Dyneins are members of the AAA+ family of ATPases that act as microtubule-based molecular motors involved in a wide variety of essential cellular functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. The ~2.0 MDa outer dynein arm from flagella of Chlamydomonas offers an excellent model system in which to study dynein structure, function and regulation as it contains components closely related to those in the cytoplasmic isozyme, is amenable to classical/molecular genetics and can be purified in large amounts for biochemical analysis. It is now clear that dyneins are subject to a wide array of regulatory inputs such as responses to redox poise, Ca2+ levels, phosphorylation etc. However, the mechanisms by which dynein motor activity is regulated at the molecular level and how these different inputs are integrated remain very poorly understood. This application proposes four specific areas of investigation. 1) We will investigate the mechanism by which outer arm dynein is regulated in response to alterations in flagellar redox poise. This will involve identification and subsequent functional analysis of the five (or more) proteins that form mixed disulfides with dynein-associated thioredoxins and a redox-sensitive Ca2+-binding protein. 2) We will determine how a leucine-rich repeat protein associated with the ATP-binding modules of the dynein heavy chain regulates motor activity. This light chain also associates with microtubules and we will define the axonemal geometry of the heavy chain / light chain / tubulin ternary complex. Combined with mutagenesis approaches to disrupt individual interactions, we will test several competing hypotheses for how this regulatory pathway is activated. 3) The lissencephaly protein is a known regulator of cytoplasmic dynein. We have now found that this protein is present in cilia and flagella, and associates with the outer dynein arm. Furthermore, Lis1 levels within the flagellum are modulated by an intraflagellar signaling pathway. Thus, we will test the hypothesis that Lis1 represents an additional flagellar dynein regulatory system that involves alteration in dynein quaternary structure. 4) Intraflagellar transport (IFT) is required for the assembly of cilia/flagella at their distal tip.
This aim will focus on the detailed analysis of the enigmatic dynein that is thought to be responsible for retrograde movement from the ciliary tip to the cell body. Using biochemical methods, we will purify this dynein, define its composition and investigate the functional contribution of previously undescribed components to retrograde transport using RNAi methods and/or genetic screens to identify mutants. Furthermore, we have devised a purification scheme that yields a multi-megadalton complex containing this dynein and the kinesin responsible for anterograde IFT. We will use in vitro assays to define motor function and to test potential mechanisms by which the activity of these two opposing motors is coordinated.

Public Health Relevance

Dyneins are microtubule-based molecular motors involved in a wide variety of essential cellular Functions including retrograde vesicle trafficking, nuclear envelope breakdown, ciliary/flagellar motility and cell division. Defects in dynein function can lead to a broad array of human genetic disorders. This project will investigate the mechanisms by which dynein motor function in cilia and flagella is regulated in response to cellular and environmental inputs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM051293-17
Application #
8249885
Study Section
Cell Structure and Function (CSF)
Program Officer
Gindhart, Joseph G
Project Start
1995-05-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
17
Fiscal Year
2012
Total Cost
$339,605
Indirect Cost
$119,082
Name
University of Connecticut
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
022254226
City
Farmington
State
CT
Country
United States
Zip Code
06030
Owa, Mikito; Furuta, Akane; Usukura, Jiro et al. (2014) Cooperative binding of the outer arm-docking complex underlies the regular arrangement of outer arm dynein in the axoneme. Proc Natl Acad Sci U S A 111:9461-6
Ide, Takahiro; Owa, Mikito; King, Stephen M et al. (2013) Protein-protein interactions between intermediate chains and the docking complex of Chlamydomonas flagellar outer arm dynein. FEBS Lett 587:2143-9
King, Stephen M (2013) Biochemical and physiological analysis of axonemal dyneins. Methods Enzymol 524:123-45
Patel-King, Ramila S; Gilberti, Renee M; Hom, Erik F Y et al. (2013) WD60/FAP163 is a dynein intermediate chain required for retrograde intraflagellar transport in cilia. Mol Biol Cell 24:2668-77
King, Stephen M (2013) A solid-state control system for dynein-based ciliary/flagellar motility. J Cell Biol 201:173-5
King, Stephen M; Patel-King, Ramila S (2012) Functional architecture of the outer arm dynein conformational switch. J Biol Chem 287:3108-22
Hom, Erik F Y; Witman, George B; Harris, Elizabeth H et al. (2011) A unified taxonomy for ciliary dyneins. Cytoskeleton (Hoboken) 68:555-65
King, Stephen M (2010) Axonemal dyneins winch the cilium. Nat Struct Mol Biol 17:673-4
Rompolas, Panteleimon; Patel-King, Ramila S; King, Stephen M (2010) An outer arm Dynein conformational switch is required for metachronal synchrony of motile cilia in planaria. Mol Biol Cell 21:3669-79
King, Stephen M (2010) Sensing the mechanical state of the axoneme and integration of Ca2+ signaling by outer arm dynein. Cytoskeleton (Hoboken) 67:207-13

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