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 execution of female meiosis and the establishment of the zygote is arguably the most critical stage of mammalian development. The egg can be arrested in the prophase of meiosis I for decades, and when it is activated, the spindle is assembled de novo. This spindle must function with the highest of fidelity and yet its assembly is unusually achieved in the absence of conventional centrosomes and with minimal influence of chromatin. Moreover, its dramatic asymmetric positioning is achieved through remarkable properties of the actin cytoskeleton to ensure elimination of the polar bodies. The second meiotic arrest marks a uniquely prolonged metaphase eventually interrupted by egg activation at fertilization to complete meiosis and mark a period of preparation of the male and female pronuclear genomes not only for their entry into the mitotic cleavage divisions but also for the imminent prospect of their zygotic expression.
Assuntos
Desenvolvimento Embrionário , Mamíferos/embriologia , Meiose , Mitose , Animais , Cromossomos de Mamíferos/metabolismo , Humanos , Fuso Acromático/metabolismoRESUMO
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
Centrioles duplicate once in each cell division cycle through so-called templated or canonical duplication. SAK, also called PLK4 (SAK/PLK4), a kinase implicated in tumor development, is an upstream regulator of canonical biogenesis necessary for centriole formation. We found that overexpression of SAK/PLK4 could induce amplification of centrioles in Drosophila embryos and their de novo formation in unfertilized eggs. Both processes required the activity of DSAS-6 and DSAS-4, two molecules required for canonical duplication. Thus, centriole biogenesis is a template-free self-assembly process triggered and regulated by molecules that ordinarily associate with the existing centriole. The mother centriole is not a bona fide template but a platform for a set of regulatory molecules that catalyzes and regulates daughter centriole assembly.
Assuntos
Centríolos/fisiologia , Centrossomo/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Embrião não Mamífero/fisiologia , Oócitos/fisiologia , Animais , Animais Geneticamente Modificados , Centríolos/ultraestrutura , Centrossomo/ultraestrutura , Drosophila/metabolismo , Proteínas de Drosophila/genética , Desenvolvimento Embrionário , Feminino , MitoseRESUMO
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
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
dsRNA of several hundred nucleotides in length is effective at interfering with gene expression in mouse oocytes, pre-implantation embryos, and embryonic stem (ES) cells but is not as efficient in differentiated cell lines. Here we describe a method to achieve RNA interference in totipotent and differentiated ES cells together with a wide range of other mammalian cell types that is both simple and efficient. It utilizes a linearized plasmid that directs the expression of a hairpin RNA with a 22-nucleotide-paired region. This molecule has a 13-nucleotide 5' overhang that would be subject to capping on its 5' phosphoryl group and thus differs from the ideal structure suggested for effective small interfering RNAs. Thus, it appears either that the structure of small inhibitory RNA molecules may not need to be as precise as previously thought or that such a transcript is efficiently processed to a form that is effective in interfering with gene expression.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento/genética , Interferência de RNA , RNA de Cadeia Dupla/metabolismo , Células-Tronco/metabolismo , Animais , Diferenciação Celular/genética , Linhagem Celular , Células Híbridas , Camundongos/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Células-Tronco/citologia , Transfecção/métodosAssuntos
Mitose , Proteínas Quinases/fisiologia , Animais , Divisão Celular , Centrossomo/fisiologiaRESUMO
We have previously shown that the molecular chaperone heat shock protein 90 (Hsp90) is required to ensure proper centrosome function in Drosophila and vertebrate cells. This observation led to the hypothesis that this chaperone could be required for the stability of one or more centrosomal proteins. We have found that one of these is Polo, a protein kinase known to regulate several aspects of cell division including centrosome maturation and function. Inhibition of Hsp90 results in the inactivation of Polo kinase activity. It also leads to a loss in the ability of cytoplasmic extracts to complement the failure of salt-stripped preparations of centrosomes to nucleate microtubules. This effect can be rescued upon addition of active recombinant POLO: We also show that Polo and Hsp90 are part of a complex and conclude that stabilization of Polo is one of the mechanisms by which Hsp90 contributes to the maintenance of functional centrosomes.
Assuntos
Centrossomo/fisiologia , Proteínas de Drosophila , Proteínas de Choque Térmico HSP90/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Divisão Celular , Linhagem Celular , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/fisiologia , Teste de Complementação Genética , Proteínas Recombinantes/metabolismo , VertebradosRESUMO
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
We present the sequence of a contiguous 2.63 Mb of DNA extending from the tip of the X chromosome of Drosophila melanogaster. Within this sequence, we predict 277 protein coding genes, of which 94 had been sequenced already in the course of studying the biology of their gene products, and examples of 12 different transposable elements. We show that an interval between bands 3A2 and 3C2, believed in the 1970s to show a correlation between the number of bands on the polytene chromosomes and the 20 genes identified by conventional genetics, is predicted to contain 45 genes from its DNA sequence. We have determined the insertion sites of P-elements from 111 mutant lines, about half of which are in a position likely to affect the expression of novel predicted genes, thus representing a resource for subsequent functional genomic analysis. We compare the European Drosophila Genome Project sequence with the corresponding part of the independently assembled and annotated Joint Sequence determined through "shotgun" sequencing. Discounting differences in the distribution of known transposable elements between the strains sequenced in the two projects, we detected three major sequence differences, two of which are probably explained by errors in assembly; the origin of the third major difference is unclear. In addition there are eight sequence gaps within the Joint Sequence. At least six of these eight gaps are likely to be sites of transposable elements; the other two are complex. Of the 275 genes in common to both projects, 60% are identical within 1% of their predicted amino-acid sequence and 31% show minor differences such as in choice of translation initiation or termination codons; the remaining 9% show major differences in interpretation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos/genética , Análise de Sequência de DNA/métodos , Cromossomo X/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos , Biologia Computacional , Elementos de DNA Transponíveis/genética , Proteínas de Ligação a DNA/genética , Feminino , Ordem dos Genes/genética , Masculino , Dados de Sequência Molecular , Mapeamento Físico do Cromossomo/métodos , Fatores de Transcrição/genéticaRESUMO
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
Plo1-associated casein kinase activity peaked during mitosis before septation. Phosphatase treatment abolished this activity. Mitotic Plo1 activation had a requirement for prior activation of M-phase promoting factor (MPF), suggesting that Plo1 does not act as a mitotic trigger kinase to initiate MPF activation during mitotic commitment. A link between Plo1 and the septum initiating network (SIN) has been suggested by the inability of plo1 Delta cells to septate and the prolific septation following plo1(+) overexpression. Interphase activation of Spg1, the G protein that modulates SIN activity, induced septation but did not stimulate Plo1-associated kinase activity. Conversely, SIN inactivation did not affect the mitotic stimulation of Plo1-associated kinase activity. plo1.ts4 cells formed a misshapen actin ring, but rarely septated at 36 degrees C. Forced activation of Spg1 enabled plo1.ts4 mutant cells, but not cells with defects in the SIN component Sid2, to convert the actin ring to a septum. The ability of plo1(+) overexpression to induce septation was severely compromised by SIN inactivation. We propose that Plo1 acts before the SIN to control septation.
Assuntos
Proteínas de Drosophila , Mitose , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Schizosaccharomyces/citologia , Proteína Quinase CDC2/metabolismo , Caseína Quinases , Proteínas de Ciclo Celular/metabolismo , Ativação Enzimática , Proteínas Fúngicas/metabolismo , Fator Promotor de Maturação/metabolismo , Fenótipo , ras-GRF1/metabolismoRESUMO
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 cloned the Drosophila gene discs degenerate-4 (dd4) and find that it encodes a component of the gamma-tubulin ring complex (gammaTuRC) homologous to Spc98 of budding yeast. This provides the first opportunity to study decreased function of a member of the gamma-tubulin ring complex, other than gamma-tubulin itself, in a metazoan cell. gamma-tubulin is no longer at the centrosomes but is dispersed throughout dd4 cells and yet bipolar metaphase spindles do form, although these have a dramatically decreased density of microtubules. Centrosomin (CNN) remains in broad discrete bodies but only at the focused poles of such spindles, whereas Asp (abnormal spindle protein) is always present at the presumptive minus ends of microtubules, whether or not they are focused. This is consistent with the proposed role of Asp in coordinating the nucleation of mitotic microtubule organizing centers. The centrosome associated protein CP190 is partially lost from the spindle poles in dd4 cells supporting a weak interaction with gamma-tubulin, and the displaced protein accumulates in the vicinity of chromosomes. Electron microscopy indicates not only that the poles of dd4 cells have irregular amounts of pericentriolar material, but also that they can have abnormal centrioles. In six dd4 cells subjected to serial sectioning centrioles were missing from one of the two poles. This suggests that in addition to its role in nucleating cytoplasmic and spindle microtubules, the gammaTuRC is also essential to the structure of centrioles and the separation of centrosomes.
Assuntos
Centrossomo/metabolismo , Proteínas de Drosophila , Drosophila/genética , Proteínas dos Microtúbulos/genética , Mutação , Tubulina (Proteína)/genética , Animais , Centrossomo/ultraestrutura , Cromossomos/metabolismo , Clonagem Molecular , Feminino , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Homeodomínio/ultraestrutura , Masculino , Metáfase/genética , Proteínas dos Microtúbulos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/imunologia , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Proteínas Nucleares/imunologia , Proteínas Nucleares/metabolismo , Subunidades Proteicas , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Tubulina (Proteína)/metabolismoRESUMO
The mutagen-sensitive-101 (mus101) gene of Drosophila melanogaster was first identified 25 years ago through mutations conferring larval hypersensitivity to DNA-damaging agents. Other alleles of mus101 causing different phenotypes were later isolated: a female sterile allele results in a defect in a tissue-specific form of DNA synthesis (chorion gene amplification) and lethal alleles cause mitotic chromosome instability that can be observed genetically and cytologically. The latter phenotype presents as a striking failure of mitotic chromosomes of larval neuroblasts to undergo condensation of pericentric heterochromatic regions, as we show for a newly described mutant carrying lethal allele mus101(lcd). To gain further insight into the function of the Mus101 protein we have molecularly cloned the gene using a positional cloning strategy. We report here that mus101 encodes a member of the BRCT (BRCA1 C terminus) domain superfamily of proteins implicated in DNA repair and cell cycle checkpoint control. Mus101, which contains seven BRCT domains distributed throughout its length, is most similar to human TopBP1, a protein identified through its in vitro association with DNA topoisomerase IIbeta. Mus101 also shares sequence similarity with the fission yeast Rad4/Cut5 protein required for repair, replication, and checkpoint control, suggesting that the two proteins may be functional homologs.
Assuntos
Proteína BRCA1/genética , Proteínas de Ciclo Celular/genética , Mapeamento Cromossômico , Reparo do DNA/genética , Replicação do DNA/genética , Proteínas de Drosophila , Drosophila melanogaster/genética , Genes de Insetos , Genes Letais , Heterocromatina/genética , Sequência de Aminoácidos , Animais , Proteína BRCA1/química , Proteínas de Ciclo Celular/química , Feminino , Genes BRCA1 , Infertilidade Feminina/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Larva , Masculino , Dados de Sequência Molecular , Mutagênese , Fenótipo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Cromossomo XRESUMO
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
The meiotic spindle of Drosophila oocytes is acentriolar but develops an unusual central microtubule organising centre (MTOC) at the end of meiosis I. In polo oocytes, this common central pole for the two tandem spindles of meiosis II was poorly organised and in contrast to wild-type failed to maintain its associated Pav-KLP motor protein. Furthermore, the polar body nuclei failed to arrest at metaphase, and the four products of female meiosis all underwent repeated haploid division cycles on anastral spindles. This was linked to a failure to form the astral array of microtubules with which the polar body chromosomes are normally associated. The MTOC associated with the male pronucleus was also defective in polo eggs, and the sperm aster did not grow. Migration of the female pronucleus did not take place and so a gonomeric spindle could not form. We discuss these findings in relation to the known roles of polo like kinases in regulating the behaviour of MTOCs.