Microtubule motor proteins are responsible for many aspects of intracellular transport and cytoplasmic dynein is a major minus end- directed motor in mammalian cells which orchestrates the movement of membranes; chromosomes and mitotic spindles. We do not understand how cytoplasmic dynein identifies and binds cargo or how this process is regulated. Several rival hypotheses have been proposed to explain cargo-binding and regulation, implicating different subunits and different receptor molecules. The goals of this proposal are to evaluate these hypotheses.
The Specific Aims of this proposal are: 1) To Map Dynein Phosphorylation Sites That Regulate Cargo Binding, 2) To Define Dynein Subunits Responsible for Cargo Binding and 3) To Determine the Mechanisms of Cargo Release.
In Aim 1, we will isolate cytoplasmic dynein that has been released from cargo during mitosis, identify phosphorylated subunits and map phosphorylated residues using mass spectrometry.
In Aim 2, we will use recombinant dynein subunits as well as disruption of the dynein complex to identify the minimal dynein subcomplex capable of binding organelles.
In Aim 3, we will test the impact of phosphorylation on subunit function by creating site-directed mutants in dynein subunits designed to mimic phosphorylated and dephosphorylated versions of the protein. These mutants will be compared to wild-type subunits in organelle binding and motility assays . These studies will test existing hypotheses of dynein targeting and regulation. Through identification of cargo binding subunits and regulatory pathways that control dynein function, this work will advance our understanding of molecular motors and provide new tools for future work.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM060560-02
Application #
6622007
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Deatherage, James F
Project Start
2002-04-01
Project End
2007-03-31
Budget Start
2003-04-01
Budget End
2004-03-31
Support Year
2
Fiscal Year
2003
Total Cost
$228,690
Indirect Cost
Name
University of Notre Dame
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
824910376
City
Notre Dame
State
IN
Country
United States
Zip Code
46556
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Hornick, Jessica E; Mader, Christopher C; Tribble, Emily K et al. (2011) Amphiastral mitotic spindle assembly in vertebrate cells lacking centrosomes. Curr Biol 21:598-605
Bader, Jason R; Vaughan, Kevin T (2010) Dynein at the kinetochore: Timing, Interactions and Functions. Semin Cell Dev Biol 21:269-75
Collins, Elizabeth S; Hornick, Jessica E; Durcan, Thomas M et al. (2010) Centrosome biogenesis continues in the absence of microtubules during prolonged S-phase arrest. J Cell Physiol 225:454-65
Towns, William L; Tauhata, Sinji B F; Vaughan, Patricia S et al. (2009) Transfection-induced defects in dynein-driven transport: evidence that ICs mediate cargo-binding. Cell Motil Cytoskeleton 66:80-9
Durcan, Thomas M; Halpin, Elizabeth S; Casaletti, Luciana et al. (2008) Centrosome duplication proceeds during mimosine-induced G1 cell cycle arrest. J Cell Physiol 215:182-91
Hornick, Jessica E; Bader, Jason R; Tribble, Emily K et al. (2008) Live-cell analysis of mitotic spindle formation in taxol-treated cells. Cell Motil Cytoskeleton 65:595-613
Whyte, Jacqueline; Bader, Jason R; Tauhata, Sinji B F et al. (2008) Phosphorylation regulates targeting of cytoplasmic dynein to kinetochores during mitosis. J Cell Biol 183:819-34
Vaughan, Kevin T (2005) TIP maker and TIP marker; EB1 as a master controller of microtubule plus ends. J Cell Biol 171:197-200
Pfister, K Kevin; Fisher, Elizabeth M C; Gibbons, Ian R et al. (2005) Cytoplasmic dynein nomenclature. J Cell Biol 171:411-3

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