Functional Exploration Of Outer Kinetochore Proteins SKAP And TIP60

The most important differences between organisms and non-living matters is whether they can grow and reproduce themselves.In mammals,the basic event of growth and reproduce is mitosis of somatic cells and meiosis of germ cells.Although mitosis and meiosis differ in form,both their implementation relies on the assembly and disassembly of macromolecular complex includes spindle,the centrosome and the kinetochore.The components of kinetochore change dynamically in cell division,which is not only a reflection of the complexity of cell division,but also determines the ultimate fate of cells.Despite the abundance of researches in this field,the composition of kinetochore still need further recognition,limited by its dynamicity.In the early work of our lab,kinetochore motor CENP-E was reported to be very essential in mitotic regulation,and my thesis was focused in the functional exploration of novel interacting parter of CENP-E.With yeast two-hybridization system,we discovered a novel kinetochore component SKAP.SKAP is recruited to kinetochore in prometaphase,whereas enrichment of SKAP is further enhanced in metaphase.We found that the C-terminal coiled-coil region was responsible for the localization of SKAP on the kinetochore fibers,which was responsible to regulate the attachment between kinetochore and microtubule.The kinetochore localization of SKAP was dependent on the stability of the kinetochore-microtubule attachment,whereas Aurora B negatively regulated SKAP at the kinetochores,which may be partly achieved through phosphorylation of Hec1.We also identified the MYST family acetyltransferase TIP60 was also a CENP-E interacting partner.With antibodies recognizing different epitope,we established that TIP60 localized to the outer kinetochore.In contrary to the SKAP,TIP60 mainly localized to the kinetochore at early mitosis and diminished from kinetochore in metaphase.We further found that the recruitment of TIP60 depended on the kinase activity of Mps1 and Ndc80.Either TIP60-depletion or TIP60 inhibition by small chemicals led to misalignment and lagging chromosome,suggesting a critical role of TIP60 in correcting the mal-attached kinetochore.Taken together,we identified two novel components of kinetochore,SKAP and TIP60.The temporal dynamics of the two proteins on kinetochore is quite different,suggesting that the dynamic change of kinetochore components is critical for mitotic regulation.The exploration of novel kinetochore components and the investigation of their function will enrich the knowledge of cell division and will help to explain cellular details in growth and reproduction of organisms.