Abstract
Cell proliferation requires the proper segregation of sister chromatids to opposite poles of the dividing cell. Proper chromosome segregation can only occur when microtubules capture kinetochores and align sister chromatids on the mitotic spindle. Initially kinetochores interact with the lateral surface of a microtubule (lateral attachment), which is subsequently converted to an end-on attachment when the tip of a shrinking microtubule reaches the kinetochore.The mitotic spindle, which consists of bipolar array of tubulin polymers (microtubules), is an essential component of mitosis and is crucial for chromosome segregation. In S. cerevisiae the chromosomes remain attached to the mitotic spindle throughout the whole cell cycle, with the exception of a brief window in S phase. Nuclear envelope breakdown never occurs and cells go through what is referred to as a “closed mitosis”.
Kinetochores are multicomponent, large protein structures that assemble on chromosomal centromeres and attach to mitotic-spindle microtubules. The two kinetochore proteins involved in the conversion from lateral attachment to end-on attachment (crucial step for bi-orientation) are the Ndc80 and Dam1 complexes. The Ndc80 complex consists of four subunits: Ndc80, Nuf2, Spc24 and Spc25 and is rod- shaped. The Dam1/DASH complex is composed of ten proteins, including Dam1, and forms a ring structure around microtubules. The Ndc80 and Dam1 complexes have been suggested to physically interact with each other and may provide an interface for stable end-on attachment. It is highly disputed as to what domain of Ndc80 protein directly interacts with Dam1. In previous studies, the internal loop and the CH-Loop of Ndc80p were suggested as sites for interaction with Dam1c.
In this study, we found that the αH helix of Ndc80p is conserved among various yeasts carrying Dam1 complex, but not conserved in higher eukaryotes. We addressed whether the CH-Loop and the αH helix of Ndc80p are involved in the interaction with the Dam1 complex, formation of end-on attachment and establishment of bi-orientation. We used two-hybrid assay for protein interaction, studied kinetochore-microtubule interaction in vivo, and investigated co-localizations of the Dam1 complex with kinetochores. Our results suggest that both the CH-Loop and the αH helix might be involved in the process mentioned above, but may play distinct roles. Our study gives new insight into the mechanism by which kinetochore- microtubule interaction is formed upon end-on attachment.
| Date of Award | 2016 |
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| Original language | English |
| Awarding Institution |
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| Sponsors | European Research Council |
| Supervisor | Tomoyuki Tanaka (Supervisor) |