Efficiency and fidelity in mitotic spindle assembly

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The goal of cell division (mitosis) is to partition genetic information, in the form of chromosomes, equally into the two daughter cells. To achieve this goal, ‘kinetochores’, specialized macromolecular complexes on chromosomes, must attach to the poles of the ‘spindle’, a macromolecular machine assembled from dynamic biopolymers called ‘microtubules’. Attachment defects lead to chromosome mis-segregation, which is a hallmark of tumorigenesis. The overarching goal of our research is to reveal the mechanisms that allow the spindle to assemble rapidly and with minimal number of errors. Our previous work demonstrates that every major step in spindle assembly can be reached via several alternative routes. Some routes are swift but error prone, others are accurate but not efficient. This multiplicity of alternative mechanisms prompts the hypothesis that a proper balance in the contributions from individual mechanisms must be maintained to ensure error-free chromosome segregation. We will test this hypothesis by quantitatively characterizing spindle assembly in normal vs. chromosomally instable (CIN) cells that frequently mis-segregate their chromosomes. Over the next five years we will focus our studies on the four major aspects of spindle assembly: 1) Identification of the mechanism(s) by which direct capture of microtubules nucleated at the spindle poles suppresses the number of segregation errors. Although only ~25% of chromosomes normally utilize direct capture, segregation errors become numerous in the absence of this mechanism. 2) Characterization of the molecular mechanisms that govern attachment to non-centrosomal microtubules nucleated in the immediate proximity of kinetochores, which is the main mode of attachment employed by ~75% of chromosomes in normal cells. Specifically, we will localize microtubule- nucleating activities to a particular domain(s) within the kinetochore and establish the role of kinesin CenpE in the formation of microtubule bundles (K-fibers) with proper polarity of microtubules. 3) We will characterize structural changes within the kinetochore that trigger removal of the ‘checkpoint proteins’. This process is essential for controlling orderly progression through mitosis. 4) Finally, we will quantify how often chromosomes in various cell types are propelled poleward by a dynein-mediated pulling force exerted at the distal end of short K-fibers instead of the more common mechanism that involves generation of the force within the kinetochore. Ultrastructural organization of kinetochores transported by the alternative force production mechanisms will be compared contributions and the contributions of these mechanisms for error-free chromosome segregation will be characterized. To achieve our goals, we employ sophisticated imaging such as laser microsurgery, precise tracking of chromosome movements, and correlative electron-microscopy analyses conducted on the kinetochores whose behavior was followed in live cells up to the moment of fixation. These approaches in conjunction with molecular and cell-biology techniques for inactivation of specific proteins will produce a significant new insight into the mechanisms that ensure high fidelity of chromosome segregation.