The transition zone (TZ) of eukaryotic cilia and flagella is a structural intermediate between the basal body and the axoneme that regulates ciliary traffic. Mutations in genes encoding TZ proteins (TZPs) cause human inherited diseases (ciliopathies). Here, we use the trypanosome to identify TZ components and localize them to TZ subdomains, showing that the Bardet-Biedl syndrome complex (BBSome) is more distal in the TZ than the Meckel syndrome (MKS) complex. Several of the TZPs identified here have human orthologs. Functional analysis shows essential roles for TZPs in motility, in building the axoneme central pair apparatus and in flagellum biogenesis. Analysis using RNAi and HaloTag fusion protein approaches reveals that most TZPs (including the MKS ciliopathy complex) show long-term stable association with the TZ, whereas the BBSome is dynamic. We propose that some Bardet-Biedl syndrome and MKS pleiotropy may be caused by mutations that impact TZP complex dynamics.

Sir William Dunn School of Pathology University of Oxford Oxford OX1 3RE United Kingdom;

Sir William Dunn School of Pathology University of Oxford Oxford OX1 3RE United Kingdom; Institute of Molecular Genetics Academy of Sciences of the Czech Republic 142 20 Prague 4 Czech Republic

Zobrazit více v PubMed

Wickstead B, Gull K. The evolution of the cytoskeleton. J Cell Biol. 2011;194(4):513–525. PubMed PMC

Alsford S, et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res. 2011;21(6):915–924. PubMed PMC

Broadhead R, et al. Flagellar motility is required for the viability of the bloodstream trypanosome. Nature. 2006;440(7081):224–227. PubMed

Reiter JF, Blacque OE, Leroux MR. The base of the cilium: Roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 2012;13(7):608–618. PubMed PMC

Chih B, et al. A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat Cell Biol. 2011;14(1):61–72. PubMed

Garcia-Gonzalo FR, et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat Genet. 2011;43(8):776–784. PubMed PMC

Williams CL, et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J Cell Biol. 2011;192(6):1023–1041. PubMed PMC

Lechtreck K-F, et al. The Chlamydomonas reinhardtii BBSome is an IFT cargo required for export of specific signaling proteins from flagella. J Cell Biol. 2009;187(7):1117–1132. PubMed PMC

Nachury MV, et al. A core complex of BBS proteins cooperates with the GTPase Rab8 to promote ciliary membrane biogenesis. Cell. 2007;129(6):1201–1213. PubMed

Ounjai P, et al. Architectural insights into a ciliary partition. Curr Biol. 2013;23(4):339–344. PubMed PMC

O’Toole ET, Giddings TH, McIntosh JR, Dutcher SK. Three-dimensional organization of basal bodies from wild-type and delta-tubulin deletion strains of Chlamydomonas reinhardtii. Mol Biol Cell. 2003;14(7):2999–3012. PubMed PMC

Gibbons IR, Grimstone AV. On flagellar structure in certain flagellates. J Biophys Biochem Cytol. 1960;7:697–716. PubMed PMC

Ringo DL. Flagellar motion and fine structure of the flagellar apparatus in Chlamydomonas. J Cell Biol. 1967;33(3):543–571. PubMed PMC

Anderson RG. The three-dimensional structure of the basal body from the rhesus monkey oviduct. J Cell Biol. 1972;54(2):246–265. PubMed PMC

Deane JA, Cole DG, Seeley ES, Diener DR, Rosenbaum JL. Localization of intraflagellar transport protein IFT52 identifies basal body transitional fibers as the docking site for IFT particles. Curr Biol. 2001;11(20):1586–1590. PubMed

Craige B, et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J Cell Biol. 2010;190(5):927–940. PubMed PMC

Gibbons IR. The relationship between the fine structure and direction of beat in gill cilia of a lamellibranch mollusc. J Biophys Biochem Cytol. 1961;11:179–205. PubMed PMC

Dute R, Kung C. Ultrastructure of the proximal region of somatic cilia in Paramecium tetraurelia. J Cell Biol. 1978;78(2):451–464. PubMed PMC

Tucker JB. Development and deployment of cilia, basal bodies, and other microtubular organelles in the cortex of the ciliate Nassula. J Cell Sci. 1971;9(3):539–567. PubMed

Höög JL, et al. Modes of flagellar assembly in Chlamydomonas reinhardtii and Trypanosoma brucei. eLife. 2014;3:e01479. PubMed PMC

Hart SR, et al. Analysis of the trypanosome flagellar proteome using a combined electron transfer/collisionally activated dissociation strategy. J Am Soc Mass Spectrom. 2009;20(2):167–175. PubMed

Ishikawa H, Thompson J, Yates JR, 3rd, Marshall WF. Proteomic analysis of mammalian primary cilia. Curr Biol. 2012;22(5):414–419. PubMed PMC

Kilburn CL, et al. New Tetrahymena basal body protein components identify basal body domain structure. J Cell Biol. 2007;178(6):905–912. PubMed PMC

Pazour GJ, Agrin N, Leszyk J, Witman GB. Proteomic analysis of a eukaryotic cilium. J Cell Biol. 2005;170(1):103–113. PubMed PMC

Subota I, et al. Proteomic analysis of intact flagella of procyclic Trypanosoma brucei cells identifies novel flagellar proteins with unique sub-localization and dynamics. Mol Cell Proteomics. 2014;13(7):1769–1786. PubMed PMC

Diener DR, Lupetti P, Rosenbaum JL. Proteomic analysis of isolated ciliary transition zones reveals the presence of ESCRT proteins. Curr Biol. 2015;25(3):379–384. PubMed PMC

Hodges ME, Scheumann N, Wickstead B, Langdale JA, Gull K. Reconstructing the evolutionary history of the centriole from protein components. J Cell Sci. 2010;123(Pt 9):1407–1413. PubMed PMC

Barker AR, Renzaglia KS, Fry K, Dawe HR. Bioinformatic analysis of ciliary transition zone proteins reveals insights into the evolution of ciliopathy networks. BMC Genomics. 2014;15:531. PubMed PMC

Bringaud F, et al. Characterization and disruption of a new Trypanosoma brucei repetitive flagellum protein, using double-stranded RNA inhibition. Mol Biochem Parasitol. 2000;111(2):283–297. PubMed

Dean S, et al. A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids. Open Biol. 2015;5(1):140197. PubMed PMC

Shiba D, et al. Localization of Inv in a distinctive intraciliary compartment requires the C-terminal ninein-homolog-containing region. J Cell Sci. 2009;122(Pt 1):44–54. PubMed

Warburton-Pitt SRF, et al. Ciliogenesis in Caenorhabditis elegans requires genetic interactions between ciliary middle segment localized NPHP-2 (inversin) and transition zone-associated proteins. J Cell Sci. 2012;125(Pt 11):2592–2603. PubMed PMC

Joglekar AP, Bloom K, Salmon ED. In vivo protein architecture of the eukaryotic kinetochore with nanometer scale accuracy. Curr Biol. 2009;19(8):694–699. PubMed PMC

Wan X, et al. Protein architecture of the human kinetochore microtubule attachment site. Cell. 2009;137(4):672–684. PubMed PMC

Woods A, et al. Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci. 1989;93(Pt 3):491–500. PubMed

Bonhivers M, Nowacki S, Landrein N, Robinson DR. Biogenesis of the trypanosome endo-exocytotic organelle is cytoskeleton mediated. PLoS Biol. 2008;6(5):e105. PubMed PMC

Emms DM, Kelly S. OrthoFinder: Solving fundamental biases in whole genome comparisons dramatically improves orthogroup inference accuracy. Genome Biol. 2015;16(1):157. PubMed PMC

Robinson DR, Sherwin T, Ploubidou A, Byard EH, Gull K. Microtubule polarity and dynamics in the control of organelle positioning, segregation, and cytokinesis in the trypanosome cell cycle. J Cell Biol. 1995;128(6):1163–1172. PubMed PMC

Los GV, et al. HaloTag: A novel protein labeling technology for cell imaging and protein analysis. ACS Chem Biol. 2008;3(6):373–382. PubMed

Absalon S, et al. Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes. Mol Biol Cell. 2008;19(3):929–944. PubMed PMC

Davidge JA, et al. Trypanosome IFT mutants provide insight into the motor location for mobility of the flagella connector and flagellar membrane formation. J Cell Sci. 2006;119(Pt 19):3935–3943. PubMed

Branche C, et al. Conserved and specific functions of axoneme components in trypanosome motility. J Cell Sci. 2006;119(Pt 16):3443–3455. PubMed

Garcia-Gonzalo FR, Reiter JF. Scoring a backstage pass: Mechanisms of ciliogenesis and ciliary access. J Cell Biol. 2012;197(6):697–709. PubMed PMC

Huang L, et al. TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone. Am J Hum Genet. 2011;89(6):713–730. PubMed PMC

Petersen C, Aumüller G, Bahrami M, Hoyer-Fender S. Molecular cloning of Odf3 encoding a novel coiled-coil protein of sperm tail outer dense fibers. Mol Reprod Dev. 2002;61(1):102–112. PubMed

Egydio de Carvalho C, et al. Molecular cloning and characterization of a complementary DNA encoding sperm tail protein SHIPPO 1. Biol Reprod. 2002;66(3):785–795. PubMed

Ghafouri-Fard S, Ghafouri-Fard S, Modarressi MH. Expression of splice variants of cancer-testis genes ODF3 and ODF4 in the testis of a prostate cancer patient. Genet Mol Res. 2012;11(4):3642–3648. PubMed

Cha P-C, et al. A genome-wide association study identifies three loci associated with susceptibility to uterine fibroids. Nat Genet. 2011;43(5):447–450. PubMed

Lambacher NJ, et al. TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome. Nat Cell Biol. 2016;18(1):122–131. PubMed PMC

Prevo B, Mangeol P, Oswald F, Scholey JM, Peterman EJG. Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia. Nat Cell Biol. 2015;17(12):1536–1545. PubMed

Langousis G, et al. Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei. Proc Natl Acad Sci USA. 2016;113(3):632–637. PubMed PMC

Poon SK, Peacock L, Gibson W, Gull K, Kelly S. A modular and optimized single marker system for generating Trypanosoma brucei cell lines expressing T7 RNA polymerase and the tetracycline repressor. Open Biol. 2012;2(2):110037. PubMed PMC

Brun R, Schönenberger MJ. Cultivation and in vitro cloning or procyclic culture forms of Trypanosoma brucei in a semi-defined medium. Short communication. Acta Trop. 1979;36(3):289–292. PubMed

Redmond S, Vadivelu J, Field MC. RNAit: An automated web-based tool for the selection of RNAi targets in Trypanosoma brucei. Mol Biochem Parasitol. 2003;128(1):115–118. PubMed

Inoue M, et al. The 14-3-3 proteins of Trypanosoma brucei function in motility, cytokinesis, and cell cycle. J Biol Chem. 2005;280(14):14085–14096. PubMed

Unnikrishnan A, Akiyoshi B, Biggins S, Tsukiyama T. An efficient purification system for native minichromosome from Saccharomyces cerevisiae. Methods Mol Biol. 2012;833:115–123. PubMed PMC

Trudgian DC, et al. Comparative evaluation of label-free SINQ normalized spectral index quantitation in the central proteomics facilities pipeline. Proteomics. 2011;11(14):2790–2797. PubMed

Woodward R, Carden MJ, Gull K. Immunological characterization of cytoskeletal proteins associated with the basal body, axoneme and flagellum attachment zone of Trypanosoma brucei. Parasitology. 1995;111(Pt 1):77–85. PubMed

Kohl L, Sherwin T, Gull K. Assembly of the paraflagellar rod and the flagellum attachment zone complex during the Trypanosoma brucei cell cycle. J Eukaryot Microbiol. 1999;46(2):105–109. PubMed

Dacheux D, et al. A MAP6-related protein is present in protozoa and is involved in flagellum motility. PLoS One. 2012;7(2):e31344. PubMed PMC

Morriswood B, et al. The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein. Mol Biochem Parasitol. 2009;167(2):95–103. PubMed

Lacomble S, et al. Three-dimensional cellular architecture of the flagellar pocket and associated cytoskeleton in trypanosomes revealed by electron microscope tomography. J Cell Sci. 2009;122(Pt 8):1081–1090. PubMed PMC

Identification of 30 transition fibre proteins in Trypanosoma brucei reveals a complex and dynamic structure

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

Genome sequencing reveals metabolic and cellular interdependence in an amoeba-kinetoplastid symbiosis

Protein diversity in discrete structures at the distal tip of the trypanosome flagellum