The overall goal of this application is to understand the mechanical basis of chromosome segregation in human cells and the monitoring system that ensures defects in this process is corrected before chromosomes missegregate. The immediate focus is on studying the molecular composition and functions of the proteins that form a macromolecular structure called the kinetochore. The kinetochore is essential for chromosome segregation as it is the site of force generation, and its activity is directly monitored be a checkpoint control.
The aims of this application are directed towards analyzing the molecular function of two proteins that reside at the kinetochore of human chromosomes. CENP-E is a molecular motor that functions to generate force for chromosome alignment. A detailed characterization of the importance of the various domains within CENP-E that contribute to kinetochore function should establish a molecular link between this protein and chromosome segregation. This study will test how specific mutations that are introduced into CENP-E will alter the behavior of kinetochores in real-time and thus dissect the complexity of kinetochore functions. The second protein that will be examined is CENP-F, a novel protein that may play a role in organizing the assembly and the structure of the kinetochore complex. The analysis of this protein will reveal how kinetochore assembly is temporally regulated during the cell cycle. Characterization of CENP-E and CENP-F will also contribute towards defining the biochemical interactions that specify the formation of the kinetochore complex. This investigation is directly relevant to human health issues as chromosome segregation maintains genome stability. Defects in this process have profound adverse cellular affects that lead to birth defects, sterility and increase risk of cancer. From a practical standpoint, the investigation of kinetochore proteins has the benefit of identifying novel targets for the development of new anti-mitotic agents that would exhibit high specificity for only mitotic cells. For example, inhibition of CENP-E function does not interfere with normal interphase events but specifically block cells that enter mitosis. These blocked mitotic cells eventually die.
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