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1.
bioRxiv ; 2023 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-37333421

RESUMO

Faithful segregation of chromosomes into daughter cells during mitosis requires formation of attachments between kinetochores and mitotic spindle microtubules. Chromosome alignment on the mitotic spindle, also referred to as congression, is facilitated by translocation of side-bound chromosomes along the microtubule surface, which allows the establishment of end-on attachment of kinetochores to microtubule plus ends. Spatial and temporal constraints hinder observation of these events in live cells. Therefore, we used our previously developed reconstitution assay to observe dynamics of kinetochores, the yeast kinesin-8, Kip3, and the microtubule polymerase, Stu2, in lysates prepared from metaphase-arrested budding yeast, Saccharomyces cerevisiae . Using total internal reflection fluorescence (TIRF) microscopy to observe kinetochore translocation on the lateral microtubule surface toward the microtubule plus end, motility was shown to be dependent on both Kip3, as we reported previously, and Stu2. These proteins were shown to have distinct dynamics on the microtubule. Kip3 is highly processive and moves faster than the kinetochore. Stu2 tracks both growing and shrinking microtubule ends but also colocalizes with moving lattice-bound kinetochores. In cells, we observed that both Kip3 and Stu2 are important for establishing chromosome biorientation, Moreover, when both proteins are absent, biorientation is completely defective. All cells lacking both Kip3 and Stu2 had declustered kinetochores and about half also had at least one unattached kinetochore. Our evidence argues that despite differences in their dynamics, Kip3 and Stu2 share roles in chromosome congression to facilitate proper kinetochore-microtubule attachment.

2.
J Cell Sci ; 136(11)2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-37305999

RESUMO

The budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and the cytoplasmic microtubules (MTs), both of which generate forces to faithfully segregate chromosomes, remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has distinct functions on MTs in each compartment. Here, we show that two proteins, Cik1 and Vik1, which form heterodimers with Kar3, regulate its localization and function within the cell, and along MTs in a cell cycle-dependent manner. Using a yeast MT dynamics reconstitution assay in lysates from cell cycle-synchronized cells, we found that Kar3-Vik1 induces MT catastrophes in S phase and metaphase, and limits MT polymerization in G1 and anaphase. In contrast, Kar3-Cik1 promotes catastrophes and pauses in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track MT motor protein motility, we observed that Cik1 is necessary for Kar3 to track MT plus-ends in S phase and metaphase but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.


Assuntos
Cinesinas , Saccharomyces cerevisiae , Cinesinas/genética , Ciclo Celular , Anáfase , Metáfase
3.
Elife ; 112022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35791811

RESUMO

During mitosis, individual microtubules make attachments to chromosomes via a specialized protein complex called the kinetochore to faithfully segregate the chromosomes to daughter cells. Translocation of kinetochores on the lateral surface of the microtubule has been proposed to contribute to high fidelity chromosome capture and alignment at the mitotic midzone, but has been difficult to observe in vivo because of spatial and temporal constraints. To overcome these barriers, we used total internal reflection fluorescence (TIRF) microscopy to track the interactions between microtubules, kinetochore proteins, and other microtubule-associated proteins in lysates from metaphase-arrested Saccharomyces cerevisiae. TIRF microscopy and cryo-correlative light microscopy and electron tomography indicated that we successfully reconstituted interactions between intact kinetochores and microtubules. These kinetochores translocate on the lateral microtubule surface toward the microtubule plus end and transition to end-on attachment, whereupon microtubule depolymerization commences. The directional kinetochore movement is dependent on the highly processive kinesin-8, Kip3. We propose that Kip3 facilitates stable kinetochore attachment to microtubule plus ends through its abilities to move the kinetochore laterally on the surface of the microtubule and to regulate microtubule plus end dynamics.


Assuntos
Cinetocoros , Proteínas de Saccharomyces cerevisiae , Cinesinas , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
J Cell Sci ; 132(4)2018 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-30185524

RESUMO

Microtubules (MTs) are important for cellular structure, transport of cargoes and segregation of chromosomes and organelles during mitosis. The stochastic growth and shrinkage of MTs, known as dynamic instability, is necessary for these functions. Previous studies to determine how individual MT-associated proteins (MAPs) affect MT dynamics have been performed either through in vivo studies, which provide limited opportunity for observation of individual MTs or manipulation of conditions, or in vitro studies, which focus either on purified proteins, and therefore lack cellular complexity, or on cell extracts made from genetically intractable organisms. In order to investigate the ensemble activities of all MAPs on MT dynamics using lysates made from a genetically tractable organism, we developed a cell-free assay for budding yeast lysates using total internal reflection fluorescence (TIRF) microscopy. Lysates were prepared from yeast strains expressing GFP-tubulin. MT polymerization from pre-assembled MT seeds adhered to a coverslip was observed in real time. Through use of cell division cycle (cdc) and MT depolymerase mutants, we found that MT polymerization and dynamic instability are dependent on the cell cycle state and the activities of specific MAPs.


Assuntos
Microtúbulos/metabolismo , Mitose/fisiologia , Tubulina (Proteína)/metabolismo , Cinesinas/metabolismo , Microscopia de Fluorescência/métodos , Proteínas Associadas aos Microtúbulos/metabolismo , Polimerização , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/metabolismo
5.
J Cell Biol ; 217(5): 1687-1700, 2018 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-29563217

RESUMO

Mitotic spindle disassembly after chromosome separation is as important as spindle assembly, yet the molecular mechanisms for spindle disassembly are unclear. In this study, we investigated how the chromosomal passenger complex (CPC), which contains the Aurora B kinase Ipl1, swiftly concentrates at the spindle midzone in late anaphase, and we researched the role of this dramatic relocalization during spindle disassembly. We showed that the kinesins Kip1 and Kip3 are essential for CPC relocalization. In cells lacking Kip1 and Kip3, spindle disassembly is severely delayed until after contraction of the cytokinetic ring. Purified Kip1 and Kip3 interact directly with the CPC and recruit it to microtubules in vitro, and single-molecule experiments showed that the CPC diffuses dynamically on microtubules but that diffusion stops when the CPC encounters a Kip1 molecule. We propose that Kip1 and Kip3 trap the CPC at the spindle midzone in late anaphase to ensure timely spindle disassembly.


Assuntos
Cinesinas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Anáfase , Proteínas Cromossômicas não Histona , Proteínas Imobilizadas/metabolismo , Microtúbulos/metabolismo , Ligação Proteica
6.
Mol Biol Cell ; 26(14): 2620-39, 2015 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-26023090

RESUMO

We observed the dynamic recruitment of spindle checkpoint proteins Mad1 and Bub1 to detached kinetochores in budding yeast using real-time live-cell imaging and quantified recruitment in fixed cells. After induced de novo kinetochore assembly at one pair of sister centromeres, Mad1 appeared after the kinetochore protein Mtw1. Detached kinetochores were not associated with the nuclear envelope, so Mad1 does not anchor them to nuclear pore complexes (NPCs). Disrupting Mad1's NPC localization increased Mad1 recruitment to detached sister kinetochores. Conversely, increasing the number of detached kinetochores reduced the amount of Mad1 per detached kinetochore. Bub1 also relocalized completely from the spindle to detached sister centromeres after kinetochore assembly. After their capture by microtubules, Mad1 and Bub1 progressively disappeared from kinetochores. Sister chromatids that arrested with a lateral attachment to one microtubule exhibited half the Mad1 of fully detached sisters. We propose that detached kinetochores compete with alternate binding sites in the nucleus to recruit Mad1 and Bub1 from available pools that are small enough to be fully depleted by just one pair of detached kinetochores and that lateral attachment licenses Mad1 removal from kinetochores after a kinetic delay.


Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Microtúbulos , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo
7.
Mol Biol Cell ; 26(13): 2505-18, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25971801

RESUMO

Casein kinase 1δ (CK1δ) family members associate with microtubule-organizing centers (MTOCs) from yeast to humans, but their mitotic roles and targets have yet to be identified. We show here that budding yeast CK1δ, Hrr25, is a γ-tubulin small complex (γTuSC) binding factor. Moreover, Hrr25's association with γTuSC depends on its kinase activity and its noncatalytic central domain. Loss of Hrr25 kinase activity resulted in assembly of unusually long cytoplasmic microtubules and defects in spindle positioning, consistent with roles in regulation of γTuSC-mediated microtubule nucleation and the Kar9 spindle-positioning pathway, respectively. Hrr25 directly phosphorylated γTuSC proteins in vivo and in vitro, and this phosphorylation promoted γTuSC integrity and activity. Because CK1δ and γTuSC are highly conserved and present at MTOCs in diverse eukaryotes, similar regulatory mechanisms are expected to apply generally in eukaryotes.


Assuntos
Caseína Quinase Idelta/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Tubulina (Proteína)/metabolismo , Caseína Quinase I/metabolismo , Ciclo Celular/fisiologia , Citoesqueleto/metabolismo , Microtúbulos/metabolismo , Fosforilação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/citologia , Saccharomycetales/metabolismo , Fuso Acromático/metabolismo
8.
Dev Cell ; 32(2): 231-40, 2015 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-25625208

RESUMO

In budding yeast, over 60 proteins functioning in at least five modules are recruited to endocytic sites with predictable order and timing. However, how sites of clathrin-mediated endocytosis are initiated and stabilized is not well understood. Here, the casein kinase 1 (CK1) Hrr25 is shown to be an endocytic protein and to be among the earliest proteins to appear at endocytic sites. Hrr25 absence or overexpression decreases or increases the rate of endocytic site initiation, respectively. Ede1, an early endocytic Eps15-like protein important for endocytic initiation, is an Hrr25 target and is required for Hrr25 recruitment to endocytic sites. Hrr25 phosphorylation of Ede1 is required for Hrr25-Ede1 interaction and promotes efficient initiation of endocytic sites. These observations indicate that Hrr25 kinase and Ede1 cooperate to initiate and stabilize endocytic sites. Analysis of the mammalian homologs CK1δ/ε suggests a conserved role for these protein kinases in endocytic site initiation and stabilization.


Assuntos
Caseína Quinase I/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animais , Humanos , Fosforilação/fisiologia
9.
Genetics ; 198(3): 1043-57, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25213170

RESUMO

Timely spindle disassembly is essential for coordination of mitotic exit with cytokinesis. In the budding yeast Saccharomyces cerevisiae, the microtubule-associated protein She1 functions in one of at least three parallel pathways that promote spindle disassembly. She1 phosphorylation by the Aurora kinase Ipl1 facilitates a role for She1 in late anaphase, when She1 contributes to microtubule depolymerization and shrinkage of spindle halves. By examining the genetic interactions of known spindle disassembly genes, we identified three genes in the environmental stress-sensing HOG (high-osmolarity glycerol response) pathway, SHO1, PBS2, and HOG1, and found they are necessary for proper localization of She1 to the anaphase spindle and for proper spindle disassembly. HOG pathway mutants exhibited spindle disassembly defects, as well as mislocalization of anillin-related proteins Boi1 and Boi2 from the bud neck. Moreover, Boi2, but not Boi1, plays a role in spindle disassembly that places Boi2 in a pathway with Sho1, Pbs2, and Hog1. Together, our data identify a process by which cells monitor events at the spindle and bud neck and describe a novel role for the HOG pathway in mitotic signaling.


Assuntos
Meio Ambiente , Mitose , Saccharomycetales/citologia , Saccharomycetales/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo , Citocinese , Mutação/genética , Polimerização , Ligação Proteica , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico
10.
PLoS One ; 8(9): e73194, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24039885

RESUMO

In Saccharomyces cerevisiae mitosis, the protein Slk19 plays an important role in the initial release of Cdc14 phosphatase from the nucleolus to the nucleus in early anaphase, an event that is critical for proper anaphase progression. A role for Slk19 in later mitotic stages of Cdc14 regulation, however, has not been demonstrated. While investigating the role of Slk19 post-translational modification on Cdc14 regulation, we found that a triple point mutant of SLK19, slk19(3R) (three lysine-to-arginine mutations), strongly affects Cdc14 localization during late anaphase and mitotic exit. Using fluorescence live-cell microscopy, we found that, similar to slk19Δ cells, slk19(3R) cells exhibit no defect in spindle stability and only a mild defect in spindle elongation dynamics. Unlike slk19Δcells, however, slk19(3R) cells exhibit no defect in Cdc14 release from the nucleolus to the nucleus. Instead, slk19(3R) cells are defective in the timing of Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase. This mutant has a novel phenotype: slk19(3R) causes premature Cdc14 movement to the cytoplasm prior to, rather than concomitant with, spindle disassembly. One consequence of this premature Cdc14 movement is the inappropriate activation of the mitotic exit network, made evident by the fact that slk19(3R) partially rescues a mutant of the mitotic exit network kinase Cdc15. In conclusion, in addition to its role in regulating Cdc14 release from the nucleolus to the nucleus, we found that Slk19 is also important for regulating Cdc14 movement from the nucleus to the cytoplasm at the end of anaphase.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Anáfase , Expressão Gênica , Proteínas Associadas aos Microtúbulos/genética , Modelos Biológicos , Mutação , Fenótipo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo
11.
J Biol Chem ; 288(32): 23203-11, 2013 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-23814063

RESUMO

The chromosomal passenger complex (CPC) is a key regulator of mitosis in eukaryotes. It comprises four essential and conserved proteins known in mammals/yeasts as Aurora B/Ipl1, INCENP/Sli15, Survivin/Bir1, and Borealin/Nbl1. These subunits act together in a highly controlled fashion. Regulation of Aurora B/Ipl1 kinase activity and localization is critical for CPC function. Although regulation of CPC localization and kinase activity in vivo has been investigated elsewhere, studies on the complete, four-subunit CPC and its basic biochemical properties are only beginning. Here we describe the biochemical characterization of purified and complete Saccharomyces cerevisiae four-subunit CPC. We determined the affinity of the CPC for microtubules and demonstrated that the binding of CPC to microtubules is primarily electrostatic in nature and depends on the acidic C-terminal tail (E-hook) of tubulin. Moreover, phosphorylation of INCENP/Sli15 on its microtubule binding region also negatively regulates CPC affinity for microtubules. Furthermore, we show that phosphorylation of INCENP/Sli15 is required for activation of the kinase Aurora B/Ipl1 and can occur in trans. Although phosphorylation of INCENP/Sli15 is essential for activation, we determined that a version of the CPC lacking the INCENP/Sli15 microtubule binding region (residues Glu-91 to Ile-631) is able to form an intact complex that retains microtubule binding activity. Thus, we conclude that this INCENP/Sli15 linker domain plays a largely regulatory function and is not essential for complex formation or microtubule binding.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Aurora Quinases , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Complexos Multiproteicos/genética , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
12.
Mol Biol Cell ; 23(2): 258-67, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-22090343

RESUMO

Incomplete mitotic spindle disassembly causes lethality in budding yeast. To determine why spindle disassembly is required for cell viability, we used live-cell microscopy to analyze a double mutant strain containing a conditional mutant and a deletion mutant compromised for the kinesin-8 and anaphase-promoting complex-driven spindle-disassembly pathways (td-kip3 and doc1Δ, respectively). Under nonpermissive conditions, spindles in td-kip3 doc1Δ cells could break apart but could not disassemble completely. These cells could exit mitosis and undergo cell division. However, the daughter cells could not assemble functional, bipolar spindles in the ensuing mitosis. During the formation of these dysfunctional spindles, centrosome duplication and separation, as well as recruitment of key midzone-stabilizing proteins all appeared normal, but microtubule polymerization was nevertheless impaired and these spindles often collapsed. Introduction of free tubulin through episomal expression of α- and ß-tubulin or introduction of a brief pulse of the microtubule-depolymerizing drug nocodazole allowed spindle assembly in these td-kip3 doc1Δ mutants. Therefore we propose that spindle disassembly is essential for regeneration of the intracellular pool of assembly-competent tubulin required for efficient spindle assembly during subsequent mitoses of daughter cells.


Assuntos
Ciclo Celular , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , Fuso Acromático/metabolismo , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Deleção de Genes , Cinesinas/genética , Cinesinas/metabolismo , Multimerização Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/metabolismo
13.
Mol Biol Cell ; 22(22): 4335-42, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21965284

RESUMO

All eukaryotic cells must segregate their chromosomes equally between two daughter cells at each division. This process needs to be robust, as errors in the form of loss or gain of genetic material have catastrophic effects on viability. Chromosomes are captured, aligned, and segregated to daughter cells via interaction with spindle microtubules mediated by the kinetochore. In Saccharomyces cerevisiae one microtubule attaches to each kinetochore, requiring extreme processivity from this single connection. The yeast Dam1 complex, an essential component of the outer kinetochore, forms rings around microtubules and in vitro recapitulates much of the functionality of a kinetochore-microtubule attachment. To understand the mechanism of the Dam1 complex at the kinetochore, we must know how it binds to microtubules, how it assembles into rings, and how assembly is regulated. We used electron microscopy to map several subunits within the structure of the Dam1 complex and identify the organization of Dam1 complexes within the ring. Of importance, new data strongly support a more passive role for the microtubule in Dam1 ring formation. Integrating this information with previously published data, we generated a structural model for the Dam1 complex assembly that advances our understanding of its function and will direct future experiments.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Divisão Celular , Segregação de Cromossomos , Cromossomos/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/química , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia
14.
J Cell Biol ; 194(1): 137-53, 2011 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-21727193

RESUMO

Dynamic microtubules facilitate chromosome arrangement before anaphase, whereas during anaphase microtubule stability assists chromosome separation. Changes in microtubule dynamics at the metaphase-anaphase transition are regulated by Cdk1. Cdk1-mediated phosphorylation of Sli15/INCENP promotes preanaphase microtubule dynamics by preventing chromosomal passenger complex (CPC; Sli15/INCENP, Bir1/Survivin, Nbl1/Borealin, Ipl1/Aurora) association with spindles. However, whether Cdk1 has sole control over microtubule dynamics, and how CPC-microtubule association influences microtubule behavior, are unclear. Here, we show that Ipl1/Aurora-dependent phosphorylation of Sli15/INCENP modulates microtubule dynamics by preventing CPC binding to the preanaphase spindle and to the central spindle until late anaphase, facilitating spatiotemporal control of microtubule dynamics required for proper metaphase centromere positioning and anaphase spindle elongation. Decreased Ipl1-dependent Sli15 phosphorylation drives direct CPC binding to microtubules, revealing how the CPC influences microtubule dynamics. We propose that Cdk1 and Ipl1/Aurora cooperatively modulate microtubule dynamics and that Ipl1/Aurora-dependent phosphorylation of Sli15 controls spindle function by excluding the CPC from spindle regions engaged in microtubule polymerization.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Aurora Quinases , Fosforilação
15.
Mol Biol Cell ; 22(15): 2680-9, 2011 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-21633108

RESUMO

Protein kinase CK2 is one of the most conserved kinases in eukaryotic cells and plays essential roles in diverse processes. While we know that CK2 plays a role(s) in cell division, our understanding of how CK2 regulates cell cycle progression is limited. In this study, we revealed a regulatory role for CK2 in kinetochore function. The kinetochore is a multi-protein complex that assembles on the centromere of a chromosome and functions to attach chromosomes to spindle microtubules. To faithfully segregate chromosomes and maintain genomic integrity, the kinetochore is tightly regulated by multiple mechanisms, including phosphorylation by Aurora B kinase. We found that a loss of CK2 kinase activity inhibits anaphase spindle elongation and results in chromosome missegregation. Moreover, a lack of CK2 activates the spindle assembly checkpoint. We demonstrate that CK2 associates with Mif2, the Saccharomyces cerevisiae homologue of human CENP-C, which serves as an important link between the inner and outer kinetochore. Furthermore, we show Mif2 and the inner kinetochore protein Ndc10 are phosphorylated by CK2, and this phosphorylation plays antagonistic and synergistic roles with Aurora B phosphorylation of these targets, respectively.


Assuntos
Caseína Quinase II/metabolismo , Segregação de Cromossomos/fisiologia , Proteínas de Ligação a DNA/metabolismo , Cinetocoros/metabolismo , Mitose/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Fuso Acromático/metabolismo , Aurora Quinase B , Aurora Quinases , Imunoprecipitação da Cromatina , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Humanos , Microscopia de Fluorescência , Microtúbulos/metabolismo , Fosforilação , Plasmídeos , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , Transfecção
16.
Mol Biol Cell ; 22(4): 457-66, 2011 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21169562

RESUMO

There has been much effort in recent years aimed at understanding the molecular mechanism by which the Dam1 kinetochore complex is able to couple microtubule depolymerization to poleward movement. Both a biased diffusion and a forced walk model have been proposed, and several key functional aspects of Dam1-microtubule binding are disputed. Here, we investigate the elements involved in tubulin-Dam1 complex interactions and directly visualize Dam1 rings on microtubules in order to infer their dynamic behavior on the microtubule lattice and its likely relevance at the kinetochore. We find that the Dam1 complex has a preference for native tubulin over tubulin that is lacking its acidic C-terminal tail. Statistical mechanical analysis of images of Dam1 rings on microtubules, applied to both the distance between rings and the tilt angle of the rings with respect to the microtubule axis, supports a diffusive ring model. We also present a cryo-EM reconstruction of the Dam1 ring, likely the relevant assembly form of the complex for energy coupling during microtubule depolymerization in budding yeast. The present studies constitute a significant step forward by linking structural and biochemical observations toward a comprehensive understanding of the Dam1 complex.


Assuntos
Microtúbulos/química , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Tubulina (Proteína)/química , Segregação de Cromossomos/genética , Difusão , Cinetocoros/química , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Modelos Químicos , Ligação Proteica/genética , Saccharomycetales/genética , Saccharomycetales/metabolismo , Tubulina (Proteína)/genética
17.
J Cell Biol ; 191(4): 795-808, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21079246

RESUMO

The mitotic spindle is a complex and dynamic structure. Although much has been learned about how spindles assemble and mediate chromosome segregation, how spindles rapidly and irreversibly disassemble during telophase is less clear. We used synthetic lethal screens in budding yeast to identify mutants defective in spindle disassembly. Real-time, live cell imaging analysis of spindle disassembly was performed on nine mutants defective in this process. Results of this analysis suggest that spindle disassembly is achieved by mechanistically distinct but functionally overlapping subprocesses: disengagement of the spindle halves, arrest of spindle elongation, and initiation of interpolar microtubule depolymerization. These subprocesses are largely governed by the anaphase-promoting complex, Aurora B kinase, and kinesin-8. Combinatorial inhibition of these subprocesses yielded cells with hyperstable spindle remnants and dramatic defects in cell cycle progression, establishing that rapid spindle disassembly is crucial for cell proliferation.


Assuntos
Cinesinas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase , Aurora Quinases , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Citocinese/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Cinesinas/genética , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/genética , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/genética , Miosina Tipo V/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Complexos Ubiquitina-Proteína Ligase/genética
18.
Mol Biol Cell ; 20(13): 3003-11, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19403691

RESUMO

Dynein is a minus-end-directed microtubule motor important for mitotic spindle positioning. In budding yeast, dynein activity is restricted to anaphase when the nucleus enters the bud neck, yet the nature of the underlying regulatory mechanism is not known. Here, the microtubule-associated protein She1p is identified as a novel regulator of dynein activity. In she1 Delta cells, dynein is activated throughout the cell cycle, resulting in aberrant spindle movements that misposition the spindle. We also found that dynactin, a cofactor essential for dynein motor function, is a dynamic complex whose recruitment to astral microtubules (aMTs) increases dramatically during anaphase. Interestingly, loss of She1p eliminates the cell-cycle regulation of dynactin recruitment and permits enhanced dynactin accumulation on aMTs throughout the cell cycle. Furthermore, localization of the dynactin complex to aMTs requires dynein, suggesting that dynactin is recruited to aMTs via interaction with dynein and not the microtubule itself. Lastly, we present evidence supporting the existence of an incomplete dynactin subcomplex localized at the SPB, and a complete complex that is loaded onto aMTs from the cytoplasm. We propose that She1p restricts dynein-dependent spindle positioning to anaphase by inhibiting the association of dynein with the complete dynactin complex.


Assuntos
Dineínas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Miosina Tipo V/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fuso Acromático/fisiologia , Anáfase/fisiologia , Ciclo Celular/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Complexo Dinactina , Dineínas/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Cadeias Pesadas de Miosina/genética , Miosina Tipo V/genética , Ligação Proteica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Proteínas de Saccharomyces cerevisiae/genética
19.
Mol Biol Cell ; 20(6): 1772-84, 2009 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-19158380

RESUMO

The Aurora kinase complex, also called the chromosomal passenger complex (CPC), is essential for faithful chromosome segregation and completion of cell division. In Fungi and Animalia, this complex consists of the kinase Aurora B/AIR-2/Ipl1p, INCENP/ICP-1/Sli15p, and Survivin/BIR-1/Bir1p. A fourth subunit, Borealin/Dasra/CSC-1, is required for CPC targeting to centromeres and central spindles and has only been found in Animalia. Here we identified a new core component of the CPC in budding yeast, Nbl1p. NBL1 is essential for viability and nbl1 mutations cause chromosome missegregation and lagging chromosomes. Nbl1p colocalizes and copurifies with the CPC, and it is essential for CPC localization, stability, integrity, and function. Nbl1p is related to the N-terminus of Borealin/Dasra/CSC-1 and is similarly involved in connecting the other CPC subunits. Distant homology searching identified nearly 200, mostly unannotated, Borealin/Dasra/CSC-1-related proteins from nearly 150 species within Fungi and Animalia. Analysis of the sequence of these proteins, combined with comparative protein structure modeling of Bir1p-Nbl1p-Sli15p using the crystal structure of the human Survivin-Borealin-INCENP complex, revealed a striking structural conservation across a broad range of species. Our biological and computational analyses therefore establish that the fundamental design of the CPC is conserved from Fungi to Animalia.


Assuntos
Proteínas de Transporte/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Aurora Quinases , Proteínas de Transporte/química , Proteínas de Transporte/genética , Ciclo Celular , Cromossomos/genética , Sequência Conservada , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência
20.
Proc Natl Acad Sci U S A ; 105(40): 15423-8, 2008 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-18824692

RESUMO

Accurate chromosome segregation during mitotic division of budding yeast depends on the multiprotein kinetochore complex, Dam1 (also known as DASH). Purified Dam1 heterodecamers encircle microtubules (MTs) to form rings that can function as "couplers," molecular devices that transduce energy from MT disassembly into the motion of a cargo. Here we show that MT depolymerization develops a force against a Dam1 ring that is sixfold larger than the force exerted on a coupler that binds only one side of an MT. Wild-type rings slow depolymerization fourfold, but rings that include a mutant Dam1p with truncated C terminus slow depolymerization less, consistent with the idea that this tail is part of a strong bond between rings and MTs. A molecular-mechanical model for Dam1-MT interaction predicts that binding between this flexible tail and the MT wall should cause a Dam1 ring to wobble, and Fourier analysis of moving, ring-attached beads corroborates this prediction. Comparison of the forces generated against wild-type and mutant complexes confirms the importance of tight Dam1-MT association for processive cargo movement under load.


Assuntos
Cromossomos Fúngicos/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Fenômenos Biomecânicos , Segregação de Cromossomos , Cinetocoros/fisiologia , Cinetocoros/ultraestrutura , Modelos Biológicos , Saccharomycetales/metabolismo
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