtDNAs from both parents by their strictly
maternal inheritance (5, 6). For example, sperm-derived mtDNA is
selectively and completely eliminated from fertilized mouse eggs before
the late pronucleus stage, suggesting that the mtDNA population of all
individuals is derived exclusively from the eggs (5, 6). Therefore, it
is difficult to prove occurrence of mitochondrial interaction by the
presence of recombinant mtDNA molecules in mammalian species
because of the difficulty in identification of recombinant molecules
between mtDNAs that have very similar sequences, even if mtDNA
recombination occurs.
However, when cell fusion techniques are applied to mammalian somatic
cells, mtDNAs with significant sequence divergences derived from
different individuals of the same species or even from those of
different species can be coexisted within the same somatic cells, which
allow us to examine the occurrence of mtDNA recombination and
mitochondrial interaction. Using somatic cell fusion techniques, the
occurrence of interaction of the mammalian mitochondrial genetic system
was proved in heteroplasmic cells by translational complementation of
mitochondrial rRNA between mitochondria with chloramphenicol-sensitive
and -resistant mtDNAs (7, 8), and by translational complementation or
competition of mitochondrial tRNAs between mitochondria with wild-type
and pathogenic deletion mutant mtDNAs (8, 9), although extensive recombination was not observed in heteroplasmic cells with rat mtDNAs
or rat and mouse mtDNAs (10). Subsequently, we found rapid penetration
of HeLa mtDNA and/or its products into mitochondria of mtDNA-less
( A recent contradictory report proposed that there is no mitochondrial
interaction between distinct organelles derived from different cells,
because the coexistence of mitochondria containing different pathogenic
mutant mtDNAs derived from different patients in single cells did not
restore reduced mitochondrial respiratory function (12). On the other
hand, we found convincing evidence for the presence of interaction
between mitochondria originating from different cell lines (8). We
constructed cybrids by introducing chloramphenicol-resistant HeLa mitochondria into cells with
respiration-deficient In this study, we examined this possibility by isolating cells with
syn Cells and Cell Culture--
Respiration-deficient
Intercellular Transfer of mtDNA--
Intercellular transfer of
mtDNA was carried out as described previously (8) by fusion of
enucleated H5 cells with CM114a cells in the presence of polyethylene
glycol 1500 (Boehringer Mannheim).
Southern Blot and Polymerase Chain Reaction Analyses of
mtDNA--
For identification and determination of the content of
Analysis of Mitochondrial Translation Activity and Cytochrome c
Oxidase Activity--
Analysis of mitochondrial translation products
labeled with [35S]methionine was carried out as described
previously (8). Cytochrome c oxidase activity was measured
as the rate of cyanide-sensitive oxidation of reduced cytochrome
c as described before (15).
We examined mitochondrial interaction by isolation of cybrids
using the nutritional requirements of two different types of respiration-deficient cell lines as parental cells. One was
cell line having mutant mitochondrial DNA
(mtDNA) with a 5,196-base pair deletion including five tRNA genes
(tRNAGly, Arg, Ser(AGY), Leu(CUN), His),
mtDNA5196, causing Kearns-Sayre syndrome. The
other was a respiration-deficient syn
cell
line having mutant mtDNA with an A to G substitution at 4,269 in
the tRNAIle gene, mtDNA4269, causing fatal
cardiomyopathy. The occurrence of mitochondrial interaction was
examined by determining whether cybrids constructed by fusion of
enucleated
cells with syn
cells became respiration competent by exchanging their tRNAs. No
cybrids were isolated in selection medium, where only
respiration-competent cells could survive, suggesting that no
interaction occurred, or that it occurred so slowly that sufficient
recovery of mitochondrial respiratory function was not attained by the
time of selection. The latter possibility was confirmed by the
observations that heteroplasmic cybrids with both mutant
mtDNA4269 and
mtDNA5196 isolated without selection
showed restored mitochondrial respiration activity. This demonstration
of transcomplementation between different respiration-deficient
mitochondria will help in understanding the relationship between
somatic mutant mtDNAs and the roles of such mutations in aging processes.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
0) HeLa cells in cybrids isolated by the fusion of
enucleated HeLa cells with
0 HeLa cells (11).
mitochondria containing
deletion mutant mtDNA (
mtDNA5196) with a 5,196-base pair
deletion including five tRNA genes and seven structural genes derived
from a patient with Kearns-Sayre syndrome (9) and found that fusion
genes newly formed around the deletion break point in
mtDNA5196 could be translated even in the presence of
chloramphenicol with the help of both chloramphenicol-resistant rRNA
and five kinds of tRNAs lacking in
mitochondria
transcribed from HeLa mtDNA. The apparent discrepancy on the
presence or absence of mitochondrial interaction might be explained by
supposing that there is no interaction when different types of
respiration-deficient mitochondria coexist in the same cells, probably
because of lack of sufficient energy supply from respiration-deficient
mitochondria required for mitochondrial fusion, subsequent mixing
of their contents, and peptide synthesis.
mitochondria, which are
respiration-deficient because of pathogenic mutant mtDNA with a point
mutation in the tRNAIle gene at 4,269 derived from a
patient with fatal cardiomyopathy (13, 14) and then creating a chance
for interaction between syn
mitochondria and
mitochondria. In this combination, because fusion
peptides were exclusively encoded by
mtDNA5196, whereas
ATP8, ATP6, COIII ND3, ND4L, ND4, and ND5 were exclusively encoded by
mtDNA4269, the interaction could be unambiguously proved by
identification of these peptides. The results suggested the occurrence
of mitochondrial interaction even between distinct respiration-deficient organelles with different types of pathogenic mutant mtDNAs derived from patients with different mitochondrial diseases.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H5 cells (8) contain only mutant
mtDNA5196 with a large-scale deletion from nucleotide
positions 8,563 to 13,758 including seven structural genes and five
tRNA genes, which was derived from a patient with Kearns-Sayre syndrome
(9). Respiration-deficient syn
CM114a and
normal CM1-9 cells were isolated by the fusion of
0 HeLa
cells with enucleated fibroblasts from a patient with fatal cardiomyopathy (13). Therefore, the nuclear genomes of CM114a and CM1-9
clones were from HeLa cells, and mtDNAs were from the patient. CM114a
cells contain only mutant mtDNA with an A to G substitution at 4,269 in
tRNAIle gene, mtDNA4269, whereas CM1-9 cells possessed
predominantly wild-type mtDNA from the patient. As these
respiration-deficient cell lines are auxotrophic for uridine and
pyruvate, cells were grown in medium (RPMI 1640 + pyruvate (0.1 mg/ml) + uridine (50 µg/ml) + 10% fetal bovine serum).
mtDNA5196, total DNA (2 µg) extracted from 2 × 105 cells was digested with XhoI, and the
fragments were separated by 1% agarose gel electrophoresis. After
blotting onto a NYTRAN membrane, the DNA fragments were hybridized with
[
-32P]dATP-labeled HeLa mtDNA. The radioactivities of
the fragments were measured with a BAS2000 instrument (Fuji Photo Film,
Tokyo). The mutant mtDNA4269 was identified by the mismatch polymerase chain reaction method as described previously (13). The products digested with SspI were separated by 4% agarose X (Wako,
Tokyo) gel electrophoresis in the presence of ethidium bromide.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
H5 cells (8), which are respiration-deficient because
of a large-scale deletion mutant
mtDNA5196 derived from
a patient with Kearns-Sayre syndrome (9). The other was
syn
CM114a cells, which are
respiration-deficient because of mutant mtDNA4269 (13) derived from a
patient with fatal cardiomyopathy. The CM114a cells without
mitochondrial translation activity (Fig. 1) were isolated by repeated recloning of
CM114 cells that showed slight translation activity (14). These two
lines are auxotrophic for pyruvate and uridine because of the complete
absence of oxidative phosphorylation activity in their mitochondria (8,
14).
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Fig. 1.
Analyses of mtDNA genotypes and phenotypic
expression of mitochondrial translation in cybrids. CM114a
contained only mtDNA4269, whereas CM1-9 contained predominantly
wild-type mtDNA. H5 contained only mtDNA5196. Clones 0A4
and 8B3 are cybrid clones. A, identification of the amount
of mtDNA4269 by SspI digestion of the polymerase chain
reaction products. Wild type mtDNA and
mtDNA5196 gave a
153-bp fragment, whereas mtDNA4269 gave a 184-bp fragment because of
the loss of an SspI site by an A to G substitution at 4,269 (13). B, identification of the amount of
mtDNA5196 by Southern blot analysis of
XhoI digests. Wild type mtDNA and mtDNA4269 gave a 16-kbp
fragment, whereas
mtDNA5196 gave an 11-kbp fragment (8).
C, analysis of mitochondrial translation products by
SDS-polyacrylamide gel electrophoresis. ND5, COI, ND4, Cytb, ND2, ND1,
COII, COIII, ATP6, ND6, ND3, ATP8, and ND4L are polypeptides assigned
to mtDNA. Genes encoding ATP8, ATP6, COIII, ND3, ND4L, ND4, and ND5 are
missing in
mtDNA5196, whereas fusion polypeptides (shown
by an arrowhead) were exclusively encoded by
mtDNA5196.
If mitochondrial interaction occurs between the respiration-deficient
and syn
mitochondria,
mitochondrial respiratory function should be restored by their
coexistence in a cell, because the missing five tRNAs in the
mitochondria and the missing wild-type
tRNAIle in the syn
mitochondria
should be supplemented by the syn
and
mitochondria, respectively. Because
syn
CM114a cells are resistant to
6-thioguanine, the coexistence of syn
CM114a
mitochondria and
H5 mitochondria could be attained by
the fusion of enucleated H5 cells with CM114a cells followed by
selection in medium with 6-thioguanine but without pyruvate and
uridine. In this nutrition-deficient selection medium, only cybrids,
i.e. CM114a cells that had acquired respiration competence
by introduction of exogenous
H5 mitochondria, could
survive. The results showed that no colonies grew in this selective
medium, suggesting that the cybrids were not respiration competent,
possibly because of the absence of mitochondrial interaction between
syn
and
mitochondria.
However, the failure to isolate cybrids by nutritional selection could
not necessarily be direct evidence for the absence of interaction. For
example, our results did not completely exclude the possibility that
transcomplementation between syn and
mitochondria occurred very slowly because of the
absence of an energy supply necessary for rapid interaction, and so
sufficient restoration of mitochondrial translation by
transcomplementation was not accomplished by the time of nutritional
selection that exclude respiration deficient cells. For examination of
this possibility, cybrids with both syn
and
mitochondria must be isolated to study whether
restoration of mitochondrial respiratory function could be attained in
the heteroplasmic cybrids during prolonged cultivation after fusion.
However, we could not use effective selection to remove host CM114a
cells from fusion mixtures of CM114a cells and enucleated H5 cells
(Table I). As selection with
6-thioguanine was incomplete and could exclude only parental H5 cells,
most of the colonies that grew in the selective medium would be either
the host CM114a cells without
mitochondria or
cybrids, i.e. CM114a cells with imported
mitochondria.
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Therefore, on day 14-20 after fusion, we picked up 98 growing colonies
randomly without using nutritional selection to exclude respiration-deficient cells and analyzed their mtDNA compositions to
find cybrid clones. Most of these colonies did not contain even a
slight amount of mtDNA5196, and both parental mtDNAs
were detectable only in 2 of 98 clones, suggesting that two
heteroplasmic clones, named 0A4 and 8B3, were cybrids but that the
others were parental CM114a cells. Cybrid clone 0A4 had 60%
mtDNA5196 imported from
H5 cells,
whereas cybrid clone 8B3 had 18%
mtDNA5196, the
remaining mtDNA being host mtDNA4269 (Fig. 1 and Table I). Thus, the
coexistence of respiration-deficient syn
and
mitochondria was attained in these cybrid clones.
Then, by [35S]methionine labeling we examined whether
mitochondrial translation activity was restored in these cybrid clones. Fig. 1 shows that mitochondrial translation activity was observed in
0A4 cybrids with 60% mtDNA5196, but not in 8B3 cybrids
with 18%
mtDNA5196. The apparent discrepancy on the
presence or absence of mitochondrial translation activity in cybrid
clones 0A4 and 8B3 could be explained by supposing that the 8B3 clone
was not a cybrid clone but was derived from parental CM114a cells
contaminated with H5 cells. Such contamination could occur, if we had
failed to isolate a single clone, and some H5 cells, which happen to
survive in the presence of 6-thioguanine during the selection period
(14-20 days), were included in the cell population of CM114a line,
although we confirmed before fusion that 6-thioguanine killed all
parental H5 cells during the 14-day cultivation period.
For examination of this possibility, five subclones were isolated from
the 8B3 clone by recloning, and their mtDNA compositions were studied.
As shown in Fig. 2, A and
B, all the subclones had both mutant mtDNA4269 and
mtDNA5196, suggesting that the 8B3 cells were not a
simple mixture of the parental CM114a and H5 cells, but were
heteroplasmic cybrids with both syn
and
mitochondria. However, during cultivation for about
30 passages for isolation and examination of subclones, the amounts of
mtDNA5196 in all the subclones progressively increased
to 40-55%. Considering that the amount of
mtDNA5196 in
the original 8B3 clone was 18% (Fig. 1A and Table I), its increase was not because of random segregation but to a propagational advantage of
mtDNA5196 of smaller size than mutant
mtDNA4269. These observations are consistent with our previous
observations that
mtDNA5196 was propagated
preferentially over wild-type mtDNA in following generations of cybrids
(9). Then, we examined the activities of mitochondrial translation and
respiratory enzymes in these subclones. All subclones showed
mitochondrial translation and cytochrome c oxidase
activities restored to 40-70% of those of cybrids repopulated with
wild-type mtDNA (Figs. 2 and 3),
suggesting the occurrence of interaction between
syn
and
mitochondria.
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Moreover, these cybrid clones synthesized fusion peptides exclusively
encoded by mtDNA5196 and seven peptides ATP8, ATP6,
COIII, ND3, ND4L, ND4, and ND5 missing in
mtDNA5196,
although their amounts seem to be slightly lower than those of the
other peptides (Figs. 1 and 2). As
mtDNA5196 had lost
seven structural genes but gained new fusion genes because of 5,196-bp
deletion with a break point between 8,563 and 13,758 (9), these results
suggest the recoveries of gene expressions of both
mtDNA5196 and mtDNA4269 by their coexistence in the
cybrids. Thus, these observations provided unambiguous evidence for the
presence of complementation between different types of
respiration-deficient mitochondria.
There seem to be two possible explanations for the apparent discrepancy
between the findings that all subclones showed restored mitochondrial
translation activity, whereas the original cybrid clone 8B3 did not
(Figs. 1 and 2). One is that interaction did occur in clone 8B3, but
that the amount of mtDNA5196 (18%) was not enough to
provide sufficient normal tRNAIle for restoring
mitochondrial translation activity in syn
mitochondria. The other is that interaction occurred, but that a long
expression time was required to restore mitochondrial translation activity particularly between respiration-deficient mitochondria. Because it seems impossible to keep the amount of
mtDNA5196 at lower levels because of its propagational
advantage over mtDNA of normal size, other mtDNA with a pathogenic
point mutation, that is expected to be neutral with respect to
propagational advantage, must be used to examine the above possibility.
The discrepancy with respect to the presence (this study) and absence
(12) of interaction between respiration-deficient mitochondria and our
inconsistent findings that cybrids were not isolated in selective
medium, whereas heteroplasmic cybrids with syn
and
mitochondria restored mitochondrial respiratory
function might also be explained by supposing as follows: a long
expression time is required to restore mitochondrial respiratory
function in cybrids isolated by the fusion of
syn
cells with enucleated
cells, or the amount of exogenously imported mtDNA required to restore
respiration deficiency in host cells might be limited to about
40-60%, particularly when imported mtDNA is not wild-type but
possesses a different type of pathogenic mutation. It is also possible
that the different types of pathogenic mtDNA mutations used in the
complementation assay allows for a different conclusion. We are testing
these possibilities by isolating cybrids with the same pathogenic mtDNA
mutations as those used by Yoneda et al. (12).
In any case, this study showed the occurrence of interaction even between different types of respiration-deficient mitochondria and provided two important aspects of mitochondrial biogenesis. One was the individuality of each mitochondrion within a cell. Mammalian cells have been thought to contain hundreds of independent mitochondria (16). However, our previous observations provided the totally different view that in living cells mitochondria function as a single dynamic cellular unit, indicating that they lose individuality (11). In this study, we again provided convincing evidence to support this idea by showing the presence of transcomplementation between respiration-deficient mitochondria originated from different cell types.
The other important aspect of mitochondrial biogenesis provided from
our findings was with respect to the relationship between age-associated mitochondrial dysfunction and age-associated
accumulation of somatic mutations in mtDNA. It is generally thought
that somatic mutations are more likely to accumulate in mtDNA than in
nuclear DNA, because mtDNA is a target of most carcinogens and mutagens and is continuously exposed to oxygen-free radicals produced in mitochondria (17-19). If there were no mitochondrial interaction, it
would be much easier to induce reduction of mitochondrial respiratory function by progressive accumulation of somatic mtDNA mutations during
aging, particularly in postmitotic oxidative tissues, such as brain and
muscles (20, 21). On the other hand, if transcomplementation occurred
continuously, mitochondrial respiration activity could be maintained
without significant reduction, even when various kinds of somatic
mutant mtDNAs were accumulated. In fact, we recently found that mtDNA
in autopsied brain tissues from aged subjects could be rescued in
0 HeLa cells by fusion of brain synaptosomal fractions
with
0 HeLa cells, and that this mtDNA transfer resulted
in complete restoration of mitochondrial respiratory function, even
though the brain tissues and their cybrids possessed mtDNAs with
various pathogenic mutations, suggesting functional integrity of the
brain mtDNA from aged subjects (22). These observations can be
explained by occurrence of transcomplementation between mitochondria.
Thus, even though somatic mutations are more likely to accumulate in mtDNA than in nuclear DNA, the occurrence of transcomplementation between mitochondria could provide stable respiratory function in cells
during aging.
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FOOTNOTES |
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* This work was supported in part by grants for Research Fellowships from the Japan Society for Promotion of Science for Young Scientists (to K. I.), University of Tsukuba Special Research grant (to D. T.), grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan, Health Sciences Research grants for research on brain science from Ministry of Health and Welfare of Japan, and University of Tsukuba Special Research grant (Superior) (to J.-I. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-53-6650; Fax: 81-298-53-6614; E-mail: jih45{at}sakura.cc.tsukuba.ac.jp.
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ABBREVIATIONS |
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The abbreviations used are: mtDNA, mitochondrial DNA; bp, base pair; kbp, kilobase pair.
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