The centrosome is the principal nucleator of the microtubule (MT) cytoskeleton, which is required for cell polarity, vesicle trafficking, and spindle formation and function. While the analogous structure in the yeast S. cerevisiae (the spindle pole body or SPB) is morphologically distinct, a conserved set of Y-tubulin complexes is used to nucleate MT assembly. In this Project we focus on the assembly and regulation of the nucleating machinery using a broad combination of structural approaches (x-ray crystallography, cryoEM single particle reconstruction, cryoEM Tomography) to determine the structures of Y-tubulin complexes in vitro and in situ, and to understand their mechanism of action through quantitative in vitro functional studies and innovative kinetic modeling. Previously we discovered that yeast Y-tubulin small complex (YTUSC) can assemble into rings and obtained a 6.5A cryoEM structure of the rings, explaining the origins of MT 13-fold symmetry and discovering unexpected modes of regulation and assembly. Based on our previous results we propose that there are three phases of regulation: Y-TUSC ring assembly restricted to the spindle pole body by requiring interactions with Spcl 10 or Spc72, ring closure to fully match MT symmetry and, activation of the Y-tubulins for efficient nucleation. The proposed experiments expand upon our previous results with the long-term goal of synthesizing an atomic resolution picture of all the relevant structural and functional interactions between aP- and Y-tubulin complexes, regulatory proteins, and how these complexes are linked to the spindle pole body or centrosome matrix. Specifically we will (i) improve the resolution of our cryoEM reconstruction of yeast YTUSC rings and, collaborate with the Bioinformatics Core to generate a complete pseudo-atomic structure. Structures of yTuSC rings bound to MTs or 1 layer of non-polymerizing yeast ap-tubulin will be determined and compared to structures of in situ capped MT minus ends from cryoEM tomography of yeast SPBs. (ii) We will use a newly developed FRET assay to efficiently measure ring assembly in vitro and determine what domains of Spcl 10 and Spc72 are required for assembly and the role of Spcl 10/72 phosphorylation, (iii) While necessary, assembly into rings is insufficient for potent MT nucleation, with a need for both YTUSC closure to match MT symmetry and an allosteric activation. The role of PTMs or other binding partners in this process will be determined.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
Project #
Application #
Study Section
Special Emphasis Panel (ZRG1)
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
University of Colorado at Boulder
United States
Zip Code
Fong, Kimberly K; Graczyk, Beth; Davis, Trisha N (2016) Purification of Fluorescently Labeled Saccharomyces cerevisiae Spindle Pole Bodies. Methods Mol Biol 1413:189-95
Jain, Saumya; Wheeler, Joshua R; Walters, Robert W et al. (2016) ATPase-Modulated Stress Granules Contain a Diverse Proteome and Substructure. Cell 164:487-98
Hsia, Yang; Bale, Jacob B; Gonen, Shane et al. (2016) Design of a hyperstable 60-subunit protein icosahedron. Nature 535:136-9
Wheeler, Joshua R; Matheny, Tyler; Jain, Saumya et al. (2016) Distinct stages in stress granule assembly and disassembly. Elife 5:
Greenberg, Charles H; Kollman, Justin; Zelter, Alex et al. (2016) Structure of γ-tubulin small complex based on a cryo-EM map, chemical cross-links, and a remotely related structure. J Struct Biol 194:303-10
Lyon, Andrew S; Morin, Geneviève; Moritz, Michelle et al. (2016) Higher-order oligomerization of Spc110p drives γ-tubulin ring complex assembly. Mol Biol Cell 27:2245-58
Peng, Yutian; Moritz, Michelle; Han, Xuemei et al. (2015) Interaction of CK1δ with γTuSC ensures proper microtubule assembly and spindle positioning. Mol Biol Cell 26:2505-18
Kollman, Justin M; Greenberg, Charles H; Li, Sam et al. (2015) Ring closure activates yeast γTuRC for species-specific microtubule nucleation. Nat Struct Mol Biol 22:132-7
Burns, Shannon; Avena, Jennifer S; Unruh, Jay R et al. (2015) Structured illumination with particle averaging reveals novel roles for yeast centrosome components during duplication. Elife 4:
Morphew, M K; O'Toole, E T; Page, C L et al. (2015) Metallothionein as a clonable tag for protein localization by electron microscopy of cells. J Microsc 260:20-9