Browsing by keyword "Kinetochores"
Now showing items 1-6 of 6
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BRCA1 foci in normal S-phase nuclei are linked to interphase centromeres and replication of pericentric heterochromatinBreast cancer-associated protein 1 (BRCA1) forms foci at sites of induced DNA damage, but any significance of these normal S-phase foci is unknown. BRCA1 distribution does not simply mirror or overlap that of replicating DNA; however, BRCA1 foci frequently abut sites of BrdU incorporation, mostly at mid-to-late S phase. Although BRCA1 does not overlap XIST RNA across the inactive X chromosome, BRCA1 foci position overwhelmingly in heterochromatic regions, particularly the nucleolar periphery where many centromeres reside. In humans and mice, including early embryonic cells, BRCA1 commonly associates with interphase centromere-kinetochore complexes, including pericentric heterochromatin. Proliferating cell nuclear antigen or BrdU labeling demonstrates that BRCA1 localizes adjacent to, or "paints," major satellite blocks as chromocenters replicate, where topoisomerase is also enriched. BRCA1 loss is often associated with proliferative defects, including postmitotic bridges enriched with satellite DNA. These findings implicate BRCA1 in replication-linked maintenance of centric/pericentric heterochromatin and suggest a novel means whereby BRCA1 loss may contribute to genomic instability and cancer.
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Direct interaction of pericentrin with cytoplasmic dynein light intermediate chain contributes to mitotic spindle organizationPericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150(Glued) subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin-dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.
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Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpointThe spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re-expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1-phosphorylation-deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.
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Mutations synthetically lethal with cep1 target S. cerevisiae kinetochore componentsCP1 (encoded by CEP1) is a Saccharomyces cerevisiae chromatin protein that binds a DNA element conserved in centromeres and in the 5'-flanking DNA of methionine biosynthetic (MET) genes. Strains lacking CP1 are defective in chromosome segregation and MET gene transcription, leading to the hypothesis that CP1 plays a general role in assembling higher order chromatin structures at genomic sites where it is bound. A screen for mutations synthetically lethal with a cep1 null allele yielded five recessive csl (cep1 synthetic lethal) mutations, each defining a unique complementation group. Four of the five mutations synergistically increased the loss rate of marker chromosomes carrying a centromere lacking the CP1 binding site, suggesting that the cep1 synthetic lethality was due to chromosome segregation defects. Three of these four CSL genes were subsequently found to be known or imputed kinetochore genes: CEP3, NDC10, and CSE4. The fourth, CSL4, corresponded to ORF YNL232w on chromosome XIV, and was found to be essential. A human cDNA was identified that encoded a protein homologous to Csl4 and that complemented the csl4-1 mutation. The results are consistent with the view that the major cellular role of CP1 is to safeguard the biochemical integrity of the kinetochore.
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Phosphorylation state defines discrete roles for monopolin in chromosome attachment and spindle elongation.BACKGROUND: It is unknown how oscillations in Cdk1 activity drive the dramatic changes in chromosome and spindle dynamics that occur at the metaphase/anaphase transition. RESULTS: We show that the Schizosaccharomyces pombe monopolin complex has distinct functions in metaphase and anaphase that are determined by the phosphorylation state of its Mde4 subunit. When Cdk1 activity is high in metaphase, Mde4 is hyperphosphorylated on Cdk1 phosphorylation sites and localizes to kinetochores. A nonphosphorylatable mutant of Mde4 does not localize to kinetochores, appears prematurely on the metaphase spindle, and interferes with spindle dynamics and chromosome segregation, illustrating the importance of Cdk1 phosphorylation in regulating metaphase monopolin activity. When Cdk1 activity drops in anaphase, dephosphorylation of Mde4 triggers monopolin localization to the mitotic spindle, where it promotes spindle elongation and integrity, coupling the late mitotic loss of Cdk1 activity to anaphase spindle dynamics. CONCLUSIONS: Together, these findings illustrate how the sequential phosphorylation and dephosphorylation of monopolin helps ensure the orderly execution of discrete steps in mitosis.
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The histone fold domain of Cse4 is sufficient for CEN targeting and propagation of active centromeres in budding yeastCentromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly. Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae. Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT). The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal. To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4DeltaNT) were analyzed. No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4. A Cse4 degron strain was used to examine targeting of a Cse4DeltaNT protein in the absence of wild-type Cse4. The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting. Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S. cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Delta129) were constructed. Despite the complete absence of the NT, mitotic chromosome loss in the cse4Delta129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4. Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S. cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast.
