The long-term focus of this research program has been microtubules (MTs) and the mechanisms of their assembly and function in mitosis, cytokinesis, and motility. Accurate chromosome segregation depends on the dynamics and polarity of spindle MTs, the activities of motor proteins associated with MT s, the activities of protein components of the mitotic spindle checkpoint, such as Mad2p, and the ability of MTs to direct the plane of cytokinesis for accurate segregation of the chromosomes. Errors in segregation can induce cancer and developmental defects. In continuing and expanding on previous research efforts, we will be analyzing the properties and functions of MTs and associated proteins that are involved with various aspects of mitosis and cytokinesis. A major strength of our work has been the development and application of new techniques in quantitative optical microscopy and dynamic digital imaging of living cells and cytoplasmic extracts. Here we propose to develop a new digital imaging microscope system that combines multiwavelength fluorescence, DIC, and fluorescence speckle microscopy with fluorescence redistribution after laser photobleaching or photoactivation and laser optical trapping. We also will improve our computer vision methods for measuring the mechanical and assembly properties of MTs. This new technology will enable us to analyze the dynamics of kinetochore protein assembly and function in the recruitment of MTs by kinetochores, chromosome motility, and the mitotic spindle checkpoint as well as the molecular mechanisms of MT poleward flux and its function in mitosis. Using budding yeast genetics and digital imaging of GFP fusion proteins, we will identify molecular components, including MT motor proteins, that are involved with the coupling of force generation to the assembly/disassembly of MTs. We intend to continue investigating the mechanisms of MT dynamic instability and the biophysical properties of MTs, including the action of anticancer tubulin drugs. Finally, we will analyze how MT dynamics may be involved in the regulation of cytokinesis.
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