This proposal focuses on the assembly and maintenance of eukaryotic cilia and flagella. These organelles are found on nearly every human cell and are also essential in the life cycles of most other eukaryotic organisms, including those that afflict Homo sapiens as disease causing organisms, such as Trypanosoma, Leishmania, Plasmodium, Giardia and Naegleria. Ciliary assembly requires transport of precursors from cytoplasmic pools to assembly sites by intraflagellar transport complexes and molecular motors, but cargo recognition by this system remains unknown. In addition, most ciliary proteins assemble as parts of larger complexes, and mechanisms that direct pre-assembly of these complexes are uncharacterized. These steps are essential for the formation of axonemes in cilia that act only as sensory antennae as well as in cilia important for their dynein-based motility. Here we use mutations in the model organism Chlamydomonas reinhardtii that affect pre-assembly and transport of an abundant complex in motile cilia, the multi-subunit axonemal outer arm dynein motor, to uncover basic mechanisms of each step and characterize genes important in this pathway.
In Aim 1 we will characterize the pre-assembly of dynein complexes in the cytoplasm, taking advantage of three mutations (pf13, pf22 and oda7) that block this step. We will test the hypothesis that these proteins act as co-chaperones for folding and/or pre-assembly of axonemal dyneins by isolating complexes containing each assembly locus gene product through biochemical approaches. Experiments proposed in Aim 2 will characterize cargo recognition by the IFT transport machinery and test the role of ODA16 as a cargo adaptor for dynein transport. To understand dynein transport we will immunolocalize cargo and adaptor under varying conditions, including during flagellar assembly, flagellar disassembly, and in cells harboring various mutations and mutant combinations that alter the assembly process.
In Aim 3 we will test the hypothesis that products of the ODA5, ODA8 and ODA10 loci form a complex that acts at a late step to make transported outer arm dynein competent for axonemal binding, taking advantage of a new in vitro assay for assembly-competency of cytoplasmic complexes. Our results will identify new human disease gene candidates and characterize conserved steps in assembly of axonemal proteins.
This proposal focuses on genes needed for the assembly and maintenance of eukaryotic cilia and flagella. These organelles are found on nearly every human cell and their absence results in sterility and life-threatening conditions including respiratory and kidney disease. They are also essential in the life cycles of many protozoan organisms that cause human diseases, such as trypanosomiasis, leishmaniasis, malaria, giardiasis, and trichosomiasis.
|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|
|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 (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-Gonzalez, Blanca I; Heuser, Thomas; Fu, Xiaofeng et al. (2013) Conserved structural motifs in the central pair complex of eukaryotic flagella. Cytoskeleton (Hoboken) 70:101-20|
|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|
|Duquesnoy, Philippe; Escudier, Estelle; Vincensini, Laetitia et al. (2009) Loss-of-function mutations in the human ortholog of Chlamydomonas reinhardtii ODA7 disrupt dynein arm assembly and cause primary ciliary dyskinesia. Am J Hum Genet 85:890-6|
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