Centrosomes organize the bipolar mitotic spindle, which is essential for chromosome segregation, and defects in centrosomes lead to chromosomal instability and cancer in humans. The yeast centrosome (Spindle Pole Body, SPB) shares many components and regulators with human centrosomes. Phosphorylation by Cyclin- dependent kinase and Mpsl kinase is essential for both centrosome and SPB assembly and activity. In this project in the PPG, we will determine structural features and phosphorylation events in core SPB components that play significant roles in their assembly, function and exchange. To ascertain important functional domains and residues, we will generate a library of mutations in SPB components based on two significant informational resources: the first is structural data from Ivan Rayment (Project 3) on full-length SPB components or particular domains. The second is phosphorylation sites mapped in vivo in our yeast SPB phosphoproteome. Mutated SPB proteins will be tested for their competence in assembly into new SPBs at duplication and into existing SPBs during mitosis when SPBs expand (mimicking centrosome maturation). They will also be evaluated for compromised interactions with other SPB components and for structural perturbations as assayed by electron microscopy and electron tomography. Candidate protein kinases, phosphatases and other regulators will be screened for function with particular SPB components. It is anticipated that this work will expand the collection of SPB regulatory molecules. Finally, despite evidence that components exchange in and out of SPBs during the cell cycle, little is known about the mechanism of homeostasis. We will first determine the extent of SPB size dynamics and component exchange in wild-type cells, using electron tomography and Fluorescent Recovery After Photobleaching (FRAP). We will then use our mutant collection to assess the contribution of particular features of individual SPB components to their exchange and attempt to identify trans-acting regulators.
This third aim will involve working closely with Davis (Project 2) on similar issues of SPB dynamics and remodeling. Overall, we will provide an in vivo analysis of SPB components, bringing to bear our strengths in electron microscopy and tomography to the PPG. We will contribute substantial in vivo analyses to test hypotheses of SPB component behavior derived from structural studies and molecular modeling. Several of the SPB components and regulators have human orthologs and studies in yeast will be critical to understanding their assembly and function in the pericentriolar material (PCM) of centrosomes.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Program Projects (P01)
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University of Colorado at Boulder
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Helgeson, Luke A; Zelter, Alex; Riffle, Michael et al. (2018) Human Ska complex and Ndc80 complex interact to form a load-bearing assembly that strengthens kinetochore-microtubule attachments. Proc Natl Acad Sci U S A 115:2740-2745
Fong, Kimberly K; Zelter, Alex; Graczyk, Beth et al. (2018) Novel phosphorylation states of the yeast spindle pole body. Biol Open 7:
Jones, Michele Haltiner; O'Toole, Eileen T; Fabritius, Amy S et al. (2018) Key phosphorylation events in Spc29 and Spc42 guide multiple steps of yeast centrosome duplication. Mol Biol Cell 29:2280-2291
Jung, Seung-Ryoung; Deng, Yi; Kushmerick, Christopher et al. (2018) Minimizing ATP depletion by oxygen scavengers for single-molecule fluorescence imaging in live cells. Proc Natl Acad Sci U S A 115:E5706-E5715
Webb, Benjamin; Viswanath, Shruthi; Bonomi, Massimiliano et al. (2018) Integrative structure modeling with the Integrative Modeling Platform. Protein Sci 27:245-258
Fernandez, Jose-Jesus; Li, Sam; Bharat, Tanmay A M et al. (2018) Cryo-tomography tilt-series alignment with consideration of the beam-induced sample motion. J Struct Biol 202:200-209
LlaurĂ³, Aida; Hayashi, Hanako; Bailey, Megan E et al. (2018) The kinetoplastid kinetochore protein KKT4 is an unconventional microtubule tip-coupling protein. J Cell Biol 217:3886-3900
Driver, Jonathan W; Geyer, Elisabeth A; Bailey, Megan E et al. (2017) Direct measurement of conformational strain energy in protofilaments curling outward from disassembling microtubule tips. Elife 6:
Viswanath, Shruthi; Chemmama, Ilan E; Cimermancic, Peter et al. (2017) Assessing Exhaustiveness of Stochastic Sampling for Integrative Modeling of Macromolecular Structures. Biophys J 113:2344-2353
Viswanath, Shruthi; Bonomi, Massimiliano; Kim, Seung Joong et al. (2017) The molecular architecture of the yeast spindle pole body core determined by Bayesian integrative modeling. Mol Biol Cell 28:3298-3314

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