Chromosome segregation must be carefully regulated because missegregation has catastrophic consequences such as genome instability and cancer. In order to segregate properly, all replicated chromosomes must orient correctly on the mitotic spindle with each pair of sister kinetochores making attachments to microtubules from opposing spindle poles. Failure of even a single chromosome to achieve this """"""""bioriented"""""""" configuration triggers a major cell cycle checkpoint response, known as the spindle assembly checkpoint (SAC), which halts the cell cycle and prevents separation of sister chromatids. Many of the downstream effectors of the SAC have been well characterized, but the mechanistic details of how misaligned chromosomes trigger the initial activation of the SAC remain poorly understood. A critical player in this regulation is the chromosomal passenger complex (CPC) that controls the activity of the essential mitotic kinase Aurora B. Highlighting the importance of the CPC to cell cycle control, members of CPC are known to be upregulated in cancer cells and are currently being used as targets in drug development. However, despite this importance a thorough understanding of the functions of CPC components is lacking. The research training plan proposed here describes three complementary lines of experimentation to investigate the mechanisms by which the CPC controls the activity of Aurora B and thereby coordinates chromosome structure and cell cycle progression. These studies will provide critical insights into chromosome biology and the regulation of the cell cycle that will have broad relevance to genome maintenance as well as specific impacts on the understanding of cancer progression. The goals of the research training plan proposed here are 1) to determine how the CPC effects the localization and dynamic turnover of Aurora B on mitotic chromosomes 2) to elucidate which targets of Aurora B kinase are effected by chromosome biorientation and 3) to uncover whether the interaction with specific binding partners confers the sensitivity of CPC function to chromosome structure. To meet these goals, three complementary lines of experimentation will be undertaken using the Xenopus egg extract system, which has been widely used to characterize the biochemistry of cell cycle regulation because of the unprecedented control it provides of cell cycle progression. The power of the Xenopus system will be combined with high-resolution microscopic analysis and recent innovations in the reconstitution and purification of mitotic chromatin. These experiments will test current hypotheses for the function of the CPC and will provide valuable training in the application of cutting edge technologies to the important fields of chromosome biology and cell cycle control.
An underlying cause of many cancers and developmental defects such as Down syndrome is a failure to coordinate cell division with the segregation of chromosomes. The research proposed here investigates the function of a group of proteins that control this coordination by monitoring the correct orientation of chromosomes at a critical point during cell division. This project will provide insights into the function of these important protens that will guide drug development and have broad impacts on our understanding of chromosome biology and cell cycle regulation and their effects on human health.
|Wynne, David J; Funabiki, Hironori (2016) Heterogeneous architecture of vertebrate kinetochores revealed by three-dimensional superresolution fluorescence microscopy. Mol Biol Cell 27:3395-3404|
|Wynne, David J; Funabiki, Hironori (2015) Kinetochore function is controlled by a phospho-dependent coexpansion of inner and outer components. J Cell Biol 210:899-916|
|Funabiki, Hironori; Wynne, David J (2013) Making an effective switch at the kinetochore by phosphorylation and dephosphorylation. Chromosoma 122:135-58|