Cell Cycle Dependent Morphology Changes and Associated Mitochondrial
DNA Redistribution in Mitochondria of Human Cell Lines
Daciana H. Margineantu*, W. Gregory Cox+, Linda Sundell, Steven W. Sherwood+, Joseph M.
Beechem+, and Roderick A. Capaldi*
*
Institute of Molecular Biology, University of Oregon, Eugene, OR
+
Molecular Probes Incorporated, Eugene, OR
Corresponding author: Roderick A. Capaldi
Email: rcapaldi@oregon.uoregon.edu
Tel: (001)-541-346-5881
Fax: (001)-541-346-5891
Running title: Mitochondrial DNA is Clustered in Nucleoids which Segregate to each
Mitochondrial Fragment.
1
Abstract
Mitochondria of osteosarcoma cells (143B) in culture have variable morphologies, classified
according to the shape and size of the organelle as reticular, fragmented or intermediate.
Synchronization and release from G0 has shown that the morphology of mitochondria oscillates
between the reticular and fragmented state in a cell cycle dependent manner. Cells in G1 have
reticular mitochondria while those in S phase have fragmented mitochondria. By using a novel
method of fluorescence in situ hybridization, the morphology of mitochondria was correlated
with mitochondrial DNA distribution. MtDNA molecules were seen in clusters of 2-4 along
mitochondrial filaments. In the fully fragmented state, each mitochondrion contained at least one
cluster. We discuss the importance of fission and fusion events in regulating the morphology of
mitochondria, segregation of mtDNA and maintenance of organelle’s functional unity.
Key Words: mitochondrial morphology, mitochondrial DNA, nucleoid, cell cycle, fluorescence
in situ hybridization.
2
1. Introduction
The morphology of mitochondria is tightly controlled by fusion and fission events that
are regulated by several proteins now being identified in yeast and mammalian cells (Hermann
and Shaw, 1998; Bereiter-Hahn and Voth, 1994; Jensen et al., 2000; van der Bliek, 2000; Danino
and Hinshaw, 2001). While the biological significance is not fully understood, it is already clear
that fusion and fission of the mitochondrial mass is a dynamic equilibrium that determines the
proper intracellular distribution of mitochondria, maintains normal mitochondrial morphology,
and determines the inheritance of mtDNA. There is ample evidence that the morphology of
mitochondria varies by cell type (Aw and Jones, 1989; Bereiter-Hahn, 1990). The classic picture
is of small cigar-shaped organelles distributed widely through the cytoplasm. In muscle,
mitochondria are seen in rows between sarcolemma, while in sperm cells they spiral through the
tail region (Bereiter-Hahn, 1990). In fibroblasts and HeLa cells in culture, a reticulum of the
mitochondrial mass has been described (Barni et al., 1996; Rizzuto et al., 1998).
We have previously described both a reticular and a fragmented morphology of
mitochondria in human osteosarcoma cell lines (Gilkerson et al., 2000) which raises the
possibility that the organelle is pleomorphic in cells depending on conditions such as cell cycle
stage or metabolic state. To examine this possibility we have now imaged human osteosarcoma
cells at defined cell cycle stages.
How mitochondrial DNA molecules segregate before and during mitosis determines the
genetic identity of daughter cells, and is particularly relevant in several human diseases where
there is heteroplasmy due to inherited or newly acquired mutations in the DNA (Wallace, 1999;
Schon, 2000). Although many studies have contributed to identifying the factors involved in the
replication, transcription and maintenance of mtDNA, particularly in yeast (Azpiroz and Butow,
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1993; Shadel and Clayton, 1997; Moraes, 2001), information about how the genetic material is
organized and distributed in mammalian mitochondria is limited. One reason is the difficulty in
optimally visualizing the relatively small mtDNA molecules. Here, we provide a modified
method of Fluorescence In Situ Hybridization (FISH) for imaging mtDNA in mammalian cells
which resolves mtDNA clearly. Importantly, the technique allows us to image the genetic
material and visualize mitochondrial morphology at the same time, using organelle-specific
stains. We show how mtDNA molecules distribute when reticular mitochondria interconvert with
small individual organelles, and we discuss the functional significance of this redistribution.
2. Materials and Methods
Cell Culture. Human osteosarcoma cells (143B) were grown in High Glucose-Dulbecco's
Modified Eagle Medium supplemented with 10% Fetal Calf Serum and 5% CO2 at  ºC.
Apoptosis was induced with 10 M staurosporine for two hours. Rho-0 143 B cells were grown
in medium supplemented with ethidium bromide (50 ng/ml) and uridine (50 g/ml). For
microscopy studies, cells were plated on glass coverslips in 35 mm diameter Petri dishes. Cells
were stained for 15 minutes with 250 M MitoTracker Red (Molecular Probes) diluted in the
culture medium. This was followed by three washings, 5 minute each, in dye free medium, all at
37ºC. Subsequently, the cells were fixed for 20 min with 4% paraformaldehyde at room
temperature. Cell permeabilization with ethanol reduced the cytoplasmic background. The cells
were dehydrated and then rehydrated back to PBS using serial dilutions of ethanol (70%, 90%
and100%), 2 minutes for each step. The coverslips were mounted on slides using a Moviol based
medium and sealed with rubber cement.
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Cell Synchronization. Cells were arrested in S phase by treatment with Aphidicolin (2 g/ml) for
16 hours. For starvation, cells were plated at very low densities on coverslips and left for 4 hours
after trypsinization to recover and to attach to the glass surface. The growth medium was
replaced with medium without FCS for 64 to 72 hours. Cells were stained with MitoTracker Red
and fixed before, and then every 2 hours after, FCS readdition for up to 12 hours. Mitochondrial
morphology was evaluated by microscopic inspection of more than 400 cells for each time point.
The numbers obtained were the percentage of cells falling in a certain category (reticular or
fragmented) calculated from the entire cell population after exclusion of cells with ambiguous
mitochondrial morphology (~10%).
DAPI and SYBR Green I Staining. Fixed cells were permeabilized with ethanol and then treated
with 0.1 mg/ml RNaseI, DNase free (Roche) for an hour at 37 ºC. Digestions with 0.1% pepsin
in 0.01 M HCl for 10 minutes at 37 ºC, followed by postfixation (in 4% paraformaldehyde for 10
minutes), were sometimes performed and resulted in a brighter and clearer mtDNA signal. Cells
were incubated with DAPI (0.5 g/ml) or SYBR Green I (1: 2000) (Molecular Probes, Inc.) for
10 minutes and then mounted.
Preparation of the Hybridization Probe. A 1.5 kb COX I DNA fragment was generated by PCR
and then purified using a Qiagen PCR purification column. Typically, 1 g COX I DNA was
used in a nick translation reaction to incorporate 5-(3-aminoallyl)-dUTP and then conjugated to
Alexa Fluor 488 fluorescent dye (ARES DNA labeling kit, Molecular Probes Inc.). We obtained
good incorporation rates for the modified nucleotide, as the resulting ratio of labeled to unlabeled
nucleotides was usually 1 to 11 - 15. The probe was ethanol precipitated with 3 g salmon sperm
5
DNA as a carrier and 5 g COT-1 DNA (Gibco BRL) as general blockant. A probe stock was
made by resuspending the Alexa Fluor 488 dye-labeled COX I DNA to a concentration of 10
ng/l in hybridization solution (50% deionized formamide, 10% Dextran Sulfate, 2 x SSC).
Before hybridization, the probe was diluted to a final concentration of 2 ng/l (1:5) using
hybridization solution, and then denatured for 5 minutes at 72 ºC and snapped cool on ice. The
hybridization volume for a 22 x 22 mm surface was 15 l.
In Situ Hybridization. Cell cultures were grown on glass coverslips, fixed, permeabilized and
sometimes digested with RNaseI as described above. The cells were equilibrated during a
prehybridization incubation for 30 - 45 minutes in 2 x sodium citrate buffer (2 x SSC: 0.3 M
NACl, 0.04 M sodium citrate.), pH 7.5, at 37°C and then dehydrated by serial ethanol dilutions.
The coverslips were left to air dry and then stored at 4°C or processed immediately by
denaturation in 70 % deionized formamide, 2 x SSC, pH 7-8 for 5 min at 72 °C (+/- 2 °C). After
denaturation the cells were dehydrated again by immersion into 70% ice cold ethanol to prevent
renaturation of the DNA (1 minute), then in 90 % ethanol (1 minute) and kept in 100% ethanol
at room temperature until hybridized. Just before the probe was applied on the coverslip, the
cells were quickly dried on a heated block (37-42 ºC). A glass slide was used as a solid support
for the coverslip which was set with the cell side up. The probe was applied to the cells, covered
with another coverslip and then sealed with rubber cement. Hybridization was performed
overnight at 37 ºC in a humid chamber in the dark. Posthybridization washings were as
following: stringent wash in 0.4 x SSC with 0.3 % Tween-20 at 72 °C (+/- 2 °C) for 5 min; mild
wash in 2 x SSC with 0.1 % Tween-20 at room temperature for 1 min. The cells were briefly
counterstained with DAPI and mounted.
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Microscopy. The cells were visualized using an upright Zeiss Axioskop2 microscope equipped
with a 100 x Neofluar objective (1.3 N.A.). FITC and Texas Red filters were obtained from
Chroma Technologies. Images were acquired using a Hamamatsu Orca digital camera (1028 x
1022) driven by OpenLab system software (Improvisions). The pixel length was calibrated by
imaging a transmission electron microscope (TEM) grid of known scale. The aspect ratio was
determined to be 1± 0.1 and the calibrated pixel dimension is 1 pixel = 0.065 mm.
3. Results
Mitochondrial morphology in cell culture
Generally cells in culture are asynchronuous and exhibit a heterogeneous mitochondrial
morphology as shown in Figure 1 for human osteosarcoma cells. This includes short, round or
ovoid mitochondria, which were classified here as fragmented; and long, branched or
interconnected mitochondria, classified as reticular. In some of the cells, mitochondria were
mostly fragmented (panel A), while in others, the organelle was predominantly a reticulum
(panel C). An intermediate morphology in which short mitochondrial fragments coexisted with
branched structures is shown in panel B. Typically this is seen in less than 10% of cells. Electron
microscopy studies showed that all of these morphologies are of mitochondria with normal
ultrastructure, including multiple cristae, the hallmark of an active and normally respiring
organelle (result not shown).
It is known that mitochondria respond rapidly to cell injury and that this induces unusual
morphologies, including donut shaped mitochondria, absence of the typical branched structures
and occasionally, mitochondrial aggregates (Funk et al., 1999; De Vos et al., 1998) . Examples
7
of altered mitochondrial morphology are shown in Figure 2 by example of Rho-0 cells, which are
defective in oxidative phosphorylation (panel A) and 143B cells undergoing apoptosis, due to
staurosporin treatment (panel B). Such abherant morphologies were not seen under the
conditions used here to study the cell cycle dependence of mitochondrial form.
Dependence of mitochondrial morphology on the cell cycle phase
Under standard culturing conditions around 40% of osteosarcoma cells were found to
contain predominantly reticular mitochondria, and 50% contained predominantly fragmented
mitochondria, while 10% were a roughly equal mix of the two states. Culturing of cells for 16
hours in the presence of Aphidicolin, a nuclear DNA replication inhibitor (Merrill, 1998), shifted
the percentage of cells with fragmented mitochondria up to 90% (result not shown), indicating
that cells in S phase have fragmented mitochondria. The same morphology changes were
observed after synchronization of cells by release from fetal calf serum (FCS) starvation. Figure
3 shows typical results with cells that started at very different levels of fragmentation (which
varies significantly from one experiment to another, probably a result of the of the prolonged
maintenance of cells at low plating density in the absence of growth factors). The key result is
the change of morphology that follows FCS addition. Between 2 to 4 hours after FCS treatment
the predominant morphology was reticular as cells left G0 and entered G1. During the following
hours there was a progressive increase in the number of cells with fragmented, i.e. small,
individual, and perinuclear-located mitochondria. During this time the cells entered the S phase
as indicated by an enlargement of their nuclei and the beginning of chromatin condensation. The
number of cells with fragmented mitochondria reached a peak of 60 -75% between 8 and 10
hours after release and this was independent of the levels of fragmentation at the start of the
8
experiment. By 12 hours, the number of cells with a reticular morphology was increasing at the
expense of those with fragmented mitochondria as the cell population went through mitosis and
into G1 again.
Mitochondrial DNA is localized in discrete regions of the mitochondrial reticulum
Fission and fusion events that break and reform the mitochondrial reticulum will lead
alternately to a separation versus a continuum of mitochondrial components. One obvious
consideration in determining the physiological roles of the different morphologies is how key
functional entities become distributed upon fission into small organelles. Particularly relevant is
the position of mtDNA; does every oval small mitochondrion get one (or more) DNA molecule
during fission?
Microscopy studies of the distribution of mtDNA in mammalian cells have been limited
because of the less than optimal staining methods available. Nucleic acid dyes such as DAPI,
SYBR Green I or Ethidium Bromide can be used to image mtDNA, but high-resolution
fluorescence micrographs and correlations to mitochondrial morphology are difficult to obtain.
Figure 4 shows fluorescence micrographs of mtDNA molecules in osteosarcoma cells stained
with DAPI (A) and SYBR Green I (B). They appear as compact structures discretely distributed
in a pattern resembling mitochondria, but the strong signal irradiating from the nucleus hides any
copies of the mitochondrial genome located perinuclearly. Moreover, the low intensity signal,
which bleaches rapidly, and the cytoplasmic background preclude detailed examination of spot
distribution. RNase I and short pepsin treatments were found to diminish the cytoplasmic
background and create a better contrast for imaging mtDNA. In rare cases, pepsin digestion
induced the loss of the nucleus so that the mtDNA could be resolved without the overwhelming
9
nuclear fluorescence. However, the number of cells in which this happened was very low and
sometimes the overall cytoplasmic architecture was affected.
Figure 5 shows cells in which mtDNA has been labeled with an Alexa Fluor 488 labeled
COXI DNA probe (1.5 kb). Punctate green spots are clearly visible with no labeling of the
nucleus and very good resolution over background fluorescence. In panel A, the cells were
treated with RNase I; so that the FISH probe labeled only mtDNA molecules. Rho-0 cells devoid
of mtDNA were negative for FISH (result not shown) as were cells that had been digested with a
combination of DNase and RNase I (Figure 5, panels G-I), confirming that the hybridization
signal is very clean and specific.
Importantly, this FISH labeling method is compatible with staining of mitochondria using
MitoTracker Red (Figure 5B), so that mtDNA distribution can be correlated directly with the
morphology of the organelle (Figure 5C) by merging the two images. MtDNA appears as spots
along the mitochondrial reticulum that in high magnification often appear as clusters of 2-4.
When RNase I treatment was excluded, the probe detected both mtRNA and mtDNA. An
example is shown in Figure 5, panels D-F. The density of spots is now much greater and the
signal more dispersed due to hybridization to RNA molecules.
Most individual mitochondria contain several mtDNA molecules
Observation of mtDNA distribution in cells in which mitochondria are found in the
fragmented state revealed that mtDNA molecules are present in essentially all of the individual
organelles (Figure 6). As in the reticular morphology, the spots representing the mtDNA are
clustered so that many individual mitochondria have multiple green spots in close proximity
10
(Figure 6 D). We interpret these clusters as nucleoids (see discussion), each with more than one
DNA molecule.
Figure 7 is an interesting image in which the mitochondrial mass has been captured
during the fission phase before the reticulum is completely broken down into small ovoid
organelles (i.e. as in Figure 1B). In many cases elongated fragments can be seen that contain two
or more clusters of mtDNA molecules. Often these clusters are at, or near the tips of, filamentous
organelles.
4. Discussion
Recently it has become clear that mitochondrial morphology is tightly controlled by
multiple proteins involved in fusion and fission and also that the distribution of the organelle is
regulated by attachments to microtubules and actin filaments (Hollenbeck, 1996; Yaffe, 1999;
Jensen et al., 2000). The two general processes are probably interconnected and the tight
regulation of mitochondrial form and distribution suggests an important relationship between
morphology and function.
Two predominating morphologies are a reticulum and many distinct small mitochondria
variously estimated between 0.5 and 5.0 m length (Perkins and Frey, 2000). One suggested role
of the reticular form is in rapid transmission of energy through propagation of membrane
potential, which would allow for generation of energy in one part of the cell and sustained use in
another part (Skulachev, 2001). Also, the reticulum, and so the fusion process which regulates its
formation by providing a continuous volume, allows the diffusion of small proteins such as GFP
(Rizzuto et al., 1998; Nunnari et al., 1997) and possible messenger RNAs which can explain
11
intramitochondrial complementation (Ono et al., 2001). Here we show that reticulum
predominates when cells are in G1 phase where metabolism is focused on energy production and
protein and lipid synthesis for the next mitotic division. In the S phase we show that the
mitochondrial mass becomes fragmented and perinuclear. These results agree with, and extend,
previous studies on fibroblast cells (Barni et al., 1996) where cell cycle dependent shifts in
morphology were observed. They are consistent with previous studies that have correlated
mitochondrial biogenesis and function with specific stages of the cell cycle (Van den Bogert et
al., 1988; Leprat et al., 1990; Karbowski et al., 2001).
There is an emerging picture of the inside of mitochondria as a highly ordered structure
where individual enzymes are associated into functional units by compartmentation to outer
membrane, inner membrane, cristal membrane, intermembrane or matrix space. Recent evidence
supports an organization of protein complexes into macromolecular functional entities in
addition to the compartmentation described above (e.g. the respirasome of Schagger and Pfeiffer,
2000). We have shown that PDH complex is not randomly distributed but located at discrete sites
within the mitochondrial matrix (Margineantu et al., 2001). Other enzymes such as Krebs cycle
proteins are probably also localized within the organelle, possibly by interactions with the inner
membrane plus cristal membrane as suggested by the 'metabolon' concept of Srere (Srere et al.,
1987; Velot at al., 1997) and the spectroscopic measurements of Waagpetersen et al. (2000).
MtDNA in humans is 16 kb and encodes 13 polypeptides all of which are components of
the OXPHOS system (reviewed in Wallace, 1999). Estimates of the number of copies of mtDNA
in human cells range from 200 to 2000 based on PCR and Southern blotting estimates as well as
counting of DAPI stained cells (Robin and Wong, 1988; Satoh and Kuroiwa, 1991). There is
good evidence, especially in S. cerevisiae, that mtDNA is organized into replication units by
12
interaction with several DNA binding proteins including Mgt1 (Lockshon et al., 1995), Mgm101
(Meeusen et al., 1999) and Abf2 (Zelenaya-Troitskaya et al., 1998) into what have been termed
nucleoids. Based on flow cytometry of isolated mitochondria and quantitative PCR it has been
proposed that one nucleoid contains on average 2 to 5 copies of the mitochondrial genome
(Cavelier et al., 2000).
We have focused on the arrangement of mtDNA in different morphological states of
human mitochondria. To image this distribution at reasonably high resolution requires better
probes that have been used to date. For reasons detailed already, general nucleic acid stains are
inadequate for this purpose. Bromodeoxyuridine (BrdU) has been successfully employed to label
mtDNA (Davis and Clayton, 1996; Karbowski et al., 2001) but its use is limited by the necessity
to use thymidine kinase negative cells, (143B TK-), to avoid nuclear incorporation of BrdU and
growth inhibition. This method labels only newly replicated DNA, so prolonged incubation times
with BrdU must be used to see the total mitochondrial DNA content and this can alter mtDNA
replication rates and mitochondrial morphology. In situ hybridization has also been used to
detect mtDNA in human cells using mainly radiographic (Nakamura et al., 1990) and enzymatic
detection methods (Nakamura et al., 1996; Vu et al., 2000). However, it has not been possible to
obtain high-resolution images with these labeling approaches.
In principle FISH methods should provide the clearest imaging of the mtDNA. However
the few such studies to date have used probes that have requested amplification (van de Corput et
al., 1997; van den Ouweland et al., 2000, Lewis et al., 2000). Using secondary detection methods
both lowers the resolution obtainable and can alter the specificity of the signal. Here we describe
a FISH method which uses probes generated using the ARES DNA kit (Molecular Probes). This
is a two-step process in which an amine-modified nucleotide, 5-(3-aminoallyl)-dUTP is first
13
incorporated into DNA via an enzymatic reaction. Subsequently the amine-modified DNA is
chemically labeled using an amine-reactive, fluorescent dye. This labeling procedure allows the
probe size to be kept to minimum and as, we show, clearly provides a high signal that does not
need secondary amplification.
Using this approach we found that the reticulum form of mitochondria contains many
punctate (green) spots which are clustered into groups along filaments. Each individual spot is
very likely a single DNA molecule with its tightly associated hybridization probe. Therefore, we
suggest that the clusters are the several DNA copies incorporated into nucleoids. The alternative,
that they are individual DNA molecules superimposed by the depth of field of the images is
unlikely, as osteosarcoma cells are very thin, particularly at the periphery.
Our results indicate that when the mitochondrial reticulum becomes fragmented to
eventually generate small ovoid mitochondria, each small organelle gets copies of mtDNA,
possibly one nucleoid. Previous studies using mitochondrial particles from mechanically
disrupted cells had claimed that a significant proportion (more than 40%) contained no DNA
(Cavelier et al., 2000). This is clearly not the case. The few images we obtained where the
mitochondrial reticulum was fragmented but still filamentous showed that the mtDNA clusters
were spread out, with fission of the organelle taking place between these. It is interesting then to
correlate the work here with the recent study of Smirnova et al. (2001) on the role and
distribution of dynamin-related Drp1 protein in mammalian cells. These workers show discrete
location of this protein contributing for the fission process. Taken together the recent data
suggest that the mitochondrial mass is made up of repeating units containing most or all of the
metabolic and biosynthetic functions of the organelle. These units come apart or fuse to a
14
reticulum to maximize either cellular bioenergetics or inheritance of the DNA in a cell cycle
dependent fashion.
15
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Legends to Figures
Figure 1. Variations of mitochondrial morphology in cells cultured under normal conditions;
fragmented (A), intermediate (B) and reticular (C) morphologies. (MitoTracker Red staining)
Figure 2. Mitochondrial morphology is altered in Rho-0 (A) and apoptotic (B) osteosarcoma
cells. (MitoTracker Red staining)
Figure 3. Changes of mitochondrial morphology in osteosarcoma cells following release from
G0 arrest. Percentages of cells with fragmented mitochondria are plotted against time.
Figure 4. Mitochondrial DNA staining using nucleic acid stains: DAPI (A) and SYBR Green I
(B).
Figure 5. Mitochondrial DNA and RNA have a discrete distribution within mitochondria. RNase
I treatment allows the specific visualization of mtDNA molecules (A). In the absence of any
pretreatment, hybridization with amino-allyl modified COXI-Alexa Fluor 488 highlights both
mtDNA and mtRNA molecules (D). Both signals are compared to the position and area of
mitochondrial profiles stained with MitoTracker Red (B and E) in the merged images (C and F).
MtDNA molecules appear discretely distributed along mitochondria, clustered in bright spots of
various size and shape. There are many more spots that are more widely dispersed along
mitochondrial filaments when hybridization is performed against both nucleic acid species. The
FISH signal is negative or very much reduced in cells treated with both DNase and RNaseI
(panels G, H and I) indicating good labeling specificity.
Figure 6. Mitochondria fragment in a manner that assures the segregation of at least one
nucleoid to each individual organelle. The distribution of mtDNA FISH signal (A) is compared
to the mitochondrial morphology - MitoTracker Red staining (B). The merged image (C) and the
magnification of one portion of the image (by a factor of 4) (D) show the colocalization of the
two structures.
Figure 7. In cells with mitochondrial filaments of intermediate size mtDNA molecules are often
localized at the tips of the fragments (arrowed). The constrictions that are apparent in the middle
of some mitochondrial profiles are most likely the sites of a previous fusion or future fission
event.
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