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
Kumar, Dhivya; Thomason, Rebecca T; Yankova, Maya et al. (2018) Microvillar and ciliary defects in zebrafish lacking an actin-binding bioactive peptide amidating enzyme. Sci Rep 8:4547
King, Stephen M; Sale, Winfield S (2018) Fifty years of microtubule sliding in cilia. Mol Biol Cell 29:698-701
Shoemark, Amelia; Moya, Eduardo; Hirst, Robert A et al. (2018) High prevalence of CCDC103 p.His154Pro mutation causing primary ciliary dyskinesia disrupts protein oligomerisation and is associated with normal diagnostic investigations. Thorax 73:157-166
King, Stephen M (2018) Turning dyneins off bends cilia. Cytoskeleton (Hoboken) 75:372-381
Kumar, Dhivya; Strenkert, Daniela; Patel-King, Ramila S et al. (2017) A bioactive peptide amidating enzyme is required for ciliogenesis. Elife 6:
Kumar, Dhivya; King, Stephen M (2017) Trainspotting in a cilium. Elife 6:
Yamamoto, Ryosuke; Obbineni, Jagan M; Alford, Lea M et al. (2017) Chlamydomonas DYX1C1/PF23 is essential for axonemal assembly and proper morphology of inner dynein arms. PLoS Genet 13:e1006996
Zhu, Xiaoyan; Poghosyan, Emiliya; Gopal, Radhika et al. (2017) General and specific promotion of flagellar assembly by a flagellar nucleoside diphosphate kinase. Mol Biol Cell 28:3029-3042
Pigino, Gaia; King, Stephen M (2017) Switching dynein motors on and off. Nat Struct Mol Biol 24:557-559
King, Stephen M; Patel-King, Ramila S (2016) Planaria as a Model System for the Analysis of Ciliary Assembly and Motility. Methods Mol Biol 1454:245-54

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