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  • Book
    Colin J. Fuller.
    Digital2013
    Faithful propagation of the genome during division is a process fundamental to all cells. Eukaryotic cells segregate their chromosomes by attaching them via the kinetochore, a large multi-protein complex, to a microtubule spindle that physically moves the chromosomes to the daughter cells. The kinetochore is assembled at the site of the centromere, a specialized chromatin domain and set of associated proteins. In most organisms, the identity of the centromere is not determined by an underlying DNA sequence but by the incorporation of a centromere-specific histone H3 variant called CENP-A. How the cell recognizes these centromere-specific histones as the site for kinetochore assembly and the site at which to replenish CENP-A during cell division remains a major unanswered question. Many experimental techniques have been used to probe the function of the centromere, but quantitative fluorescence imaging has proved especially useful because of its molecular specificity, high sensitivity, and ability to examine single cells and single centromeres. Accurate quantitative analysis of images is critical to being able to apply fluorescence imaging to centromeres, where often meaningful intensity differences between treatments can be a factor of two or smaller, and meaningful spatial distances are far smaller than the diffraction limit of light. Empirical determination of the accuracy of an image analysis method is necessary to have confidence in these subtle measurements. We first present a series of quantitative image metaanalysis tools whose aim is to provide a quantitative metric to compare different analysis methods, and we develop an accurate and linear automated method for quantifying the intensities of centromeres in fluorescence images. Next, we describe a new superresolution microscopy methodology that can be used to measure and correct the optical aberrations introduced by cells, a critical step for measuring spatial distances at centromeres. Finally, we combine fluorescence imaging with a system for biochemical reconstitution of centromeric chromatin to examine the question of how CENP-A is recognized as the site at which the cell will incorporate additional CENP-A during cell division. We find that the six amino acids at the CENP-A C-terminus are both necessary and sufficient for this recognition.