CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Department of Cell Biology, University of Massachusetts Medical School, Worcester, MA 01655, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 09/2010; 190(5):927-40. DOI: 10.1083/jcb.201006105
Source: PubMed

ABSTRACT Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule-membrane links there. Ultrastructural analysis revealed defects in these microtubule-membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule-membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.

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    • "In support of a conserved role for the TZ, orthologues of the genes encoding components of the MKS and NPHP complexes are present in other ciliated organisms, including Caenorhabditis elegans and Chamydomonas reinhardtii (Craige et al., 2010; Williams et al., 2011; Reiter et al., 2012). Inherited defects in ciliary function underlie a diverse set of diseases called ciliopathies. "
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    ABSTRACT: The Meckel syndrome (MKS) complex functions at the transition zone, located between the basal body and axoneme, to regulate the localization of ciliary membrane proteins. We investigated the role of Tmem231, a two-pass transmembrane protein, in MKS complex formation and function. Consistent with a role in transition zone function, mutation of mouse Tmem231 disrupts the localization of proteins including Arl13b and Inpp5e to cilia, resulting in phenotypes characteristic of MKS such as polydactyly and kidney cysts. Tmem231 and B9d1 are essential for each other and other complex components such as Mks1 to localize to the transition zone. As in mouse, the Caenorhabditis elegans orthologue of Tmem231 localizes to and controls transition zone formation and function, suggesting an evolutionarily conserved role for Tmem231. We identified TMEM231 mutations in orofaciodigital syndrome type 3 (OFD3) and MKS patients that compromise transition zone function. Thus, Tmem231 is critical for organizing the MKS complex and controlling ciliary composition, defects in which cause OFD3 and MKS. © 2015 Roberson et al.
    The Journal of Cell Biology 04/2015; 209(1):129-142. DOI:10.1083/jcb.201411087 · 9.83 Impact Factor
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    • "The current model proposes two modules; the MKS/TCTN/B9 complex and the NPHP complex [21, 22], which are linked in C. elegans by RPGRIP1L [20]. Given that several of these proteins contain transmembrane domains, and that disruption of TZ complex proteins often causes loss of ciliary Y-links [20, 23, 24], the model also suggests that TZ complex proteins may make up the Y-links connecting the axonemal doublets to the membrane [21, 22]. "
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    ABSTRACT: Background Cilia are critical for diverse functions, from motility to signal transduction, and ciliary dysfunction causes inherited diseases termed ciliopathies. Several ciliopathy proteins influence developmental signalling and aberrant signalling explains many ciliopathy phenotypes. Ciliary compartmentalisation is essential for function, and the transition zone (TZ), found at the proximal end of the cilium, has recently emerged as a key player in regulating this process. Ciliary compartmentalisation is linked to two protein complexes, the MKS and NPHP complexes, at the TZ that consist largely of ciliopathy proteins, leading to the hypothesis that ciliopathy proteins affect signalling by regulating ciliary content. However, there is no consensus on complex composition, formation, or the contribution of each component. Results Using bioinformatics, we examined the evolutionary patterns of TZ complex proteins across the extant eukaryotic supergroups, in both ciliated and non-ciliated organisms. We show that TZ complex proteins are restricted to the proteomes of ciliated organisms and identify a core conserved group (TMEM67, CC2D2A, B9D1, B9D2, AHI1 and a single TCTN, plus perhaps MKS1) which are present in >50% of all ciliate/flagellate organisms analysed in each supergroup. The smaller NPHP complex apparently evolved later than the larger MKS complex; this result may explain why RPGRIP1L, which forms the linker between the two complexes, is not one of the core conserved proteins. We also uncovered a striking correlation between lack of TZ proteins in non-seed land plants and loss of TZ-specific ciliary Y-links that link microtubule doublets to the membrane, consistent with the interpretation that these proteins are structural components of Y-links, or regulators of their formation. Conclusions This bioinformatic analysis represents the first systematic analysis of the cohort of TZ complex proteins across eukaryotic evolution. Given the near-ubiquity of only 6 proteins across ciliated eukaryotes, we propose that the MKS complex represents a dynamic complex built around these 6 proteins and implicated in Y-link formation and ciliary permeability. Electronic supplementary material The online version of this article (doi:10.1186/1471-2164-15-531) contains supplementary material, which is available to authorized users.
    BMC Genomics 06/2014; 15(1):531. DOI:10.1186/1471-2164-15-531 · 3.99 Impact Factor
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    • "Dikaryon experiments were performed to ask if the unlabeled protein is exchanged with tagged CEP290 protein from the dikaryon pool in a wild-type 3 by-wild-type cross. In dikaryons between a cep290; CEP290HA and a wild-type parent, the tagged CEP290 protein is initially observed only on two transition zones, but within 20 min, signal is seen on all four of the transition zones [Craige et al., 2010] (Fig. 3C). This suggests that there is a pool of this protein and the protein can undergo turnover. "
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    ABSTRACT: Cilia/flagella and basal bodies/centrioles play key roles in human health and homeostasis. Among the organisms used to study these microtubule-based organelles, the green alga Chlamydomonas reinhardtii has several advantages. One is the existence of a temporary phase of the life cycle, termed the dikaryon. These cells are formed during mating when the cells fuse and the behavior of flagella from two genetically distinguishable parents can be observed. During this stage the cytoplasms mix allowing for a defect in the flagella of one parent to be rescued by proteins from the other parent. This offers the unique advantage of adding back wild-type gene product or labeled protein at endogenous levels that can used to monitor various flagellar and basal body phenotypes. Mutants that show rescue and ones that fail to show rescue are both informative about the nature of the flagella and basal body defects. When rescue occurs, it can be used to determine the mutant gene product and to follow the temporal and spatial patterns of flagellar assembly. This review describes many examples of insights into basal body and flagellar proteins' function and assembly that have been discovered using dikaryons and discusses the potential for further analyses. © 2013 Wiley Periodicals, Inc.
    Cytoskeleton 02/2014; 71(2). DOI:10.1002/cm.21157 · 3.12 Impact Factor
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