1 Department of NanoBiophotonics, Max Planck Institute for Biophysical
Chemistry, Am Fassberg 11, 37077 Göttingen, Germany
2 Institut für Physiologische Chemie, Universität München,
Butenandtstrasse 5, 81377 München, Germany
Author for correspondence (e-mail:
sjakobs{at}gwdg.de)
Accepted 11 February 2003
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Summary |
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Key words: Saccharomyces cerevisiae, Fluorescence microscopy, Glucose repression, Membrane fission, Organelle morphology
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Introduction |
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The dynamin-related GTPase Dnm1p is a key component of the yeast
mitochondrial fission machinery (Bleazard
et al., 1999; Otsuga et al.,
1998
; Sesaki and Jensen,
1999
). It interacts with the WD40-repeat protein Mdv1p
(Cerveny et al., 2001
;
Fekkes et al., 2000
;
Mozdy et al., 2000
;
Tieu and Nunnari, 2000
;
Tieu et al., 2002
). Dnm1p
colocalizes with Mdv1p in punctate structures on the mitochondrial outer
membrane. These structures have been proposed to mediate mitochondrial
membrane constriction and/or division. Assembly and proper distribution of
Dnm-p/Mdv1p complexes on the mitochondrial surface depend on Fis1p, an
integral component of the outer membrane
(Mozdy et al., 2000
;
Tieu and Nunnari, 2000
).
Similar to dnm1 and mdv1 mutants
(Mozdy et al., 2000
;
Otsuga et al., 1998
;
Tieu and Nunnari, 2000
),
fis1 mutant cells frequently display fenestrated mitochondria that
are often reminiscent of miniaturized fishing nets. It has been suggested that
these fenestrated mitochondria are generated because mitochondrial fission is
severely compromised in these mutants while fusion is still going on
(Jensen et al., 2000
;
Shaw and Nunnari, 2002
;
Yaffe, 1999
).
Two outer membrane proteins, Fzo1p and Ugo1p, are involved in the fusion of
mitochondria (Hermann et al.,
1998; Rapaport et al.,
1998
; Sesaki and Jensen,
2001
). Yeast mutants lacking functional Fzo1p contain fragmented
mitochondria, presumably because fusion is blocked while fission is going on.
Remarkably, double mutants of fzo1 and fis1 display tubular
or net-like mitochondria rather than fragmented organelles
(Mozdy et al., 2000
;
Tieu and Nunnari, 2000
). This
finding led to the proposal that deletion of the FIS1 gene prevents
fragmentation of mitochondria in fzo1 mutants by blocking the fission
pathway, which further supports a central role for Fis1p in mitochondrial
fission.
Mitochondria as double-membrane-bounded organelles have to fuse and divide
their inner and outer membranes in a coordinated manner
(Westermann, 2002). It has
been proposed that the outer membrane fusion machinery is in contact with yet
unknown factors in the inner membrane
(Fritz et al., 2001
). Recently,
the inner membrane protein Mdm33p has been suggested to be involved in inner
membrane fission (Messerschmitt et al.,
2003
). To date, the functional characterization of components of
the mitochondrial fission machinery in yeast has relied mostly on genetic and
biochemical data and observations of morphology mutants under steady-state
conditions. Owing to the dynamic nature of mitochondria, important aspects of
mutant phenotypes have most probably been overlooked.
Here, we report on a detailed analysis of mitochondrial dynamics resolved
in time and space, focusing on wild-type and fis1 mutant
cells. Using confocal microscopy, the dynamical behaviour of mitochondria
labelled with GFP targeted to the matrix has been followed over a time period
of up to 5 hours. To follow mitochondrial changes over time, we employed the
structural adaptations of mitochondria occurring upon the exchange of the
non-fermentable carbon source glycerol by glucose in the growth medium. The
three-dimensional confocal time-lapse data sets provide insight into the
complex structural adaptations of mitochondria, enabling us to accurately
count apparent matrix separation and fusion events. We provide evidence that
some of these matrix separations in fis1
mutants are due to
genuine tubule fissions, whereas others do not involve fission of the outer
membrane. The analysis of mitochondrial dynamics using four-dimensional
microscopy gives new insight into the function of proteins involved in
mitochondrial fission that would have been missed otherwise.
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Materials and Methods |
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Plasmid constructions
Standard cloning procedures were used
(Sambrook et al., 1989). PCR
was performed using Pfu polymerase (Stratagen, La Jolla, USA) according to the
manufacturer's instructions. For labelling the matrix with GFP the plasmid
pVT100U-mtGFP was employed. This plasmid, containing a DNA fragment encoding
GFP fused to subunit 9 (aa1-69) of the F0-ATPase of Neurospora
crassa under control of the constitutive alcohol dehydrogenase promoter,
is described elsewhere (Westermann and
Neupert, 2000
). The plasmid pHS12-DsRed.T4 encoding pCox4-DsRed
fusion proteins is published elsewhere
(Bevis and Glick, 2002
). To
label the outer membrane with GFP, pAS43 was constructed. This vector, a
2µ-URA3 plasmid that expresses OM45p-GFP
(Cerveny et al., 2001
), was
constructed as follows. A 1223 base pair (bp) DNA fragment encoding the OM45
ORF and 41 bp of upstream sequences was amplified from yeast genomic DNA using
the oligonucleotides 5'-GCGAAGCTTGGCCAGTAACGTTAATCA3' and
5'-GCGGGTACCGTCCTTTTTCGAGCTCCA-3'. The PCR fragment was digested
with HindIII and KpnI and inserted into pVT100U-mtGFP
(Westermann and Neupert, 2000
)
replacing the Su9 presequence. To label the matrix with DsRed the plasmid
pSJ55 was constructed. For this vector the coding sequence of DsRed.4 was
amplified by PCR using pHS12-DsRed.T4
(Bevis and Glick, 2002
) as a
template. The PCR fragment was inserted into pYX142-mtGFP
(Westermann and Neupert, 2000
)
replacing the DNA sequence encoding GFP, resulting in a vector encoding
Su9(1-69)-DsRed.4 under control of the constitutive triosephosphate isomerase
promoter (TPI). Finally the TPI-Su9(1-69)-DsRed.4 cassette was inserted into
the SmaI restriction site of the 2µ-based vector pRS323
(Sikorski and Hieter, 1989
),
resulting in pSJ55.
Beam-scanning confocal microscopy
For Fig. 1A-C,
Fig. 2A-C and
Fig. 3A-C, cells were collected
by centrifugation, resuspended in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM
Na2HPO4, 1.4 mM KH2PO4, pH 7.3)
and immobilized in PBS with 1% low melting point agarose on a microscope
slide. For Fig. 3D-J cells were
grown in PMGal medium to mid-log phase and placed in a chamber as described
for time-lapse confocal microscopy (see below). For image acquisition a
beam-scanning microscope (Leica TCS SP2, Leica Lasertechnik, Heidelberg,
Germany) equipped with a 1.4 numerical aperture oil immersion lens (Leica
100X, Planapo, Wetzlar, Germany) was employed. GFP- and DsRed-expressing cells
were imaged as described previously
(Jakobs et al., 2000). All
imaging was performed at ambient temperature (
22°C).
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Time-lapse confocal microscopy
Cells expressing mtGFP were grown in YPGlycerol to mid-log phase and
subsequently transferred to YPD. Following further incubation in YPD at
30°C (45 minutes, wildtype or 70 minutes, deletion mutants) cells were
harvested. They were embedded in a solution of one part YPD with two parts PBS
and 1% low melting point agarose and filled in a microscopic chamber. For the
duration of the experiment, the chamber was constantly flushed with YPD
(100 µl/minute) and kept at ambient temperature. Images were acquired
digitally with a multi-focal single-photon excitation confocal system
(UltraView confocal scanner; PerkinElmer, Boston, USA) attached to an inverted
Leica DMIRBE microscope. Three-dimensional image data of the mitochondrial
compartment were acquired at each time point. The three-dimensionality of the
data permitted the recognition of matrix separation and fusion events, some of
which would have been missed otherwise (in case of separation) or spuriously
included (in case of fusion). Data collection was carried out with a
100x/1.40 oil immersion lens (Leica, Planapo, Wetzlar, Germany) and a
cooled CCD camera (Imager, LaVision, Göttingen, Germany). The shutters,
stage motion and image acquisition were computer controlled. Three-dimensional
stacks of mitochondria were acquired by recording focal plane images (xy) and
moving the stage in 0.25 µm steps along the optic axis. Three dimensional
datasets of mitochondria from fis1
and dnm1
cells were deconvolved by non-linear image restoration using a maximum
likelihood algorithm (Lucy,
1974
; Nagorni and Hell,
2001
; Richardson,
1972
). In addition to providing a slightly higher resolution this
algorithm is particularly suitable for disproving disconnections in
mitochondrial tubules with weak GFP fluorescence. For collecting the images of
wild-type mitochondria we used an exposure time of 550 milliseconds; for the
deletion mutants the exposure time was 1000 milliseconds. Quadratic focal
plane pixel areas were 0.04 µm2 and 0.01 µm2 for
wild-type and mutant cells, respectively. Individual stacks were recorded
every 78 seconds (wild-type cells), 116 seconds (fis1
) or 60
seconds (dnm1
). For presentation, maximum intensity
projections were generated, that is, optical planes were added along the optic
axis. With the exception of contrast stretching, no further image processing
was employed.
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Results |
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90% of the mitochondria of wild-type cells grown on glycerol were highly
branched, whereas on glucose more than 90% of the organelles were simple
networks (Table 1). In contrast
to fis1 cells, the structure of wild-type mitochondria was
largely homogenous on the same carbon source.
Carbon-source-dependent simplification of mitochondria is delayed in
fis1 cells
As fis1 mutants have been proposed to be deficient in
mitochondrial fission, it was surprising to discover similar
carbon-source-dependent morphology adaptation processes in
fis1
and wild-type mitochondria. To identify a possibly subtle
effect of a lack of Fis1p on the restructuring process of the organelle, the
adaptation process of mitochondria was followed over time. Unsynchronised
cultures of wild-type cells were grown to mid-log phase in glycerol-containing
medium, transferred to glucose-containing medium and mitochondrial networks
were examined by conventional fluorescence microscopy at various time points
(Table 2). As shown above, in
glycerol-containing medium, the vast majority of wild-type mitochondria could
be attributed to the highly branched mitochondria type. After merely 1 hour in
glucose-containing medium, only about one third of the cells contained a
highly branched mitochondrial network. After three hours the share of highly
branched networks was less than 10% (Table
2).
|
A corresponding analysis of the fis1 strain was performed
(Table 2). For the analysis the
same categories of fis1
mitochondria as above were used. In
contrast to the wild-type situation the distribution of mitochondrial
morphologies within the fis1
population remained almost
unchanged for two hours after transfer to glucose. However, after four hours,
more than half of the large fenestrated nets had been converted to more simple
structures. Hence, structural adaptation of the mitochondrial compartment
induced by a change of the carbon source is markedly delayed in cells lacking
Fis1p.
Temporal dynamics of the restructuring process of the wild-type
mitochondrial network
To obtain three-dimensional information on the dynamics of the
mitochondrial compartments we employed confocal time-lapse microscopy.
Wild-type cells harbouring mitochondrial matrix-targeted GFP were grown to log
phase in glycerol-containing medium, transferred to glucose-containing medium
and incubated for an additional 45 minutes. After this time, the majority of
mitochondrial networks were expected in the initial phase of the restructuring
process (compare Table 2). For
all experiments described in this study the cells were kept in a microscope
chamber at ambient temperature. The chamber was continuously flushed with
medium to ensure a constant nutrient supply. Since we employed matrix-targeted
GFP, our observations of the mitochondrial networks were restricted to the
observation of the matrix compartment. In this context we use the term
`separation' to describe an apparent discontinuity of the GFP-labelled matrix
compartment. Such a separation could be due to a local matrix constriction, a
fission of the inner membrane or a fission of both inner and outer membranes.
We use the term `tubule fission' to describe complete fission of both
membranes.
To follow the restructuring process we chose wild-type cells displaying highly ramified mitochondrial morphologies. We followed shape changes in the mitochondrial network in a total number of 15 wild-type cells. Nine of these cells budded during the imaging period. In each of these dividing cells we observed an unambiguous simplification of the mitochondrial network. The remaining six cells did not bud during the observation period. In spite of ongoing matrix separations and fusions, mitochondria of these cells did not simplify. The latter observation suggests a connection between organellar remodelling events and the cell cycle in wild-type cells.
The overall pattern of mitochondrial metamorphoses was similar in all examined budding wild-type cells. Representative cells imaged at various intermediate stages of the process are displayed in Fig. 1A-C. A newly formed daughter cell was always invaded by a mitochondrial tubule shortly after emergence of the bud. The invading tubule appeared to move without restrictions within the bud for about 10-15 minutes. This was followed by attachment to one site of the cortex of the daughter cell. During subsequent outgrowth of the bud the mitochondrion remained attached to this point, whereas the remaining mitochondrial tubules continued to move freely. Tubules connecting the mitochondrial compartments of mother and daughter cells frequently underwent fusion and fission (Fig. 1D-I, Movie 1and Movie 2, available at jcs.biologists.org/supplemental). The amount of mitochondrial tubules in the bud appeared to increase after each fission event, suggesting that mitochondrial tubules were efficiently transported from the mother into the daughter cell. Movement of mitochondria into the bud was paralleled by ongoing matrix separation and fusion in the mother cell. In addition, numerous tubule fissions that were followed by fusion at a different site were recorded (e.g. Fig. 1D-F, upper arrows). It is conceivable that the latter separation and fusion events were responsible for simplification of the network within the mother cell.
Remodelling of the fenestrated fis1 mitochondrial
network is accompanied by mitochondrial tubule fusion and fission
To analyse whether tubule fission in mutants lacking Fis1p occurs upon
glucose repression as seen in wild-type cells, we used the same experimental
set-up as described above.
For time-lapse analysis we chose cells that displayed large fenestrated
nets. We followed mitochondrial shape changes in a total number of 17
fis1 cells, some of which were observed for more than 3 hours.
In this context we will use the term `simplification' to describe a size
reduction of the fenestrated fis1
net concomitant with an
enlargement of long tubules connected to the net. Three of the observed cells
budded during the imaging period and simultaneously simplified the
mitochondrial compartment. Seven cells displayed a marked simplification of
the mitochondrial network without any signs of budding. Hence in cells lacking
Fis1p simplification appears to be partly disconnected from the cell cycle. In
case of budding, invasion of mitochondria into the bud
(Fig. 2A-C) followed a pattern
similar to that of wild-type cells. An emerging bud was invaded by a
mitochondrial tubule that remained connected to the fenestrated net of the
mother cell. Later, the tubule was attached to one point at the cell cortex of
the bud where it remained fixed during outgrowth of the daughter cell.
The simplification process upon glucose repression followed a similar
pattern in all examined fis1 cells, irrespective of budding
(Fig. 2D-I, Movie 3 and Movie
4, available at
jcs.biologists.org/supplemental).
Surprisingly, we observed frequent separation and fusion events of the
GFP-labelled matrix. In many cases matrix separations were followed by fusions
occurring at the same site of the organelle
(Fig. 2G, lower arrow). We also
recorded some tubule fission events that were either followed by fusion events
occurring at a different site (Fig.
2G-I, upper arrows) or that were not followed by refusion at all
(Fig. 2D). Separation and
fusion events were also detected within the core of the fenestrated nets
(Fig. 2E).
Matrix separation can occur in the absence of outer membrane fission
in fis1 mutants
We assumed that separations followed by re-fusion were due to a
constriction of the matrix or fission of the inner membrane without involving
fission of the outer membrane. To test this hypothesis we labelled the outer
mitochondrial membrane with GFP fused to the C-terminus of the mitochondrial
outer membrane protein OM45 (OM-GFP)
(Cerveny et al., 2001) and
simultaneously expressed DsRed fused to the presequence of subunit 9 of the
F0-ATPase of Neurospora crassa. This presequence targets
DsRed to the mitochondrial matrix (data not shown). Mitochondria of cells
grown in medium containing galactose as a carbon source were imaged. Generally
we found a strict colocalization of the OM-GFP and the DsRed matrix label.
However, occasionally we observed continuous tubules as proven by the OM-GFP
label, which enclosed separated matrix compartments
(Fig. 3A-C). This finding
demonstrates that a separation of the matrix without fission of the outer
membrane can occur in fis1
mutants. Hence, matrix separation
and outer membrane fission appear to be separable processes. We note that the
term matrix separation does not necessarily imply a fission of the inner
membrane. A matrix separation could also be the result of a mere matrix
constriction with a continuous inner membrane.
Complete tubule fission is not abolished in fis1
mutants
Tubule fissions that were not followed by fusion demonstrated that complete
mitochondrial fissions occur in fis1 mutants
(Fig. 2). To validate this
finding we employed mitochondria labelled at the outer membrane with OM-GFP
and at the inner membrane with DsRed fused to the presequence of Cox4p
(Bevis and Glick, 2002
).
Biochemical analysis revealed that the latter fusion protein is directed to
the inner membrane (data not shown). In time-lapse series of galactose-grown
cells double-labelled with these constructs, we recorded simultaneous
separations of the outer and the inner membrane
(Fig. 3D-J). This demonstrates
that in the absence of Fis1p complete tubule fissions involving both membranes
can occur.
The frequency of matrix separation and fusion events is similar in
fis1 and wild-type mitochondria
Next, we counted the number of separation and fusion events in single
wild-type and fis1 cells during glucose-repression-induced
remodelling of the organelle. We recorded three-dimensional stacks every 78 or
116 seconds (wild-type or fis1
cells, respectively). By
scrutinizing each optical section within a three-dimensional stack consisting
of about 20-25 single optical sections, we recorded all recognizable matrix
separation and fusion incidents in the mother cells. As we might have
overlooked some events, especially in the core of fenestrated
fis1
mitochondria, we might have slightly underestimated the
frequency of separation and fusion.
We performed a detailed analysis based on the data stacks of a wild-type
cell and a fis1 cell depicted in
Fig. 1D-I and
Fig. 2D-I. For both cells, the
ratio of matrix separation to fusion was balanced. We counted 104 separation
and 103 fusion events in the wild-type cell, and 85 separation and 80 fusion
events in the fis1
cell over a time frame of 105 minutes
(Fig. 4A). In the wild-type
cell the number of matrix separation and fusion events per minute decreased
from 2.5 to 0.6 during remodelling. Similarly, the frequencies of events in
the mitochondrial compartment of the cell lacking Fis1p decreased from 2.4 to
1.2 separation and fusion events per minute during simplification
(Fig. 4A).
|
To verify whether the analysed cells were representative we counted
separation and fusion events in three additional cells at characteristic time
periods during glucose adaptation (Fig.
4B,C). Similar numbers of separation and fusion events were
counted; the frequencies of separation and fusion decreased during
simplification in the analysed wild type and fis1 cells upon
change of the carbon source. This suggests that a decrease in the number of
matrix separation and fusion events is a characteristic process during the
simplification of mitochondrial networks. The frequency of separation and
fusion remained constant at about 1.7 events per minute in mitochondria of
cells that were continuously kept on glucose-containing medium
(Fig. 4B).
Mitochondrial tubule fusion and fission is reduced in the dnm1
mitochondrial network
Our data demonstrate that the ability of mitochondria of
fis1 cells to sever inner and outer mitochondrial membranes is
not completely absent. The GTPase Dnm1p has been suggested to act as a key
component of the mitochondrial fission machinery
(Bleazard et al., 1999
;
Otsuga et al., 1998
;
Sesaki and Jensen, 1999
).
Dnm1p complexes present on mitochondrial tubules are reduced in
fis1
cells but not completely absent
(Mozdy et al., 2000
;
Tieu and Nunnari, 2000
). Thus,
it seemed possible that residual Dnm1p-containing complexes were responsible
for the observed tubule fission activity during remodelling of
fis1
mitochondria.
To verify this hypothesis, we analysed mitochondria of cells lacking Dnm1p
for their ability to separate their matrix. The same experimental conditions
were employed as for the analysis of mitochondria of wild-type and
fis1 cells. Confocal time-lapse microscopy following glucose
repression revealed that mitochondria of dnm1
cells perform
matrix separations and fusions (Fig.
5A-C, Movie 5). In contrast to mitochondria of
fis1
mutants, separation was apparently always followed by a
fusion event at the same site in dnm1
mitochondria (see Movie
5). Intriguingly, we did not observe the generation of free mitochondrial tips
by fission. Although we cannot rule out rare tubule fission events, complete
mitochondrial division appears to be severely hampered in dnm1
cells. We conclude that the matrix can still be separated in
dnm1
mutants; however, unlike Fis1p, the presence of Dnm1p is
apparently essential for complete mitochondrial tubule fission.
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Discussion |
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Upon glucose repression in wild-type cells the size reduction of the mitochondrial network occurs concomitantly with the budding of the cell. It is conceivable that an important part of the size reduction of the mitochondrial compartment is due to a partitioning of the mitochondrial tubules between mother cell and bud. Although this does not preclude additional mechanisms like autophagy from being involved in the size regulation of mitochondrial networks.
The dynamin-like GTPase Dnm1p is another key component of mitochondrial
fission. It is conceivable that the tubule fission activity in
fis1 mutants is due to Dnm1p-containing complexes, which are
still able to assemble on mitochondria lacking Fis1p, albeit at a reduced
number and with an altered distribution
(Mozdy et al., 2000
;
Tieu and Nunnari, 2000
;
Tieu et al., 2002
). Indeed, we
were not able to identify unequivocally any tubule fission events in cells
lacking Dnm1p. In contrast to cells lacking Fis1p, virtually all matrix
separation events are followed by re-fusion in dnm1
mutant
cells. This suggests that Dnm1p might be essential for outer membrane fission.
However, as proven by the observed matrix separations in dnm1
mutants, constriction of the mitochondrial matrix or fission of the
inner membrane occurs in mitochondria lacking Dnm1p. A similar
observation has been made in Caenorhabditis elegans mutants, which
expressed dominant interfering mutant versions of DRP-1, a homolog of yeast
Dnm1p. These worms harboured mitochondria with separated matrix compartments
that were still connected by the outer membrane, presumably because inner
membrane fission or matrix constriction persisted in the
absence of outer membrane fission
(Labrousse et al., 1999
). Our
observations support the idea that constriction of the matrix space and/or
fission of the inner membrane can occur in the absence of outer membrane
fission activity. This suggests that in S. cerevisiae matrix
constriction is a prerequisite for tubule fission and that these two processes
are mediated by independent molecular machineries.
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Acknowledgments |
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Footnotes |
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