3-Sub-Cellular Localisation of GFP-Tagged Tobacco Mitotic Cyclins During the Cell Cycle and After Spindle Checkpoint Activation

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 [email protected]).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

The Plant Journal (2001) 28(5), 569581

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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).

570 Marie Claire Criqui et al.

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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.

Localization of GFP-tagged cyclins in tobacco cells 571



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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




12/13

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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3-Sub-Cellular Localisation of GFP-Tagged Tobacco Mitotic Cyclins During the Cell Cycle and After Spindle Checkpoint Activation