Download Plant mitochondria move on F-actin, but their positioning in the

Journal of Experimental Botany, Vol. 53, No. 369, pp. 659–667, April 2002
Plant mitochondria move on F-actin, but their positioning
in the cortical cytoplasm depends on both F-actin and
microtubules
K. Van Gestel1, R.H. Köhler 2 and J-P. Verbelen1,3
1
2
Department of Biology, University of Antwerp UIA, Universiteitsplein 1, 2610 Wilrijk, Belgium
Lion Bioscience Research Inc., Cambridge, MA 02139, USA
Received 27 August 2001; Accepted 26 November 2001
Abstract
Introduction
Mitochondrion movement and positioning was
studied in elongating cultured cells of tobacco
(Nicotiana tabacum L.), containing mitochondrialocalized green fluorescent protein. In these cells
mitochondria are either actively moving in strands
of cytoplasm transversing or bordering the vacuole,
or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell’s ATP stock with the
uncoupling agent DNP shows that the movement is
much more energy demanding than the positioning.
The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin
B, the myosin ATPase inhibitor 2,3-butanedione
2-monoxime and the sulphydryl-modifying agent
N-ethylmaleimide. The microtubule disrupting drug
oryzalin did not affect the movement of mitochondria
itself, but it slightly stimulated the recruitment of
cytoplasmic strands, along which mitochondria
travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to
the cell axis, in the cortical cytoplasm of elongated
cells. This positioning is mainly microtubule based.
After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous
parallel arrays transverse or oblique to the cell axis
or clustered around chloroplasts and around patches
and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel
arrays.
Displacements and changes in shape of mitochondria
are thought to depend on driving cytoskeletal forces
as well as on intrinsic factors (Bereiter-Hahn and Vöth,
1994). In animal cells mitochondrion movement is
mainly associated with microtubules (MTs) (Heggeness
et al., 1987; Hirokawa, 1998). Only in neuronal
axons (Kuznetsov et al., 1992) and in specific insect cells
(Sturmer et al., 1995) do actin filaments (AFs) serve as a
track for mitochondrial transport. In yeasts, the MTcytoskeleton is indispensable for mitochondrial distribution in Schizosaccharomyces pombe (Yaffe et al., 1996),
while in Saccharomyces cerevisiae only AFs and intermediate filaments have been implicated in mitochondrial
translocations (reviewed by Hermann and Shaw, 1998).
For plant cells, the basic model for AF-based cytoplasmic movement of organelles (Williamson, 1993) is
still valid, also for mitochondria. Structural associations
of organelles with AFs have been described in fixed
material (Menzel, 1987; Lichtscheidl et al., 1990), while
AF-based movement of GFP-labelled Golgi stacks has
been demonstrated in vivo in tobacco leaf epidermis
(Boevink et al., 1998). Evidence for the mechanism of
mitochondrion movement and positioning is, however,
scarce and mostly based on comparison with other organelles. In onion (Allium cepa L.) epidermal cells using
the fluorochrome DiOC6(3), mitochondria and spherosomes were found to follow the same pathway as the
ER-strands, whose movement was shown to be dependent
on F-actin (Quader et al., 1987, 1989; Allen and Brown,
1988). In the same tissue DiOC6(3)-stained mitochondria
were shown to co-localize with rhodamine–phalloidin
labelled AFs (Olyslaegers and Verbelen, 1998). In tobacco
BY-2 cells Nebenführ et al. observed GFP-labelled
Key words: Actin, green fluorescent protein, mitochondrion,
Nicotiana tabacum L., tubulin.
3
To whom correspondence should be addressed. Fax: q32 3 8202271. E-mail: [email protected]
ß Society for Experimental Biology 2002
660
Van Gestel et al.
Golgi stacks and fluorochrome-labelled mitochondria
following the same travelling paths and showing similar
stop-and-go-movements (Nebenführ et al., 1999).
Stably transformed tobacco (Köhler et al., 1997) and
Arabidopsis (Logan and Leaver, 2000) plant lines
with GFP targeted to the mitochondria were developed.
In Arabidopsis the heterogeneity in morphology and
dynamics of mitochondria was described. Using the
transformed tobacco plants (Köhler et al., 1997), mitochondrion movement and positioning in cells was
analysed by means of different inhibitor treatments.
In these cells it is demonstrated that fast directional
movement of mitochondria is dependent on an intact
F-actin–myosin system while the positioning of immobile
mitochondria in the cortical cytoplasm is both AF- and
MT-based.
in DMSO, 2,3-butanedione 2-monoxime (BDM) (Sigma) was
prepared as a 1 M stock in distilled H2O (50 8C). Appropriate
amounts of stock solutions were dissolved in K3A-culture
medium (Potrykus and Shillito, 1986) to reach the following
concentrations: 1.25 mM latrunculin B (Gibbon et al., 1999),
10 mM oryzalin (Hugdahl and Morejohn, 1993), 50 mM NEM
(Liebe and Quader, 1994), 40 mM DNP (Markova et al., 1990),
and 20 mM BDM (May et al., 1998). Incubation times were
1–5 h for latrunculin B and oryzalin, 1–2 h for NEM, BDM and
DNP.
Microscopy
Fluorescence of GFP, DiOC6(3), and Alexa-phalloidin were
imaged using the 488 nm laser line of a Bio-Rad MRC 600
confocal system mounted on a Zeiss Axioskop microscope,
equipped with a 40 3 (NA 0.9) water-immersion objective.
Part of the micrographs are z-series projections of optical
sections, made with the standard COMOS software (Bio-Rad
Microscience Ltd.). Brightfield and phase-contrast images were
acquired using the transmission mode of the confocal system.
Materials and methods
Plant material and labelling
Elongated tobacco cells were regenerated from protoplasts
(Verbelen et al., 1992) which were isolated (Potrykus and
Shillito, 1986) from leaves of sterile-grown, transgenic plants of
Nicotiana tabacum L. cv. Petite Havana expressing mitochondrion targeted GFP (Köhler et al., 1997). This targeting is
mediated by the yeast cytochrome oxidase subunit IV (coxIV)
transit peptide, which is removed upon import of the GFP
molecule into the mitochondrion. The specificity and the
non-toxicity of the GFP targeting have been demonstrated
(Köhler et al., 1997). For visualization of the ER, AFs and MTs,
tobacco cells without mitochondrion targeted GFP were used.
The ER, together with the mitochondria, was stained with
0.87 mM 3,39-dihexyloxa-carbocyanine iodide (DiOC6(3))
(Molecular Probes). Alexa-phalloidin (Molecular Probes) was
used to label AFs after permeabilization of the cells with
detergents, and MTs were labelled by immunocytochemistry
(Vissenberg et al., 2000).
Drug treatments
Stock concentrations of 2.5 mM latrunculin B (Calbiochem),
100 mM oryzalin (Merck), 50 mM N-ethylmaleimide (NEM)
(Sigma), and 40 mM dinitrophenol (DNP) (Merck) were made
Results
The protoplast derived cells used in these experiments are
a model system for cell elongation (Vissenberg et al.,
2000). After the regeneration of a wall, cells elongate and
thereby increase in volume 10–15 times.
Regarding mitochondrion movement, the cytoplasm of
elongating tobacco cells can be divided in two domains:
fast and directional movement is concentrated in conspicuous strands of cytoplasm, bordering or transversing
the vacuole, while the more static mitochondria reside
in the very thin cortical layer of cytoplasm appressed
against the plasma membrane.
Mitochondrion movement in cytoplasmic strands
Mitochondria move vigorously through transvacuolar
and vacuole bordering cytoplasmic strands and they often
stop, change direction, fuse or divide. This activity is most
easily illustrated with pictures of the transvacuolar
strands. In Fig. 1, a sequence of four fluorescence images,
taken 3 s apart, demonstrates the directional movement
Fig. 1. Sequence of four confocal images, taken 3 s apart, demonstrating the directional movement of GFP-tagged mitochondria in a transvacuolar
cytoplasmic strand of a tobacco cell. Arrowheads indicate the fast moving mitochondria while the arrow points to immobile mitochondria in the
cortical cytoplasm layer. Scale bar ¼ 10 mm.
Movement of plant mitochondria
661
Fig. 2. The effect of latrunculin B on cytoplasmic architecture. The figure represents a bright-field image (left side) and a single plane confocal image
(right side) of two tobacco cells. (A) In a control cell cytoplasmic strands containing mitochondria radiate from the nucleus (n) towards the cortical
layer. (B) Latrunculin B (1 h) has a disruptive effect on the cytoplasmic strands. Scale bar ¼ 20 mm.
of mitochondria in a transvacuolar strand from top right
to bottom left of the image. Note also the immobile
mitochondria in the cortical layer of cytoplasm at the
right side of the images. The speed of this directional
movement is similar to that of cytoplasmic streaming
and can reach up to 10 mm s1. A movie of this dynamic
transvacuolar mitochondrion movement can be seen at
http:uubio-www.uia.ac.beubioufymoukvg. In Fig. 2A, the
multiple transvacuolar strands radiating from the nucleus
to the cortical layer (brightfield image) contain many
moving mitochondria (fluorescence image).
Treatment of the cells with 1.25 mM latrunculin B
causes an abrupt cessation of directional mitochondrion
movement and a breakdown of most cytoplasmic strands
within 1 h (Fig. 2B). Mitochondria from the broken
transvacuolar strands become incorporated in the cortical
cytoplasm. Both NEM (50 mM) and BDM (20 mM)
cause a cessation of mitochondrial tracking accompanied
by a disturbance of the cytoplasmic strands (results not
shown). Recovery of structure and movement after
removal of the drugs only occurred in the case of
latrunculin B and BDM.
Oryzalin (10 mM) did not affect the movement of the
individual mitochondria. On the contrary, after several
hours of incubation in oryzalin, a stimulation of the
mobility was noticed in the population of mitochondria
due to an increase in the number of cytoplasmic strands
within the cells. In a sample of 50 cells, oryzalin-treated
cells contain about 9.2 transvacuolar strands against
7.5 in non-treated control cells (P-0.01, Student’s t-test).
Mitochondria in the cortical layer of cytoplasm
The proportion of mitochondria being immobile depends
on the developmental stage of the cells.
In freshly isolated protoplasts (Fig. 3A) and young
(2–5 d) cells in culture (Fig. 3B) most mitochondria move
actively throughout the cortical cytoplasm and very few
are immobile (see http:uubio-www.uia.ac.beubioufymoukvg
for a movie). No particular positioning of these cortical
mitochondria was observed besides an association with
chloroplasts. In the protoplasts, latrunculin B causes all
mitochondria to cluster around chloroplasts and small
disc-shaped structures (Fig. 3F). In the young cells part of
the mitochondria form additional aggregates, showing no
particular orientation (Fig. 3G).
From the onset of elongation on, cells contain many
more mitochondria (Fig. 3C). Active movement in the
cortical cytoplasm is restricted to vacuole bordering
cytoplasmic strands and in the areas which are devoid
of these strands, numerous mitochondria are immobile.
The position of the immobile mitochondria in the
thin cortical layer is not immediately affected by
latrunculin B. After sustained incubation (1–2 h) these
mitochondria cluster around chloroplasts, disc-shaped
and strand-like structures, but it is their positioning in
parallel transverse or oblique arrays (Fig. 3H) that is
conspicious.
In older well-elongated cells, part of the immobile
mitochondria is already arranged along parallel lines
transverse or oblique to the cell axis (Fig. 3D). In these
cells too, latrunculin B causes a redistribution of mitochondria to the circumference of the chloroplasts, the
disc-shaped and the strand-like structures. But it is again
their positioning into clear parallel arrays, transverse or
oblique to the cell axis which is the most striking feature
(Fig. 3I). In cells treated with oryzalin, all transverse
arrays of mitochondria are lost (Fig. 3E) and mitochondria are positioned evenly throughout the cortical cytoplasm. A simultaneous incubation in latrunculin B and
oryzalin, causes the mitochondria to cluster around
chloroplasts and around disc-shaped and strand-like
structures with an obvious absence of any clear transverse
orientation (Fig. 3J).
The typical patterns of mitochondria localization in
elongated cells, as described above, are strongly amplified
by incubating the cells in the uncoupling agent
2,4-dinitrophenol (DNP). This DNP treatment results
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Van Gestel et al.
Fig. 3. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of tobacco cells at different stages of
development (A–D) and (F–I), and after different drug treatments (E-J). The grey discs (arrow in A) represent autofluorescent chloroplasts.
The sequence of development from protoplast (A), after regeneration of a cell wall (B), at the onset of elongation (C), to a well-elongated cell (D),
demonstrates that mitochondria increase in number and become quantitatively less mobile and finally adopt a parallel orientation transverse to the cell
axis. Latrunculin B (1 h) makes the mitochondria cluster around chloroplasts and disc-shaped ER (arrow) in protoplasts (F) and young spherical cells
(G). At the onset of elongation (H) mitochondria additionally lie in parallel transverse arrays (1–2 h treatment). In fully elongated cells (I) these
Movement of plant mitochondria
in a situation where all cortical mitochondria lie immobile
and head-to-tail on parallel transverse arrays (Fig. 4A).
All mitochondria are immobile in the strands as well.
This situation is, however, fully reversible as mitochondria regain their former behaviour directly after washing
the cells with DNP-free culture medium. In latrunculin
B-treated cells, the DNP effect is very similar, the
array-like head-to-tail positioning of mitochondria is
663
accentuated (Fig. 4B). In oryzalin-treated cells, however,
DNP did not induce any array-like positioning of cortical
mitochondria (Fig. 4C).
BDM (20 mM) and NEM (50 mM) cause the immobile
mitochondria of elongated cells to aggregate along short
lines, with an average transverse orientation (Fig. 5A, B).
In NEM-treated cells, however, more irregular clumping
of the mitochondria was observed.
Fig. 4. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of DNP-treated (1–2 h) tobacco cells. (A) In
normal cells DNP induces head-to-tail parking of immobile mitochondria on parallel arrays transverse to the cell axis. (B) In latrunculin B-treated
(4 h) cells DNP has a similar effect on those mitochondria which are not clustered around chloroplasts or ER-structures. (C) In oryzalin-treated (4 h)
cells DNP could not induce transverse array-like positioning of the mitochondria. Scale bar ¼ 50 mm.
Fig. 5. Z-series projections of confocal images of GFP-tagged mitochondria in the cortical cytoplasm of BDM- and NEM-treated (1–2 h) tobacco
cells. Both BDM (A) and NEM (B) induce clustering of mitochondria along short lines, NEM has, however, a more clumping effect. Scale
bar ¼ 15 mm.
transverse arrays (right side of the picture) and the clustering along ER-strands (arrowheads) are predominant (2– 4 h treatment). Oryzalin (4 h)
destroys the parallel positioning of the mitochondria in elongated cells (E). Simultaneous treatment with latrunculin B and oryzalin (4 h) results in
clustering around chloroplasts and disc-shaped ER, without any parallel transverse orientation (J). Scale bar ¼ 25 mm.
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Van Gestel et al.
To evaluate the proper effects of the inhibitors
latrunculin B, oryzalin, NEM, and BDM on the
cytoskeleton, AFs and MTs were visualized in the
cells after the different treatments. AFs were unaffected by treatment with BDM and oryzalin, NEM
caused a conspicuous decrease in number, intactness
and thickness of the filaments and latrunculin B
resulted in a complete breakdown of the AFs (data
not shown). MTs were unaffected by BDM and
latrunculin B, NEM had a slight disrupting effect,
while oryzalin destroyed them completely (data not
shown).
The disc-shaped and strand-like structures around
which the mitochondria cluster in cells treated with
latrunculin B, with or without oryzalin, are very probably
of ER nature. They are visible using phase-contrast
optics (Fig. 6) and can be labelled with the fluorochrome
DiOC6(3) (Fig. 7). These ER-structures are far more
fluorescent than the cortical ER network, which is not
visible in Fig. 7, but are comparable in fluorescence
intensity to the mobile ER-stretches in cytoplasmic
strands of control cells (data not shown). The transverse
orientation of the strand-like ER bordered by the mitochondria is very conspicuous in elongated cells (Fig. 7A).
Part of the mitochondria in latrunculin B-treated cells
are, however, positioned in transverse arrays without
association with these conspicuous ER structures
(Fig. 7B).
Fig. 6. The strand-like (A) and disc-shaped (B) ER-structures (arrows) along which GFP-tagged mitochondria cluster in the cortical cytoplasm after
latrunculin B treatment (4 h) are visible with phase-contrast microscopy (right side). The left part of the pictures is a single plane confocal image
indicating the position of the mitochondria. Scale bar ¼ 10 mm.
Fig. 7. Single plane confocal images of mitochondria and ER, both stained with DiOC6(3), in latrunculin B-treated (4 h) elongated tobacco cells.
(A) Mitochondria are clustered around disc-shaped and strand-like parts of the ER. (B) Part of the mitochondria in latrunculin B-treated cells
are, however, positioned in transverse arrays without association with these transverse ER structures. Scale bar ¼ 10 mm.
Movement of plant mitochondria
Discussion
In elongating tobacco cells, there is a clear distinction
between mitochondria in the cytoplasmic strands—
through and along the vacuole—and those in the very
thin cortical layer of cytoplasm. While the former
display a fast directional tracking, the latter are often
hardly moving. A similar difference between the inner and
the cortical cytoplasm has been described for the movement of the ER (Allen and Brown, 1988) and of the Golgi
stacks (Nebenführ et al., 1999).
The ratio between mobile and immobile mitochondria
depends on the physiological status of the cell. In
protoplasts and cells that have just generated a cell
wall, very few mitochondria are immobile. When the
cells elongate, the number of mitochondria increases,
especially in the non-mobile part of the population.
Mitochondrion movement in the cytoplasmic strands
depends on F-actin–myosin interaction while the
recruitment of strands is negatively controlled by MTs
Mitochondria move through the cytoplasmic strands in a
directional but non-uniform way. There is a great variation in mobility within a cell, between cells and in time.
Similar movements of fluorochromically labelled mitochondria have been reported and called ‘stop-and-go
movements’ (Quader et al., 1987; Nebenführ et al., 1999).
Mitochondial mobility is a very energy-demanding
process. The uncoupling agent DNP abolishes the proton gradient over the inner mitochondrial membrane,
leading to a depletion of ATP in the cells (Rawn, 1989).
In cells treated with DNP, movement of mitochondria
was totally abolished. This movement of mitochondria
involves F-actin as it was totally arrested by the F-actin
disrupting drug latrunculin B. The myosin ATPase
inhibitor BDM and the sulphydryl-modifying agent
NEM were used to confirm the involvement of myosins
in mitochondrial mobility, as reported previously for
onion epidermal cells (Liebe and Quader, 1994). In
the tobacco cells both drugs inhibited mitochondrial
movement. Recently, it has been demonstrated that
myosin is associated with the surface of mitochondria
prepared from etiolated wheat (Triticum aestivum L.)
seedlings (Zhao et al., 1999).
The MT-depolymerizing drug oryzalin did not affect
the movement of mitochondria itself, but it caused a
slight increase in the frequency of cytoplasmic strands
with moving mitochondria, hence leading to a slightly
higher number of mobile mitochondria.
This study thus confims the results obtained previously
with fluorochromically labelled mitochondria (Quader
et al., 1989): mitochondrion movement is dependent on
an intact F-actin–myosin system. The negative control
by MTs resembles the observations by Nebenführ et al.
who found that the AF-based mobility of Golgi stacks
665
was negatively controlled by the presence of MTs
(Nebenführ et al., 1999). Examples of interactions
between MTs and AFs are numerous, both in animal
and in plant cells (Gavin, 1997; Blancaflor, 2000; Šamai
et al., 2000). In plant cells especially, the mechanism at
the base of these interactions is not yet understood.
In maize (Zea mays L.) root cells, Šamai et al. found
evidence for interaction between AFs and MTs via
BDM-sensitive myosins (Šamai et al., 2000). It is unclear
which mechanism lies at the base of the observed
increase in AF-containing cytoplasmic strands upon
depolymerization of the MTs.
Mitochondrial positioning in the cortical cytoplasm
depends on AFs and MTs
The involvement of AFs is most obvious in protoplasts
and young cells. MT-dependence is predominant in
elongating cells.
Freshly prepared protoplasts and young cells are
roughly spherical and isotropic in growth and in
cellular organization; their cytoskeleton contains relatively few and randomly oriented cortical MTs and AFs
(Vissenberg et al., 2000). The immobile mitochondria
are positioned throughout the cortical cytoplasm. After
incubation in latrunculin B the majority of mitochondria
clustered around chloroplasts and ER-patches. Mitochondria found in close association with chloroplasts
have been reported for Vallisneria and Arabidopsis
(Liebe and Menzel, 1995; Logan and Leaver, 2000). In
Arabidopsis, baskets of F-actin were visualized surrounding the chloroplasts (Kandasamy and Meagher, 1999).
Although no F-actin was detected around the chloroplasts of the tobacco cells after latrunculin B treatment,
it is clear that mitochondria become trapped there as a
result of the F-actin breakdown in the cytoplasmic
strands. The disc-shaped ER-patches, around which the
mitochondria clustered in AF-depleted cells, are probably
derived from previously mobile tubular ER of the cytoplasmic strands, according to the fluorescence intensity
when stained with DiOC6(3). Similar cortical ER‘patches’ were observed in cytochalasin D-treated onion
epidermal cells (Knebel et al., 1990).
Cells that elongate have very prominent transverse
parallel arrays of cortical MTs, while arrays of parallel
AFs are also present (Vissenberg et al., 2000). In these
cells, cytoplasmic strands bordering the vacuole are
scarce and most mitochondria are immobile in the very
thin cortical layer of cytoplasm. Part of these mitochondria are often positioned in parallel transverse or
oblique arrays. In latrunculin B-treated cells, where all
the cortical AFs were lost, the mitochondria remained
and even gathered on parallel transverse arrays. Part
of the mitochondria clustered around the chloroplasts,
ER-patches and ER-strands which act as lifebuoys.
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Van Gestel et al.
After treatment with oryzalin, however, all transverse
ordering was lost. Thus the immobile part of the mitochondrion population is preferentially positioned along
MTs. Clearly this positioning is less energy-demanding
than actin-based mobility. DNP, which stops mobility,
does not affect the positioning along microtubules.
Indeed, when treated with DNP, mitochondria were frozen head-to-tail on parallel transverse or oblique arrays
in the cortical cytoplasm of normal and AF-depleted cells,
but failed to do so in MT-depleted cells.
All these data together make it possible to conclude
that, first, in elongating cells of tobacco, mitochondrion
travelling depends on intact F-actin–myosin interactions
whereby MTs seem to regulate the frequency of travelling
tracks. Secondly, depending on the physiology of the
cells, the positioning of mitochondria in the cortical
cytoplasm is based on AFs or on MTs.
Co-operation between MT- and AF-based motor
proteins for the transport of membranous organelles has
recently come into focus for animal cells. Organelles are
supposed to possess both myosins and microtubule
motor proteins (Allan and Schroer, 1999; Brown, 1999).
These data also suggest that plant mitochondria could
well possess multiple types of motor proteins allowing
them to be associated with AFs as well as with MTs. The
latter association would only be used for immobilization.
Acknowledgements
The work of K Van Gestel was financially supported by the
Research program of the Fund for Scientific Research, Flanders
(grants 3.0028.90 and G.0034.97). R Köhler acknowledges the
support from the grant from the US Department of Energy
Biosciences Program DOE DE-FG02-89ER14030.
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