In the bi-flagellate alga Chlamydomonas, flagellar detachment signals the immediate, coordinate upregulation of over 200 genes encoding flagellar proteins, and, within minutes, the synthesis of these proteins and their assembly into new flagella. This project is directed toward determining (a) where in the cytoplasm the mRNAs encoding these flagellar proteins are located and where these proteins are synthesized; (b) to what extent these polypeptides are pre-assembled in the cytoplasm prior to entering the nascent flagella; (c) how these proteins are targeted to the flagella; (d) the role of the basal body/centriole in this targeting process; and (e) how, once in the flagella, the precursors are transported to the assembly site at the flagellar tips. Using a variety of assembly mutants, these studies examine the synthesis, assembly and transport of flagellar radial spokes, which are complexes composed of 17 polypeptides that pre-assemble in the cytoplasm, yet function solely in the flagella. Studies on transport focus on intraflagellar transport (IFT), a motility characterized by the rapid anterograde and retrograde movement of particles between the flagellar membrane and axoneme. IFT particles are composed of ca. 15 polypeptides, the genes for which are being cloned. Mutations in C. elegans homologues of 3 of these polypeptides have been identified that result in defects in assembly of cilia found on sensory dendrites. Likewise, mutations in the microtubule motors that power anterograde and retrograde IFT in Chlamydomonas and their homologues in C. elegans result in the inability to assemble functional flagella or sensory cilia. IFT is, therefore, one of the only microtubule-based motilities in which mutants are available in both the anterograde and retrograde transport of defined, isolable particles. Particles, ultrastructurally similar to IFT particles, are found in the connecting cilium of vertebrate retinal rod inner-outer segments, and kinesins, homologous to those associated with IFT, have been localized to this region. These observations suggest that IFT is universally required for assembly and maintenance of both motile and non-motile sensory cilia and, therefore, may be involved in male fertility, the assembly and maintenance of respiratory and oviduct cilia, and diseases of the retinal rod and cone outer segments.

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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Cellular Biology and Physiology Subcommittee 1 (CBY)
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Yale University
Schools of Arts and Sciences
New Haven
United States
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Long, Huan; Zhang, Fan; Xu, Nannan et al. (2016) Comparative Analysis of Ciliary Membranes and Ectosomes. Curr Biol 26:3327-3335
Diener, Dennis R; Lupetti, Pietro; Rosenbaum, Joel L (2015) Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins. Curr Biol 25:379-384
Wood, Christopher R; Rosenbaum, Joel L (2014) Proteins of the ciliary axoneme are found on cytoplasmic membrane vesicles during growth of cilia. Curr Biol 24:1114-20
Mencarelli, Caterina; Mitchell, Aaron; Leoncini, Roberto et al. (2013) Isolation of intraflagellar transport trains. Cytoskeleton (Hoboken) 70:439-52
Satish Tammana, Trinadh V; Tammana, Damayanti; Diener, Dennis R et al. (2013) Centrosomal protein CEP104 (Chlamydomonas FAP256) moves to the ciliary tip during ciliary assembly. J Cell Sci 126:5018-29
Yuan, Shiaulou; Zhao, Lu; Sun, Zhaoxia (2013) Dissecting the functional interplay between the TOR pathway and the cilium in zebrafish. Methods Enzymol 525:159-89
Wood, Christopher R; Huang, Kaiyao; Diener, Dennis R et al. (2013) The cilium secretes bioactive ectosomes. Curr Biol 23:906-11
Gupta, Anjali; Diener, Dennis R; Sivadas, Priyanka et al. (2012) The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly. J Cell Biol 198:115-26
Yuan, Shiaulou; Li, Jade; Diener, Dennis R et al. (2012) Target-of-rapamycin complex 1 (Torc1) signaling modulates cilia size and function through protein synthesis regulation. Proc Natl Acad Sci U S A 109:2021-6
Kohno, Takahiro; Wakabayashi, Ken-ichi; Diener, Dennis R et al. (2011) Subunit interactions within the Chlamydomonas flagellar spokehead. Cytoskeleton (Hoboken) 68:237-46

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