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 ProteicaRESUMO
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 , MutagêneseRESUMO
Eukaryotic chromosome segregation requires the kinetochore, a megadalton-sized machine that forms on specialized centromeric chromatin containing CENP-A, a histone H3 variant. CENP-A deposition requires a chaperone protein HJURP that targets it to the centromere, but it has remained unclear whether HJURP has additional functions beyond CENP-A targeting and why high AT DNA content, which disfavors nucleosome assembly, is widely conserved at centromeres. To overcome the difficulties of studying nucleosome formation in vivo, we developed a microscopy assay that enables direct observation of de novo centromeric nucleosome recruitment and maintenance with single molecule resolution. Using this assay, we discover that CENP-A can arrive at centromeres without its dedicated centromere-specific chaperone HJURP, but stable incorporation depends on HJURP and additional DNA-binding proteins of the inner kinetochore. We also show that homopolymer AT runs in the yeast centromeres are essential for efficient CENP-A deposition. Together, our findings reveal requirements for stable nucleosome formation and provide a foundation for further studies of the assembly and dynamics of native kinetochore complexes.
Assuntos
Proteínas Cromossômicas não Histona , Nucleossomos , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Centrômero/genética , Centrômero/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
The spindle checkpoint ensures proper chromosome segregation during cell division. Unravelling checkpoint signalling has been a long-standing challenge owing to the complexity of the structures and forces that regulate chromosome segregation. New reports have now substantially advanced our understanding of checkpoint signalling mechanisms at the kinetochore, the structure that connects microtubules and chromatin. In contrast to the traditional view of a binary checkpoint response - either completely on or off - new findings indicate that the checkpoint response strength is variable. This revised perspective provides insight into how checkpoint bypass can lead to aneuploidy and informs strategies to exploit these errors for cancer treatments.
Assuntos
Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Cromossomos/metabolismo , Mitose , Fuso Acromático/metabolismo , Aneuploidia , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica , Segregação de Cromossomos , Cromossomos/ultraestrutura , Regulação da Expressão Gênica , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Ligação Proteica , Transdução de Sinais , Análise Espaço-Temporal , Fuso Acromático/ultraestruturaRESUMO
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.
Centromeres are essential chromosomal regions that do not encode gene products and instead ensure the accurate partitioning of chromosomes during cell division. Despite the lack of genes, transcription has been detected at centromeres. It has not been clear where this centromeric RNA comes from and how it is regulated. In this study, the authors identified all of the centromeric RNAs at and around budding yeast centromeres during the cell cycle. Unlike RNAs that encode for proteins, centromeric RNAs are a complex mixture of transcripts that result from adjacent RNAs that continue into the centromere. The authors found that most transcription is blocked at the centromere border by a protein called Cbf1. This mechanism shields the centromere from untimely transcription to ensure genome stability.
Assuntos
Centrômero , Proteínas de Saccharomyces cerevisiae , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Centrômero/genética , Centrômero/metabolismo , Segregação de Cromossomos/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
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 , Proteínas Serina-Treonina Quinases/metabolismo , Humanos , Cinetocoros/química , Microtúbulos/metabolismo , Modelos Biológicos , Fosforilação , Ligação Proteica , Saccharomycetales/metabolismoRESUMO
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/genéticaRESUMO
The spindle checkpoint is a conserved signaling pathway that ensures genomic integrity by preventing cell division when chromosomes are not correctly attached to the spindle. Checkpoint activation depends on the hierarchical recruitment of checkpoint proteins to generate a catalytic platform at the kinetochore. Although Mad1 kinetochore localization is the key regulatory downstream event in this cascade, its receptor and mechanism of recruitment have not been conclusively identified. Here, we demonstrate that Mad1 kinetochore association in budding yeast is mediated by phosphorylation of a region within the Bub1 checkpoint protein by the conserved protein kinase Mps1. Tethering this region of Bub1 to kinetochores bypasses the checkpoint requirement for Mps1-mediated kinetochore recruitment of upstream checkpoint proteins. The Mad1 interaction with Bub1 and kinetochores can be reconstituted in the presence of Mps1 and Mad2. Together, this work reveals a critical mechanism that determines kinetochore activation of the spindle checkpoint.
Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Cinetocoros/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Fuso Acromático/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Escherichia coli/genética , Proteínas Nucleares/genética , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fuso Acromático/genéticaRESUMO
Centromere identity and its epigenetic maintenance require the incorporation of a histone H3 variant called CENP-A at centromeres. CENP-A mislocalization to ectopic sites may disrupt chromatin-based processes and chromosome segregation, so it is important to uncover the mechanisms by which this variant is exclusively localized to centromeres. Here, we identify a role for the conserved chromatin-modifying complex FACT (facilitates chromatin transcription/transactions) in preventing budding yeast CENP-A(Cse4) mislocalization to euchromatin by mediating its proteolysis. The Spt16 subunit of the FACT complex binds to Psh1 (Pob3/Spt16/histone), an E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. The interaction between Psh1 and Spt16 is critical for both CENP-A(Cse4) ubiquitylation and its exclusion from euchromatin. We found that Psh1 cannot efficiently ubiquitylate CENP-A(Cse4) nucleosomes in vitro, suggesting that additional factors must facilitate CENP-A(Cse4) removal from chromatin in vivo. Consistent with this, a Psh1 mutant that cannot associate with FACT has a reduced interaction with CENP-A(Cse4) in vivo. Together, our data identify a previously unknown mechanism to maintain centromere identity and genomic stability through the FACT-mediated degradation of ectopically localized CENP-A(Cse4).
Assuntos
Autoantígenos/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiologia , Fatores de Elongação da Transcrição/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Centromérica A , Eucromatina/metabolismo , Mutação , Nucleossomos/metabolismo , Fatores de Alongamento de Peptídeos/genética , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Proteólise , Saccharomyces cerevisiae/enzimologia , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
The exclusive localization of the histone H3 variant CENP-A to centromeres is essential for accurate chromosome segregation. Ubiquitin-mediated proteolysis helps to ensure that CENP-A does not mislocalize to euchromatin, which can lead to genomic instability. Consistent with this, overexpression of the budding yeast CENP-A(Cse4) is lethal in cells lacking Psh1, the E3 ubiquitin ligase that targets CENP-A(Cse4) for degradation. To identify additional mechanisms that prevent CENP-A(Cse4) misincorporation and lethality, we analyzed the genome-wide mislocalization pattern of overexpressed CENP-A(Cse4) in the presence and absence of Psh1 by chromatin immunoprecipitation followed by high throughput sequencing. We found that ectopic CENP-A(Cse4) is enriched at promoters that contain histone H2A.Z(Htz1) nucleosomes, but that H2A.Z(Htz1) is not required for CENP-A(Cse4) mislocalization. Instead, the INO80 complex, which removes H2A.Z(Htz1) from nucleosomes, promotes the ectopic deposition of CENP-A(Cse4). Transcriptional profiling revealed gene expression changes in the psh1Δ cells overexpressing CENP-A(Cse4). The down-regulated genes are enriched for CENP-A(Cse4) mislocalization to promoters, while the up-regulated genes correlate with those that are also transcriptionally up-regulated in an htz1Δ strain. Together, these data show that regulating centromeric nucleosome localization is not only critical for maintaining centromere function, but also for ensuring accurate promoter function and transcriptional regulation.
Assuntos
Autoantígenos/genética , Centrômero/genética , Proteínas Cromossômicas não Histona/genética , Histonas/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína Centromérica A , Proteínas de Ligação a DNA/genética , Eucromatina/genética , Regulação Fúngica da Expressão Gênica , Nucleossomos/metabolismo , Regiões Promotoras Genéticas , Proteólise , Saccharomyces cerevisiae/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genéticaRESUMO
Proper centromere function is critical to maintain genomic stability and to prevent aneuploidy, a hallmark of tumors and birth defects. A conserved feature of all eukaryotic centromeres is an essential histone H3 variant called CENP-A that requires a centromere targeting domain (CATD) for its localization. Although proteolysis prevents CENP-A from mislocalizing to euchromatin, regulatory factors have not been identified. Here, we identify an E3 ubiquitin ligase called Psh1 that leads to the degradation of Cse4, the budding yeast CENP-A homolog. Cse4 overexpression is toxic to psh1Δ cells and results in euchromatic localization. Strikingly, the Cse4 CATD is a key regulator of its stability and helps Psh1 discriminate Cse4 from histone H3. Taken together, we propose that the CATD has a previously unknown role in maintaining the exclusive localization of Cse4 by preventing its mislocalization to euchromatin via Psh1-mediated degradation.
Assuntos
Centrômero/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Histonas/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Eucromatina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Fatores de Alongamento de Peptídeos/química , Ligação Proteica , Isoformas de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Estabilidade Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Ubiquitina-Proteína Ligases/química , UbiquitinaçãoRESUMO
The kinetochore is a macromolecular complex that controls chromosome segregation and cell cycle progression. When sister kinetochores make bioriented attachments to microtubules from opposite poles, the spindle checkpoint is silenced. Biorientation and the spindle checkpoint are regulated by a balance between the Ipl1/Aurora B protein kinase and the opposing activity of protein phosphatase I (PP1). However, little is known about the regulation of PP1 localization and activity at the kinetochore. Here, we developed a method to purify centromere-bound kinetochores and used quantitative proteomics to identify the Fin1 protein as a PP1 regulatory subunit. The Fin1/PP1 complex is regulated by phosphorylation and 14-3-3 protein binding. When Fin1 is mislocalized, bipolar spindles fail to assemble but the spindle checkpoint is inappropriately silenced due to PP1 activity. These data suggest that Fin1 is a PP1 regulatory subunit whose spatial and temporal activity must be precisely controlled to ensure genomic stability.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Regulação Fúngica da Expressão Gênica , Cinetocoros/metabolismo , Proteína Fosfatase 1/metabolismo , Proteômica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas 14-3-3/metabolismo , Ciclo Celular/fisiologia , Cromossomos Fúngicos/genética , Ligação Proteica , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genéticaRESUMO
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
Assuntos
Cromossomos/metabolismo , Cinetocoros/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas Fúngicas/isolamento & purificação , Proteínas Fúngicas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
During mitosis, multiprotein complexes called kinetochores orchestrate chromosome segregation by forming load-bearing attachments to dynamic microtubule tips, and by participating in phosphoregulatory error correction. The conserved kinase Aurora B phosphorylates the major microtubule-binding kinetochore subcomplexes, Ndc80 and (in yeast) Dam1, to promote release of erroneous attachments, giving another chance for proper attachments to form. It is unknown whether Aurora B phosphorylation promotes release directly, by increasing the rate of kinetochore detachment, or indirectly, by destabilizing the microtubule tip. Moreover, the relative importance of phosphorylation of Ndc80 vs. Dam1 in the context of whole kinetochores is unclear. To address these uncertainties, we isolated native yeast kinetochore particles carrying phosphomimetic mutations on Ndc80 and Dam1, and applied advanced laser-trapping techniques to measure the strength and stability of their attachments to individual dynamic microtubule tips. Rupture forces were reduced by phosphomimetic mutations on both subcomplexes, in an additive manner, indicating that both subcomplexes make independent contributions to attachment strength. Phosphomimetics on either subcomplex reduced attachment lifetimes under constant force, primarily by accelerating detachment during microtubule growth. Phosphomimetics on Dam1 also increased the likelihood of switches from microtubule growth into shortening, further promoting release in an indirect manner. Taken together, our results suggest that, in vivo, Aurora B releases kinetochores via at least two mechanisms: by weakening the kinetochore-microtubule interface and also by destabilizing the kinetochore-attached microtubule tip.
Assuntos
Cinetocoros/metabolismo , Microtúbulos/metabolismo , Saccharomyces cerevisiae/metabolismo , Fenômenos Biomecânicos , Cinética , Mimetismo Molecular , Complexos Multiproteicos/metabolismo , Mutação/genética , Fosforilação , Fosfosserina/metabolismo , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The kinetochore is the macromolecular complex that assembles onto centromeric DNA and orchestrates the segregation of duplicated chromosomes. More than 60 components make up the budding yeast kinetochore, including inner kinetochore proteins that bind to centromeric chromatin and outer proteins that directly interact with microtubules. However, little is known about how these components assemble into a functional kinetochore and whether there are quality control mechanisms that monitor kinetochore integrity. We previously developed a method to isolate kinetochore particles via purification of the conserved Dsn1 kinetochore protein. We find that the Mub1/Ubr2 ubiquitin ligase complex associates with kinetochore particles through the CENP-C(Mif2) protein. Although Mub1/Ubr2 are not stable kinetochore components in vivo, they regulate the levels of the conserved outer kinetochore protein Dsn1 via ubiquitylation. Strikingly, a deletion of Mub1/Ubr2 restores the levels and viability of a mutant Dsn1 protein, reminiscent of quality control systems that target aberrant proteins for degradation. Consistent with this, Mub1/Ubr2 help to maintain viability when kinetochores are defective. Together, our data identify a previously unknown regulatory mechanism for the conserved Dsn1 kinetochore protein. We propose that Mub1/Ubr2 are part of a quality control system that monitors kinetochore integrity, thus ensuring genomic stability.
Assuntos
Proteínas de Transporte , Proteínas Cromossômicas não Histona , Cinetocoros/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitina-Proteína Ligases , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sobrevivência Celular/genética , Centrômero/genética , Centrômero/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and there has been progress in structural studies on recombinant subassemblies. However, there is limited structural information on native kinetochore architecture. To address this, we purified functional, native kinetochores from the thermophilic yeast Kluyveromyces marxianus and examined them by electron microscopy (EM), cryoelectron tomography (cryo-ET), and atomic force microscopy (AFM). The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder, Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
RESUMO
Eukaryotic chromosome segregation requires kinetochores, multi-megadalton protein machines that assemble on the centromeres of chromosomes and mediate attachments to dynamic spindle microtubules. Kinetochores are built from numerous complexes, and understanding how they are arranged is key to understanding how kinetochores perform their multiple functions. However, an integrated understanding of kinetochore architecture has not yet been established. To address this, we purified functional, native kinetochores from Kluyveromyces marxianus and examined them by electron microscopy, cryo-electron tomography and atomic force microscopy. The kinetochores are extremely large, flexible assemblies that exhibit features consistent with prior models. We assigned kinetochore polarity by visualizing their interactions with microtubules and locating the microtubule binder Ndc80c. This work shows that isolated kinetochores are more dynamic and complex than what might be anticipated based on the known structures of recombinant subassemblies, and provides the foundation to study the global architecture and functions of kinetochores at a structural level.
RESUMO
The kinetochore is the proteinaceous complex that governs the movement of duplicated chromosomes by interacting with spindle microtubules during mitosis and meiosis. Faithful chromosome segregation requires that kinetochores form robust load-bearing attachments to the tips of dynamic spindle microtubules, correct microtubule attachment errors, and delay the onset of anaphase until all chromosomes have made proper attachments. To understand how this macromolecular machine operates to segregate duplicated chromosomes with exquisite accuracy, it is critical to reconstitute and study kinetochoremicrotubule interactions in vitro using defined components. Here, we review the current status of reconstitution as well as recent progress in understanding the microtubule-binding functions of kinetochores in vivo.
Assuntos
Segregação de Cromossomos/genética , Cinetocoros/fisiologia , Microtúbulos/fisiologia , Animais , Aurora Quinases , Proteínas de Caenorhabditis elegans/fisiologia , Fracionamento Celular , Histonas/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Microtúbulos/genética , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Saccharomycetales/genética , UltracentrifugaçãoRESUMO
The spindle checkpoint ensures accurate chromosome segregation by delaying cell-cycle progression until all sister kinetochores capture microtubules from opposite poles and come under tension (for reviews, see refs 1, 2). Although the checkpoint is activated by either the lack of kinetochore-microtubule attachments or defects in the tension exerted by microtubule-generated forces, it is not clear whether these signals are linked. We investigated the connection between tension and attachment by studying the conserved budding yeast Ipl1Aurora protein kinase that is required for checkpoint activation in the absence of tension but not attachment. Here, we show that spindle-checkpoint activation in kinetochore mutants that seem to have unattached kinetochores depends on Ipl1 activity. When Ipl1 function was impaired in these kinetochore mutants, the attachments were restored and the checkpoint was turned off. These data indicate that Ipl1 activates the checkpoint in response to tension defects by creating unattached kinetochores. Moreover, although the Dam1 kinetochore complex has been implicated as a key downstream target, we found the existence of unidentified Ipl1 sites on Dam1 or additional important substrates that regulate both microtuble detachment and the checkpoint.