Centrosomes and their functional equivalents in yeast, the spindle pole bodies (SPBs), are microtubule organizing centers that orchestrate mitotic spindle assembly, chromosome segregation, nuclear positioning, and numerous other aspects of cell motility and morphogenesis. To uncover how they function, we are developing biophysical tools to manipulate attachments between individual organizing centers and single microtubules. It is assumed that centrosomes and SPBs sustain considerable forces in vivo, and that these forces vary during the cell cycle. However, forces on microtubule organizing centers have never been directly measured in any organism, and the effects offeree on SPB/centrosome function are unknown. SPBs and centrosomes in vivo assemble and disassemble dynamically, and the number of microtubule attachments is regulated in correlation with cell cycle-dependent forces. This correlation suggests a form of mechanical feedback whereby SPB/centrosome-microtubule attachments are selectively stabilized when microtubules become forcefullyi.e., productivelyengaged with intracellular targets. We have recently developed several assays in which microtubules are nucleated in vitro from isolated budding yeast SPBs. By adapting these reconstituted assays for laser trapping and total internal reflection fluorescence (TIRF) microscopy, we will:
(Aim 1) measure the rupture strength and bending stiffness for individual SPB-microtubule attachments, and determine the molecular basis ofthese mechanical properties;
(Aim 2) determine whether and how SPBs are mechanically regulated by measuring how phosphorylation of specific components affects attachment strength, by comparing strengths across various cell cycle stages, and by testing whether mechanical tension affects attachment stability;
(Aim 3) quantify the forces sustained by SPB-microtubule attachments in vivo during various cell cycle stages by developing a force sensor based on fluorescence resonance energy transfer (FRET) for use in living yeast. Our efforts will also include comparative measurements using centrosomes isolated from animal sources, in order to begin identifying conserved aspects of centrosome mechanics and mechanoregulation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Program Projects (P01)
Project #
1P01GM105537-01A1
Application #
8668224
Study Section
Special Emphasis Panel (ZRG1)
Project Start
2014-09-01
Project End
2019-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
1
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Colorado at Boulder
Department
Type
DUNS #
City
Boulder
State
CO
Country
United States
Zip Code
80303
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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