The long-range goal of this project is to define a high-resolution structural map that explains how mammalian kinetochores coordinate and control microtubule dynamics. The mammalian kinetochore is a distinctive structure that functions to attach chromosomes to spindle microtubules, produce motion required for chromosome alignment, and prevent cell division before proper alignment is achieved. This function is crucial for reliable transmission of genetic code, and thereby vital to human health and the survival of all organisms. In this application, we propose to use high-throughput electron tomography to investigate the critical link between the kinetochore function and the dynamic transitions between assembly and disassembly that take place at microtubule plus-ends. Our approach takes advantage of the striking structural changes of microtubule plus-ends that accompany changes in dynamic state.
In Specific Aim 1 we establish high-throughput data processing by optimizing specimen preparation for maximize contrast; implementing the latest advances in automated tomographic data collection; developing algorithms for noise reduction and automated feature extraction; and developing a scheme for classification analysis.
In Specific aim 2 we will use this high-throughput analysis to create a large database of kinetochore microtubule plus-end structures from different stages of mitosis. Classification analysis of this database will be used to test current hypotheses and models concerning kinetochore interactions with microtubule plus-ends during different directions of kinetochore movement.
In Specific Aim 3 we will use the same approach to test the hypotheses that treatment with select anti-mitotic drugs alters the plus-end conformations and that removal of kinetochore protein CENP-E alters plus-end conformation and the pattern of microtubule attachments. The proposed studies will advance our knowledge of kinetochore functional mechanisms and thereby enhance our ability to understand and treat human disorders arising from mitotic and meiotic malfunction, including birth defects, miscarriages, and many forms of cancer. In addition, technical development in Specific Aim 1 will benefit other investigators using electron tomography.
