Mitochondria are morphologically heterogeneous within cells
Laboratory of Molecular Signalling, The Babraham Institute, Babraham, Cambridge, CB2 4AT, UK
* Author for correspondence (e-mail: tony.collins{at}bbsrc.ac.uk)
Accepted 15 January 2003
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Summary |
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Key words: mitochondrial morphology, mitochondrial network, fluorescence-recovery-after-photobleaching (FRAP), tetra-methyl rhodamine ethyl ester (TMRE)
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Introduction |
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Several lines of evidence have been presented to suggest that mitochondria
are physically interconnected and functionally homogenous. Real-time
monitoring of mitochondrial membrane potential indicated electrical continuity
across large parts of a mitochondrial network in COS-7 cells
(De Giorgi et al., 2000), human
skin fibroblasts and neonatal rat cardiac myocytes
(Amchenkova et al., 1988
).
However, using the same approach multiple individual mitochondria were
observed in adult rat cardiac myocytes
(Zorov et al., 2000
) and
various non-electrically excitable cell types
(Collins et al., 2002
).
Reconstruction of electron micrographs revealed apparent mitochondrial
networks in rat hepatocytes (Brandt et al.,
1974
) and a single large mitochondrion in yeast cells
(Hoffman and Avers, 1973
);
however, the number of mitochondria within yeast cells is thought to vary from
one to ten (Koning et al.,
1993
; Nunnari et al.,
1997
).
Evidence for the existence of a largely interconnected mitochondrial
network in HeLa cells was presented using non-confocal, deconvolution imaging
and `fluorescence-recovery-after-photobleaching' (FRAP)
(Rizzuto et al., 1998). These
authors found that the fluorescence of mitochondrially targeted green
fluorescent protein (GFP) recovered after irradiation of a subcellular region,
and suggested that this indicated continuity of the mitochondrial matrix.
However, the slow recovery of fluorescence observed by Rizzuto et al.
(1998
) is not compatible with
the rapid translocation of fluorescent proteins within the mitochondrial
matrix (Collins et al., 2002
;
Partikian et al., 1998
), and
their data are more easily reconciled with the concept of multiple physically
discrete mitochondria undergoing infrequent fusion.
Early electron microscopy revealed populations of mitochondria with
different matrix densities within single cells
(Ord, 1979;
Simon et al., 1969
), thought
to reflect differences in metabolic states
(Ord, 1979
). In cardiac cells
(Jahangir et al., 1999
) and
skeletal muscle cells (Lombardi et al.,
2000
; Battersby and Moyes,
1998
), two distinct populations of mitochondria are proposed to
exist with differing biochemical and respiratory properties. Subcellular
heterogeneity in Ca2+ sequestration by mitochondria has been
reported for pancreatic acinar cells (Park
et al., 2001
), chromaffin cells
(Montero et al., 2002
), CHO.T
cells (Rutter et al., 1996
)
and HeLa cells (Collins et al.,
2002
). Also, asynchronous permeability transition pore opening
within single cells has been demonstrated in response to oxidant stress
(Collins et al., 2002
). These
data suggest that mitochondria can behave as functionally discrete entities,
consistent with their physical segregation.
The aim of the present study was to compliment previous work
(Collins et al., 2002)
addressing the connectivity of mitochondria in living mammalian cells, using a
range of microscopic techniques. Our conclusions are that, typically,
mitochondria exist as lumenally and electrically discontinuous organelles
within cells.
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Materials and methods |
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For imaging studies, the culture medium was replaced with an extracellular medium (EM) containing (mmol l-1): NaCl, 121; KCl, 5.4; MgCl2, 0.8; CaCl2, 1.8; NaHCO3, 6.0; D-glucose, 5.5; Hepes, 25; pH 7.3. All fluorescent dyes were obtained from Molecular Probes (Oregon, USA).
Cells were transfected with mitochondrially targeted DsRed1 (mito-DsRed1) from Clontech (Palo Alto, CA, USA) with Effectene transfection reagent (Qiagen, Crawley, UK), following the manufacturers' recommended protocol.
3-D reconstruction
z-series stacks of mito-DsRed1-expressing and tetra-methyl
rhodamine ethyl ester (TMRE)-loaded cells were acquired with a Bio-Rad MRC1024
LSCM (Hemel Hempsted, UK). Subsequent image restoration was achieved with the
deconvolution software AutoDeblur (Autoquant, New York, USA) using the `Power
accelerated' blind deconvolution algorithm. Image analysis and processing was
performed with the public domain software ImageJ (NIH,
http://rsb.info.nih.gov/ij).
Single channel surface-rendered images were processed with ImageJ running the
VolumeJ plugin (M. Abràmoff;
http://www.isi.uu.nl/people/michael/vr.htm).
FRAP
For the FRAP experiments illustrated in
Fig. 2, cellular areas of
perinuclear mitochondria (typically 25100 µm2) were
bleached with a Bio-Rad MRC1024 LSCM by briefly digitally zooming into the
region of interest with an enhanced laser intensity. The bleaching procedure
was continued until the fluorescence intensity of the region being bleached
had reached zero. This typically took 515 s.
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Results |
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In all cell types, individual mitochondria were discernible in the
periphery of the cell. Both thread-like and grain-like mitochondria were
apparent. This differs from primary hepatocytes
(Collins et al., 2002) and
primary adult ventricular myocytes (Fig.
4D; Zorov et al.,
2000
), which seem to have predominantly, if not exclusively,
grain-like mitochondria.
FRAP of fluorophores in the mitochondrial matrix
Whilst individual mitochondria were clearly visible in the periphery of
cells, it was less clear whether the mitochondria in the perinuclear region
were continuous or only densely aggregated. To probe this we used the
technique of FRAP, which involved bleaching mitochondrially targeted DsRed1 in
a subcellular region using brief high-intensity illumination with the confocal
zoom increased. The bleaching procedure was continued until fluorescence in
the bleached area was reduced to zero. Any subsequent recovery of fluorescence
in the bleached region occurs due to inward diffusion of unbleached
fluorophore molecules. In HeLa cells (Fig.
2A), the fluorescence failed to significantly recover in the
centre of the photobleached area for up 1 h post irradiation. The bleached
mitochondria were still intact and functional since they loaded with TMRE, a
membrane potential-sensitive indicator, demonstrating that they were still
polarised (Fig. 2Av). DsRed1
fluorescence recovered slightly more rapidly in cortical astrocytes, where 5%
and 60% of the original fluorescence recovered after 20 min and 1 h,
respectively (Fig. 2B).
A critical control for the FRAP experiments is the demonstration that the fluorophore is sufficiently mobile to have reached the bleached cellular areas during the recovery phase. We therefore examined the rate of diffusion of DsRed1 within the mitochondrial matrix. The end portion of a clearly distinct long mitochondrion (Fig. 3A, arrowhead) was photobleached using the same protocol described earlier. Following irradiation, the rate of fluorescence recovery along the mitochondrion was measured and a linear diffusion rate for DsRed1 was estimated at approximately 1 µm s-1 (Fig. 3). DsRed1 therefore has a rapid mobility within the mitochondrial matrix.
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These data indicate that if mitochondria form a lumenally interconnected
network, DsRed1 can diffuse sufficiently fast for FRAP to be apparent within a
few minutes following photobleaching. In all the cells we have tested
(Fig. 2;
Collins et al., 2002),
irradiation of a subcellular region that wholly encompassed several
mitochondria caused a bleach that persisted for at least several tens of
minutes. This indicates a lack of mitochondrial lumenal connectivity. This
slow recovery of fluorescence in such bleached subcellular regions is due to
organelle movement or fusion of unbleached and bleached mitochondria, rather
than to diffusion of DsRed1 along interconnected mitochondria.
Mitochondria are electrically discontinuous
The electrical continuity of mitochondria was determined using the
phenomenon of irradiation-induced depolarisation. The combination of high
mitochondrial concentrations of TMRE (1 µmol 1-1 for 20 min),
plus moderate to high laser irradiation during confocal imaging, results in
the rapid depolarisation of mitochondria. This is thought to be due to the
generation of reactive oxygen species (ROS) that trigger the mitochondrial
permeability transition pore (PTP). In many cases, PTP occurs initially as
transient stochastic events, usually followed by a permanent opening.
Mitochondria that are electrically continuous show synchronous
depolarisations.
In HeLa cells, HUVEC cells, pancreatic acinar cells and adult ventricular myocytes, depolarisation of individual electrically discrete mitochondria was clearly visible (Fig. 4). The TMRE fluorescence in individual mitochondria usually flickered several times, indicative of transient PTP events. Eventually, the TMRE fluorescence was lost from individual mitochondria, presumably when an irreversible opening of the PTP occurred. The flickering and loss of TMRE florescence was randomly observed throughout the mitochondrial population of individual cells.
With HeLa cells and ventricular myocytes, the majority of mitochondria displayed transient PTP events. Within pancreatic acinar cells, rapid depolarisations were obvious in a subset of the mitochondria. A significant number showed no electrical activity, and the TMRE fluorescence in these organelles was lost due a progressive bleach of the indicator (Fig. 4Aii).
Atypical interconnected mitochondrial networks
The data presented above and in our previous study
(Collins et al., 2002), clearly
establish that mitochondria are lumenally and electrically discontinuous in
several primary cell types and cell lines. In a very few instances, however,
we have observed cells that seemed to posses a single large interconnected
mitochondrion (Fig. 5). Having
visualised mitochondria in many thousands of cells of many different types, we
have so far seen only three individual cells (one HeLa, one cortical astrocyte
and one HUVEC cell) displaying an interconnected mitochondrion. This phenotype
is striking and is readily identifiable in cells expressing
mitochondrial-specific fluorophores. In each situation where cells with an
interconnected mitochondrion were observed, it was atypical in that all the
surrounding cells had clearly discontinuous mitochondria. We would estimate
that the frequency of cells bearing an interconnected mitochondrion is
significantly less than 0.1%. Although they are extremely rare, the instances
of cells with interconnected mitochondria provide a situation where we could
examine whether mitochondria can behave as electrically continuous entities,
and if DsRed1 can diffuse throughout a large mitochondrial matrix.
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We examined the electrical continuity of the apparently interconnected mitochondrion in the HUVEC cell using the TMRE- and laser-induced PTP activation protocol described above. Essentially, we observed that the majority of the mitochondrial network flickered simultaneously throughout the cell (Fig. 5A). Note the dramatic decline in fluorescence in Fig. 5Aii and its recovery to the previous level 8 s later in Fig. 5Aiii. Eventually, after three of these depolarisation and recovery events, the TMRE fluorescence was lost following an irreversible PTP opening. Although this cell appeared to possess a largely interconnected mitochondrion, there were also a few smaller mitochondria that retained TMRE fluorescence after the large mitochondrion had irreversibly depolarised (Fig. 5Aiv).
The cortical astrocyte possessing the interconnected mitochondrion was used to examine the movement of DsRed1 within the mitochondrial matrix. Photobleaching a small region of the cell (Fig. 5Bi,ii) reduced the mitochondrial DsRed1 fluorescence within that area to negligible levels. Within 10 s of the irradiation, the fluorescence had visibly recovered within the bleached area (Fig. 5Biii), and after 2 min the fluorescence had fully recovered (Fig. Biv). Simultaneous with the recovery of fluorescence in the bleached area, the DsRed1 emission declined in the other portions of the interconnected mitochondrion, until equilibrium was established.
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Discussion |
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Individual mitochondria can be clearly seen in the three-dimensional reconstructions shown in Fig. 1. Physically isolated mitochondria are easiest to see in the periphery of cells, where they are usually sparsely arranged. The exception to this is pancreatic acinar cells, where a dense uninterrupted layer of mitochondria was found beneath the plasma membrane. Even in the case of pancreatic acinar cells, however, individual mitochondria are visible. The dense perinuclear aggregates of mitochondria in PAE cells, SH-SY5Y cells, COS-7 cells and cortical astrocytes appear as a lumenally continuous network, due to the inability to see the ends of individual organelles. However, when the lumenal continuity of this aggregation was tested using the FRAP technique, it was found to be discontinuous (Fig. 2).
The slow recovery of DsRed1 fluorescence following photobleaching of
subcellular areas in HeLa cells and astrocytes
(Fig. 2) contrasts with rapid
diffusion of this fluorophore within the mitochondrial matrix (Figs
3,
5B). We therefore consider that
the slow recovery of fluorescence is not due to lumenal tunnelling of DsRed1,
but rather to movement of mitochondria or fusion of unbleached to bleached
mitochondria. Interestingly, astrocytes and HeLa cells differed significantly
in their slow recovery from photobleaching
(Fig. 2), possibly reflecting
different mitochondrial fusion rates in these cell types. Our empirical
observations also suggest that HeLa cell mitochondria appeared to be less
mobile than astrocyte mitochondria (data not shown). Since DsRed1 is can
diffuse quickly within the mitochondrial matrix
(Fig. 3), a lumenally
continuous mitochondrial network would have recovered within 2 min
(Fig. 5B). Our measured rate of
diffusion of DsRed1 within the mitochondrial matrix (approx. 1 µm
s-1) is comparable to that of EGFP within the ER and mitochondrial
matrixes (Partikian et al.,
1998; Subramanian and Meyer,
1997
).
As previously reported, the mitochondria in HeLa cells
(Collins et al., 2002) and
adult cardiac myocytes (Zorov et al.,
2000
) were found to be electrically discontinuous
(Fig. 4B,C). In adult cardiac
myocytes the mitochondrial depolarisation events, whilst asynchronous, seemed
to occur in a wave across the cell (Fig.
4Dii). This wave was too slow to represent electrical continuity,
which rapidly spreads between interconnected mitochondria (see
Fig. 5) and is consistent with
a diffusable messenger being released upon mitochondrial depolarisation acting
to trigger depolarisation in neighbouring mitochondria. We have also seen a
similar phenomenon in primary rat hepatocytes (C.T.J. and M.D.B., unpublished
data). Most likely, the diffusable messenger is a ROS, released upon PTP
opening (Zorov et al., 2000
).
These ROS act upon neighbouring mitochondria, already under ROS stress from
TMRE irradiation, precipitating PTP opening and generation of more ROS. The
wave of depolarisation therefore represents successive PTP induction and
ROS-induced ROS release (Zorov et al.,
2000
).
In a detailed study of Ca2+ sequestration by mitochondria in
pancreatic acinar cells, it was demonstrated that the mitochondria are
segregated in three regions: sub-plasmalemmal, perigranular and perinuclear
(Park et al., 2001). We have
also visualised such sub-plasmalemmal (Fig.
1A) and perigranular (Fig.
4Ai) mitochondria. Rings of perinuclear mitochondria were not as
obvious since we did not specifically determine the location of nuclei. The
study by Park et al. (2001
)
demonstrated the lack of Ca2+ tunnelling between the mitochondria
in an individual acinar cell. We now report that the mitochondria within
pancreatic acinar cells are also electrically discontinuous
(Fig. 4A). These data,
therefore, strongly support the hypothesis that pancreatic acinar cells have
morphologically and functionally discrete mitochondria. The perigranular
mitochondria form a dense aggregation between the apical and basal poles of
the acinar cells and have been shown to limit the spread of Ca2+
waves inside hormone-stimulated cells
(Tinel et al., 1999
). The
sub-plasmalemmal mitochondria also form a dense sheet of mitochondria that
extends completely around the cells (Figs
1A,
4A1). These mitochondria may
have significant roles in controlling functions of the basal membrane in
acinar cells.
There is a striking difference in the distribution of mitochondria in
pancreatic acinar cells compared to the other cell types studied here. In most
cell types, mitochondria are found to be most densely aggregated in the cell
centre (Fig. 1) where they are
often intertwined with the most concentrated strands of the endoplasmic
reticulum (Collins et al.,
2002) or regularly spaced around myofibrils and the sarcoplasmic
reticulum in the case of striated muscle
(Fig. 4D). However, in
pancreatic acinar cells, the mitochondria appear to be sparse in the basal
region where the nucleus and rough endoplasmic reticulum is located. In a
sense, pancreatic acinar cell mitochondria appear to be largely excluded from
regions where other organelles are densely packed.
Very rarely, we have observed cells possessing mitochondria that seemingly
formed a continuous network. These cells were immediately obvious, with very
few mitochondrial `ends' visible (Fig.
5). These may be cells in which the mitochondrial fission/fusion
machinery has shifted its balance towards fusion. The mitochondrial structure
in these cells is reminiscent of COS-7 cells expressing the dominant negative
mutated Drp1 protein, Drp1-K38A, and the mitofusin protein, Mfn2
(Santel and Fuller, 2001). In
these cells, fission is inhibited by Drp1-K38A and fusion promoted by Mfn2.
The infrequency of cells with an interconnected mitochondrion suggest to us
that this phenotype is not representative of a stage in the cell cycle, as in
our asynchronous cultures we would expect a higher percentage to show such
morphology. Similarly, in a homogenous culture we would rule out a response to
an environmental signal.
Fission and fusion of mitochondria over the lifetime of a cell means that
in certain respects mitochondria will effectively act as a single continuous
network. This appears to be the case with lumenal proteins
(Nunnari et al., 1997).
Describing mitochondria as a single continuous network, however, likens them
to the ER, which is also a dynamic organelle constantly undergoing fission and
fusion (Lee and Chen, 1988
),
but unlike mitochondria, the ER is a demonstrably continuous organelle (C.T.J.
and M.D.B., unpublished data; Subramanian
and Meyer, 1997
; Terasaki et
al., 1994
), at least for the majority of interphase cells.
Clearly, mitochondria vary substantially in their morphology and distribution
between different cell types. Despite such differences, it is obvious that
unless perturbed, the balance lies in favour of fission into morphologically
and functionally distinct entities.
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Acknowledgments |
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