1 Institut de Biologie Moléculaire des Plantes, Centre National de la
Recherche Scientifique UPR 2357, Université Louis Pasteur, 12 rue du
Général Zimmer F-67084, Strasbourg Cedex, France
2 Institut für Allgemeine Botanik, Hamburg, Ohnhorststr. 18, D-22609
Hamburg, Germany
* Present address: Laboratoire de Physiologie Cellulaire Végétale,
UMR 5019 CEA/CNRS/UJF, CEA Grenoble, 17 Avenue des Martyrs 38054 Grenoble
cedex 9, France
Author for correspondence (e-mail:
anne-catherine.schmit{at}ibmp-ulp.u-strasbg.fr
)
Accepted 13 March 2002
![]() |
Summary |
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Key words: -TuSCs,
-TuRCs, Arabidopsis, Tobacco BY-2 cells
![]() |
Introduction |
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Compared with other eukaryotes, the situation in higher plants is unique
and surprising. Two major features of plant cells should be underlined. First,
diverse MT arrays assemble successively, with different orientations during
the cell cycle and/or developmental controls. The cortical MTs, the
preprophase band and the phragmoplast are not found in other eukaryotic cells
(Staiger and Lloyd, 1991;
Lambert and Lloyd, 1994
;
Shibaoka and Nagai, 1994
). It
is not known whether a single MTOC or whether multiple MTOCs are involved in
generating the plant MT cytoskeleton
(Canaday et al., 2000
;
Vantard et al., 2000
), and it
is not clear how assembly of an acentrosomal spindle could be regulated
(Marc, 1997
;
Vaughn and Harper, 1998
).
Secondly, in higher plant cells, -tubulin is distributed along all
MT arrays (Liu et al., 1993
;
Joshi and Palevitz, 1996
;
Endlé et al., 1997
;
Canaday et al., 2000
). This
localization is enigmatic as
-tubulin, which is considered to be a
universal nucleator in other eukaryotes
(Oakley, 1992
), would not be
expected to nucleate along MTs. The nuclear surface is the only functionally
characterized MT nucleation site in plants
(Mizuno, 1993
;
Stoppin et al., 1994
), and
-tubulin is detected there. Our aim was to identify proteins involved
in MT nucleation in higher plant cells and to use them as markers of plant MT
nucleation sites.
In the present report, we have identified and characterized SPC98
orthologues in rice (Oryza sativa) and Arabidopsis thaliana,
indicating that higher plants possess -tubulin complex components,
although no centrosome-like organelle is present. Unlike
-tubulin,
plant Spc98p is not detected along MTs, suggesting that plant
-tubulin
may have a role that is independent of MT nucleation at these sites. We show
that Spc98p and
-tubulin colocalize at MT nucleation sites on the
nuclear surface. In addition, both proteins are also found close to the cell
membrane, suggesting that cortical MTs are either nucleated at these sites or
stabilized by a minus-end anchorage complex as suggested for pericentriolar
MTs in animal cells (Mogensen et al.,
2000
).
![]() |
Materials and Methods |
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Antibody production
To generate rabbit polyclonal antibodies against plant -tubulin, two
peptides were selected. The first was a maize peptide EDFATQGGDRKDVFFYQ, (p1),
which is conserved in eukaryotes (Joshi et
al., 1992
), and the second, CESPDYIKWGMEDP, (p2), is a
plant-specific sequence from the C-terminus. Antibody specificity was shown
using extracts from tobacco BY-2 cells, mammalian 3T3 cells and from
Escherichia Coli expressing plant
-tubulin.
Rabbit polyclonal antibodies were raised against three rice Spc98p peptides. The first peptide, (pA), LETAIRASNAQYDDRDIL, is from the central domain, which is conserved in eukaryotes (Fig. 1, residue 625 to 647). The second peptide, (pB), DLDSIAKDYTSSLDA, is a plant-specific peptide from the C-terminus. The third peptide, (pC), FRLDFTEYYSRVSSNK, is from the C-terminal domain and is less conserved than pA or pB. All antibodies were affinity purified against the corresponding peptide.
|
For MT labeling, commercial mouse monoclonal antibodies against
-tubulin (Amersham RPN 356) were used. Alexa-488- and -568-labeled
anti-rabbit or anti-mouse IgGs were purchased from Molecular Probes (A-11029
and A-11036).
Cell extracts and immunoblotting
A suspension of the tobacco BY-2 cell line derived from Nicotiana
tabaccum L. cv. Bright Yellow 2 was subcultured as described by Nagata et
al. (Nagata et al., 1981).
Transient expression of SPC98::GFP was obtained after bombardment of
cDNA-coated tungsten particles using a particle inflow gun. 1 µg of cDNA,
mixed with 1 mg of particles in the presence of 0.75 M CaCl2 and
1.5 M spermidin, was used per assay. Cells expressing Spc98p-GFP fusion
proteins were collected under a fluorescence-equipped binocular microscope.
Cells were centrifuged at 60 g for 3 minutes and mixed (v/v)
with sample buffer (250 mM Tris, 20% glycerol, 100 mM DTT and 1.5 M SDS).
After 3 minutes of sonication, the sample was boiled for 5 minutes and stored
in aliquots at -20°C. Cell extracts were analyzed by SDS-polyacrylamide
gel electrophoresis (SDS-PAGE) according to Laemmli
(Laemmli, 1970
) using 0.75 or
1 mm thick mini-slab gels and an acrylamide concentration of 6.5% or 12.5%.
After electrotransfer to Immobilon (Millipore) membranes, samples were probed
using various antibodies. Non-specific binding sites were saturated with 5%
dry milk, 1% acetylated BSA (Aurion) and 0.2% Triton X-100 in 20 mM Tris-HCl
pH 7.4 for 1 hour at 37°C. Primary antibodies were diluted in the
saturating buffer. Secondary horseradish-peroxidase-conjugated antibodies
(Pierce) were detected using a chemiluminescence kit (Roche).
Nuclei isolation and in vitro MT nucleation assays
Nuclei were isolated from 3.5-day-old cultured tobacco BY-2 cells
(Stoppin et al., 1996). BY-2
cells were grown with 1.5% sucrose for 24 hours. To avoid plastid
contamination, cells were grown without sucrose for the last 12 hours before
harvesting. Protoplasts were then prepared using 3% cellulase RS (Onozuka),
0.2% macerozyme R10 (Serva 28302) and 0.15% pectolyase Y23 (Seishin), 0.45 M
mannitol, 8 mM CaCl2 in 25 mM MES buffer pH 5.5 for 2 hours at
30°C with slow agitation. After three washes with 250 mM sucrose, 0.5 mM
EDTA, 1 mM EGTA, 1 mM MgCl2 in 25 mM MES pH 5.5, protoplasts were
suspended in washing medium supplemented with 0.025% NP40, 10 µM leupeptin,
10 µM pepstatin, 1 mM PMSF, 1 mM DTT, 10 µg/ml aprotinin for 20 minutes
at 4°C and agitated slowly. Protoplasts were then broken by passage
through a 10 µm nylon mesh. Purified nuclei were centrifuged for 5 minutes
at 100 g at 4°C and conserved in 50% (v/v) glycerol in
liquid nitrogen.
MT nucleation assays were done (Stoppin
et al., 1994; Stoppin et al.,
1996
) in the presence of 10-5 M oryzalin to avoid
elongation of plant MT seeds on the nuclei. Oryzalin is a potent inhibitor of
the assembly of plant MTs but does not interfere with neurotubulin assembly.
The concentration of purified neurotubulin used (7 to 10 µM) was below the
critical concentration for autoassembly. Isolated nuclei were observed by DIC
and fluorescence microscopy.
Antibody inhibition assays
Isolated nuclei were incubated in the presence of anti-Spc98pA, pB or
anti--tubulin antibodies (diluted 1/50-1/200) for 20 minutes at 4°C
before the nucleation assays. Incubation with anti-GFP or anti-Spc98pC
antibodies, which do not crossreact with tobacco Spc98p, were used as negative
controls. Competition was performed by incubation of anti-Spc98p antibodies
together with their corresponding peptides for 1 hour at room temperature
before the nucleation assays. Each experiment was done three times. 200 nuclei
were counted in each assay and compared with controls done simultaneously
using the same set of tubulin and nuclei. The proportion of nuclei unable to
nucleate MTs in controls was subtracted from the inhibition measurements. This
allows direct comparison between the 100% nucleation in controls and the
various nucleation rates shown in inhibition assays.
Indirect immunofluorescence microscopy
Arabidopsis or BY-2 cells were fixed for 15 minutes in 3.7%
formaldehyde in MT-stabilizing buffer (5 mM EGTA, 2 mM MgCl2, 50 mM
PIPES, pH 6.9), post-fixed for 5 minutes in cold methanol and washed three
times with stabilizing buffer. Cell walls were permeabilized for 5 minutes in
a 1/10 dilution of the enzyme mixture used for protoplast isolation and
incubated overnight with anti--tubulin (1/1000 in PBS: 0.14 M NaCl, 2.7
mM KCl, 1.5 mM KH2PO4, 8,1 mM
Na2HPO4, pH 7.4) or anti-Spc98p antibodies (1/500),
followed by secondary antibodies (1/300 in PBS supplemented with 0.1%
acetylated bovine serum albumin from Aurion). Controls were performed both
using preimmune sera and by preincubation of the antibodies with their
respective corresponding peptides.
Immunofluorescence images were obtained using a Sony camera connected to Visolab 200 software adapted to a Leica DMBR microscope equipped with various filters (SP505-550 and LP560), which avoid crosstalk. TIFF images were treated using Adobe Photoshop software.
Microscopy imaging
Living BY-2 cells were attached to polylysine-coated glass coverslips,
mounted in a perfusion chamber and perfused for 15 to 30 minutes with culture
medium in the presence or absence of 0.2 M mannitol. Cells were monitored
during plasmolysis and recovery. Distribution of AtSpc98p-GFP fusion protein
in living or fixed cells was analysed using a Zeiss LSM 510 Confocal Laser
Scanning Microscope equipped with argon and helium/neon lasers. Pinholes were
adjusted to obtain 0.4 µm optical sections. Projections of serial optical
sections using Z-series of 0.4 µm intervals were obtained using the LSM
software. Optical planes were scanned with the 488 nm ray of the Argon laser
and using a 505-550 nm barrier filter to detect GFP fluorescence. For dual
imaging of AtSpc98p-GFP and Alexa-568-labeled MTs, the 543 nm ray of the
helium/neon laser was used in a multitrack configuration to avoid crosstalk. A
dichroic mirror at 545 nm separated GFP (short pass filter, 505-545 nm) and
Alexa (long pass filter >560 nm) channels. Time-lapse series of the same
confocal plane were taken during 30 minutes with 10 second intervals. LSM
images were converted into TIFFs for treatment with Adobe Photoshop software.
A Zeiss microscope equipped for conventional fluorescence microscopy was used
to capture the corresponding Dapi images using a 3CCD digital camera (Axiocam)
associated with Axiovision software.
![]() |
Results |
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Spc98p and -tubulin antibodies recognize specific bands by
immunoblotting
Nicotiana tabaccum L. cv. Bright Yellow 2 (BY-2) culture cells
were used for transient expression of Spc98p-GFP. Protein extracts were
prepared from wild-type and bombarded populations of cells. Immunoblots were
probed with antibodies directed against , ß and
-tubulin,
and polypeptides of the expected sizes (50 to 55 kDa) were detected
(Fig. 2A, lane 1-3). Monoclonal
anti-GFP antibodies labeled the 125 kDa Spc98p-GFP fusion protein
(Fig. 2A, lane 4). Free GFP was
not detected, indicating the absence of proteolysis in Spc98p-GFP cells.
Antibodies were raised against three rice peptides (pA, pB and pC) and used to
probe BY-2 cell extracts (Fig.
2B) and extracts from cells expressing Spc98p-GFP
(Fig. 2A, lane 5). Anti-Spc98pB
labeled the 125 kDa Spc98p-GFP fusion protein and revealed endogenous tobacco
Spc98p (Fig. 2A, lane 5;
Fig. 2B, lanes 7), as did the
anti-Spc98pA antibodies (Fig.
2B, lanes 6). The third one, anti-Spc98pC, did not crossreact with
the tobacco Spc98p orthologue and was used as negative control
(Fig. 2B, lanes 8).
Anti-Spc98pA labels centrosomes in human HEP cells (data not shown).
|
An Spc98p homologue is co-distributed with -tubulin at the
nuclear surface
To investigate the role of plant Spc98p, we used a functional assay for MT
nucleation. Isolated tobacco BY-2 nuclei were incubated with purified
neurotubulin, which was below the critical concentration for MT autoassembly
in the absence of stabilizing agents such as taxol. Oryzalin, a specific
inhibitor of plant MT assembly, was used to prevent elongation from MT seeds,
which could be present on the nuclear surface after nuclei isolation.
Neurotubulin assembly is not affected by oryzalin at the concentration used.
In our in vitro assay, MTs are specifically nucleated on the surface of plant
nuclei and form a sun-like MT pattern (Fig.
3B,C,E,F). The average nucleation efficiency reached 68%.
MT-nucleated nuclei were labeled with the same antibodies used for
immunoblots, either polyclonal antibodies raised against plant -tubulin
or a mixture of anti-Spc98pA and pB. MTs were labeled simultaneously using
commercial monoclonal anti-
-tubulin. Optical sections obtained by laser
scanning microscopy showed that the plant Spc98p homologue
(Fig. 3A) as well as
-tubulin (Fig. 3D) are
distributed over the entire surface of the plant nucleus. Both proteins are
found at sites where MTs are nucleated, showing that they remain at the
nuclear surface during nuclei isolation and that their MT nucleation activity
is conserved (Fig. 3C,F).
However, Spc98p and
-tubulin immunolabeling is decreased by treating
nuclei with detergent or salt (data not shown). When isolated nuclei were
preincubated with antibodies against
-tubulin
(Fig. 3G) or against Spc98pB
(Fig. 3H,I), sun-like nucleated
nuclei decreased dramatically, showing 63 to 96% inhibition
(Fig. 4). Each inhibition assay
was simultaneously performed, without antibodies, using the same batch of
purified brain tubulin and isolated tobacco nuclei. 200 nuclei were analysed
in each experiment, and the proportion of nucleated nuclei was compared with
the corresponding controls in which inactive nuclei were subtracted from
active ones. Thus, the control nucleation rate was adjusted to 100%, and
nucleation inhibition appeared as a decrease in the nucleation rate.
|
|
Increasing antibody concentration proportionally decreased the nucleation
capacity. 70 to 93% inhibition was observed using -tubulin antibodies,
and 80 to 85% inhibition was observed using Spc98pA antibodies. Increasing the
time of incubation of antibodies before nucleation assays also enhanced the
inhibition of MT nucleation, which varied from 72% after 20 minutes of
incubation to 96% after 60 minutes. Controls using either non-crossreactive
anti-Spc98pC antibody or an anti-GFP antibody at comparable levels of
concentration and time of incubation did not significantly affect the
nucleation process.
The plant homologue of Spc98p is localized at MT nucleation sites in
situ but is not co-distributed with -tubulin along plant MTs
To determine whether the plant Spc98p is located at MT nucleation sites, we
labeled tobacco BY-2 and cultured A. thaliana cells at different
stages of the cell cycle with anti-Spc98p, anti--tubulin and
anti-
-tubulin antibodies, as shown for BY-2 cells in
Fig. 5. Both anti-Spc98p
(Fig. 5A) and
anti-
-tubulin (Fig. 5C)
densely label the nuclear surface that functions as a MT nucleation site in
plants. The anti-Spc98p antibody gives some punctate cytoplasmic staining.
Neither preimmune sera nor antibody depletion by peptide competition produced
such labeling.
|
-tubulin was found at the nuclear surface and along all MT arrays:
nuclear-associated MTs, cortical MTs and the preprophase band
(Fig. 5C,D). Spc98p is not
co-distributed with the
-tubulin associated with MTs. The same results
were obtained in both tobacco and Arabidopsis cells.
AtSpc98p-GFP in vivo localization
The AtSPC98::GFP fusion construct was introduced by bombarding
tobacco BY-2 cells or by electroporating protoplasts. In both BY-2 cells and
protoplasts, the fusion protein was found mainly on the nuclear surface
(Fig. 6C), whereas, when
unfused GFP is transiently expressed in BY-2 cells, a diffuse distribution is
detected in both the cytoplasm and the nucleus
(Fig. 6A). In addition to
perinuclear labeling, AtSpc98p-GFP was regularly distributed in the cortical
cytoplasm, close to the plasma membrane
(Fig. 6C,G-L). These cortical
labelings were particularly visible in elongated cells where the cortical MTs
are well organized. To determine whether the cortical labelling is linked to
the plasma membrane, we followed the movement of fluorescent fusion proteins.
Time-lapse images were taken every 10 seconds for 30 minutes on turgescent
cells. Then, cells were plasmolyzed for 15 minutes, and recovery was analysed
during the following 15 minutes. The GFP fluorescent signals moved slightly
during plasmolysis within the focal area and followed the speed of membrane
displacement during the 6 minutes necessary for turgescence recovery, as shown
by the increasing distance between fluorescent dots marked by an arrow and an
arrowhead (Fig. 6G-M). Some of
the GFP signals observed using confocal microscopy coincide with ends of MT
bundles as observed after immunolabeling
(Fig. 6M,N). These data suggest
that Spc98p is linked to the plasma membrane and localized at sites that could
be involved in cortical MT nucleation and/or anchoring.
|
The subcellular distribution of Spc98p-GFP confirms the results obtained by immunolabeling, although some fusion protein aggregates were observed when the protein was overexpressed. Using both techniques, a punctate labeling is observed at the nuclear surface and in the cytoplasm. The cortical labeling at the plasma membrane is clearer when the Spc98p-GFP fusion protein is expressed than when immunolabeled, which is mainly because of membrane permeabilization in the latter case.
![]() |
Discussion |
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The presence of both -tubulin and Spc98p suggests that plant cells
contain
-TuSC-like nucleation complexes. In addition, Spc97, another
-TuSC complex component, has a putative homologue in the
Arabidopsis thaliana database. Do plant cells possess larger MT
nucleating complexes, such as the
-TuRCs
(Murphy et al., 1998
;
Jeng and Stearns, 1999
;
Wiese and Zheng, 1999
;
Moritz and Agard, 2001
), found
in other eukaryotes? Large soluble
-tubulin-containing complexes were
identified in plants (Stoppin-Mellet et
al., 2000
), but their activity has not yet been characterized.
Further experiments will be necessary to isolate and characterize
-tubulin complexes containing plant Spc97p and Spc98p and to determine
whether additional plant-specific proteins are present in these nucleation
complexes.
Colocalization of Spc98p and -tubulin at MT nucleation sites
on isolated nuclei
The colocalization of Spc98p and -tubulin on isolated BY-2 nuclei
where MT nucleation is initiated favors the hypothesis that MT nucleation
complexes are present at the nuclear surface. Inhibition of MT nucleation by
Spc98p and
-tubulin antibodies argues that
Spc98p/
-tubulin-containing complexes are involved in MT nucleation.
Isolated centrosomes, like plant nuclei, are capable of MT nucleation in
vitro. Addition of plant cellular extracts to urea-inactivated mammalian
centrosomes rescues nucleation activity
(Stoppin-Mellet et al., 1999
),
suggesting that cytosolic
-tubulin complexes containing Spc98p may be
recruited and activated at MT nucleation sites, as in mammalian cells
(Moudjou et al., 1996
).
AtSpc98p-GFP in living cells: identification of cortical MT
nucleation sites
AtSpc98p-GFP was detected in living tobacco BY-2 cells on the nuclear
surface as expected. In addition, in G1 phase and in elongated cells, a
fluorescent signal is detected close to the plasma membrane, at ends of MT
bundles. -tubulin is also present at sites where cortical MTs contact
the cell membrane (McDonald et al.,
1993
; Canaday et al.,
2000
), and perinuclear MTs do not directly form cortical arrays
(Nagata et al., 1994
). The
co-distribution of Spc98p and
-tubulin suggests that cortical MTs are
nucleated in the cortex at the plasma membrane. A 49 kDa component of the
centrosphere, identified as elongating factor E2F-1
, is also found both
at the nuclear envelope and at the plasmalemma
(Hasezawa and Nagata, 1993
).
This result is indicative of multiple MT nucleation sites, but E2F-1
could be involved in signal transduction rather than nucleation.
Cortical MTs turn over more rapidly than centrosome-nucleated MTs in animal
cells, suggesting that cortical MTs are assembled by de novo nucleation
(Wasteneys et al., 1993;
Yuan et al., 1994
;
Hush et al., 1994
). This
cortical MT instability and the localization of AtSpc98p-GFP at sites where MT
bundles start, strongly argue for nucleation at the cortex. Spc98p was not
clearly immunodetected at cortical sites, but the fixation and
permeabilization procedures used could affect the plasma membrane, leading to
the loss of small structures such as
-tubulin-nucleating complexes. In
vivo detection using GFP fusion protein enhances the resolution of membrane
nucleation sites and provides evidence to support the hypothesis that there
are multiple MT nucleation sites in plant cells.
Plant Spc98p is not co-distributed with -tubulin along
MTs
Neither AtSpc98p-GFP nor antibody-labeled endogenous Spc98p are
co-distributed with -tubulin along MT arrays. This indicates that the
-tubulin associated along the length of MTs has an alternative activity
that may affect MT properties. Cytosol plant
-tubulin is present in
high molecular weight complexes containing Hsp70 and TCP1 chaperones
(Stoppin-Mellet et al., 2000
),
suggesting that the different
-tubulin complexes present in higher
plant cells may have different functions.
-tubulin may be involved in
MT stabilization instead of nucleation or be maintained in a storage form
before its recruitment at nucleation sites
(Dibbayawan et al., 2001
).
Perinuclear and perhaps cortical sites where
-tubulin and Spc98p
colocalize would correspond to activatable nucleation sites.
A functional model for recruitment and activation of
Spc98p/-tubulin complexes in higher plants
On the basis of our present data, we suggest that plant
-tubulin/Spc98p-containing complexes are involved in nucleation of
plant MTs and are functionally homologous to the
-TuRCs found in
metazoans and fungi. The localization of Spc98p-GFP in vivo in combination
with the results of our MT nucleation assays leads us to propose a model for
the dynamics of plant MT nucleating complexes during the cell cycle and
development (Fig. 7).
|
Cytoplasmic MT-nucleating complexes containing -tubulin and plant
Spc98p could be recruited to various MT nucleation sites during the cell cycle
or development. Complexes situated at cortical sites could be activated during
G0 and G1, that is, at stages where cortical MTs are assembled. The complexes
located at perinuclear sites would be activated in G2, when MTs radiating from
the nuclear surface are predominant. The pre-prophase band assembled at this
stage could originate from perinuclear and/or cortical nucleation sites. In
most eukaryotes, the
-TuRC is targeted to a structured organelle such
as the centrosome or the SPB, which nucleates and organizes all MTs. In higher
plants,
-TuRC-like complexes could be recruited to different sites and
coordinately activated to organize the successive MT arrays.
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Acknowledgments |
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