From MitoKor, Inc., San Diego, California 92121
Received for publication, November 18, 2002
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ABSTRACT |
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We report the inducible, stable expression of a
dominant negative form of mitochondria-specific DNA polymerase- Mitochondria are unique among cellular organelles, in that
their constituent proteins are encoded by two separate genomes. The
nuclear and mitochondrial genomes are structurally and functionally distinct and use different genetic codes and separate systems for
replication and expression (1-3). Most of the estimated 1500 mitochondrial proteins are encoded by nuclear DNA, translated on
cytoplasmic ribosomes, and imported into the organelle. The remaining
13 mitochondrial proteins, all subunits of protein complexes involved
in electron transport and oxidative phosphorylation, are encoded by the
mtDNA.1 Because electron
transport complexes are composed of both nuclear and mitochondrial
encoded subunits, normal oxidative metabolism is dependent upon the
integrity and coordinated expression of both genomes. Indeed, disease
states are associated with mutations of the mtDNA (4-8), as well as
with nuclear genes that encode respiratory complex subunits (9,
10).
Determining whether a disease state is associated with respiratory
chain dysfunction or is caused by a nuclear or mitochondrial mutation(s) can be challenging. Cybrid cell technology addresses this
challenge by evaluating foreign mitochondrial genomes against a common
nuclear background (11, 12), allowing one to determine whether specific
mtDNA alterations have functional consequences. The construction of
cybrid cell lines typically entails fusing donor platelets, which
contain mitochondria but no nuclei, with cultured cells from which all
resident mtDNA has been eliminated: so-called Mitochondrial DNA replication is catalyzed by DNA polymerase- We employed an alternate expression and selection strategy to stably
incorporate into cultured human HEK293 cells a dominant negative POLG
sequence in which aspartate 1135 was replaced with alanine. By using an
inducible expression system, we incorporated the construct and selected
clones before subjecting the transfected cells to the additional stress
imposed by expression of the dominant negative POLG. Induced expression
of the dominant negative POLG gene caused rapid depletion of mtDNA with
concomitant reduction of gene products and mitochondrial bioenergetic
function. Cell populations devoid of mtDNA were produced by this method
and used to create cybrid cell lines harboring a foreign mitochondrial genome.
Vector Construction--
cDNA encoding human POLG was
obtained by reverse transcription-PCR (SuperScript first-strand
synthesis system; Invitrogen, Carlsbad, CA) using human heart total RNA
(Clontech, Palo Alto, CA) as template, and
oligonucleotide primers complementary to the 5' and 3' ends of the
published coding sequence (20). The 3' primer included a FLAG sequence.
The resulting PCR product was subcloned into the vector pBluescript II
SK+ (Stratagene, La Jolla, CA) and sequence verified by standard
techniques using an ABI 3700 sequencer. A dominant negative form of
POLG (22), which we term POLGdn, was produced by altering codon 1135 from GAC (Asp) to GCG (Ala) using site-directed mutagenesis (Stratagene QuikChange site-directed mutagenesis system). The finished POLGdn sequence was subcloned into pCDNA4/TO (Invitrogen), which contains a tet operator and a zeocin resistance gene, and verified by sequencing.
Cell Culture and Stable Clone Generation--
T-REx293 cells
(Invitrogen) were grown in Immunofluorescent Localization of Recombinant POLGdn--
Cells
expressing POLGdn were grown on polylysine-coated coverslips and
stained with MitoTracker Red dye (Molecular Probes, Eugene, OR), then
fixed in 3% paraformaldehyde for 15 min at 37 °C. Fixed cells were
permeabilized for 15 min at room temperature in phosphate-buffered
saline containing 0.1% Tween 20, 0.3% Triton X-100, 3% bovine serum
albumin prior to incubation with primary and secondary antibodies.
Hoechst stain (Molecular Probes) was applied after the secondary
antibody incubation. Coverslips were mounted onto glass slides with
ProLong Antifade mounting medium (Molecular Probes), dried overnight,
and sealed with clear nail polish.
Quantitative PCR (qPCR) Measurement of DNA and RNA--
Total
DNA was isolated from cell samples using a DNAeasy kit (Invitrogen)
according to the instructions from the manufacturer. Total RNA was
isolated using TRIzol reagent (Invitrogen), followed by RQ1 DNase
treatment (Promega, Madison, WI) at 1 unit/µg of RNA for 30 min at
37 °C. The RNA was then purified by phenol chloroform extraction and
ammonium acetate/ethanol precipitation. First strand cDNA was then
generated by reverse transcription (Invitrogen SuperScript first-strand
synthesis system). Quantitative PCR was carried out using a Prism 7700 sequence detection system with primers and fluorescence-labeled probes
specific for various genes. Standard curves were constructed for each
primer/probe set to verify performance. Each sample was run in
triplicate for each measurement. A relative quantification method was
employed for analysis, comparing the signal of mitochondrial gene
probes to a nuclear gene probe signal as standard.
Western Blotting--
Protein (30 µg) from whole cell lysates
was subjected to SDS-PAGE on NuPAGE 4-12% gels. After transfer to
nitrocellulose membrane, blots were blocked with Tris-buffered saline
(plus 0.2% Tween 20) containing 2.5% nonfat milk solids and 2.5%
bovine serum albumin, and probed with primary antibody diluted in
blocking buffer. Primary antibodies used were: mouse anti-human COII,
mouse anti-human COIV, and mouse anti-human ATP Visualization of Mitochondrial Membrane Potential in Intact
Cells--
T-REx293/POLGdn cells grown with or without tetracycline
for 10 days were incubated in buffer (120 mM NaCl, 3.1 mM KCl, 0.4 mM KH2PO4,
5 mM NaHCO3, 1.2 mM
Na2SO4, 1.3 mM CaCl2,
and 20 mM HEPES) containing 100 nM
tetramethylrhodamine methylester (TMRM) for 30 min before being mounted
in a perfusion chamber and placed on a 37 °C temperature-controlled
stage. TMRM (100 nM) was maintained in the medium
throughout the experiment. Imaging was performed using a 75-watt xenon
lamp-based monochromator (T.I.L.L. Photonics GmbH, Martinsried,
Germany). The emission light was detected using a CCD camera
(Hamamatsu, Japan), and data acquisition was controlled using Simple
PCI software (Compix, Cranberry, PA). Cells were excited at 540 nm, and
the emission was collected through a 610/75-nm filter. Images were
collected every 20 s for a period of 10 min.
Cell Fusion and Cybrid Selection--
Restriction Fragment Length Polymorphism (RFLP) Detection of
Marker Mitochondrial Genome--
Total DNA from platelets and cell
lines was isolated with a DNAeasy kit (Qiagen) and used as template to
PCR-amplify the mitochondrial ATPase 6 gene. PCR fragments were
purified with a PCR Clean-up kit (Roche Applied Science, Indianapolis,
IN) and digested with the restriction endonuclease Fnu4HI
(New England Biolabs, Beverly, MA), and the resulting
fragments were separated on a 4% TBE agarose gel containing ethidium
bromide and visualized under UV light.
Assays of Mitochondrial Function in Permeabilized
Cells--
Basal medium for mitochondrial functional assays contained
250 mM sucrose, 20 mM HEPES-KOH, pH 7.4, 2 mM potassium phosphate, 100 µM EGTA, and was
supplemented as indicated either by 5 mM glutamate plus 5 mM malate, 10 mM succinate plus 2 µM rotenone, or 2 mM ascorbate plus 2 mM
N,N,N',N'-tetramethyl-p-phenylenediamine. Membrane potential, respiration, and optical density of the cell suspension were recorded simultaneously in a 1-ml custom-made multiparameter chamber (Boris F. Krasnikov, Burke Medical Research Institute, White Plains, NY). Membrane potential was followed in the
presence of 2 µM tetraphenyl phosphonium
(TPP+) using a TPP+-sensitive electrode
connected to an amplifier (Vernier Software, Beaverton, OR). Oxygen
consumption was measured using a Clark-type electrode (Diamond General,
Ann Arbor, MI) connected to an oxygen meter (Yellow Springs
Instruments, Yellow Springs, OH). Optical density of the cell
suspension was measured at 660 nm and served as an independent
confirmation of the consistency of cell counts. For these experiments,
cells were harvested, resuspended in the growth medium, and maintained
during the experimental day in suspension under vigorous shaking at
room temperature. For the experimental run, an aliquot containing
2.7 × 107 cells was withdrawn, quickly pelleted (1 min at 200 × g), resuspended in 1 ml of basal assay
medium to remove traces of the growth medium, pelleted again,
transferred into the chamber, permeabilized with 0.015% digitonin, and
measurements were begun.
Measurement of Mitochondrial Enzyme Activities--
Citrate
synthase activity was measured according to standard procedures (15).
Briefly, 500,000 cells were pre-incubated for 3 min in Tris buffer (125 mM, pH 8.0) containing 10% Triton X-100, 2 mM
5,5'-dithiobis(2-nitrobenzoic acid), and 6 mM acetyl CoA.
The reaction was started by the addition of 10 mM
oxalacetic acid, and the linear rate was recorded for 3 min. Samples
were analyzed in triplicate in each experiment.
Complex I activity was measured by following the oxidation of NADH
fluorimetrically. Cells were suspended at 200,000 cells/ml in sodium
phosphate (10 mM, pH 7.4, 1 mM EDTA) and
subjected to three freeze/thaw cycles. A suspension of 400,000 cells
was added to a cuvette together with 2 µg/ml antimycin A, 1 mM KCN, and 13 mM NADH. Samples were
pre-incubated for 5 min until a flat base line was observed.
Decyloubiquinone (600 mM) was added to the sample, and the
rate was recorded for 6 min. Rotenone (2 µg/ml) was added and the
rate recorded for an additional 6 min. The rotenone sensitivity was
routinely >80%. The rotenone-inhibited rate was subtracted from the
non-rotenone inhibited rate, and values were presented as relative
fluorescence units/min × 106 cells. Samples were
analyzed in duplicate in each experiment.
Complex II activity was measured according to Birch-Machin et
al. (23) with minor modifications. Cells were suspended at 200,000 cells/ml in sodium phosphate (10 mM, pH 7.4, 1 mM EDTA) and subjected to three freeze/thaw cycles. Cells
were then pre-incubated with 20 mM succinate for 10 min at
30 °C. Antimycin A (2 µg/ml), rotenone (2 µg/ml), KCN (1 mM), and dichlorophenolindophenol (50 µM)
were added. The reaction was carried out at 30 °C and a base line
recorded at 600 nm for 3 min. The reaction was then started with 65 µM ubiquinol and monitored for 5 min. To obtain the
complex II specific activity, samples were analyzed in parallel with
the addition of 20 mM malonate. Samples were analyzed in
triplicate in each experiment.
Complex IV activity was measured by following the oxidation of reduced
cytochrome c at 550 nm with 580 nm as a reference
wavelength, using a modification of previously reported methods (23,
24). Reduced cytochrome c (15 µM) was added to
a MES buffer (100 mM MES, pH 6.0, 10 µM EDTA,
30 °C) containing 30 mM
n-dodecyl-
Protein was measured using the BCA Protein Assay reagent kit from Pierce.
mtDNA Depletion System--
We produced a dominant negative form
of DNA polymerase- Localization of POLGdn to Mitochondria--
To determine the
location of recombinant POLGdn in the stable clones, immunofluorescent
staining was performed. T-REx293/POLGdn cells were induced by
tetracycline addition for 2 days, followed by staining with anti-FLAG
antibody linked to Alexa Fluor 488 to visualize POLGdn. Mitotracker Red
was also applied to visualize mitochondria, and Hoechst dye was added
to stain the nucleus as a reference (Fig.
2). The POLGdn pattern precisely
overlapped the Mitotracker Red pattern, confirming localization of the
overexpressed recombinant protein to the mitochondria. In uninduced
cells, no POLGdn signal was observed (data not shown).
Expression of POLGdn Impedes Growth Rate--
The average doubling
time of uninduced T-REx293/POLGdn cells was ~26 h. After the addition
of tetracycline, the growth rate remained normal for the first 5 days,
but then slowed abruptly (Fig. 3). A
marked increase in the acidification rate of the growth media also
occurred after 5 days, necessitating daily media replacement for the
duration of the experiment. The doubling time of cells during days 6 through 14 of tetracycline treatment was approximately one fifth of the
normal rate. Treatment of non-transfected parental T-REx293 cells with
tetracycline for up to 21 days had no effect on growth rate (data not
shown).
POLGdn Expression Rapidly Depletes Mitochondrial DNA, RNA, and
Protein--
qPCR was used to monitor the copy number of mtDNA in
T-REx293/POLGdn cells. We used a relative quantitation method in this analysis, comparing the signal produced by mitochondrial gene probes to
the signal from nuclear gene probes. In uninduced cells, the mtDNA copy
number was typically 400-500 times higher than the nuclear gene value,
consistent with the presence of multiple mitochondrial genomes per
cell. Addition of tetracycline caused a time-dependent
reduction of the mtDNA copy number, as indicated by the decreases in
the copy numbers relative to the nuclear actin gene of three selected
mitochondrial encoded genes, NADH dehydrogenase subunit 1 (ND1),
cytochrome oxidase subunit II (COII), and ATP synthase subunit 8 (ATPase 8; Fig. 4A). The
marked decrease in the growth rate of induced cells at day 6 (Fig. 3)
coincided with the reduction of mtDNA content to ~10% of the
starting level (Fig. 4A). By day 10 of induction, the
cellular content of mtDNA was reduced by ~99%. The persistence of a
normal doubling time until mtDNA copy number was reduced by 90% is
consistent with previous reports that most of the mtDNA population of a
cell must be compromised before a measurable phenotypic change occurs
(5, 26).
To assess the impact of POLGdn on mitochondrial encoded mRNA
levels, total RNA was extracted from T-REx293/ POLGdn cells at various time points after induction, reverse transcribed into cDNA,
and measured by qPCR. The decline in the mRNA levels of ND1, COII,
and ATPase 8 in induced cells closely paralleled the reduction in DNA
copy number (Fig. 4B). The content of a mitochondrially encoded protein, COII, was also measured in these cells. Equal amounts
of cell lysates collected at various time points of induction were
analyzed by Western blots probed for COII and actin (Fig. 5A). The cellular content of
COII was reduced in a time-dependent manner, whereas the
amount of actin did not change significantly when POLGdn expression was
induced. These results confirm that POLGdn-dependent
reduction of mtDNA was accompanied by depletion of the corresponding
gene products (Fig. 5B).
mtDNA Loss Is Reversible--
We tested T-REx293/POLGdn cells to
determine whether mtDNA depletion could be reversed when tetracycline
was withdrawn. The ability of the cells to repopulate mtDNA after
repression of POLGdn expression was critical for the production of
cybrids, which require functional endogenous POLG to amplify the
foreign mtDNA introduced during the cybrid fusion. If POLGdn expression
could not be fully repressed after eliminating mtDNA, cybrid production
would not be possible. T-REx293/POLGdn cells were exposed to
tetracycline for 3 days and subsequently maintained in culture without
tetracycline. Samples were taken each day for a total of 12 days, and
DNA and protein extracts were prepared and analyzed. qPCR measurements revealed a sharp decline in mtDNA content through day 4 of the experiment, followed by recovery to a normal level by day 12 (Fig. 6). The rate of mtDNA restoration during
the final 4 days of the experiment was similar to the rate of mtDNA
depletion during the initial 4 days (t1/2 ~ 2-3
days). Western blot analysis of POLGdn expression in parallel samples revealed significant accumulation of POLGdn protein within 24 h,
with maximal content observed at day 3 (Fig. 6). POLGdn protein disappeared rapidly after removal of tetracycline, and remained undetectable from day 4 to 10 (Fig. 6, inset). The results
of this experiment demonstrated that mtDNA depletion was reversible in
this system and predicted that production of cybrids that are dependent
upon endogenous POLG was feasible.
Production of Platelet Fusion to Create Cybrids--
mtDNA Loss Compromises Cellular Respiration--
The bioenergetic
status of T-REx293/POLGdn cells was analyzed using
digitonin-permeabilized cells (27, 28). The lowest concentration of
digitonin that sustained maximal succinate-supported respiration in the
presence of rotenone was 0.015% (data not shown). This concentration,
which completely permeabilized plasma membranes based on trypan blue
dye exclusion, was used for all subsequent studies. The metabolic state
of mitochondria from uninduced permeabilized T-REx293/POLGdn cells
(Fig. 9) was between states 3 and 4, as established by addition of ADP and atractyloside, respectively. Mitochondria isolated from cells that had expressed the POLGdn gene for
10 days exhibited some respiratory compromise, whereas oxygen
consumption of
The contributions of discrete respiratory complexes to overall oxygen
consumption were measured in two ways. First, respiratory complex
activities were measured by varying the respiratory substrates in
experiments similar to the one shown in Fig. 9. This approach allowed
measurement of maximal respiration rates (in the presence of uncoupler
DNP) as well as state 3 and state 4 rates. Although in normal
mitochondria maximal respiration rates are limited by other segments of
metabolic pathways, under the conditions of severely compromised
respiratory chain activity, oxygen consumption rates can be used as a
measure of activity of separate complexes in intact mitochondria.
Because ascorbate plus
N,N,N',N'-tetramethyl-p-phenylenediamine feed electrons directly to complex IV, succinate (plus rotenone) to
complex III (via complex II), and NAD-linked substrates glutamate plus malate to complex I, these combinations of substrates were used to
assay the activities of the respective complexes. Complex V (ATP
synthase) activity was determined by measuring the
ADP-dependent respiratory rate (state 3 minus state 4 in
the presence of succinate). Table II
summarizes the data from these experiments. The activities of complexes
I, III, IV, and V were significantly reduced by induction of POLGdn for
10 days and were essentially eliminated in
To confirm these results, the catalytic activities of several enzymes
were also measured. Complexes I and IV, which contain mitochondrial
encoded subunits, showed normal activities in cybrid cells as compared
with uninduced parental T-REx293/POLGdn cells, confirming that
expression of the mitochondrial encoded subunits had been restored to
normal (Table III). Citrate synthase and
complex II, which are entirely encoded by nuclear genes, also had equal activities in parental cells and in cybrid cells.
mtDNA Loss Decreases Mitochondrial Membrane Potential and Changes
Mitochondrial Morphology--
The impact of mtDNA loss on
mitochondrial membrane potential was determined in intact
T-REx293/POLGdn cells using the potentiometric dye TMRM, and in
permeabilized cells by using a TPP+-sensitive electrode.
Cells grown without tetracycline displayed a tubular network of
mitochondria that fluoresced brightly following addition of TMRM and
excitation at 540 nm (Fig.
10A). Exposure to
tetracycline for 10 days, which eliminated almost all of the mtDNA,
resulted in a greatly reduced signal, indicating a reduction in
mitochondrial membrane potential (Fig. 10B). Measurement of membrane potential using the TPP+ electrode showed a
similar result (Fig. 9). Despite the respiratory compromise in cells
that had been induced with tetracycline for 10 days (partial mtDNA
depletion), the cells retained the ability to generate a measurable
membrane potential and to synthesize ATP, as indicated by the
hyperpolarizing effect of atractyloside (Fig. 9, upper
tracings). In contrast, the functional state of mitochondria
in
Time-lapse photography of the uninduced T-REx293/POLGdn cells revealed
a dynamic mitochondrial network in which interconnected mitochondria
undulated within the cells (Fig. 10A, and supplemental materials available in the on-line version of this article). In contrast, the mitochondrial network was more fragmented and less mobile
in mtDNA-depleted cells (Fig. 10B, and supplemental materials).
Controlled depletion of mtDNA from cultured cells has been used
both to study disease mechanisms and to define normal biochemical mechanisms in a variety of cell types. Because the mitochondrial genome
encodes only proteins of the electron transport chain and ATP synthase,
manipulations of the mtDNA are primarily useful for the study of
mitochondrial oxidative metabolism and the physiological functions that
are regulated by oxidative metabolism. By removing all mtDNA from
cultured cells and replacing the endogenous mitochondrial genome with
exogenous mtDNA from human donors, investigators have identified a
variety of mtDNA mutations that have adverse effects. Such cybrid
studies have elucidated the pathogenetic bases of such syndromes as
MELAS (for myoclonic epilepsy,
lactic acidosis, and strokelike
episodes; Ref. 29) and Leber's hereditary optic neuropathy (30).
Depletion of mtDNA has also been used to define the role of oxidative
phosphorylation in normal biological processes. For example, inhibition
of mtDNA synthesis in cell culture models of the pancreatic mtDNA synthesis is carried out by a multiprotein replication complex,
of which DNA polymerase- Stable expression of POLGdn harboring a D1135A transition in HEK293
cells under the control of a tetracycline-regulated promoter resulted
in a tightly regulated system for the study of mtDNA depletion. The
recombinant DNA polymerase- The decline of mtDNA and mitochondrial encoded protein to undetectable
levels during POLGdn expression indicates that the endogenous POLG was
unable to compensate for the catalytically inactive recombinant
protein. RNA dot-blot analysis showed that the POLGdn mRNA levels
were at least 20 times those of the endogenous POLG mRNA at 6 days
after induction (data not shown). The apparent lack of a compensatory
increase in the endogenous POLG in our system is consistent with
previous reports that POLG expression is not regulated developmentally
(36), nor by tissue metabolic activity (36) or mtDNA copy number (37).
Spelbrink and colleagues (22) postulated that the overexpressed
dominant negative polymerase forms "dead end" replication
complexes, thereby blocking mtDNA synthesis. If this hypothesis is
correct, then mtDNA replication should resume once POLGdn synthesis
ceases, and the residual POLGdn protein dissociates from the
replication complex and is degraded. Indeed, mtDNA content of
T-REx293/POLGdn cells returned to normal after tetracycline was removed
from the media following brief (<10 days) periods of induction. A lag
period of 2-4 days following removal of tetracycline preceded a
measurable increase in mtDNA content.
We assessed the functional consequences of POLGdn expression by
monitoring mitochondrial membrane potential ( The defects in respiratory function following induction of the
T-REx293/POLGdn cells were reversed by repletion of the mtDNA with a
foreign mtDNA. Restoration of function to the cells was the result
of replication and expression of the donor mtDNA (as opposed to
residual HEK293 mtDNA), as indicated by the RFLP analysis of the fused
cells, as well as by the failure of parental In summary, ectopic expression of a dominant negative DNA
polymerase- to
eliminate mitochondrial DNA (mtDNA) from human cells in culture. HEK293 cells were transfected with a plasmid encoding inactive DNA
polymerase-
harboring a D1135A substitution (POLGdn). The cells
rapidly lost mtDNA (t1/2 = 2-3 days) when
expression of the transgene was induced. Concurrent reduction of
mitochondrial encoded mRNA and protein, decreased cellular growth
rate, and compromised respiration and mitochondrial membrane potential
were observed. mtDNA depletion was reversible, as demonstrated by
restoration of mtDNA copy number to normal within 10 days when the
expression of POLGdn was suppressed following a 3-day induction period.
Long term (20 days) expression of POLGdn completely eliminated mtDNA from the cells, resulting in
0 cells that were
respiration-deficient, lacked electron transport complex activities,
and were auxotrophic for pyruvate and uridine. Fusion of the
0 cells with human platelets yielded clonal cybrid cell
lines that were populated exclusively with donor-derived mtDNA.
Respiratory function, mitochondrial membrane potential, and electron
transport activities were restored to normal in the cybrid cells.
Inducible expression of a dominant negative DNA polymerase-
can
yield mtDNA-deficient cell lines, which can be used to study the impact
of specific mtDNA mutations on cellular physiology, and to investigate
mitochondrial genome function and regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0 cells.
The cells are repopulated by the donor mtDNA and ultimately express a
phenotype that is determined in part by the mitochondrial genome of the
donor.
0 cells are most often produced by exposing cells
in culture to ethidium bromide, a dye that intercalates into DNA and
prevents replication (12-16). Alternate agents such as rhodamine 6G
(17), ditercalinium (18), and dideoxycytidine (19) have also been used
to deplete cells of mitochondrial DNA. Two significant problems have
been observed when such toxins are used to produce
0
cells. First, some cell types have proven to be resistant to the
agents. Second, ethidium bromide and other intercalating agents can
damage nuclear DNA, complicating the interpretation of phenotypic changes that occur in the mtDNA-depleted cell lines or in cybrids constructed with these
0 cells. These limitations
prompted us to explore a more specific method to deplete mtDNA from
cultured cells.
(POLG), a mitochondria-specific enzyme that is part of a larger mitochondrial replication complex (20, 21). Because POLG is spatially
and functionally restricted to the mitochondria, inhibition of the POLG
activity should result in loss of mtDNA without affecting nuclear DNA.
Moreover, because all cell types that have mitochondria are dependent
upon POLG for mtDNA replication, inhibition of POLG should deplete
mtDNA from any cell type that can adapt to the need for anaerobic
metabolism. Spelbrink and colleagues (22) identified specific residues
that are required for the catalytic activity of mammalian POLG, and
demonstrated significant reduction of mtDNA copy number in HEK293 cells
that transiently expressed inactive POLG, in which one of two key
aspartate residues (position 890 or 1135) was replaced by asparagine or
alanine, respectively (22). They did not produce stable clones of the
mtDNA-deficient cells, perhaps because constitutive expression of
dominant negative POLG throughout the transfection and selection
processes prevented the cells from adapting to the need for anaerobic metabolism.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0 media (Dulbecco's modified
Eagle's medium containing 4.5 g/liter glucose, 4 mM L-glutamine, 10% heat-inactivated fetal bovine serum, 2 mM sodium pyruvate, 50 µg/ml uridine, 1000 units/ml
penicillin, and 1000 µg/ml streptomycin) at 37 °C with 5%
CO2. For transfections, cells were plated at ~50%
confluence in six-well culture dishes (~106 cells/well)
24 h prior to the addition of 1 µg of plasmid DNA/well with
Effectene reagent (Qiagen, Valencia, CA). Transfected cultures were
grown an additional 3 days and then replated in 100-cm dishes and put
under selection by addition of 200 µg/ml zeocin to the culture
medium. Clones were tested for expression of POLG by addition of 1 µg/ml tetracycline followed by Western blotting with FLAG antibody.
(Molecular Probes);
rabbit-anti-human ATP8 (raised against amino acids 39-58; MITOP
sequence data base, mips.gsf.de/proj/medgen/mitop/); and anti-actin.
Blots were developed using horseradish peroxidase-conjugated sheep
anti-mouse or donkey anti-rabbit antibody and chemiluminescent
substrate (Amersham Biosciences); proteins of interest were
detected by autoradiography.
0 cells
were fused with donor platelets to create cybrid cells as previously
reported (15). Briefly, a venous blood sample was collected from a
volunteer after obtaining informed consent. Platelets were separated by
differential centrifugation and fused with
0 cells as
previously described (15). Unfused
0 cells were
eliminated from cultures by omission of pyruvate and uridine from the
culture medium. Surviving colonies were retrieved by trypsinization in
glass cloning rings after 3 weeks of selection. In "mock fusion"
samples, in which
0 cells were taken through the fusion
protocol in the absence of platelets, cells did not survive in
selection medium.
-D-maltoside. The non-enzymatic
rate was recorded for 1 min, and then 50,000 cells were added to the
assay. Control experiments with the addition of 1 mM KCN
were performed in parallel. Samples were analyzed in triplicate in each experiment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
in which aspartate 1135 was replaced by alanine
(POLGdn), and spliced it into pCDNA4/TO, a mammalian expression
vector regulated by the tet operator. This plasmid was used to
transfect the cell line T-REx293, which is a derivative of the HEK293
cell line that expresses the tet repressor in a constitutive manner. In
the absence of tetracycline, expression of POLGdn was repressed in
these cells. Addition of tetracycline de-repressed transcription, and
production of the mutant polymerase ensued under the control of the
cytomegalovirus promoter. Cells stably propagating a copy of this
construct were selected with zeocin in the absence of tetracycline,
which maintained repression of the POLGdn gene. Using this strategy,
the transfected cells were taken through the selection process without
the added metabolic stress caused by mtDNA loss resulting from POLGdn
expression. Surviving clones were isolated, amplified, and then induced
for POLGdn expression with tetracycline (Fig.
1). Positive clones exhibited tight
regulation of the transgene, with no detectable POLGdn in uninduced
cultures.
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Fig. 1.
Stable, regulated expression of POLGdn in
HEK293 cells. T-REx293/POLGdn clonal cell lines were cultured for
48 h with or without tetracycline (TET; 1 µg/ml) and
then harvested and analyzed by Western blotting. The blots were probed
sequentially with anti-FLAG and anti-actin antibodies.
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Fig. 2.
POLGdn is localized to mitochondria.
T-REx293/POLGdn cells were induced with tetracycline for 2 days and
then analyzed using immunocytochemical techniques. A,
Hoechst dye shows DNA in the nucleus of a single cell. B,
Mitotracker Red shows mitochondria. C, anti-FLAG antibody
linked to Alexa 488 shows location of POLGdn. D, composite
overlay shows precise overlap of POLGdn and mitochondrial images.
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Fig. 3.
mtDNA depletion by expression of POLGdn
impairs cellular growth rate. T-REx293/POLGdn cells were grown in
culture with (TET+) or without (TET )
tetracycline for 14 days. Every 2 days, the cellular replication was
monitored by manually counting the cells.
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Fig. 4.
Expression of POLGdn in T-REx293 cells causes
a time-dependent loss of mtDNA and mRNA.
A, T-REx293/POLGdn cells grown in the presence of
tetracycline were harvested at several time points. mtDNA content was
monitored by measuring the relative copy numbers of three mitochondrial
encoded genes using quantitative PCR. Mitochondrial gene content is
expressed relative to actin gene content and as a proportion of the
uninduced level. B, mitochondrial mRNA level declines
with POLGdn expression. T-REx293/POLGdn cells grown in the presence of
tetracycline were harvested at various time points, and total RNA was
extracted, converted to cDNA by reverse transcription-PCR, and then
assayed by quantitative PCR. mRNA content is expressed relative to
actin mRNA content and as a proportion of the uninduced
level.
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Fig. 5.
POLGdn expression causes a coordinate decline
in mitochondrial protein, DNA, and mRNA. A, Western
blot analysis of the decline in COII protein content of T-REx293 cells.
T-REx293/ POLGdn cells grown with tetracycline were harvested
at various time points. Cell lysates were analyzed by SDS-PAGE and
Western blotting using anti-COII and anti-actin antibodies.
B, COII protein content was quantified by densitometric
scanning of the blots in A. In parallel cultures, COII
mRNA, and COII gene (mtDNA) copy number were measured as described
in the legend to Fig. 4. All values are expressed relative to the
levels in uninduced cells.
View larger version (13K):
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Fig. 6.
POLGdn expression and mtDNA depletion are
reversible. T-REx293/POLGdn cells were exposed to tetracycline
(TET) for 3 days, then grown without tetracycline for an
additional 9 days. Samples were taken daily, and DNA was extracted and
analyzed by qPCR. mtDNA content was determined by the level of ND1 gene
DNA relative to the actin gene and expressed as a percentage of the
uninduced level. Inset, Western blot analysis (anti-FLAG
antibody) of POLGdn protein content in parallel samples.
0 Cells--
To produce cells devoid
of mtDNA, we exposed T-REx293/POLGdn cultures to tetracycline
continuously for 20 days. The cultures were then replated in fresh
medium without tetracycline. After 15 days of culture in the absence of
tetracycline, a condition that represses POLGdn expression, two
distinct cellular morphologies were evident. In most of the wells, the
total cell number remained low and individual cells grew separately or
in small clusters. However, a few wells developed large colonies
containing hundreds of tightly packed cells in addition to the
background of scattered cells present in all the wells. mtDNA content
of wells containing only scattered cells was very low as measured by
qPCR, whereas mtDNA copy numbers in wells containing a large colony
were normal or near normal (Table I). The
large colony phenotype probably represented cells with one or more
residual copies of mtDNA, which was replicated following repression of
POLGdn expression. The sharp contrast in growth characteristics of
cells with or without mtDNA provided an easy means of identifying
candidate
0 populations, which were confirmed by qPCR
analysis (Table I). "Clone 7A-1" was deemed to be
0
and was used for subsequent analyses.
mtDNA levels of 0 candidate cell lines
0 cells
produced by prolonged POLGdn expression (clone 7A-1) were used to
construct cybrid cells. To allow identification of the donor mtDNA in
the cybrids, we fused
0 clone 7A-1 T-REx293/POLGdn cells
with platelets from a donor with an A9007 to G polymorphism in the
mtDNA. This polymorphism created an additional restriction endonuclease
cleavage site in the ATPase 6 gene that we used to distinguish the
donor genome from the endogenous mtDNA sequence of HEK293 cells (Fig.
7A). Following the fusion and
selection protocol, several individual surviving cybrid clones were
isolated and identified by RFLP analysis (Fig. 7B). All 10 cybrid clones tested contained the foreign mtDNA, confirming
incorporation of the donor sequence into the mitochondria of the host
clone 7A-1 HEK293 cells. Western blot analysis demonstrated repletion
of the mitochondrial encoded COII and ATP8 proteins to normal levels in
the cybrid cells (Fig. 8). In contrast,
the nuclear encoded mitochondrial proteins COIV and ATP
were
unchanged by POLGdn expression, by mtDNA depletion, or by cybrid fusion (Fig. 8).
View larger version (54K):
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Fig. 7.
Donor mtDNA can be distinguished from
T-REx293 cell mtDNA by RFLP analysis in
0 and cybrid cells. A,
diagram of the mitochondrial ATP6 gene. A single Fnu4HI site
is present in HEK mtDNA, whereas a second Fnu4HI site is
present in mtDNA with an alternate base at position 9007. B,
PCR-amplified ATP6 gene from DNA extracted from HEK293 cells
(HEK), A9007G platelets, and cybrid cells. Undigested and
restricted DNA samples are stained with ethidium bromide on a 4% TBE
agarose gel.
View larger version (24K):
[in a new window]
Fig. 8.
The cellular content of mitochondrial encoded
proteins is decreased following POLGdn induction, undetectable in
0 cells, and is restored to normal
after cybrid fusion. A, equivalent amounts of cellular
protein from uninduced T-REx293/POLGdn cells (TET
),
T-REx293/POLGdn that had been induced with tetracycline for 10 days
(TET+),
0 T-REx293/POLGdn cells (clone 7A-1;
0), and cybrid cells (Cyb) were
subjected to SDS-PAGE and transferred to nitrocellulose. Replicate
blots were probed with antibodies against FLAG (to detect POLGdn),
mitochondrial encoded proteins (COII, ATP8), nuclear encoded
mitochondria proteins (COIV, ATPb), and actin. B, the blots
were analyzed by densitometric scanning. Protein content is expressed
relative to that in TET
cells.
0 cells was nearly abolished (Fig. 9,
lower tracings). Oxygen consumption in cybrid
cells was restored to a normal level.
View larger version (18K):
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Fig. 9.
Cellular respiration and mitochondrial
membrane potential are compromised by expression of POLGdn. Oxygen
consumption (lower tracings) and
m (upper tracings) were
recorded simultaneously as described under "Materials and
Methods." Additions were as follows: Cells, 2.7 × 107 cells/ml; Dig, 0.015% digitonin;
ADP, 200 µM ADP; Atr, 100 µM atractyloside; DNP 100, 100 µM DNP; DNP 40, 40 µM DNP.
0 cells. The
low residual rates of oxygen consumption in
0 cells were
insensitive to rotenone and cyanide and therefore were
non-mitochondrial (data not shown). Fusion of the respiration-deficient
0 cells with platelets to create cybrid cells restored
the activities of all four complexes to essentially normal values.
Respiratory activities of permeabilized T-REx293/POLGdn
cells
Cybrid cells exhibit electron transport chain complex catalytic
activities similar to those of the parental cell line
0 T-REx293/POLGdn cells was dramatically different.
Mitochondrial membrane potential was extremely low and was not affected
by ADP, atractyloside, or DNP (Fig. 9). The magnitude of the membrane potential was dependent on ATP availability and could be modestly increased if exogenous ATP and an ATP regenerating system were added
(data not shown). In the cybrid cell line, mitochondrial membrane
potential and respiration were restored to normal levels (Fig. 9).
View larger version (18K):
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Fig. 10.
Mitochondrial membrane potential is
decreased, and mitochondrial morphology altered, by expression of
POLGdn. T-REx293/POLGdn cells were incubated with or without
tetracycline for 9 days. TMRM was then added to the cultures, and the
cells examined as described under "Materials and Methods." See
also the videos in the supplemental material, available in the on-line
version of this article.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell
has demonstrated that nutrient-stimulated insulin secretion is
regulated by, and is dependent upon oxidative metabolism (31-33).
mtDNA has historically been depleted from cells by the addition of
intercalating agents like ethidium bromide. Although effective, these
agents lack specificity for mtDNA and act relatively slowly (12-16).
The present study describes a highly specific, tightly controlled,
rapid, and reversible method to deplete mtDNA from cells and tissues.
is an essential component (21). DNA
polymerase-
is believed to be the only DNA polymerase present
in mammalian mitochondria and is specific for mtDNA (20-22). Both the
localization of polymerase-
to the mitochondria and the disparity
between nuclear and mitochondrial genetic codes (34) preclude activity
of the polymerase on chromosomal DNA. Spelbrink and colleagues (22)
identified two aspartic acid residues at positions 1135 and 890 in the
C-terminal portion of the protein as critical for polymerase activity.
We hypothesized that mtDNA replication could be controlled by ectopic
expression of a dominant negative DNA polymerase-
made by mutation
of a critical aspartic acid residue, and expressed under the control of
an inducible promoter.
was appropriately localized to the
mitochondria, as indicated by immunofluorescent staining. The rapid
induction of POLGdn protein synthesis after addition of tetracycline to
the cells was correlated with rapid and dramatic decreases in mtDNA
content, mitochondria-specific mRNA content, and mitochondrial
encoded protein content. mtDNA disappeared with a half-life of ~2-3
days during the initial induction period, when the cellular growth rate
remained normal (doubling time ~ 26 h). This observation
suggests that some residual mtDNA synthesis persisted during the first
few days of POLGdn induction, because complete inhibition of mtDNA
replication would result in halving of the mtDNA content with each cell
division. However, as the rate of cell growth declined after day 5, and
the expression level of POLGdn increased through day 3, the rate of
loss of mtDNA per cell division likely reflected complete absence of
new mtDNA synthesis. The rate of loss of COII protein, a cytochrome
oxidase subunit encoded by the mitochondrial genome, was slower than
the loss of mtDNA or mRNA. This finding was expected, because
cytochrome oxidase has a relatively long half-life of more than 1 week
(35).
m),
mitochondrial movement, cellular respiration, and catalytic activities
of selected electron transport complexes. In addition, we produced
cybrid cell lines using a healthy donor to determine whether
complementation of
0 T-REx293/POLGdn cells with normal
mtDNA could restore normal mitochondrial oxidative activity. Partial or
complete depletion of mtDNA from T-REx293/POLGdn cells dramatically
altered the cellular phenotype. The morphological changes observed in
the
0 T-REx293/POLGdn cells were similar to those
reported in other models of mtDNA depletion, with a change from the
normal reticular mitochondrial network to a more punctate pattern (38,
39). The loss of oxidative capacity in the tetracycline-induced cells was associated with a decrease in the motility of the mitochondria, as
has been reported in models of acute respiratory inhibition (40, 41).
Depletion of mtDNA in our T-REx293/POLGdn cells decreased but did not
abolish the
m. Similar findings have been described
previously in
0 143B osteosarcoma cells (25), in
0 MRC5 fibroblast cells (38), and in
0
INS-1
cells (31). Buchet and Godinot (25) proposed that
0 cells sustain
m by translocating
ATP4
into the mitochondria through reverse functioning of
the adenine nucleotide translocator, and subsequent hydrolysis of
ATP4
to ADP3
by the
F1F0-ATPase. In the present study, the
dependence of
m on exogenous ATP supports the model
by Buchet and Godinot.
0 cells
that were grown in parallel with the cybrids to repopulate with mtDNA.
This finding indicates that endogenous DNA polymerase-
activity
returned to normal levels in the
0 cells following
removal of the tetracycline, and that provision of a mtDNA template
through cybrid fusion allowed rapid restoration of mitochondrial
encoded proteins and their associated functions in the cells.
in human cultured cells reproducibly and reversibly depletes mtDNA. Because all cells with mitochondria are dependent upon
DNA polymerase-
, this approach should be applicable to essentially any cell type, enabling much broader studies of the role of
mitochondrial oxidative metabolism in physiological processes than has
previously been possible. It may also be possible to interrogate the
role of mitochondrial oxidative metabolism in specific tissues in
vivo by targeted expression of the inducible POLGdn in transgenic animals.
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FOOTNOTES |
---|
* 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.
The on-line version of this article (available at
http://www.jbc.org) contains videos that accompany Fig. 10 of
the main text.
To whom correspondence should be addressed: MitoKor, 11494 Sorrento Valley Rd., San Diego, CA 92121. Tel.: 858-509-5613;
Fax: 858-793-7805; E-mail: andersonc@mitokor.com.
Published, JBC Papers in Press, January 6, 2003, DOI 10.1074/jbc.M211730200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
mtDNA, mitochondrial
DNA;
POLG, DNA polymerase-;
POLGdn, dominant negative D1135A DNA
polymerase-
;
COII, cytochrome oxidase subunit 2;
ND1, NADH
dehydrogenase subunit 1;
TMRM, tetramethylrhodamine methylester;
RFLP, restriction fragment length polymorphism;
TPP+, tetraphenyl phosphonium;
DNP, dinitrophenol;
ATP8, ATP synthase subunit
8;
COIV, cytochrome oxidase subunit IV;
ATP6, ATP synthase subunit 6;
m, mitochondrial membrane potential;
qPCR, quantitative PCR;
MES, 4-morpholineethanesulfonic acid.
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