RESUMEN
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.
Asunto(s)
Proteína Quinasa CDC2/metabolismo , Microtúbulos/metabolismo , Mitosis , Nicotiana/enzimología , Plantas Tóxicas , Proteína Quinasa CDC2/genética , Cromatina/metabolismo , Prueba de Complementación Genética , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Microtúbulos/ultraestructura , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Nicotiana/citologíaRESUMEN
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.
Asunto(s)
Núcleo Celular/química , Mitosis , Plantas/metabolismo , Tubulina (Proteína)/metabolismo , Ciclo Celular , Centriolos , Fabaceae/química , Fabaceae/metabolismo , Técnica del Anticuerpo Fluorescente , Microscopía Confocal , Plantas/química , Plantas MedicinalesRESUMEN
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.
Asunto(s)
Citoesqueleto/enzimología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas Quinasas Activadas por Mitógenos/fisiología , Células Vegetales , Fenómenos Fisiológicos de las Plantas , División Celular/fisiología , Plantas/enzimologíaRESUMEN
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.
Asunto(s)
Medicago sativa/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Mitosis/genética , Proteínas de Plantas , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , División Celular/genética , División Celular/fisiología , Clonación Molecular , Técnica del Anticuerpo Fluorescente , Medicago sativa/metabolismo , Microtúbulos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mitosis/fisiología , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional/fisiología , Homología de Secuencia de AminoácidoRESUMEN
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.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Proteínas de Ciclo Celular , Proteínas Quinasas Activadas por Mitógenos , Mitosis , Nicotiana/citología , Proteínas de Plantas/metabolismo , Plantas Tóxicas , Proteínas de Schizosaccharomyces pombe , Anafase , Benzamidas/farmacología , Proteínas Quinasas Dependientes de Calcio-Calmodulina/análisis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/inmunología , Línea Celular , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente Indirecta , Proteínas Fúngicas/metabolismo , Immunoblotting , Metafase , Microtúbulos/fisiología , Nitrobencenos , Compuestos Organotiofosforados/farmacología , Paclitaxel/farmacología , Proteínas de Plantas/análisis , Proteínas de Plantas/inmunología , Proteínas Quinasas/metabolismo , Purinas/farmacología , Roscovitina , Telofase , Nicotiana/efectos de los fármacos , Nicotiana/enzimologíaRESUMEN
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.
Asunto(s)
Proteína Quinasa CDC2/genética , Fabaceae/citología , Microtúbulos/ultraestructura , Raíces de Plantas/efectos de los fármacos , Plantas Medicinales , Purinas/farmacología , Huso Acromático/efectos de los fármacos , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclina A/genética , Ciclina B/genética , Inhibidores Enzimáticos/farmacología , Meristema/citología , Meristema/efectos de los fármacos , Meristema/ultraestructura , Microtúbulos/efectos de los fármacos , Raíces de Plantas/citología , Raíces de Plantas/ultraestructura , Proteínas Quinasas/metabolismo , Roscovitina , Transcripción GenéticaRESUMEN
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.
RESUMEN
MPM-2 antibody reacts with a subset of mitotic phosphoproteins. We followed localization of MPM-2 immunoreactive material and localization of microtubules during cell cycle progression in a highly synchronous population of Vicia faba root meristem cells and isolated nuclei. The MPM-2 antibody labelling showed significant cell cycle dependence. MPM-2 nuclear reactivity was weak and homogeneous in G1 and S phase of the cell cycle and became stronger and heterogeneous during G2, resembling staining of the nuclear matrix, with maximum staining at the G2/M interface. Similarly the staining intensity of nucleoli increased from late G1 phase to nucleoli dispersion in early prophase. During mitosis MPM-2 immunoreactivity was associated with spindle configurations and the brightest signal was localized in kinetochores from prophase to metaphase.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Fabaceae/citología , Fosfoproteínas/análisis , Proteínas de Plantas/análisis , Plantas Medicinales , Tubulina (Proteína)/análisis , Ciclo Celular , Núcleo Celular/química , Núcleo Celular/ultraestructura , Células Cultivadas , Fabaceae/química , Fabaceae/inmunología , Fosfoproteínas/inmunología , Proteínas de Plantas/inmunología , Huso Acromático/química , Huso Acromático/ultraestructura , Células Madre/química , Células Madre/ultraestructura , Tubulina (Proteína)/inmunologíaRESUMEN
The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.