Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis.

A-type cyclin-dependent kinases (CDKs), also known as cdc2, are central to the orderly progression of the cell cycle. We made a functional Green Fluorescent Protein (GFP) fusion with CDK-A (Cdc2-GFP) and followed its subcellular localization during the cell cycle in tobacco cells. During interphase, the Cdc2-GFP fusion protein was found in both the cytoplasm and the nucleus, where it was highly resistant to extraction. In premitotic cells, a bright and narrow equatorial band appeared on the cell surface, resembling the late preprophase band, which disintegrated within 10 min as followed by time-lapse images. Cdc2-GFP was not found on prophase spindles but left the chromatin soon after this stage and associated progressively with the metaphase spindle in a microtubule-dependent manner. Arresting cells in mitosis through the stabilization of microtubules by taxol further enhanced the spindle-localized pool of Cdc2-GFP. Toward the end of mitosis, Cdc2-GFP was found at the midzone of the anaphase spindle and phragmoplast; eventually, it became focused at the midline of these microtubule structures. In detergent-extracted cells, the Cdc2-GFP remained associated with mitotic structures. Retention on spindles was prevented by pretreatment with the CDK-specific inhibitor roscovitine and was enhanced by the protein phosphatase inhibitor okadaic acid. Furthermore, we demonstrate that both the endogenous CDK-A and Cdc2-GFP were cosedimented with taxol-stabilized plant microtubules from cell extracts and that Cdc2 activity was detected together with a fraction of polymerized tubulin. These data provide evidence that the A-type CDKs associate physically with mitotic structures in a microtubule-dependent manner and may be involved in regulating the behavior of specific microtubule arrays throughout mitosis.

Sub-cellular localisation of GFP-tagged tobacco mitotic cyclins during the cell cycle and after spindle checkpoint activation.

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 in the nucleus in interphase cells and disappeared earlier than the cyclin B1 proteins during mitosis.

Stressing the role of MAP kinases in mitogenic stimulation.

In yeast and animal cells, distinct subfamilies of mitogen-activated protein kinases (MAPKs) have evolved for transmitting different types of signals, such as the extracellular signal-regulated kinase (ERK) for mitogenic stimuli and differentiation, p38 and JUN kinase (JNK) for stress factors. Based on sequence analysis, the presently known plant MAPKs are most similar to ERKs, even though compelling evidence implies a role in various forms of biotic and abiotic stress responses. However, knowledge of their involvement in controlling proliferation is just emerging. A subgroup of the plant MAPKs, containing the alfalfa MMK3 and tobacco NTF6, are only active in mitotic cells and their localisation to the cell plate suggests a role in cytokinesis. An upstream regulator of MAPKs, the tobacco NPK1, appears to be also activated during mitosis. NPK1 might be associated and regulated by a microtubule motor protein. The localisation of NPK1 to the cell plate and its mitosis-specific activation suggest that together with NTF6 it could constitute a mitotic MAPK signalling module in tobacco. NPK1 appears to have a second role in repression of auxin-induced gene expression. MAPKs might also be involved in signalling within the meristems as suggested by the recruitement of a small G-protein to the CLAVATA 1 receptor-like protein kinase upon activation. In animal and yeast cells some of the small G-proteins relay signals from receptors to MAPK pathways.

SIMKK, a mitogen-activated protein kinase (MAPK) kinase, is a specific activator of the salt stress-induced MAPK, SIMK.

In eukaryotes, mitogen-activated protein kinases (MAPKs) play key roles in the transmission of external signals, such as mitogens, hormones, and different stresses. MAPKs are activated by MAPK kinases through phosphorylation of MAPKs at both the threonine and tyrosine residues of the conserved TXY activation motif. In plants, several MAPKs are involved in signaling of hormones, stresses, cell cycle, and developmental cues. Recently, we showed that salt stress-induced MAPK (SIMK) is activated when alfalfa cells are exposed to hyperosmotic conditions. Here, we report the isolation and characterization of the alfalfa MAPK kinase SIMKK (SIMK kinase). SIMKK encodes an active protein kinase that interacts specifically with SIMK, but not with three other MAPKs, in the yeast two-hybrid system. Recombinant SIMKK specifically activates SIMK by phosphorylating both the threonine and tyrosine residues in the activation loop of SIMK. SIMKK contains a putative MAPK docking site at the N terminus that is conserved in mammalian MAPK kinases, transcription factors, and phosphatases. Removal of the MAPK docking site of SIMKK partially compromises but does not completely abolish interaction with SIMK, suggesting that other domains of SIMKK also are involved in MAPK binding. In transient expression assays, SIMKK specifically activates SIMK but not two other MAPKs. Moreover, SIMKK enhances the salt-induced activation of SIMK. These data suggest that the salt-induced activation of SIMK is mediated by the dual-specificity protein kinase SIMKK.

Nuclear gamma-tubulin during acentriolar plant mitosis.

Neither the molecular mechanism by which plant microtubules nucleate in the cytoplasm nor the organization of plant mitotic spindles, which lack centrosomes, is well understood. Here, using immunolocalization and cell fractionation techniques, we provide evidence that gamma-tubulin, a universal component of microtubule organizing centers, is present in both the cytoplasm and the nucleus of plant cells. The amount of gamma-tubulin in nuclei increased during the G(2) phase, when cells are synchronized or sorted for particular phases of the cell cycle. gamma-Tubulin appeared on prekinetochores before preprophase arrest caused by inhibition of the cyclin-dependent kinase and before prekinetochore labeling of the mitosis-specific phosphoepitope MPM2. The association of nuclear gamma-tubulin with chromatin displayed moderately strong affinity, as shown by its release after DNase treatment and by using extraction experiments. Subcellular compartmentalization of gamma-tubulin might be an important factor in the organization of plant-specific microtubule arrays and acentriolar mitotic spindles.

Regulation of cell division and the cytoskeleton by mitogen-activated protein kinases in higher plants.

The microtubule-associated protein 2 kinase (MAP2-kinase), now better known as mitogen-activated protein kinase (MAPK), was initially discovered in association with the cytoskeleton, and was later also implicated in cell division. The importance of mitogenic stimulation in plant development roused interest in finding the plant homologues of MAPKs. However, data on plant MAPKs in cell division are rather sparse and fragmentary. Therefore we place the available information on cell cycle control of MAPKs in plants into a broader context. We discuss four aspects of cell division control: cell proliferation and the G1/S-phase transition, G2-phase and mitosis, cytokinesis, and cytoskeletal reorganisation. Future work will reveal to what extent plants use signalling pathways that are similar or different to those of animal or yeast cells in regulating cell divisions.

A MAP kinase is activated late in plant mitosis and becomes localized to the plane of cell division.

In eukaryotes, mitogen-activated protein kinases (MAPKs) are part of signaling modules that transmit diverse stimuli, such as mitogens, developmental cues, or various stresses. Here, we report a novel alfalfa MAPK, Medicago MAP kinase 3 (MMK3). Using an MMK3-specific antibody, we detected the MMK3 protein and its associated activity only in dividing cells. The MMK3 protein could be found during all stages of the cell cycle, but its protein kinase activity was transient in mitosis and correlated with the timing of phragmoplast formation. Depolymerization of microtubules by short treatments with the drug amiprophosmethyl during anaphase and telophase abolished MMK3 activity, indicating that intact microtubules are required for MMK3 activation. During anaphase, MMK3 was found to be concentrated in between the segregating chromosomes; later, it localized at the midplane of cell division in the phragmoplast. As the phragmoplast microtubules were redistributed from the center to the periphery during telophase, MMK3 still localized to the whole plane of division; thus, phragmoplast microtubules are not required to keep MMK3 at this location. Together, these data strongly support a role for MMK3 in the regulation of plant cytokinesis.

A cell cycle regulated MAP kinase with a possible role in cytokinesis in tobacco cells.

Mitogen-activated protein (MAP) kinases have been demonstrated to have a role in meiosis but their involvement in mitotic events is less clear. Using a peptide antibody raised against the tobacco MAP kinase p43(Ntf6) and extracts from synchronized tobacco cell suspension cultures, we show that this kinase is activated specifically during mitosis. Entry into mitosis appears to be necessary for the activation of the kinase, which occurs as a post-translational event. The activation of the kinase occurs in late anaphase/early telophase. The p43(Ntf6) protein shows a transient localization to the cell plate in anaphase cells, in the middle of the two microtubule arrays characteristic of the phragmoplast, a plant-specific structure involved in laying down the new cell wall. The combined data support a role for the MAP kinase p43(Ntf6) in cytokinesis in tobacco cells.

MP2C, a plant protein phosphatase 2C, functions as a negative regulator of mitogen-activated protein kinase pathways in yeast and plants.

By interference of the yeast pheromone mitogen-activated protein kinase (MAPK) pathway with an alfalfa cDNA expression library, we have isolated the MP2C gene encoding a functional protein phosphatase type 2C. Epistasis analysis in yeast indicated that the molecular target of the MP2C phosphatase is Ste11, a MAPK kinase kinase that is a central regulator of the pheromone and osmosensing pathways. In plants, MP2C functions as a negative regulator of the stress-activated MAPK (SAMK) pathway that is activated by cold, drought, touch, and wounding. Although activation of the SAMK pathway occurs by a posttranslational mechanism, de novo transcription and translation of protein factor(s) are necessary for its inactivation. MP2C is likely to be this or one of these factors, because wound-induced activation of SAMK is followed by MP2C gene expression and recombinant glutathione S-transferase-MP2C is able to inactivate extracts containing wound-induced SAMK. Wound-induced MP2C expression is a transient event and correlates with the refractory period, i.e., the time when restimulation of the SAMK pathway is not possible by a second stimulation. These data suggest that MP2C is part of a negative feedback mechanism that is responsible for resetting the SAMK cascade in plants.

Treatment of Vicia faba root tip cells with specific inhibitors to cyclin-dependent kinases leads to abnormal spindle formation.

Many events during cell division are triggered by an evolutionary conserved regulator, the cyclin-dependent kinase (Cdk). Here we used two novel drugs, the purine analogues bohemine and roscovitine, to study the role of Cdks in cell cycle progression and microtubule organisation in Vicia faba root tip cells. Both drugs inhibited the activity of immunopurified Vicia faba and alfalfa Cdc2-kinase. The transcript levels of an A- and B-type cyclin, as well as of the cdc2 genes, declined in treated root tips, while the mRNA level of a D-type cyclin gene was not affected. An observed transient arrest at the G1/S and G2/M regulatory points indicated that inhibition of the Cdc2-kinase had an effect on both transitions. In contrast to the regular bipolar spindle in untreated cell, in drug-treated metaphase cells abnormally short and dense kinetochore microtubule fibres were observed. These microtubules were randomly arranged in the vicinity of the kinetochores and connected the chromosomes. Thus, the chromosomes were not aligned on the metaphase plate but were arranged in a circle, with kinetochores pointing inwards and chromosome arms pointing outwards. gamma-Tubulin, which plays a role in microtubule nucleation, also localised to the centre of the monopolar spindle. The observed abnormalities in mitosis, after inhibition of Cdc2-kinase by specific drugs, suggest a role for this enzyme in regulating some of the steps leading to a bipolar spindle structure.

The cdc2Ms Kinase Is Differently Regulated in the Cytoplasm and in the Nucleus.

To study a cyclin-dependent kinase (CDK) from alfalfa (Medicago sativa L.), an antibody was raised against the C-terminal 16 amino acids of the protein cdc2aMs. The cdc2Ms protein was immunopurified with this antibody and its histone kinase activity was measured. The cdc2Ms kinase is activated at the G1/S transition when phosphate-starved cells from the G0 phase re-enter the cell cycle and remain active as cells transit the S, G2, and M phases, indicating that the same CDK regulates all of these phases in alfalfa. In contrast, when cdc2Ms kinase was purified by binding to p13suc1, it was active only in the G2 and M phases. In immunoblots the C-terminal antibody detected an equal amount of the cdc2Ms protein in the cytoplasm and in the nucleus. By indirect immunofluorescence, however, the cytoplasmic form of cdc2Ms could not be found in the S phase of the cells, indicating that the epitope for the cdc2 antibody is not accessible. Binding of putative inhibitor proteins to cdc2 was shown by inactivation of purified plant CDK when cell extracts were added. Furthermore, purified CDK inhibitors, such as the mouse p27kip1 and the yeast p40sic1, blocked the purified plant CDK activity.

Wounding Induces the Rapid and Transient Activation of a Specific MAP Kinase Pathway.

Mechanical injury in plants induces responses that are involved not only in healing but also in defense against a potential pathogen. To understand the intracellular signaling mechanism of wounding, we have investigated the involvement of protein kinases. Using specific antibodies, we showed that wounding alfalfa leaves specifically induces the transient activation of the p44MMK4 kinase, which belongs to the family of mitogen-activated protein kinases. Whereas activation of the MMK4 pathway is a post-translational process and was not blocked by [alpha]-amanitin and cycloheximide, inactivation depends on de novo transcription and translation of a protein factor(s). After wound-induced activation, the MMK4 pathway was subject to a refractory period of 25 min, during which time restimulation was not possible, indicating that the inactivation mechanism is only transiently active. After activation of the p44MMK4 kinase by wounding, transcript levels of the MMK4 gene increased, suggesting that the MMK4 gene may be a direct target of the MMK4 pathway. In contrast, transcripts of the wound-inducible MsWIP gene, encoding a putative proteinase inhibitor, were detected only several hours after wounding. Abscisic acid, methyl jasmonic acid, and electrical activity are known to mediate wound signaling in plants. However, none of these factors was able to activate the p44MMK4 kinase in the absence of wounding, suggesting that the MMK4 pathway acts independently of these signals.

Developmental and cell cycle regulation of alfalfa nucMs1, a plant homolog of the yeast Nsr1 and mammalian nucleolin.

We report here the isolation and characterization of the nucMs1 alfalfa cDNA, whose predicted amino acid sequence structurally resembles the yeast Nsr1 protein and animal nucleolins. These proteins consist of an N-terminal acidic domain, centrally located RNA recognition motifs (RRMs), and a C-terminal glycine- and arginine-rich domain. In comparison with animal nucleolins that contain four RRMs, NucMs1 more closely resembles the yeast Nsr1 protein, which contains only two RRMs. A NucMs1 C-terminal peptide antibody specifically recognized a 95-kD nucleolar protein in alfalfa cells that changed its localization in a cell cycle-dependent manner. The nucMs1 transcript and p95nucMs1 protein levels correlated with cell proliferation, and nucMs1 gene expression was found to be induced in the G1 phase upon mitogenic stimulation of G0-arrested leaf cells. In situ hybridization analysis of different alfalfa organs during various developmental stages showed that nucMs1 gene expression is highest in root meristematic cells, but it is also found in other meristematic cells of the plant body. nucMs1 expression is tightly linked to cell proliferation but does not depend on a particular cell cycle phase. No nucMs1 expression was observed in cells that had exited the cell cycle and were undergoing differentiation or polar growth, indicating that nucMs1 may not be necessary for processes other than cell proliferation.

The D-type alfalfa cyclin gene cycMs4 complements G1 cyclin-deficient yeast and is induced in the G1 phase of the cell cycle.

Cyclins are key regulators of the cell cycle in all eukaryotes. In alfalfa, we have previously isolated three B-type cyclins. The closely related cycMs1 and cycMs2 genes are expressed primarily during the G2 and M phases and are most likely mitotic cyclins; expression of the cycMs3 gene is induced in the G0-to-G1 transition, when cells reenter the cell cycle. By complementation of G1 cyclin-deficient yeast cells, a novel alfalfa cyclin, designated cycMs4, was isolated. The predicted amino acid sequence of the cycMs4 gene is most similar to that of the Arabidopsis cyclin delta 3 gene. CycMs4 and cyclin delta 3 belong to the class of D-type cyclins and contain PEST-rich regions and a retinoblastoma binding motif. When comparing expression levels in different organs, cycMs4 transcripts were present predominantly in roots. Whereas expression of the cycMs4 gene was cell cycle-regulated in suspension-cultured cells, transcription in roots was observed to depend also on the positional context of the cell. When differentiated G0-arrested leaf cells were induced to resume cell division by treatment with plant hormones, cycMs4 transcription was induced before the onset of DNA synthesis. Whereas this induction was preceded by that of the cycMs3 gene, cycMs2 expression occurred later and at the same time as mitotic activity. These data suggest that cycMs4 plays a role in the G1-to-S transition and provide a model to investigate the plant cell cycle at the molecular level.

cycMs3, a novel B-type alfalfa cyclin gene, is induced in the G0-to-G1 transition of the cell cycle.

Cyclins are key regulators of the cell cycle in all eukaryotes. We have previously isolated two B-type cyclin genes, cycMs1 and cycMs2, from alfalfa that are primarily expressed during the G2-to-M phase transition and are most likely mitotic cyclin genes. Here, we report the isolation of a novel alfalfa cyclin gene, termed cycMs3 (for cyclin Medicago sativa), by selecting for mating type alpha-pheromone-induced cell cycle arrest suppression in yeast. The central region of the predicted amino acid sequence of the cycMs3 gene is most similar to the cyclin box of yeast B-type and mammalian A- and B-type cyclins. In situ hybridization showed that cycMs3 mRNA can be detected only in proliferating cells and not in differentiated alfalfa cells. When differentiated G0-arrested cells were induced to reenter the cell cycle in the G1 phase and resume cell division by treatment with plant hormones, cycMs3 transcript levels increased long before the onset of DNA synthesis. In contrast, histone H3-1 mRNA and cycMs2 transcripts were not observed before DNA replication and mitosis, respectively. In addition, cycMs3 mRNA was found in all stages of the cell cycle in synchronously dividing cells, whereas the cycMs2 and histone H3-1 genes showed a G2-to-M phase- or S phase-specific transcription pattern, respectively. These data suggest that the role of cyclin CycMs3 differs from that of CycMs1 and CycMs2. We propose that CycMs3 helps control reentry of quiescent G0-arrested cells into the G1 phase of the cell cycle.

Isolation and characterization of phosphoprotein phosphatase 1 from alfalfa.

Protein phosphatases are central regulatory components of diverse processes in eukaryotes and are among the most highly conserved proteins known. In this paper, we report the cloning and sequencing of a type 1 protein phosphatase (pp1Ms) cDNA from alfalfa. Southern analysis indicates the presence of a gene family of PP1 proteins in alfalfa. The pp1Ms open reading frame is very similar to one of five predicted Arabidopsis type 1 protein phosphatases, indicating that different subtypes are individually conserved. Expression of the alfalfa pp1Ms in a temperature-sensitive Schizosaccharomyces pombe PP1 mutant, dis2-11, revealed no complementation, suggesting that PP1Ms is not involved in mitotic regulation. In different plant organs, different pp1Ms transcript levels were observed; in contrast, mRNA levels remained constant in all phases of the cell cycle and in logarithmically growing cells. However, when cells entered stationary phase pp1Ms transcript levels decreased considerably.

cdc2MsB, a cognate cdc2 gene from alfalfa, complements the G1/S but not the G2/M transition of budding yeast cdc28 mutants.

The product of the cdc2 gene encodes the p34cdc2 protein kinase that controls entry of yeast cells into S phase and mitosis. In higher eukaryotes, at least two cdc2-like genes appear to be involved in these processes. A cdc2 homologous gene has previously been isolated from alfalfa and shown to complement a fission yeast cdc2ts mutant. Here the isolation of cdc2MsB, a cognate cdc2 gene from alfalfa (Medicago sativa) is reported. Southern blot analysis shows that cdc2MsA and cdc2MsB are present as single copy genes in different tetraploid Medicago species. cdc2MsB encodes a slightly larger mRNA (1.5 kb) than cdc2MsA (1.4 kb). Both genes were found to be expressed at similar steady state levels in different alfalfa organs. Expression levels of both cdc2Ms genes correlate with the proliferative state of the organs. Complementation studies revealed that in contrast to cdc2MsA, cdc2MsB was not able to rescue a cdc2ts fission yeast mutant. cdc2MsB was also unable to rescue a G2/M-arrested cdc28ts budding yeast mutant which could be rescued by expression of the cdc2MsA gene. Conversely, cdc2MsB but not cdc2MsA was found to complement the G1/S block of another cdc28ts budding yeast mutant. These results suggest that cdc2MsA and cdc2MsB function at different control points in the cell cycle.

The MsK family of alfalfa protein kinase genes encodes homologues of shaggy/glycogen synthase kinase-3 and shows differential expression patterns in plant organs and development.

This paper reports on the isolation of a novel class of plant serine/threonine protein kinase genes, MsK-1, MsK-2 and MsK-3. They belong to the superfamily of cdc2-like genes, but show highest identity to the Drosophila shaggy and rat GSK-3 proteins (65-70%). All of these kinases share a highly conserved catalytic protein kinase domain. Different amino-terminal extensions distinguish the different proteins. The different plant kinases do not originate from differential processing of the same gene as is found for shaggy, but are encoded by different members of a gene family. Similarly to the shaggy kinases, the plant kinases show different organ-specific and stage-specific developmental expression patterns. Since the shaggy kinases play an important role in intercellular communication in Drosophila development, the MsK kinases are expected to perform a similar function in plants.

The plant homologue of MAP kinase is expressed in a cell cycle-dependent and organ-specific manner.

In animals, MAP kinase plays a key role in growth factor-stimulated signalling and in mitosis. The isolation of a Medicago sativa cDNA clone MsK7 which shows 52% identity to animal MAP kinases is reported. The deduced protein sequence shows all the important structural features of MAP kinases and also contains the highly conserved Thr-183 and Tyr-185 residues. Northern analysis of synchronized alfalfa cells showed that the MsK7 kinase gene is expressed at low levels in G1 phase but at higher levels in S and G2 phases of the cell cycle. In the plant, only stems and roots were found to contain MAP kinase MsK7 mRNA. Southern and PCR analyses indicated that alfalfa contains at least four highly related MAP kinase genes.