3D Structure and Function of the Mammalian Kinetochore

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In eukaryotic cell division, the partitioning of daughter chromosomes into the daughter cells is achieved by attachment of the chromosomes to microtubules which then move the chromosomes apart prior to cytokinesis. The site of attachment of the microtubules to the chromosomes is a morphologically and biochemically distinctive chromosomal region called the kinetochore. Recent work by Dr. McEwen, employing state-of-the-art preservation and high resolution imaging technologies, has challenged the "trilaminar" morphological model of the kinetochore that has been accepted in the field for the past thiry years, and which was probably inaccurate due to fixation artifacts inherent in conventional fixation protocols. Now,Dr. McEwen's long-range goal is to define a high-resolution structural map of the mammalian kinetochore that explains and predicts functional mechanisms. The research combines state-of-the-art methods of light and electron microscopy with molecular approaches to compute the structural map and obtain functional characterization of key kinetochore components. The current project has four objectives, listed below.Research Objective 1. Establish a prototype high-resolution 3D structural map of the mammalian kinetochore. 1a: Identify key features of the kinetochore and look for evidence of a subunit structure. 1b. Test the hypothesis that the kinetochore has discrete structural domains that dynamically respond to microtubule attachment and deprivation, and that an enlarged outer domain is correlated with promiscuous microtubule capture and a high incidence of aneuploidy.Research Objective 2. Test three hypotheses concerning the molecular role of CENP-E in mitosis. 2a: CENP-E has a role in maintaining the stability of kinetochore microtubule attachments. 2b: CENP-E prevents monooriented chromosomes from over-traveling poleward into the spindle pole. 2c: CENP-E is not required for release of the anaphase onset checkpoint.Research objective 3. Test the hypotheses that CENP-E function is redundant with ZW10/dynein in binding kinetochore microtubules, and opposes dynein in poleward motion.Research objective 4. Test the hypothesis that the kinetochore is sequentially assembled, with kinetochore proteins CENP-C and BUB1 being required for complete assembly.This research project will provide important and significant new information on the complexities of kinetochore structure and function. It will fill a major gap in our present knowledge of how molecular components of the kinetochore are arranged and how they interact with other components of the cell division apparatus.