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 , Cinesinas/metabolismo , Proteína Fosfatase 1/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos , Regulação Enzimológica da Expressão Gênica , Cinesinas/genética , Cinetocoros , Proteína Fosfatase 1/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The Ndc80 complex (Ndc80, Nuf2, Spc24 and Spc25) is a highly conserved kinetochore protein essential for end-on anchorage to spindle microtubule plus ends and for force generation coupled to plus-end polymerization and depolymerization. Spc24/Spc25 at one end of the Ndc80 complex binds the kinetochore. The N-terminal tail and CH domains of Ndc80 bind microtubules, and an internal domain binds microtubule-associated proteins (MAPs) such as the Dam1 complex. To determine how the microtubule- and MAP-binding domains of Ndc80 contribute to force production at the kinetochore in budding yeast, we have inserted a FRET tension sensor into the Ndc80 protein about halfway between its microtubule-binding and internal loop domains. The data support a mechanical model of force generation at metaphase where the position of the kinetochore relative to the microtubule plus end reflects the relative strengths of microtubule depolymerization, centromere stretch and microtubule-binding interactions with the Ndc80 and Dam1 complexes.
Assuntos
Centrômero/metabolismo , Cromossomos Fúngicos/metabolismo , Cinetocoros/metabolismo , Proteínas Luminescentes , Microtúbulos/metabolismo , Sítios de Ligação/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Biológicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomycetales/genética , Saccharomycetales/metabolismo , Imagem com Lapso de TempoRESUMO
The Ndc80 complex, which mediates end-on attachment of spindle microtubules, is linked to centromeric chromatin in human cells by two inner kinetochore proteins, CENP-T and CENP-C. Here to quantify their relative contributions to Ndc80 recruitment, we combine measurements of kinetochore protein copy number with selective protein depletion assays. This approach reveals about 244 Ndc80 complexes per human kinetochore (â¼14 per kinetochore microtubule), 215 CENP-C, 72 CENP-T and only 151 Ndc80s as part of the KMN protein network (1:1:1 Knl1, Mis12 and Ndc80 complexes). Each CENP-T molecule recruits â¼2 Ndc80 complexes; one as part of a KMN network. In contrast, â¼40% of CENP-C recruits only a KMN network. Replacing the CENP-C domain that binds KMN with the CENP-T domain that recruits both an Ndc80 complex and KMN network yielded functional kinetochores. These results provide a quantitative picture of the linkages between centromeric chromatin and the microtubule-binding Ndc80 complex at the human kinetochore.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Cinetocoros/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas do Citoesqueleto , Imunofluorescência , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Immunoblotting , Imagem ÓpticaRESUMO
Constitutive centromere-associated network (CCAN) proteins, particularly CENP-C, CENP-T, and the CENP-H/-I complex, mechanically link CENP-A-containing centromeric chromatin within the inner kinetochore to outer kinetochore proteins, such as the Ndc80 complex, that bind kinetochore microtubules. Accuracy of chromosome segregation depends critically upon Aurora B phosphorylation of Ndc80/Hec1. To determine how CCAN protein architecture mechanically constrains intrakinetochore stretch between CENP-A and Ndc80/Hec1 for proper Ndc80/Hec1 phosphorylation, we used super-resolution fluorescence microscopy and selective protein depletion. We found that at bi-oriented chromosomes in late prometaphase cells, CENP-T is stretched â¼16 nm to the inner end of Ndc80/Hec1, much less than expected for full-length CENP-T. Depletion of various CCAN linker proteins induced hyper-intrakinetochore stretch (an additional 20-60 nm) with corresponding significant decreases in Aurora B phosphorylation of Ndc80/Hec1. Thus, proper intrakinetochore stretch is required for normal kinetochore function and depends critically on all the CCAN mechanical linkers to the Ndc80 complex.