Project Summary: Rapid and organized assembly of meiotic and mitotic spindles is essential for ensuring proper segregation of chromosomes during cell division. A majority of spindle microtubules are generated via branching microtubule nucleation. In this microtubule nucleation pathway, new microtubules are nucleated from pre-existing microtubules in shallow angles, which causes exponential amplification of microtubules. Augmin, a large hetero-octameric complex conserved throughout eukaryotes, is essential for branching microtubule nucleation. It is required both to recruit nucleation factors to the pre-existing microtubule and, by orienting the nascent microtubule at an acute angle to the mother, to ensure that spindle polarity is maintained. Yet how augmin carries out these two functions remains poorly understood, both because we lack a complete picture of augmin's direct binding partners and because we have no high resolution structural information about the augmin complex. I will use a combination of single particle cryo-electron microscopy, reconstitution experiments using purified proteins, and ex vivo Xenopus laevis meiotic egg extract assays to determine how the augmin complex enables branching microtubule nucleation and establishes correct microtubule branching geometry in the spindle. In addition to revealing the mechanism that underlies this key aspect of spindle formation, insight into the structure and function of the augmin complex will also provide us with a foundation to understand how other complex microtubule nucleation sites are organized within the cell. In addition to giving me the biological background I need to pursue my ultimate scientific interest of how the microtubule cytoskeleton cooperates with vesicle trafficking, this proposed research plan will also train me in two key technical areas that I will need to establish my own independent research group. The first of these is a TIRF-based assay to study microtubule nucleation at the single molecule level using Xenopus laevis meiotic egg extract. This ex vivo system is an incredibly powerful tool that allows precise depletion or replacement of protein components to quantitatively determine their contribution to microtubule nucleation, bridging the gap between bottom-up reconstitution approaches and live-cell imaging. One of my primary motivations for working with Dr. Petry was to be trained in this method. The second technique, which I will use extensively throughout this proposal, is single particle cryo-electron microscopy, especially in solving the structures of extended and dynamic molecules. In learning this technique, I will benefit from the mentorship of my co-sponsor Dr. Nieng Yan and the cutting-edge vibrant structural biology community at Princeton as a whole. Finally, I will learn from Dr. Petry and Dr. Yan how to successfully lead a research group that fearlessly leverages a broad range of techniques and disciplines to answer critical scientific questions.