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

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation
Marie Claire Criqui1,, Magdalena Weingartner2,, Arnaud Capron1, Yves Parmentier1, Wen-Hui Shen1, Erwin Heberle-Bors2, Laszlo Bo gre3 and Pascal Genschik1,* 1 culaire des Plantes du CNRS, 12 rue du Ge ne ral Zimmer, 67084 Strasbourg Ce dex, France, Institut de Biologie Mole 2 Vienna Biocenter, Institute of Microbiology and Genetics, Dr. Bohrgasse 9, 1030 Vienna, Austria, and, 3 School 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 GFPtagged 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 in the 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 et al., 1995; Kreutzer et al., 1995; Minshull et al., 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 complex 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 et al., 1998;
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Murphy et al., 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 et al., 1998). The sub-cellular localisation of both A- and B-type cyclins has been documented in animal cells. In HeLa cells, 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 569

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Marie Claire Criqui et al. 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 acetyltransferase (CAT) reporter protein at the exit of mitosis and that this degradation is proteasome-dependent (Genschik et al., 1998). More recently we showed that endogenous cyclin B1 (Nicta;CycB1;1) protein is subjected to cell cycle dependent-proteolysis (Criqui et al., 2000). Here, we investigated 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

Figure 1. Schematic representation of the constructs used for the localisation 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 diamondshaped (native in white and mutated in black).

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 cyclins requires a motif of nine amino acids in the N-terminal domain of the cyclins called the destruction box (Dbox) (Glotzer et al., 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 et al., 1999; Renaudin et al., 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 experiments 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 et al., 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

The coding sequences of tobacco cyclin B1;1 (Nicta;CycB1;1) and cyclin A3;1 (Nicta;CycA3;1) were expressed 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 immunoprecipitation 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 et al., 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 et al., 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 noninduced transgenic cell cultures.
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Localization of GFP-tagged cyclins in tobacco cells 571

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.

From S-phase (1 h after aphidicolin removal) to G2 (46 h after aphidicolin removal, depending on the synchronisation 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 et al., 2000), these results indicate that both cyclins are transported to the nucleus by an
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active mechanism. GFP alone was found in both cytoplasmic and nuclear compartments with a stronger staining in the nucleus (data not shown and Grebenok et al., 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

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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-toanaphase. 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.

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;1GFP 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 associated with chromosomes during anaphase (Figure 2j). During telophase, the protein was still detectable on the decondensing chromosomes, but never in the phragmoplast region at cytokinesis (Figure 2k). Furthermore, the overexpression of the non-degradable cyclin inhibited
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Localization of GFP-tagged cyclins in tobacco cells 573 neither chromosome decondensation nor nuclear envelope 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 et al., 2000). Nevertheless, after a longer period of Dex induction (48 h of 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 et al., 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;2 and 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 GFPtagged cyclin B1;3 under the control of the tetracycline inducible promoter, were used to generate a cell suspension 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 demonstrate, that CycB1;3-GFP starts to degrade already in metaphase cells, we made time-lapse images from beginning of mitosis with a strong chromatin-bound GFPuorescence until the signal started to disappear during metaphase, as illustrated in Figure 3(g) in which images
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were sequentially recorded in 10-min time intervals starting from prophase. These results were very similar to the cyclin B1;1-GFP localisation studies (see above) suggesting 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 et al., 2001). GFP was 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 separation and cyclin B degradation (Whiteld et al., 1990; Clute and Pines, 1999; Hunt et al., 1992). We previously showed that the use of such drugs block the Dbox pathway in plant cells (Genschik et al., 1998). We thus investigated endogenous 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 4. Detergent extraction assays. (ab) Fluorescent microscopic images of non-extracted (a) and detergent extracted (b) cells expressing GFP and the corresponding DIC images (below). (c) Fluorescent microscopic image of detergent extracted cells expressing CDKA-GFP and the corresponding DIC images (below). (df) Fluorescent microscopic images of degradable Cyclin B1;1-GFP (d), nondegradable Cyclin B1;1-GFP (e) and Cyclin B1;3-GFP (f) in detergent extracted cells and the corresponding DIC images (below).

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Figure 7. Legend on facing page

Localization of GFP-tagged cyclins in tobacco cells 575 (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 accumulated 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 et al., 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 (Figure 6). 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, indicating that it was degraded as previously published (Criqui et al., 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 et al., 1998). This cell-cycle arrest can be explained by proteolytic inhibition of securin proteins (Nasmyth et al., 2000). In the blocked cells, cyclin B1;1-GFP protein was found in association 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 et al., 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 asterisk indicates an aspecic protein band (Criqui et al., 2000). The blot was subsequently striped and immunoblotted with the polyclonal antiPSTAIRE 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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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.

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.
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Localization of GFP-tagged cyclins in tobacco cells 577 control, we used a mitotic extract prepared from transgenic 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 CyclinGFP 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 reinvestigated in real time of the cell cycle in animal cells using 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 et al., 1999). Here we decided to investigate the cyclin stability in unxed living cells using Cyclin-GFP fusions. During interphase, animal cyclin B1 is found predominantly 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 B1type cyclins in the nucleus, it might be that in some cells this nuclear-cytoplasmic transport is less efcient. Timelapse 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 et al., 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 et al., 2000). However during interphase, the cyclin is efciently exported from the nucleus due to a leucine-rich nuclear export sequence (NES) (See Hagting et al., 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 p13suc1-sepharose bound protein fractions. Phosphorylated histone H1 was visualised by autoradiography (upper panel) and histone H1 loading was controlled by Coomassie brilliant blue R250 staining (bottom panel).

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
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Marie Claire Criqui et al. the CDK partner(s) of cyclin B1 proteins has not yet been clearly determined (Mironov et al., 1999; Stals et al., 2000). Immunolocalisation of A-type CDKs has been assayed in different plant systems (Mironov et al., 1999) and has sometimes led to contradictory results. For example, in maize an A-type CDK does not bind the chromosomes (Mews et al., 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 chromosomes at the metaphase-anaphase transition (Stals et al., 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 et al., 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 during metaphase 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 et al., 1995; Ookata et al., 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 cyclinGFP 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 et al., 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 chromosomes. 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 et al., 1992; Minshull et al., 1990; Whiteld et al., 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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et al., 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 anaphase. 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 et al., 1999). Indirect immunouorescence was used in maize root tip cells to localise three different B-type cyclins (Mews et al., 1997). The localisation of the tobacco cyclin B1-GFP fusion proteins is consistent with the localisation of one maize Btype 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 et al., 1997). Interestingly, only Zeama;CYCB1;2, like the tobacco B1-type cyclins, is subjected 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 phosphorylationdecient mutant of cyclin B1, which is unable to translocate into the nucleus, cannot promote germinal-vesicle breakdown in maturing frog oocytes (Li et al., 1997). In addition, it has been documented that cyclin B-Cdk1 kinase is involved in mitotic chromosome condensation (Kimura et al., 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 chromosomes 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 et al., 1998; Leresche et al., 1996). In animal cells, cyclin B1 only associates with CDK1 (cdc2) to full its mitotic function. In plants the identity of

Localization of GFP-tagged cyclins in tobacco cells 579 (Medsa;CycA2) is also nuclear localised and its proteolysis seems to start in early M phase (Roudier et al., 2000). 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 et al. (2000). The handling of the BY2 cell culture, as well as the synchronisation experiments, were performed according to Nagata et al. (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 et al., 1992) was transformed with plasmid pBinHygTX-TMV-CycB1;3-GFP by Agrobacterium mediated leaf disk transformation. Several independent transgenic 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.

Experimental procedures
Unless stated otherwise, all procedures for manipulating DNA and RNA were carried out according to Sambrook et al. (1989) and Ausubel et al. (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-leucinal (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 et al. (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-AAACCACTCGAGTGAATGGCGAACGAAGAAAATAAG-3) and oligonucleotide 2 (5-TTCTTTGGATCCAGCATCGTCAAAAAAACAAGC-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 in plasmids 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 PCRbased site directed mutagenesis a NcoI-site upstream of the coding region of cycB1;3 using oligonucleotide 3 (5-AAAGGATCCCTGCCATGGCTTCAAGAAACGTTCTTCAACAG-3) and a NotI site just upstream of the STOP codon allowing in frame fusion with the GFP followed by a BamHI site using oligonucleotide 4 (5-AAGGATCCTAGCGGCCGCCTTCATATGAAAGAGCAGCCAAGAG-3). The resulting PCR product was placed as a NcoIBamHI fragment into plasmid pSP72-TMV in frame with a TMVomega 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 fragment 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-GFPMAP4 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 GFPMAP4 coding sequence (Marc et al., 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 et al., 2001). Blackwell Science Ltd, The Plant Journal, (2001), 28, 569581

Detergent extraction of cells

Cells were detergent-extracted essentially as has been previously described for preparing cytoskeletons (Chan et al., 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 0.45 M 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 et al. (2000). The histone H4 probe corresponds to the 196bp restriction fragment AccI-DdeI of the coding region of the gene H4A748 (Chaboute et al., 1987). The cyclin B1;1 probe corresponds to the Nicta;CycB1;1 cDNA (Qin et al., 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, p13Suc1sepharose afnity binding and histone H1 kinase assays

The production of the polyclonal antibody against tobacco cyclin B1;1 is described in Criqui et al. (2000). Samples of 15 mg of proteins were separated by 10% SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis) gels and transferred to Immobilon-P membrane (Millipore, Bedford, MA, USA). The

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Brandeis, M., Rosewell, I., Carrington, M., Crompton, T., Jacobs, M.A., Kirk, J., Gannon, J. and Hunt, T. (1998) Cyclin B2-null mice develop normally and are fertile whereas cyclin B1-null mice die in utero. Proc. Natl Acad. Sci. USA, 95, 43444349. Chaboute, M.E., Chaubet, N., Philipps, G., Ehling, M. and Gigot, C. (1987) Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana. Plant Mol. Biol. 8, 179191. Chan, J., Rutten, T. and Lloyd, C. (1996) Isolation of microtubuleassociated proteins from carrot cytoskeletons: a 120 kDa map decorates all four microtubule arrays and the nucleus. Plant J. 10, 251259. Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S.M., Lambert, G.M. and Galbraith, D.W. (2000) Nuclear dynamics in Arabidopsis thaliana. Mol. Biol. Cell, 11, 27332741. Clute, P. and Pines, J. (1999) Temporal and spatial control of cyclin B1 destruction in metaphase. Nat. Cell Biol. 1, 8287. Criqui, M.-C., Parmentier, Y., Derevier, A., Shen, W.-H., Dong, A. and Genschik, P. (2000) Cell cycle-dependent proteolysis and ectopic overexpression of cyclin B1 in tobacco BY2 cells. Plant J. 24, 763773. Gallant, P. and Nigg, E.A. (1994) Identication of a novel vertebrate cyclin: cyclin B3 shares properties with both A- and B-type cyclins. EMBO J. 13, 595605. Gatz, C., Frohberg, C. and Wendenburg, R. (1992) Stringent repression and homogeneous de-repression by tetracycline of a modied CaMV 35S promoter in intact transgenic tobacco plants. Plant J. 2, 397404. Genschik, P., Criqui, M.C., Parmentier, Y., Derevier, A. and Fleck, J. (1998) Cell cycle-dependent proteolysis in plants. Identication of the destruction box pathway and metaphase arrest produced by the proteasome inhibitor MG132. Plant Cell, 10, 20632076. Glotzer, M., Murray, A.W. and Kirschner, M.W. (1991) Cyclin is degraded by the ubiquitin pathway. Nature, 349, 132138. Granger, C.L. and Cyr, R.J. (2000) Microtubule reorganization in tobacco BY-2 cells stably expressing GFP-MBD. Planta, 210, 502509. Grebenok, R.J., Lambert, G.M. and Galbraith, D.W. (1997) Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. Plant J. 12, 685696. Hagting, A., Karlsson, C., Clute, P., Jackman, M. and Pines, J. (1998) MPF localization is controlled by nuclear export. EMBO J. 17, 41274138. Heix, J., Vente, A., Voit, R., Budde, A., Michaelidis, T.M. and Grummt, I. (1998) Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation. EMBO J. 17, 73737381. Howe, J.A., Howell, M., Hunt, T. and Newport, J.W. (1995) Identication of a developmental timer regulating the stability of embryonic cyclin A and a new somatic A-type cyclin at gastrulation. Genes Dev. 9, 11641176. Hunt, T., Luca, F.C. and Ruderman, J.V. (1992) The requirements for protein synthesis and degradation, and the control of destruction of cyclins A and B in the meiotic and mitotic cell cycles of the clam embryo. J. Cell Biol. 116, 707724. Ito, M. (2000) Factors controlling cyclin B expression. Plant Mol. Biol. 43, 677690. Jacobs, H.W., Knoblich, J.A. and Lehner, C.F. (1998) Drosophila Cyclin B3 is required for female fertility and is dispensable for mitosis like Cyclin B. Genes Dev. 12, 37413751. Kimura, K., Hirano, M., Kobayashi, R. and Hirano, T. (1998) Phosphorylation and activation of 13S condensin by Cdc2 in vitro. Science, 282, 487490. Blackwell Science Ltd, The Plant Journal, (2001), 28, 569581

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-conjugated 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 et al. (2000). Histone H1 kinase reactions were performed as described by Magyar et al. (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 differential interference contrast (Nomarski) optics and argon laser illumination 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 gion Alsace for founding the confocal microscope, Philippe and Re 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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