Dissecting the Spatiotemporal Regulation of Kinetochore Interactions

Kriti S Thapa, Purdue University

Abstract

Kinetochores are conserved protein complexes that bind the centromeres in replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we investigated the phospho-regulated dynamic interaction between the conserved kinetochore protein Cnn1CENP-T, the centromere region and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization is unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain that associates with centromere region, and a N-terminal Spc24/25-interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that a previously established Ndc80 binding site in the N-terminus of Cnn1, Cnn160-84, should be extended to include flanking residues, Cnn125-91, to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74 based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that in G1 through metaphase, Cnn1 localizes via the histone-fold domain or a N-terminal Spc24/25- interaction sequence because deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. Endogenous expression of the N-terminal region is sufficient to localize to the kinetochore demonstrating the availability to bind to Cnn1 and indicating the presence of the Cnn1-kinetchore linkages throughout the cell cycle. At anaphase onset (when Mps1 kinase activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/25-interaction sequence confirming previous studies using full-length Cnn1. In sum, we provide the first in vivo evidence of Cnn1 pre-anaphase linkages with the kinetochore and enrichment of the linkages during anaphase with this interaction sequence. Cse4 is a centromere-specific nucleosomal protein that has a similar motif organization to Cnn1 in that it has a histone-fold and a N-terminal tail sequence. Shugoshin (Sgo1) is also reported to localize to the centromere and pericentromere and is a key component in tension-sensing as a result of bi-polar attachment. We demonstrate a novel interaction between Sgo1 and Cse4 (yeast ortholog of CENP-A) at the centromere using yeast two-hybrid, live cell imaging and co-immunoprecipitation assays. We mapped this interaction to the first 132 residues of Sgo1 and the Cse4 N-terminal tail. Using mutational analysis, we identified several regions including a basic patch, S105 site and a putative coiled-coil region in Cse4 involved in the Sgo1 interaction. In sum, the identification of the Cse4-Sgo1 interaction is a key determinant in recruiting Sgo1 to the centromere and controlling the tension-sensing mechanism.

Degree

Ph.D.

Advisors

Hazbun, Purdue University.

Subject Area

Molecular biology|Cellular biology|Biochemistry

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