Institut für Physiologische Chemie der Universität München, Butenandtstr. 5, D-81377 München, Germany
* Author for correspondence (e-mail: benedikt.westermann{at}bio.med.uni-muenchen.de )
Accepted 20 February 2002
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Summary |
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Key words: Green fluorescent protein, Microtubules, Mitochondria, Neurospora, Organelle biogenesis
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Introduction |
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Transport of mitochondria has been most extensively studied in the budding
yeast Saccharomyces cerevisiae. In this organism, the actin
cytoskeleton is of major importance for mitochondrial movement, whereas the
microtubule system is not involved
(Hermann and Shaw, 1998). In
contrast, mitochondria are associated with microtubules in mammalian cells
(Heggeness et al., 1978
) and
transport depends on kinesin-like motor proteins in Drosophila and in
mice (Nangaku et al., 1994
;
Pereira et al., 1997
;
Tanaka et al., 1998
).
Similarly, cytoplasmic microtubules are required for mitochondrial
distribution in the fission yeast Schizosaccharomyces pombe
(Yaffe et al., 1996
). It was
shown that mitochondrial movement in the filamentous fungus Neurospora
crassa is a microtubule-dependent process
(Steinberg and Schliwa, 1993
)
and that disruption of microfilaments has no effect on mitochondrial
distribution and morphology (Prokisch et
al., 2000
). This, combined with its distinct genetic and
biochemical advantages, makes Neurospora an ideal model organism to
study microtubule-dependent organelle transport processes.
In a pioneering study, Steinberg and Schliwa observed vectorial saltatory
movement of mitochondria with a mean velocity of 1.4 µm/second in
Neurospora hyphae, protoplasts, cell fragments and a cell wall-less
mutant. In all cell systems tested, disruption of microtubules with nocodazole
impaired directional mitochondrial motility
(Steinberg and Schliwa, 1993).
The molecular components mediating mitochondrial movement in
Neurospora are not known. Nkin, a distantly related member of the
`conventional' kinesin family (Steinberg
and Schliwa, 1995
), is not involved in movement and positioning of
mitochondria (Seiler et al.,
1997
). Recently, we described the outer membrane protein MMM1, a
component required for mitochondrial morphogenesis in Neurospora
(Prokisch et al., 2000
).
However, it was not known whether MMM1 was required for an interaction of
mitochondria with microtubules.
Previous work using Neurospora to study mitochondrial behaviour
relied on the use of electron microscopy
(Alberghina et al., 1974;
Grad et al., 1999
;
Hawley and Wagner, 1967
),
computer-enhanced video microscopy
(Steinberg and Schliwa, 1993
)
or fluorescent dyes (Minke et al.,
1999
; Prokisch et al.,
2000
). In recent years, the green fluorescent protein (GFP) from
the jellyfish Aequorea victoria has been developed as a vital marker
for the specific labelling of intracellular structures
(Tsien, 1998
). Although
labelling of mitochondria by expression of mitochondria-targeted GFP is a
straight forward approach in many experimental systems, including mammalian
cells and yeast (Rizzuto et al.,
1995
; Westermann and Neupert,
2000
), expression of GFP turned out to be much more difficult in
other organisms including many plants, fungi and protozoa (e.g.
Fernandez-Abalos et al., 1998
;
Haseloff et al., 1997
;
Hauser et al., 2000
;
Spellig et al., 1996
). Only
very recently could expression of GFP be demonstrated in Neurospora;
however, GFP labelling of specific subcellular structures was never reported
(Freitag et al., 2001
).
We constructed a mitochondria-targeted GFP (mtGFP) and used this protein to examine the behaviour of mitochondria in vivo and in vitro and to show directly binding of isolated mitochondria to microtubules. Furthermore, we characterised the biochemical basis of the interaction of mitochondria with microtubules in vitro. Interaction of mitochondria is ATP-sensitive and depends on peripherally associated mitochondrial proteins. MMM1 is not required for this process, ascribing to this component a more general role for mitochondrial morphogenesis.
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Materials and Methods |
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The mgfp5 allele (Siemering et
al., 1996) (GenBank U87973) was amplified by PCR using primers
GFP-N (5' CGG GTA CCA GAT CTA TGA GTA AGG GTG AAG AAC TTT TC) and GFP-C
(5' CGG AAT TCT TAT TTG TAT AGT TCA TCC) and cloned into the
KpnI/EcoRI sites of pGEM3Su9(1-69)
(Rapaport et al., 1998
)
yielding plasmid pGEM3Su9(1-69)-GFP5. A BamHI/EcoRI fragment
containing the GFP-coding region plus some upstream restriction sites was
transferred to vector pBluescript KS(-) (Stratagene) yielding plasmid
pBS-GFP5. The promoter and presequence-coding region of the atp-1
gene (Bowman and Knock, 1992
)
(GenBank M84191) was amplified by PCR from genomic Neurospora DNA
using primers F1a1 (5' AAA TCT AGA GAT ATC TTG GAA CGG CCC GG) and F1a2
(5' AAA GGA TCC GGC GTA GGT GCG GGC CTG) and cloned into the
XbaI/BamHI sites of pBS-GFP5 yielding plasmid pBS-mtGFPa.
The 3' coding and terminator region of the atp-1 gene was
amplified by PCR from genomic Neurospora DNA using primers F1a3
(5' AAA AGT CGA CGT GGT GAG CGT GTA AGT GC) and F1a4 (5' AAA GGG
CCC TAC TGT GAT CCG CAA ATT CAG) and cloned into the
SalI/ApaI sites of pBS-mtGFPa yielding plasmid pBS-mtGFPb.
Finally, a hygromycin-resistance-conferring cassette was amplified by PCR from
plasmid pCB1179 (Sweigard et al.,
1997
) using primers NotI-Hyg (5' AAA GCG GCC GCA
GGG AAT AAG GGC GAC ACG G) and Hyg-NotI (5' AAA GCG GCC GCT GCC
GAT TTC GGC CTA TTG G) and cloned into the NotI site of pBS-mtGFPb
yielding plasmid pNc-mtGFP.
Neurospora genetic methods and isolation of
mitochondria
Neurospora wild-type strain was St. Lawrence 74A (Fungal
Genetics Stock Center, Kansas City, KS), and mmm-1 mutant strain was
mmm-1RIP23 (Prokisch
et al., 2000). Standard genetic and microbiological techniques
were used for the growth and manipulation of Neurospora strains
(Davis and de Serres, 1970
).
Neurospora was grown at 25°C in Vogel's minimal medium under
continuous aeration and illumination with white light, with the exception that
cultures for isolation of GFP-labelled mitochondria were grown in the dark in
order to avoid extensive photobleaching. Transformation of Neurospora
was carried out as described (Staben et
al., 1989
; Vollmer and
Yanofsky, 1986
). Selection of hygromycin-resistant strains was on
Vogel's minimal medium supplemented with 150 µg/ml hygromycin B
(Boehringer, Mannheim, Germany). Isolation of microconidia was according to
published procedures (Ebbole and Sachs,
1990
). Mitochondria were isolated by differential centrifugation
as described (Sebald et al.,
1979
).
Preparation of microtubules
Preparation of tubulin from porcine brain was according to published
procedures (Williams and Lee,
1982). To prepare microtubules, 1 mg tubulin was thawed on ice and
centrifuged for 10 minutes at 250,000 g in a Beckman TLA 100 rotor at
4°C. The supernatant was transferred to a new centrifuge tube,
supplemented with 1 mM GTP and incubated for 12 minutes at 37°C. After
addition of 20 µM taxol (Molecular Probes, Eugene, OR) to stabilise
microtubules, polymerisation was continued for 30 minutes. Microtubules were
purified by sedimentation through a sucrose cushion (10 mM MOPS, pH 7.2, 1 mM
EDTA, 1 mM PMSF, 4 mM MgCl2, 40% w/v sucrose) by centrifugation for
10 minutes at 250,000 g in a Beckman TLA 100 rotor at 25°C.
Pelleted microtubules were washed carefully and resuspended in buffer A (10 mM
MOPS, pH 7.2, 1 mM EDTA, 1 mM PMSF, 4 mM MgCl2, 250 mM sucrose)
supplemented with 20 µM taxol. For rhodamine-labelled microtubules, tubulin
was mixed with rhodamine-labelled tubulin (a kind gift of Günther
Woehlke, Universität München) in a ratio of 5:1 and polymerisation
was performed as above.
Incubation of microtubules with mitochondria for fluorescence
microscopy
Rhodamine-labelled microtubules (1 mg/ml) were incubated together with
GFP-labelled mitochondria (1 mg/ml) for 15 minutes on ice in buffer A (see
above) in the presence of 20 µM taxol and 20 U/ml apyrase. Samples were
examined directly by fluorescence microscopy.
Floatation of microtubules with mitochondria
For pretreatment of mitochondria with high salt concentration, isolated
organelles were incubated for 10 minutes on ice in buffer A (see above)
supplemented with 1 M KCl. Then, organelles were pelleted by centrifugation
for 8 minutes at 12,000 g, washed twice with SEM (250 mM sucrose, 1
mM EDTA, 10 mM MOPS, pH 7.2) and resuspended in SEM at a final concentration
of 10 mg/ml. For pretreatment of mitochondria with protease, isolated
organelles were incubated for 20 minutes on ice in buffer A supplemented with
100 µg/ml trypsin (PMSF was omitted). Protease treatment was stopped by the
addition of 1 mg/ml soybean trypsin inhibitor (STI) and 15 minutes incubation
on ice. Then, mitochondria were washed and resuspended as above.
To allow binding of mitochondria to microtubules, 40 µg mitochondria were incubated with 40 µg taxol-stabilised microtubules in 250 µl buffer A supplemented with 20 µM taxol and 20 U/ml apyrase and incubated for 20 minutes on ice (for trypsin-pretreated mitochondria 0.5 mg/ml STI were added). To study adenine nucleotide-dependent interactions, 2 mM ATP or ADP were supplemented instead of apyrase.
For floatation, samples were adjusted to a sucrose concentration of 1.8 M by addition of 650 µl 2.5 M sucrose and loaded at the bottom of a SW60 ultracentrifugation tube. This was overlayed with 2 ml buffer A adjusted to 1.7 M sucrose and 1 ml buffer A. The gradient was centrifuged for 4 hours at 200,000 g in a Beckman SW60 rotor at 4°C. After centrifugation, a 900 µl fraction from the upper part of the gradient was removed. Four fractions of 0.5 ml and one fraction of 1 ml were harvested, and sedimented material at the bottom of the tube was resuspended in SEM. Proteins were precipitated with TCA and analysed by Western blotting. Floatation of mitochondria was controlled using a polyclonal antibody against porin, whereas floatation of microtubules was detected using monoclonal anti-tubulin antibody WA3 (kindly provided by Ursula Euteneuer and Manfred Schliwa, Universität München). Signals were quantified by densitometry (Pharmacia ImageScanner with Image Master 1D Elite software package, Amersham Bioscience, Uppsala, Sweden).
Miscellaneous
Standard fluorescence and phase contrast microscopy and processing of
images were performed as described
(Prokisch et al., 2000).
SDS-PAGE and blotting of proteins to nitrocellulose were performed according
to standard methods. The ECL detection system (Amersham Pharmacia Biotech,
Uppsala, Sweden) was used for western blotting.
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Results |
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It was reported that mRNA transcribed from the wild-type GFP coding region
of Aequorea victoria is aberrantly spliced in Arabidopsis.
Successful GFP expression in plants was only achieved with a gene that was
optimised to the codon usage of Arabidopsis, whereby the cryptic
intron was removed (Haseloff et al.,
1997). Arabidopsis-optimised alleles were used for
expression of GFP in the basidiomycete fungus Ustilago maydis
(Spellig et al., 1996
) and the
filamentous ascomycete fungus Aspergillus nidulans
(Suelmann et al., 1997
). We
chose the mgfp5 allele (Siemering
et al., 1996
) to construct a mitochondria-targeted GFP for
expression in Neurospora. In addition to altered codons, this variant
contains some amino acid exchanges that improve protein folding at elevated
temperatures.
We reasoned that optimal expression of GFP will be best achieved if all
regulatory elements are derived from a highly expressed Neurospora
gene. The subunit of the F1 ATP synthase is an abundant
protein of mitochondria. In Neurospora it is encoded by the nuclear
atp-1 gene (Bowman and Knock,
1992
). The protein is synthesized in the cytosol as a precursor
with a cleavable presequence that directs it to the mitochondrial matrix
space. The coding sequence is interrupted by five introns, one of which is
located within the region coding for the presequence. Several reports propose
that introns may have an expression-augmenting activity (e.g.
Brinster et al., 1988
;
Buchman and Berg, 1988
;
Choi et al., 1991
;
Korb et al., 1993
;
Palmiter et al., 1991
).
Therefore we decided to include the first and fifth intron of atp-1
in the chimeric gene.
A DNA fragment covering 1130 bp of the atp-1 upstream region and
the first 40 codons including intron 1 were fused in frame with the
gfp5 coding region including a stop codon. At the 3' end we
added a DNA fragment covering the last 91 codons including intron 5 and 441 bp
of the terminator region. The encoded protein, mtGFP, consists of the
F1 presequence (including the processing site for the matrix
processing peptidase), two amino acids of the mature F1
protein and seven additional amino acids (present due to cloning reasons),
fused to GFP. The mtGFP expression cassette was combined with a cassette
conferring resistance to hygromycin B. The resulting plasmid,
pNc-mtGFP, is shown in Fig.
1.
|
Expression of mitochondria-targeted GFP in Neurospora
Upon transformation of pNc-mtGFP into Neurospora crassa
wildtype, 27 hygromycin B-resistant strains were isolated. Mycelia of 25
transformants exhibited significant green fluorescence. As expected,
fluorescent staining was restricted to mitochondria, virtually without
background staining. However, for unknown reasons only a small fraction of
cells contained fluorescent mitochondria. One strain that showed intensely
labelled mitochondria in single hyphae was selected for further analysis. In
order to enrich the cells that expressed the mtGFP construct, this strain was
passaged seven times on hygromycin B-containing medium. It should be noted
that repeated passage on selective media also enriched the fraction of
mtGFP-expressing cells in other transformants. After this procedure, the
majority of cells contained green mitochondria. We reasoned that most of the
progeny that germinated from conidia of a strain that was sufficiently
enriched in mtGFP-expressing cells should also express mtGFP. To obtain a
homokaryotic strain (i.e. a strain containing only a single type of nuclei),
microconidia were isolated at this stage and germinated on hygromycin
B-containing medium. This step resulted in strains containing fluorescent
mitochondria in more than 90% of the cells. Specific labelling of mitochondria
was observed at all stages of the asexual life cycle. Mitochondria were
observed as small thread-like organelles in conidia
(Fig. 2A) and newly germinated
conidia (Fig. 2B), as well as
in hyphal tips (Fig. 2C), old
hyphae (Fig. 2D) and at hyphal
branch points (Fig. 2E).
|
Behaviour of isolated mitochondria
In order to examine the behaviour of isolated organelles, we prepared
mitochondria from the mtGFP-expressing Neurospora strain. Isolated
mitochondria were small spherical organelles of about 0.5 µm diameter or
less (Fig. 3A). In contrast to
mitochondria observed in vivo (see above), the diameter of the isolated
organelles was rather diverse, but all organelles were spherical, indicating
that the isolation procedure resulted in a loss of structural features. Under
the conditions used, no co-isolated tubulin could be detected by Western
blotting (not shown), which indicates that the interaction of mitochondria
with the cytoskeleton was lost upon isolation. We consider it likely that this
is the reason for a collapse of the thread-like organelles seen in vivo to
round spherical structures. When rhodamine-labelled microtubules were added,
specific binding of mitochondria was observed
(Fig. 3B), indicating that
isolated mitochondria retain their ability to interact with the cytoskeleton
without the requirement of soluble cytosolic factors.
|
Interaction of mitochondria with microtubules in vitro
To define the biochemical basis of mitochondrial binding to microtubules we
developed an assay that allows this interaction to be monitored in vitro.
Isolated organelles were incubated with taxol-stabilised porcine brain
microtubules and then floated in a sucrose density gradient. In the absence of
ATP, a significant amount of microtubules floated together with mitochondria
to the top of the gradient, which indicates binding to the organelle
(Fig. 4A). Under these
conditions, 40-60% of the recovered tubulin fractionated with mitochondria, as
determined by quantification of western blot signals
(Fig. 4B). Binding of
mitochondria to microtubules could not be observed when the organelles were
pretreated with protease (Fig.
4B), indicating that the interaction is mediated by proteins on
the organellar surface. Similarly, mitochondria that were pre-treated with a
high-salt wash and then incubated with microtubules under standard (i.e.
low-salt) conditions did not show stable binding to microtubules
(Fig. 4B). This suggests that
proteins peripherally associated with the mitochondrial outer membrane are
required. Addition of cytosol or dialysed salt extract did not restore binding
of salt-washed mitochondria to microtubules (not shown), suggesting that the
proteins mediating this interaction are not cycling on and off the organelle.
Also, the addition of ATP- or ADP-dissociated microtubules from mitochondria
(Fig. 4B) suggesting an
involvement of adenine nucleotide-dependent factors. We conclude that the
binding of mitochondria to microtubules is mediated by ATP-dependent proteins
that are peripherally associated with the organellar surface.
|
Interaction of mmm-1 mutant mitochondria with
microtubules
MMM1 is integral to the mitochondrial outer membrane. Null mutants in yeast
and Neurospora exhibit giant mitochondria that are largely immotile
(Boldogh et al., 1998;
Prokisch et al., 2000
). It has
been proposed that the Mmm1 protein of yeast is required for coupling of the
organelle to the actin cytoskeleton
(Boldogh et al., 1998
). We
asked whether Neurospora mitochondria lacking MMM1 retain their
ability to interact with microtubules. Mitochondria isolated from an
mmm-1 mutant strain were incubated with microtubules and floated in a
sucrose density gradient as above. Microtubules were observed to bind to the
organelles in an ATP-dependent manner similar to wild-type mitochondria
(Fig. 5). We conclude that the
interaction of mitochondria with microtubules is independent of MMM1.
|
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Discussion |
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Our results indicate that binding of mitochondria to microtubules depends
on peripherally associated organellar proteins and is sensitive to adenine
nucleotides. It does not require binding of soluble cytosolic factors to the
organelle. This interaction might be mediated by a microtubule-dependent motor
protein, such as a kinesin family member or a dynein-related protein. Also GTP
and GDP were observed to dissociate microtubules from mitochondria in the
binding assay (not shown). This can be explained by the fact that kinesin
proteins are capable of using different nucleotides, as demonstrated, for
example, by in vitro microtubule gliding assays
(Steinberg and Schliwa, 1996).
Alternatively, this observation may point to an involvement of GTPases that
might regulate organelle binding to the cytoskeleton. Homology searches on the
completely sequenced Neurospora genome (Neurospora
Sequencing Project, Whitehead Institute/MIT Center for Genome Research;
www-genome.wi.mit.edu
) reveal the existence of at least 10 putative kinesin-related proteins (F.F.
and B.W., unpublished). We consider it likely that one of these predicted
proteins is involved in mitochondrial transport, as it was demonstrated for
the kinesin family members KIF1B, KLP67A and KIF5B in animal cells
(Nangaku et al., 1994
;
Pereira et al., 1997
;
Tanaka et al., 1998
). The
availability of genomic sequence data should aid in the identification of the
Neurospora mitochondria motor.
What are the cellular mechanisms that determine the shape of an organelle?
The outer membrane protein MMM1 is a key component for maintenance of normal
mitochondrial morphology (Burgess et al.,
1994; Prokisch et al.,
2000
). It was suggested that it establishes mitochondrial
structure by recruiting actin binding proteins to the organelle
(Boldogh et al., 1998
).
Surprisingly, it was found that mmm-1 null mutants of
Neurospora display giant mitochondria very similar to the yeast
mutant (Prokisch et al.,
2000
). At this time it could not be excluded that MMM1 acts as a
receptor for microtubule-binding proteins in Neurospora and thereby
determines mitochondrial structure
(Prokisch et al., 2000
). The
data presented here argue against this possibility because mitochondria
lacking MMM1 can still bind microtubules. Our results favour a function of
MMM1 in an internal organellar scaffold-like structure, as recently proposed
(Aiken Hobbs et al., 2001
).
This study also proposed a role for Mmm1p in mitochondrial DNA inheritance in
yeast. However, unlike budding yeast, Neurospora is an obligate
aerobic organism. As mmm-1 is not essential for viability in
Neurospora (Prokisch et al.,
2000
), mechanisms must exist that facilitate transmission of the
mitochondrial genome and, therefore, maintenance of respiratory competence, in
the absence of MMM1.
Two lines of evidence suggest that organellar morphology is determined by a complex interplay of internal and external factors. First, isolated mitochondria, which have lost their interaction with the cytoskeleton, collapse into round spherical structures. This indicates that intrinsic organellar factors are not sufficient to maintain an elongated shape of mitochondria and, furthermore, suggests that an alignment along cytoskeletal tracks is important. Second, an elongated shape is apparently not a prerequisite for a mitochondria/cytoskeleton interaction because isolated wild-type and mmm-1 mutant mitochondria are still able to (re)bind to microtubules. By contrast, null mutants of mmm-1 have a severely affected mitochondrial structure, although their capability to interact with microtubules is maintained. These observations suggest that the ability to bind to the cytoskeleton is not sufficient to establish normal mitochondrial morphology. It will be a challenge in the future to identify the organellar and cytoskeletal components involved. This will certainly improve our understanding of the molecular mechanisms that contribute to the establishment of the 3D structure of eukaryotic cells.
Another achievement reported here is the use of GFP as a fluorescent marker
in Neurospora crassa, one of the classic model organisms used by
geneticists, biochemists and cell biologists. While this manuscript was in
preparation, another group described the visualisation of cytosolic GFP
expressed from the heterologous ToxA promoter in Neurospora
(Freitag et al., 2001).
However, this group reported that it was not possible to find
Neurospora promotors sufficiently strong to drive high level
expression of GFP, and that GFP had to accumulate for at least 16 hours in
hyphae before visualisation was possible. Our approach demonstrates that: (1)
it is possible to express sufficient amounts of GFP from the endogenous
Neurospora atp-1 promoter; (2) GFP labelling can be observed in
conidia and in newly germinated hyphae; and (3) GFP can be used as a marker of
specific intracellular structures. The availability of molecular genetic tools
and the possibility of producing large amounts of biomass at low cost make
Neurospora an attractive model in which to study many different
topics ranging from organelle biogenesis to molecular clocks or developmental
processes (Davis, 2000
;
Perkins, 1992
). The strategies
described here and in previous studies
(Freitag et al., 2001
),
preferably together with additional improvements, might help to establish GFP
as a valuable tool for the exploration of a variety of different aspects of
Neurospora cell biology.
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Acknowledgments |
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