TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome

Journal name:
Nature Cell Biology
Volume:
18,
Pages:
122–131
Year published:
DOI:
doi:10.1038/ncb3273
Received
Accepted
Published online

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel–Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome1 (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral–facial–digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.

At a glance

Figures

  1. A weighted coexpression approach to discover TZ genes identifies TMEM107 as a TZ protein.
    Figure 1: A weighted coexpression approach to discover TZ genes identifies TMEM107 as a TZ protein.

    (a) Frequency histogram of binned human gene coexpression scores, derived from weighted analyses of gene expression data sets using a training set of 20 known TZ genes (Supplementary Table 1). Frequencies normalized to compare different distributions. SYSCILIA gold standard genes21 in yellow; TZ gene training in blue; all other genes in grey hatched. Box plots show median and quartiles for histogram distributions. Whiskers (hashed lines) denote the minimum and maximum extent of the data set. (b) Recall performance (also known as sensitivity) of the coexpression approach retrieves known TZ (blue lines) and ciliary (yellow lines) genes. The graph shows that TZ genes can be preferentially retrieved compared with ciliary genes. Inset: recall performance for top 200 ranked genes. Ciliary genes taken from the SYSCILIA gold standard21. (c,dC. elegans TMEM-107::GFP localizes at the TZ. Shown are fluorescence images from worms expressing TMEM-107::GFP alone (c) or together with an ARL-13::tdTomato reporter (d). (c) Accumulation of TMEM-107::GFP at the ciliary base region of 12 bilateral amphid cilia (amp; brackets), labial and CEP cilia (subset denoted by arrowheads), bilateral phasmid cilia (arrows) and the right-sided PQR cilium (asterisk) in the tail. Note that the head schematic shows only a subset of the hermaphrodites ciliated head neurons. (d) TMEM-107::GFP localizes immediately proximal to middle segment (ms)-restricted ARL-13::tdTomato. Image shows all four phasmid cilia (left and right). Schematic denotes major subcompartments in phasmid cilia with microtubule doublets (only two shown) in the TZ and middle segments, and microtubule singlets in the distal segment (ds). Den, dendrite. Scale bars, 2μm (left two images), 1μm (right images). (e) Human TMEM107 localizes at the TZ. Shown are images of hTERT-RPE1 cells stably expressing GFP-tagged human TMEM107 (green) at a low level, co-stained with antibodies for ciliary axonemes (polyglutamylated tubulin, PolyGluTub) and the TZ (RPGRIP1L, TMEM67). Scale bars, 5μm.

  2. TMEM107 regulates mammalian ciliogenesis and is mutated in OFDVI and JBTS individuals.
    Figure 2: TMEM107 regulates mammalian ciliogenesis and is mutated in OFDVI and JBTS individuals.

    (a) IMCD3 cells transfected with Tmem107 siRNA possess reduced Tmem107 mRNA expression (versus scrambled siRNA control; qPCR data) and reduced mean ciliary frequency. Data represent mean ± s.e.m. (n = 350 cells, 1 experiment). P < 0.05 (unpaired t-test; versus control). (b) When grown in three-dimensional culture, IMCD3 cells transfected with Tmem107 siRNA form spheroids with a reduced mean size. Cilia (orange) stained for acetylated α-tubulin; cell junctions (green) stained for beta-catenin. Data represent mean ± s.e.m. (n = 25 spheroids pooled from 2 independent experiments). P < 0.05 (unpaired t-test; versus control). Scale bar, 5μm. (c) Schematic of the human TMEM107 protein showing the position of identified patient mutations. Grey rectangles correspond to the transmembrane domains. Mat, maternal; Pat, paternal; NA, not available. (d) Brain magnetic resonance imaging (MRI) images (axial views) showing the molar tooth sign, linked to elongated, thick and mal-oriented superior peduncles (white arrows) and hypoplastic vermis. (e) Brain MRI showing a dysplastic and highly hypoplastic vermis in sagittal view. A secondary enlargement of the fourth ventricle with displacement of the fastigium is also evident. (f) Brain MRI (axial view) showing heterotopias, enlarged lateral ventricles and polymicrogyria. (g) Brain MRI (sagittal view) showing enlarged posterior fossa (asterisk) with a cystic dilation of the fourth ventricle, a severe midbrain dysplasia and a thin corpus callosum with enlarged ventricles. (h) Fibroblasts derived from skin biopsies of healthy control (wild type, WT) and patient 3 (JBTS) immunostained for cilia using antibodies against ARL13B (red; ciliary membrane) and acetylated tubulin (white; axonemal microtubules). (i,j) Compared with control cells, JBTS cell cilia possess increased lengths (i) and reduced frequencies (j). Data represent mean ± s.e.m. (n = 10 (i) and 25 (j) cells; data represent 1 of 3 independent experiments). P < 0.05, P < 0.01 (unpaired t-test; versus WT); scale bars, 5μm.

  3. C. elegans tmem-107 controls diffusion barrier integrity and functions with nphp-4 to regulate ciliary and TZ structural integrity.
    Figure 3: C. elegans tmem-107 controls diffusion barrier integrity and functions with nphp-4 to regulate ciliary and TZ structural integrity.

    (a) Schematic of oq100 indel mutation in the tmem-107 gene. Exons denoted by grey rectangles (numbers show nucleotide positions). del., deletion; ins., insertion. (b) oq100 mutation disrupts TMEM-107 expression. Shown are amphid cilia TZs in worms expressing GFP-tagged wild-type or mutant (oq100) TMEM-107. Scale bar, 2μm (images identically scaled). (c) Dye-filling assay (measure of cilium integrity for 6 amphid (head) and 2 phasmid (tail) ciliated neurons) showing dye-filling defects (Dyf) in tmem-107(oq100);nphp-4(tm925) double mutants, but not single mutants, or a tmem-107(oq100);mkrs-1(tm3083) double mutant. The Dyf phenotype is rescued by expression of wild-type tmem-107 (GFP-tagged; see Fig. 1c, d). Scale bars, 10μm. (d) Images of ASER neuronal cilia from worms expressing a gcy-5p::gfp that stains the ASER neuron. Numbers refer to cilium length measurements; mean ± s.e.m. (n = 28 (N2), 44 (tmem-107), 46 (nphp-4) and 81 (tmem-107;nphp-4) cilia). Brackets denote ciliary axonemes (cil). Arrowhead indicates occasional break in GFP staining observed only in double mutant. den; dendrite. P < 0.01 (unpaired t-test; versus WT), P = 0.01 (unpaired t-test; versus nphp-4); scale bars, 3μm. (etmem-107(oq100);nphp-4(tm925) double mutants possess defects in cilia-related behaviours. Shown are population assays of isoamyl alcohol (IAA) attraction and single-worm foraging assays. Data represent mean ± s.e.m. For IAA assays, n = 30 (N2), 20 (tmem-107), 22 (nphp-4) and 29 (tmem-107;nphp-4); for foraging assays, n = 44 (N2), 43 (tmem-107), 63 (nphp-4), 54 (tmem-107;nphp-4) and 37 (tmem-107;nphp-4;Ex[tmem-107(wt)]) independent experiments, respectively; P < 0.01 (unpaired t-test; versus WT), P < 0.01 (unpaired t-test; versus tmem-107;nphp-4). CI, chemotaxis index. (f) TZ composition is altered in tmem-107(oq100);nphp-4 (tm925) double mutants. Shown are phasmid cilia from worms expressing TZ-localized MKS-2::GFP and periciliary membrane-localized, TRAM-1::tdTomato (asterisk). Scale bars, 2μm. (g) tmem-107(oq100);nphp-4(tm925) double mutants possess short phasmid (PHA/B) dendrites and misplaced cilia. Neurons stained with OSM-6(IFT52)::GFP. cil, ciliary axonemes; den, dendrite; cb, cell bodies (also denoted by asterisks). Brackets denote PHA/B cilia. Scale bars, 5μm. (h) TZ membrane diffusion barrier is selectively disrupted in tmem-107(oq100) mutants. Shown are phasmid cilia from worms expressing TRAM-1::tdTomato (and MKS-2::GFP; marks TZ) (left images) or RPI-2::GFP (and XBX-1::tdTomato; marks cilia) (right images). TRAM-1 (translocon subunit) and RPI-2 (retinitis pigmentosa 2) are excluded from wild-type (WT) cilia, whereas TRAM-1 (but not RPI-2) leaks into tmem-107(oq100) cilia. Asterisk, TZ localization of MKS-2; pcm, periciliary membrane; cil, ciliary axoneme. Scale bars, 2μm.

  4. Evolutionarily conserved association of TMEM107 with the TZ-localized MKS module.
    Figure 4: Evolutionarily conserved association of TMEM107 with the TZ-localized MKS module.

    (a) Phasmid TZ localizations of GFP-tagged MKS and NPHP module proteins in WT and tmem-107(oq100) mutant worms, and TMEM-107::GFP in MKS and NPHP mutants. Scale bar, 1μm (all images similarly scaled). mis-loc., mislocalized. (b) Schematic summarizing TZ localization dependencies in a. TMEM-107 positioned at an intermediate level within a hierarchical three-layer (L1-3) MKS module assembly model (drawn on the basis of refs 10,13,15,19; MKS-1 ‘unassigned because hierarchical analysis has not yet been conducted using an mks-1 null allele). Human orthologues denoted in brackets. (c) Expression of TMEM-107::RFP with disrupted cytosolic N or C termini (nTMEM-107, cTMEM-107; see Methods) rescues mislocalized TMEM-17::GFP and TMEM-231::GFP in tmem-107(oq100) mutants. Shown are phasmid cilia TZs. Scale bars, 0.5μm. (d) Tmem107 depletion (siRNA) in IMCD3 cells disrupts relative localizations of endogenous MKS module proteins. Cells double-stained as indicated and co-localization determined as an Rtotal Pearson correlation value (FIJI ‘Co-localization Threshold plug-in). In Tmem107-depleted cells, Rpgrip1l localization is unaffected (relative to basal body (BB) γ-tubulin), whereas Tmem231 and Tmem237 proteins shift (black arrows) relative to γ-tubulin or Rpgrip1l. Data in graph represent mean ± s.e.m. (n = 150 cells pooled from 3 independent experiments). Scr siRNA, siRNA scrambled control. P < 0.01, P < 0.05 (unpaired t-test; versus Scr siRNA control). Scale bar, 1μm. (e) Co-immunoprecipitation (coIP) assays in IMCD3 cells. Upper panels, lanes 1–4: input material from whole cell extracts (WCEs) transfected with the indicated constructs and immunoblotted (IB) with anti-GFP or anti-FLAG. Lanes 5–8: proteins immunoprecipitated (IP) by an irrelevant antibody (irr. Ab; anti-MICU3) or anti-GFP, and then immunoblotted for FLAG or GFP. IgG heavy chain (HC) and light chain (LC) in co-immunoprecipitates are indicated. Asterisks () mark nonspecific proteins. Lower panels, lanes 9–12: input WCE showing expression of FLAG–TMEM231, FLAG–TMEM17 and c myc–MKS1. Lanes 13–21: IPs with antibodies against MKS1 (lane 14), TMEM231 (231; lane 17) and TMEM17 (17; lane 20) and then immunoblotted as indicated. Note that although TMEM107 co-immunoprecipitates TMEM231, TMEM231 does not co-immunoprecipitate detectable levels of TMEM107. Unprocessed original scans of blots are shown in Supplementary Fig. 6. (f) Co-evolution relationships between MKS components using differential Dollo parsimony that counts along a phylogenetic tree how often two genes are lost independently from each other. Thickness and colour gradient indicate strong co-evolution. Edges with differential Dollo parsimony scores >11 are not shown. Dashed rectangle: co-evolving MKS submodule.

  5. Anchoring and periodic distributions of MKS module proteins within the TZ.
    Figure 5: Anchoring and periodic distributions of MKS module proteins within the TZ.

    (a) GFP-tagged TMEM-107, MKS-2 and MKS-6 are immobile within the C. elegans TZ. Shown are FRAP curves and representative time-lapse images after photobleaching one half of a TZ signal (outlined region). Data points represented as mean ± s.e.m. (n = 3 (MKS-6) or 4 (TMEM-107, MKS-2) independent experiments). Scale bars, 500nm. (bC. elegans MKS-2 immobility depends on MKS module proteins. Shown is a FRAP curve and representative time-lapse images (phasmid cilia) after photobleaching MKS-2::GFP signals (boxed region) in an mksr-1 mutant. Asterisk, periciliary membrane. Data points represented as mean ± s.e.m. (n = 4 independent experiments). a.u., arbitrary units; scale bar, 2μm. (c) STED super-resolution microscopy images of C. elegans MKS and NPHP module protein (all GFP-tagged) distributions in transition zones. Shown are single focal plane images of TZs in axial orientation (from the side) taken from head (amphid, labial) and tail (phasmid) ciliated neurons. Note the smaller size of the labial cilium TZ. Schematics indicate ring-like or spiral-like domains formed by the C. elegans MKS/NPHP module proteins. Arrowheads show independent signal clusters within a ring-like domain. Scale bars, 200nm (high-magnification images), 500nm (low-magnification images). (d) STED images of endogenous human RPGRIP1L and TMEM67 in renal RPTEC cells showing clusters (arrowheads) of protein in a single ring of differing diameters (mean ± s.d.) at the TZ. Corresponding confocal images co-stained for cilia with polyglutamylated tubulin antibody. P = 0.001 (unpaired t-test; versus TMEM67). Scale bars, 100nm. (e) dSTORM of human RPGRIP1L (visualized with Alexa Fluor 647) with 10nm binning, image smoothing and contrast enhancement in FIJI (raw images shown in Supplementary Fig. 5d), showing periodic localization (arrowheads) in a loose ring at the TZ. Image depth-coded by colour. Z-axis scale bar (nm) on right. Scale bar, 100nm. (f) Models. MKS module proteins (and C. elegans NPHP-1) occupy periodic radial and axial TZ subdomains. Mammalian RPGRIP1L and TMEM67 localize as independent clusters, forming a single ring domain at the TZ core (RPGRIP1L) or membrane (TMEM67). C. elegans MKS and NPHP proteins also localize as discrete independent clusters, forming multiple ring domains (or possible spiral domains) along the TZ length. The nematode axial distribution may correspond to the ciliary necklace (TEM example from ref. 12). Periodicity and immobility of MKS module proteins suggests association with Y-links, which form extended sheets in C. elegans (Supplementary Video 1) and are implicated in necklace formation.

  6. Phylogenetic and bioinformatics screening data of candidate TZ genes.
    Supplementary Fig. 1: Phylogenetic and bioinformatics screening data of candidate TZ genes.

    (a) Frequency histogram of binned mouse gene co-expression scores, derived from weighted analyses of gene expression datasets using a training set of 20 known TZ genes (Supplementary Table 1). This graph is the equivalent of the human gene co-expression dataset presented in Fig. 1a. Frequencies normalised to compare different distributions. Grey hatched; all human genes, yellow; ciliary genes in the SysCilia gold standard21, blue; TZ gene training set. Box-plots display median and quartiles for histogram distributions. (b) Presence and absence of candidate and known TZ genes in 52 eukaryotic species. The presence of orthologues for the 20 TZ training set genes and the five candidate TZ genes were determined by bi-directional best hits using BLAST and PSI-BLAST, as well as custom built hidden Markov models, HHPred, and intermediate sequence searches using PSI-BLAST and TBLASTN. Species are ordered according to their phylogenetic relationship as shown by the phylogenetic tree at the top. The top row indicates which species possess cilia or flagella. Grey columns indicate species lacking a (canonical) TZ. Ciliated species that have lost MKS genes appear to lack well defined Y-shaped linkers22. (c) Model of the four transmembrane helix topology of human TMEM107. Predicted transmembrane regions for TMEM107 and three known TZ proteins (TMEM216, TMEM138, and TMEM17) using TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM). Alignment of TMEM107 sequences to the homologous TMEM216, TMEM138, and TMEM17 suggests TMEM107 is homologous to these three TZ proteins (not shown). (d) To model the transmembrane helices we used a standard existing helix obtained from the PDB. We swapped the amino acid side chains one by one using YASARA. The transmembrane topology of TMEM107 was predicted with TMHMM2.0. Helices are ordered anti-clockwise, starting with helix 1 in the right-rear, (bottom to top), helix 2 at the left-rear (top to bottom), helix 3 at the front-left (bottom to top) and helix 4 at the front-right (top to bottom). On the right side the four helices are depicted from a downwards viewpoint. The evolutionary conserved, charged residues (in red, a histidine and an arginine in helix 1, a glutamate in helix 2, a histidine in helix 3 and a glutamate in helix 4) are at the same height in the four helices, suggesting interactions, and therefore a four helix bundle model of the proteins transmembrane structure. The conserved non-charged residues are in cyan. The mouse Schlei (E125G) mutation (the human equivalent is E131G)23 and the human F106Del and L134Ffs mutations found in this study are indicated by arrows. E45G lies within the extracellular loop between helix 1 and helix 2 and is not depicted here.

  7. Expression and localisation analyses of C. elegans and human TMEM107 constructs (wild type and variants).
    Supplementary Fig. 2: Expression and localisation analyses of C. elegans and human TMEM107 constructs (wild type and variants).

    (a) C. elegans tmem-107 is expressed exclusively in ciliated sensory neurons. Shown are fluorescence images of worms expressing a transcriptional tmem-107p::GFP reporter (P). DiI costain identifies a subset of ciliated neurons, namely 6 amphid cells (ADL, ASH, ASJ, ASK, AWB and ASI (not shown)) and both phasmid cells (PHA/B). Bars; 25μm (large whole worm panels), 6μm (small head and tail panels). den; dendrites, cil; cilia. (b) Schematics showing candidate X-box sequences in the promoters of human and nematode TMEM107. (c) DAF-19 RFX transcription factor is required for TMEM-107::GFP expression in C. elegans. Shown are head (left panels) and tail (right panels) regions of N2 (wild-type) and daf-19(m86);daf-12(sa204) double mutant worms expressing a translational tmem-107::gfp transgene (see Fig. 1c). Bars; 6μm. (d) Analysis of TMEM-107 (wild type and variants) localisation in C. elegans. Shown are fluorescence images of the amphid and phasmid TZ regions (see also bottom schematic) in worms expressing various GFP tagged (C-terminus) TMEM-107 proteins. Top schematic shows the predicted topology of the tetraspan TMEM-107 C. elegans protein and indicates the disrupted domains and sequences. Linker 1 replacement sequence taken from SNG-1, and linker 2 and 3 replacement sequences taken from SPE-38 (see Methods section for further details). The coloured residues denote amino acids mutated in the TMEM107 patients (see Methods section for descriptions). TZ; transition zone. Bars; 1μm. (e) Analysis of human TMEM107 patient variant protein localisation. Images of hTERT-RPE1 cells expressing GFP-tagged human TMEM107(E45G) or TMEM107(F106del), costained with antibodies for ciliary axonemes (acetylated tubulin; AcTub) and basal bodies (pericentrin). Bars; 10μm.

  8. Sequencing details for the three cases of mutated TMEM107 and clinical details of TMEM107 patient phenotypes.
    Supplementary Fig. 3: Sequencing details for the three cases of mutated TMEM107 and clinical details of TMEM107 patient phenotypes.

    Integrative genomics viewer data showing: (a) compound heterozygous TMEM107 mutations in case 3 consisting of one frameshift deletion (NM_032354.3: g.8077560delT; p.Leu134Phefs8) and one in-frame deletion (NM_032354.3: g.8077890_ 8077893delGAA; p.Phe106del), and (b) homozygous TMEM107 missense variant (NM_183065: g.8079298T > C; p.Glu45Gly) in cases 1 and 2. Clinical details of the three TMEM107-mutated cases are presented in (c), leading to OFDVI and JBTS diagnoses. Cases 1 and 2 had previously been reported40.

  9. Effect of tmem-107 mutations on cilium ultrastructure and function.
    Supplementary Fig. 4: Effect of tmem-107 mutations on cilium ultrastructure and function.

    (a) Cilium ultrastructure is highly disrupted in tmem-107;nphp-4 double mutants. Low (large panels) and high (small panels) magnification TEM images of cilia from serial cross sections taken from the distal (1), middle (2) and proximal (3) regions of the amphid pore (position of section in pore denoted by numbers in schematic). Wild-type pores consist of 10 ciliary axonemes (only three shown in schematics for simplicity), each consisting of a distal segment (DS; singlet A microtubules), a middle segment (MS; doublet A/B microtubules), a transition zone (TZ; with membrane-microtubule connecting Y-links) and a periciliary membrane compartment (PCMC). In tmem-107(oq100);nphp-4(tm925) double mutants (also harbours the him-5(e1490) mutation linked to nphp-4), multiple axonemes are missing in the middle and distal pore regions, TZ Y-links (Ys) are reduced or missing, and vesicles frequently accumulate in the TZ and PCMC regions. Also, the majority of double mutant TZs are partially or fully disconnected (undocked) from the ciliary membrane, extending from ectopic anterior positions within the PCMC. In contrast, most or all of the ciliary axonemes are present in tmem-107(oq100) and nphp-4 single mutants. However, nphp-4 worms carrying the tm925 deletion (with or without him-5(e1490)) or the gk529336 nonsense mutation show consistent defects in Y-link integrity and TZs are undocked in two neurons (ADF, ADL). Images are representative of at least 4 analysed amphid pores for all strains except nphp-4(tm925) and nphp-4(gk529336) where 2 pores were analysed. Bars; 200nm (low magnification images), 100nm (high magnification images). (b) Compendium of TZ images and associated schematics showing the TZ defects outlined above in (a). Bars; 100nm. (c) Dye filling assay (DiI) of tmem-107(oq100);nphp-4(tm925) worms transgenically expressing various GFP-tagged TMEM-107 constructs (wild type, E46G, F96del, L120G). Shown are fluorescence images of the head region. Non-transgenic worms are strongly dye-filling defective, whereas dye filling is restored in worms expressing TMEM-107 constructs (wild type or mutant versions). Bars; 10μm.

  10. Supplementary FRAP and super resolution imaging data.
    Supplementary Fig. 5: Supplementary FRAP and super resolution imaging data.

    (a) FRAP curve and representative time lapse images following quenching of 100% of MKS-2::GFP and TMEM-107::GFP signals at the TZ (boxed region shows the bleached TZ of an amphid channel cilium). Data points represented as mean ± s.d.n = 3 (MKS-2::GFP) or 4 (TMEM-107::GFP) independent experiments. Bar; 500nm. (b) Raw and deconvolved (decon.) STED and confocal images of C. elegans MKS and NPHP module proteins (GFP-tagged). Bars; 500nm. (c) Raw and deconvolved (decon.) STED and confocal images of renal RPTEC cells stained for polyglutamylated tubulin (ciliary axonemes; red; confocal only) and either endogenous human RPGRIP1L or TMEM67 (green; confocal and STED). STED imaging reveals that RPGRIP1L and TMEM67 form clusters of discrete signals arranged as a hollow ring at the TZ. Bars; 500nm. (d) Super-resolution dSTORM microscopy of RPGRIP1L in the ciliary transition zone of human hTERT-RPE1 cells. The loose, tilted ring TZ organisation of RPGRIP1L shown in Fig. 5e (i) and examples from additional cells (ii-iv). dSTORM image reconstruction used 10nm histogram bins, Gaussian image smoothing in the palm3d reconstruction output and contrast enhancement in FIJI. Dashed circles and ovals circumscribe TZ localisations which form the identified hollow loose ring structure, with discrete clusters of protein denoted by white arrowheads. Localisation density at individual points on the ring varied between samples, and was highest in (i). The distribution of signals in iv deviates significantly from an oval and could represent a partial spiral or helical arrangement. In some images (I, ii), some signal appears to enter the ciliary axoneme (ax) distal to the TZ. Images depth-coded by colour, with the z axis scale bar in nm indicated on the right. Representative bright-field and epifluoresence images from cells stained for RPGRIP1L and TMEM67, with the transition zone acquired and reconstructed in (iv) indicated by the white arrow. Red arrowheads indicate fiducials. Scale bars; 100nm (dSTORM images; all images identically scaled), 10μm (bright-field and epifluorescence images).

  11. Uncropped scans of western blots shown in Fig. 4e.
    Supplementary Fig. 6: Uncropped scans of western blots shown in Fig. 4e.

    Red boxes denote the cropped regions shown in Fig. 4e.

Videos

  1. Electron Tomogram of the C. elegans TZ.
    Video 1: Electron Tomogram of the C. elegans TZ.
    Reconstruction derived from a 200nm section of a C. elegans amphid channel ciliary TZ. Arrow denotes a Y-link density throughout the tomogram, indicating that the Y-link structures are continuous sheets along the axial plane. Bar; 100nm.

References

  1. Reiter, J., Blacque, O. & Leroux, M. The base of the cilium: roles for transition fibres and the transition zone in ciliary formation, maintenance and compartmentalization. EMBO Rep. 13, 608618 (2012).
  2. Goetz, S. C. & Anderson, K. V. The primary cilium: a signalling centre during vertebrate development. Nat. Rev. Genet. 11, 331344 (2010).
  3. Blacque, O. E. & Sanders, A. A. Compartments within a compartment: what C. elegans can tell us about ciliary subdomain composition, biogenesis, function, and disease. Organogenesis 10, 126137 (2014).
  4. Hsiao, Y. C., Tuz, K. & Ferland, R. J. Trafficking in and to the primary cilium. Cilia 1, 4 (2012).
  5. Chih, B. et al. A ciliopathy complex at the transition zone protects the cilia as a privileged membrane domain. Nat. Cell Biol. 14, 6172 (2011).
  6. Craige, B. et al. CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content. J. Cell Biol. 190, 927940 (2010).
  7. Garcia-Gonzalo, F. R. et al. A transition zone complex regulates mammalian ciliogenesis and ciliary membrane composition. Nat. Genet. 43, 776784 (2011).
  8. Hu, Q. et al. A septin diffusion barrier at the base of the primary cilium maintains ciliary membrane protein distribution. Science 329, 436439 (2010).
  9. Kee, H. L. et al. A size-exclusion permeability barrier and nucleoporins characterize a ciliary pore complex that regulates transport into cilia. Nat. Cell Biol. 14, 431437 (2012).
  10. Williams, C. L. et al. MKS and NPHP modules cooperate to establish basal body/transition zone membrane associations and ciliary gate function during ciliogenesis. J. Cell Biol. 192, 10231041 (2011).
  11. Gilula, N. B. & Satir, P. The ciliary necklace. A ciliary membrane specialization. J. Cell Biol. 53, 494509 (1972).
  12. Heller, R. F. & Gordon, R. E. Chronic effects of nitrogen dioxide on cilia in hamster bronchioles. Exp. Lung Res. 10, 137152 (1986).
  13. Roberson, E. C. et al. TMEM231, mutated in orofaciodigital and Meckel syndromes, organizes the ciliary transition zone. J. Cell Biol. 209, 129142 (2015).
  14. Cevik, S. et al. Active transport and diffusion barriers restrict Joubert syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain. PLoS Genet. 9, e1003977 (2013).
  15. Huang, L. et al. TMEM237 is mutated in individuals with a Joubert syndrome related disorder and expands the role of the TMEM family at the ciliary transition zone. Am. J. Hum. Genet. 89, 713730 (2011).
  16. Jauregui, A. R., Nguyen, K. C., Hall, D. H. & Barr, M. M. The Caenorhabditis elegans nephrocystins act as global modifiers of cilium structure. J. Cell Biol. 180, 973988 (2008).
  17. Williams, C. L., Winkelbauer, M. E., Schafer, J. C., Michaud, E. J. & Yoder, B. K. Functional redundancy of the B9 proteins and nephrocystins in Caenorhabditis elegans ciliogenesis. Mol. Biol. Cell 19, 21542168 (2008).
  18. Schouteden, C., Serwas, D., Palfy, M. & Dammermann, A. The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans. J. Cell Biol. 210, 3544 (2015).
  19. Jensen, V. L. et al. Formation of the transition zone by Mks5/Rpgrip1L establishes a ciliary zone of exclusion (CIZE) that compartmentalises ciliary signalling proteins and controls PIP2 ciliary abundance. EMBO J. 34, 25372556 (2015).
  20. Baughman, J. M. et al. A computational screen for regulators of oxidative phosphorylation implicates SLIRP in mitochondrial RNA homeostasis. PLoS Genet. 5, e1000590 (2009).
  21. van Dam, T. J., Wheway, G., Slaats, G. G., Huynen, M. A. & Giles, R. H. The SYSCILIA gold standard (SCGSv1) of known ciliary components and its applications within a systems biology consortium. Cilia 2, 7 (2013).
  22. Barker, A. R., Renzaglia, K. S., Fry, K. & Dawe, H. R. Bioinformatic analysis of ciliary transition zone proteins reveals insights into the evolution of ciliopathy networks. BMC Genomics 15, 531 (2014).
  23. Christopher, K. J., Wang, B., Kong, Y. & Weatherbee, S. D. Forward genetics uncovers Transmembrane protein 107 as a novel factor required for ciliogenesis and Sonic hedgehog signaling. Dev. Biol. 368, 382392 (2012).
  24. Giles, R. H., Ajzenberg, H. & Jackson, P. K. 3D spheroid model of mIMCD3 cells for studying ciliopathies and renal epithelial disorders. Nat. Protoc. 9, 27252731 (2014).
  25. Friedland, A. E. et al. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat. Methods 10, 741743 (2013).
  26. Starich, T. A. et al. Mutations affecting the chemosensory neurons of Caenorhabditis elegans. Genetics 139, 171188 (1995).
  27. Williams, C. L., Masyukova, S. V. & Yoder, B. K. Normal ciliogenesis requires synergy between the cystic kidney disease genes MKS-3 and NPHP-4. J. Am. Soc. Nephrol. 21, 782793 (2010).
  28. Valente, E. M. et al. Mutations in TMEM216 perturb ciliogenesis and cause Joubert, Meckel and related syndromes. Nat. Genet. 42, 619625 (2010).
  29. Kensche, P. R., van Noort, V., Dutilh, B. E. & Huynen, M. A. Practical and theoretical advances in predicting the function of a protein by its phylogenetic distribution. J. R. Soc. 5, 151170 (2008).
  30. Iglesias, A. et al. The usefulness of whole-exome sequencing in routine clinical practice. Genet. Med. 16, 922931 (2014).
  31. Shaheen, R. et al. Identification of a novel MKS locus defined by TMEM107 mutation. Hum. Mol. Genet. 24, 52115218 (2015).
  32. Nakada, C. et al. Accumulation of anchored proteins forms membrane diffusion barriers during neuronal polarization. Nat. Cell Biol. 5, 626632 (2003).
  33. Xu, K., Zhong, G. & Zhuang, X. Actin, spectrin, and associated proteins form a periodic cytoskeletal structure in axons. Science 339, 452456 (2013).
  34. van Dam, T. J. et al. Evolution of modular intraflagellar transport from a coatomer-like progenitor. Proc. Natl Acad. Sci. USA 110, 69436948 (2013).
  35. Sanders, A. A., Kennedy, J. & Blacque, O. E. Image analysis of Caenorhabditis elegans ciliary transition zone structure, ultrastructure, molecular composition, and function. Methods Cell Biol. 127, 323347 (2015).
  36. Hobert, O. PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. BioTechniques 32, 728730 (2002).
  37. Arts, H. H. et al. Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome. Nat. Genet. 39, 882888 (2007).
  38. Dawe, H. R. et al. Nesprin-2 interacts with meckelin and mediates ciliogenesis via remodelling of the actin cytoskeleton. J. Cell Sci. 122, 27162726 (2009).
  39. York, A. G., Ghitani, A., Vaziri, A., Davidson, M. W. & Shroff, H. Confined activation and subdiffractive localization enables whole-cell PALM with genetically expressed probes. Nat. Methods 8, 327333 (2011).

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Author information

  1. These authors contributed equally to this work.

    • Nils J. Lambacher,
    • Ange-Line Bruel &
    • Teunis J. P. van Dam

Affiliations

  1. School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland

    • Nils J. Lambacher,
    • Stefanie Kuhns,
    • Julie E. Kennedy,
    • Karl Gaff &
    • Oliver E. Blacque
  2. EA4271 GAD, Genetics of Development Abnormalities, Burgundy University, 21078 Dijon, France

    • Ange-Line Bruel,
    • Jean-Baptiste Rivière,
    • Laurence Faivre &
    • Christel Thauvin-Robinet
  3. Centre for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Geert Grooteplein 26-28, 6525 GA Nijmegen, Netherlands

    • Teunis J. P. van Dam,
    • Robin van der Lee &
    • Martijn A. Huynen
  4. Section of Ophthalmology and Neurosciences, Leeds Institute of Biomolecular & Clinical Sciences, University of Leeds, Leeds LS9 7TF, UK

    • Katarzyna Szymańska &
    • Colin A. Johnson
  5. Department of Nephrology and Hypertension, University Medical Center Utrecht, 3584 CX Utrecht, The Netherlands

    • Gisela G. Slaats,
    • Ka Man Wu &
    • Rachel H. Giles
  6. School of Biochemistry and Immunology, Microscopy Facility, Trinity Biomedical Sciences Institute, Trinity College Dublin, 152-160 Pearse Street, Dublin 2, Ireland

    • Gavin J. McManus
  7. Centre de référence des malformations et maladies congénitales du cervelet et Service de Génétique, APHP, Hôpital Trousseau, 75012 Paris, France

    • Lydie Burglen &
    • Diane Doummar
  8. INSERM U1141, 75019 Paris, France

    • Lydie Burglen
  9. FHU TRANSLAD, CHU Dijon, 21079 Dijon, France

    • Jean-Baptiste Rivière,
    • Laurence Faivre &
    • Christel Thauvin-Robinet
  10. INSERM UMR1163, Hôpital Necker-Enfants Malades, 75015 Paris, France

    • Tania Attié-Bitach &
    • Sophie Saunier
  11. Université Paris Descartes, Sorbonne Paris Cité, 75006 Paris, France

    • Tania Attié-Bitach &
    • Sophie Saunier
  12. Institut IMAGINE, 75015 Paris, France

    • Tania Attié-Bitach &
    • Sophie Saunier
  13. Département de Génétique, Hôpital Necker-Enfants Malades, AP-HP, 75015 Paris, France

    • Tania Attié-Bitach
  14. School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, UK

    • Alistair Curd &
    • Michelle Peckham

Contributions

N.J.L., J.E.K., K.G. and O.E.B. performed and interpreted experiments with C. elegans. T.J.P.v.D., R.v.d.L. and M.A.H. performed all bioinformatics analyses. A.-L.B., L.B., D.D., T.A.-B., S.S. and C.T.-R. collected and purified patient samples, performed exome sequencing and analysed sequencing data. N.J.L., S.K. and G.J.M. performed the STED imaging. A.C., M.P. and C.A.J. conducted the dSTORM imaging and processing. K.S., S.K., G.G.S., K.M.W. and R.H.G. conducted transfection and immunofluorescence microscopy in mammalian cells. K.S. and C.A.J. contributed the co-immunoprecipitation experiments. J.-B.R., L.F. and C.T.-R. diagnosed and referred patients. The co-corresponding authors shared supervision of the work. M.A.H. uncovered TMEM107 as a candidate ciliary gene, and directed the bioinformatics work. C.T.-R. collated JBTS and OFD patient samples, performed clinical characterization and directed the sequencing. O.E.B. directed research, analysed and collated data for the manuscript. O.E.B., M.A.H., R.H.G. and C.A.J. conceived and executed the study, and O.E.B., N.J.L., T.J.v.D. and M.A.H. wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Phylogenetic and bioinformatics screening data of candidate TZ genes. (831 KB)

    (a) Frequency histogram of binned mouse gene co-expression scores, derived from weighted analyses of gene expression datasets using a training set of 20 known TZ genes (Supplementary Table 1). This graph is the equivalent of the human gene co-expression dataset presented in Fig. 1a. Frequencies normalised to compare different distributions. Grey hatched; all human genes, yellow; ciliary genes in the SysCilia gold standard21, blue; TZ gene training set. Box-plots display median and quartiles for histogram distributions. (b) Presence and absence of candidate and known TZ genes in 52 eukaryotic species. The presence of orthologues for the 20 TZ training set genes and the five candidate TZ genes were determined by bi-directional best hits using BLAST and PSI-BLAST, as well as custom built hidden Markov models, HHPred, and intermediate sequence searches using PSI-BLAST and TBLASTN. Species are ordered according to their phylogenetic relationship as shown by the phylogenetic tree at the top. The top row indicates which species possess cilia or flagella. Grey columns indicate species lacking a (canonical) TZ. Ciliated species that have lost MKS genes appear to lack well defined Y-shaped linkers22. (c) Model of the four transmembrane helix topology of human TMEM107. Predicted transmembrane regions for TMEM107 and three known TZ proteins (TMEM216, TMEM138, and TMEM17) using TMHMM2.0 (http://www.cbs.dtu.dk/services/TMHMM). Alignment of TMEM107 sequences to the homologous TMEM216, TMEM138, and TMEM17 suggests TMEM107 is homologous to these three TZ proteins (not shown). (d) To model the transmembrane helices we used a standard existing helix obtained from the PDB. We swapped the amino acid side chains one by one using YASARA. The transmembrane topology of TMEM107 was predicted with TMHMM2.0. Helices are ordered anti-clockwise, starting with helix 1 in the right-rear, (bottom to top), helix 2 at the left-rear (top to bottom), helix 3 at the front-left (bottom to top) and helix 4 at the front-right (top to bottom). On the right side the four helices are depicted from a downwards viewpoint. The evolutionary conserved, charged residues (in red, a histidine and an arginine in helix 1, a glutamate in helix 2, a histidine in helix 3 and a glutamate in helix 4) are at the same height in the four helices, suggesting interactions, and therefore a four helix bundle model of the proteins transmembrane structure. The conserved non-charged residues are in cyan. The mouse Schlei (E125G) mutation (the human equivalent is E131G)23 and the human F106Del and L134Ffs mutations found in this study are indicated by arrows. E45G lies within the extracellular loop between helix 1 and helix 2 and is not depicted here.

  2. Supplementary Figure 2: Expression and localisation analyses of C. elegans and human TMEM107 constructs (wild type and variants). (601 KB)

    (a) C. elegans tmem-107 is expressed exclusively in ciliated sensory neurons. Shown are fluorescence images of worms expressing a transcriptional tmem-107p::GFP reporter (P). DiI costain identifies a subset of ciliated neurons, namely 6 amphid cells (ADL, ASH, ASJ, ASK, AWB and ASI (not shown)) and both phasmid cells (PHA/B). Bars; 25μm (large whole worm panels), 6μm (small head and tail panels). den; dendrites, cil; cilia. (b) Schematics showing candidate X-box sequences in the promoters of human and nematode TMEM107. (c) DAF-19 RFX transcription factor is required for TMEM-107::GFP expression in C. elegans. Shown are head (left panels) and tail (right panels) regions of N2 (wild-type) and daf-19(m86);daf-12(sa204) double mutant worms expressing a translational tmem-107::gfp transgene (see Fig. 1c). Bars; 6μm. (d) Analysis of TMEM-107 (wild type and variants) localisation in C. elegans. Shown are fluorescence images of the amphid and phasmid TZ regions (see also bottom schematic) in worms expressing various GFP tagged (C-terminus) TMEM-107 proteins. Top schematic shows the predicted topology of the tetraspan TMEM-107 C. elegans protein and indicates the disrupted domains and sequences. Linker 1 replacement sequence taken from SNG-1, and linker 2 and 3 replacement sequences taken from SPE-38 (see Methods section for further details). The coloured residues denote amino acids mutated in the TMEM107 patients (see Methods section for descriptions). TZ; transition zone. Bars; 1μm. (e) Analysis of human TMEM107 patient variant protein localisation. Images of hTERT-RPE1 cells expressing GFP-tagged human TMEM107(E45G) or TMEM107(F106del), costained with antibodies for ciliary axonemes (acetylated tubulin; AcTub) and basal bodies (pericentrin). Bars; 10μm.

  3. Supplementary Figure 3: Sequencing details for the three cases of mutated TMEM107 and clinical details of TMEM107 patient phenotypes. (514 KB)

    Integrative genomics viewer data showing: (a) compound heterozygous TMEM107 mutations in case 3 consisting of one frameshift deletion (NM_032354.3: g.8077560delT; p.Leu134Phefs8) and one in-frame deletion (NM_032354.3: g.8077890_ 8077893delGAA; p.Phe106del), and (b) homozygous TMEM107 missense variant (NM_183065: g.8079298T > C; p.Glu45Gly) in cases 1 and 2. Clinical details of the three TMEM107-mutated cases are presented in (c), leading to OFDVI and JBTS diagnoses. Cases 1 and 2 had previously been reported40.

  4. Supplementary Figure 4: Effect of tmem-107 mutations on cilium ultrastructure and function. (2,742 KB)

    (a) Cilium ultrastructure is highly disrupted in tmem-107;nphp-4 double mutants. Low (large panels) and high (small panels) magnification TEM images of cilia from serial cross sections taken from the distal (1), middle (2) and proximal (3) regions of the amphid pore (position of section in pore denoted by numbers in schematic). Wild-type pores consist of 10 ciliary axonemes (only three shown in schematics for simplicity), each consisting of a distal segment (DS; singlet A microtubules), a middle segment (MS; doublet A/B microtubules), a transition zone (TZ; with membrane-microtubule connecting Y-links) and a periciliary membrane compartment (PCMC). In tmem-107(oq100);nphp-4(tm925) double mutants (also harbours the him-5(e1490) mutation linked to nphp-4), multiple axonemes are missing in the middle and distal pore regions, TZ Y-links (Ys) are reduced or missing, and vesicles frequently accumulate in the TZ and PCMC regions. Also, the majority of double mutant TZs are partially or fully disconnected (undocked) from the ciliary membrane, extending from ectopic anterior positions within the PCMC. In contrast, most or all of the ciliary axonemes are present in tmem-107(oq100) and nphp-4 single mutants. However, nphp-4 worms carrying the tm925 deletion (with or without him-5(e1490)) or the gk529336 nonsense mutation show consistent defects in Y-link integrity and TZs are undocked in two neurons (ADF, ADL). Images are representative of at least 4 analysed amphid pores for all strains except nphp-4(tm925) and nphp-4(gk529336) where 2 pores were analysed. Bars; 200nm (low magnification images), 100nm (high magnification images). (b) Compendium of TZ images and associated schematics showing the TZ defects outlined above in (a). Bars; 100nm. (c) Dye filling assay (DiI) of tmem-107(oq100);nphp-4(tm925) worms transgenically expressing various GFP-tagged TMEM-107 constructs (wild type, E46G, F96del, L120G). Shown are fluorescence images of the head region. Non-transgenic worms are strongly dye-filling defective, whereas dye filling is restored in worms expressing TMEM-107 constructs (wild type or mutant versions). Bars; 10μm.

  5. Supplementary Figure 5: Supplementary FRAP and super resolution imaging data. (498 KB)

    (a) FRAP curve and representative time lapse images following quenching of 100% of MKS-2::GFP and TMEM-107::GFP signals at the TZ (boxed region shows the bleached TZ of an amphid channel cilium). Data points represented as mean ± s.d.n = 3 (MKS-2::GFP) or 4 (TMEM-107::GFP) independent experiments. Bar; 500nm. (b) Raw and deconvolved (decon.) STED and confocal images of C. elegans MKS and NPHP module proteins (GFP-tagged). Bars; 500nm. (c) Raw and deconvolved (decon.) STED and confocal images of renal RPTEC cells stained for polyglutamylated tubulin (ciliary axonemes; red; confocal only) and either endogenous human RPGRIP1L or TMEM67 (green; confocal and STED). STED imaging reveals that RPGRIP1L and TMEM67 form clusters of discrete signals arranged as a hollow ring at the TZ. Bars; 500nm. (d) Super-resolution dSTORM microscopy of RPGRIP1L in the ciliary transition zone of human hTERT-RPE1 cells. The loose, tilted ring TZ organisation of RPGRIP1L shown in Fig. 5e (i) and examples from additional cells (ii-iv). dSTORM image reconstruction used 10nm histogram bins, Gaussian image smoothing in the palm3d reconstruction output and contrast enhancement in FIJI. Dashed circles and ovals circumscribe TZ localisations which form the identified hollow loose ring structure, with discrete clusters of protein denoted by white arrowheads. Localisation density at individual points on the ring varied between samples, and was highest in (i). The distribution of signals in iv deviates significantly from an oval and could represent a partial spiral or helical arrangement. In some images (I, ii), some signal appears to enter the ciliary axoneme (ax) distal to the TZ. Images depth-coded by colour, with the z axis scale bar in nm indicated on the right. Representative bright-field and epifluoresence images from cells stained for RPGRIP1L and TMEM67, with the transition zone acquired and reconstructed in (iv) indicated by the white arrow. Red arrowheads indicate fiducials. Scale bars; 100nm (dSTORM images; all images identically scaled), 10μm (bright-field and epifluorescence images).

  6. Supplementary Figure 6: Uncropped scans of western blots shown in Fig. 4e. (254 KB)

    Red boxes denote the cropped regions shown in Fig. 4e.

Video

  1. Video 1: Electron Tomogram of the C. elegans TZ. (4.99 MB, Download)
    Reconstruction derived from a 200nm section of a C. elegans amphid channel ciliary TZ. Arrow denotes a Y-link density throughout the tomogram, indicating that the Y-link structures are continuous sheets along the axial plane. Bar; 100nm.

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