ANALYSIS AND CONTROL OF CHROMOSOME MOVEMENT

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DESCRIPTION (Verbatim from the applicant's abstract): The goal is to understand precise chromosome movement and the accurate distribution of chromosomes to the daughter cells in mitosis and meiosis. Errors in distribution can lead to cancer and to Down syndrome and other chromosome disorders in humans. How cells avoid errors is the subject of this project. Mechanical tension from mitotic forces is the key. Early in mitosis, chromosomes move to a position quite precisely midway between the spindle poles. The movement is powered by motile kinetochores (the structures that attach the chromosome to spindle microtubules). Kinetochores switch between pulling and an inactive, 'neutral' state. The switch may be regulated by tension, which will be tested directly by pushing on chromosomes with a micromanipulation needle to relax the tension and allow switching to occur. The motors dynein and CENP-E are present at kinetochores when chromosomes begin to move but are later lost from the chromosomes. The possible uses for such transitory kinetochore motors will be tested. The common errors in chromosome distribution are of two sorts, and tension is involved in avoiding both of them. Avoiding errors of the first sort depends on an anchorage of chromosomes to the spindle that is sensitive to tension. The possibility that the poles are the sensitive site will be tested by micromanipulation. Errors of the second sort are avoided by a checkpoint that detects errors and delays the completion of mitosis. Tension-sensitive kinetochore protein phosphorylation may be the signal to the checkpoint. Tension certainly causes kinetochore dephosphorylation, but the effect may be direct (deformation of some component) or indirect (tension increases the number of microtubules, which may lead to dephosphorylation). This ambiguity will be resolved by creating a situation in which tension can be manipulated yet does not increase the number of microtubules. The effect of tension on the structure of the kinetochore and its components will be explored by combining micromanipulation to vary the tension force with electron microscopy to view the consequences. Kinetochores will be reconstructed by threedimensional tomography. The ultimate goal is to understand how tension, by altering structure, can lead to chemical changes such as dephosphorylation.