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 ciliogenesis28–29
. 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
are associated with JBTS and COACH syndromes9–10
. 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 (). Consistent with this model, mouse Tctn1
null mutants display phenotypes resembling human MKS, whereas Tmem67
mutants have a less severe phenotype, suggesting partial loss of Tectonic complex function25
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 ()9–10
. 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 locations16–17
. 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
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.