The long-terin goals of this research are to understand the structure, assembly, and function of cilia and flagella. The studies will utilize Chlamydomonas and mice as model organisms, and will focus on processes and proteins that are highly conserved among ciliated organisms. A combination of genetic, biochemical, and cell biological approaches will be taken. Intraflagellar transport (IFT), which is necessary for assembly of cilia, will be investigated. IFT involves the movement of particles and cargo within the cilium. Studies are proposed to learn more about the motors that transport the IFT particles, the particles themselves, and the BBSome ~ an IFT cargo adapter. Experiments will use existing Chlamydomonas mutants to determine the specific function of the IFT-particle protein IFT74, and of FAP133 ~ the intermediate chain for the retrograde IFT motor, dynein 1B. Studies will Investigate the specific roles of several BBSome proteins and establish the basis for BBSome-IFT particle interaction. The hypotiiesis that the BBSome exports proteins from the flagella will be tested. The relationship between the BBSome and phosphollpase D, a putative cargo of the BBSome, will be investigated. Studies will explore how the transition zone protein CEP290 regulates entry of IFT particles and BBSomes into the flagellum. Studies will define the domains of CEP290 necessary for assembly into the transition zone, assign specific functions to the domains, and Identify interacting proteins. An existing mutant defective In an uncharacterized motility regulating protein will be studied to determine the location of the protein In the axoneme and to Identify Its interacting partners. To discover novel proteins involved in flagellar assembly, in flagellar motility, and In the induction of genes encoding flagellar proteins, new insertlonal mutants will be made by a method that allows rapid identification of the genomic DNA flanking both ends of the insert, so that the mutated gene is quickly identified. Characterization of selected mutants will be facilitated by rescuing the mutants with constructs expressing HA- or GFP-tagged proteins so that the proteins can be quickly localized, obsen/ed by TIRF microscopy in living flagella, and followed in biochemical experiments.
Defects in the processes/proteins under Investigation cause disease in humans. For example, defects in BBSome proteins cause Bardet-Biedl syndrome, defects in CEP290 cause blindness, and defects in proteins involved in ciliary motility cause primary ciliary dyskinesia. The research will provide new information on the roles of specific ciliary proteins in human health, and why defects in these proteins are pathogenic.
|Kubo, Tomohiro; Hou, Yuqing; Cochran, Deborah A et al. (2018) A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1). Mol Biol Cell 29:1060-1074|
|Wingfield, Jenna L; Mengoni, Ilaria; Bomberger, Heather et al. (2017) IFT trains in different stages of assembly queue at the ciliary base for consecutive release into the cilium. Elife 6:|
|Hou, Yuqing; Witman, George B (2017) The N-terminus of IFT46 mediates intraflagellar transport of outer arm dynein and its cargo-adaptor ODA16. Mol Biol Cell 28:2420-2433|
|Brown, Jason M; Mosley, Matthew; Montes-Berrueta, Daniela et al. (2017) Characterization of a new oda3 allele, oda3-6, defective in assembly of the outer dynein arm-docking complex in Chlamydomonas reinhardtii. PLoS One 12:e0173842|
|Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R et al. (2016) Corrigendum: TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport. Nat Commun 7:11270|
|Kubo, Tomohiro; Brown, Jason M; Bellve, Karl et al. (2016) Together, the IFT81 and IFT74 N-termini form the main module for intraflagellar transport of tubulin. J Cell Sci 129:2106-19|
|Damerla, Rama Rao; Cui, Cheng; Gabriel, George C et al. (2015) Novel Jbts17 mutant mouse model of Joubert syndrome with cilia transition zone defects and cerebellar and other ciliopathy related anomalies. Hum Mol Genet 24:3994-4005|
|Schmidts, Miriam; Hou, Yuqing; Cortés, Claudio R et al. (2015) TCTEX1D2 mutations underlie Jeune asphyxiating thoracic dystrophy with impaired retrograde intraflagellar transport. Nat Commun 6:7074|
|Song, Kangkang; Awata, Junya; Tritschler, Douglas et al. (2015) In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography. J Biol Chem 290:5341-53|
|Yang, T Tony; Su, Jimmy; Wang, Won-Jing et al. (2015) Superresolution Pattern Recognition Reveals the Architectural Map of the Ciliary Transition Zone. Sci Rep 5:14096|
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