Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 64
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Nat Commun ; 13(1): 2152, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35443757

RESUMO

Chromosome segregation requires sister kinetochores to attach microtubules emanating from opposite spindle poles. Proper attachments come under tension and are stabilized, but defective attachments lacking tension are released, giving another chance for correct attachments to form. This error correction process depends on Aurora B kinase, which phosphorylates kinetochores to destabilize their microtubule attachments. However, the mechanism by which Aurora B distinguishes tense versus relaxed kinetochores remains unclear because it is difficult to detect kinase-triggered detachment and to manipulate kinetochore tension in vivo. To address these challenges, we apply an optical trapping-based assay using soluble Aurora B and reconstituted kinetochore-microtubule attachments. Strikingly, the tension on these attachments suppresses their Aurora B-triggered release, suggesting that tension-dependent changes in the conformation of kinetochores can regulate Aurora B activity or its outcome. Our work uncovers the basis for a key mechano-regulatory event that ensures accurate segregation and may inform studies of other mechanically regulated enzymes.


Assuntos
Segregação de Cromossomos , Cinetocoros , Aurora Quinase B/genética , Microtúbulos , Polos do Fuso
2.
Genes Dev ; 36(7-8): 495-510, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-35483740

RESUMO

The identity of human protein-coding genes is well known, yet our in-depth knowledge of their molecular functions and domain architecture remains limited by shortcomings in homology-based predictions and experimental approaches focused on whole-gene depletion. To bridge this knowledge gap, we developed a method that leverages CRISPR-Cas9-induced mutations across protein-coding genes for the a priori identification of functional regions at the sequence level. As a test case, we applied this method to 48 human mitotic genes, revealing hundreds of regions required for cell proliferation, including domains that were experimentally characterized, ones that were predicted based on homology, and novel ones. We validated screen outcomes for 15 regions, including amino acids 387-402 of Mad1, which were previously uncharacterized but contribute to Mad1 kinetochore localization and chromosome segregation fidelity. Altogether, we demonstrate that CRISPR-Cas9-based tiling mutagenesis identifies key functional domains in protein-coding genes de novo, which elucidates separation of function mutants and allows functional annotation across the human proteome.


Assuntos
Sistemas CRISPR-Cas , Sistemas CRISPR-Cas/genética , Humanos , Mutagênese
3.
Nucleic Acids Res ; 2022 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-35253883

RESUMO

Centromeres are the chromosomal loci essential for faithful chromosome segregation during cell division. Although centromeres are transcribed and produce non-coding RNAs (cenRNAs) that affect centromere function, we still lack a mechanistic understanding of how centromere transcription is regulated. Here, using a targeted RNA isoform sequencing approach, we identified the transcriptional landscape at and surrounding all centromeres in budding yeast. Overall, cenRNAs are derived from transcription readthrough of pericentromeric regions but rarely span the entire centromere and are a complex mixture of molecules that are heterogeneous in abundance, orientation, and sequence. While most pericentromeres are transcribed throughout the cell cycle, centromere accessibility to the transcription machinery is restricted to S-phase. This temporal restriction is dependent on Cbf1, a centromere-binding transcription factor, that we demonstrate acts locally as a transcriptional roadblock. Cbf1 deletion leads to an accumulation of cenRNAs at all phases of the cell cycle which correlates with increased chromosome mis-segregation that is partially rescued when the roadblock activity is restored. We propose that a Cbf1-mediated transcriptional roadblock protects yeast centromeres from untimely transcription to ensure genomic stability.

4.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
5.
J Cell Biol ; 220(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34647959

RESUMO

Dividing cells detect and correct erroneous kinetochore-microtubule attachments during mitosis, thereby avoiding chromosome missegregation. The Aurora B kinase phosphorylates microtubule-binding elements specifically at incorrectly attached kinetochores, promoting their release and providing another chance for proper attachments to form. However, growing evidence suggests that the Mps1 kinase is also required for error correction. Here we directly examine how Mps1 activity affects kinetochore-microtubule attachments using a reconstitution-based approach that allows us to separate its effects from Aurora B activity. When endogenous Mps1 that copurifies with kinetochores is activated in vitro, it weakens their attachments to microtubules via phosphorylation of Ndc80, a major microtubule-binding protein. This phosphorylation contributes to error correction because phospho-deficient Ndc80 mutants exhibit genetic interactions and segregation defects when combined with mutants in other error correction pathways. In addition, Mps1 phosphorylation of Ndc80 is stimulated on kinetochores lacking tension. These data suggest that Mps1 provides an additional mechanism for correcting erroneous kinetochore-microtubule attachments, complementing the well-known activity of Aurora B.


Assuntos
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Cinetocoros/química , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Nucleares/química , Fosforilação , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Transdução de Sinais
6.
Mol Biol Cell ; 31(26): 2868-2870, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33320706

RESUMO

Fifty years ago, the first isolation of conditional budding yeast mutants that were defective in cell division was reported. Looking back, we now know that the analysis of these mutants revealed the molecular mechanisms and logic of the cell cycle, identified key regulatory enzymes that drive the cell cycle, elucidated structural components that underly essential cell cycle processes, and influenced our thinking about cancer and other diseases. Here, we briefly summarize what was concluded about the coordination of the cell cycle 50 years ago and how that relates to our current understanding of the molecular events that have since been elucidated.


Assuntos
Ciclo Celular , Segregação de Cromossomos , Replicação do DNA , Mutação/genética , Saccharomyces cerevisiae/citologia , Proteínas de Saccharomyces cerevisiae/genética
7.
Elife ; 92020 12 30.
Artigo em Inglês | MEDLINE | ID: mdl-33377866

RESUMO

Accurate chromosome segregation requires kinetochores on duplicated chromatids to biorient by attaching to dynamic microtubules from opposite spindle poles, which exerts forces to bring kinetochores under tension. However, kinetochores initially bind to microtubules indiscriminately, resulting in errors that must be corrected. While the Aurora B protein kinase destabilizes low-tension attachments by phosphorylating kinetochores, low-tension attachments are intrinsically less stable than those under higher tension in vitro independent of Aurora activity. Intrinsic tension-sensitive behavior requires the microtubule regulator Stu2 (budding yeast Dis1/XMAP215 ortholog), which we demonstrate here is likely a conserved function for the TOG protein family. The human TOG protein, chTOG, localizes to kinetochores independent of microtubules by interacting with Hec1. We identify a chTOG mutant that regulates microtubule dynamics but accumulates erroneous kinetochore-microtubule attachments that are not destabilized by Aurora B. Thus, TOG proteins confer a unique, intrinsic error correction activity to kinetochores that ensures accurate chromosome segregation.


Assuntos
Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/fisiologia , Segregação de Cromossomos , Sequência Conservada/genética , Células HCT116 , Células HeLa , Humanos , Imunoprecipitação , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Mitose/genética , Mutação/genética , Saccharomyces cerevisiae
8.
Curr Biol ; 30(22): 4491-4499.e5, 2020 11 16.
Artigo em Inglês | MEDLINE | ID: mdl-32946748

RESUMO

To ensure the faithful inheritance of DNA, a macromolecular protein complex called the kinetochore sustains the connection between chromosomes and force-generating dynamic microtubules during cell division. Defects in this process lead to aneuploidy, a common feature of cancer cells and the cause of many developmental diseases [1-4]. One of the major microtubule-binding activities in the kinetochore is mediated by the conserved Ndc80 complex (Ndc80c) [5-7]. In budding yeast, the retention of kinetochores on dynamic microtubule tips also depends on the essential heterodecameric Dam1 complex (Dam1c) [8-15], which binds to the Ndc80c and is proposed to be a functional ortholog of the metazoan Ska complex [16, 17]. The load-bearing activity of the Dam1c depends on its ability to oligomerize, and the purified complex spontaneously self-assembles into microtubule-encircling oligomeric rings, which are proposed to function as collars that allow kinetochores to processively track the plus-end tips of microtubules and harness the forces generated by disassembling microtubules [10-15, 18-22]. However, it is unknown whether there are specific regulatory events that promote Dam1c oligomerization to ensure accurate segregation. Here, we used a reconstitution system to discover that Cdk1, the major mitotic kinase that drives the cell cycle, phosphorylates the Ask1 component of the Dam1c to increase its residence time on microtubules and enhance kinetochore-microtubule attachment strength. We propose that Cdk1 activity promotes Dam1c oligomerization to ensure that kinetochore-microtubule attachments are stabilized as kinetochores come under tension in mitosis.


Assuntos
Proteína Quinase CDC28 de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Segregação de Cromossomos , Ensaios Enzimáticos , Proteínas Associadas aos Microtúbulos/genética , Mitose , Mutação , Fosforilação/fisiologia , Multimerização Proteica/fisiologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética
9.
Nat Commun ; 11(1): 809, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041946

RESUMO

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.


Assuntos
Replicação do DNA , Histonas/metabolismo , Transcrição Genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Cromatina/metabolismo , DNA Polimerase II/metabolismo , Genoma Fúngico/genética , Instabilidade Genômica , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/genética , Metilação , Modelos Genéticos , Mutação , Pontos de Checagem da Fase S do Ciclo Celular/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
10.
PLoS Genet ; 15(10): e1008423, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31584935

RESUMO

Accurate segregation of chromosomes to daughter cells is a critical aspect of cell division. It requires the kinetochores on duplicated chromosomes to biorient, attaching to microtubules from opposite poles of the cell. Bioriented attachments come under tension, while incorrect attachments lack tension and must be released to allow proper attachments to form. A well-studied error correction pathway is mediated by the Aurora B kinase, which destabilizes low tension-bearing attachments. We recently discovered that in vitro, kinetochores display an additional intrinsic tension-sensing pathway that utilizes Stu2. The contribution of kinetochore-associated Stu2 to error correction in cells, however, was unknown. Here, we identify a Stu2 mutant that abolishes its kinetochore function and show that it causes biorientation defects in vivo. We also show that this Stu2-mediated pathway functions together with the Aurora B-mediated pathway. Altogether, our work indicates that cells employ multiple pathways to ensure biorientation and the accuracy of chromosome segregation.


Assuntos
Aurora Quinases/metabolismo , Segregação de Cromossomos , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Aurora Quinases/genética , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos , Mutação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
11.
Proc Natl Acad Sci U S A ; 116(35): 17355-17360, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31405987

RESUMO

Accurate mitosis depends on a surveillance system called the spindle assembly checkpoint. This checkpoint acts at kinetochores, which attach chromosomes to the dynamic tips of spindle microtubules. When a kinetochore is unattached or improperly attached, the protein kinase Mps1 phosphorylates kinetochore components, catalyzing the generation of a diffusible "wait" signal that delays anaphase and gives the cell time to correct the error. When a kinetochore becomes properly attached, its checkpoint signal is silenced to allow progression into anaphase. Recently, microtubules were found to compete directly against recombinant human Mps1 fragments for binding to the major microtubule-binding kinetochore element Ndc80c, suggesting a direct competition model for silencing the checkpoint signal at properly attached kinetochores. Here, by developing single-particle fluorescence-based assays, we tested whether such direct competition occurs in the context of native kinetochores isolated from yeast. Mps1 levels were not reduced on kinetochore particles bound laterally to the sides of microtubules or on particles tracking processively with disassembling tips. Instead, we found that Mps1 kinase activity was sufficient to promote its release from the isolated kinetochores. Mps1 autophosphorylation, rather than phosphorylation of other kinetochore components, was responsible for this dissociation. Our findings suggest that checkpoint silencing in yeast does not arise from a direct competition between Mps1 and microtubules, and that phosphoregulation of Mps1 may be a critical aspect of the silencing mechanism.


Assuntos
Proteínas Fúngicas/metabolismo , Cinetocoros/metabolismo , /metabolismo , Humanos , Cinetocoros/química , Microtúbulos/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Saccharomycetales/metabolismo
12.
Curr Biol ; 29(14): 2407-2414.e7, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31257143

RESUMO

The spindle checkpoint monitors kinetochore-microtubule interactions and generates a "wait anaphase" delay when any defects are apparent [1-3]. This provides time for cells to correct chromosome attachment errors and ensure high-fidelity chromosome segregation. Checkpoint signals are generated at unattached chromosomes during mitosis. To activate the checkpoint, Mps1Mph1 kinase phosphorylates the kinetochore component KNL1Spc105/Spc7 on conserved MELT motifs to recruit Bub3-Bub1 complexes [4-6] via a direct Bub3 interaction with phospho-MELT motifs [7, 8]. Mps1Mph1 then phosphorylates Bub1, which strengthens its interaction with Mad1-Mad2 complexes to produce a signaling platform [9, 10]. The Bub1-Mad1 platform is thought to recruit Mad3, Cdc20, and Mad2 to produce the mitotic checkpoint complex (MCC), which is the diffusible wait anaphase signal [9, 11, 12]. The MCC binds and inhibits the mitotic E3 ubiquitin ligase, known as Cdc20-anaphase promoting complex/cyclosome (APC/C), and stabilizes securin and cyclin to delay anaphase onset [13-17]. Here we demonstrate, in both budding and fission yeast, that kinetochores and KNL1Spc105/Spc7 can be bypassed; simply inducing heterodimers of Mps1Mph1 kinase and Bub1 is sufficient to trigger metaphase arrest that is dependent on Mad1, Mad2, and Mad3. We use this to dissect the domains of Bub1 necessary for arrest, highlighting the need for Bub1-CD1, which binds Mad1 [9], and Bub1's highly conserved N-terminal tetratricopeptide repeat (TPR) domain [18, 19]. We demonstrate that the Bub1 TPR domain is both necessary and sufficient to bind and recruit Mad3. We propose that this brings Mad3 into close proximity to Mad1-Mad2 and Mps1Mph1 kinase, enabling efficient generation of MCC complexes.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/genética , Repetições de Tetratricopeptídeos/genética , Proteínas de Ciclo Celular/metabolismo , Pontos de Checagem da Fase M do Ciclo Celular , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo
13.
Curr Biol ; 28(17): 2697-2704.e3, 2018 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-30174190

RESUMO

Kinesin-5 is a highly conserved homo-tetrameric protein complex responsible for crosslinking microtubules and pushing spindle poles apart. The budding yeast Kinesin-5, Cin8, is highly concentrated at kinetochores in mitosis before anaphase, but its functions there are largely unsolved. Here, we show that Cin8 localizes to kinetochores in a cell-cycle-dependent manner and concentrates near the microtubule binding domains of Ndc80 at metaphase. Cin8's kinetochore localization depends on the Ndc80 complex, kinetochore microtubules, and the Dam1 complex. Consistent with its kinetochore localization, a Cin8 deletion induces a loss of tension at the Ndc80 microtubule binding domains and a major delay in mitotic progression. Cin8 associates with Protein Phosphatase 1 (PP1), and mutants that inhibit its PP1 binding also induce a loss of tension at the Ndc80 microtubule binding domains and delay mitotic progression. Taken together, our results suggest that Cin8-PP1 plays a critical role at kinetochores to promote accurate chromosome segregation by controlling Ndc80 attachment to microtubules.


Assuntos
Segregação de Cromossomos/fisiologia , Regulação Fúngica da Expressão Gênica/fisiologia , Proteína Fosfatase 1/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos , Regulação Enzimológica da Expressão Gênica , Cinetocoros , Proteína Fosfatase 1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Elife ; 72018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-30117803

RESUMO

Chromosome segregation depends on the kinetochore, the machine that establishes force-bearing attachments between DNA and spindle microtubules. Kinetochores are formed every cell cycle via a highly regulated process that requires coordinated assembly of multiple subcomplexes on specialized chromatin. To elucidate the underlying mechanisms, we developed an assay to assemble kinetochores de novo using centromeric DNA and budding yeast extracts. Assembly is enhanced by mitotic phosphorylation of the Dsn1 kinetochore protein and generates kinetochores capable of binding microtubules. We used this assay to investigate why kinetochores recruit the microtubule-binding Ndc80 complex via two receptors: the Mis12 complex and CENP-T. Although the CENP-T pathway is non-essential in yeast, we demonstrate that it becomes essential for viability and Ndc80c recruitment when the Mis12 pathway is crippled by defects in Dsn1 phosphorylation. Assembling kinetochores de novo in yeast extracts provides a powerful and genetically tractable method to elucidate critical regulatory events in the future.


Assuntos
Cromatina/genética , Cinetocoros , Proteínas Associadas aos Microtúbulos/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Segregação de Cromossomos/genética , Microtúbulos/genética , Fosforilação , Saccharomyces cerevisiae/genética , Transdução de Sinais
15.
Elife ; 72018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29897335

RESUMO

Stu2/XMAP215 microtubule polymerases use multiple tubulin-binding TOG domains and a lattice-binding basic region to processively promote faster elongation. How the domain composition and organization of these proteins dictate polymerase activity, end localization, and processivity is unknown. We show that polymerase activity does not require different kinds of TOGs, nor are there strict requirements for how the TOGs are linked. We identify an unexpected antagonism between the tubulin-binding TOGs and the lattice-binding basic region: lattice binding by the basic region is weak when at least two TOGs engage tubulins, strong when TOGs are empty. End-localization of Stu2 requires unpolymerized tubulin, at least two TOGs, and polymerase competence. We propose a 'ratcheting' model for processivity: transfer of tubulin from TOGs to the lattice activates the basic region, retaining the polymerase at the end for subsequent rounds of tubulin binding and incorporation. These results clarify design principles of the polymerase.


Assuntos
Proteínas Associadas aos Microtúbulos/química , Engenharia de Proteínas/métodos , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Tubulina (Proteína)/química , Sítios de Ligação , Clonagem Molecular , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo
16.
Methods Cell Biol ; 144: 349-370, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29804677

RESUMO

Chromosome segregation relies on forces generated by spindle microtubules that are translated into chromosome movement through interactions with kinetochores, highly conserved macromolecular machines that assemble on a specialized centromeric chromatin structure. Kinetochores not only have to stably attach to growing and shrinking microtubules, but they also need to recruit spindle assembly checkpoint proteins to halt cell cycle progression when there are attachment defects. Even the simplest kinetochore in budding yeast contains more than 50 unique components that are present in multiple copies, totaling more than 250 proteins in a single kinetochore. The complex nature of kinetochores makes it challenging to elucidate the contributions of individual components to its various functions. In addition, it is difficult to manipulate forces in vivo to understand how they regulate kinetochore-microtubule attachments and the checkpoint. To address these issues, we developed a technique to purify kinetochores from budding yeast that can be used to analyze kinetochore functions and composition as well as to reconstitute kinetochore-microtubule attachments in vitro.


Assuntos
Técnicas Citológicas/métodos , Cinetocoros/metabolismo , Saccharomyces cerevisiae/metabolismo , Benomilo , Ciclo Celular , Imunoprecipitação , Mutação/genética
17.
Cell Syst ; 4(6): 645-650.e5, 2017 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-28601560

RESUMO

Proper segregation of the replicated genome requires that kinetochores form and maintain bioriented, amphitelic attachments to microtubules from opposite spindle poles and eliminate erroneous, syntelic attachments to microtubules from the same spindle pole. Phosphorylation of kinetochore proteins destabilizes low-tension kinetochore-microtubule attachments, yet tension stabilizes bioriented attachments. This conundrum for forming high-tension amphitelic attachments is recognized as the "initiation problem of biorientation (IPBO)." A delay before kinetochore-microtubule detachment solves the IPBO, but it lacks a mechanistic framework. We developed a stochastic mathematical model for kinetochore-microtubule error correction in yeast that reveals: (1) under low chromatin tension, requiring a large number of phosphorylation events at multiple sites to achieve detachment provides the necessary delay; and (2) kinetochore-induced microtubule depolymerization generates tension in amphitelic, but not syntelic, attachments. With these requirements, the model provides a mechanistic framework for the delay before detachment to solve the IPBO and demonstrates the high degree of amphitely observed experimentally for wild-type spindles under optimal conditions.


Assuntos
Mitose/genética , Saccharomyces cerevisiae/genética , Saccharomycetales/genética , Polos do Fuso/genética , Cromatina/genética , Segregação de Cromossomos/genética , Cinetocoros/fisiologia , Microtúbulos/genética
18.
Genetics ; 205(1): 113-124, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27794026

RESUMO

The incorporation of histone variants into nucleosomes can alter chromatin-based processes. CENP-A is the histone H3 variant found exclusively at centromeres that serves as an epigenetic mark for centromere identity and is required for kinetochore assembly. CENP-A mislocalization to ectopic sites appears to contribute to genomic instability, transcriptional misregulation, and tumorigenesis, so mechanisms exist to ensure its exclusive localization to centromeres. One conserved process is proteolysis, which is mediated by the Psh1 E3 ubiquitin ligase in Saccharomyces cerevisiae (budding yeast). To determine whether there are features of the CENP-A nucleosome that facilitate proteolysis, we performed a genetic screen to identify histone H4 residues that regulate CENP-ACse4 degradation. We found that H4-R36 is a key residue that promotes the interaction between CENP-ACse4 and Psh1 Consistent with this, CENP-ACse4 protein levels are stabilized in H4-R36A mutant cells and CENP-ACse4 is enriched in the euchromatin. We propose that the defects in CENP-ACse4 proteolysis may be related to changes in Psh1 localization, as Psh1 becomes enriched at some 3' intergenic regions in H4-R36A mutant cells. Together, these data reveal a key residue in histone H4 that is important for efficient CENP-ACse4 degradation, likely by facilitating the interaction between Psh1 and CENP-ACse4.


Assuntos
Autoantígenos/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Histonas/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/genética , Autoantígenos/genética , Proteína Centromérica A , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Eucromatina , Histonas/genética , Cinetocoros/metabolismo , Nucleossomos/metabolismo , Ligação Proteica , Proteólise , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitinação
20.
Cell ; 165(6): 1428-1439, 2016 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-27156448

RESUMO

The development and survival of all organisms depends on equal partitioning of their genomes during cell division. Accurate chromosome segregation requires selective stabilization of kinetochore-microtubule attachments that come under tension due to opposing pulling forces exerted on sister kinetochores by dynamic microtubule tips. Here, we show that the XMAP215 family member, Stu2, makes a major contribution to kinetochore-microtubule coupling. Stu2 and its human ortholog, ch-TOG, exhibit a conserved interaction with the Ndc80 kinetochore complex that strengthens its attachment to microtubule tips. Strikingly, Stu2 can either stabilize or destabilize kinetochore attachments, depending on the level of kinetochore tension and whether the microtubule tip is assembling or disassembling. These dichotomous effects of Stu2 are independent of its previously studied regulation of microtubule dynamics. Altogether, our results demonstrate how a kinetochore-associated factor can confer opposing, tension-dependent effects to selectively stabilize tension-bearing attachments, providing mechanistic insight into the basis for accuracy during chromosome segregation.


Assuntos
Segregação de Cromossomos , Cinetocoros/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Fenômenos Biomecânicos , Humanos , Proteínas Nucleares/fisiologia , Ligação Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...