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A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition.
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PMID:  21725307     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Francesc R Garcia-Gonzalo; Kevin C Corbit; María Salomé Sirerol-Piquer; Gokul Ramaswami; Edgar A Otto; Thomas R Noriega; Allen D Seol; Jon F Robinson; Christopher L Bennett; Dragana J Josifova; José Manuel García-Verdugo; Nicholas Katsanis; Friedhelm Hildebrandt; Jeremy F Reiter
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't     Date:  2011-07-03
Journal Detail:
Title:  Nature genetics     Volume:  43     ISSN:  1546-1718     ISO Abbreviation:  Nat. Genet.     Publication Date:  2011 Aug 
Date Detail:
Created Date:  2011-07-27     Completed Date:  2011-10-03     Revised Date:  2014-09-22    
Medline Journal Info:
Nlm Unique ID:  9216904     Medline TA:  Nat Genet     Country:  United States    
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Languages:  eng     Pagination:  776-84     Citation Subset:  IM    
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Membrane / physiology*
Cerebellar Diseases / genetics
Chickens
Cilia / metabolism*,  pathology*
Ciliary Motility Disorders / genetics
Encephalocele / genetics
Eye Abnormalities / genetics
Humans
Kidney Diseases, Cystic / genetics
Membrane Proteins / genetics,  metabolism,  physiology*
Mice
Mice, Inbred C57BL
Mice, Knockout
Morphogenesis
Mutation / genetics*
Organ Specificity
Peptide Fragments / immunology
Polycystic Kidney Diseases / genetics
Rabbits
Retina / abnormalities
Signal Transduction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Grant Support
ID/Acronym/Agency:
AR054396/AR/NIAMS NIH HHS; DK072301/DK/NIDDK NIH HHS; DK075972/DK/NIDDK NIH HHS; DK1068306/DK/NIDDK NIH HHS; DK1069274/DK/NIDDK NIH HHS; HD04260/HD/NICHD NIH HHS; R01 DK068306/DK/NIDDK NIH HHS; R01 DK068306-06/DK/NIDDK NIH HHS; R01 DK068306-07/DK/NIDDK NIH HHS; R01 DK072301/DK/NIDDK NIH HHS; R01 HD042601/HD/NICHD NIH HHS; R25-GM56847/GM/NIGMS NIH HHS; RC4 DK090917/DK/NIDDK NIH HHS; RC4 DK090917-01/DK/NIDDK NIH HHS; RC4-K090917/RC/CCR NIH HHS; //Howard Hughes Medical Institute
Chemical
Reg. No./Substance:
0/Membrane Proteins; 0/Peptide Fragments; 0/Tctn1 protein, human; 0/Tctn1 protein, mouse; 0/tectonin I; 0/tectonin II
Comments/Corrections
Comment In:
Nat Genet. 2011 Aug;43(8):723-4   [PMID:  21792235 ]

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Journal ID (nlm-journal-id): 9216904
Journal ID (pubmed-jr-id): 2419
Journal ID (nlm-ta): Nat Genet
ISSN: 1061-4036
ISSN: 1546-1718
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nihms-submitted publication date: Day: 13 Month: 6 Year: 2011
Electronic publication date: Day: 3 Month: 7 Year: 2011
pmc-release publication date: Day: 1 Month: 2 Year: 2012
Volume: 43 Issue: 8
First Page: 776 Last Page: 784
ID: 3145011
PubMed Id: 21725307
DOI: 10.1038/ng.891
ID: nihpa300973

A Transition Zone Complex Regulates Mammalian Ciliogenesis and Ciliary Membrane Composition
Francesc R. Garcia-Gonzalo12*
Kevin C. Corbit12*
María Salomé Sirerol-Piquer3
Gokul Ramaswami45
Edgar A. Otto45
Thomas R. Noriega1
Allen D. Seol12
Jon F. Robinson67
Christopher L. Bennett67
Dragana J. Josifova8
José Manuel García-Verdugo39
Nicholas Katsanis67
Friedhelm Hildebrandt4510
Jeremy F. Reiter12
1 Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158, USA
2 Cardiovascular Research Institute, University of California, San Francisco, San Francisco, CA 94158, USA
3 Laboratorio de Morfología Celular, Unidad Mixta CIPF-UVEG, CIBERNED, Valencia, Spain
4 Department of Pediatrics, University of Michigan, Ann Arbor, MI 48109, USA
5 Department of Human Genetics, University of Michigan, Ann Arbor, MI 48109, USA
6 Center for Human Disease Modeling, Department of Cell Biology, Duke University, Durham, NC 27710, USA
7 Department of Pediatrics, Duke University, Durham, NC 27710, USA
8 Department of Clinical Genetics, Guy’s Hospital, London, SE1, 9RT, UK
9 Department of Comparative Neurobiology, Cavanilles Institute of Biodiversity and Evolutionary Biology, University of Valencia, Spain
10 Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA
*These authors contributed equally to this work.

INTRODUCTION

Cilia are microtubule-based cell surface projections, likely possessed by the eukaryotic cenancestor1. Some mammalian cells possess motile cilia, whose functions include pulmonary mucous clearance and left-right axis determination. In contrast, most primary cilia are immotile, instead functioning in signal transduction during embryogenesis and adult tissue homeostasis2.

An early ciliogenic event is the docking of the mother centriole to a vesicle3. Following docking, the centriole extends an axoneme that invaginates the vesicle. Fusion of the vesicle with the plasma membrane exposes the nascent cilium to the extracellular environment. Vesicle trafficking is required for ciliogenesis, and thereafter contributes lipids and transmembrane proteins to the ciliary membrane4.

Once transported to the ciliary base, ciliary-bound transmembrane proteins must cross the transition zone, a region between the basal body and axoneme. Although little is known about the transition zone, three nephronophthisis (NPHP)-associated proteins, Nphp1, Nphp4 and Rpgrip1l, form a complex that localizes to this compartment5. Furthermore, transition zone components regulate ciliary protein composition in Chlamydomonas, C. elegans, and mammalian cells68.

Ciliary dysfunction underlies a group of diseases, termed ciliopathies, that include JBTS, characterized by cerebellar and brainstem malformations, MKS, characterized by occipital encephalocele, kidney cysts and polydactyly, and NPHP, characterized by medullary kidney cysts910. The genes underlying these ciliopathies display extensive allelism. For example, mutations in six MKS genes, TMEM216, TMEM67, CEP290, RPGRIP1L, CC2D2A and TCTN2, also cause JBTS or COACH, which resembles JBTS but includes liver fibrosis5,911. Some of these loci also cause or modify the manifestations of ciliopathies such as Bardet-Biedl syndrome (BBS), characterized by obesity, polydactyly and retinitis pigmentosa1013.

Polycystic kidney disease (PKD), a common ciliopathy, is caused by mutations in ciliary transmembrane proteins, including Pkd2910. Targeting of transmembrane proteins to cilia involves ciliary localization sequences (CLSs)4. For example, Pkd2 relies upon an amino-terminal RVxP sequence, whereas Rhodopsin uses a carboxy-terminal VxPx motif1415. Other CLSs, such as that of SSTR3, bind the BBSome, a protein complex involved in BBS1618. The BBSome acts as a membrane coat trafficking G-protein-coupled receptors (GPCRs) to the cilium16. Despite its important role in GPCR targeting, the BBSome is not required for ciliogenesis in most tissues19.

Abrogating vertebrate ciliogenesis disrupts Hedgehog (Hh) signaling, resulting in embryonic neural tube dorsalization and polydactyly2. Many Hh pathway components, including Patched1 (Ptch1), Smoothened (Smo), Sufu, and Gli transcription factors, localize to cilia, and ciliary translocation of Smo is required to robustly activate the pathway2,20. Cilia also participate in other pathways, such as Pdgfr-α, Wnt and mTOR signaling2,4,21.

We previously described a novel gene, Tctn1, that modulates mouse Hh signal transduction22. Tctn1 belongs to a family of signal sequence-containing proteins conserved among eukaryotes that generate cilia or flagella. We demonstrate that Tctn1 forms a membrane-spanning transition zone complex with JBTS and MKS proteins, and identify human TCTN1 as a novel JBTS gene. The Tectonic ciliopathy complex controls ciliogenesis and ciliary protein composition, suggesting that JBTS, COACH and MKS result from transition zone dysfunction.


RESULTS
Tctn1 is required for ciliogenesis in a tissue-dependent manner

We previously demonstrated that Tctn1 is expressed specifically in the node of late headfold-stage mouse embryos and is required for correct establishment of left-right asymmetry22. Consistently, nodal flow and left lateral plate induction of Nodal gene expression are disrupted in Tctn1−/− mouse embryos (Figure S1a and data not shown). Because left-right axis determination depends on leftward flow generated by nodal cilia22, we examined these cilia in Tctn1−/− embryos. Staining of E8.5 embryonic nodes for Arl13b, a ciliary membrane-associated protein2324, revealed abundant cilia in wild type nodes, but not Tctn1−/− nodes (Figure 1a). Scanning electron microscopy (SEM) confirmed the absence of cilia in Tctn1−/− nodes, and transmission electron microscopy (TEM) demonstrated that basal bodies of mutant node cells attach to the plasma membrane but fail to extend axonemes (Figures 1b-c).

In addition to left-right axis specification, Tctn1 is required for Sonic hedgehog (Shh) patterning of the ventral neural tube22. Cilia are essential for interpreting Shh signals during vertebrate neural development2. Immunostaining of wild type and Tctn1−/− E10.5 neural tubes for Arl13b and γ-tubulin, a basal body component, revealed a normal number and localization of basal bodies, coupled with a dramatic reduction in the number of cilia (Figure 1d). By SEM, cilia were numerous in control, but scarce in Tctn1−/− neural tubes. Of the few neural cilia that formed in mutants, most were short and bulbous, while some displayed swellings (Figure 1e). TEM of Tctn1−/− neural tubes confirmed that basal bodies dock to the apical membrane in the absence of Tctn1, but mostly fail to extend axonemes (Figures 1f and S1b).

Thus, Tctn1 mutant phenotypes, including laterality defects and neural tube dorsalization, can be explained by ciliary defects in the node and neural tube, respectively. However, as reported for Mks1, Tctn1 is not essential for ciliogenesis in all tissues25. For example, anterior to the node, the notochord of Tctn1−/− embryos produces cilia, as does the early gut epithelium (Figure 1g and data not shown). Similarly, mesenchymal cells surrounding the neural tube possess acetylated tubulin (AcTub) containing cilia, as do limb bud mesenchymal cells (Figure 1h-i). Nevertheless, these cilia display markedly reduced levels of Arl13b (Figure 1h-i). Therefore, loss of Tctn1 has different effects in different tissues, with tissues such as the node and neural tube requiring Tctn1 for ciliogenesis, and tissues such as the perineural and limb bud mesenchyme requiring Tctn1 for ciliary localization of Arl13b.

The differential requirements for Tctn1 may reflect different ciliogenic mechanisms in the node and neural tube versus the mesenchyme. In support of this hypothesis, Ift20, a ciliogenic component of the intraflagellar transport complex B (IFT-B), localizes differently in neural tube and perineural mesenchyme cells (Figure S1c)26. Consistent with previous reports, Ift20 is Golgi-localized in mesenchymal cells, but not in the neural tube (Figure S1c)27. In contrast, Ift88, another IFT-B component, is present in both mesenchymal and neural cilia (Figure S1d). Thus, differential localization of ciliogenic proteins in mesenchymal and neural cells may signify different mechanisms of ciliogenesis, one of which is dependent on Tctn1.

To assess whether Tctn1 participates in ciliary function even where it is dispensable for ciliogenesis, we examined patterning of the limb bud, which contains cilia in Tctn1 mutants (Figure 1i). Shh specifies vertebrate digit number and identity by preventing cilium-dependent formation of Gli3 repressor (Gli3-R)2829. Consequently, both increased Hh signaling or decreased ciliary function in the limb bud result in polydactyly2.

Tctn1−/− embryos developed an extra preaxial digit 1 on one or both hindlimbs (Figure 1j). Consistent with a role for Tctn1 in Hh signal transduction, Shh expression was unaffected in Tctn1−/− limb buds, but Shh-regulated genes, including Fgf4, Bmp2, Gremlin and Hoxd11 were expanded anteriorly (Figure 1k)30. Similarly, two general Hh transcriptional targets, Gli1 and Ptch1, were also expanded anteriorly in Tctn1−/− limb buds (Figure 1k). In contrast, expression of Fgf8, a Shh-independent gene, was unaltered, indicating that patterning, not formation, of the apical ectodermal ridge depends on Tctn1 (Figure 1k).

As shown previously for the neural tube, Tctn1 was epistatic to Shh in limb bud patterning (Figure 1l)22. Compared to controls, Tctn1−/− embryos had markedly increased levels of full length Gli3 (Gli3-FL), suggesting that Gli3 processing is compromised (Figure 1m). These results indicate that, although Tctn1 is not required for ciliogenesis in the limb bud, it is required for cilium-dependent Hh signal transduction.


Tctn1 is part of a ciliopathy-associated protein complex

To elucidate the role of Tctn1 in ciliogenesis, we generated NIH-3T3 cells stably expressing Tctn1 fused to a localization and affinity purification (LAP) tag31. These cells were ciliated and Tctn1-LAP was at the base of some cilia, although most Tctn1-LAP was retained in the endoplasmic reticulum (ER) (Figure 2a). To identify Tctn1-associated proteins, we isolated Tctn1-LAP by tandem affinity purification and analyzed copurifying interactors by mass spectrometry. Using a cutoff of three unique peptides, we identified 154 proteins specifically in the Tctn1-LAP purification (Table S1). Among the proteins with the highest number of mass spectra, a measure of the amount of copurified protein, were the products of several genes associated with MKS, JBTS and other ciliopathies, including Mks1, Cep290, Cc2d2a, Tctn2 and B9d1 (Figure 2b)5,911,32. The two strongest Tctn1 interactors were Tctn2 and Tctn3, indicating that the three Tectonics are part of the same complex. None of these proteins were detected in a control purification from LAP-only expressing NIH-3T3 cells (Table S1).

Proteins represented by fewer mass spectra included several ER chaperones, possibly reflecting ER processing of Tctn1-LAP (Table S1). However, as Tctn1 colocalizes with ciliopathy proteins at the ciliary transition zone (shown below), it is likely that some of these interactions reflect ER retention of overexpressed Tctn1.

To confirm the interactions of Tctn1 with ciliopathy proteins, we performed coimmunoprecipitation assays using tagged proteins expressed in COS1 cells. Tctn1 interacted with Tctn2, Tctn3, Mks1, Cc2d2a and B9d1 (Figure 2c). Other ciliary or basal body proteins, including Smo, Rab8a and Rab11a, did not interact with Tctn1 (Figure 2c)4. Intriguingly, Tctn1 and Cep290 did not reproducibly interact by coimmunoprecipitation in COS1 cells, possibly indicating that the interaction of Tctn1 with Cep290 is compromised by tagging Cep290, or depends on immunoprecipitation conditions or cell type.

As our proteomic analysis identified five of the eight known MKS proteins, we tested whether Tctn1 associates with the other three: Tmem67, Tmem216 and Rpgrip1l910. In transfected COS1 cells, Tctn1 interacted with Tmem67 and Tmem216, but not Rpgrip1l (Figure 2c-d). Thus, Tctn1 interacts with Mks1, Cc2d2a, B9d1, Tctn2, Tctn3 and, under some conditions, Tmem216, Tmem67 and Cep290. In addition, using antibodies against endogenous proteins, we found that Tctn2 interacts with Mks1 and Tctn3 in ciliated hTERT-RPE1 cells (Figure S2).

To investigate the size of the Tectonic ciliopathy complex, we subjected Tctn1-LAP purified from stably expressing NIH-3T3 cells to gel filtration chromatography (Figure 2e). We analyzed the fractions for protein concentration and the presence of Tctn1, Tctn2, Mks1, Cc2d2a and Cep290. Interestingly, these proteins co-eluted in fractions 15–19, corresponding to a molecular weight between 0.7 and 2 MDa, suggesting that they comprise one or a set of related large complexes.

Localization of Mks1 and Tmem67 to the ciliary transition zone depends upon Tctn1

To investigate the function of Tectonic and MKS proteins in ciliogenesis, we examined their subcellular localization. The specificity of antibodies against Tctn1–3, Tmem67 and Cc2d2a was confirmed using siRNA-treated cells or cells harboring null alleles for these genes (Figures S2a, S3a-c). In hTERT-RPE1 cells, Tctn1 localized to the transition zone, between the ciliary axoneme and basal body (Figure 3a, S3d). The Tctn1 interactors Tctn2, Tctn3, Mks1, Tmem67, Cep290, Cc2d2a and B9d1 also localized to the transition zone (Figure 3a, S3d). In addition, Nphp4, not detected as a Tctn1 interactor, localized to the transition zone (Figure 3a, S3d). Some of these proteins also localized to the basal body (B9d1), cilium proper (Tctn2, Tctn3, Tmem67), or centriolar satellites (Cep290). Tmem216 occasionally localized to the transition zone, but more typically localized to the basal body or axoneme. Rpgrip1l, the only MKS protein that did not biochemically interact with Tctn1, localized to the basal body, similar to BBSome component Bbs5.

To understand how Tctn1 contributes to transition zone function, we examined whether Tcn1 controls the organization of the transition zone (Figure 3b, S3e-g). Several transition zone proteins, including Nphp1, Nphp4, RP2 and Septin2, localized independently of Tctn15,8,3334. The same was true of Bbs5, Ift88 and Ift139, components of the BBSome, IFT-B and IFT-A ciliary protein complexes, respectively, and of Rpgrip1l, which ocalized to the transition zone of MEFs. Tmem216 localization to the basal body and, variably, to the transition zone was also unaffected. Similarly, Cc2d2a was at the transition zone of Tctn1−/− MEFs, indicating that at least some Tctn1 interactors localize to the transition zone independently of Tctn1. In contrast, Tctn1−/− MEFs exhibited decreased Mks1 and Tmem67 localization to the transition zone (Figure 3b-c, S3e). These results reveal that Tctn1 is required for transition zone localization of a subset of interacting proteins.

Tctn1 interactors Tctn2, Cc2d2a and Tmem67 also promote tissue-specific ciliogenesis

To assess Tectonic complex function, we analyzed mouse Tctn2, Cc2d2a and Tmem67 mutants. Mice homozygous for the Tctn2 mutant allele displayed phenotypes closely resembling those of Tctn1 mutants5. Like Tctn1−/− embryos, Tctn2−/− embryos lacked nodal cilia, as determined by immunostaining and SEM (Figure 4a-b). Cilia in Tctn2−/− neural tubes were scarce, morphologically defective, and failed to elongate axonemes (Figures 4c-d, S4). Also similar to Tctn1 mutants, basal bodies docked to the plasma membrane in Tctn2−/− neural epithelium (Figures 4d, S4b). In the limb bud mesenchyme, cilia numbers were reduced, yet the remaining cilia appeared normal by AcTub and γ-tubulin staining (Figure 4e). In contrast, no Arl13b ciliary staining was seen in Tctn2−/− perineural mesenchyme, suggesting that, as in Tctn1−/− mutants, Tctn2−/− cilia lack Arl13b (Figure 4f). These data suggest that Tctn1 and Tctn2 share a common function, with both affecting ciliogenesis in a tissue-specific manner.

We also generated mice mutant for Cc2d2a (Figure S5a). Cc2d2a−/− embryos exhibit randomized left-right axes, holoprosencephaly, microphthalmia, and a variably expressive curved body axis, phenotypes also exhibited by Tctn1 and Tctn2 mutants (Figure 5a and data not shown)22. Cc2d2a−/− embryos also have cilia defects, exemplified by the absence of Arl13b staining in the neural tube and surrounding mesenchyme (Figure 5b-c). Furthermore, like Tctn1−/− MEFs, Cc2d2a−/− MEFs can generate cilia (Figure 5d). Thus, Cc2d2a shares tissue-specific ciliogenic functions with Tctn1 and Tctn2.

Finally, we analyzed mice homozygous for a targeted allele of Tmem67 (Figures 5e-g, S5b-d). Unlike Tctn1, Tctn2 or Cc2d2a mutants, Tmem67−/− mice survived to birth with no overt morphological abnormalities. However, these mice died soon after birth (Figure S5c-d). These results are similar to those of bpck mutants, which harbor a large genomic deletion including Tmem67 and five other genes35. bpck mutants develop kidney cysts, and cilia of bpck mutant kidney tubules are dysmorphic and vary in length35.

We examined the kidneys of E18.5 Tmem67−/− embryos (Figure 5e-f). Consistent with the bpck phenotype, Tmem67−/− embryonic kidneys develop cysts (Figure 5e), and kidney tubules have fewer cilia than controls (Figure 5f). Tmem67 siRNA-treated IMCD3 kidney cells also display ciliogenesis defects36. In contrast, Tmem67−/− MEFs were not defective in ciliogenesis (Figure 5g). Hence, Tmem67 also has tissue-specific roles in ciliogenesis, although the ciliogenic defects are milder than those of Tctn1, Tctn2 or Cc2d2a mutants.

Given that Tmem67 and Tctn1 mutants display distinct phenotypes, we generated Tmem67−/−Tctn1−/− double mutant embryos to examine the genetic relationship between both genes. Double mutant embryos were indistinguishable from Tctn1−/− single mutants (Figure 5h). As compared to Tctn1, Tctn2, and Cc2d2a mutants, the weaker phenotypes of Tmem67 null mutants suggest that Tmem67 has an ancillary role in promoting the ciliogenic functions of the core Tctn1, Tctn2, and Cc2d2a complex.

The Tectonic ciliopathy complex controls ciliary membrane protein localization

All Tctn genes encode signal peptides and are thus predicted to be translated at the ER. Consistently, Tctn1 undergoes N-glycosylation and, when overexpressed, is seen at the ER (Figures 2a, S6a). Tctn2 and 3, but not Tctn1, have predicted transmembrane helices in their carboxy-termini. Despite the absence of a transmembrane domain, Tctn1 is not secreted and functions cell autonomously (Figure S6b-c and data not shown).

Given the association of Tectonics with membranes and the transition zone, we hypothesized that Tctn1 regulates ciliary membrane composition. We examined the localization of GFP-tagged SSTR3, a G protein-coupled receptor that can localize to cilia16. This protein localized to cilia equally in wild type and Tctn1−/− MEFs (Figure S6d).

Adenylyl cyclase III (AC3) is another multipass transmembrane protein that localizes to primary cilia of adult mouse brains37. AC3 also localizes to cilia in wild type MEFs and E14.5 embryos, but not in Tctn1−/− MEFs or embryos (Figure 6). We examined whether Tctn1 interacting proteins are similarly required for ciliary membrane protein localization. Strikingly, MEFs lacking Tctn2, Cc2d2a or Tmem67 lacked AC3 in their cilia (Figure 6). Thus, the Tectonic complex is required for the ciliary localization of AC3.

Pkd2, the product of a gene mutated in autosomal dominant polycystic kidney disease, is another ciliary transmembrane protein910,14. Pkd2 localizes to cilia in some wild type MEFs but, like AC3, its ciliary localization is abrogated in Tctn1−/−, Tctn2−/− and Cc2d2a−/− MEFs, and in Tctn1−/− E14.5 mesenchyme (Figure 6). Pkd2 also localizes to cilia in some wild type E18.5 kidney tubules, and this was unchanged in Tmem67−/− kidneys, indicating that Tmem67, unlike other complex components, is not required for ciliary localization of Pkd2 (Figure 6f)38.

Smo is a seven transmembrane protein that moves to cilia in response to Hh pathway activation20. Smo displayed reduced ciliary localization in Tctn1−/− and Tctn2−/− MEFs treated with the Hh pathway agonist SAG, an effect that was even stronger in Cc2d2a−/− MEFs (Figure 6)39. In contrast, SAG-treated Tmem67−/− MEFs displayed normal levels of ciliary Smo, again consistent with an ancillary role for Tmem67 in the Tectonic complex.

Tctn1−/−, Tctn2−/− and Cc2d2a−/− MEFs also displayed dramatically reduced levels of ciliary Arl13b, whereas Tmem67−/− MEFs had a more modest reduction (Figure 6). Although Arl13b is not an integral membrane protein, it is membrane-associated via palmitoylation24. Consistent with a role for Tctn1 in controlling ciliary protein composition, transfection of Tctn1−/− MEFs with myc- or LAP-tagged Tctn1 rescued ciliary localization of Arl13b and AC3 (Figure S6e-f and data not shown).

Taken together, these results suggest that the Tectonic ciliopathy complex acts at the transition zone to control ciliary membrane protein composition.

A human TCTN1 mutation is a cause of JBTS

Given the connections between Tctn1 and recognized ciliopathy proteins, we examined whether human TCTN1 gene mutations also cause ciliopathies. We used homozygosity mapping in consanguineous kindreds with consecutive exon sequencing to test whether TCTN1 mutations are associated with ciliopathies41.

In kindred A2090 from Bangladesh, the parents of two sisters with JBTS were first degree cousins. In both sisters (II-1 and II-2), brain MRI revealed cerebellar vermis hypoplasia and the “molar tooth sign”, the characteristic radiographic feature of JBTS. Sibling II-1 also had bilateral frontotemporal pachygyria, a coarsening of the cerebral gyri. Ophthalmologic assessment and renal ultrasounds of siblings II-1 and II-2 were normal at ages 7 and 4 yrs, respectively.

Homozygosity mapping of this kindred identified nine segments of homozygosity by descent, representing candidate regions for a recessive disease-causing mutation (Figure 7a)41. Because none of these segments coincided with known ciliopathy loci, we hypothesized that JBTS in family A2090 is caused by a recessive mutation in an unrecognized ciliopathy gene. TCTN1 mapped to a homozygous candidate region on chromosome 12 (Figure 7a). Direct Sanger sequencing of all TCTN1 exons revealed a homozygous obligatory splice-acceptor mutation (IVS1–2a>g) in both affected siblings of family A2090 (Figure 7b). This mutation, predicted to disrupt domains of the TCTN1 protein required for its function (Figure S7), was absent from 96 healthy control individuals and from the 1,000 Genomes Project, thus confirming TCTN1 mutation as a new cause of JBTS. Investigation of four families homozygous at the TCTN1 locus and 48 families with JBTS revealed no additional mutations, suggesting that TCTN1 mutations are a rare cause of JBTS.


DISCUSSION

By combining developmental, genetic, and biochemical analyses, we have identified a complex containing Tctn1, Tctn3 and seven MKS proteins (Mks1, Tmem216, Tmem67, Cep290, Cc2d2a, B9d1 and Tctn2). This complex localizes to the ciliary transition zone, where it regulates ciliogenesis and ciliary membrane composition in a tissue-dependent manner. Together with the MKS-like phenotypes exhibited by mouse Tectonic mutants, our data suggest transition zone dysfunction is the cell biological defect underlying human MKS.

Tctn1 modulates Hh signaling in the mouse neural tube22. As both Gli activator and repressor activity depend on cilia, the role of Tctn1 in neural tube patterning is likely due to its requirement in neural ciliogenesis2829. Tctn1 also participates in Hh signaling in the limb bud. However, Tctn1 is not required for limb ciliogenesis, but promotes ciliary translocation of Arl13b and Smo, both involved in Hh signaling20,23. Therefore, the role of Tctn1 in regulating ciliary membrane composition may underlie its function in limb patterning.

The mechanisms underlying tissue specificity of Tectonic complex function remain unclear. Cilia from different tissues possess different functions and compositions, perhaps reflecting different ciliogenic mechanisms. In support of this hypothesis, the ciliogenic protein Ift20 localizes to the Golgi in mesenchymal but not neural cells. Given that the transition zone is formed early during ciliogenesis, the transition zone may regulate the entrance of IFT particles into the nascent axoneme in some cell types6.

Cep290 was identified as a Tctn1 interactor by mass spectrometry and chromatography, but not coimmunoprecipitation. In contrast, Tmem67 and Tmem216 were not detected by mass spectrometry, but did coimmunoprecipitate with Tctn1. Thus, Cep290, Tmem67 and Tmem216 may be substoichiometric or peripheral components of the Tectonic complex. Besides ciliopathy proteins, our mass spectrometric analysis identified many additional potential Tctn1 interactors. Although most of these were considerably less abundant in the Tctn1 purification than the ciliopathy proteins, they may still participate in Tectonic complex function.

We identified a TCTN1 mutation causing JBTS, an MKS-related ciliopathy. TCTN2 mutations also lead to MKS and JBTS, underscoring the connection between TCTN genes and human ciliopathies5,11. Moreover, mutations in ARL13B, whose ciliary localization depends on Tectonics, similarly cause JBTS42. Apart from MKS, TMEM216, TMEM67 and CC2D2A are associated with JBTS and COACH syndromes910. How do these genes display such a high degree of allelism? We propose that different degrees of complex dysfunction manifest as different syndromes, with severe loss of function causing MKS, and progressively less severe impairment causing COACH and JBTS (Figure 7c). Consistent with this model, mouse Tctn1, Tctn2, Cc2d2a and Mks1 null mutants display phenotypes resembling human MKS, whereas Tmem67 mutants have a less severe phenotype, suggesting partial loss of Tectonic complex function25.

Like Tctn mutants, Rpgrip1l mutant mice display MKS-like phenotypes43. However, Rpgrip1l does not interact with Tctn1, and instead forms a transition zone complex with Nphp1 and Nphp45. This NPHP complex functions together with the Tectonic complex, as the nematode orthologs of Nphp genes genetically interact with components of the Tectonic complex6. Moreover, C. elegans Rpgrip1l regulates transition zone localization of both complexes6. In contrast, mammalian Tctn1 is required for proper localization of some Tectonic complex members, but not for the NPHP complex. Thus, transition zone integrity depends upon both complexes operating in a partially redundant manner.

In contrast to the Tectonic complex, severe dysfunction of the NPHP complex may result in Senior-Løken syndrome (SLSN), characterized by NPHP with retinitis pigmentosa. Some ciliopathies may also result from abrogated activity of both the NPHP and Tectonic complexes, as exemplified by the minority of JBTS patients who also have NPHP and retinitis pigmentosa (Figure 7c)910. The phenotypic diversity caused by transition zone dysfunction may relate to whether mutations abrogate or spare ciliogenesis, in addition to disrupting ciliary membrane composition. The former may result in MKS, COACH or JBTS, while the latter may cause SLSN or NPHP.

In BBS, BBSome disruption also leads to ciliary composition defects, raising the question of how BBSome and Tectonic complexes are functionally connected16,18. Interestingly, AC3 entrance into cilia is BBSome-independent16,18, but Tectonic-dependent. Hence, the BBSome and Tectonic complexes may control ciliary localization of different membrane proteins. Moreover, BBSome subunits do not interact with Tctn1 and do not localize to the transition zone, indicating that BBSome and Tectonic complexes operate at different suborganellar locations1617. The BBSome acts as a coat that traffics membrane proteins to the cilium, and is itself an IFT cargo, suggesting that BBSome-associated proteins enter the cilium aboard IFT trains16,44. If so, such trains need to pass the transition zone station, which might be the site of a crossing gate mediated by the Tectonic complex.

The transition zone is best known in Chlamydomonas, whose Cep290 ortholog controls flagellar protein content7,45. The similarity of Chlamydomonas Cep290 and vertebrate Tectonic complex mutants suggests that the ciliary functions of the Tectonic complex are conserved deeply through evolution. Accordingly, Tmem67 and Cc2d2a orthologs are found in all Tectonic-possessing eukaryotes for which genomic information exists, and these Tectonic complex components were likely to be possessed by the eukaryotic cenancestor (Figure S8). Other transition zone components, such as Cep290, are not present in the genomes of many ciliated unicellular organisms, or even all bilaterians, suggesting that their roles in the complex are dispensable for some ciliary functions1.

In summary, we have identified a transition zone complex of MKS and JBTS proteins that regulates ciliogenesis and ciliary membrane composition. The identification of physical interactions and functional similarities between Tctn1 and other complex components suggests that the transition zone is compromised in human MKS and JBTS. In support of this conclusion, mutations in human TCTN1 are a rare cause of JBTS.



Notes

FN2SUPPLEMENTAL INFORMATION

Supplemental information includes eight figures and one table.

FN3AUTHOR CONTRIBUTIONS

F.R.G-G. and K.C.C. performed most experiments and wrote the manuscript. M.S.S did most electron microscopy, supervised by J.M.G-V. Human genetics were supervised by F.H. and N.K. and performed by G.R., E.A.O., D.J.J., C.L.B. and J.F.Robinson. T.R.N performed the gel filtration chromatography. A.D.S. helped with mouse experiments. J.F.Reiter wrote the manuscript and supervised the work.

The authors thank the IRM and Nikon Imaging Centers at UCSF. The Servei Central de Suport a la Investigació Experimental and Mario Soriano-Navarro assisted with EM. Members of the Reiter lab provided valuable discussions. Kathryn Anderson, David Beier, Joseph Gleeson, Colin Johnson, Hemant Khanna, Robert Molday, Kirk Mykytyn Gregory Pazour, Suzie Scales, James Sillibourne, and Stefan Somlo provided antibodies or plasmids. T.R.N. is supported by an NSF predoctoral grant and an NIGMS-IMSD grant (R25-GM56847). F.H. is an Investigator of the Howard Hughes Medical Institute, a Doris Duke Distinguished Clinical Scientist and a Frederick G. L. Huetwell Professor. This work was funded by grants from the National Institutes of Health to F.H. (DK1069274, DK1068306, RC4-K090917) and to J.F.Reiter (AR054396), and from the March of Dimes, the Burroughs Wellcome Fund, the Packard Foundation, and the Sandler Family Supporting Foundation to J.F.Reiter.


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