RESUMEN
Despite over one hundred years of research, the duplication of the centrosome is a poorly understood process. Three recent papers--exploring three different kinases--may have provided the answer.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Caenorhabditis elegans , Centrosoma/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Animales , Ciclo Celular/fisiología , Ciclina E/metabolismo , Ciclina E/fisiología , Quinasa 2 Dependiente de la Ciclina , Quinasas Ciclina-Dependientes/fisiología , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Región Organizadora del Nucléolo/metabolismo , Nucleofosmina , Proteínas Quinasas/genética , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/fisiología , Receptor EphA1Asunto(s)
Ciclo Celular/fisiología , Centrosoma/fisiología , Replicación del ADN/fisiología , Animales , Polaridad Celular , Segregación Cromosómica/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Humanos , Interfase/fisiología , Mitosis/fisiología , Proteínas Nucleares/metabolismo , Nucleofosmina , Huso Acromático/fisiología , Ubiquitinas/metabolismoRESUMEN
Centrosomes were microsurgically removed from BSC-1 African green monkey kidney cells before the completion of S phase. Karyoplasts (acentrosomal cells) entered and completed mitosis. However, postmitotic karyoplasts arrested before S phase, whereas adjacent control cells divided repeatedly. Postmitotic karyoplasts assembled a microtubule-organizing center containing gamma-tubulin and pericentrin, but did not regenerate centrioles. These observations reveal the existence of an activity associated with core centrosomal structures-distinct from elements of the microtubule-organizing center-that is required for the somatic cell cycle to progress through G1 into S phase. Once the cell is in S phase, these core structures are not needed for the G2-M phase transition.
Asunto(s)
Centrosoma/fisiología , Fase G1 , Fase S , Animales , Antígenos/metabolismo , División Celular/efectos de los fármacos , Línea Celular , Centriolos/fisiología , Chlorocebus aethiops , Gránulos Citoplasmáticos/fisiología , Gránulos Citoplasmáticos/ultraestructura , Interfase , Microscopía por Video , Centro Organizador de los Microtúbulos/fisiología , Microtúbulos/fisiología , Microtúbulos/ultraestructura , Mitosis , Paclitaxel/farmacología , Huso Acromático/fisiología , Huso Acromático/ultraestructura , Tubulina (Proteína)/metabolismoAsunto(s)
Ciclo Celular/fisiología , Centrosoma/fisiología , Animales , Núcleo Celular/fisiología , HumanosRESUMEN
Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/fisiología , Dictyostelium/fisiología , Endocitosis , Neuropéptidos/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Actinas/inmunología , Animales , Adhesión Celular , División Celular , Membrana Celular/metabolismo , Movimiento Celular , Células Cultivadas , Quimiotaxis , Dictyostelium/citología , Dictyostelium/genética , Dictyostelium/crecimiento & desarrollo , Humanos , Mutagénesis Sitio-Dirigida , Neuropéptidos/genética , Proteínas Recombinantes de Fusión , Homología de Secuencia , Transducción de Señal , Proteínas de Unión al GTP rac/genética , Proteína de Unión al GTP rac1RESUMEN
It is of great importance for the cell to precisely coordinate the doubling of the interphase centrosome with nuclear events during the cell cycle and limit the number of centrosomes it contains at the onset of mitosis to two and only two. The penalties for mistakes are abnormal spindle assembly, inappropriate chromosome distribution, and consequently, genomic instability. We review the functional properties of the mechanisms that control when the centrosome duplicates in the cell cycle and the controls for centrosome copy number. We look to limits that are intrinsic to the centrosome itself and controls imposed by cell cycle linked changes in cytoplasmic conditions. Control of centrosome reproduction is exercised at both levels.
Asunto(s)
Ciclo Celular/fisiología , Células/enzimología , Centrosoma/fisiología , Quinasas Ciclina-Dependientes/metabolismo , Animales , Células/citologíaRESUMEN
In sea urchin zygotes and mammalian cells nuclear envelope breakdown (NEB) is not driven simply by a rise in cytoplasmic cyclin dependent kinase 1-cyclin B (Cdk1-B) activity; the checkpoint monitoring DNA synthesis can prevent NEB in the face of mitotic levels of Cdk1-B. Using sea urchin zygotes we investigated whether this checkpoint prevents NEB by restricting import of regulatory proteins into the nucleus. We find that cyclin B1-GFP accumulates in nuclei that cannot complete DNA synthesis and do not break down. Thus, this checkpoint limits NEB downstream of both the cytoplasmic activation and nuclear accumulation of Cdk1-B1. In separate experiments we fertilize sea urchin eggs with sperm whose DNA has been covalently cross-linked to inhibit replication. When the pronuclei fuse, the resulting zygote nucleus does not break down for >180 minutes (equivalent to three cell cycles), even though Cdk1-B activity rises to greater than mitotic levels. If pronuclear fusion is prevented, then the female pronucleus breaks down at the normal time (average 68 minutes) and the male pronucleus with cross-linked DNA breaks down 16 minutes later. This male pronucleus has a functional checkpoint because it does not break down for >120 minutes if the female pronucleus is removed just prior to NEB. These results reveal the existence of an activity released by the female pronucleus upon its breakdown, that overrides the checkpoint in the male pronucleus and induces NEB. Microinjecting wheat germ agglutinin into binucleate zygotes reveals that this activity involves molecules that must be actively translocated into the male pronucleus.
Asunto(s)
Citoplasma/metabolismo , Mitosis/fisiología , Membrana Nuclear/metabolismo , Fosfatasas cdc25 , Animales , Afidicolina/farmacología , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Reactivos de Enlaces Cruzados/farmacología , Inhibidores Enzimáticos/farmacología , Femenino , Fertilización/fisiología , Ficusina/farmacología , Masculino , Modelos Genéticos , Fosfoproteínas Fosfatasas/metabolismo , Erizos de Mar , Albúmina Sérica Bovina/metabolismo , Factores Sexuales , Factores de Tiempo , Aglutininas del Germen de Trigo/metabolismoRESUMEN
The abnormally high number of centrosomes found in many human tumor cells can lead directly to aneuploidy and genomic instability through the formation of multipolar mitotic spindles. To facilitate investigation of the mechanisms that control centrosome reproduction, a frog egg extract arrested in S phase of the cell cycle that supported repeated assembly of daughter centrosomes was developed. Multiple rounds of centrosome reproduction were blocked by selective inactivation of cyclin-dependent kinase 2-cyclin E (Cdk2-E) and were restored by addition of purified Cdk2-E. Confocal immunomicroscopy revealed that cyclin E was localized at the centrosome. These results demonstrate that Cdk2-E activity is required for centrosome duplication during S phase and suggest a mechanism that could coordinate centrosome reproduction with cycles of DNA synthesis and mitosis.
Asunto(s)
Quinasas CDC2-CDC28 , Proteínas de Ciclo Celular , Centrosoma/metabolismo , Ciclina E/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Fase S , Proteínas Supresoras de Tumor , Animales , Afidicolina/farmacología , Blastómeros/química , Extractos Celulares , Centrosoma/química , Ciclina E/análisis , Quinasa 2 Dependiente de la Ciclina , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , ADN/biosíntesis , Inhibidores Enzimáticos/farmacología , Microscopía Confocal , Microscopía Fluorescente , Microscopía por Video , Proteínas Asociadas a Microtúbulos/farmacología , Óvulo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas , Proteínas Recombinantes/farmacología , Xenopus , Proteínas de XenopusRESUMEN
Biochemical fractionation of axonemal microtubules yields the protofilament ribbon (pf-ribbon), an insoluble structure of 3-4 longitudinal protofilaments composed primarily of alpha/beta tubulin, tektins A, B and C, and two previously uncharacterized polypeptides of 77 kDa and 83 kDa. We have isolated the 77/83 kDa polypeptides (termed Sp77 and Sp83) from sperm flagella of the sea urchin Stronglyocentrotus purpuratus and raised polyclonal antibodies against them. Sp77 and Sp83 copurify exclusively with the pf-ribbon. Both the anti-Sp77 and anti-Sp83 antibodies detected the nine outer doublets and the basal bodies of sea urchin sperm by immunofluorescence microscopy. In addition, the anti-Sp83 antibody, but not the anti-Sp77 antibody, detected a single 83 kDa polypeptide on immunoblots of unfertilized sea urchin egg cytoplasm, and a single polypeptide of 80 kDa on blots of isolated mitotic spindles from Chinese hamster ovary (CHO) cells. Previous studies have shown that tektins are present in the basal bodies and centrosomes/centrioles of cells ranging from clam to human. We found that anti-Sp83 decorates the spindle poles in sea urchin zygotes, and the interphase centrosome and spindle poles in CHO cells. In CHO cells arrested in S phase with aphidicolin, anti-Sp83 detects multiple centrosomes. The staining of the centrosome was not disrupted by prolonged nocodazole treatment, suggesting that the 80 kDa polypeptide is associated with the centrioles themselves. Our observations demonstrate that, like tektins, Sp77 and Sp83 are structural proteins associated with stable doublet microtubules, and may be components of basal bodies and centrioles of sea urchins and mammalian cells.
Asunto(s)
Centriolos/química , Flagelos/química , Proteínas Asociadas a Microtúbulos/aislamiento & purificación , Microtúbulos/ultraestructura , Cola del Espermatozoide/química , Espermatozoides/química , Animales , Reacciones Antígeno-Anticuerpo , Células CHO , Cricetinae , Flagelos/ultraestructura , Masculino , Peso Molecular , Erizos de Mar , Espermatozoides/ultraestructura , Cigoto/químicaRESUMEN
Centrosomes repeatedly reproduce in sea urchin zygotes arrested in S phase, whether cyclin-dependent kinase 1-cyclin B (Cdk1-B) activity remains at prefertilization levels or rises to mitotic values. In contrast, when zygotes are arrested in mitosis using cyclin B Delta-90, anaphase occurs at the normal time, yet centrosomes do not reproduce. Together, these results reveal the cell cycle stage specificity for centrosome reproduction and demonstrate that neither the level nor the cycling of Cdk1-B activity coordinate centrosome reproduction with nuclear events. In addition, the proteolytic events of the metaphase-anaphase transition do not control when centrosomes duplicate. When we block protein synthesis at first prophase, the zygotes divide and arrest before second S phase. Both blastomeres contain just two complete centrosomes, which indicates that the cytoplasmic conditions between mitosis and S phase support centrosome reproduction. However, the fact that these daughter centrosomes do not reproduce again under such supportive conditions suggests that they are lacking a component required for reproduction. The repeated reproduction of centrosomes during S phase arrest points to the existence of a necessary "licensing" event that restores this component to daughter centrosomes during S phase, preparing them to reproduce in the next cell cycle.
Asunto(s)
Núcleo Celular/fisiología , Centrosoma/fisiología , Fase S/fisiología , Cigoto/citología , Anafase/fisiología , Animales , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , Centrosoma/ultraestructura , Femenino , Masculino , Metafase/fisiología , Microscopía Electrónica , Mitosis/fisiología , Profase/fisiología , Biosíntesis de Proteínas , Proteínas Quinasas/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Reproducción/fisiología , Fase S/efectos de los fármacos , Erizos de Mar , Cigoto/enzimologíaRESUMEN
Through association with CDK1, cyclin B accumulation and destruction govern the G2/M/G1 transitions in eukaryotic cells. To identify CDK1 inactivation-dependent events during late mitosis, we expressed a nondestructible form of cyclin B (cyclin BDelta90) by microinjecting its mRNA into prometaphase normal rat kidney cells. The injection inhibited chromosome decondensation and nuclear envelope formation. Chromosome disjunction occurred normally, but anaphase-like movement persisted until the chromosomes reached the cell periphery, whereupon they often somersaulted and returned to the cell center. Injection of rhodamine-tubulin showed that this movement occurred in the absence of a central anaphase spindle. In 82% of cells cytokinesis was inhibited; the remainder split themselves into two parts in a process reminiscent of Dictyostelium cytofission. In all cells injected, F-actin and myosin II were diffusely localized with no detectable organization at the equator. Our results suggest that a primary effect of CDK1 inactivation is on spindle dynamics that regulate chromosome movement and cytokinesis. Prolonged CDK1 activity may prevent cytokinesis through inhibiting midzone microtubule formation, the behavior of proteins such as TD60, or through the phosphorylation of myosin II regulatory light chain.