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Mol Biol Cell. Dec 2008; 19(12): 5338–5346.
PMCID: PMC2592648

Sentan: A Novel Specific Component of the Apical Structure of Vertebrate Motile Cilia

Francis A. Barr, Monitoring Editor

Abstract

Human respiratory and oviductal cilia have specific apical structures characterized by a narrowed distal portion and a ciliary crown. These structures are conserved among vertebrates that have air respiration systems; however, the molecular components of these structures have not been defined, and their functions are unknown. To identify the molecular component(s) of the cilia apical structure, we screened EST libraries to identify gene(s) that are exclusively expressed in ciliated tissues, are transcriptionally up-regulated during in vitro ciliogenesis, and are not expressed in testis (because sperm flagella have no such apical structures). One of the identified gene products, named sentan, was localized to the distal tip region of motile cilia. Using anti-sentan polyclonal antibodies and electron microscopy, sentan was shown to localize exclusively to the bridging structure between the cell membrane and peripheral singlet microtubules, which specifically exists in the narrowed distal portion of cilia. Exogenously expressed sentan showed affinity for the membrane protrusions, and a protein–lipid binding assay revealed that sentan bound to phosphatidylserine. These findings suggest that sentan is the first molecular component of the ciliary tip to bridge the cell membrane and peripheral singlet microtubules, making the distal portion of the cilia narrow and stiff to allow for better airway clearance or ovum transport.

INTRODUCTION

Motile cilia and flagella are membrane-bound, microtubule-containing cell surface projections that are differentiated by their length, movement characteristics, and numbers per cell (Haimo and Rosenbaum, 1981 blue right-pointing triangle; Mitchell, 2007 blue right-pointing triangle; Satir and Christensen, 2007 blue right-pointing triangle). Flagella-containing cells generally have only one or two flagella, which are long motile structures that beat independently and exhibit an undulating motion. Conversely, cilia are generally much more numerous and are shorter motile projections with an oscillating to-and-fro motion. In humans, flagella are observed only in sperm cells, whereas motile cilia are observed on the respiratory epithelium, along the female reproductive tract, and on the ependymal cells lining the ventricles of the brain.

Early studies using electron microscopy showed that the terminal end of mammalian motile cilia is quite distinct from that of sperm flagella (see Figure 6). At the terminal end of mammalian sperm flagella, peripheral doublet microtubules lose the outer B subfibers or separate into two singlet microtubules. Each singlet microtubule approaches the cell membrane, becomes electron dense and terminates successively. Only a few peripheral singlet subfibers continue to the tip of the sperm tail (Woolley and Nickels, 1985 blue right-pointing triangle; Afzelius et al., 1995 blue right-pointing triangle; Suzuki and Nagano, 2002 blue right-pointing triangle). In contrast, the apical structure of mammalian cilia has a distinct fine structure at the termination site of the axonemal microtubules. The outer B subfibers of peripheral doublet microtubules terminate at the distal portion of cilia, and only the singlet A subfibers continue to the tip. The cell membrane and lateral surface of the singlet A subfibers are connected by filamentous structures that Foliguet and Puchelle (1986) blue right-pointing triangle called “lateral spokes.” The peripheral A subfibers terminate in an electron-dense capping structure, together with a central pair of singlet microtubules. The capping structure is connected with the cell membrane, and claw-like structures called ciliary crowns are elongated at the surface of the ciliary tip. These specialized capping structures and narrowed distal portions have been reported in not only tracheal and oviductal cilia of various mammals but also chicken tracheal cilia and frog palate cilia (Dirksen and Satir, 1972 blue right-pointing triangle; Kuhn and Engleman, 1978 blue right-pointing triangle; Dentler and LeCluyse, 1982 blue right-pointing triangle; Dalen, 1983 blue right-pointing triangle; LeCluyse and Dentler, 1984 blue right-pointing triangle; Foliguet and Puchelle, 1986 blue right-pointing triangle). These evolutionarily conserved apical structures are considered to make the distal portion stiff to provide better propulsion, promote debris clearance from the trachea, and support ovum transport along the oviduct. To date, many ciliogenesis-related genes have been identified via intensive proteomic studies (Ostrowski et al., 2002 blue right-pointing triangle), comparative genomics studies (Li et al., 2004 blue right-pointing triangle; Baron et al., 2007 blue right-pointing triangle), and genome-wide transcriptional analyses of flagellar regeneration or mucocilliary differentiation (Stolc et al., 2005 blue right-pointing triangle; Hayes et al., 2007 blue right-pointing triangle; Ross et al., 2007 blue right-pointing triangle). Nevertheless, the molecular components of these specific apical structures have not been identified.

Figure 6.
Schematic of the cilia tip structure. Microtubule termination differs between the sperm end piece (A) and the tracheal or oviductal cilia tip (B). Longitudinal sections are represented on the left and transverse sections, on the right (Woolley and Nickels, ...

We hypothesize that the specialized tip structural protein(s) of cilia are not expressed in flagellated cells such as sperm. In this study focused on ciliogenesis-related genes that are not regulated in the testis, we isolated the first component of the specific apical structure of vertebrate tracheal and oviductal cilia. Based on its exclusive localization to the distal tip region of cilia, we have named the protein “sentan,” which means “tip” in Japanese.

MATERIALS AND METHODS

Digital Differential Display

Digital differential display (http://www.ncbi.nlm.nih.gov/UniGene/ddd.cgi) was performed on the NCBI UniGene website. In all, 17 EST libraries (190,220 ESTs) of multiple-ciliated epithelia-containing tissues originating from lung, uterus, and trachea were compared with 31 libraries (123,804 ESTs) of simple epithelia-containing tissues originating from colon, liver, kidney, stomach, and intestine or with 62 libraries (250,757 ESTs) originating from those same tissues and brain.

Phylogenetic Tree

The amino acid alignment of sentan was constructed using the ClustalW multiple alignment program and the Boxshade program. The UPGMA phylogenetic tree for sentan was constructed using PROTDIST and NEIGHBOR programs contained in the Phylogeny Inference Package (PHYLIP, ver. 3.6a; Felsenstein, 2003 blue right-pointing triangle). The following amino acid sequences were used: cow, Bos taurus: NP_001071606; horse, Equus caballus: ENSECAP00000008198; dog, Canis familiaris: ENSCAFP00000010240; human, Homo sapiens: ENSP00000341442; chimpanzee, Pan troglodytes: ENSPTRP00000026032; mouse, Mus musculus: ENSMUSP00000062092; rat, Rattus norvegicus: NP_001102568; opossum, Monodelphis domestica: ENSMODP00000003611; chicken, Gallus gallus: ENSGALP00000011789; anole, Anolis carolinensis: the amino acid sequence was assembled using the reverse strand of the genomic sequence of the scaffold_315: 1476029-0475890 and 1473763-1473611 from the A. carolinensis draft assembly that was available in February 2007 in the BLAT system of the University of California, Santa Cruz (UCSC); and frog, Xenopus tropicalis: the X. tropicalis amino acid sequence was assembled using the reverse strand of the genomic sequence of the scaffold_369: 439038-438998, 437962-437823, and 431447-431289 from the X. tropicalis whole genome shotgun assembly version 4.1 that was available in August 2005 in the BLAT system of UCSC.

Constructs of Mouse Sentan and Adenovirus Preparation

The EST clone IMAGp998J0313722Q, which contains the full-length mouse sentan [amino acid (aa) 1-147] cDNA, was purchased from RZPD (Berlin, Germany). Mouse sentan (aa 2-147) was tagged with hemagglutinin (HA)-peptide at the NH2-terminus according to a previously described method (Yuba-Kubo et al., 2005 blue right-pointing triangle). To construct an expression vector for HA-sentan (pCAG-HA-sentan), an NheI site was introduced at the start codon of the sentan cDNA by PCR, and the PCR fragment and an adapter DNA encoding an HA peptide were introduced into pCAGGS-neodelEcoRI (Niwa et al., 1991 blue right-pointing triangle), provided by Dr. J. Miyazaki (Osaka University). To make an adenovirus for expressing HA-sentan, the HA-sentan cDNA was subcloned into pShuttle-CMV vector (Stratagene, La Jolla, CA). Recombinant adenovirus containing HA-sentan (rAd-HA-sentan) was then generated by incorporating the expression cassette into pAdEasy-1 vector (Stratagene) according to the manufacturer's instructions (Stratagene). A control adenovirus (rAd-Ctrl) with no transgene was also constructed. The recombinant adenovirus was propagated in AD-293 cells according to the manufacturer's instructions (Stratagene).

Animals

C57BL/6J mice and Japanese white rabbits were purchased from Japan Clea (Tokyo, Japan) and Shimizu Laboratory Supplies (Kyoto, Japan), respectively. For the primary culture of mouse tracheal epithelial cells (MTECs) for the immunofluorescence studies of mouse tissues and for the mRNA and protein preparation from mouse tissues, C57BL/6J mice were killed by intraperitoneal injection of pentobarbital sodium (Nembutal; Dainippon Sumitomo Pharma, Tokyo, Japan). All animal protocols were approved by the animal ethics review board of Keio University (Tokyo, Japan) and Kyoto University (Kyoto, Japan) and conformed to National Institutes of Health guidelines.

Generation of Polyclonal Antibodies

The cDNA encoding amino acids 1-147 of mouse sentan was subcloned into pGEX 5X-1 (Amersham Pharmacia Biotech, Piscataway, NJ) to produce a fusion protein with glutathione S-transferase (GST). The GST fusion protein was expressed in Escherichia coli, purified using glutathione Sepharose 4B columns (Amersham Pharmacia Biotech; Smith and Johnson, 1988 blue right-pointing triangle), and used as an antigen to generate polyclonal antibodies (pAb) in rabbits. Rabbit serum was affinity-purified on a PVDF membrane containing the fusion protein.

Cell Culture and Transfection

Human HeLa cells were cultured in DMEM (Invitrogen-BRL, Rockville, MD) supplemented with 10% fetal calf serum. Human hTERT-RPE-1 retinal pigment epithelial cells were purchased from BD Biosciences Clontech (Palo Alto, CA) and cultured in DMEM/Ham's F12 (Invitrogen-BRL) supplemented with 10% fetal calf serum. Primary cilia were induced by culturing hTERT-RPE-1 cells in medium with 0.25% serum for 48 h. Cultured HeLa and hTERT-RPE-1 cells were transfected with expression vectors by using LipofectAmine2000 (Invitrogen-BRL, Gaithersburg, MD) according to the manufacturer's instructions. C57BL/6J MTECs were harvested from isolated tracheas and grown in primary cultures on support membranes (Transwell Clear; Corning-Costar, Corning, NY) as described previously (You et al., 2002 blue right-pointing triangle). When the cells were confluent and the transmembrane resistance increased to more than 1000 Ω/cm2, the medium was removed from the upper chamber to establish an air-liquid interface (ALI). The ciliogenesis induction medium was DMEM/Ham's F12 (1:1) supplemented with 2% NuSerum (Becton-Dickinson, Bedford, MA). For gene transfer to MTECs, the medium was removed from the upper chamber 5 d after initiating the culture (just before ALI day 0). The support membranes were turned upside-down, and rAd-HA-sentan or rAd-Ctrl was delivered for 2 h through the basal surface of the support membrane.

Real-Time PCR

Total RNA was prepared from adult C57BL/6J mouse tissue and cultured MTECs at ALI days 0, 4, 8, and 16 using a RNeasy mini-kit and QIAshredder according to the manufacturer's instructions (QIAGEN, Valencia, CA). The cDNA were reverse-transcribed from total RNA and used as a template for quantitative real-time PCR analysis in duplicate, as described previously (Matsui et al., 2004 blue right-pointing triangle). Primers were designed to be compatible with a single real-time PCR thermal profile (95°C for 15 min; 40 cycles of 95°C for 15 s, and 60°C for 1 min), such that multiple transcripts could be analyzed simultaneously. All data were normalized to an internal standard [glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA, ΔCt method, User Bulletin 2, Applied Biosystems]. Primer sets were as follows: mouse sentan (NM_177624) forward (5′-AAATGCTTCTGACCCTGATGGTAAAC-3′) and reverse (5′-ATTTTGGCTTGGTTTCTTCTCTCTCTG-3′); mouse PCM-1 (AB029291) forward (5′-TGCCACAGTCAGTAATTCAGAAGAAAC-3′) and reverse (5′-GGGACGGCAGAAACATCACTTATAG-3′); mouse centrin4 (NM_145825) forward (5′-AAAGTTGAACTGAATGACACCCAGAAG-3′) and reverse (5′-GCCCTCATTGCAATCTTTAGTTCTTTC-3′); and mouse GAPDH (NM008084) forward (5′-AAGGTGGTGAAGCAGGCATCTGAG-3′) and reverse (5′-GGAAGAGTGGGAGTTGCTGTTGAAGTC-3′).

Immunoblotting

Trachea and testes were dissected from adult C57BL/6J mice, minced, homogenized in SDS sample buffer (Laemmli, 1970 blue right-pointing triangle), and boiled for 10 min. HeLa cells were mechanically collected from culture dishes, minced in PBS, homogenized in SDS sample buffer, and boiled for 5 min. Denatured proteins in the boiled samples were separated by SDS-PAGE (5–20% precast gradient gels; Daiichi Kagaku, Tokyo, Japan) and transferred onto Immobilon transfer membranes (Millipore, Bedford, MA). Membranes were then incubated with rabbit anti-sentan pAb or mouse anti-α-tubulin mAb (DM1A; Sigma, St. Louis, MO). Bound antibodies were detected with HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG (Amersham), respectively, and visualized using chemiluminescence (Western Lightning Chemiluminescent Reagent Plus; Perkin Elmer-Cetus, Boston, MA).

Immunofluorescence Microscopy

Fluorescein isothiocyanate (FITC)-conjugated mouse anti-α-tubulin mAb (FITC-DM1A) and mouse anti-acetylated tubulin mAb were purchased from Sigma. Mouse anti-β-tubulin IV mAb, rat anti-HA mAb (3F10), and Texas Red-X-phalloidin were purchased from BioGenex (San Ramon, CA), Roche (Basel, Switzerland), and Molecular Probes (Eugene, OR), respectively. MTECs cultured on Transwell membranes (Corning) were fixed with methanol for 5 min at −20°C, washed in PBS, incubated with 0.12% glycine/PBS for 20 min, and then processed for immunofluorescence microscopy as described previously (Kubo et al., 1999 blue right-pointing triangle). Mouse tissues were dissected and fixed in 3.7% formaldehyde/PBS for 30 min. Samples were mounted in Tissue-Tek and frozen using liquid nitrogen. Frozen sections, ~12 μm thick, were cut on a cryostat, mounted on poly-l-lysine–coated glass coverslips, air-dried, and soaked in PBS containing 1% Triton X-100 for 10 min. The slides were then rinsed in PBS and processed for immunofluorescence microscopy as described previously (Kubo et al., 1999 blue right-pointing triangle). HeLa cells and hTERT-RPE-1 cells grown on poly-l-lysine–coated coverslips were fixed with Zamboni's fixative (Stefanini et al., 1967 blue right-pointing triangle) for 2 h, permeabilized with 0.05% saponin/PBS for 30 min, and then processed for immunofluorescence microscopy as described previously (Kubo et al., 1999 blue right-pointing triangle). Alexa Fluor 488–conjugated anti-mouse IgG or anti-rat IgG and Alexa Fluor 594–conjugated anti-rabbit IgG were purchased from Molecular Probes and used as secondary antibodies. After washing with PBS, the samples were mounted in Mowiol (Calbiochem) and observed under a DeltaVision optical sectioning microscope (Applied Precision Instruments, Issaquah, WA). Whole-cell images were obtained at 0.2-μm intervals in z section, deconvolved, and integrated with DeltaVision software (Applied Precision).

Electron Microscopy

For ultrathin-section electron microscopy, mouse tracheas were fixed in 0.1 M cacodylate buffer containing 0.5% Triton X-100 and 4% paraformaldehyde for 10 min and processed as described previously (Kubo et al., 1999 blue right-pointing triangle). Goat anti-rabbit IgG coupled to 10-nm gold particles (Nycomed Amersham, Westbury, NY) was used as a secondary antibody. Samples were examined with an electron microscope (JEM 1010; JEOL, Peabody, MA) at an accelerating voltage of 100 kV.

Protein–Lipid Overlay Assay

Protein binding to phospholipids was investigated in a protein–lipid overlay assay using PIP Strips and a membrane lipid array (Echelon Biosciences, Salt Lake City, UT) according to the manufacturer's protocol. In brief, the purified GST-sentan fusion protein was incubated with the membrane for 1 h at room temperature. After intensive washing of the membrane, proteins were detected by incubation with a 1:1000 dilution of anti-GST mouse mAb (GST 3–4C, Zymed, San Francisco, CA), followed by incubation with a 1:10,000 dilution of HRP-conjugated anti-rat IgG (Amersham). Immunoreactive proteins were detected by chemiluminescence (Western Lightning Chemiluminescent Reagent Plus; Perkin Elmer-Cetus).

RESULTS

Identification of Sentan

To identify cilia tip component candidate(s), we first performed digital differential display (http://www.ncbi.nlm.nih.gov/UniGene/ddd.cgi) to compare EST libraries from human multiple-ciliated epithelia-containing tissues and simple epithelia-containing tissues. Trachea, lung, and oviduct-containing uterus were selected for ciliated epithelia-containing tissues. Tissues of the gastrointestinal tract, kidney, and liver, or these tissues plus brain tissue were selected for simple epithelia-containing tissues. As every group includes ESTs derived from primary cilium-bearing epithelial, fibroblastic, mesenchymal, or neuronal cells, we expected motile cilia-specific genes to be isolated. A number of genes were overrepresented in ciliated epithelia-containing tissues (Supplemental Table S1). Among the 96 genes identified, those with the highest enrichment were Bardet-Biedl syndrome 1 (BBS1), Tektin1, and EF-hand domain C-terminal containing 1 (EFHC1); these genes have been experimentally identified as cilia-related genes (Larsson et al., 2000 blue right-pointing triangle; Ikeda et al., 2005 blue right-pointing triangle; Badano et al., 2006 blue right-pointing triangle; Marshall, 2008 blue right-pointing triangle). Next, we examined the tissue expression profiles of these candidates using the EST database. We identified one uncharacterized gene, A430083B19Rik, as being expressed in ciliated epithelia-containing tissues and not in testis tissue, which contained mRNAs of sperm cells whose flagella have no specific tip structures. The protein encoded by A430083B19Rik was a predicted cytoplasmic protein of 147 amino acids with a calculated molecular mass of 16.4 kDa. It comprised conserved S-100–related EF-hand domains (cd00213 of the NCBI conserved domain database) and a hydrophobic domain at its carboxy-terminus (Figure 1A). The genomic database search and phylogenetic analysis revealed that the protein is conserved among tetrapods such as Xenopus, anole, chicken, and various mammals, including both eutherians and metatherians, but does not exist in the genomes of fish, invertebrates, or microorganisms (Figure 1B and Supplemental Figure S1). The protein thus appears to be a unique variant of S-100–related protein that is conserved among vertebrates that have air respiration systems. We named the protein “sentan.” Quantitative real-time PCR analysis using various mouse tissues confirmed the ciliated-tissue-specific expression of sentan (Figure 1C).

Figure 1.
Gene and protein profiles of sentan. (A) Predicted domain structure of mouse sentan protein. (B) Phylogenetic tree of sentan calculated with the program UPGMA, using homologous regions of each species with the corresponding residues of amino acids 49-147 ...

Sentan Is Up-Regulated during Ciliogenesis

Next, we analyzed sentan gene expression during ciliogenesis. Primary MTECs were cultured on a membrane until confluent, and the membrane was transferred to an ALI to induce ciliogenesis. Cultured cells were processed for immunofluorescence studies or harvested for total RNA extraction just before ALI day 0 and on ALI days 4, 8, and 16. On ALI day 0, no cilia were observed at the surface of any cultured cells. The induction of pericentriolar material-1 (PCM-1) protein, a molecular component of fibrous granules that appears in the earliest phases of ciliogenesis (Kubo et al., 1999 blue right-pointing triangle), was observed in a group of cells within 4 d after ALI transfer. Cilia were observed on ALI day 8, and many mature ciliated cells were observed on ALI day 16 (data not shown). The gene expression of PCM-1 and forkhead box J1 (FoxJ1), an f-box transcription factor essential for ciliogenesis (You et al., 2004 blue right-pointing triangle), markedly increased on ALI day 4 and was maintained at a high level throughout the ALI culture period (Figure 1D). In contrast, the expression of centrin4, a structural protein of the basal body expressed specifically in ciliated cells (Gavet et al., 2003 blue right-pointing triangle), gradually increased and exhibited its highest expression level on ALI day 16 (Figure 1D). The expression of sentan was not observed before ALI transfer and was gradually up-regulated during in vitro ciliogenesis, similar to the differentiation-specific gene centrin4 (Figure 1D).

Sentan Is Localized at the Ciliary Tip

We next analyzed the subcellular localization of sentan in ciliated epithelial cells. Full-length cDNA encoding mouse sentan was cloned by PCR using the EST clone IMAGp998J0313722Q as a template. HA-tagged sentan was exogenously expressed in cultured primary MTECs via adenovirus-mediated gene transfer. On day 16, the cultured ALI cells were doubly stained with anti-HA rat mAb and anti-acetylated tubulin mouse mAb (Figure 2A). The acetylated tubulin signal was detected throughout the cilia structure, whereas the HA-tagged sentan signal was concentrated exclusively on the tip of each cilium. We then raised a pAb against recombinant mouse sentan produced in E. coli. This pAb recognized a ~15-kDa band in total lysate of mouse trachea but not in the lysate from testis (Figure 2B). The pAb was also immunoreactive with other bands observed in both the trachea and testis. Judging from the calculated molecular mass of mouse sentan (16.4 kDa) and from the reactivity of this pAb with recombinant mouse sentan produced in E. coli and with HA-tagged sentan produced in HeLa cells, we concluded that this pAb recognizes mouse sentan.

Figure 2.
Identification of sentan as a ciliary tip protein. (A) HA-sentan expressed via adenovirus localized to the tip of cilia in cultured primary mouse tracheal cells. Cells were doubly stained with anti-HA rat mAb (green) and anti-acetylated tubulin mouse ...

Next, to examine the subcellular localization of sentan by immunofluorescence microscopy, freshly prepared mouse trachea was immunostained with the anti-sentan pAb and a mAb to βIV-tubulin, a cilia-specific tubulin subtype (Renthal et al., 1993 blue right-pointing triangle), in a whole-mount manner (Figure 2C). The sentan pAb was immunoreactive with protein found exclusively at the tip of each cilium. Taken together with the cilia tip localization of the exogenously expressed HA-tagged sentan protein, we concluded that this pAb specifically recognizes sentan.

Sentan Is Exclusively Expressed in Ciliated Epithelial Cells

Mouse tracheas from 16-d-old embryos were subjected to immunofluorescence microscopy and showed evidence of ciliogenesis. In the ciliogenetic cells, sentan appeared to localize to the tip of normal- and short-length cilia (Figure 3A). However, sentan did not appear in the very short cilia (Figure 3B), indicating that it localizes to the tip of motile cilia in the growing phase. Next, we investigated sentan localization in other types of cilia. In the multiple-ciliated cells of the oviduct, which have cilia with the same morphology as tracheal cilia, sentan was again observed at the cilial tip (Figure 3C). In contrast, the expression of sentan could not be detected in testis by real-time PCR (Figure 1C), and no specific signal of sentan was detected in the distal portion of sperm flagella (data not shown). To examine the presence or absence of sentan in primary cilia, mouse kidney, and cultured human hTERT-RPE cells were stained with anti-sentan pAb and anti-acetylated tubulin mAb (Figure 3, D and E). No specific sentan signal was detected in primary cilia. These observations indicated that sentan is a component of the specific apical structure of tracheal and oviductal motile cilia.

Figure 3.
Sentan expression in various types of cilia. (A and B) Sentan localization in the growing cilia of mouse fetal trachea. Vertical sections of trachea were triply stained with anti-sentan pAb (green pseudocolor), FITC-conjugated anti-α-tubulin mAb ...

Sentan Localizes between the Cell Membrane and Peripheral A-Subfibers

We next investigated the subcellular localization of sentan using immunoelectron microscopy. When the apical structure was examined in Triton X-100–treated tracheal epithelial cells by immunoelectron microscopy, anti-sentan pAb was seen to specifically label the membrane side of the singlet peripheral A-tubules, at the distal narrowed portion of the axoneme (Figure 4A). No sentan signal was detected on the A-tubules where they form doublets with B-subfibers. The longitudinal section of the tip showed no sentan label at the electron-dense capping structure. The sentan signal began to be detected on the membrane side of the A-tubules, next to the microtubule plug-in site of the capping structure, in both the transverse and longitudinal sections (Figure 4, A and B).

Figure 4.
Subcellular localization of sentan at the tip of mouse tracheal cilia. Mouse trachea was treated with 4% paraformaldehyde containing 0.5% Triton X-100 and labeled with anti-sentan pAb. (A) Transverse sections of cilia demonstrating 9 doublets + 2 and ...

Affinity of Sentan for the Cell Membrane

These observations indicated that sentan is a component of the bridging structure between the cilia membrane and peripheral singlet microtubules. We next investigated the affinity of sentan for microtubules or the cell membrane. When sentan was exogenously overexpressed in hTERT cells, sentan showed a dotted signal along the axoneme and some concentration to the apical portion of the primary cilia, but no exclusive localization to the tip was observed (Figure 5A). When sentan was overexpressed in HeLa cells, it was localized exclusively to the peripheral membrane protrusions (Figure 5B). Coimmunostaining with actin filaments and microtubules showed that these sentan-localized protrusions were actin-based filopodia and did not include microtubules. No sentan colocalization was observed with the actin stress fibers or with microtubules in the cytoplasm (Figure 5B and data not shown).

Figure 5.
Affinity of sentan for membrane protrusions and phospholipids. (A) Exogenously overexpressed sentan localization in hTERT-RPE cells. Cells were doubly stained with anti-sentan pAb (green) and anti-acetylated tubulin mAb (red). The boxed area is enlarged ...

We next performed a protein–lipid overlay assay to specify the membrane lipid affinity of sentan. GST-fused sentan showed strong binding with phosphatidylserine and very weak binding with phosphatidylinositol-3-monophosphate and phosphatidylglycerol sphingomyelin; GST did not produce any signal in this assay (Figure 5C).

DISCUSSION

Tracheal cilia are thought to play a crucial role in the clearance of mucus and foreign particles from the respiratory tract; disorders of ciliary movement cause immotile cilia syndrome, characterized by chronic sinusitis and bronchiectasis (Ibañez-Tallon et al., 2003 blue right-pointing triangle; Eley et al., 2005 blue right-pointing triangle; Zariwala et al., 2007 blue right-pointing triangle). In patients with immotile cilia syndrome, there is also increased incidence of both female infertility and ectopic pregnancy, because the cilia lining the oviduct normally transport the ovum toward the uterus (Ibañez-Tallon et al., 2003 blue right-pointing triangle; Eley et al., 2005 blue right-pointing triangle). Electron micrographs showed that the tips of mammalian tracheal and oviductal cilia have some structural features that are not observed in mammalian sperm flagella or in cilia and flagella of other organisms (Figure 6, A and B). To date, no molecular component of the apical cilial structure has been identified, although a 97-kDa kinetochore antigen was suggested to localize to the microtubule capping structure (Miller et al., 1990 blue right-pointing triangle).

In this study, we identified sentan as the first molecular component of the apical structure of tracheal and oviductal cilia. Sentan localized exclusively between the ciliary membrane and the peripheral singlet microtubules at the distal narrowed portion of cilia, suggesting that sentan is a molecular component of the linker structure that bridges the ciliary membrane and the peripheral singlet microtubules (Figure 6B; called “lateral spokes” in Foliguet and Puchelle, 1986 blue right-pointing triangle). According to the genomic database, sentan is found in most mammals, including eutherians and metatherians, and in birds, reptiles, and amphibians. However, the genomic database does not suggest its presence in invertebrates or fish such as Tetraodon nigroviridis, Takifugu rubripes, Danio rerio, and Oryzias latipes. Consistent with in silico analyses, a narrowed distal portion has been observed in cilia in the trachea of various mammals, in chickens, in the palate of frogs, and in the ciliated grooves of frogs between the choanae (nasal openings) and the esophageal opening (Kuhn and Engleman, 1978 blue right-pointing triangle; Dentler and LeCluyse, 1982 blue right-pointing triangle; LeCluyse and Dentler, 1984 blue right-pointing triangle; Foliguet and Puchelle, 1986 blue right-pointing triangle); however, it has not been observed in fish or invertebrates (Dentler, 1980 blue right-pointing triangle). We speculate that sentan is indispensable for the morphogenesis of the narrowed distal portion of cilia and that sentan and this specific structure appeared early in the evolution of air respiratory systems. These structures are most likely widely conserved among tetrapods because of the need to clear mucus and debris from the airway. We propose naming the sentan-positive distal narrowed portion of cilia the “sentan domain” and the sentan-positive microtubule-membrane bridges “A-tubule–membrane linkers” (Figure 6B).

Early electron microscopy studies of growing cilia of the frog palate showed that a disk-shaped, electron-dense plate forms at the tip of cilia during the first micron of growth; the maturation of the capping structure is complete in cilia longer than 2 μm (Portman et al., 1987 blue right-pointing triangle). In human cilia, the peripheral singlet microtubules and the sentan domain are observed when cilia are longer than 3 μm (Foliguet and Puchelle, 1986 blue right-pointing triangle). The formation of the capping structure suggests that only the singlet A-tubules are capped and possibly stabilized, making it reasonable to hypothesize that the sentan domain is formed after the formation of the capping structure. In agreement with this conjecture, sentan begins to localize to the tip only after cilia of the fetal trachea reach ~3 μm in length. These results indicate that the formation of the sentan domain with peripheral singlet microtubules and the recruitment of sentan occur simultaneously.

Overexpressed sentan localized to cell peripheral protrusions in hTERT-RPE cells; however, a dotted signal along the axoneme and some concentration at the apical portion of primary cilia were observed. This dotted pattern suggests that exogenous sentan is transported on intraflagellar transport particles and, as a result, concentrates at the apical portion of cilia (Pazour and Rosenbaum, 2002 blue right-pointing triangle; Cole, 2005 blue right-pointing triangle; Sloboda, 2005 blue right-pointing triangle; Follit et al., 2006 blue right-pointing triangle; Hou et al., 2007 blue right-pointing triangle; Omori et al., 2008 blue right-pointing triangle; Scholey, 2008 blue right-pointing triangle). The dotted pattern and the fact that only a very small portion of exogenously expressed sentan localized to primary cilia also indicated that sentan is not recruited to the spaces between the cell membrane and peripheral doublet microtubules in primary cilia or in mature or growing tracheal cilia. When sentan was overexpressed in HeLa cells, it clearly localized to actin-related membrane protrusions such as filopodia. As sentan did not colocalize with actin stress fibers and because an immunoprecipitation assay of HA-sentan in HeLa cells failed to detect any cosedimentation proteins, including actin (unpublished data), sentan localization to the membrane protrusion appears to be independent of actin fibers. The strong affinity of sentan for phosphatidylserine, one of the major inner leaflet phospholipids, indicates that sentan may localize to the protrusion by direct binding to the cell membrane.

Why does sentan localize only around the peripheral singlet microtubules and not around the peripheral doublet microtubules in motile cilia? One hypothesis is that the cell membrane affinity of sentan is dependent on the curvature of the membrane; as a result, sentan localization to the protrusions could be dependent on their diameter. Interestingly, the diameter of the distal portion of cilia is almost equivalent to the diameter of HeLa cell protrusions: 150 and ~120–130 nm, respectively (Fisher and Cooper, 1967 blue right-pointing triangle; Porter et al., 1974 blue right-pointing triangle; Foliguet and Puchelle, 1986 blue right-pointing triangle; Hosaka et al., 1993 blue right-pointing triangle). These diameters are smaller than that of cilia axoneme (~250–300 nm) and larger than that of microvilli (~80 nm) present on the apical surface of tracheal epithelial cells (Simionescu and Simionescu, 1976 blue right-pointing triangle; Dalen, 1983 blue right-pointing triangle); neither of these recruited adenovirus-overexpressed or native sentan, which supports the idea that sentan localization is dependent on the curvature of the membrane. Another hypothesis is that sentan localization is dependent on specifically modified tubulin found in peripheral singlet microtubules. Axonemal tubulin has been reported to harbor multiple posttranslational modifications (e.g., polyglutamylation by tubulin tyrosine ligase-like proteins or monomeric or polymeric glycylation by unknown enzymes; Redeker et al., 1994 blue right-pointing triangle; Million et al., 1999 blue right-pointing triangle; Ikegami et al., 2006 blue right-pointing triangle; Pathak et al., 2007 blue right-pointing triangle), and ciliary tip tubulin has been shown to be excluded from monomeric glycylation (Dossou et al., 2007 blue right-pointing triangle). It is possible that specifically modified singlet peripheral microtubules recruit sentan via direct binding or via binding with specific microtubule-binding proteins. The elucidation of other components associated with singlet microtubules in the sentan domain of cilia could provide an understanding of the interplay between sentan and singlet peripheral microtubules.

In conclusion, we identified sentan as the first molecular component of the apical structure of vertebrate tracheal and oviductal cilia. Sentan is also the first protein to be identified as linking ciliary microtubules to the ciliary membrane in mammalian tissue. The identification of sentan provides valuable information regarding the molecular structure of the characteristic sentan domain of vertebrate motile cilia and is an important step in understanding the contribution of the sentan domain to cilia function. Further detailed analysis of the sentan domain of cilia and the sentan molecule should help elucidate the molecular mechanism of ciliogenesis and the evolution of air respiration systems.

Supplementary Material

[Supplemental Materials]

ACKNOWLEDGMENTS

We thank all of the members of our laboratories (Department of Cell Biology, Faculty of Medicine, Kyoto University and Department of Dermatology, Keio University School of Medicine) for helpful discussions. We also thank Hiromi Doi for her excellent technical assistance. This manuscript is dedicated to the memory of Professor Shoichiro Tsukita. This work was supported in part by a Grant-in-Aid for Young Scientists (B) to A.K. from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and Keio University Research Grants for Life Science and Medicine to A.K. from the Keio University Medical Science Fund.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-07-0691) on October 1, 2008.

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