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1.
J Cell Sci ; 136(16)2023 08 15.
Article in English | MEDLINE | ID: mdl-37519149

ABSTRACT

Accurate genome segregation in mitosis requires that all chromosomes are bioriented on the spindle. Cells monitor biorientation by sensing tension across sister centromeres. Chromosomes that are not bioriented have low centromere tension, which allows Aurora B (yeast Ipl1) to perform error correction that locally loosens kinetochore-microtubule attachments to allow detachment of microtubules and fresh attempts at achieving biorientation. However, it is not known whether low tension recruits Aurora B to centromeres or, alternatively, whether low tension directly activates Aurora B already localized at centromeres. In this work, we experimentally induced low tension in metaphase Saccharomyces cerevisiae yeast cells, then monitored Ipl1 localization. We find low tension recruits Ipl1 to centromeres. Furthermore, low tension-induced Ipl1 recruitment depended on Bub1, which is known to provide a binding site for Ipl1. In contrast, Top2, which can also recruit Ipl1 to centromeres, was not required. Our results demonstrate cells are sensitive to low tension at centromeres and respond by actively recruiting Ip1l for error correction.


Subject(s)
Kinetochores , Saccharomyces cerevisiae , Aurora Kinase B/genetics , Aurora Kinase B/metabolism , Centromere/metabolism , Chromosome Segregation , Fungal Proteins/metabolism , Kinetochores/metabolism , Metaphase , Microtubules/metabolism , Mitosis , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism
2.
J Cell Biol ; 213(6): 651-64, 2016 06 20.
Article in English | MEDLINE | ID: mdl-27325791

ABSTRACT

Faithful chromosome segregation depends on the precise timing of chromatid separation, which is enforced by checkpoint signals generated at kinetochores. Here, we provide evidence that the C-terminal domain (CTD) of DNA topoisomerase IIα (Topo II) provides a novel function at inner centromeres of kinetochores in mitosis. We find that the yeast CTD is required for recruitment of the tension checkpoint kinase Ipl1/Aurora B to inner centromeres in metaphase but is not required in interphase. Conserved CTD SUMOylation sites are required for Ipl1 recruitment. This inner-centromere CTD function is distinct from the catalytic activity of Topo II. Genetic and biochemical evidence suggests that Topo II recruits Ipl1 via the Haspin-histone H3 threonine 3 phosphorylation pathway. Finally, Topo II and Sgo1 are equally important for Ipl1 recruitment to inner centromeres. This indicates H3 T3-Phos/H2A T120-Phos is a universal epigenetic signature that defines the eukaryotic inner centromere and provides the binding site for Ipl1/Aurora B.


Subject(s)
Antigens, Neoplasm/metabolism , Aurora Kinase B/metabolism , Centromere/metabolism , Centromere/physiology , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Metaphase/physiology , Cell Cycle Proteins/metabolism , Chromatids/metabolism , Chromosome Segregation/physiology , Fungal Proteins/metabolism , Histones/metabolism , Kinetochores/metabolism , Kinetochores/physiology , Phosphorylation/physiology , Protein Serine-Threonine Kinases/metabolism , Sumoylation/physiology , Yeasts/metabolism , Yeasts/physiology
3.
Mol Microbiol ; 95(3): 442-57, 2015 Feb.
Article in English | MEDLINE | ID: mdl-25417844

ABSTRACT

The spindle assembly checkpoint (SAC) plays a critical role in preventing mitotic errors by inhibiting anaphase until all kinetochores are correctly attached to spindle microtubules. In spite of the economic and medical importance of filamentous fungi, relatively little is known about the behavior of SAC proteins in these organisms. In our efforts to understand the role of γ-tubulin in cell cycle regulation, we have created functional fluorescent protein fusions of four SAC proteins in Aspergillus nidulans, the homologs of Mad2, Mps1, Bub1/BubR1 and Bub3. Time-lapse imaging reveals that SAC proteins are in distinct compartments of the cell until early mitosis when they co-localize at the spindle pole body. SAC activity is, thus, spatially regulated in A. nidulans. Likewise, Cdc20, an activator of the anaphase-promoting complex/cyclosome, is excluded from interphase nuclei, but enters nuclei at mitotic onset and accumulates to a higher level in mitotic nuclei than in the surrounding nucleoplasm before leaving in anaphase/telophase. The activity of this critical cell cycle regulatory complex is likely regulated by the location of Cdc20. Finally, the γ-tubulin mutation mipAD159 causes a nuclear-specific failure of nuclear localization of Mps1 and Bub1/R1 but not of Cdc20, Bub3 or Mad2.


Subject(s)
Anaphase-Promoting Complex-Cyclosome , Aspergillus nidulans/metabolism , Cell Cycle Proteins/metabolism , Fungal Proteins/metabolism , M Phase Cell Cycle Checkpoints , Mad2 Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Anaphase , Aspergillus nidulans/genetics , Cdc20 Proteins/genetics , Cell Cycle Proteins/genetics , Fungal Proteins/genetics , Kinetochores/metabolism , M Phase Cell Cycle Checkpoints/genetics , Mad2 Proteins/genetics , Mitosis , Mutation , Protein Serine-Threonine Kinases/genetics , Recombinant Fusion Proteins/metabolism , Repressor Proteins/metabolism , Time-Lapse Imaging , Tubulin/genetics , Tubulin/metabolism
4.
Nat Protoc ; 1(6): 3111-20, 2006.
Article in English | MEDLINE | ID: mdl-17406574

ABSTRACT

We describe a rapid method for the production of fusion PCR products that can be used, generally without band purification, to transform Aspergillus nidulans. This technique can be used to replace genes; tag genes with fluorescent moeties or epitope tags; or replace endogenous promoters with regulatable promoters, by introducing an appropriate selective cassette (e.g., fluorescent protein + selectable marker). The relevant genomic fragments and cassette are first amplified separately by PCR using primers that produce overlapping ends. A second PCR using 'nested' primers fuses the fragments into a single molecule with all sequences in the desired order. This procedure allows a cassette to be amplified once, frozen and used subsequently in many fusion PCRs. Transformation of nonhomologous recombination deficient (nkuADelta) strains of A. nidulans with fusion PCR products results in high frequencies of accurate gene targeting. Fusion PCR takes less than 2 d. Protoplast formation and transformation takes less than 1 d.


Subject(s)
Aspergillus nidulans/genetics , Gene Targeting/methods , Polymerase Chain Reaction/methods , Gene Transfer Techniques , Protoplasts
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