The long-term 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 hypothesis 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 insertional 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, observed by TIRF microscopy in living flagella, and followed in biochemical experiments.

Public Health Relevance

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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
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Special Emphasis Panel (NSS)
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Gindhart, Joseph G
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University of Massachusetts Medical School Worcester
Anatomy/Cell Biology
Schools of Medicine
United States
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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; Cort├ęs, 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
Hou, Yuqing; Witman, George B (2015) Dynein and intraflagellar transport. Exp Cell Res 334:26-34
San Agustin, Jovenal T; Pazour, Gregory J; Witman, George B (2015) Intraflagellar transport is essential for mammalian spermiogenesis but is absent in mature sperm. Mol Biol Cell 26:4358-72
McKenzie, Casey W; Craige, Branch; Kroeger, Tiffany V et al. (2015) CFAP54 is required for proper ciliary motility and assembly of the central pair apparatus in mice. Mol Biol Cell 26:3140-9
Brown, Jason M; Cochran, Deborah A; Craige, Branch et al. (2015) Assembly of IFT trains at the ciliary base depends on IFT74. Curr Biol 25:1583-93

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