Kinetochores are multiprotein machines that drive mitotic chromosome segregation by harnessing energy released during microtubule tip disassembly to generate force and movement. Kinetochores also ensure the accuracy of mitosis. They sense and release improper microtubule attachments and, when unattached or improperly attached, they also recruit checkpoint proteins that generate `wait' signals, delaying anaphase and giving more time for proper attachments to form. To uncover how these vital functions occur, we are reconstituting kinetochore activities using pure components and applying state-of-the-art biophysical tools for manipulating and tracking individual molecules. Our unique in vitro approach allows long-standing questions about kinetochore function to be answered in direct ways that would be impossible in living cells. Specifically, we will: (1) measure the force-generating capacity of protofilaments as they curl out from a disassembling tip and determine the contribution that this `conformational wave' can make to force production at kinetochores; (2) determine how the conserved microtubule polymerase, Stu2, stabilizes kinetochore-microtubule attachments and how its activity at kinetochores is regulated in a tension-dependent manner; (3) directly observe the association of individual checkpoint proteins with single kinetochores and distinguish whether their binding is directly inhibited by lateral attachment to the side of a microtubule, by end-attachment, or by mechanical tension. Together this work will elucidate how kinetochores generate force to move chromosomes, and how their attachments to spindle microtubules are regulated. Understanding the basis for these kinetochore functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore-microtubule attachments or disrupt kinetochore regulation. Promising new chemotherapeutics are also being developed to target kinetochore and spindle checkpoint components, and these efforts will benefit substantially from a more complete knowledge of the roles of specific components and the mechanisms by which they operate.

Public Health Relevance

During cell division, duplicated chromosomes are organized and separated by an exquisite molecular machine, the mitotic spindle. This project will bring us closer to a complete understanding of how the spindle works. Having a mechanistic understanding of the spindle promises to revolutionize the design of chemotherapeutic drugs that target spindle components. Ultimately, it may also guide efforts to develop useful man-made nanomachines, which thus far cannot match the remarkable abilities of naturally occurring protein machines.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM079373-10A1
Application #
9103625
Study Section
Macromolecular Structure and Function C Study Section (MSFC)
Program Officer
Deatherage, James F
Project Start
2006-09-29
Project End
2020-03-31
Budget Start
2016-05-01
Budget End
2017-03-31
Support Year
10
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Washington
Department
Physiology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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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
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:
Kim, Jae Ook; Zelter, Alex; Umbreit, Neil T et al. (2017) The Ndc80 complex bridges two Dam1 complex rings. Elife 6:
Asbury, Charles L (2017) Anaphase A: Disassembling Microtubules Move Chromosomes toward Spindle Poles. Biology (Basel) 6:
Deng, Yi; Asbury, Charles L (2017) Simultaneous Manipulation and Super-Resolution Fluorescence Imaging of Individual Kinetochores Coupled to Microtubule Tips. Methods Mol Biol 1486:437-467
Fong, Kimberly K; Sarangapani, Krishna K; Yusko, Erik C et al. (2017) Direct measurement of the strength of microtubule attachment to yeast centrosomes. Mol Biol Cell 28:1853-1861
Kudalkar, Emily M; Deng, Yi; Davis, Trisha N et al. (2016) Coverslip Cleaning and Functionalization for Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085548
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Single-Molecule Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.top077800

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