Chromosomes are organized and separated during cell division by a microtubule-based molecular machine, the mitotic spindle. Our overall goal is to reconstitute spindle functions using pure components and apply new tools for manipulating and tracking single molecules to uncover how this machine operates. Here we request support for an ultrasensitive fluorescence microscope capable of simultaneously recording interactions between individual protein molecules, multiprotein complexes, and dynamic microtubule filaments. The instrument is based on an Olympus IX51 inverted microscope equipped with total internal reflection fluorescence (TIRF) illumination and with three Andor emCCD cameras, enabling fast, multicolor molecular tracking. It will be used by four NIH-funded investigators, each with complementary expertise, working in close collaboration with one another. The Davis lab will study super-complexes assembled from pure, recombinant forms of the six protein subcomplexes that make up kinetochores to determine how their interactions allow kinetochores to link chromosomes to spindle microtubules. The Asbury and Biggins labs will take a complementary approach, studying whole kinetochores isolated from wild-type yeast and from mutants with targeted defects to assess quantitatively the contribution each kinetochore subcomplex makes to chromosome-microtubule coupling. The Wordeman lab will study how the activities of microtubule regulatory enzymes implicated in kinetochore function are modulated through interactions with plus end binding proteins (+TIPs) and other microtubule binding factors. The Wordeman lab will also observe nucleotide turnover in a single microtubule-depolymerizing enzyme (MCAK) to test whether it enzymatically removes multiple tubulin subunits before detaching from the filament. These projects will bring us closer to a complete understanding of spindle function by elucidating the mechanisms underlying attachment of chromosomes to spindle microtubules, correction of attachment errors, and regulation of chromosome and spindle movements. Ultimately, having a mechanistic understanding of the spindle promises to revolutionize the design of chemotherapeutic drugs that target spindle components.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biomedical Research Support Shared Instrumentation Grants (S10)
Project #
1S10RR026406-01
Application #
7791455
Study Section
Special Emphasis Panel (ZRG1-CB-Q (30))
Program Officer
Birken, Steven
Project Start
2010-05-06
Project End
2011-05-05
Budget Start
2010-05-06
Budget End
2011-05-05
Support Year
1
Fiscal Year
2010
Total Cost
$214,157
Indirect Cost
Name
University of Washington
Department
Physiology
Type
Schools of Medicine
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
Jung, Seung-Ryoung; Kushmerick, Christopher; Seo, Jong Bae et al. (2017) Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding. Proc Natl Acad Sci U S A 114:E5579-E5588
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Jung, Seung-Ryoung; Seo, Jong Bae; Deng, Yi et al. (2016) Contributions of protein kinases and ?-arrestin to termination of protease-activated receptor 2 signaling. J Gen Physiol 147:255-71
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Single-Molecule Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.top077800
Asbury, Charles L (2016) Data Analysis for Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085571
Kudalkar, Emily M; Davis, Trisha N; Asbury, Charles L (2016) Preparation of Reactions for Imaging with Total Internal Reflection Fluorescence Microscopy. Cold Spring Harb Protoc 2016:pdb.prot085563
Kudalkar, Emily M; Scarborough, Emily A; Umbreit, Neil T et al. (2015) Regulation of outer kinetochore Ndc80 complex-based microtubule attachments by the central kinetochore Mis12/MIND complex. Proc Natl Acad Sci U S A 112:E5583-9

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