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1.
J Cell Sci ; 126(Pt 15): 3441-51, 2013 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-23704354

RESUMO

Basal bodies and centrioles are conserved microtubule-based organelles the improper assembly of which leads to a number of diseases, including ciliopathies and cancer. Tubulin family members are conserved components of these structures that are integral to their proper formation and function. We have identified the ε-tubulin gene in Tetrahymena thermophila and detected the protein, through fluorescence of a tagged allele, to basal bodies. Immunoelectron microscopy has shown that ε-tubulin localizes primarily to the core microtubule scaffold. A complete genomic knockout of ε-tubulin has revealed that it is an essential gene required for the assembly and maintenance of the triplet microtubule blades of basal bodies. We have conducted site-directed mutagenesis of the ε-tubulin gene and shown that residues within the nucleotide-binding domain, longitudinal interacting domains, and C-terminal tail are required for proper function. A single amino acid change of Thr150, a conserved residue in the nucleotide-binding domain, to Val is a conditional mutation that results in defects in the spatial and temporal assembly of basal bodies as well as their stability. We have genetically separated functions for the domains of ε-tubulin and identified a novel role for the nucleotide-binding domain in the regulation of basal body assembly and stability.


Assuntos
Corpos Basais/fisiologia , Infecções por Cilióforos/metabolismo , Tetrahymena thermophila/fisiologia , Tubulina (Proteína)/fisiologia , Corpos Basais/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiologia , Centríolos/genética , Centríolos/metabolismo , Infecções por Cilióforos/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Microtúbulos/fisiologia , Tetrahymena thermophila/genética , Tetrahymena thermophila/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
2.
Science ; 340(6134): 871-5, 2013 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-23579499

RESUMO

Scaffold-assisted signaling cascades guide cellular decision-making. In budding yeast, one such signal transduction pathway called the mitotic exit network (MEN) governs the transition from mitosis to the G1 phase of the cell cycle. The MEN is conserved and in metazoans is known as the Hippo tumor-suppressor pathway. We found that signaling through the MEN kinase cascade was mediated by an unusual two-step process. The MEN kinase Cdc15 first phosphorylated the scaffold Nud1. This created a phospho-docking site on Nud1, to which the effector kinase complex Dbf2-Mob1 bound through a phosphoserine-threonine binding domain, in order to be activated by Cdc15. This mechanism of pathway activation has implications for signal transmission through other kinase cascades and might represent a general principle in scaffold-assisted signaling.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Desoxirribonucleases/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Mitose , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , tRNA Metiltransferases/metabolismo , Anáfase , Proteínas de Ciclo Celular/química , Desoxirribonucleases/química , Ativação Enzimática , Fosfoproteínas/química , Fosforilação , Conformação Proteica , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais , tRNA Metiltransferases/química
3.
Annu Rev Biochem ; 81: 561-85, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22482908

RESUMO

MPS1 protein kinases are found widely, but not ubiquitously, in eukaryotes. This family of potentially dual-specific protein kinases is among several that regulate a number of steps of mitosis. The most widely conserved MPS1 kinase functions involve activities at the kinetochore in both the chromosome attachment and the spindle checkpoint. MPS1 kinases also function at centrosomes. Beyond mitosis, MPS1 kinases have been implicated in development, cytokinesis, and several different signaling pathways. Family members are identified by virtue of a conserved C-terminal kinase domain, though the N-terminal domain is quite divergent. The kinase domain of the human enzyme has been crystallized, revealing an unusual ATP-binding pocket. The activity, level, and subcellular localization of Mps1 family members are tightly regulated during cell-cycle progression. The mitotic functions of Mps1 kinases and their overexpression in some tumors have prompted the identification of Mps1 inhibitors and their active development as anticancer drugs.


Assuntos
Mitose , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Animais , Citocinese , Humanos , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Fuso Acromático/metabolismo , Leveduras/citologia , Leveduras/enzimologia
4.
Genetics ; 190(4): 1197-224, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22491889

RESUMO

The Saccharomyces cerevisiae mitotic spindle in budding yeast is exemplified by its simplicity and elegance. Microtubules are nucleated from a crystalline array of proteins organized in the nuclear envelope, known as the spindle pole body in yeast (analogous to the centrosome in larger eukaryotes). The spindle has two classes of nuclear microtubules: kinetochore microtubules and interpolar microtubules. One kinetochore microtubule attaches to a single centromere on each chromosome, while approximately four interpolar microtubules emanate from each pole and interdigitate with interpolar microtubules from the opposite spindle to provide stability to the bipolar spindle. On the cytoplasmic face, two to three microtubules extend from the spindle pole toward the cell cortex. Processes requiring microtubule function are limited to spindles in mitosis and to spindle orientation and nuclear positioning in the cytoplasm. Microtubule function is regulated in large part via products of the 6 kinesin gene family and the 1 cytoplasmic dynein gene. A single bipolar kinesin (Cin8, class Kin-5), together with a depolymerase (Kip3, class Kin-8) or minus-end-directed kinesin (Kar3, class Kin-14), can support spindle function and cell viability. The remarkable feature of yeast cells is that they can survive with microtubules and genes for just two motor proteins, thus providing an unparalleled system to dissect microtubule and motor function within the spindle machine.


Assuntos
Mitose , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/fisiologia , Cromossomos Fúngicos/genética , Cromossomos Fúngicos/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Genes Fúngicos , Cinesinas/genética , Cinesinas/metabolismo , Cinetocoros/metabolismo , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Membrana Nuclear/fisiologia , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/genética , Fuso Acromático/fisiologia
5.
J Biol Chem ; 286(51): 43660-43667, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22045814

RESUMO

Ufd2 is a U-box-containing ubiquitylation enzyme that promotes ubiquitin chain assembly on substrates. The physiological function of Ufd2 remains poorly understood. Here, we show that ubiquitylation and degradation of the cell cycle kinase Mps1, a known target of the anaphase-promoting complex E3, require Ufd2 enzyme. Yeast cells lacking UFD2 exhibit altered chromosome stability and several spindle-related phenotypes, expanding the biological function of Ufd2. We demonstrate that Ufd2-mediated Mps1 degradation is conserved in humans. Our results underscore the significance of Ufd2 in proteolysis and further suggest that Ufd2-like enzymes regulate far more substrates than previously envisioned.


Assuntos
Candida albicans/genética , Proteínas de Ciclo Celular/metabolismo , Regulação Fúngica da Expressão Gênica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Animais , Células da Medula Óssea/metabolismo , Candida albicans/metabolismo , Linhagem Celular Tumoral , Humanos , Lectinas/química , Masculino , Camundongos , Mitose , Proteólise , Ubiquitina/química , Complexos Ubiquitina-Proteína Ligase/química , Ubiquitina-Proteína Ligases/química
6.
F1000 Biol Rep ; 2: 58, 2010 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-21173875

RESUMO

Centrioles organize both centrosomes and cilia. Centriole duplication is tightly regulated and coordinated with the cell cycle to limit duplication to only once per cell cycle. Defects in centriole number and structure are commonly found in cancer. Plk4/SAK and the functionally related Caenorhabditis elegans ZYG-1 kinases initiate centriole duplication. Several recent studies have elucidated the regulated activity of these kinases and potential downstream targets for centriole assembly.

7.
Cell ; 135(5): 894-906, 2008 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-19041752

RESUMO

During mitosis, sister chromatids congress to the spindle equator and are subsequently segregated via attachment to dynamic kinetochore microtubule (kMT) plus ends. A major question is how kMT plus-end assembly is spatially regulated to achieve chromosome congression. Here we find in budding yeast that the widely conserved kinesin-5 sliding motor proteins, Cin8p and Kip1p, mediate chromosome congression by suppressing kMT plus-end assembly of longer kMTs. Of the two, Cin8p is the major effector and its activity requires a functional motor domain. In contrast, the depolymerizing kinesin-8 motor Kip3p plays a minor role in spatial regulation of yeast kMT assembly. Our analysis identified a model where kinesin-5 motors bind to kMTs, move to kMT plus ends, and upon arrival at a growing plus end promote net kMT plus-end disassembly. In conclusion, we find that length-dependent control of net kMT assembly by kinesin-5 motors yields a simple and stable self-organizing mechanism for chromosome congression.


Assuntos
Cinesinas/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares , Mutação , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
8.
Nat Cell Biol ; 10(6): 665-75, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18500339

RESUMO

Separation of duplicated centrosomes (spindle-pole bodies or SPBs in yeast) is a crucial step in the biogenesis of the mitotic spindle. In vertebrates, centrosome separation requires the BimC family kinesin Eg5 and the activities of Cdk1 and polo kinase; however, the roles of these kinases are not fully understood. In Saccharomyces cerevisiae, SPB separation also requires activated Cdk1 and the plus-end kinesins Cin8 (homologous to vertebrate Eg5) and Kip1. Here we report that polo kinase has a role in the separation of SPBs. We show that adequate accumulation of Cin8 and Kip1 requires inactivation of the anaphase-promoting complex-activator Cdh1 through sequential phosphorylation by Cdk1 and polo kinase. In this process, Cdk1 functions as a priming kinase in that Cdk1-mediated phosphorylation creates a binding site for polo kinase,which further phosphorylates Cdh1. Thus, Cdh1 inactivation through the synergistic action of Cdk1 and polo kinase provides a new model for inactivation of cell-cycle effectors.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático , Ciclossomo-Complexo Promotor de Anáfase , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdh1 , Ciclo Celular , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Modelos Genéticos , Proteínas Motores Moleculares , Fosforilação , Proteínas Repressoras/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Quinase 1 Polo-Like
9.
Genes Dev ; 22(1): 91-105, 2008 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-18086858

RESUMO

beta-Catenin plays important roles in cell adhesion and gene transcription, and has been shown recently to be essential for the establishment of a bipolar mitotic spindle. Here we show that beta-catenin is a component of interphase centrosomes and that stabilization of beta-catenin, mimicking mutations found in cancers, induces centrosome splitting. Centrosomes are held together by a dynamic linker regulated by Nek2 kinase and its substrates C-Nap1 (centrosomal Nek2-associated protein 1) and Rootletin. We show that beta-catenin binds to and is phosphorylated by Nek2, and is in a complex with Rootletin. In interphase, beta-catenin colocalizes with Rootletin between C-Nap1 puncta at the proximal end of centrioles, and this localization is dependent on C-Nap1 and Rootletin. In mitosis, when Nek2 activity increases, beta-catenin localizes to centrosomes at spindle poles independent of Rootletin. Increased Nek2 activity disrupts the interaction of Rootletin with centrosomes and results in binding of beta-catenin to Rootletin-independent sites on centrosomes, an event that is required for centrosome separation. These results identify beta-catenin as a component of the intercentrosomal linker and define a new function for beta-catenin as a key regulator of mitotic centrosome separation.


Assuntos
Centrossomo/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , beta Catenina/metabolismo , Animais , Proteínas do Domínio Armadillo/metabolismo , Células Cultivadas , Centrossomo/enzimologia , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Cães , Humanos , Interfase , Quinases Relacionadas a NIMA , Proteínas/análise , Proteínas/metabolismo , beta Catenina/análise , tRNA Metiltransferases
10.
Mol Biol Cell ; 18(11): 4457-69, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17804818

RESUMO

Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrossomo/metabolismo , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Centrossomo/efeitos dos fármacos , Sequência Conservada , Ciclina A/metabolismo , Quinase 2 Dependente de Ciclina/metabolismo , Humanos , Dados de Sequência Molecular , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma , Proteínas Tirosina Quinases , Alinhamento de Sequência , Transdução de Sinais
11.
Genes Cells ; 12(6): 797-810, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17573779

RESUMO

Mortalin is a member of Hsp70 chaperoning protein family involved in various cellular functions. Through the search of the kinases that mortalin physically interact with, we identified Mps1 as such a kinase. Mps1 kinase has been implicated in the regulation of centrosome duplication and mitotic checkpoint response. Mortalin binds to Mps1, and is phosphorylated by Mps1 on Thr62 and Ser65. The phosphorylated mortalin then super-activates Mps1 in a feedback manner. Mortalin has been previously shown to localize to centrosomes, and to be involved in the regulation of centrosome duplication. We found that centrosomal localization of mortalin depends on the presence of Mps1. Moreover, Mps1-associated acceleration of centrosome duplication depends on the presence of mortalin and super-activation by the Thr62/Ser65 phosphorylated mortalin.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Centrossomo/metabolismo , Regulação da Expressão Gênica , Humanos , Hidroxiureia/farmacologia , Microscopia de Fluorescência , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas Tirosina Quinases , Serina/química , Treonina/química
12.
Nat Genet ; 39(6): 727-9, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17468754

RESUMO

Jeune asphyxiating thoracic dystrophy, an autosomal recessive chondrodysplasia, often leads to death in infancy because of a severely constricted thoracic cage and respiratory insufficiency; retinal degeneration, cystic renal disease and polydactyly may be complicating features. We show that IFT80 mutations underlie a subset of Jeune asphyxiating thoracic dystrophy cases, establishing the first association of a defective intraflagellar transport (IFT) protein with human disease. Knockdown of ift80 in zebrafish resulted in cystic kidneys, and knockdown in Tetrahymena thermophila produced shortened or absent cilia.


Assuntos
Asfixia/genética , Doenças do Desenvolvimento Ósseo/genética , Proteínas de Transporte/genética , Doenças Renais Císticas/genética , Mutação/genética , Tetrahymena thermophila/genética , Doenças Torácicas/genética , Peixe-Zebra/genética , Animais , Feminino , Humanos , Recém-Nascido , Masculino , Linhagem , Polidactilia/genética , Tetrahymena thermophila/crescimento & desenvolvimento , Peixe-Zebra/crescimento & desenvolvimento
13.
Results Probl Cell Differ ; 42: 111-46, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-16903210

RESUMO

Centrosomes are dynamic organelles involved in many aspects of cell function and growth. Centrosomes act as microtubule organizing centers, and provide a site for concerted regulation of cell cycle progression. While there is diversity in microtubule organizing center structure among eukaryotes, many centrosome components, such as centrin, are conserved. Experimental analysis has provided an outline to describe centrosome duplication, and numerous centrosome components have been identified. Even so, more work is needed to provide a detailed understanding of the interactions between centrosome components and their roles in centrosome function and duplication. Precise duplication of centrosomes once during each cell cycle ensures proper mitotic spindle formation and chromosome segregation. Defects in centrosome duplication or function are linked to human diseases including cancer. Here we provide a multifaceted look at centrosomes with a detailed summary of the centrosome cycle.


Assuntos
Ciclo Celular , Centrossomo/metabolismo , Animais , Proteínas de Ciclo Celular , Segregação de Cromossomos , Humanos , Centro Organizador dos Microtúbulos/metabolismo , Mitose , Modelos Biológicos
14.
Science ; 313(5787): 680-4, 2006 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-16825537

RESUMO

The spindle checkpoint delays cell cycle progression until microtubules attach each pair of sister chromosomes to opposite poles of the mitotic spindle. Following sister chromatid separation, however, the checkpoint ignores chromosomes whose kinetochores are attached to only one spindle pole, a state that activates the checkpoint prior to metaphase. We demonstrate that, in budding yeast, mutual inhibition between the anaphase-promoting complex (APC) and Mps1, an essential component of the checkpoint, leads to sustained inactivation of the spindle checkpoint. Mps1 protein abundance decreases in anaphase, and Mps1 is a target of the APC. Furthermore, expression of Mps1 in anaphase, or repression of the APC in anaphase, reactivates the spindle checkpoint. This APC-Mps1 feedback circuit allows cells to irreversibly inactivate the checkpoint during anaphase.


Assuntos
Anáfase/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Fuso Acromático/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Sequência de Aminoácidos , Ciclossomo-Complexo Promotor de Anáfase , Proteínas Cdc20 , Proteínas de Ciclo Celular/metabolismo , Cromossomos Fúngicos/fisiologia , Retroalimentação Fisiológica , Proteínas de Ligação ao GTP/metabolismo , Cinetocoros/fisiologia , Proteínas Mad2 , Mitose , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Securina
15.
EMBO J ; 25(11): 2551-63, 2006 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-16688214

RESUMO

In yeast, separation of duplicated spindle pole bodies (SPBs) (centrosomes in higher eukaryotes) is an indispensable step in the assembly of mitotic spindle and is triggered by severing of the bridge that connects the sister SPBs. This process requires Cdk1 (Cdc28) activation by Tyrosine 19 dephosphorylation. We show that cells that fail to activate Cdk1 are devoid of spindles due to persistently active APCCdh1, which targets microtubule-associated proteins Cin8, Kip1 and Ase1 for degradation. Tyrosine 19 dephosphorylation of Cdk1 is necessary to specifically prevent proteolysis of these proteins. Interestingly, SPB separation is dependent on the microtubule-bundling activity of Cin8 but not on its motor function. Since ectopic expression of proteolysis-resistant Cin8, Kip1 or Ase1 is sufficient for SPB separation even in the absence of Cdc28-Clb activity, we suggest that stabilization of these mechanical force-generating proteins is the predominant role of Cdc28-Clb in centrosome separation.


Assuntos
Proteína Quinase CDC2/metabolismo , Centrossomo/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Proteína Quinase CDC2/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas Cdh1 , Ciclo Celular/fisiologia , Ativação Enzimática , Cinesinas , Proteínas Associadas aos Microtúbulos/genética , Proteínas Motores Moleculares , Complexo de Endopeptidases do Proteassoma/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
16.
RNA ; 12(1): 94-101, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16373495

RESUMO

Retroviruses and retrotransposons assemble intracellular immature core particles around a RNA genome, and nascent particles collect in association with membranes or as intracellular clusters. How and where genomic RNA are identified for retrovirus and retrotransposon assembly, and how translation and assembly processes are coordinated is poorly understood. To understand this process, the subcellular localization of Ty3 RNA and capsid proteins and virus-like particles was investigated. We demonstrate that mRNAs, proteins, and virus-like particles of the yeast Ty3 retrotransposon accumulate in association with cytoplasmic P-bodies, which are sites of mRNA translation repression, storage, and degradation. Deletions of genes encoding P-body proteins decreased Ty3 transposition and caused changes in the pattern of Ty3 foci, underscoring the biological significance of the association of Ty3 virus-like protein components and P-bodies. These results suggest the hypothesis that P-bodies may serve to segregate translation and assembly functions of the Ty3 genomic RNA to promote assembly of virus-like particles. Because Ty3 has features of a simple retrovirus and P-body functions are conserved between yeast and metazoan organisms, these findings may provide insights into host factors that facilitate retrovirus assembly.


Assuntos
Corpos de Inclusão/metabolismo , DNA Polimerase Dirigida por RNA/metabolismo , Retroelementos/genética , Retroviridae/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Vírion , Montagem de Vírus , DNA Polimerase Dirigida por RNA/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/virologia , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência
17.
Mol Biol Cell ; 16(3): 1178-88, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15635095

RESUMO

Meiotic chromosome segregation leads to the production of haploid germ cells. During meiosis I (MI), the paired homologous chromosomes are separated. Meiosis II (MII) segregation leads to the separation of paired sister chromatids. In the budding yeast Saccharomyces cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four-spored ascus. We have modeled the microtubules in 20 MI and 15 MII spindles by using reconstruction from electron micrographs of serially sectioned meiotic cells. Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles. It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization. Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule. The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells. Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B and that these movements are concurrent. This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process.


Assuntos
Cromossomos Fúngicos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/química , Fuso Acromático/ultraestrutura , Anáfase , Núcleo Celular/metabolismo , Cromátides/ultraestrutura , Segregação de Cromossomos , Proteínas Fúngicas/metabolismo , Proteínas de Fluorescência Verde/química , Haploidia , Processamento de Imagem Assistida por Computador , Cinetocoros/metabolismo , Meiose , Microscopia Eletrônica , Microtúbulos/metabolismo , Modelos Teóricos , Mutação , Fenótipo
18.
Proc Natl Acad Sci U S A ; 100(25): 14875-80, 2003 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-14657364

RESUMO

The mitotic spindle is essential for the maintenance of genetic stability, and in budding yeast its assembly and function depend on the Mps1 protein kinase. Mps1p is required for centrosome duplication and the spindle checkpoint. Several recent reports demonstrate that vertebrate Mps1 proteins regulate the spindle checkpoint, but reports conflict regarding their role in centrosome duplication. Here we provide multiple lines of evidence that the human Mps1 protein (hMps1) is required for centrosome duplication. A recently described rabbit polyclonal antibody against hMps1 specifically recognizes centrosomes in a variety of human cell types. Overexpression of a dominant-negative version of hMps1 (hMps1KD) can prevent centrosome duplication in a variety of cell types, and active hMps1 accelerates centrosome reduplication in U2OS cells. Finally, we demonstrate that disruption of hMps1 function with pools of hMps1-specific small interfering RNAs causes a pleiotropic phenotype resulting from the combination of severe mitotic abnormalities and failures in centrosome duplication. This approach demonstrates that hMps1 is required for centrosome duplication and for the normal progression of mitosis, and suggests that the threshold level of hMps1 function required for centrosome duplication is lower than that required for hMps1 mitotic functions.


Assuntos
Proteínas de Ciclo Celular , Centrossomo , Mitose , Proteínas Quinases , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Centrossomo/ultraestrutura , Técnica Indireta de Fluorescência para Anticorpo , Genes Dominantes , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Immunoblotting , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Fenótipo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
19.
Curr Opin Cell Biol ; 14(6): 700-5, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12473342

RESUMO

Centrosomes are microtubule organising centres that act as spindle poles during mitosis. Recent work implicates centrosomes in many other processes, and shows that centrosome defects can cause genetic instability. Many regulators of mammalian centrosome function were predicted from studies of model systems. Surprisingly, some well-known tumour suppressors have recently been found at centrosomes, where they influence centrosome duplication and function, suggesting that control of centrosome function is central to genetic stability.


Assuntos
Centrossomo/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Centrossomo/química , Centrossomo/ultraestrutura , Modelos Biológicos , Mutação , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genética
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