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

Abstract

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 syndrome (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.

Figure 1. A weighted co-expression approach to discover TZ genes identifies TMEM107 as a TZ protein.

(a) Frequency histogram of binned human gene co-expression scores, derived from weighted analyses of gene expression datasets using a training set of 20 known TZ genes (Supplementary Table 1). Frequencies normalised to compare different distributions. SYSCILIA gold standard genes in yellow; TZ gene training in blue; all other genes in grey hatched. Box-plots display median and quartiles for histogram distributions. Whiskers (hashed lines) denote the minimum and maximum extent of the dataset. (b) Recall performance (also known as sensitivity) of the co-expression approach retrieves known TZ (blue lines) and ciliary (yellow lines) genes. The graph shows that TZ genes can be retrieved compared to ciliary genes. Inset: recall performance for top 200 ranked genes. Ciliary genes taken from the SYSCILIA gold standard. (c) C. elegans TMEM-107::GFP localises at the TZ. Shown are fluorescence images from worms expressing TMEM-107::GFP alone (left panels) or together with an ARL-13::tdTomato reporter (right panels). Left panels; 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 (arrowheads) and the right-sided PQR cilium (asterisk) in the tail. Note that head schematic only shows a subset of the hermaphrodite’s ciliated head neurons. Right panels; TMEM-107::GFP localises 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. Bars; 2 μm (left two images), 1 μm (right images). (d) Human TMEM107 localises at the TZ. Shown are images of hTERT-RPE1 cells stably expressing GFP-tagged human TMEM107 (green) at a low level, costained with antibodies for ciliary axonemes (polyglutamylated tubulin; PolyGluTub) and the TZ (RPGRIP1L, TMEM67). Bars; 5 μm.

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 (vs scrambled siRNA control; qPCR data) and reduced mean ciliary frequency. Data represents mean ± S.E.M (n=350 cells, 1 experiment). *p<0.05 (unpaired t-test; vs control). (b) When grown in 3-D culture, IMCD3 cells transfected with Tmem107 siRNA form spheroids with a reduced mean size. Cilia (orange) stained for acetylated alpha-tubulin; cell junctions (green) stained for beta-catenin. Data represents mean ± S.E.M (n=25 spheroids 2 independent experiments). *p<0.05 (unpaired t-test; vs control). Bar; 5 μm. (c) Schematic of human TMEM107 protein showing the position of identified patient mutations. Grey boxes correspond to the transmembrane domains. Mat; maternal, Pat; paternal, NA; not available. (d) Brain MRIs (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-j) Shown in h are 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). Compared to control cells, JBTS cell cilia possess reduced lengths (i) and frequencies (j). Data represents mean ± S.E.M (n=10 (i) and 25 (j) cells pooled from 3 independent experiments).* p<0.05 (unpaired t-test; vs WT), ** p<0.01 (unpaired t-test; vs WT), bars; 5 μm.

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 tmem-107 gene. Exons denoted by grey boxes (numbers; 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. Bar; 2 μm (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. Dyf phenotype is rescued by expression of wild-type tmem-107 (GFP-tagged; see Figure 1c). Bars; 10 μm. (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; occasional break in GFP staining observed only in double mutant. den; dendrite. * p<0.01 (unpaired t-test; vs WT), ** p=0.01 (unpaired t-test; vs nphp-4), Bars; 3 μm. (e) tmem-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 represents 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)] * p<0.01 (unpaired t-test; vs WT), ** p<0.01 (unpaired t-test; vs tmem-107;nphp-4). CI; chemotaxis index. (f) TZ composition is altered in tmem-107;nphp-4 double mutants. Shown are phasmid cilia from worms expressing TZ-localised MKS-2::GFP and periciliary membrane-localised, TRAM-1::tdTomato (asterisk). Bars; 2 μm. (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. Bars; 5 μm. (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. Bars; 2 μm.

Figure 4. Evolutionary conserved association of TMEM107 with the TZ-localised MKS module.

(a) Phasmid TZ localisations 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. Bar; 1 μm (all images similarly scaled). mis-loc.; mislocalised. (b) Schematic summarising TZ localisation dependencies in (a). TMEM-107 positioned at an intermediate level within a hierarchical three layer (L1-3) MKS module assembly model (drawn based on refs, , , ; 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 mislocalised TMEM-17::GFP and TMEM-231::GFP in tmem-107(oq100) mutants. Shown are phasmid cilia TZs. Bar; 0.5 μm. (d) Tmem107 depletion (siRNA) in IMCD3 cells disrupts relative localisations of endogenous MKS module proteins. Cells double-stained as indicated and colocalisation determined as an R_total Pearson correlation value (FIJI “Colocalization Threshold” plugin). In Tmem107 depleted cells, Rpgrip1l localisation is unaffected (relative to basal body (BB) γ-tubulin), whereas Tmem231and Tmem237 proteins shift (black arrows) relative to γ-tubulin or Rpgrip1l. Data in graph represents mean ± S.E.M (n=150 cells pooled from 3 independent experiments). siScr; siRNA scrambled control. ** p<0.01, *p<0.05 (unpaired t-test; vs siScr control). Bar; 1 μm. (e) Coimmunoprecipitation (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 coIPs are indicated. Asterisks (*) mark non-specific 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-IP’s TMEM231, TMEM231 does not co-IP detectable levels of TMEM107. (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 color gradient indicate strong co-evolution. Edges with differential Dollo parsimony scores >11 are not shown. Dashed box: co-evolving MKS submodule.

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 fluorescence recovery after photobleaching (FRAP) curves and representative time-lapse images after photobleaching one half of a TZ signal (boxed region). Data points represented as mean ± S.E.M. (n=3 (MKS-6) or 4 (TMEM-107, MKS-2) independent experiments). Bar; 500 nm. (b) C. 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). au; arbitrary units, Bar; 2 μm. (c) Arrowheads; independent signal clusters within a ring-like domain. Bars; 200 nm (high magnification images), 500 nm (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; vs TMEM67). Bars; 100 nm. (e) dSTORM of human RPGRIP1L (visualised with AlexaFluor647) with 10 nm binning, image smoothing and contrast enhancement in FIJI (raw images shown in Supplementary Figure 5d), showing periodic localisation (arrowheads) in a loose ring at the TZ. Image depth-coded by colour. Z-axis scale bar (nm) on right. Bar; 100 nm. (f) Models. MKS module proteins (and C. elegans NPHP-1) occupy periodic radial and axial TZ subdomains. Mammalian RPGRIP1L and TMEM67 localise as independent clusters, forming a single ring domain at the TZ core (RPGRIP1L) or membrane (TMEM67). C. elegans MKS and NPHP proteins also localise 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 ref12). 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.