RESUMO
BACKGROUND: The RZZ complex, composed of the proteins Rough-Deal (Rod), Zw10 and Zwilch, plays a central role in the spindle assembly checkpoint (SAC), which assures proper sister chromatid segregation during mitosis. RZZ contributes to the regulation of the spindle assembly checkpoint by helping to recruit Mad1-Mad2 and the microtubule motor dynein to unattached kinetochores. It is an important component of the outer kinetochore and specifically the fibrous corona whose expansion is believed to facilitate microtubule capture. How RZZ carries out its diverse activities is only poorly understood. The C-terminal region of the Rod subunit is relatively well-conserved across metazoan phylogeny, but no function has been attributed to it. RESULTS: To explore the importance of the Rod_C domain in RZZ function in Drosophila, we generated a series of point mutations in a stretch of 200 residues within this domain and we report here their phenotypes. Several of the mutations profoundly disrupt recruitment of RZZ to kinetochores, including one in a temperature-sensitive manner, while still retaining the capacity to assemble into a complex with Zw10 and Zwilch. Others affect aspects of dynein activity or recruitment at the kinetochore. CONCLUSIONS AND SIGNIFICANCE: These results suggest that the Rod_C domain participates in the protein interactions necessary for RZZ recruitment and functionality at kinetochores.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/fisiologia , Drosophila , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Mad2/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Fuso Acromático/metabolismoRESUMO
Proper kinetochore recruitment and regulation of dynein and the Mad1-Mad2 complex requires the Rod-Zw10-Zwilch (RZZ) complex. Here, we describe rod(Z3), a maternal-effect Drosophila mutation changing a single residue in the Rough Deal (Rod) subunit of RZZ. Although the RZZ complex containing this altered subunit (denoted R(Z3)ZZ) is present in early syncytial stage embryos laid by homozygous rod(Z3) mothers, it is not recruited to kinetochores. Consequently, the embryos have no spindle assembly checkpoint (SAC), and syncytial mitoses are profoundly perturbed. The polar body (residual meiotic products) cannot remain in its SAC-dependent metaphase-like state, and decondenses into chromatin. In neuroblasts of homozygous rod(Z3) larvae, R(Z3)ZZ recruitment is only partially reduced, the SAC is functional and mitosis is relatively normal. R(Z3)ZZ nevertheless behaves abnormally: it does not further accumulate on kinetochores when microtubules are depolymerized; it reduces the rate of Mad1 recruitment; and it dominantly interferes with the dynein-mediated streaming of RZZ from attached kinetochores. These results suggest that the mutated residue of rod(Z3) is required for normal RZZ kinetochore recruitment and function and, moreover, that the RZZ recruitment pathway might differ in syncytial stage embryos and post-embryonic somatic cells.
Assuntos
Animais Geneticamente Modificados/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação/genética , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/embriologia , Animais Geneticamente Modificados/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Feminino , Processamento de Imagem Assistida por Computador , Dados de Sequência Molecular , Neurônios/citologia , Neurônios/metabolismo , Homologia de Sequência de Aminoácidos , Fuso Acromático/fisiologiaRESUMO
The presence or absence of Mad1 at kinetochores is a major determinant of spindle assembly checkpoint (SAC) activity, the surveillance mechanism that delays anaphase onset if one or more kinetochores remain unattached to spindle fibers. Among the factors regulating the levels of Mad1 at kinetochores is the Rod, Zw10, and Zwilch (RZZ) complex, which is required for Mad1 recruitment through a mechanism that remains unknown. The relative dynamics and interactions of Mad1 and RZZ at kinetochores have not been extensively investigated, although Mad1 has been reported to be stably recruited to unattached kinetochores. In this study, we directly compare Mad1-green fluorescent protein (GFP) turnover dynamics on unattached Drosophila kinetochores with that of RZZ, tagged either with GFP-Rod or GFP-Zw10. We find that nearly 40 % of kinetochore-bound Mad1 has a significant dynamic component, turning over with a half-life of 12 s. RZZ in contrast is essentially stable on unattached kinetochores. In addition, we report that a fraction of RZZ and Mad1 can co-immunoprecipitate, indicating that the genetically determined recruitment hierarchy (in which Mad1 depends on RZZ) may reflect a physical association of the two complexes.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila/genética , Drosophila/metabolismo , Cinetocoros/metabolismo , Animais , Animais Geneticamente Modificados , Expressão Gênica , Ordem dos Genes , Genes Reporter , Loci Gênicos , Pontos de Checagem da Fase M do Ciclo Celular , Ligação Proteica , Transporte Proteico , Fuso Acromático/metabolismo , TransgenesRESUMO
The functions of Beclin-1 in macroautophagy, tumorigenesis and cytokinesis are thought to be mediated by its association with the PI3K-III complex. Here, we describe a new role for Beclin-1 in mitotic chromosome congression that is independent of the PI3K-III complex and its role in autophagy. Beclin-1 depletion in HeLa cells leads to a significant reduction of the outer kinetochore proteins CENP-E, CENP-F and ZW10, and, consequently, the cells present severe problems in chromosome congression. Beclin-1 associates with kinetochore microtubules and forms discrete foci near the kinetochores of attached chromosomes. We show that Beclin-1 interacts directly with Zwint-1-a component of the KMN (KNL-1/Mis12/Ndc80) complex-which is essential for kinetochore-microtubule interactions. This suggests that Beclin-1 acts downstream of the KMN complex to influence the recruitment of outer kinetochore proteins and promotes accurate kinetochore anchoring to the spindle during mitosis.
Assuntos
Proteínas Reguladoras de Apoptose/fisiologia , Cromossomos Humanos/metabolismo , Cinetocoros/metabolismo , Proteínas de Membrana/fisiologia , Proteína Beclina-1 , Segregação de Cromossomos , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Microscopia de Fluorescência , Mitose , Proteínas Nucleares/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , Imagem com Lapso de TempoRESUMO
Mad2 has a key role in the spindle-assembly checkpoint (SAC) - the mechanism delaying anaphase onset until all chromosomes correctly attach to the spindle. Here, we show that unlike every other reported case of SAC inactivation in metazoans, mad2-null Drosophila are viable and fertile, and their cells almost always divide correctly despite having no SAC and an accelerated 'clock', which is caused by premature degradation of cyclin B. Mitosis in Drosophila does not need the SAC because correct chromosome attachment is achieved very rapidly, before even the cell lacking Mad2 can initiate anaphase. Experimentally reducing spindle-assembly efficiency renders the cells Mad2-dependent. In fact, the robustness of the SAC may generally mask minor mitotic defects of mutations affecting spindle function. The reported lethality of other Drosophila SAC mutations may be explained by their multifunctionality, and thus the 'checkpoint' phenotypes previously ascribed to these mutations should be considered the consequence of eliminating both the checkpoint and a second mitotic function.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Mitose/fisiologia , Fuso Acromático/metabolismo , Anáfase/fisiologia , Ciclossomo-Complexo Promotor de Anáfase , Animais , Proteínas de Ciclo Celular/genética , Colchicina/farmacologia , Ciclina B/metabolismo , Drosophila/efeitos dos fármacos , Drosophila/embriologia , Drosophila/genética , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Homeodomínio/metabolismo , Cinetocoros/metabolismo , Larva/fisiologia , Proteínas Mad2 , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/efeitos dos fármacos , Mutação , Fenótipo , Fuso Acromático/efeitos dos fármacos , Fatores de Tempo , Moduladores de Tubulina/farmacologia , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Unattached kinetochores generate an anaphase inhibitor, through the spindle assembly checkpoint (SAC), that allows cells more time to establish proper kinetochore-microtubule (K-MT) linkages and thus avoid aneuploidy. Mad1 is the receptor for Mad2 at kinetochores, where it catalyzes the formation of Mad2-Cdc20 complexes, an essential part of the anaphase inhibitor, but whether it has any other mitotic function is unknown. We have generated a mad1-null mutation in Drosophila. This mutant is SAC defective and Mad2 is no longer localized to either nuclear envelope or kinetochores, but it displays normal basal mitotic timing. Unlike mad2 mutants, which have relatively normal mitoses, mad1 anaphases show high frequencies of lagging chromatids, at least some of which are caused by persistent merotelic linkages. A transgene expressing GFP-Mad1 rescues both the SAC and the anaphase defects. In an attempt to separate the SAC function from the mitotic function, we made a mad1 transgene with a mutated Mad2-binding domain. Surprisingly, this transgene failed to complement the anaphase phenotype. Thus, Mad1 has activity promoting proper K-MT attachments in addition to its checkpoint function. This activity does not require the presence of Mad2, but it does depend in some unknown way on key residues in the Mad2-binding domain of Mad1.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/fisiologia , Mitose/fisiologia , Proteínas Nucleares/fisiologia , Fuso Acromático/fisiologia , Transgenes , Anáfase , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Cinetocoros/metabolismo , Proteínas Mad2 , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fenótipo , Transdução de Sinais , Fuso Acromático/genética , Fuso Acromático/metabolismoRESUMO
AMP-activated protein kinase (AMPK, also known as SNF1A) has been primarily studied as a metabolic regulator that is activated in response to energy deprivation. Although there is relatively ample information on the biochemical characteristics of AMPK, not enough data exist on the in vivo function of the kinase. Here, using the Drosophila model system, we generated the first animal model with no AMPK activity and discovered physiological functions of the kinase. Surprisingly, AMPK-null mutants were lethal with severe abnormalities in cell polarity and mitosis, similar to those of lkb1-null mutants. Constitutive activation of AMPK restored many of the phenotypes of lkb1-null mutants, suggesting that AMPK mediates the polarity- and mitosis-controlling functions of the LKB1 serine/threonine kinase. Interestingly, the regulatory site of non-muscle myosin regulatory light chain (MRLC; also known as MLC2) was directly phosphorylated by AMPK. Moreover, the phosphomimetic mutant of MRLC rescued the AMPK-null defects in cell polarity and mitosis, suggesting MRLC is a critical downstream target of AMPK. Furthermore, the activation of AMPK by energy deprivation was sufficient to cause dramatic changes in cell shape, inducing complete polarization and brush border formation in the human LS174T cell line, through the phosphorylation of MRLC. Taken together, our results demonstrate that AMPK has highly conserved roles across metazoan species not only in the control of metabolism, but also in the regulation of cellular structures.
Assuntos
Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Metabolismo Energético , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Proteínas Quinases Ativadas por AMP , Animais , Linhagem Celular , Polaridade Celular , Proteínas de Drosophila/deficiência , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Humanos , Masculino , Mitose , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Cadeias Leves de Miosina/metabolismo , Fenótipo , Fosforilação , Proteínas Quinases/deficiência , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Introduction. Candida spp. may cause opportunistic infections called vulvovaginal candidiasis (VVC), which is estimated to be the second most common cause of vaginitis worldwide.Gap Statement. Under various circumstances, VVC could compromise pregnancy outcomes. Emerging data suggests that VVC during pregnancy may be associated with increased risk of complications and congenital cutaneous candidiasis.Aim. To assess the prevalence of Candida spp. in asymptomatic pregnant women and determine the susceptibility of the isolates to antifungal drugs.Methodology. In a prospective cohort, 65 high vaginal swab samples of consented pregnant women. Candida isolates were identified using both microbiological and molecular tools and drug susceptibilities were profiled.Results. The prevalence of VVC among our study participants was 37â%, 24 of the 65 asymptomatic pregnant women show Candida spp. colonization. C. albicans was the most common species 61â%, followed by C. glabrata 39 %. In addition, a significant fraction of the isolated colonies showed resistance to Fluconazole, with a ratio of 63 % for C. albicans isolates and 16â% for Candida glabrata isolates. Moreover, relative quantification of genes related to resistance to fluconazole, CDR1, ERG11 as well as HWP1, showed a significant change compared to controls.Conclusion. Monitoring of vaginal Candida colonization before the third trimester of pregnancy, that could reduce congenital Candida colonization and risk of pregnancy complications.
Assuntos
Candida , Candidíase Vulvovaginal , Antifúngicos/farmacologia , Candida albicans/genética , Candidíase Vulvovaginal/epidemiologia , Candidíase Vulvovaginal/microbiologia , Feminino , Fluconazol/farmacologia , Humanos , Recém-Nascido , Gravidez , Gestantes , Estudos Prospectivos , Vagina/microbiologiaRESUMO
Drosophila testes are a powerful model system for studying biological processes including stem cell biology, nuclear architecture, meiosis and sperm development. However, immunolabeling of the whole Drosophila testis is often associated with significant non-uniformity of staining due to antibody penetration. Squashed preparations only partially overcome the problem since it decreases the 3D quality of the analyses. Herein, we describe a whole-mount protocol using NP40 and heptane during fixation together with immunolabeling in liquid media. It preserves the volume suitable for confocal microscopy together with reproducible and reliable labeling. We show different examples of 3D reconstitution of spermatocyte nuclei from confocal sections. The intra- and inter-testes reproducibility allows 3D quantification and comparison of fluorescence between single cells from different genotypes. We used different components of the intranuclear MINT structure (Mad1-containing Intra Nuclear Territory) as well as two components associated with the nuclear pore complex to illustrate this protocol and its applications on the largest cells of the testis, the S4-S5 spermatocytes.
Assuntos
Drosophila melanogaster/citologia , Imageamento Tridimensional , Microscopia Confocal/métodos , Espermatócitos/citologia , Testículo/citologia , Animais , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imunofluorescência , Masculino , Interferência de RNA , Reprodutibilidade dos Testes , Espermatócitos/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Fixação de TecidosRESUMO
Compromising the activity of the spindle checkpoint permits mitotic exit in the presence of unattached kinetochores and, consequently, greatly increases the rate of aneuploidy in the daughter cells. The metazoan checkpoint mechanism is more complex than in yeast in that it requires additional proteins and activities besides the classical Mads and Bubs. Among these are Rod, Zw10, and Zwilch, components of a 700 Kdal complex (Rod/Zw10) that is required for recruitment of dynein/dynactin to kinetochores but whose role in the checkpoint is poorly understood. The dynamics of Rod and Mad2, examined in different organisms, show intriguing similarities as well as apparent differences. Here we simultaneously follow GFP-Mad2 and RFP-Rod and find they are in fact closely associated throughout early mitosis. They accumulate simultaneously on kinetochores and are shed together along microtubule fibers after attachment. Their behavior and position within attached kinetochores is distinct from that of BubR1; Mad2 and Rod colocalize to the outermost kinetochore region (the corona), whereas BubR1 is slightly more interior. Moreover, Mad2, but not BubR1, Bub1, Bub3, or Mps1, requires Rod/Zw10 for its accumulation on unattached kinetochores. Rod/Zw10 thus contributes to checkpoint activation by promoting Mad2 recruitment and to checkpoint inactivation by recruiting dynein/dynactin that subsequently removes Mad2 from attached kinetochores.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/metabolismo , Animais , Western Blotting , Encéfalo/metabolismo , Clonagem Molecular , Primers do DNA , Drosophila , Dineínas/metabolismo , Gânglios dos Invertebrados/metabolismo , Proteínas de Fluorescência Verde , Larva/metabolismo , Proteínas Luminescentes , Microtúbulos/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteína Vermelha FluorescenteRESUMO
The Drosophila Mad1 spindle checkpoint protein helps organize several nucleoplasmic components, and flies lacking Mad1 present changes in gene expression reflecting altered chromatin conformation. In interphase, checkpoint protein Mad1 is usually described as localizing to the inner nuclear envelope by binding the nucleoporin Tpr, an interaction believed to contribute to proper mitotic regulation. Whether Mad1 has other nuclear interphase functions is unknown. We found in Drosophila that Mad1 is present in nuclei of both mitotic and postmitotic tissues. Three proteins implicated in various aspects of chromatin organization co-immunoprecipitated with Mad1 from fly embryos: Mtor/Tpr, the SUMO peptidase Ulp1 and Raf2, a subunit of a Polycomb-like complex. In primary spermatocytes, all four proteins colocalized in a previously undescribed chromatin-associated structure called here a MINT (Mad1-containing IntraNuclear Territory). MINT integrity required all four proteins. In mad1 mutant spermatocytes, the other proteins were no longer confined to chromatin domains but instead dispersed throughout the nucleoplasm. mad1 flies also presented phenotypes indicative of excessive chromatin of heterochromatic character during development of somatic tissues. Together these results suggest that Drosophila Mad1, by helping organize its interphase protein partners in the nucleoplasm, contributes to proper chromatin regulation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Interfase/fisiologia , Animais , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Masculino , Poro Nuclear/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Espermatócitos/citologia , Espermatócitos/metabolismoRESUMO
Cenp-E is a kinesin-like motor protein required for efficient end-on attachment of kinetochores to the spindle microtubules. Cenp-E immunodepletion in Xenopus mitotic extracts results in the loss of mitotic arrest and massive chromosome missegregation, whereas its depletion in mammalian cells leads to chromosome segregation defects despite the presence of a functional spindle assembly checkpoint (SAC). Cenp-meta has previously been reported to be the Drosophila homolog of vertebrate Cenp-E. In this study, we show that cenp-metaΔ mutant neuroblasts arrest in mitosis when treated with colchicine. cenp-metaΔ mutant cells display a mitotic delay. Yet, despite the persistence of the two checkpoint proteins Mad2 and BubR1 on unattached kinetochores, these cells eventually enter anaphase and give rise to highly aneuploid daughter cells. Indeed, we find that cenp-metaΔ mutant cells display a slow but continuous degradation of cyclin B, which eventually triggers the mitotic exit observed. Thus, our data provide evidence for a role of Cenp-meta in sustaining the SAC response.
RESUMO
BubR1 is a critical component of the spindle assembly checkpoint, the surveillance mechanism that helps maintain the high fidelity of mitotic chromosome segregation by preventing cells from initiating anaphase if one or more kinetochores are not attached to the spindle. BubR1 also helps promote the establishment of stable kinetochore-microtubule attachments during prometaphase. In this chapter, we review the structure, functions, and regulation of BubR1 in these "classical roles" at the kinetochore. We discuss its recruitment to kinetochores, its assembly into the inhibitor of anaphase progression, and the importance of its posttranslational modifications. We also consider the evidence for its participation in other roles beyond mitosis, such as the meiosis-specific processes of recombination and prophase arrest of the first meiotic division, the cellular response to DNA damage, and in the regulation of centrosome and basal body function. Finally, studies are presented linking BubR1 dysfunction or misregulation to aging and human disease, particularly cancer.
Assuntos
Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Envelhecimento , Animais , Proteínas de Ciclo Celular , Humanos , Meiose , Mitose/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genéticaRESUMO
Selection of the cleavage plane during cytokinesis in dividing cells is linked to the position of the mitotic spindle. A major player in cleavage plane positioning is believed to be the anaphase central spindle and its associated signaling complex called centralspindlin, composed of MgcRacGap and MKLP1. Centralspindlin has the capacity to induce furrowing of the cell cortex by promoting the localized activation of RhoA, which in turn promotes assembly of the contractile ring. We have found a way to induce a cytokinesis-like process in unfertilized Drosophila eggs and very early embryos, when spindle structures are few and located far from invaginating egg cortex. The simple injection of a small molecule inhibitor of Cdk1/Cyclin B (either Roscovitin or RO3306) is sufficient to promote membrane invagination near the site of injection. The furrow generated is in many respects similar to a classical cleavage furrow. Actin, myosin, anillin and MKLP1 are all associated with the forming furrow, which in some cases can entirely circumscribe the unfertilized egg. A similar furrow can also be generated by the localized injection of constitutively active RhoA protein, suggesting that Cdk1 is normally an upstream inhibitor of RhoA activation. We show further that this process apparently is not associated with microtubules. Since simple localized inhibition of Cdk1 is sufficient to induce a furrow, we suggest that in real cytokinesis in normal cells, the localized downregulation of Cdk1 activity at the metaphase-anaphase transition may contribute, along with the spindle, to the positioning of the cleavage furrow.
Assuntos
Proteína Quinase CDC2/antagonistas & inibidores , Ciclina B/antagonistas & inibidores , Citocinese , Drosophila/fisiologia , Óvulo/citologia , Actinas/metabolismo , Animais , Divisão Celular , Células Cultivadas , Proteínas Contráteis/metabolismo , Citocinese/efeitos dos fármacos , Drosophila/embriologia , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/citologia , Proteínas Ativadoras de GTPase , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose , Miosinas/metabolismo , Óvulo/fisiologia , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Quinolinas/farmacologia , Roscovitina , Fuso Acromático/metabolismo , Tiazóis/farmacologia , Proteína rhoA de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/farmacologiaRESUMO
Monopolar spindle 1 (Mps1) is essential for the spindle assembly checkpoint (SAC), which prevents anaphase onset in the presence of misaligned chromosomes. Moreover, Mps1 kinase contributes in a SAC-independent manner to the correction of erroneous initial attachments of chromosomes to the spindle. Our characterization of the Drosophila homologue reveals yet another SAC-independent role. As in yeast, modest overexpression of Drosophila Mps1 is sufficient to delay progression through mitosis during metaphase, even though chromosome congression and metaphase alignment do not appear to be affected. This delay in metaphase depends on the SAC component Mad2. Although Mps1 overexpression in mad2 mutants no longer causes a metaphase delay, it perturbs anaphase. Sister kinetochores barely move apart toward spindle poles. However, kinetochore movements can be restored experimentally by separase-independent resolution of sister chromatid cohesion. We propose therefore that Mps1 inhibits sister chromatid separation in a SAC-independent manner. Moreover, we report unexpected results concerning the requirement of Mps1 dimerization and kinase activity for its kinetochore localization in Drosophila. These findings further expand Mps1's significance for faithful mitotic chromosome segregation and emphasize the importance of its careful regulation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Proteínas de Drosophila/metabolismo , Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Fuso Acromático/metabolismo , Anáfase , Animais , Animais Geneticamente Modificados , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Cinetocoros/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas Mad2 , Metáfase , Microscopia de Fluorescência , Mutação , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteína Vermelha FluorescenteRESUMO
BubR1 performs several roles during mitosis, affecting the spindle assembly checkpoint (SAC), mitotic timing, and spindle function, but the interdependence of these functions is unclear. We have analyzed in Drosophila melanogaster the mitotic phenotypes of kinase-dead (KD) BubR1 and BubR1 lacking the N-terminal KEN box. bubR1-KD individuals have a robust SAC but abnormal spindles with thin kinetochore fibers, suggesting that the kinase activity modulates microtubule capture and/or dynamics but is relatively dispensable for SAC function. In contrast, bubR1-KEN flies have normal spindles but no SAC. Nevertheless, mitotic timing is normal as long as Mad2 is present. Thus, the SAC, timer, and spindle functions of BubR1 are substantially separable. Timing is shorter in bubR1-KEN mad2 double mutants, yet in these flies, lacking both critical SAC components, chromosomes still segregate accurately, reconfirming that in Drosophila, reliable mitosis does not need the SAC.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/metabolismo , Mitose/fisiologia , Fuso Acromático/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Fuso Acromático/ultraestruturaRESUMO
Nuclear pore complexes (NPCs) are multisubunit protein entities embedded into the nuclear envelope (NE). Here, we examine the in vivo dynamics of the essential Drosophila nucleoporin Nup107 and several other NE-associated proteins during NE and NPCs disassembly and reassembly that take place within each mitosis. During both the rapid mitosis of syncytial embryos and the more conventional mitosis of larval neuroblasts, Nup107 is gradually released from the NE, but it remains partially confined to the nuclear (spindle) region up to late prometaphase, in contrast to nucleoporins detected by wheat germ agglutinin and lamins. We provide evidence that in all Drosophila cells, a structure derived from the NE persists throughout metaphase and early anaphase. Finally, we examined the dynamics of the spindle checkpoint proteins Mad2 and Mad1. During mitotic exit, Mad2 and Mad1 are actively imported back from the cytoplasm into the nucleus after the NE and NPCs have reformed, but they reassociate with the NE only later in G1, concomitantly with the recruitment of the basket nucleoporin Mtor (the Drosophila orthologue of vertebrate Tpr). Surprisingly, Drosophila Nup107 shows no evidence of localization to kinetochores, despite the demonstrated importance of this association in mammalian cells.