A Transition Zone Complex Regulates Mammalian Ciliogenesis and Ciliary Membrane Composition

Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California, USA.
Nature Genetics (Impact Factor: 29.35). 07/2011; 43(8):776-84. DOI: 10.1038/ng.891
Source: PubMed

ABSTRACT Mutations affecting ciliary components cause ciliopathies. As described here, we investigated Tectonic1 (Tctn1), a regulator of mouse Hedgehog signaling, and found that it is essential for ciliogenesis in some, but not all, tissues. Cell types that do not require Tctn1 for ciliogenesis require it to localize select membrane-associated proteins to the cilium, including Arl13b, AC3, Smoothened and Pkd2. Tctn1 forms a complex with multiple ciliopathy proteins associated with Meckel and Joubert syndromes, including Mks1, Tmem216, Tmem67, Cep290, B9d1, Tctn2 and Cc2d2a. Components of this complex co-localize at the transition zone, a region between the basal body and ciliary axoneme. Like Tctn1, loss of Tctn2, Tmem67 or Cc2d2a causes tissue-specific defects in ciliogenesis and ciliary membrane composition. Consistent with a shared function for complex components, we identified a mutation in TCTN1 that causes Joubert syndrome. Thus, a transition zone complex of Meckel and Joubert syndrome proteins regulates ciliary assembly and trafficking, suggesting that transition zone dysfunction is the cause of these ciliopathies.

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Available from: Dragana J Josifova, Sep 27, 2015
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    • "This suggests that these six proteins are likely to have critical conserved functions. All are implicated in Meckel-Gruber and/or Joubert syndromes [18, 27–35], two severe ciliopathies, highlighting their importance for cilium function. A 7th protein, MKS1, is present in >50% of five of the six supergroups and is only absent from Rhizaria; this supergroup is currently represented by a single sequenced genome in this analysis, and as more genomes become available it may become apparent that MKS1 is also core. "
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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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    • "In other systems, MKS3 (TMEM67) functions as part of the filter or as a gatekeeper in the transition zone, which is the region between the basal body and the ciliary necklace [13,42-44]. Failure of transition zone function to control ciliary structure and membrane composition can lead to short and bulbous cilia [45], which is similar to our observed blebby cilia on cells depleted of MKS3 by RNAi. Our immunofluorescence data of paramecia expressing FLAG-MKS3 suggest that FLAG-MKS3p is in the transition zone, which in Paramecium has been defined as an area that spans from the epiplasm to the base of the cilium below the ciliary necklace [25,26] (Figure 1 and Additional file 5: Movie S1). "
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    ABSTRACT: Meckelin (MKS3), a conserved protein linked to Meckel Syndrome, assists in the migration of centrioles to the cell surface for ciliogenesis. We explored for additional functions of MKS3p using RNA interference (RNAi) and expression of FLAG epitope tagged protein in the ciliated protozoan Paramecium tetraurelia. This cell has a highly organized cell surface with thousands of cilia and basal bodies that are grouped into one or two basal body units delineated by ridges. The highly systematized nature of the P. tetraurelia cell surface provides a research model of MKS and other ciliopathies where changes in ciliary structure, subcellular organization and overall arrangement of the cell surface can be easily observed. We used cells reduced in IFT88 for comparison, as the involvement of this gene's product with cilia maintenance and growth is well understood. FLAG-MKS3p was found above the plane of the distal basal body in the transition zone. Approximately 95% of those basal bodies observed had staining for FLAG-MKS3. The RNAi phenotype for MKS3 depleted cells included global shortening and loss of cilia. Basal body structure appeared unaffected. On the dorsal surface, the basal bodies and their associated rootlets appeared rotated out of alignment from the normal anterior-posterior rows. Likewise, cortical units were abnormal in shape and out of alignment from normal rows. A GST pull down using the MKS3 coiled-coil domain suggests previously unidentified interacting partners. Reduction of MKS3p shows that this protein affects development and maintenance of cilia over the entire cell surface. Reduction of MKS3p is most visible on the dorsal surface. The anterior basal body is attached to and moves along the striated rootlet of the posterior basal body in preparation for duplication. We propose that with reduced MKS3p, this attachment and guidance of the basal body is lost. The basal body veers off course, causing basal body rows to be misaligned and units to be misshapen. Rootlets form normally on these misaligned basal bodies but are rotated out of their correct orientation. Our hypothesis is further supported by the identification of novel interacting partners of MKS3p including a kinetodesmal fiber protein, KdB2.
    Cilia 01/2014; 3(1):2. DOI:10.1186/2046-2530-3-2
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    • "The CEP290 gene was first isolated from human brain cDNA libraries, and encompasses 54 exons that encode a 2479 amino acid protein [12]. CEP290 has been localized to the centrosome and to the transition zone of cilia [13,14]. The exact physiological role of CEP290 remains unclear, although it has been shown that it plays an important role in the regulation of ciliary protein trafficking and cilium assembly [13,15]. "
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    ABSTRACT: Leber congenital amaurosis (LCA) is the most severe form of retinal dystrophy with an onset in the first year of life. The most frequent genetic cause of LCA, accounting for up to 15% of all LCA cases in Europe and North-America, is a mutation (c.2991+1655AG) in intron 26 of CEP290. This mutation generates a cryptic splice donor site resulting in the insertion of an aberrant exon (exon X) containing a premature stop codon to CEP290 mRNA. In order to study the pathophysiology of the intronic CEP290 mutation, we generated two humanized knock-in mouse models each carrying ~6.3 kb of the human CEP290 gene, either with or without the intronic mutation. Transcriptional characterization of these mouse models revealed an unexpected splice pattern of CEP290 mRNA, especially in the retina. In both models, a new cryptic exon (coined exon Y) was identified in ~5 to 12% of all Cep290 transcripts. This exon Y was expressed in all murine tissues analyzed but not detected in human retina or fibroblasts of LCA patients. In addition, exon x that is characteristic of LCA in humans, was expressed at only very low levels in the retina of the LCA mouse model. Western blot and immunohistochemical analyses did not reveal any differences between the two transgenic models and wild-type mice. Together, our results show clear differences in the recognition of splice sites between mice and humans, and emphasize that care is warranted when generating animal models for human genetic diseases caused by splice mutations.
    PLoS ONE 11/2013; 8(11):e79369. DOI:10.1371/journal.pone.0079369 · 3.23 Impact Factor
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