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Vol. 19, Issue 12, 5338-5346, December 2008
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*Department of Dermatology, Keio University School of Medicine, Tokyo 160-8582, Japan; Mitsubishi Kagaku Institute of Life Sciences, Tokyo 194-8511, Japan; Biological Science Laboratory, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871, Japan; and Department of Cell Biology, Kyoto University Faculty of Medicine, Kyoto 606-8501, Japan
Submitted July 7, 2008;
Revised August 11, 2008;
Accepted September 24, 2008
Monitoring Editor: Francis A. Barr
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Mitchell, 2007; Satir and Christensen, 2007). 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; Afzelius et al., 1995; Suzuki and Nagano, 2002). 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) 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; Kuhn and Engleman, 1978; Dentler and LeCluyse, 1982; Dalen, 1983; LeCluyse and Dentler, 1984; Foliguet and Puchelle, 1986). 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), comparative genomics studies (Li et al., 2004; Baron et al., 2007), and genome-wide transcriptional analyses of flagellar regeneration or mucocilliary differentiation (Stolc et al., 2005; Hayes et al., 2007; Ross et al., 2007). Nevertheless, the molecular components of these specific apical structures have not been identified.
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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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). 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). 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), 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), 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). When the cells were confluent and the transmembrane resistance increased to more than 1000 
/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). 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, 
Ct method, User Bulletin 2, Applied Biosystems]. Primer sets were as follows: mouse sentan (NM_177624
[GenBank]
) forward (5'-AAATGCTTCTGACCCTGATGGTAAAC-3') and reverse (5'-ATTTTGGCTTGGTTTCTTCTCTCTCTG-3'); mouse PCM-1 (AB029291
[GenBank]
) forward (5'-TGCCACAGTCAGTAATTCAGAAGAAAC-3') and reverse (5'-GGGACGGCAGAAACATCACTTATAG-3'); mouse centrin4 (NM_145825
[GenBank]
) 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), 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-
-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--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). 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). HeLa cells and hTERT-RPE-1 cells grown on poly-L-lysine–coated coverslips were fixed with Zamboni's fixative (Stefanini et al., 1967) 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). 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). 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).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ikeda et al., 2005; Badano et al., 2006; Marshall, 2008). 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).
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), 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), 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), 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.
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), 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.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Eley et al., 2005; Zariwala et al., 2007). 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; Eley et al., 2005). 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).
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). 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; Dentler and LeCluyse, 1982; LeCluyse and Dentler, 1984; Foliguet and Puchelle, 1986); however, it has not been observed in fish or invertebrates (Dentler, 1980). 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). In human cilia, the peripheral singlet microtubules and the sentan domain are observed when cilia are longer than 3 µm (Foliguet and Puchelle, 1986). 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; Cole, 2005; Sloboda, 2005; Follit et al., 2006; Hou et al., 2007; Omori et al., 2008; Scholey, 2008). 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; Porter et al., 1974; Foliguet and Puchelle, 1986; Hosaka et al., 1993). 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; Dalen, 1983); 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; Million et al., 1999; Ikegami et al., 2006; Pathak et al., 2007), and ciliary tip tubulin has been shown to be excluded from monomeric glycylation (Dossou et al., 2007). 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.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Akiharu Kubo (akiharukubo{at}gmail.com)
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Badano, J. L., Mitsuma, N., Beales, P. L., and Katsanis, N. (2006). The ciliopathies: an emerging class of human genetic disorders. Annu. Rev. Genomics Hum. Genet 7, 125–148.[CrossRef][Medline]
Baron, D. M., Ralston, K. S., Kabututu, Z. P., and Hill, K. L. (2007). Functional genomics in Trypanosoma brucei identifies evolutionarily conserved components of motile flagella. J. Cell Sci 120, 478–491.
Cole, D. G. (2005). Intraflagellar transport: keeping the motors coordinated. Curr. Biol 15, R798–R801.[CrossRef][Medline]
Dalen, H. (1983). An ultrastructural study of the tracheal epithelium of the guinea-pig with special reference to the ciliary structure. J. Anat 136, 47–67.[Medline]
Dentler, W. L. (1980). Structures linking the tips of ciliary and flagellar microtubules to the membrane. J. Cell Sci 42, 207–220.[Abstract]
Dentler, W. L., and LeCluyse, E. L. (1982). Microtubule capping structures at the tips of tracheal cilia: evidence for their firm attachment during ciliary bend formation and the restriction of microtubule sliding. Cell Motil 2, 549–572.[CrossRef][Medline]
Dirksen, E. R., and Satir, P. (1972). Ciliary activity in the mouse oviduct as studied by transmission and scanning electron microscopy. Tiss. Cell 4, 389–403.[CrossRef]
Dossou, S. J., Bré, M. H., and Hallworth, R. (2007). Mammalian cilia function is independent of the polymeric state of tubulin glycylation. Cell Motil. Cytoskelet 64, 847–855.[CrossRef][Medline]
Eley, L., Yates, L. M., and Goodship, J. A. (2005). Cilia and disease. Curr. Opin. Genet. Dev 15, 308–314.[CrossRef][Medline]
Felsenstein, J. (2003). PHYLIP (Phylogeny Inference Package), version 3.6a, Seattle: Department of Genetics, University of Washington, Distributed by the author.
Fisher, H. W., and Cooper, T. W. (1967). Electron microscope studies of the microvilli of HeLa cells. J. Cell Biol 34, 569–576.
Foliguet, B., and Puchelle, E. (1986). Apical structure of human respiratory cilia. Bull. Eur. Physiopathol. Respir 22, 43–47.[Medline]
Follit, J. A., Tuft, R. A., Fogarty, K. E., and Pazour, G. J. (2006). The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol. Biol. Cell 17, 3781–3792.
Gavet, O., Alvarez, C., Gaspar, P., and Bornens, M. (2003). Centrin4p, a novel mammalian centrin specifically expressed in ciliated cells. Mol. Biol. Cell 14, 1818–1834.
Haimo, L. T., and Rosenbaum, J. L. (1981). Cilia, flagella, and microtubules. J. Cell Biol 91, 125s–130s.
Hayes, J. M., Kim, S. K., Abitua, P. B., Park, T. J., Herrington, E. R., Kitayama, A., Grow, M. W., Ueno, N., and Wallingford, J. B. (2007). Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development. Dev. Biol 312, 115–130.[CrossRef][Medline]
Hosaka, S., Usuda, N., and Nagata, T. (1993). An ultrastructural study on HeLa cells cultured in roller bottles forming aggregates. Med. Electron Microsc 26, 125–131.[CrossRef]
Hou, Y., Qin, H., Follit, J. A., Pazour, G. J., Rosenbaum, J. L., and Witman, G. B. (2007). Functional analysis of an individual IFT protein: IFT46 is required for transport of outer dynein arms into flagella. J. Cell Biol 176, 653–665.
Ibañez-Tallon, I., Heintz, N., and Omran, H. (2003). To beat or not to beat: roles of cilia in development and disease. Hum. Mol. Genet 12, R27–R35.
Ikeda, T., Ikeda, K., Enomoto, M., Park, M. K., Hirono, M., and Kamiya, R. (2005). The mouse ortholog of EFHC1 implicated in juvenile myoclonic epilepsy is an axonemal protein widely conserved among organisms with motile cilia and flagella. FEBS Lett 579, 819–822.[CrossRef][Medline]
Ikegami, K., Mukai, M., Tsuchida, J., Heier, R. L., Macgregor, G. R., and Setou, M. (2006). TTLL7 is a mammalian beta-tubulin polyglutamylase required for growth of MAP2-positive neurites. J. Biol. Chem 281, 30707–30716.
Kubo, A., Sasaki, H., Yuba-Kubo, A., Tsukita, S., and Shiina, N. (1999). Centriolar satellites: molecular characterization, ATP-dependent movement toward centrioles and possible involvement in ciliogenesis. J. Cell Biol 147, 969–980.
Kuhn, C., and Engleman, W. (1978). The structure of the tips of mammalian respiratory cilia. Cell Tiss. Res 186, 491–498.[Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Larsson, M., Norrander, J., Gräslund, S., Brundell, E., Linck, R., Ståhl, S., and Höög, C. (2000). The spatial and temporal expression of Tekt1, a mouse tektin C homologue, during spermatogenesis suggest that it is involved in the development of the sperm tail basal body and axoneme. Eur. J. Cell Biol 79, 718–725.[CrossRef][Medline]
LeCluyse, E. L., and Dentler, W. L. (1984). Asymmetrical microtubule capping structures in frog palate cilia. J. Ultrastruct. Res 86, 75–85.[CrossRef][Medline]
Li, J. B. et al. (2004). Comparative genomics identifies a flagellar and basal body proteome that includes the BBS5 human disease gene. Cell 117, 541–552.[CrossRef][Medline]
Marshall, W. F. (2008). The cell biological basis of ciliary disease. J. Cell Biol 180, 17–21.
Matsui, T. et al. (2004). Identification of novel keratinocyte-secreted peptides dermokine-alpha/-beta and a new stratified epithelium-secreted protein gene complex on human chromosome 19q13.1. Genomics 84, 384–397.[CrossRef][Medline]
Miller, J. M., Wang, W., Balczon, R., and Dentler, W. L. (1990). Ciliary microtubule capping structures contain a mammalian kinetochore antigen. J. Cell Biol 110, 703–714.
Million, K., Larcher, J., Laoukili, J., Bourguignon, D., Marano, F., and Tournier, F. (1999). Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells. J. Cell Sci 112, 4357–4366.[Abstract]
Mitchell, D. R. (2007). The evolution of eukaryotic cilia and flagella as motile and sensory organelles. Adv. Exp. Med. Biol 607, 130–140.[CrossRef][Medline]
Niwa, H., Yamamura, K., and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199.[CrossRef][Medline]
Omori, Y., Zhao, C., Saras, A., Mukhopadhyay, S., Kim, W., Furukawa, T., Sengupta, P., Veraksa, A., and Malicki, J. (2008). Elipsa is an early determinant of ciliogenesis that links the IFT particle to membrane-associated small GTPase Rab8. Nat. Cell Biol 10, 437–444.[CrossRef][Medline]
Ostrowski, L. E., Blackburn, K., Radde, K. M., Moyer, M. B., Schlatzer, D. M., Moseley, A., and Boucher, R. C. (2002). A proteomic analysis of human cilia: identification of novel components. Mol. Cell Proteomics 1, 451–465.
Pathak, N., Obara, T., Mangos, S., Liu, Y., and Drummond, I. A. (2007). The zebrafish fleer gene encodes an essential regulator of cilia tubulin polyglutamylation. Mol. Biol. Cell 18, 4353–4364.
Pazour, G. J., and Rosenbaum, J. L. (2002). Intraflagellar transport and cilia-dependent diseases. Trends Cell Biol 12, 551–555.[CrossRef][Medline]
Porter, K. R., Fonte, V., and Weiss, G. (1974). A scanning microscope study of the topography of HeLa cells. Cancer Res 34, 1385–1394.
Portman, R. W., LeCluyse, E. L., and Dentler, W. L. (1987). Development of microtubule capping structures in ciliated epithelial cells. J. Cell Sci 87, 85–94.[Abstract]
Redeker, V., Levilliers, N., Schmitter, J. M., Le Caer, J. P., Rossier, J., Adoutte, A., and Bré, M.H. (1994). Polyglycylation of tubulin: a posttranslational modification in axonemal microtubules. Science 266, 1688–1691.
Renthal, R., Schneider, B. G., Miller, M. M., and Ludueña, R. F. (1993). Beta IV is the major beta-tubulin isotype in bovine cilia. Cell Motil. Cytoskelet 25, 19–29.[CrossRef][Medline]
Ross, A. J., Dailey, L. A., Brighton, L. E., and Devlin, R. B. (2007). Transcriptional profiling of mucociliary differentiation in human airway epithelial cells. Am. J. Respir. Cell. Mol. Biol 37, 169–185.
Satir, P., and Christensen, S. T. (2007). Overview of structure and function of mammalian cilia. Annu. Rev. Physiol 69, 377–400.[CrossRef][Medline]
Scholey, J. M. (2008). Intraflagellar transport motors in cilia: moving along the cell's antenna. J. Cell Biol 180, 23–29.
Simionescu, N., and Simionescu, M. (1976). Galloylglucoses of low molecular weight as mordant in electron microscopy. I. Procedure, and evidence for mordanting effect. J. Cell Biol 70, 608–621.
Sloboda, R. D. (2005). Intraflagellar transport and the flagellar tip complex. J. Cell Biochem 94, 266–272.[CrossRef][Medline]
Smith, D. B., and Johnson, K. S. (1988). Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene 67, 31–40.[CrossRef][Medline]
Stefanini, M., De Martino, C., and Zamboni, L. (1967). Fixation of ejaculated spermatozoa for electron microscopy. Nature 216, 173–174.[CrossRef][Medline]
Stolc, V., Samanta, M. P., Tongprasit, W., and Marshall, W. F. (2005). Genome-wide transcriptional analysis of flagellar regeneration in Chlamydomonas reinhardtii identifies orthologs of ciliary disease genes. Proc. Natl. Acad. Sci. USA 102, 3703–3707.
Suzuki, F., and Nagano, T. (2002). Morphological relationship between the plasma membrane and the microtubules in the end piece of the boar spermatozoon. J. Electron Microsc 29, 190–192.
Woolley, D. M., and Nickels, S. N. (1985). Microtubule termination patterns in mammalian sperm flagella. J. Ultrastruct. Res 90, 221–234.[CrossRef][Medline]
You, Y., Richer, E. J., Huang, T., and Brody, S. L. (2002). Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population. Am. J. Physiol. Lung Cell Mol. Physiol 283, L1315–L1321.
You, Y., Huang, T., Richer, E. J., Schmidt, J. E., Zabner, J., Borok, Z., and Brody, S. L. (2004). Role of f-box factor foxj1 in differentiation of ciliated airway epithelial cells. Am. J. Physiol. Lung Cell Mol. Physiol 286, L650–L657.
Yuba-Kubo, A., Kubo, A., Hata, M., and Tsukita, S. (2005). Gene knockout analysis of two gamma-tubulin isoforms in mice. Dev. Biol 282, 361–373.[CrossRef][Medline]
Zariwala, M. A., Knowles, M. R., and Omran, H. (2007). Genetic defects in ciliary structure and function. Annu. Rev. Physiol 69, 423–450.[CrossRef][Medline]
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