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 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, 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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37GM030626-33
Application #
8505484
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gindhart, Joseph G
Project Start
1981-08-01
Project End
2017-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
33
Fiscal Year
2013
Total Cost
$675,351
Indirect Cost
$269,735
Name
University of Massachusetts Medical School Worcester
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
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
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
Hou, Yuqing; Witman, George B (2015) Dynein and intraflagellar transport. Exp Cell Res 334:26-34
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
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
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
Awata, Junya; Song, Kangkang; Lin, Jianfeng et al. (2015) DRC3 connects the N-DRC to dynein g to regulate flagellar waveform. Mol Biol Cell 26:2788-800
Kubo, Tomohiro; Hirono, Masafumi; Aikawa, Takumi et al. (2015) Reduced tubulin polyglutamylation suppresses flagellar shortness in Chlamydomonas. Mol Biol Cell 26:2810-22
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
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

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