Role and Mechanism of Microtubule Nucleation within the Mitotic Spindle

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DESCRIPTION (provided by applicant): The microtubule(MT)-based mitotic spindle is the cellular apparatus responsible for reliable chromosome segregation during eukaryotic cell division. Failure in this process is associated with many cancers. Spindle assembly is initiated by MT nucleation through the gamma tubulin ring complex (gTuRC) at centrosomes, chromatin and the spindle itself, yet it is unknown how gTuRC is localized and specifically activated there. The recently identified eight-subunit protein complex Augmin localizes gTuRC to spindle MTs for MT generation, and thus represents the first defined gTuRC effector. My immediate research goal is to understand the mechanism of Augmin in MT generation, and its exact role in the chromosome segregation machinery and other noncentrosomal nucleation sites. By employing an interdisciplinary approach, my long-term goal is to elucidate how MT nucleation is locally activated and coordinated. Since I arrived at UCSF as an HHMI Fellow of the Life Science Research Foundation, I characterized Augmin's function in meiotic spindle assembly, purified both native and recombinant Augmin as well as gTuRC, and thus developed unique molecular tools to study MT nucleation in vitro. Here, I propose to (i) determine how Augmin and gTuRC generate MTs by reconstituting MT nucleation in vitro and analyzing it dynamically by fluorescence microscopy (mentor Dr. Ron Vale). (ii) I will investigate the currently unknown structures of Augmin and its MT-bound complexes to understand how it activates MT nucleation at a molecular level using electron microscopy (mentor Dr. David Agard) and X-ray crystallography (major technique of Ph.D., independent phase). (iii) By adding fluorescent Augmin to Xenopus spindles, I will identify and quantify MT generation events during the spindle assembly pathway (independent phase). These results seek to answer the major unresolved questions of when, where and how MTs are nucleated to constitute the self-assembling spindle, and is likely to be relevant for MT nucleation during interphase. To achieve these aims, I will need to learn high-resolution light microscopy and electron microscopy. This will complement my training in cell biology, biochemistry and X-ray crystallography and prepare me to study complex macromolecular systems, such as the mitotic spindle, from any angle necessary. Based on a rigorous career development plan, the outstanding mentoring team I have found will support me in expanding my personal and lab management skills in preparation to complete a successful U.S. job search and lead a research laboratory. Combined with state-of-the-art equipment, an interactive spirit, and excellent career training activities, UCSF provides the optimal environment for the mentored phase of this research proposal. The K99/R00 award will provide me the opportunity to acquire the necessary skills to transition into an independent tenure-track position. Elucidating the molecular mechanism of MT nucleation will pave the way to understanding a fundamental process in biology. ) PUBLIC HEALTH RELEVANCE: In order to divide, a cell needs to equally segregate its chromosomes into two daughter cells, a process which is coordinated by the microtubule-based mitotic spindle. Failure in this process is detrimental for a cell and a leading cause for cancer. The protein complex Augmin plays an important role in generating spindle microtubules, and elucidating its exact function and mechanism is of medical importance and can be used as a new target for cancer therapy.