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7/28/2019 3-Sub-Cellular Localisation of GFP-Tagged Tobacco Mitotic Cyclins During the Cell Cycle and After Spindle Checkpoi
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Sub-cellular localisation of GFP-tagged tobacco mitoticcyclins during the cell cycle and after spindle checkpointactivation
Marie Claire Criqui1,, Magdalena Weingartner2,, Arnaud Capron1, Yves Parmentier1, Wen-Hui Shen1,
Erwin Heberle-Bors2, Laszlo Bo gre3 and Pascal Genschik1,*
1Institut de Biologie Moleculaire des Plantes du CNRS, 12 rue du General Zimmer, 67084 Strasbourg Cedex, France,2Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohrgasse 9, 1030 Vienna, Austria, and,3School of Biological Sciences, Royal Holloway College, University of London, Egham TW20 0EX, UK
Received 11 July 2001; revised 3 September 2001; accepted 7 September 2001.*For correspondence (fax +33 3 88 61 44 42; e-mail Pascal.Genschik@ibmp-ulp.u-strasbg.fr).These authors contributed equally to this work.
Summary
We have previously shown that the tobacco cyclin B1;1 protein accumulates during the G2 phase of the
cell cycle and is subsequently destroyed during mitosis. Here, we investigated the sub-cellular
localisation of two different B1-types and one A3-type cyclin during the cell cycle by using confocal
imaging and differential interference contrast (DIC) microscopy. The cyclins were visualised as GFP-
tagged fusion proteins in living tobacco cells. Both B1-type cyclins were found in the cytoplasm and in
the nucleus during G2 but when cells entered into prophase, both cyclins became associated with
condensing chromatin and remained on chromosomes until metaphase. As cells exited metaphase, the
B1-type cyclins became degraded, as shown by time-lapse images. A stable variant of cyclin B1;1-GFP
fusion protein, in which the destruction box had been mutated, maintained its association with the
nuclear material at later phases of mitosis such as anaphase and telophase. Furthermore, we
demonstrated that cyclin B1;1 protein is stabilised in metaphase-arrested cells after microtubule
destabilising drug treatments. In contrast to the B1-type cyclins, the cyclin A3;1 was found exclusively inthe nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.
Keywords: cell cycle, localisation, cyclin, GFP, Dbox, tobacco.
Introduction
The cyclins and cyclin-dependent kinases (CDKs) are key
regulators of the eukaryotic cell cycle (Nigg, 1995). A- and
B-type cyclins can be distinguished by characteristic
signature sequences within the cyclin box, the conserved
CDK-binding domain. In animal cells two different A-type
cyclins (A1 and A2) and three different B-type cyclins (B1,B2 and B3) have been reported (Gallant and Nigg, 1994;
Howe etal., 1995; Kreutzer etal., 1995; Minshull etal., 1989;
Pines and Hunter, 1989). In vertebrates, A-type cyclin binds
to both CDK2 and Cdc2 kinases and is required for
progression through the S-phase and for early mitotic
events, whereas activation of Cyclin B/Cdc2 kinase com-
plex triggers the entry into mitosis. The deletion of either
cyclin A2 (the somatic A-type cyclin) or cyclin B1 genes in
mice resulted in embryonic lethality (Brandeis etal., 1998;
Murphy etal., 1997). However, in the y, in contrast to
cyclin A, which is essential for mitotic events (Lehner and
O'Farrell, 1989), neither cyclin B1 nor B3 are essential for
viability (Jacobs etal., 1998).
The sub-cellular localisation of both A- and B-type
cyclins has been documented in animal cells. In HeLacells, cyclin A accumulates predominantly in the nucleus
from the time of its appearance in G1 to the mitotic
prophase, when it is degraded (Pines and Hunter, 1991). In
contrast, cyclin B1 is mainly in the cytoplasm before
prophase and than enters precipitously in the nucleus until
the meta- to anaphase transition, when it is destroyed
(Pines and Hunter, 1991; Pines and Hunter, 1994). By using
a GFP fusion protein, Clute and Pines (1999) demonstrated
in living HeLa cells, that cyclin B1 destruction starts
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precisely at the end of prometaphase. Proteolysis of both
A- and B-type cyclins depends on multi-subunit ubiquitin
ligase called the anaphase promoting complex (APC/C) or
cyclosome (Townsley and Ruderman, 1998; Zachariae and
Nasmyth, 1999). The destruction of A- and B-type cyclinsrequires a motif of nine amino acids in the N-terminal
domain of the cyclins called the destruction box (Dbox)
(Glotzer etal., 1991), but how these cyclins are recognised
by the ubiquitin ligase is not fully understood.
In plants, three groups of A-type cyclins (A1, A2 and A3)
and two groups of B-type cyclins (B1 and B2) have been
identied (Mironov etal., 1999; Renaudin etal., 1998).
There are multiple members belonging to each of these
groups in a single species. For example in tobacco, both
the cyclin B1 and the cyclin A3 groups contain at least
three members each. However, little is known regarding
the localisation, stability and function of these different
mitotic cyclins. Sub-cellular immunolocalisation experi-
ments performed in maize root tip cells indicated that the
maize B1 cyclin, Zeama;CYCB1;2, behaves like animal B1
cyclins since it relocates to the nucleus in prophase and
disappears at anaphase (Mews etal., 1997). Surprisingly
another B1 cyclin, Zeama;CYCB1;1, is predominantly
nuclear localised during the entire cell cycle and does
not seem to be degraded at the exit of mitosis.
Previously, we demonstrated the existence of the Dbox
pathway in plants by showing that the N-terminal domains
of two tobacco mitotic cyclins (Nicta;CycA3;1 of cyclin
group A3 and Nicta;CycB1;1 of cyclin group B1) direct the
specic degradation of the chloramphenicol acetyltrans-
ferase (CAT) reporter protein at the exit of mitosis and that
this degradation is proteasome-dependent (Genschik etal.,
1998). More recently we showed that endogenous cyclinB1 (Nicta;CycB1;1) protein is subjected to cell cycle
dependent-proteolysis (Criqui etal., 2000). Here, we inves-
tigated the sub-cellular localisation, during the time course
of the cell cycle, of two B1-type cyclins and one A3-type
cyclin in tobacco.
Results
Subcellular localisation of the GFP-tagged cyclin B1;1
and cyclin A3;1 during the cell cycle
The coding sequences of tobacco cyclin B1;1
(Nicta;CycB1;1) and cyclin A3;1 (Nicta;CycA3;1) wereexpressed as fusion proteins with the Green Fluorescent
Protein (GFP) in transgenic BY2 cells. Mutations were
introduced to the destruction box of Cyclin B1;1 and also
fused to GFP (Figure 1). Visualisation of GFP-tagged
proteins in living cells has many advantages compared
with the immunouorescence techniques, since it does not
require the permeabilisation and xation of the cells (Clute
and Pines, 1999). We believe that the fusion proteins are
good reporters for the endogenous cyclins since (1) we
have shown that the cyclin B1;1-GFP fusion protein was
destroyed at the exit of mitosis with a similar kinetic than
the endogenous Cyclin B1 protein; and (2) after immuno-
precipitation using a polyclonal anti-GFP antibody, we
could demonstrate with histone H1 kinase assays, that the
fusion protein was able to bind and activate CDK(s) (Criqui
etal., 2000).
The tobacco cyclin B1;1-GFP and cyclin A3;1-GFP (as
well as native GFP as a control) were put under the control
of the dexamethasone (Dex) inducible promoter (Figure 1).
Clonal cell cultures were established from transgenic BY2
tobacco lines. For the cyclin B1;1-GFP expression and
localisation studies presented in this paper, we used clonal
transgenic cell cultures expressing the fusion protein at a
similar level to endogenous cyclin B1;1 (data not shown),
but the same results were obtained if the fusion protein
was expressed at higher levels. Synchronisation of the cell
cultures were conducted as previously published (Criqui
etal., 2000) and repeated at least twice for each construct.
In order to accumulate the fusion proteins, 5 mM of Dex
was added to the cultures during the 24 h aphidicolin
treatment and this concentration of the glucocorticoid was
renewed in the culture medium after aphidicolin release.
Under the conditions used, no detectable uorescence was
found in the untransformed BY2 cells or in the non-
induced transgenic cell cultures.
Figure 1. Schematic representation of the constructs used for thelocalisation studies.
The cyclin B1;1 and A3;1 constructs, as well as GFP alone, were put
under the control of the Dexamethasone inducible promoter of pTA7002
binary vector. The cyclin B1;3 construct was put under the control of the
tetracycline inducible 35S promoter. Open boxes represent the cyclin
domains and the grey boxes the GFP sequence. The Dbox is diamond-
shaped (native in white and mutated in black).
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From S-phase (1 h after aphidicolin removal) to G2 (46 h
after aphidicolin removal, depending on the synchronisa-
tion experiment), the cyclin B1-GFP fusion protein was
found in some cells more abundantly in the cytoplasm
whereas in others more in the nucleus (Figure 2a,b).
Contrary to the cyclin B1;1-GFP fusion proteins, cyclin
A3;1-GFP chimeric protein was found exclusively in the
nucleus and curiously also in the nucleoli, but never in the
cytoplasm during S-phase and G2 (Figure 2c). Since the
Cyclin-GFP fusions are above the size exclusion of the
nuclear pores (Chytilova etal., 2000), these results indicate
that both cyclins are transported to the nucleus by an
active mechanism. GFP alone was found in both cytoplas-
mic and nuclear compartments with a stronger staining in
the nucleus (data not shown and Grebenok etal., 1997).
As soon as the cells entered mitosis, Cyclin B1;1-GFP
fusion protein was found on the condensing nuclear
material (Figure 2d). This binding to the chromosomes
could be detected until metaphase (Figure 2e), whereas
native GFP could never be detected on condensed
chromatin, such as on metaphase chromosomes (Figure
2f). No mitotic spindle-like structures were found in these
cells, indicating that the chromosomes are the major
targets of the Cyclin B1;1 protein. The mitotic spindle can
Figure 2. Sub-cellular localisation of cyclin B1;1-GFP, cyclin A3;1-GFP and non-degradable cyclin B1;1-GFP fusion proteins during the cell cycle in
transgenic BY2 cells.
Each uorescent focal plane is shown with its corresponding transmitted light reference image viewed by DIC.
(ab) Cyclin B1;1-GFP fusion protein in Dex induced cells 4 h (a) and 6 h (b) after aphidicolin removal.
(c) Cyclin A3;1-GFP localisation in Dex induced cells 5 h after aphidicolin removal.
(de) Localisation of cyclin B1;1-GFP protein during prophase (D) and metaphase (e).
(f) Cellular localisation of the GFP protein in Dex induced cells during metaphase.
(g) Cellular localisation of the GFP-MAP4 fusion protein during metaphase.
(h) Localisation of cyclin B1;1-GFP protein during anaphase.
(ik) non-degradable cyclin B1;1-GFP fusion protein localisation in Dex induced cells during metaphase (i), during anaphase (j) and during telophase (k).
For image (i): projection of 32 optical sections taken 0,45 mm apart.
(l) Non-degradable cyclin B1;1-GFP fusion protein localisation in a cell after 48 h induction by Dex observed by epiuorescence. Bars = 10 mm.
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be readily visualised in BY2 cells expressing a fusion
protein between GFP and the microtubule-binding domain
of mammalian microtubule-associated protein 4 (MAP4)
(Figure 2g; Granger and Cyr, 2000). In anaphase and
telophase cells, no cyclin B1;1-GFP protein was detectable
any more, indicating that the protein had been degraded
(Figure 2h). In contrast to the B1-type cyclin, cyclin A3;1-
GFP could never be detected in cells undergoing mitosis.
Non-degradable cyclin B1;1-GFP fusion protein remained
on the chromosomes after metaphase.
Sub-cellular localisation of a non-degradable version of
the cyclin B1;1-GFP fusion protein, in which the destruc-
tion box had been mutated, was also analysed during the
time course of the cell cycle. The non-degradable fusion
protein behaved very similarly to cyclin B1;1-GFP until
metaphase. Thus the cyclin B1;1(DDbox)-GFP protein was
found both in the cytoplasm and in the nucleus during G2
(data not shown) and was also found to localise with
chromosomes during mitosis (Figure 2i). However, in
contrast to wild-type cyclin B1;1-GFP, the fusion protein
was not degraded after metaphase and remained associ-
ated with chromosomes during anaphase (Figure 2j).
During telophase, the protein was still detectable on the
decondensing chromosomes, but never in the phragmo-
plast region at cytokinesis (Figure 2k). Furthermore, the
overexpression of the non-degradable cyclin inhibited
Figure 3. Real time localisation of cyclin B1;3-GFP fusion protein in a tobacco cell suspension culture from G2-to-prometaphase and from metaphase-to-
anaphase.
Each uorescent focal plane (left panels) is shown with its corresponding transmitted light reference image viewed by DIC (right panels).
(ac) Visualisation of the Cyclin B1;3-GFP fusion protein in a tetracycline induced cell followed during late G2 (a), prophase (b), and prometaphase (c).
(df) Visualisation of the Cyclin B1;3-GFP fusion protein in a tetracycline induced cell followed during metaphase (d), early anaphase (e) and
late anaphase (f).
(g) Visualisation of CyclinB1;3-GFP in a tetracycline induced cell during progression from prophase to metaphase until the onset of anaphase with
micrographs taken every 10 min. Bars = 10 mm.
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neither chromosome decondensation nor nuclear envel-
ope reformation (data not shown). These data are in
agreement with our previously published results showing
that mitotic exit is possible without cyclin B1 degradation,
at least to a certain extent (Criqui etal., 2000).
Nevertheless, after a longer period of Dex induction (48 hof treatment) we observed a number of abnormal cells
having either enlarged nuclei or several nuclei within the
same cell (Figure 2l). Such cells were never found in the
Dex-induced cell culture expressing GFP alone. Some of
these abnormal cells looked similar to MG132-treated
anaphase cells previously reported (Genschik etal., 1998)
and will be analysed in more details.
Nicta;CycB1;3 the third member of the tobacco B1-type
cyclins, also binds to the chromosomes and is destroyed
at the onset of anaphase.
There are at least two other B1-type cyclins (Nicta;CycB1;2and Nicta;CycB1;3) in tobacco which share strong
sequence similarities. Like Nicta;CycB1;1, both cyclins are
also expressed during G2 and early mitosis (Ito, 2000).
Thus we decided to investigate the localisation and
stability of another member of the B1-type cyclins from
tobacco. Transgenic tobacco plants, expressing the GFP-
tagged cyclin B1;3 under the control of the tetracycline
inducible promoter, were used to generate a cell suspen-
sion culture. The CycB1;3-GFP localisation studies were
performed in the asynchronously growing cell suspension
culture, 12 h after tetracycline induction (0,1 mg l1).
In interphase cells, the fusion protein was found both in
the cytoplasm and in the nucleus (data not shown). We
used time-lapse uorescence imaging to follow the cyclin
localisation and proteolysis in cells undergoing mitosis
(Figure 3). In order to avoid photobleaching, the cells were
followed for less than 1 h and uorescence and DIC images
were captured sequentially. Two periods of the cell cycle
were particularly investigated: the entry into mitosis and
the metaphase to anaphase transition. In late G2, the
CycB1;3-GFP was found both in the cytoplasm and in the
nucleus with a stronger staining in the proximity to the
nuclear envelope (Figure 3a). This ring-like structure of
uorescence around the nucleus was transient and could
only be detected before it moved on the condensing
chromosomes (Figure 3b,c). When cells were followed
from metaphase to anaphase, we found that the signal
became considerably weaker, but still detectable, at the
onset of anaphase but completely disappeared as cells
progressed in anaphase (Figure 3df). To further demon-
strate, that CycB1;3-GFP starts to degrade already in
metaphase cells, we made time-lapse images from begin-
ning of mitosis with a strong chromatin-bound GFP-
uorescence until the signal started to disappear during
metaphase, as illustrated in Figure 3(g) in which images
were sequentially recorded in 10-min time intervals start-
ing from prophase. These results were very similar to the
cyclin B1;1-GFP localisation studies (see above) suggest-
ing that both cyclins may play similar roles during mitosis.
B1-type cyclin binding to chromatin in non-ionic
detergent extracted cells
To further characterise the association of B1-type cyclins
with chromatin and the mitotic spindle, we extracted cells
expressing the CyclinB1-GFP fusion proteins with a mild
non-ionic detergent. Under these conditions membranes
and the nuclear envelope are dissolved and soluble
proteins extracted but microtubules are stabilised (Figure
4). As a positive control for the extraction assays, we used
tobacco cells expressing GFP as a fusion with a CDKA
protein (Medsa;CDKA;2), which is strongly bound to the
nuclear material during interphase and to the mitotic
spindle in metaphase cells (Weingartner etal., 2001). GFPwas completely extracted by this treatment (Figure 4ab)
whereas a strong binding was retained in the nucleus and
on the mitotic spindle in cells expressing CDKA-GFP
(Figure 4c). After extraction, all the cyclin-GFP fusion
proteins were still detected on the chromosomes in
metaphase or anaphase cells (Figure 4df). A very weak
signal also persisted in the spindle zone, suggesting that a
low amount of the cyclin could be associated with this
structure. Co-immunoprecipitation experiments, using
either anti-GFP or anti-cyclin B1;1 antibodies, failed to
detect the a tubulin protein by Western blotting (data not
shown). Thus the B1-type cyclins are tightly bound to the
chromosomes, but we cannot exclude the possibility that
low amounts of these proteins are also bound to the
mitotic spindle.
Endogenous cyclin B1 protein accumulates in oryzalin
and propyzamide treated cells
Microtubule destabilising drugs are known to activate the
spindle checkpoint which blocks sister chromatid separ-
ation and cyclin B degradation (Whiteld etal., 1990; Clute
and Pines, 1999; Hunt etal., 1992). We previously showed
that the use of such drugs block the Dbox pathway in plant
cells (Genschik etal., 1998). We thus investigated endo-
genous cyclin B1 stability in BY2 cells upon treatments
with the antitubulin drugs: oryzalin or propyzamide. The
cells were rst synchronised with aphidicolin and when
20% of cells entered prophase (5 h after aphidicolin
removal), the cell culture was subdivided and treated
either by oryzalin or propyzamide. Three hours after the
treatments (8 h after aphidicolin removal), 70% of the cells
in both cultures reached a metaphase-like arrest (Figure
5a). Although the level of cyclin B1 mRNA strongly
decreased during the course of the drug treatments
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Figure
7.Legendonfacingpage
Figure
4.
Detergentextractionassays.
(ab)Fluorescentmicroscopicimagesofnon-extracted
(a)anddetergentextracted
(b)cellsexpressingGFPandthecorre
spondingDICimages(below).
(c)FluorescentmicroscopicimageofdetergentextractedcellsexpressingCDKA-GFP
andthecorrespondingDICimages(below).
(df)Fluorescentmicroscopic
images
ofdegradable
Cyclin
B1;1-GFP
(d),non-
degradableCyclinB1;1-GFP(e)andC
yclinB1;3-GFP(f)indetergentextractedcells
andthecorrespondingDICimages(below).
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(Figure 5b), cyclin B1 protein accumulated and was stable
in both drug-treated cultures (Figure 5c). Because the
cyclin B1 mRNA declined whereas CycB1 protein accumu-
lated after 5 h of drug treatments (lanes 10 h after
aphidicolin removal, Figure 5b) we conclude that Cyclin
B1 becomes stabilised when the spindle checkpoint is
activated.
Sub-cellular localisation of cyclin-GFP fusion proteins in
oryzalin and MG132-treated cells
Since we showed that endogenous cyclin B1;1 degradation
is inhibited by both microtubule destabilising drugs (see
above) and by the proteasome inhibitor MG132 (Criqui
etal., 2000), we investigated the sub-cellular localisation of
the fusion proteins after drug-treatments. Cyclin B1;1-GFP
and cyclin A3;1-GFP transgenic cell cultures were rst
synchronised with aphidicolin in the presence of 5 mM Dex.
After the removal of the drug, the cell cultures were split
into three subcultures, which were either not treated or
treated with 10 mM oryzalin or with 100 mM MG132 (Figure6). The drugs were added when the cell cultures reached
around 10% of mitotic gures (mainly prophases).
As expected the cyclin B1;1-GFP mRNA and fusion
protein accumulated in both drug-treated cells (Figure
6b,c). At the exit of mitosis, the CycB1-GFP fusion protein
was barely detectable in the untreated cell culture, indi-
cating that it was degraded as previously published (Criqui
etal., 2000), but it accumulated when cells were arrested in
metaphase by activating the spindle checkpoint or when
the proteolysis was inhibited by MG132 (Figure 6).
Contrary to this, the Cyclin A3-GFP fusion protein did not
accumulate in the metaphase arrested cells, with MT drugs
or with the proteosome inhibitor, MG132. This might
indicate that Cyclin A3 degradation is more efcient than
Cyclin B1, and not completely blocked by MG132.
In MG132 metaphase-arrested cells, the chromosomes
are congregated at the equatorial plate (Genschik etal.,
1998). This cell-cycle arrest can be explained by proteolytic
inhibition of securin proteins (Nasmyth etal., 2000). In the
blocked cells, cyclin B1;1-GFP protein was found in asso-
ciation with the chromosomes (Figure 7a). However when
the cells were blocked for longer time, the signal started to
diffuse (Figure 7b) and even to diminish in some cells
(Figure 7c). Furthermore, like in untreated cells, the cyclin
B1-GFP fusion protein binds mainly to the chromosomes
and not to the mitotic spindle, which is present in the
MG132-metaphase-blocked cells (Genschik etal., 1998 and
data not shown).
Figure 5. Accumulation patterns of endogenous cyclin B1;1 mRNA and
protein in spindle checkpoint activated BY2 cells.
(a) Mitotic index was determined after aphidicolin removal. In early
mitosis (5 h after aphidicolin removal) 10 mM oryzalin or 6 mM
propyzamide were added to the cell culture. The percentage of mitotic
gures was determined at different times during the culture in the
presence of the antitubulin drugs (grey bars for oryzalin and hatched
bars for propyzamide).
(b) Gel blot analysis of RNAs extracted at different time points. Twenty
micrograms of total RNA was separated by electrophoresis on an
agarose-formaldehyde gel, transferred to a nylon membrane, and
hybridized successively with different probes as indicated.
(c) 15 mg of total proteins extracted at indicated time points were
separated by 10% SDS-PAGE and immunoblotted with afnity-puried
polyclonal anti-N-terminal cyclin B1;1 peptide antibody. Cyclin B1;1
protein band is indicated by an open arrow, whereas the asteriskindicates an aspecic protein band (Criqui etal., 2000). The blot was
subsequently striped and immunoblotted with the polyclonal anti-
PSTAIRE antibody.
Figure 7. Subcellular localisation of both cyclin B1;1-GFP and cyclin A3;1-GFP fusion proteins in oryzalin- and MG132-treated cells. Each uorescent focal
plane is shown with its corresponding transmitted light reference image viewed by DIC.
(a) Cyclin B1;1-GFP fusion protein in MG132-metaphase arrested cell, 2.5 h after the drug was added.
(b,c) Same as in (a), but 4.5 h after the drug addition.
(d,e) Cyclin B1;1-GFP localisation in oryzalin metaphase-arrested cells, 2.5 h after the drug addition.
(f) Cyclin A3;1-GFP localisation in MG132 metaphase-arrested cells, 5.5 h after the drug addition.
(g) Cyclin A3;1-GFP localisation in oryzalin blocked cells, 5.5 h after the drug addition. Bars =10 mm.
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In cells arrested in metaphase by oryzalin, the mitotic
spindle has been destroyed and the chromosomes are
dispersed throughout the cells. Again the fusion protein
strongly attached to the condensed chromosomes (Figure
7d,e).
In contrast to cyclin B1-GFP, the cyclin A3-GFP could not
be detected in the metaphase-arrested cells during the rst
hours of drug treatments (data not shown). More than 50
metaphase arrested cells, after either oryzalin or MG132
treatments, were analysed for cyclin A3-GFP localisation
and none of these cells exhibited a GFP signal above the
background. In accordance with this observation, no cyclin
A3-GFP protein could be detected during the rst hours of
drug treatments (Figure 6f). However at later stages some
cyclin A3-GFP could be detected (at least after 46 h of
drug treatments), which probably resulted from newly
synthesised protein accumulating in cells in which the
Dbox pathway has been switched off (Figure 7f,g). In those
cells, the uorescence was very low and never associated
with the chromosomes. These results suggest that the
degradation of cyclin A3;1-GFP fusion protein is switched
on during early mitosis.
Figure 6. Accumulation patterns of cyclin B1;1-GFP and cyclin A3;1-GFP mRNAs and fusion proteins in metaphase-arrested cells after oryzalin or MG132
treatments.
(a,d) Mitotic indexes were determined after aphidicolin removal for the cyclin B1;1-GFP (a) and cyclin A3;1-GFP (d) cell cultures. In early mitosis 10 mM
oryzalin or 100 mM MG132 were added to the cell cultures. The percentage of mitotic gures was determined at different times during the culture in the
presence of the antitubulin drug (grey bars) or the proteasome inhibitor (hatched bars) or no drug treatment (open bares).
(b,e) Gel blot analysis of RNAs extracted from cyclin B1;1-GFP (b) and cyclin A3;1-GFP (e) transgenic cell cultures at different time points of the
synchronisation experiment. Twenty micrograms of total RNA was separated by electrophoresis on an agarose-formaldehyde gel, transferred to a nylon
membrane, and hybridised successively with different probes as indicated. The mRNA bands corresponding to the ectopically expressed cyclins are
indicated by asterisks whereas the endogenous cyclin mRNA bands are indicated by arrows.
(c,f) 15 mg of total proteins, extracted from cyclin B1;1-GFP (c) and cyclin A3;1-GFP (f) transgenic cell cultures at indicated time points, were separated by
10% SDS-PAGE and immunoblotted with either a polyclonal anti-GFP antibody or an afnity-puried polyclonal anti-N-terminal cyclin B1;1 peptide
antibody. The blots were subsequently stripped and immunoblotted with the polyclonal anti-PSTAIRE antibody.
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Histone H1 kinase activity in oryzalin and MG132-treated
cells
In order to see if the Cyclin B1;1-GFP protein is bound to
and activates a CDK in the oryzalin and MG132-mitotic
cells, we performed immunoprecipitation experiments
using a polyclonal anti-GFP antibody (Figure 8). As a
control, we used a mitotic extract prepared from trans-
genic BY2 cells expressing GFP alone. H1 kinase activity
could be detected in fractions puried by the GFP-specic
antibody from Cyclin B1;1-GFP-expressing cells, but not
from GFP-expressing cells, demonstrating that the Cyclin-
GFP fusion specically associates with an active kinase,similar to the endogenous cyclin. Interestingly, MG132
was less effective at increasing Cyclin B1;1-GFP-associated
H1-kinase activity than oryzalin, while the H1 kinase
activity puried by p13suc1 was similarly high both in
MG132 and oryzaline-treated cells (Figure 8c). This might
indicate the presence of another mitotic cyclin than Cyclin
B1, the degradation of which is effectively inhibited by
MG132.
Discussion
The temporal destruction of cyclin B1 protein has been re-
investigated in real time of the cell cycle in animal cellsusing cyclin B1-GFP fusion protein (Clute and Pines, 1999).
This technique allowed the authors to demonstrate that
the degradation of cyclin B1 starts as soon as the last
chromosome aligns on metaphase plate. Surprisingly,
immuno-uorescence studies indicated that some of the
plant A-type and B-type cyclins might remain stable until
the end of mitosis (Mironov etal., 1999). Here we decided
to investigate the cyclin stability in unxed living cells
using Cyclin-GFP fusions.
During interphase, animal cyclin B1 is found predomin-
antly in the cytoplasm, but transits precipitously into the
nucleus at the G2/M transition. In plant cells, we have not
found such a clear relocalisation of the cyclin B1 proteins.
The cytoplasmic localisation of the animal cyclin B1 is a
result of its dynamic shuttle in between the nucleus and
cytoplasm. Because we could nd a variable amount of B1-
type cyclins in the nucleus, it might be that in some cells
this nuclear-cytoplasmic transport is less efcient. Time-
lapse experiments revealed the dynamic re-localisation of
cyclin B1, as G2 cells entered into mitosis. In late G2-cells
the cyclin B1;3-GFP fusion accumulated around the nuclear
envelope, as indicated by the phase-contrast image.
Subsequently, all cyclin B1;3-GFP became accumulated
on the chromatin. This suggests that the plant B1-type
cyclin proteins are also actively transported into the
nucleus just as cells enter into mitosis. Interestingly,
plant and animal B1-type cyclins differ by the presence of
a nuclear localisation signal (NLS) (Renaudin etal., 1998).
Thus animal B1-type cyclins do not carry the NLS motifs,
but at least in humans, it seems that the interaction of
cyclin B1 with cyclin F (carrying the NLS) is required for the
nuclear localisation of cyclin B1 (Kong etal., 2000).
However during interphase, the cyclin is efciently
exported from the nucleus due to a leucine-rich nuclear
export sequence (NES) (See Hagting etal., 1998; Yang
Figure 8. Histone H1 kinase activity in untreated, oryzalin and MG132
treated synchronised cyclin B1;1-GFP and GFP cells.
(a) Cyclin B1;1-GFP cells were synchronised by aphidicolin. When cells
entered mitosis 10 mM oryzalin or 100 mM MG132 was added to the cell
culture. The percentage of mitotic gures was determined 3 h after drug
addition in the untreated and oryzalin and MG132 treated cell cultures. A
transgenic cell culture expressing GFP alone was also synchronised and
used here has a control for the immunoprecipitation assays.
(b) Histone H1 kinase activity detected upon immunoprecipitation with
anti-GFP antibodies. One hundred mg of extracted proteins was
precipitated with the anti-GFP antibodies. The bound kinase activities
were assayed in presence of histone H1 and the phosphorylated proteins
were resolved by SDS-PAGE (upper panel). Histone H1 loading was
controlled by Coomassie brilliant blue R250 staining (bottom panel).
(c) Histone H1 kinase activity detected in the p13
suc1
-sepharose boundprotein fractions. Phosphorylated histone H1 was visualised by
autoradiography (upper panel) and histone H1 loading was controlled by
Coomassie brilliant blue R250 staining (bottom panel).
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etal., 1998). The inspection of the tobacco cyclin B1;1
sequence indicates the presence of both a putative
bipartite NLS and NES sequences in the N-terminal
domain of the protein. Additional experiments will have
to be performed in order to determine if and how these
motifs are involved in the localisation of the plant cyclin.During mitosis, we found the cyclin B1-GFP fusion
proteins strongly associated with the chromosomes until
metaphase while it gradually disappeared during ana-
phase. These results are similar to the human cyclin B1 as
reported by Clute and Pines (1999). However, whereas the
non-degradable HeLa cyclin B1-GFP fusion protein was
unable to bind the chromosomes (Clute and Pines, 1999),
we found the plant non-degradable cyclin B1;1-GFP fusion
protein associated with the chromosomes all along the
mitotic events. Whether cyclin B1 degradation starts
already on the chromosomes remains to be demonstrated.
Localisation of the APC/C complex in relation to cyclin B1,
is currently under investigation.In plants, only a limited number of cyclin localisation
studies have been reported so far (Mironov etal., 1999).
Indirect immunouorescence was used in maize root tip
cells to localise three different B-type cyclins (Mews etal.,
1997). The localisation of the tobacco cyclin B1-GFP fusion
proteins is consistent with the localisation of one maize B-
type cyclin studied (e.g. Zeama; CYCB1; 2), but not with the
two others (Zeama;CYCB1;1 and Zeama; CYCB2;1) which
were never found to associate with the condensing
chromosomes (Mews etal., 1997). Interestingly, only
Zeama;CYCB1;2, like the tobacco B1-type cyclins, is sub-
jected to proteolysis.
In human cells the cyclin B1 is involved in different
mitotic events. Among them, it has been proposed that the
role of cyclin B1/Cdc2 kinase is to phosphorylate and
disassemble the nuclear lamina in order to promote
nuclear envelope breakdown. Indeed, a phosphorylation-
decient mutant of cyclin B1, which is unable to translo-
cate into the nucleus, cannot promote germinal-vesicle
breakdown in maturing frog oocytes (Li etal., 1997). In
addition, it has been documented that cyclin B-Cdk1 kinase
is involved in mitotic chromosome condensation (Kimura
etal., 1998; Zachariae, 1999). Both the localisation of
tobacco B1 cyclins around the nuclear envelope at the
G2-to-M transition and their strong binding to chromo-
somes argue that this class of plant cyclins may play
similar functions. However, it is also possible that the plant
B1-type cyclins have some other functions associated with
the nuclear material. It was demonstrated, at least in
animal cells, that cdc2/cyclin B can inhibit the transcription
of both RNA polymerase I and II by direct phosphorylation
of components of the transcription machinery (Heix etal.,
1998; Leresche etal., 1996).
In animal cells, cyclin B1 only associates with CDK1
(cdc2) to full its mitotic function. In plants the identity of
the CDK partner(s) of cyclin B1 proteins has not yet been
clearly determined (Mironov etal., 1999; Stals etal., 2000).
Immunolocalisation of A-type CDKs has been assayed in
different plant systems (Mironov etal., 1999) and has
sometimes led to contradictory results. For example, in
maize an A-type CDK does not bind the chromosomes(Mews etal., 1997) whereas in alfalfa root tip cells, in
addition to a number of cytoskeletal structures, a member
of this CDK class also binds transiently to the chromo-
somes at the metaphase-anaphase transition (Stals etal.,
1997). The signicance of this localisation on metaphase
chromosomes remains to be determined. Visualisation of
the GFP-tagged CDK Medsa;CDK;A;2 in living cells allowed
the authors to show that the kinase associates strongly
with condensing chromosomes but leaves the chromatin
before metaphase (Weingartner etal., 2001; Figure 4).
Thus, B1-type cyclins may well interact with A-type CDKs
to eventually trigger chromosome condensation and NEB,
nevertheless the CDK partner(s) of these cyclins duringmetaphase remains to be determined.
Human cyclin B1 strongly binds to the mitotic spindle
(mainly the poles). At least in animal cells, the interaction
of cyclin B1 with microtubules is believed to be important
for the M-phase microtubule dynamic and this may be
controlled through phosphorylation of MAP4 by p34cdc2
kinase (Ookata etal., 1995; Ookata etal., 1997). From our
sub-cellular localisation experiments conducted in live
cells or in cells extracted with non-ionic detergent, no clear
association of the B1-type cyclins with the mitotic spindle
could be demonstrated. Nevertheless, since some cyclin-
GFP signal persisted in the spindle zone in the extracted
cells, we do not rule out the possibility that a low amount
of the tobacco cyclin B1-GFP fusion proteins bound this
structure. Furthermore, it is possible that another B1-type
cyclin variant or a B2-type cyclin (which are also expressed
during mitosis) binds more specically to this structure.
Class 3 of A-type cyclins, to which belongs Nicta;CycA3;1
studied here, are expressed very early during the cell cycle,
at the G1/S transition, and their transcripts declined to low
levels when cell entered mitosis (Reichheld etal., 1996).
Our data show that this cyclin is predominantly in the
nucleus and also the nucleoli. Furthermore, we were never
able to detect the cyclin A3;1-GFP fusion protein during
mitosis, suggesting that the fusion protein had already
disappeared in late G2 or early prophase. If the fusion
protein was allowed to accumulate during mitosis, using
proteasome inhibitors, it remained diffused and was not
found associated with the mitotic spindle or chromo-
somes. Our data suggest that the A3-type cyclins play a
role in S-phase or G2/M but not during mitosis.
Furthermore, as in animal cells (Hunt etal., 1992;
Minshull etal., 1990; Whiteld etal., 1990), the plant
A-type cyclin is destroyed during the cell cycle earlier
than cyclin B1. Interestingly, another A-type cyclin
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(Medsa;CycA2) is also nuclear localised and its proteolysis
seems to start in early M phase (Roudier etal., 2000).
Experimental procedures
Unless stated otherwise, all procedures for manipulating DNA
and RNA were carried out according to Sambrook etal. (1989) and
Ausubel etal. (1994).
Chemicals
Propyzamide was from Sumitomo Chemical Co. (Osaka, Japan).
Dexamethasone (Sigma, Saint Quentin Fallaver, France) was
dissolved in ethanol and kept at a concentration of 30 mM.
Tetracycline (Sigma) was dissolved in water and kept at a
concentration of 1 mg ml1, Carbobenzoxyl-leucinyl-leucinyl-leuc-
inal (MG132) was from PEPTIDES International, Inc (Louisville,
KY, USA). The drug was dissolved in DMSO and was never kept
for longer than 1 month at 20C.
Cyclin constructs
Constructs pTACycB1;1-GFP, pTAmutDboxCycB1;1-GFP and
pTAGFP are described in Criqui etal. (2000). For construct
pTACycA3;1-GFP, we used PCR-based site-directed mutagenesis
to introduce Xho1 and BamH1 restriction sites upstream and
downstream of the coding region of cyclin A3;1 (Nicta; CycA3;1),
using oligonucleotide 1 (5-AAACCACTCGAGTGAATGGCGAACG-
AAGAAAATAAG-3) and oligonucleotide 2 (5-TTCTTTGGATCC-
AGCATCGTCAAAAAAACAAGC-3), respectively. A BamH1-Spe1
GFP fragment, obtained by PCR, was subsequently introduced in
frame at the C-terminus of the cyclin A protein and resulted inplasmids pSKCycA-GFP. The Xho1-Spe1 DNA fragments was
subcloned into the dexamethasone inducible vector pTA7002
(Aoyama and Chua, 1997; resulting in plasmid pTACycA3;1-GFP.
To construct BinHygTX-CycB1;3-GFP we introduced by PCR-
based site directed mutagenesis a NcoI-site upstream of the
coding region of cycB1;3 using oligonucleotide 3 (5-AAAGG-
ATCCCTGCCATGGCTTCAAGAAACGTTCTTCAACAG-3) and a
NotI site just upstream of the STOP codon allowing in frame
fusion with the GFP followed by a BamHI site using oligonucleo-
tide 4 (5-AAGGATCCTAGCGGCCGCCTTCATATGAAAGAGCAG-
CCAAGAG-3). The resulting PCR product was placed as a NcoI-
BamHI fragment into plasmid pSP72-TMV in frame with a TMV-
omega 5 untranslated leader sequence for enhanced translation.
A GFPs65T sequence was subsequently introduced in frame at the
3-end of the CycB1; 3 sequence as a NotI-NotI fragment resulting
in plasmid pSP72-TMV-CycB1;3-GFP. The TMV-CycB1;3-GFP frag-
ment was subcloned as a KpnI-XbaI fragment into the tetracycline
inducible plant expression vector pBinHyg-TX resulting in the
plasmid pBinHygTX-TMV-CycB1;3-GFP. Plasmid pCAMBIA-GFP-
MAP4 was a gift of Ton Timmers (Laboratoire de Biologie
Moleculaire des Relations Plantes-Microorganisms, CNRS/INRA,
31326 Castanet Tolosan Cedex, France) and consists of the GFP-
MAP4 coding sequence (Marc etal., 1998) put under the control of
the constitutive 35S promoter of pCAMBIA1390 vector (CAMBIA).
Plasmid pBinHyg-TX-CDKA-GFP allowing the expression of the
Medsa;CDK;A;2-GFP fusion protein under tetracycline control is
described in (Weingartner etal., 2001).
Tobacco cell culture, transformation and synchronisation
Clonal transgenic BY2 (Nicotiana tabacum cv Bright Yellow 2)
cell cultures for pTACycB;1-GFP, pTAmutDboxCycB;1-GFP,
pTACycA;1-GFP, pCAMBIA-GFP-MAP4 and pTAGFP constructs
were established. The Agrobacterium tumefaciens-mediated
transformation protocol of the BY2 cells is described in Criqui
etal. (2000). The handling of the BY2 cell culture, as well as the
synchronisation experiments, were performed according to
Nagata etal. (1992). In order to produce a strong accumulation
of the cyclin-GFP fusion proteins into the transgenic cell lines,
5 mM of dexamethasone was already added during the 24 h
aphidicolin treatment and the cells were re-induced with the same
concentration of Dex, just after the washing step.
The transgenic tobacco cell suspension culture was generated
as follows: A Nicotiana tabaccum Petit Hsavanna Samsun line
harbouring the tet-repressor (Gatz etal., 1992) was transformed
with plasmid pBinHygTX-TMV-CycB1;3-GFP by Agrobacterium
mediated leaf disk transformation. Several independent trans-
genic lines were grown up and propagated on solid MS medium
containing 40 mg l1 Hygromycin B. Two of them were used to
generate suspension cultured cell lines: stem segments of sterile
grown plants were placed on MS medium containing 0.5 mg l1
2,4D and 40 mg l1 Hygromycin B for induction of calli, which
were transferred into liquid MS medium, supplemented with
1 mg l1 2,4D and 40 mg l1 Hygromycin B. The cultures were
maintained in the dark under continuous shaking.
Detergent extraction of cells
Cells were detergent-extracted essentially as has been previously
described for preparing cytoskeletons (Chan etal., 1996). Cells
were incubated in the enzyme solution (1% [w/v] Cellulase R10,
0.2% [w/v] Mazerozyme, 0.45 M Sorbitol, made in PME (0.1 M
Pipes, 1 mM MgSO4, 1 mM EGTA, pH = 6.9) for 15 min. Cells with
partially digested cell walls were washed twice in PME with
0.45M
Sorbitol and incubated in Extraction buffer (10% [w/v]DMSO, 0.05% [w/v] NP40, 0.45 M Sorbitol made in PME) for
15 min. The resulting detergent-extracted cells were washed
twice in wash 2 (10% DMSO in PME) and directly observed in
the uorescent microscope.
RNA gel blotting
RNA gels were realised with 20 mg of total RNA per lane. The RNA
extraction and RNA gel blotting procedures are described in
Criqui etal. (2000). The histone H4 probe corresponds to the 196-
bp restriction fragment AccI-DdeI of the coding region of the gene
H4A748(Chaboute etal., 1987). The cyclin B1;1 probe corresponds
to the Nicta;CycB1;1 cDNA (Qin etal., 1996). The integrity and the
amount of RNA applied to each lane were veried by EtBr staining
and control hybridisations using an Arabidopsis 25S rRNA probe(GenBank Acc. T44938).
Immunoblotting, immunoprecipitations, p13Suc1-
sepharose afnity binding and histone H1 kinase assays
The production of the polyclonal antibody against tobacco cyclin
B1;1 is described in Criqui etal. (2000). Samples of 15 mg of
proteins were separated by 10% SDS-PAGE (sodium dodecylsul-
fate-polyacrylamide gel electrophoresis) gels and transferred to
Immobilon-P membrane (Millipore, Bedford, MA, USA). The
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membranes were probed with the afnity puried anticyclin
antibody diluted 1: 4000. The Cdc2 (PSTAIRE) afnity puried
polyclonal rabbit antibody (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA) was used at dilution of 1: 4500. The GFP
polyclonal rabbit antibody was used at dilution of 1: 8000. The
immunoreactive proteins were detected using peroxidase-conju-
gated goat antirabbit antibodies (Dianova, Hamburg, Germany)
and the ECL Western blot analysis system from Amersham.
Immunoprecipitation with polyclonal anti-GFP antibodies and
p13Suc1-sepharose afnity binding procedures are described in
Criqui etal. (2000). Histone H1 kinase reactions were performed as
described by Magyar etal. (1993).
Confocal imaging and time lapse imaging
Confocal images were obtained by a Zeiss (Jena, Germany)
LSM510 laser-scanning confocal microscope with argon laser
exitation at 488 nm and through 505550 emission lter-set and
using a C-APOCHROMAT (63 Q 1,2 W Korr) water objective lens.
The images are presented as single sections or stacks of
neighbouring sections as stated in the gure legends.
Transmitted light reference images were taken using differentialinterference contrast (Nomarski) optics and argon laser illumin-
ation at 488 nm. LSM 510 3D reconstruction functions were
employed to compute projections of serial confocal sections.
Time-lapse images were taken using a charge coupled device
camera (Diagnostic instruments, model SPOT) mounted on an
upright uorescence microscope (Zeiss Axioplan) equipped with
a GFP lter (AF; HQ480/20X; HQ510/20 M) for uorescence
images. Differential interference contrast (Nomarski) was used
for transmission light images. Images were contrast enhanced
using image-processing software (Photoshop; Adobe Systems
Inc., Mountain View, CA, USA).
Acknowledgements
We thank Nam-Hai Chua for the inducible vector pTA7002, Henry
Wintz for the pCK-GFP3 vector, Catherine Bergounioux for the
cyclin Nicta;CycB1;1 cDNA, Nicole Chaubet for the cyclin Nicta;
CycA3;1 cDNA, Masaki Ito for the Nicta;CycB1;3 cDNA, Ton
Timmers for the pCAMBIA-GFP-MAP4 construct, Marc Boutry
and Geoffrey Duby for the GFP polyclonal antibody, Tobacco
Science Research Laboratory, Japan Tobacco, Inc, for allowing us
to use the TBY2 cell suspension, the Arabidopsis Biological
Resource Centre for providing the 25SRNA clone, l'ULP de
Strasbourg, CNRS, ARC, La Ligue Nationale Contre le Cancer
and Region Alsace for founding the confocal microscope, Philippe
Hammann for DNA sequencing, and Anne-Marie Lambert for
critical reading of the manuscript. M. W. was funded by a PhD
stipend from the Austrian Academy of Sciences. The work in L. B.
laboratory was funded by a Biotechnology and Biological Science
Research Council grant 111/P13340 and the work in E. E.-B.
laboratory was funded by the EU Framework 5 project, ECCO.
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