(Received for publication, September 3, 1996, and in revised form, December 4, 1996)
From the For the examination of possible
intermitochondrial interaction of human mitochondria from different
cells, cybrids were constructed by introducing HeLa mitochondria into
cells with respiration-deficient ( Mammalian cells have been proposed to possess hundreds of
independent mitochondria, each containing several mitochondrial DNA
(mtDNA) molecules (1-3). Variation in the number and morphology of
mitochondria in different cell types (4) and even in the same cell type
(5) has been suggested. The interaction and fusion between mitochondria
in yeast and plant cells have received support from morphological
findings (6) and from the molecular and genetic evidence of mtDNA
recombination (7, 8). In mammalian species, however, the coexistence of
mitochondria and mtDNA from different individuals is completely
inhibited by their strictly maternal inheritance (9, 10); sperm-derived
mtDNA was selectively and completely eliminated from fertilized mouse
eggs, suggesting that the mtDNA population of mammalian individuals is
derived exclusively from eggs (10) and is extremely homoplasmic
throughout an individual (11). Therefore, it seems reasonable to
suppose that mammalian mtDNAs consequently have lost the ability to
recombine with each other. In fact, we previously showed the absence of extensive mtDNA recombination in mammalian cells, even when polymorphic mtDNA molecules from different individuals and from different species
were mixed within a cell by the fusion of somatic cells (12).
On the other hand, metabolic cooperation of the mammalian mitochondrial
genetic system was suggested by the translational complementation of
mitochondrial rRNA observed in heteroplasmic cells with
chloramphenicol-sensitive
(CAPs)1 and -resistant
(CAPr) mtDNA (13). Our previous studies provided
biochemical evidence for intermitochondrial interactions through
translational complementation and competition of mitochondrial tRNAs
using cybrids with mutant and wild type mtDNA from patients with
mitochondrial diseases (14, 15) and also provided molecular evidence
for the rapid penetration of mtDNA and/or its products into
mitochondria of mtDNA-less ( Recently, the absence of intermitochondrial interaction was proposed,
particularly when mitochondria containing different pathogenic mutant
mtDNAs derived from different patients coexisted in single cells (17).
One explanation for the apparent discrepancy between the observations
of the presence (13-16) and absence (17) of mitochondrial interactions
might be that the occurrence of interactions of mutant and wild type
mtDNA molecules could be limited only when they coexist in the
mitochondria from the time of the mutation event, but not when once
they have been separated into different mitochondria by random
segregation, or originate in distinct mitochondria of different cells
(17). If this is so, intermitochondrial cooperation followed by their
fusion would not occur in mammalian cells.
In this study, using enucleation and cell fusion techniques, we created
an environment suitable for determining whether intermitochondrial cooperation occurs even between distinct organelles derived from different cells. First, we isolated respiration-deficient
The human skin fibroblast line 197 with 21% Intercellular transfer of HeLa nuclei to 197 fibroblasts
was achieved by fusion of the fibroblasts with Intercellular transfer of HeLa mitochondria was carried
out as described previously (18) by fusion of enucleated
CAPr HeLa cells with the nuclear hybrid clone H5 containing
only For determination of the
content of Mitochondrial translation products were labeled with
[35S]methionine as described previously (18) with slight
modifications. Briefly, semiconfluent 2 × 106 cells
in a dish were incubated in methionine-free medium containing 2%
bovine serum for 30 min at 37 °C. Then, the cells were labeled with
[35S]methionine for 2 h in the presence of 0.2 mg/ml
emetine. The mitochondrial fraction was obtained by homogenization in
0.25 M sucrose, 1 mM EGTA, and 10 mM HEPES-NaOH, pH 7.4, followed by differential
centrifugation. Proteins in the mitochondrial fraction (30 mg) were
separated by SDS-polyacrylamide gradient gel (12-20%) electrophoresis. The dried gel was exposed to an imaging plate (Fuji
Photo Film) for 6 h, and the labeled polypeptides were located with a bioimaging analyzer, Fujix BAS 2000 (Fuji Photo Film).
Cells grown
on coverslips were fixed in 2% glutaraldehyde, 0.05 M
phosphate buffer, pH 7.4, for 15 min and stained for COX by the
procedure of Seligman et al. (20) with slight modifications (21) to detect the COX activities of individual mitochondria at the
ultrastructural level. The cells were then postfixed in OsO4 for 15 min and embedded in epoxy resin.
To determine whether there
is intermitochondrial cooperation between distinct organelles derived
from different cells, we first isolated Southern blot analysis showed that of the 12 nuclear hybrid clones
isolated, one (H5) had
Somatic cell genetic characteristics of parent cells and their nuclear
hybrid clones
Institute of Biological Sciences,
) mitochondria.
Respiration deficiency was due to the predominance of mutant mtDNA with
a 5,196-base pair deletion including five tRNA genes
(
mtDNA5196). The HeLa mtDNA and
mtDNA5196
encoded chloramphenicol-resistant (CAPr) and
chloramphenicol-sensitive (CAPs) 16 S rRNA, respectively.
The first evidence for the interaction was that polypeptides
exclusively encoded by
mtDNA5196 were translated on the
introduction of HeLa mitochondria, suggesting supplementation of the
missing tRNAs by
mitochondria from HeLa mitochondria.
Second, the exchange of mitochondrial rRNAs was observed; even in the
presence of CAP, CAPs
mtDNA5196-specific
polypeptides as well as those encoded by CAPr HeLa mtDNA
were translated in the cybrids. These phenomena can be explained
assuming that the translation in
mitochondria was
restored by tRNAs and CAPr 16 S rRNA supplied from HeLa
mitochondria, unambiguously indicating interorganellar interaction.
These observations introduce a new concept of the dynamics of the
mitochondrial genetic system and help in understanding the relationship
among mtDNA mutations and expression of human mitochondrial diseases
and aging.
0) HeLa cells (16). Based on
these observations, we proposed that the mitochondria and mitochondrial
genetic system in living mammalian cells have lost their individuality
and thus function as a single dynamic cellular unit (16).
cells containing more than 95% of disease-related
mutant mtDNA with a 5,196-base pair deletion
(
mtDNA5196), which have no mitochondrial translation
activity because of the deletion of five tRNA genes. Then, mitochondria
of CAPr HeLa cells were introduced into the
cells, and the translation activity of the
mitochondria was examined. The results showed that even in the presence
of CAP, the fusion proteins exclusively encoded by
mtDNA5196 were translated with the help of the tRNAs and
CAPr rRNA supplied from the imported HeLa mitochondria,
suggesting unambiguously the occurrence of intermitochondrial
cooperation between distinct organelles of
mitochondria and HeLa mitochondria.
Cells and Cell Culture
mtDNA5196 was isolated from a patient with
chronic progressive external ophthalmoplegia, a subgroup of
mitochondrial encephalomyopathies. The
0 HeLa cells and
CAPr HeLa cells used are resistant to 20 µM
6-thioguanine and 2 mM ouabain. These cell lines were grown
in normal medium (RPMI 1640, 0.1 mg/ml pyruvate, 50 µg/ml uridine,
10% fetal bovine serum).
0 HeLa
cells using polyethylene glycol 1500 as described previously (18) with
slight modifications. Briefly, the fusion mixtures were cultivated in
selective medium (RPMI 1640, 0.1 mg/ml pyruvate, 50 µg/ml uridine,
10% fetal bovine serum, hypoxanthine/aminopterin/thymidine (HAT;
Sigma), 2 mM ouabain), and on day 10 after fusion colonies grown in the selection medium were cloned by the cylinder method. Nuclear hybrids were then cultivated in normal medium.
mtDNA5196, and cybrid clones were isolated in
selection medium (DM170 + HAT). Briefly, CAPr HeLa cells
grown on round glass discs were enucleated by centrifugation (23,000 × g, at 34 °C for 10 min) in the presence
of cytochalasin B (Sigma; 10 µg/ml). The resulting cytoplasts were
mixed with H5 cells, and fusion was carried out in the presence of 50%
(w/v) polyethylene glycol 1500 (Boehringer Mannheim). The fusion
mixture was cultivated in selection medium without glucose, pyruvate, and uridine (DM170, Kyokuto Kagaku, Tokyo) supplemented with HAT (9).
On day 14-20 after fusion, cybrid clones grown in selective medium
were harvested and cloned by the cylinder method. Cybrids were
cultivated in DM170 medium without HAT.
mtDNA5196, total DNA (2 µg) extracted from
2 × 105 cells was digested with a single cut
restriction enzyme, PvuII, and the restriction fragments
were separated by 0.6% agarose gel electrophoresis. After blotting
onto a Nytran membrane, the DNA fragments were hybridized with
[
-32P]dATP-labeled HeLa mtDNA. The contents of HeLa
mtDNA in cybrid clones were determined from the HeLa mtDNA-specific
polymorphisms of HaeIII restriction patterns, which can
distinguish HeLa mtDNA from other human mtDNAs (19). The total DNA (2 µg) extracted from 2 × 105 cells was digested with
HaeIII, and the fragments were separated by 2% agarose gel
electrophoresis. After blotting onto a Nytran membrane, the DNA
fragments were hybridized with [
-32P]dATP-labeled
polymerase chain reaction products amplified using two primers
(15,756-15,775 and 15,916-15,897). The 1,284-base pair fragment was
cleaved into two fragments of 711 and 573 base pairs due to one
HaeIII site gain in HeLa mtDNA (19). The membrane was washed
and exposed to an imaging plate (Fuji Photo Film, Tokyo), and the
radioactivity of the fragments was measured with a BAS2000 instrument
(Fuji Photo Film).
Isolation of Nuclear Hybrid Clones Containing
Predominantly
mtDNA5196
nuclear
hybrid cells, which show no mitochondrial translation or
oxidative phosphorylation activities due to predominance of patient-derived
mtDNA5196 with a deletion of five tRNA
genes (14). Previously, cybrid clones were isolated by fusion of
0 HeLa cells with enucleated fibroblasts from a chronic
progressive external ophthalmoplegia patient with
mtDNA5196 in selective medium without pyruvate and
uridine to remove
0 HeLa cells (14). In this selective
medium, however,
cybrids containing
mtDNA5196 predominantly must also be removed because of
their overall respiration deficiency. Therefore, we tried to isolate
nuclear hybrid clones by fusing
0 HeLa
cells with the fibroblasts followed by HAT selection, which does not
have any selective pressure upon growth of
cells but
can remove
0 HeLa cells.
mtDNA5196 predominantly, four
(H2, H8, H10, and H12) had both
mtDNA5196 and wild type
mtDNA, and seven (H1, H3, H4, H6, H7, H9, and H11) had only wild type
mtDNA (Table I). Using three clones, H9 without
mtDNA5196, H8 with 41%
mtDNA5196, and H5
with only
mtDNA5196, we analyzed the influence of the
amount of
mtDNA5196 on the activity of mitochondrial
translation using [35S]methionine labeling.
Cell line
Drug resistance
Selection
%
mt
DNA5196
Parent cell lines
197 fibroblasts
(mitochondria donor)a
21
0
HeLa
6-Thioguanine, ouabain
b
Nuclear hybrid clones
H1
Ouac + HAT
0
H2
Oua + HAT
87
H3
Oua + HAT
0
H4
Oua + HAT
0
H5
Oua + HAT
>95
H6
Oua + HAT
0
H7
Oua + HAT
0
H8
Oua + HAT
41
H9
Oua + HAT
0
H10
Oua + HAT
72
H11
Oua + HAT
0
H12
Oua + HAT
79
a
Fibroblast line derived from a chronic progressive
external ophthalmoplegia patient.
b
No mtDNA molecules were detected even by polymerase chain
reaction techniques.
c
Oua, ouabain.
No mitochondrial translation was observed in H5 cells with only
mtDNA5196, suggesting that they are
cells (Fig. 1). On the other hand, quantitative
estimation of mtDNA translation products showed that the overall
translation capacity of H8 cells with 59%
mtDNA5196 was
comparable to that of H9 cells without
mtDNA5196.
Moreover, because of the 5,196-base pair long deletion with a
breakpoint between 8,563 and 13,788,
mtDNA5196 newly
acquired a unique fusion gene that encoded two fusion proteins, ATP8/ND5 (FA) and ATP6/ND5 (FB) (14), which were exclusively translated
in H8 cells (Fig. 1). These features indicate that polypeptides encoded
by
mtDNA5196 were translated using the tRNAs supplied
from the endogenous wild type mtDNA of the patient, suggesting
intramitochondrial cooperation.
Analysis of Intermitochondrial Cooperation between Distinct Organelles Derived from Different Cells
For determination of
whether intermitochondrial cooperation occurs even between distinct
organelles from different cell types, the chance of interaction between
mitochondria from H5 cells and
+
mitochondria from HeLa cells was created by fusion of enucleated CAPr HeLa cells with H5 cells. All cybrid clones isolated
in selective HAT medium without pyruvate and uridine contained both
mtDNA5196 and HeLa mtDNA (Table II).
|
In these cybrids, the fusion proteins (FA and FB) can be used as
specific markers of the translation of mtDNA5196. On the
other hand, since ND3 of HeLa cells, ND3
, migrated slightly faster in
SDS-polyacrylamide gel than the corresponding ND3 of other human cells
(13, 17, 22) because of a single amino acid exchange (Asp to Asn) (23),
ND3
could be used as a specific marker of the translation of HeLa
mtDNA.
First, as a negative control of intermitochondrial interaction, we
examined mitochondrial translation in a simple mixture of H5 and HeLa
cells with 53% mtDNA5196. In this case, as
mitochondria in H5 cells and
+
mitochondria in HeLa cells were completely separated and confined to
each type of cell, cooperation of the
and
+ mitochondria could not occur, and thus their
translation should be limited to mitochondria in HeLa cells. As
expected, the results showed that ND3
but not fusion proteins was
translated and that the total amount of [35S]methionine
incorporation corresponded exactly to the amount of HeLa mtDNA (Figs.
2 and 3).
Then mitochondrial translation activity was examined by
[35S]methionine labeling using a cybrid clone CH5-2 with
53% mtDNA5196 (Fig. 2A), in which the
and
+ mitochondria coexisted by cell
fusion. Figs. 2B and 3 show that the total amount of
[35S]methionine incorporation into mtDNA-encoded
polypeptides in CH5-2 cells was comparable to that in HeLa cells.
Furthermore, the fusion proteins FA and FB exclusively encoded on
mtDNA5196 were observed in the CH5-2 cells (Fig.
2B). Thus, in contrast to the case in the simple mixture,
mtDNA5196-encoded polypeptides were translated using the
tRNAs supplied from the imported HeLa mitochondria in CH5-2 cells,
showing unambiguously the occurrence of intermitochondrial cooperation
between distinct organelles of the
and
+ mitochondria derived from H5 cells and HeLa cells,
respectively.
By use of CH5-2 cells, the occurrence of intermitochondrial
cooperation could be confirmed by examining the effects of CAP on the
translations of both CAPs mtDNA5196 and
CAPr HeLa mtDNA. If intermitochondrial cooperation
occurred, the fusion proteins encoded by CAPs
mtDNA5196 should be translated by the use of
CAPr 16 S rRNA transcribed from CAPr HeLa
mtDNA.
First, we carried out a negative control experiment by testing the effect of CAP on translation of mitochondria in a simple mixture of 53% H5 and 47% HeLa cells, in which no intermitochondrial interaction occurred. In the presence of CAP, translation in CAPr HeLa mitochondria was slightly inhibited (Fig. 2B). The amount of mitochondrial translation in the mixture of H5 and HeLa cells corresponded exactly to that expected from the proportion of HeLa cells in the mixture (Fig. 2B), and no fusion proteins were translated (Fig. 2B), reflecting the absence of cooperation.
If there is no interaction between mitochondria from different cells,
the mitochondrial translation profiles in CH5-2 cells with 47% HeLa
mtDNA must correspond to those in the simple parental cell mixture with
47% HeLa mtDNA. However, the translation of the fusion proteins was
observed in CH5-2 cells even in the presence of CAP (Fig.
2B). Furthermore, the amount of translation of ND3 exclusively encoded by CAPr HeLa mtDNA, as well as those of
CAPs
mtDNA5196-specific fusion proteins, was
reduced simultaneously to much lower levels than those in the simple
parental cell mixture with 47% HeLa mtDNA (Figs. 2B and
3).
The translation of a small amount of fusion proteins in the presence of
CAP represents the occurrence of intermitochondrial cooperation, since
CAPr 16 S rRNA transcribed from the imported
CAPr HeLa mtDNA should be necessary for translation of the
fusion protein in the presence of CAP, suggesting rRNA complementation. The progressive inhibition of the translation of CAPr HeLa
mtDNA-encoded polypeptides including ND3 in CH5-2 cells by CAP might
be explained by supposing that the CAPs allele is dominant
over CAPr in mammalian cells. This phenomenon could not
occur when mitochondria with CAPs
mtDNA5196
and with CAPr HeLa mtDNA did not cooperate with each other
as in the simple mixture of mitochondria prepared from the cells (Figs.
2 and 3).
The observed complementation
of mitochondrial tRNA and rRNA excludes the possibility that
mtDNA5196 and HeLa mtDNA remained separated in
respective mitochondria. Accordingly, the
mtDNA5196 and
HeLa mtDNA in cells of the cybrid clone CH5-2 should mix homogeneously
throughout the mitochondria. This prediction can be tested by electron
microscopic analysis of COX activity in mitochondria using CH5-2 cells
with 53%
mtDNA5196, since this technique clearly
identifies COX activity of individual mitochondria in single cells.
No individual mitochondria with predominantly mtDNA5196
in H5 cells showed COX activity, whereas most mitochondria in HeLa
cells showed clear COX activity (Fig. 4). Therefore, the
COX activity of individual mitochondria could be used as a probe to
identify mitochondria with
mtDNA5196 and with HeLa
mtDNA. If they do not interact with each other and remain unfused in
CH5-2 cells after the import of HeLa mitochondria, they can be
distinguished unambiguously by COX electron microscopy, and the ratio
of COX-negative to COX-positive mitochondria in a cell should be
proportional to the ratio of
mtDNA5196 to HeLa mtDNA
(53:47).
COX activity of individual mitochondria in single CH5-2 cells was
analyzed immediately after clonal isolation to minimize intercellular
variations in the mtDNA5196 contents of different cells
in the clone. Fig. 4 shows that most mitochondria in CH5-2 cells were
COX-positive, suggesting that mitochondria from H5 cells and from HeLa
cells did not remain segregated, but interacted with each other in the
CH5-2 cells. This uniform distribution of COX-positive mitochondria
within single CH5-2 cells containing 53%
mtDNA5196 can
be explained by rapid diffusion of
mtDNA5196, HeLa
mtDNA, and their products throughout the mitochondria.
Accordingly, all observations in this study support the idea that
intermitochondrial interaction occurs even between distinct organelles
with mtDNA5196 from a patient with mitochondrial disease
and wild type mtDNA of HeLa cells.
In this study, to provide direct evidence for the presence of
interaction between distinct mitochondria originating in different cells, we created using cell fusion technique the chance of their interaction by isolating CH5-2 cells containing both
mitochondria with
mtDNA5196 derived from a patient with
a mitochondrial disease and
+ mitochondria with
CAPr mtDNA derived from HeLa cells. We demonstrated
intermitochondrial interaction unambiguously in three different
ways.
First, we demonstrated complementation of mitochondrial tRNAs
between mitochondria originating from different cells. Polypeptides encoded by mtDNA5196, which has a deletion of five tRNA
genes, were translated when exogenous normal mitochondria imported from
HeLa cells coexisted within CH5-2 cells (Fig. 2). This suggests the
mitochondrial fusion and subsequent diffusion of the tRNAs transcribed
from the imported HeLa mitochondria into the host
mitochondria with
mtDNA5196.
Second, we observed complementation of mitochondrial rRNAs. Even in the
presence of CAP, fusion proteins exclusively encoded by
CAPs mtDNA5196 were translated in the CH5-2
cells. Furthermore, the mitochondrial translations of all polypeptides
including the CAPr HeLa mtDNA-specific polypeptide ND3
in
CH5-2 cells were reduced simultaneously to much lower levels than those
in a simple mixture of the parental cells with an equivalent amount of
HeLa mtDNA (47%) (Figs. 2B and 3). These results could be
explained by supposing that intermitochondrial interaction,
i.e. complementation of mitochondrial 16 S rRNAs, occurs
between mitochondria with CAPs and CAPr mtDNA
derived from different cells and that the CAPs allele is
dominant over the CAPr allele.
The third line of evidence for intermitochondrial cooperation was
obtained by COX electron microscopy. The observed mitochondrial tRNA
and rRNA complementation could not occur if mtDNA5196
and HeLa mtDNA remained confined to their respective mitochondria after
the introduction of HeLa mitochondria. This interpretation predicts
that coexisting
mtDNA5196 and HeLa mtDNA in single CH5-2
cells should mix homogeneously throughout the mitochondria. This
prediction was examined by electron microscopic analysis of COX
activity in individual mitochondria within CH5-2 cells containing 53%
mtDNA5196, and the results demonstrated that all of the
individual mitochondria showed COX activity (Fig. 4).
These observations consistently suggest the coexistence and cooperation
of mtDNA5196 and HeLa mtDNA within the same organelles,
supporting the occurrence of interaction between distinct organelles.
Such intermitochondrial interaction could not occur if mitochondria did
not fuse and exchange mtDNA and its transcripts between fused
mitochondria. Therefore, the presence of intermitochondrial interaction
provides evidence not only for the mitochondrial fusion but also for
subsequent mixing of their contents and supports the idea we reported
previously that mitochondria and mitochondrial genome function as a
single dynamic cellular unit in living human cells (16). Moreover, this
concept will help in understanding the relationship between mtDNA
mutations and expression of human mitochondrial diseases and aging. For
example, although age-associated accumulations of various somatic
deletion mutant mtDNAs and age-associated mitochondrial dysfunction
were observed in mammalian tissues (3, 18, 24), our results show that
mtDNA mutations can complement each other, and thus they do not have a
serious influence on age-associated mitochondrial dysfunction.
With respect to the dominant behavior of the CAPs allele in
CH5-2 cells, however, it has been proposed that the CAPs
allele could not be dominant for the following reasons (13, 17). First,
it is reported that in bacteria CAPs ribosomes were able to
move along mRNA without forming polypeptides in the presence of CAP
(25). However, this simple observation does not exclude the possibility
of the dominant behavior of the CAPs allele. The second
reason is that both CAPs and CAPr mtDNA-encoded
polypeptides can be translated even in the presence of CAP (13, 17).
However, the amount of the translation of CAPr
mtDNA-encoded polypeptide ND3 in the presence of CAP was reduced progressively to a much lower level than that expected from the amount
of CAPr mtDNA in the cells (Fig. 2), suggesting the
presence of intermitochondrial cooperation in which the
CAPs allele behaves dominantly.
If the CAPs phenotype is dominant, the question arises of why a small amount of CAPs mtDNA-encoded polypeptides, fusion proteins, was translated in the presence of CAP, as observed in Fig. 2B. This question can be explained by supposing that CAPs rRNA and CAPr rRNA are distributed randomly throughout the mitochondria of cells by intermitochondrial interaction. In this case, most polysomes in CH5-2 cells would have more than one CAPs rRNA, and in these translation would be inhibited by CAP. On the other hand, a small proportion of polysomes happened to contain no CAPr rRNA, and in these both CAPs and CAPr mtDNA-encoded polypeptides were translated proportionally in the presence of CAP. Accordingly, all of the results in this study are consistent with the occurrence of intermitochondrial cooperation between organelles derived from different cells.
Recently, the absence of intermitochondrial interaction on mixing
mitochondria derived from different cells by the cell fusion techniques
was reported (17); interactions of mutant and wild type mtDNA molecules
occur only when they have been coexisting in the mitochondria from the
time of the mutation event, but not when they originate in distinct
organelles in different cells. However, these possibilities are
unlikely, since originally coexisting mutant and wild type mtDNA within
a mitochondrion would eventually segregate stochastically during
culture of the cells, if exchange of mtDNA between mitochondria did not
occur (26) as in the case between cells. It is well known that
coexisting wild type and mutant mtDNA (27-29) and coexisting mtDNAs of
different species (30) in a cell segregate in a stochastic way.
Furthermore, this study provided direct evidence for the presence of
cooperation even between organelles originating from different cells.
However, it is still possible that cooperation does not occur between
respiration-deficient mitochondria (17), possibly because of a lack of
a sufficient energy supply for mitochondrial fusion and mixing of their
contents. We are now investigating this possibility by isolating cells
with syn mitochondria, which are
respiration-deficient due to point mutations of the
tRNALeu(UUR) or tRNAIle gene (15), and then
creating a chance for interaction between syn
mitochondria or syn
and
mitochondria.