This project uses both molecular biology and genetics to study the structure and function of flagellar dynein ATPases, which are large, multi- subunit complexes that support motility along both cytoplasmic and flagellar microtubule frameworks. The long-term objective of this work are to determine the mechanism of dynein force generation and its regulation. To determine primary structure and predict higher-order structure in the catalytic heavy chain subunits, genomic clones for the Chlamydomonas reinhardtii outer arm dynein alpha and beta heavy chains will be sequenced. Sequence analysis will also be used to look for nucleotide and microtubule binding domains and homology with other mechanochemical enzymes. Transformation of Chlamydomonas dynein assembly mutants will be developed as a method to express cloned dynein genes. Expression of hybrids between cloned wild-type and sup-1 mutant beta heavy chain genes will be used to pinpoint the sup-1 mutation, which defines a region of this heavy chain involved in motility regulation by radial spoke-central pair microtubule interactions. Sequences analysis will reveal the molecular defect responsible for the suppressor phenotype. Genomic alpha heavy chain and 70 k intermediate chain clones will be used to map the genetic loci encoding each protein by restriction fragment length polymorphism (RFLP) analysis. Previously-identified dynein assembly mutants will then be tested for allelism to these structural gene loci by a combination of RFLP mapping and transformation with cloned genes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM044228-04
Application #
3303410
Study Section
Molecular Cytology Study Section (CTY)
Project Start
1990-06-01
Project End
1995-05-31
Budget Start
1993-06-01
Budget End
1994-05-31
Support Year
4
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Upstate Medical University
Department
Type
Schools of Medicine
DUNS #
058889106
City
Syracuse
State
NY
Country
United States
Zip Code
13210
Desai, Paurav B; Freshour, Judy R; Mitchell, David R (2015) Chlamydomonas axonemal dynein assembly locus ODA8 encodes a conserved flagellar protein needed for cytoplasmic maturation of outer dynein arm complexes. Cytoskeleton (Hoboken) 72:16-28
Dean, Anudariya B; Mitchell, David R (2015) Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex. Mol Biol Cell 26:3596-605
Dean, Anudariya B; Mitchell, David R (2013) Chlamydomonas ODA10 is a conserved axonemal protein that plays a unique role in outer dynein arm assembly. Mol Biol Cell 24:3689-96
Carbajal-González, Blanca I; Heuser, Thomas; Fu, Xiaofeng et al. (2013) Conserved structural motifs in the central pair complex of eukaryotic flagella. Cytoskeleton (Hoboken) 70:101-120
Mitchison, Hannah M; Schmidts, Miriam; Loges, Niki T et al. (2012) Mutations in axonemal dynein assembly factor DNAAF3 cause primary ciliary dyskinesia. Nat Genet 44:381-9, S1-2
Hom, Erik F Y; Witman, George B; Harris, Elizabeth H et al. (2011) A unified taxonomy for ciliary dyneins. Cytoskeleton (Hoboken) 68:555-65
Mitchell, David R (2010) Polyglutamylation: the GLU that makes microtubules sticky. Curr Biol 20:R234-6
Wei, Mei; Sivadas, Priyanka; Owen, Heather A et al. (2010) Chlamydomonas mutants display reversible deficiencies in flagellar beating and axonemal assembly. Cytoskeleton (Hoboken) 67:71-80
Gao, Chunlei; Wang, Guangliang; Amack, Jeffrey D et al. (2010) Oda16/Wdr69 is essential for axonemal dynein assembly and ciliary motility during zebrafish embryogenesis. Dev Dyn 239:2190-7
Mitchell, David R; Smith, Brandon (2009) Analysis of the central pair microtubule complex in Chlamydomonas reinhardtii. Methods Cell Biol 92:197-213

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