Chlamydomonas ARMC2/PF27 is an obligate cargo adapter for IFT of radial spokes

Intraflagellar transport (IFT) carries proteins into flagella but how IFT trains interact with the large number of diverse proteins required to assemble flagella remains largely unknown. Here, we show that IFT of radial spokes in Chlamydomonas requires ARMC2/PF27, a conserved armadillo repeat protein associated with male infertility and reduced lung function. Chlamydomonas ARMC2 was highly enriched in growing flagella and tagged ARMC2 and the spoke protein RSP3 comigrated on anterograde trains. In contrast, a cargo and an adapter of inner and outer dynein arms moved independently of ARMC2, indicating that unrelated cargoes distribute stochastically onto the IFT trains. After concomitant unloading at the flagellar tip, RSP3 attached to the axoneme whereas ARMC2 diffused back to the cell body. In armc2/pf27 mutants, IFT of radial spokes was abolished and the presence of radial spokes was limited to the proximal region of flagella. We conclude that ARMC2 is a cargo adapter required for IFT of radial spokes to ensure their assembly along flagella. ARMC2 belongs to a growing class of cargo-specific adapters that enable flagellar transport of preassembled axonemal substructures by IFT.

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Intraflagellar transport (IFT) cargo

The Journal of Cell Biology ◽

10.1083/jcb.200308132 ◽

2004 ◽

Vol 164 (2) ◽

pp. 255-266 ◽

Cited By ~ 244

Author(s):

Hongmin Qin ◽

Dennis R. Diener ◽

Stefan Geimer ◽

Douglas G. Cole ◽

Joel L. Rosenbaum

Keyword(s):

Cell Body ◽

Soluble Fraction ◽

Intraflagellar Transport ◽

Radial Spokes ◽

Flagellar Assembly ◽

Protein Particles ◽

Bidirectional Movement

Intraflagellar transport (IFT) is the bidirectional movement of multisubunit protein particles along axonemal microtubules and is required for assembly and maintenance of eukaryotic flagella and cilia. One posited role of IFT is to transport flagellar precursors to the flagellar tip for assembly. Here, we examine radial spokes, axonemal subunits consisting of 22 polypeptides, as potential cargo for IFT. Radial spokes were found to be partially assembled in the cell body, before being transported to the flagellar tip by anterograde IFT. Fully assembled radial spokes, detached from axonemal microtubules during flagellar breakdown or turnover, are removed from flagella by retrograde IFT. Interactions between IFT particles, motors, radial spokes, and other axonemal proteins were verified by coimmunoprecipitation of these proteins from the soluble fraction of Chlamydomonas flagella. These studies indicate that one of the main roles of IFT in flagellar assembly and maintenance is to transport axonemal proteins in and out of the flagellum.

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Late steps in cytoplasmic maturation of assembly-competent axonemal outer arm dynein in Chlamydomonas require interaction of ODA5 and ODA10 in a complex

Molecular Biology of the Cell ◽

10.1091/mbc.e15-05-0317 ◽

2015 ◽

Vol 26 (20) ◽

pp. 3596-3605 ◽

Cited By ~ 13

Author(s):

Anudariya B. Dean ◽

David R. Mitchell

Keyword(s):

Intraflagellar Transport ◽

Proximal Region ◽

Genetic Interactions ◽

In Vitro Binding ◽

Vitro Binding ◽

Immuno Electron Microscopy ◽

Assembly Factors ◽

Dynein Arms ◽

Cytoplasmic Maturation

Axonemal dyneins are multisubunit enzymes that must be preassembled in the cytoplasm, transported into cilia by intraflagellar transport, and bound to specific sites on doublet microtubules, where their activity facilitates microtubule sliding-based motility. Outer dynein arms (ODAs) require assembly factors to assist their preassembly, transport, and attachment to cargo (specific doublet A-tubule sites). In Chlamydomonas, three assembly factors—ODA5, ODA8, and ODA10—show genetic interactions and have been proposed to interact in a complex, but we recently showed that flagellar ODA8 does not copurify with ODA5 or ODA10. Here we show that ODA5 and ODA10 depend on each other for stability and coexist in a complex in both cytoplasmic and flagellar extracts. Immunofluorescence and immuno–electron microscopy reveal that ODA10 in flagella localizes strictly to a proximal region of doublet number 1, which completely lacks ODAs in Chlamydomonas. Studies of the in vitro binding of ODAs to axonemal doublets reveal a role for the ODA5/ODA10 assembly complex in cytoplasmic maturation of ODAs into a form that can bind to doublet microtubules.

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In vivo imaging of radial spoke proteins reveals independent assembly and turnover of the spoke head and stalk

10.1101/240143 ◽

2017 ◽

Author(s):

Karl F. Lechtreck ◽

Ilaria Mengoni ◽

Batare Okivie ◽

Kiersten B. Hilderhoff

Keyword(s):

Exchange Rate ◽

Cell Body ◽

In Vivo Imaging ◽

Fluorescent Protein ◽

De Novo ◽

Intraflagellar Transport ◽

Wild Type ◽

Radial Spokes ◽

Head Protein

AbstractRadial spokes (RSs) are multiprotein complexes regulating dynein activity. In the cell body and ciliary matrix, RS proteins are present in a 12S precursor, which is converted into axonemal 20S spokes consisting of a head and stalk. To study RS assembly in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pfl, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). Transport was elevated during ciliary assembly. IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RS proteins were mated to untagged cells with, without, or with partial RSs. RSP4-FP is added a tip-to-base fashion to preexisting pf1 spoke stalks while de novo RS assembly occurred lengthwise. In wild-type cilia, the exchange rate of head protein RSP4 exceeded that of the stalk protein RSP3 suggesting increased turnover of spoke heads. The data indicate that RSP3 and RSP4 while transported together separate inside cilia during RS repair and maintenance. The 12S RS precursor encompassing both proteins could represent transport form of the RS ensuring stoichiometric delivery by IFT. (196 of 200)

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FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia

10.1101/688291 ◽

2019 ◽

Author(s):

Jianfeng Lin ◽

Thuc Vy Le ◽

Katherine Augspurger ◽

Douglas Tritschler ◽

Raqual Bower ◽

...

Keyword(s):

Insertional Mutagenesis ◽

Spatial Organization ◽

Electron Tomography ◽

Coiled Coil ◽

Intraflagellar Transport ◽

Extrinsic Factors ◽

Ciliary Motility ◽

Genes Encoding ◽

Radial Spokes ◽

Dynein Arms

AbstractCiliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat, and highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for pre-assembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus,IDA8/BOP2, required for targeting the assembly of a subset of inner dynein arms to a specific location in the 96 nm repeat.IDA8encodes FAP57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple inner dynein arms and regulatory complexes.

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Bronchial stereocilia in the immotile cilia syndrome: A case report

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100110635 ◽

1984 ◽

Vol 42 ◽

pp. 52-53

Author(s):

George Price ◽

Lizardo Cerezo

Keyword(s):

Surface Modifications ◽

Respiratory Epithelium ◽

Immotile Cilia ◽

Kartagener's Syndrome ◽

Membrane Blebs ◽

Radial Spokes ◽

Males And Females ◽

Dynein Arms ◽

Ultrastructural Finding ◽

Ultrastructural Findings

Ultrastructural defects of ciliary structure have been known to cause recurrent sino-respiratory infection concurrent with Kartagener's syndrome. (1,2,3) These defects are also known to cause infertility in both males and females. (4) Overall, the defects are defined as the Immotile, or Dyskinetic Cilia Syndrome (DCS). Several ultrastructural findings have been described, including decreased number of cilia, multidirection orientation, fused and compound cilia, membrane blebs, excess matrix in the axoneme, missing outer tubular doublets, translocated doublets, defective radial spokes and dynein arms. A rare but noteworthy ultrastructural finding in DCS is the predominance of microvilli-like structures on the luminal surface of the respiratory epithelium. (5,6) These permanent surface modifications of the apical respiratory epithelium no longer resemble cilia but reflect the ultrastructure of stereocilia, similar to that found in the epidydimal epithelium. Like microvilli, stereocilia are devoid of microtubular ultrastructure in comparison with true cilia.

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Normal Ciliary Ultrastructure in Children with Kartagener's Syndrome

Annals of Otology Rhinology & Laryngology ◽

10.1177/000348948008900120 ◽

1980 ◽

Vol 89 (1) ◽

pp. 81-83 ◽

Cited By ~ 43

Author(s):

Fred S. Herzon ◽

Shirley Murphy

Keyword(s):

Immotile Cilia Syndrome ◽

Normal Limits ◽

Immotile Cilia ◽

Kartagener's Syndrome ◽

Radial Spokes ◽

Dynein Arms ◽

Ciliary Ultrastructure

Kartagener's syndrome has been found to be associated with the immotile cilia syndrome (lack of dynein arms and defective radial spokes in cilia). The ultrastructure of cilia of a child with Kartagener's syndrome was examined and found to be within normal limits. The implications of this are discussed.

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Regulation of sperm motility at the axonemal level

Reproduction Fertility and Development ◽

10.1071/rd9950847 ◽

1995 ◽

Vol 7 (4) ◽

pp. 847 ◽

Cited By ~ 15

Author(s):

C Gagnon

Keyword(s):

Direct Action ◽

Mammalian Species ◽

Basic Structure ◽

Structure And Function ◽

Structural Components ◽

Long Period ◽

Different Types ◽

Radial Spokes ◽

Dynein Arms ◽

And Function

With very few exceptions, the basic structure of the 9+2 axoneme has been well preserved over a very long period of evolution from protozoa to mammais. This stability indicates that the basic structural components of the axoneme visible by electron microscopy, as well as most of the other unidentified components, have withstood the passage of time. It also means that components of the 9+2 axoneme have sufficient diversity in function to accommodate the various types of motility patterns encountered in different species of flagella. Several of the 200 polypeptides that constitute the axoneme have been identified as components of the dynein arms, radial spokes etc. but many more remain to be identified and their function(s) remain to be determined. Because this review deals with the regulation of flagellar movement at the axonemal level, it does not include regulation of flagella by extracellular factors unless these factors have a direct action on axonemal components. In this context, it is very important firstly to understand the structural components of the axoneme and how they influence and regulate axonemal movement. Different primitive organisms are mentioned in this review since major breakthroughs in our understanding of how an axoneme generates different types of movement have been made through their study. Despite some variations in structure and function of axonemal components, the basic mechanisms involved in the regulation of flagella from Chlamydomonas or sea urchin spermatozoa should also apply to the more evolved mammalian species, including human spermatozoa.

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δ-Catenin/NPRAP (neural plakophilin-related armadillo repeat protein) interacts with and activates sphingosine kinase 1

Biochemical Journal ◽

10.1042/bj20040141 ◽

2004 ◽

Vol 382 (2) ◽

pp. 717-723 ◽

Cited By ~ 39

Author(s):

Toshitada FUJITA ◽

Taro OKADA ◽

Shun HAYASHI ◽

Saleem JAHANGEER ◽

Noriko MIWA ◽

...

Keyword(s):

Cell Motility ◽

Hippocampal Neurons ◽

Mdck Cells ◽

Specific Inhibitor ◽

Sphingosine Kinase ◽

Dependent Manner ◽

Repeat Protein ◽

Sphingosine 1 Phosphate ◽

Key Enzyme ◽

Armadillo Repeat

Sphingosine kinase (SPHK) is a key enzyme catalysing the formation of sphingosine 1-phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events acting through intracellular, as well as extracellular, mechanisms. However, the molecular mechanism of intracellular actions of SPP remains unclear. Here, we have identified δ-catenin/NPRAP (neural plakophilin-related armadillo repeat protein) as a potential binding partner for SPHK1 by yeast two-hybrid screening. From co-immunoprecipitation analyses, the C-terminal portion of δ-catenin/NPRAP containing the seventh to tenth armadillo repeats was found to be required for interaction with SPHK1. Endogenous δ-catenin/NPRAP was co-localized with endogenous SPHK1 and transfected δ-catenin/NPRAP was co-localized with transfected SPHK1 in dissociated rat hippocampal neurons. MDCK (Madin–Darby canine kidney) cells stably expressing δ-catenin/NPRAP contained elevated levels of intracellular SPP. In a purified system δ-catenin/NPRAP stimulated SPHK1 in a dose-dependent manner. Furthermore, δ-catenin/NPRAP-induced increased cell motility in MDCK cells was completely inhibited by dimethylsphingosine, a specific inhibitor of SPHK1. These results strongly suggest that at least some of δ-catenin/NPRAP functions, including increased cell motility, are mediated by an SPHK–SPP signalling pathway.

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ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

The Journal of Cell Biology ◽

10.1083/jcb.200802025 ◽

2008 ◽

Vol 183 (2) ◽

pp. 313-322 ◽

Cited By ~ 108

Author(s):

Noveera T. Ahmed ◽

Chunlei Gao ◽

Ben F. Lucker ◽

Douglas G. Cole ◽

David R. Mitchell

Keyword(s):

Binding Site ◽

Intraflagellar Transport ◽

Site Formation ◽

Wild Type ◽

Yeast Two Hybrid ◽

Hybrid Analysis ◽

B Subunit ◽

Dynein Arms ◽

Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

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