RESUMO
From concatenated chromosomes to polyploidization, large-scale genome changes are known to occur in parthenogenetic animals. Here, we report mosaic aneuploidy in larval brains of facultatively parthenogenetic Drosophila. We identified a background of aneuploidy in D. mercatorum strains and found increased levels of aneuploidy in the larval brain tissue of animals arising parthenogenetically versus those arising from sexual reproduction. There is also intra-individual variation in germline-derived aneuploidy within the same strain. To determine if this is a general feature of facultative parthenogenesis in drosophilids, we compared sexually reproduced and parthenogenetic offspring from an engineered facultative parthenogenetic strain of D. melanogaster. In addition to germline-derived aneuploidy, this revealed somatic aneuploidy that increased by up to fourfold in parthenogens compared to sexually reproduced offspring. Therefore, the genetic combination identified in D. mercatorum that causes facultative parthenogenesis in D. melanogaster results in aneuploidy, which indicates that the loss of mitotic control resulting in parthenogenesis causes subsequent genome variation within the parthenogenetic offspring. Our findings challenge the assumption that parthenogenetic offspring are near genetic replicas of their mothers.
Assuntos
Drosophila melanogaster , Drosophila , Animais , Drosophila/genética , Drosophila melanogaster/genética , Reprodução/genética , Comportamento Sexual Animal , Partenogênese/genéticaRESUMO
Parthenogenesis has been documented in almost every phylum of animals, and yet this phenomenon is largely understudied. It has particular importance in dipterans since some parthenogenetic species are also disease vectors and agricultural pests. Here, we present a catalogue of parthenogenetic dipterans, although it is likely that many more remain to be identified, and we discuss how their developmental biology and interactions with diverse environments may be linked to different types of parthenogenetic reproduction. We discuss how the advances in genetics and genomics have identified chromosomal loci associated with parthenogenesis. In particular, a polygenic cause of facultative parthenogenesis has been uncovered in Drosophila mercatorum, allowing the corresponding genetic variants to be tested for their ability to promote parthenogenesis in another species, Drosophila melanogaster. This study probably identifies just one of many routes that could be followed in the evolution of parthenogenesis. We attempt to account for why the phenomenon has evolved so many times in the dipteran order and why facultative parthenogenesis appears particularly prevalent. We also discuss the significance of coarse genomic changes, including non-disjunction, aneuploidy, and polyploidy and how, together with changes to specific genes, these might relate to both facultative and obligate parthenogenesis in dipterans and other parthenogenetic animals.
Assuntos
Drosophila melanogaster , Reprodução , Animais , Reprodução/genética , Drosophila/genética , Partenogênese/genética , AgriculturaRESUMO
The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.
Assuntos
Autoantígenos/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Autoantígenos/metabolismo , Centrômero , Proteína Centromérica A , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Humanos , Cinetocoros , Escleroderma Sistêmico/genética , Terminologia como AssuntoRESUMO
Interfering with the activity of polo-like kinases can lead to the formation of monopolar spindles. Polo-like kinases also regulate mitotic entry, activation of the anaphase-promoting complex and the necessary preconditions for cytokinesis. Similarities between the phenotypes of the Drosophila mutants asp and polo point towards a common role in spindle pole function. The abnormal spindles of asp mutants are bipolar but have disorganized broad poles at which gamma-tubulin has an abnormal distribution. Moreover, the synergism or of polo1 aspE3 double mutants indicates a possible involvement of these genes in a common process. Asp is a microtubule-associated protein of relative molecular mass 220,000 (Mr 220K) found at the face of the centrosome that contacts spindle microtubules. In partially purified centrosomes, it is required with gamma-tubulin to organize microtubule asters. Here, we show that Asp is the previously identified Mr 220K substrate of Polo kinase. Polo phosphorylates Asp in vitro, converting it into an MPM2 epitope. Polo and Asp proteins immunoprecipitate together and exist as part of a 25-38S complex. Extracts of polo-derived embryos are unable to restore the ability of salt-stripped centrosomes to nucleate microtubule asters. This can be rescued by addition of phosphorylated Asp or active Polo kinase.
Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila , Proteínas dos Microfilamentos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Proteína Quinase CDC2/metabolismo , Drosophila/embriologia , Drosophila/metabolismo , Epitopos de Linfócito B/imunologia , Camundongos , Proteínas dos Microfilamentos/imunologia , Proteínas Associadas aos Microtúbulos/imunologia , Fosforilação , RatosRESUMO
Cycles of protein phosphorylation are fundamental in regulating the progression of the eukaryotic cell through its division cycle. Here we test the complement of Drosophila protein kinases (kinome) for cell cycle functions after gene silencing by RNA-mediated interference. We observed cell cycle dysfunction upon downregulation of 80 out of 228 protein kinases, including most kinases that are known to regulate the division cycle. We find new enzymes with cell cycle functions; some of these have family members already known to phosphorylate microtubules, actin or their associated proteins. Additionally, depletion of several signalling kinases leads to specific mitotic aberrations, suggesting novel roles for familiar enzymes. The survey reveals the inter-digitation of systems that monitor cellular physiology, cell size, cellular stress and signalling processes with the basic cell cycle regulatory machinery.
Assuntos
Ciclo Celular/fisiologia , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Genoma , Proteínas Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Ciclo Celular/genética , Proliferação de Células , Citocinese , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Fase G2 , Genômica , Mitose/fisiologia , Mutação/genética , Estado Nutricional , Proteínas Quinases/genética , Interferência de RNA , Fase S , Transdução de Sinais , Fuso Acromático/fisiologia , Estresse Fisiológico/genética , Estresse Fisiológico/fisiopatologiaRESUMO
Aurora/Ipl1-related kinases are a conserved family of enzymes that have multiple functions during mitotic progression. Although it has been possible to use conventional genetic analysis to dissect the function of aurora, the founding family member in Drosophila (Glover, D.M., M.H. Leibowitz, D.A. McLean, and H. Parry. 1995. Cell. 81:95-105), the lack of mutations in a second aurora-like kinase gene, aurora B, precluded this approach. We now show that depleting Aurora B kinase using double-stranded RNA interference in cultured Drosophila cells results in polyploidy. aurora B encodes a passenger protein that associates first with condensing chromatin, concentrates at centromeres, and then relocates onto the central spindle at anaphase. Cells depleted of the Aurora B kinase show only partial chromosome condensation at mitosis. This is associated with a reduction in levels of the serine 10 phosphorylated form of histone H3 and a failure to recruit the Barren condensin protein onto chromosomes. These defects are associated with abnormal segregation resulting from lagging chromatids and extensive chromatin bridging at anaphase, similar to the phenotype of barren mutants (Bhat, M.A., A.V. Philp, D.M. Glover, and H.J. Bellen. 1996. Cell. 87:1103-1114.). The majority of treated cells also fail to undertake cytokinesis and show a reduced density of microtubules in the central region of the spindle. This is accompanied by a failure to correctly localize the Pavarotti kinesin-like protein, essential for this process. We discuss these conserved functions of Aurora B kinase in chromosome transmission and cytokinesis.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Caenorhabditis elegans , Cromossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila , Drosophila/citologia , Histonas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Animais , Aurora Quinases , Proteínas de Ciclo Celular/metabolismo , Divisão Celular , Células Cultivadas , Centrômero , Segregação de Cromossomos , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multiproteicos , Fosforilação , Poliploidia , Proteínas Serina-Treonina Quinases/genética , RNA Antissenso/metabolismo , RNA Interferente PequenoRESUMO
We have microinjected aphidicolin, a specific inhibitor of DNA polymerase alpha, into syncytial Drosophila embryos. This treatment inhibits DNA synthesis and, as a consequence, nuclear replication. We demonstrate that under these conditions several cycles of both centrosome replication and cortical budding continue, although the cycles have a longer periodicity than is normally found. As in uninjected embryos, when the cortical buds are present, the embryos have nuclei containing decondensed chromatin surrounded by nuclear membranes as judged by bright annular staining with an anti-lamin antibody. As the buds recede, the unreplicated chromatin condenses and lamin staining becomes weak and diffuse. Thus, both cytoplasmic and nuclear aspects of the mitotic cycle continue following the inhibition of DNA replication in the Drosophila embryo.
Assuntos
Diterpenos/farmacologia , Mitose/efeitos dos fármacos , Animais , Afidicolina , Compartimento Celular/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/ultraestrutura , Cromatina/efeitos dos fármacos , Cromatina/ultraestrutura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Replicação do DNA/efeitos dos fármacos , Drosophila melanogaster/efeitos dos fármacos , Drosophila melanogaster/embriologia , Laminas , Proteínas Nucleares/fisiologiaRESUMO
We describe a genetic locus rough deal (rod) in Drosophila melanogaster, identified by mutations that interfere with the faithful transmission of chromosomes to daughter cells during mitosis. Five mutant alleles were isolated, each associated with a similar set of mitotic abnormalities in the dividing neuroblasts of homozygous mutant larvae: high frequencies of aneuploid cells and abnormal anaphase figures, in which chromatids may lag, form bridges, or completely fail to separate. Surviving homozygous adults are sterile, and show cuticular defects associated with cell death, i.e., roughened eyes, sparse abdominal bristles, and notched wing margins. The morphological process of spermatogenesis is largely unaffected and motile sperm are produced, but meiocyte aneuploidy is common. The nature of the observed abnormalities in mitotic cells suggests that the reduced fidelity of chromosome transmission to the daughter cells is due to a failure in a mechanism involved in assuring the proper release of sister chromatids.
Assuntos
Mapeamento Cromossômico , Drosophila melanogaster/genética , Genes , Mitose , Alelos , Anáfase , Animais , Drosophila melanogaster/citologia , Masculino , Metáfase , Mutação , Fenótipo , EspermatogêneseRESUMO
Maternally contributed cyclin A and B proteins are initially distributed uniformly throughout the syncytial Drosophila embryo. As dividing nuclei migrate to the cortex of the embryo, the A and B cyclins become concentrated in surface layers extending to depths of approximately 30-40 microns and 5-10 microns, respectively. The initiation of nuclear envelope breakdown, spindle formation, and the initial congression of the centromeric regions of the chromosomes onto the metaphase plate all take place within the surface layer occupied by cyclin B on the apical side of the blastoderm nuclei. Cyclin B is seen mainly, but not exclusively, in the vicinity of microtubules throughout the mitotic cycle. It is most conspicuous around the centrosomes. Cyclin A is present at its highest concentrations throughout the cytoplasm during the interphase periods of the blastoderm cycles, although weak punctate staining can also be detected in the nucleus. It associates with the condensing chromosomes during prophase, segregates into daughter nuclei in association with chromosomes during anaphase, to redistribute into the cytoplasm after telophase. In contrast to the cycles following cellularization, neither cyclin is completely degraded upon the metaphase-anaphase transition.
Assuntos
Anáfase , Blastoderma/metabolismo , Cromatina/metabolismo , Ciclinas/metabolismo , Fuso Acromático/metabolismo , Animais , Núcleo Celular/metabolismo , Cicloeximida/farmacologia , Drosophila/embriologia , Embrião não Mamífero/metabolismo , Microtúbulos/metabolismo , MitoseRESUMO
We describe a new Drosophila gene, mini spindles (msps) identified in a cytological screen for mitotic mutant. Mutation in msps disrupts the structural integrity of the mitotic spindle, resulting in the formation of one or more small additional spindles in diploid cells. Nucleation of microtubules from centrosomes, metaphase alignment of chromosomes, or the focusing of spindle poles appears much less affected. The msps gene encodes a 227-kD protein with high similarity to the vertebrate microtubule-associated proteins (MAPs), human TOGp and Xenopus XMAP215, and with limited similarity to the Dis1 and STU2 proteins from fission yeast and budding yeast. Consistent with their sequence similarity, Msps protein also associates with microtubules in vitro. In the embryonic division cycles, Msps protein localizes to centrosomal regions at all mitotic stages, and spreads over the spindles during metaphase and anaphase. The absence of centrosomal staining in interphase of the cellularized embryos suggests that the interactions between Msps protein and microtubules or centrosomes may be regulated during the cell cycle.
Assuntos
Drosophila melanogaster/genética , Proteínas de Insetos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Schizosaccharomyces pombe , Fuso Acromático/metabolismo , Proteínas de Xenopus , Sequência de Aminoácidos , Animais , Ciclo Celular , Centrossomo/efeitos dos fármacos , Centrossomo/metabolismo , Segregação de Cromossomos/efeitos dos fármacos , Clonagem Molecular , Colchicina/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Humanos , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mitose/genética , Dados de Sequência Molecular , Mutação/genética , Paclitaxel/farmacologia , Homologia de Sequência de Aminoácidos , Fuso Acromático/efeitos dos fármacos , Fuso Acromático/genéticaRESUMO
The Drosophila gene polo encodes a conserved protein kinase known to be required to organize spindle poles and for cytokinesis. Here we report two strongly hypomorphic mutations of polo that arrest cells of the larval brain at a point in metaphase when the majority of sister kinetochores have separated by between 20-50% of the total spindle length in intact cells. In contrast, analysis of sister chromatid separation in squashed preparations of cells indicates that some 83% of sisters remain attached. This suggests the separation seen in intact cells requires the tension produced by a functional spindle. The point of arrest corresponds to the spindle integrity checkpoint; Bub1 protein and the 3F3/2 epitope are present on the separated kinetochores and the arrest is suppressed by a bub1 mutation. The mutant mitotic spindles are anastral and have assembled upon centrosomes that are associated with Centrosomin and the abnormal spindle protein (Asp), but neither with gamma-tubulin nor CP190. We discuss roles for Polo kinase in recruiting centrosomal proteins and in regulating progression through the metaphase-anaphase checkpoint.
Assuntos
Centrômero/fisiologia , Proteínas de Drosophila , Metáfase/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase/fisiologia , Animais , Encéfalo/citologia , Proteínas Cdc20 , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Ciclina A/metabolismo , Ciclina B/metabolismo , Drosophila , Larva/citologia , Mutação/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Troca de Cromátide Irmã/fisiologia , Fuso Acromático/metabolismo , Telômero/fisiologiaRESUMO
abnormal spindle, a gene required for normal spindle structure and function in Drosophila melanogaster, lies immediately adjacent the gene tolloid at 96A/B. It encodes a 220-kD polypeptide with a predicted pI of 10.8. The recessive mutant allele asp1 directs the synthesis of a COOH terminally truncated or internally deleted peptide of approximately 124 kD. Wild-type Asp protein copurifies with microtubules and is not released by salt concentrations known to dissociate most other microtubule-associated proteins. The bacterially expressed NH2-terminal 512-amino acid peptide, which has a number of potential phosphorylation sites for p34(cdc2) and MAP kinases, strongly binds to microtubules. The central 579-amino acid segment of the molecule contains one short motif homologous to sequences in a number of actin bundling proteins and a second motif present at the calmodulin binding sites of several proteins. Immunofluorescence studies show that the wild-type Asp protein is localized to the polar regions of the spindle immediately surrounding the centrosome. These findings are discussed in relation to the known spindle abnormalities in asp mutants.
Assuntos
Proteínas de Drosophila , Genes de Insetos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/ultraestrutura , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a Calmodulina/genética , Proteínas de Ligação a Calmodulina/metabolismo , Clonagem Molecular , Drosophila melanogaster , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Dados de Sequência MolecularRESUMO
We describe a Drosophila gene, orbit, that encodes a conserved 165-kD microtubule-associated protein (MAP) with GTP binding motifs. Hypomorphic mutations in orbit lead to a maternal effect resulting in branched and bent mitotic spindles in the syncytial embryo. In the larval central nervous system, such mutants have an elevated mitotic index with some mitotic cells showing an increase in ploidy. Amorphic alleles show late lethality and greater frequencies of hyperploid mitotic cells. The presence of cells in the hypomorphic mutant in which the chromosomes can be arranged, either in a circular metaphase or an anaphase-like configuration on monopolar spindles, suggests that polyploidy arises through spindle and chromosome segregation defects rather than defects in cytokinesis. A role for the Orbit protein in regulating microtubule behavior in mitosis is suggested by its association with microtubules throughout the spindle at all mitotic stages, by its copurification with microtubules from embryonic extracts, and by the finding that the Orbit protein directly binds to MAP-free microtubules in a GTP-dependent manner.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/citologia , Proteínas de Insetos/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Alelos , Sequência de Aminoácidos , Animais , Sistema Nervoso Central/citologia , Sistema Nervoso Central/crescimento & desenvolvimento , Cromossomos/genética , Cromossomos/metabolismo , Sequência Conservada/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Genes de Insetos/genética , Guanosina Trifosfato/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva/citologia , Larva/crescimento & desenvolvimento , Masculino , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Mapeamento Físico do Cromossomo , Poliploidia , RNA Mensageiro/análise , RNA Mensageiro/genética , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
A number of lines of evidence point to a predominance of cytokinesis defects in spermatogenesis in hypomorphic alleles of the Drosophila polo gene. In the pre-meiotic mitoses, cytokinesis defects result in cysts of primary spermatocytes with reduced numbers of cells that can contain multiple centrosomes. These are connected by a correspondingly reduced number of ring canals, structures formed by the stabilization of the cleavage furrow. The earliest defects during the meiotic divisions are a failure to form the correct mid-zone and mid-body structures at telophase. This is accompanied by a failure to correctly localize the Pavarotti kinesin- like protein that functions in cytokinesis, and of the septin Peanut and of actin to be incorporated into a contractile ring. In spite of these defects, cyclin B is degraded and the cells exit M phase. The resulting spermatids are frequently binuclear or tetranuclear, in which case they develop either two or four axonemes, respectively. A significant proportion of spermatids in which cytokinesis has failed may also show the segregation defects previously ascribed to polo1 mutants. We discuss these findings in respect to conserved functions for the Polo-like kinases in regulating progression through M phase, including the earliest events of cytokinesis.
Assuntos
Divisão Celular/fisiologia , Proteínas de Drosophila , Proteínas de Insetos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Anáfase , Animais , Diferenciação Celular , Ciclina B/metabolismo , Drosophila/metabolismo , Proteínas de Insetos/genética , Masculino , Meiose , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Mutagênese , Proteínas Serina-Treonina Quinases/genética , Espermátides/metabolismo , Espermatócitos/metabolismo , Fuso AcromáticoRESUMO
The product of the abnormal spindle (asp) gene was found to be an asymmetrically localized component of the centrosome during mitosis, required to focus the poles of the mitotic spindle in vivo. Removing Asp protein function from Drosophila melanogaster embryo extracts, either by mutation or immunodepletion, resulted in loss of their ability to restore microtubule-organizing center activity to salt-stripped centrosome preparations. This was corrected by addition of purified Asp protein. Thus, Asp appears to hold together the microtubule-nucleating gamma-tubulin ring complexes that organize the mitotic centrosome.
Assuntos
Centrossomo/fisiologia , Proteínas de Drosophila , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Mitose , Fuso Acromático/fisiologia , Animais , Anticorpos , Fracionamento Celular , Centrossomo/química , Centrossomo/ultraestrutura , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/fisiologia , Imunofluorescência , Proteínas Associadas aos Microtúbulos/análise , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/farmacologia , Mutação , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/análise , Tubulina (Proteína)/fisiologiaRESUMO
One of the rewards of having a Drosophila melanogaster whole-genome sequence will be the potential to understand the molecular bases for structural features of chromosomes that have been a long-standing puzzle. Analysis of 2.6 megabases of sequence from the tip of the X chromosome of Drosophila identifies 273 genes. Cloned DNAs from the characteristic bulbous structure at the tip of the X chromosome in the region of the broad complex display an unusual pattern of in situ hybridization. Sequence analysis revealed that this region comprises 154 kilobases of DNA flanked by 1.2-kilobases of inverted repeats, each composed of a 350-base pair satellite related element. Thus, some aspects of chromosome structure appear to be revealed directly within the DNA sequence itself.
Assuntos
Drosophila melanogaster/genética , Cromossomo X/genética , Animais , Bandeamento Cromossômico , Biologia Computacional , Cosmídeos , Elementos de DNA Transponíveis , DNA Satélite , Genes de Insetos , Hibridização In Situ , Sequências Repetitivas de Ácido Nucleico , Análise de Sequência de DNA , Cromossomo X/ultraestruturaRESUMO
BACKGROUND: SAK/PLK4 is a distinct member of the polo-like kinase family. SAK-/- mice die during embryogenesis, whereas SAK+/- mice develop liver and lung tumors and SAK+/- MEFs show mitotic abnormalities. However, the mechanism underlying these phenotypes is still not known. RESULTS: Here, we show that downregulation of SAK in Drosophila cells, by mutation or RNAi, leads to loss of centrioles, the core structures of centrosomes. Such cells are able to undergo repeated rounds of cell division, but display broad disorganized mitotic spindle poles. We also show that SAK mutants lose their centrioles during the mitotic divisions preceding male meiosis but still produce cysts of 16 primary spermatocytes as in the wild-type. Mathematical modeling of the stereotyped cell divisions of spermatogenesis can account for such loss by defective centriole duplication. The majority of spermatids in SAK mutants lack centrioles and so are unable to make sperm axonemes. Finally, we show that depletion of SAK in human cells also prevents centriole duplication and gives rise to mitotic abnormalities. CONCLUSIONS: SAK/PLK4 is necessary for centriole duplication both in Drosophila and human cells. Drosophila cells tolerate the lack of centrioles and undertake mitosis but cannot form basal bodies and hence flagella. Human cells depleted of SAK show error-prone mitosis, likely to underlie its tumor-suppressor role.
Assuntos
Centríolos/fisiologia , Flagelos/fisiologia , Mitose/fisiologia , Modelos Biológicos , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/fisiologia , Animais , Células Cultivadas , Centríolos/genética , Centríolos/ultraestrutura , Drosophila , Flagelos/genética , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNARESUMO
The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability [1-3] and are a hallmark of certain transformed cells and human cancer [4-6], the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A [7-9]. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins [10-12]. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus [13]. Here, we report that Slimb, an F-box protein that targets proteins to the SCFcomplex [14,15], plays a role in limiting centrosome replication. We found that, in the fruit fly Drosophila, the hypomorphic mutation slimb(crd) causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas de Drosophila , Drosophila/metabolismo , Proteínas de Insetos/metabolismo , Ubiquitina-Proteína Ligases , Animais , Encéfalo/citologia , Proteínas de Ciclo Celular/genética , Drosophila/genética , Imunofluorescência , Proteínas de Insetos/genética , Larva/citologia , Microscopia Confocal , Mitose/fisiologia , Mutação , Peptídeo Sintases/genética , Poliploidia , Proteínas Ligases SKP Culina F-BoxRESUMO
BACKGROUND: p21WAF1 is a potent inhibitor of the cell-cycle regulatory cyclin-dependent kinases (Cdks). It acts on Cdks in the G1 and S phases of the cell cycle, and also binds to proliferating cell nuclear antigen (PCNA), blocking DNA replication in vitro. Transcription of p21WAF1 can be induced by the human tumour suppressor protein p53, suggesting that the action of p21WAF1 may be important in cancer prevention. We have investigated the interaction between p21WAF1 and PCNA using a genetic two-hybrid screen and with arrays of synthetic peptides derived from the p21WAF1 protein sequence. RESULTS: We have established that the carboxy-terminal region of p21WAF1 interacts with PCNA in a yeast two-hybrid screen. Interaction with p21WAF1 involves the central loop of PCNA, which connects the two domains of the PCNA monomer. The interaction was finely mapped using peptides derived from the entire sequence of the p21WAF1 protein, and the critical residues were found to be QTSMTDFY (amino acids 144-151 of p21WAF1). Remarkably, a 20-residue peptide containing this sequence inhibited replication of simian virus 40 (SV40) DNA in vitro and could capture PCNA from whole cell extracts, demonstrating that small molecules can retain the biological activity characteristic of the whole protein. Sequential alanine-scan mutations of the peptide demonstrated that its ability to block replication correlates with its affinity for binding PCNA. CONCLUSIONS: We have shown that PCNA and the cell-cycle regulator p21WAF1 interact in vivo, and that this interaction requires the central loop of PCNA and an eight amino-acid motif from the carboxyl terminus of p21WAF1.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Ciclinas/química , Ciclinas/metabolismo , Replicação do DNA , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Inibidores de Proteínas Quinases , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Sequência de Aminoácidos , Sítios de Ligação , Ciclo Celular , Clonagem Molecular , Inibidor de Quinase Dependente de Ciclina p21 , Escherichia coli , Humanos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae , Proteína Supressora de Tumor p53/metabolismoRESUMO
Increasing levels of regulation act upon mitosis as the Drosophila embryo develops. The first 13 rapid cycles in the syncytial embryo rely on maternal gene products and lack feedback regulation to monitor the completion of S phase. Such regulation is introduced together with a G2 phase in cycle 14, and the network of universal mitotic regulators comes under the overall control of string, a cdc25 homologue, whose transcription is activated within mitotic domains.