Molecular characterization of centriole assembly in ciliated epithelial cells

Ciliated epithelial cells have the unique ability to generate hundreds of centrioles during differentiation. We used centrosomal proteins as molecular markers in cultured mouse tracheal epithelial cells to understand this process. Most centrosomal proteins were up-regulated early in ciliogenesis, initially appearing in cytoplasmic foci and then incorporated into centrioles. Three candidate proteins were further characterized. The centrosomal component SAS-6 localized to basal bodies and the proximal region of the ciliary axoneme, and depletion of SAS-6 prevented centriole assembly. The intraflagellar transport component polaris localized to nascent centrioles before incorporation into cilia, and depletion of polaris blocked axoneme formation. The centriolar satellite component PCM-1 colocalized with centrosomal components in cytoplasmic granules surrounding nascent centrioles. Interfering with PCM-1 reduced the amount of centrosomal proteins at basal bodies but did not prevent centriole assembly. This system will help determine the mechanism of centriole formation in mammalian cells and how the limitation on centriole duplication is overcome in ciliated epithelial cells.     

In vitro effects of colchicine and nocodazole on ciliogenesis in quail oviduct

Oviduct implants from quails which were primarily stimulated in vivo by estrogen so as to induce ciliogenesis in some epithelial cells were cultured in vitro in the presence or absence of colchicine or nocodazole. After 24 or 48 hr of culture, implants were examined by transmission and scanning electron microscopy to determine drug-induced alterations in ciliogenesis. After 24 hr of 10(-5) M colchicine treatment, the formation of basal bodies was totally inhibited, though the precursor material of generative complexes was unchanged. The inhibitory effect was not reversed when colchicine was removed in a 24 hr recovery culture. Treatment with 10(-6) M nocodazole for 24 hr, partially inhibited the assembly of basal bodies, which exhibited altered morphology. The assembly of basal bodies was restored during the 24 hr recovery period, after removal of nocodazole. Colchicine and nocodazole did not prevent polarized migration towards the apical surface of basal bodies formed prior to drug treatment. They anchored to the plasma membrane, but the formation of cilia was strongly disturbed in the presence of the drug. Numerous cells possessed anchored basal bodies which failed to induce the formation of cilia. The elongation of cilia was inhibited, as seen by their abnormal capping structure. In the enlarged tip, microtubules diverged. In contrast, these very short cilia possessed a mature ciliary necklace which was constructed during drug treatment. Differentiation of this membrane ciliary structure appeared to be unrelated to axoneme growth.     

Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity,Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.     

Chibby promotes ciliary vesicle formation and basal body docking during airway cell differentiation

Airway multiciliated epithelial cells play crucial roles in the mucosal defense system, but their differentiation process remains poorly understood. Mice lacking the basal body component Chibby (Cby) exhibit impaired mucociliary transport caused by defective ciliogenesis, resulting in chronic airway infection. In this paper, using primary cultures of mouse tracheal epithelial cells, we show that Cby facilitates basal body docking to the apical cell membrane through proper formation of ciliary vesicles at the distal appendage during the early stages of ciliogenesis. Cby is recruited to the distal appendages of centrioles via physical interaction with the distal appendage protein CEP164. Cby then associates with the membrane trafficking machinery component Rabin8, a guanine nucleotide exchange factor for the small guanosine triphosphatase Rab8, to promote recruitment of Rab8 and efficient assembly of ciliary vesicles. Thus, our study identifies Cby as a key regulator of ciliary vesicle formation and basal body docking during the differentiation of airway ciliated cells.     

The fine structure of atypical ciliated cells in the human gastric epithelium

Gastric mucosa obtained from the body and pyloric portions of the human stomach were observed by light and transmission electron microscopy. Ciliated cells were found in two of 18 subjects examined, one patient with gastric ulcer and the other one with gastric adenocarcinoma. The ciliated cells were found in epithelia at sites away from the main lesions. The tissues containing ciliated cells showed intestinal metaplasia combined with mild chronic gastritis in both cases. The epithelial layer facing the gastric lumen was composed of columnar cells with numerous uniform microvilli and goblet cells. This epithelium extended to the superficial parts of the tubules surrounded by the lamina propria. The deeper portions of the tubules were composed of mucous secretory, endocrine, and rarely ciliated cells. These ciliated cells were provided with numerous cilia the numbers of which varied considerably from cell to cell. This was in contrast to the primary cilium which is usually single. The central part of the apical cell membrane was sometimes concave in the area from where cilia tended to arise. It was also observed that numerous basal bodies as well as mucus-like granules were contained in the same cell. The axonemal pattern was different from that of ordinary cilia and showed 9 + 0 and 8 + 1 patterns. In longitudinal sections it was found that one peripheral doublet was displaced to the center of the axoneme as it left the basal body.     

Cilia-lacking respiratory cells in ciliary aplasia

This report describes the ultrastructural alterations observed in the nasal and bronchial mucosa of an 11-yr-old male suffering from immotile cilia syndrome (ICS). The morphological features observed in this patient are consistent with a ciliary aplasia. In fact, ciliated cells appeared to be replaced by columnar cells lacking cilia and basal bodies, and bearing on their surface cilium-like projections without any internal axonemal structure. In spite of the absence of basal bodies, centrioles, and kinocilia, these cells unexpectedly showed mature striated roots and centriolar precursor material scattered throughout the apical cytoplasm. These data suggest that control over basal body assembly is distinct from control over striated root formation. The presence of the above-reported structures in cells otherwise presenting many morphological features of normal ciliated cells is discussed on the basis of current knowledge of respiratory cilia biogenesis.     

Caveolin-3 and eNOS colocalize and interact in ciliated airway epithelial cells in the rat

In ciliated airway epithelial cells endothelial nitric oxide synthase as well as several other membrane bound proteins are located in the apical cell pole. To date, mechanisms that serve to target and to keep these proteins in this region are unknown. Endothelial nitric oxide synthase is known to target to caveolae by interaction with caveolin-1 or caveolin-3. Since caveolin-1 is found only in a subpopulation of ciliated cells at the basolateral cell membrane, we examined if caveolin-3 could be responsible for the apical localization of endothelial nitric oxide synthase in ciliated cells. We used real-time RT-PCR, laser-assisted microdissection, Western blotting and double-labeling immunohistochemistry to examine the presence of caveolin-3 in the airway epithelium of the rat. Indeed, we found caveolin-3-mRNA as well as protein in ciliated cells throughout the trachea and the bronchial tree. Caveolin-3-immunoreactivity was confined to the apical region and was colocalized with endothelial nitric oxide synthase and the high affinity choline transporter in a compartment distinct from the plasma membrane at the light microscopic level. No caveolae were found in the apical plasma membrane of ciliated cells but a tubulovesicular network was present in the apical region that reached up to the basal bodies of the cilia and was in close contact with mitochondria. Co-immunoprecipitation of caveolin-3 with endothelial nitric oxide synthase verified that both proteins interact in airway ciliated cells. These findings indicate that caveolin-3 is responsible to keep endothelial nitric oxide synthase in a membrane compartment in the apical region of ciliated cells.     

An ultrastructural study of morphogenesis of fibrogranular complex and centriole in ductuli efferentes of Chinese hamster.

An ultrastructural study of ciliated epithelial cells in the ductuli efferentes of young and adult hamsters has revealed that these cells possess dense granules, dense granule clusters, dense bodies and fibrogranular complexes as reservoirs or precursors for ciliogenesis. The dense granules are first seen in the centrosomal region. Later, many dense granules and dense granule clusters appear in the apical portion of the epithelial cells where, subsequently, dense bodies are also found. Finally, the fibrogranular complexes are formed in adults. Morphological evidence strongly suggests that cilia are formed from diplosomal centrioles, de novo centrioles, dense body centrioles, and fibrogranular complex centrioles. Ciliogenesis begins in the fourth day after birth and increases rapidly in the fifth day. After the sixth day, cilia appear to be generated mostly from dense bodies and the total ciliogenesis activities gradually decrease as the animal ages.     

Centriole and basal body formation during ciliogenesis revisited.

This review is concerned with the formation during ciliogenesis of centrioles and basal bodies, primarily in epithelial multi-ciliated cells from the developing vertebrate respiratory and reproductive tracts. During ciliated cell differentiation, in these as well as in other cell types, cilium formation is preceded by the formation of centrioles assembled from precursor structures having little resemblance to the mature organelle. The origin, composition and function of the centriole precursor structures in generating large numbers of centrioles in a short period of time during ciliogenesis is discussed. This review also focuses on the biochemistry of centrioles and basal bodies and on recent experimental evidence that DNA might be associated with these structures.     

RACK1, a PKC targeting protein, is exclusively localized to basal airway epithelial cells.

The novel isoform of protein kinase C (PKC), PKCepsilon, is an important regulator of ciliated cell function in airway epithelial cells, including cilia motility and detachment of ciliated cells after environmental insult. However, the mechanism of PKCepsilon signaling in the airways and the potential role of the PKCepsilon-interacting protein, receptor for activated C kinase 1 (RACK1), has not been widely explored. We used immunohistochemistry and Western blot analysis to show that RACK1 is localized exclusively to basal, non-ciliated (and non-goblet) bovine and human bronchial epithelial cells. Our immunohistochemistry experiments used the basal body marker pericentrin, a marker for cilia, beta-tubulin, and an airway goblet cell marker, MUC5AC, to confirm that RACK1 was excluded from differentiated airway cell subtypes and is only expressed in the basal cells. These results suggest that PKCepsilon signaling in the basal airway cell may involve RACK1; however, PKCepsilon regulation in ciliated cells uses RACK1-independent pathways.     

Otitis Media in Sperm-Associated Antigen 6 (Spag6)-Deficient Mice

Mammalian SPAG6 protein is localized to the axoneme central apparatus, and it is required for normal flagella and cilia motility. Recent studies demonstrated that the protein also regulates ciliogenesis and cilia polarity in the epithelial cells of brain ventricles and trachea. Motile cilia are also present in the epithelial cells of the middle ear and Eustachian tubes, where the ciliary system participates in the movement of serous fluid and mucus in the middle ear. Cilia defects are associated with otitis media (OM), presumably due to an inability to efficiently transport fluid, mucus and particles including microorganisms. We investigated the potential role of SPAG6 in the middle ear and Eustachian tubes by studying mice with a targeted mutation in the Spag6 gene. SPAG6 is expressed in the ciliated cells of middle ear epithelial cells. The orientation of the ciliary basal feet was random in the middle ear epithelial cells of Spag6-deficient mice, and there was an associated disrupted localization of the planar cell polarity (PCP) protein, FZD6. These features are associated with disordered cilia orientation, confirmed by scanning electron microscopy, which leads to uncoordinated cilia beating. The Spag6 mutant mice were also prone to develop OM. However, there were no significant differences in bacterial populations, epithelial goblet cell density, mucin expression and Eustachian tube angle between the mutant and wild-type mice, suggesting that OM was due to accumulation of fluid and mucus secondary to the ciliary dysfunction. Our studies demonstrate a role for Spag6 in the pathogenesis of OM in mice, possibly through its role in the regulation of cilia/basal body polarity through the PCP-dependent mechanisms in the middle ear and Eustachian tubes.     

Mechanosensory-defective, male-sterile unc mutants identify a novel basal body protein required for ciliogenesis in Drosophila

uncoordinated (unc) mutants of Drosophila, which lack transduction in ciliated mechanosensory neurons, do not produce motile sperm. Both sensory and spermatogenesis defects are associated with disrupted ciliary structures: mutant sensory neurons have truncated cilia, and sensory neurons and spermatids show defects in axoneme ultrastructure. unc encodes a novel protein with coiled-coil segments and a LisH motif, which is expressed in type I sensory neurons and in the male germline - the only ciliogenic cells in the fly. A functional UNC-GFP fusion protein specifically localizes to both basal bodies in differentiating sensory neurons. In premeiotic spermatocytes it localizes to all four centrioles in early G2, remaining associated with them through meiosis and as they become the basal bodies for the elongating spermatid flagella. UNC is thus specifically required for normal ciliogenesis. Its localization is an early marker for the centriole-basal body transition, a central but enigmatic event in eukaryotic cell differentiation.     

Identification of novel ciliogenesis factors using a new in vivo model for mucociliary epithelial development

Mucociliary epithelia are essential for homeostasis of many organs and consist of mucus-secreting goblet cells and ciliated cells. Here, we present the ciliated epidermis of Xenopus embryos as a facile model system for in vivo molecular studies of mucociliary epithelial development. Using an in situ hybridization-based approach, we identified numerous genes expressed differentially in mucus-secreting cells or in ciliated cells. Focusing on genes expressed in ciliated cells, we have identified new candidate ciliogenesis factors, including several not present in the current ciliome. We find that TTC25-GFP is localized to the base of cilia and to ciliary axonemes, and disruption of TTC25 function disrupts ciliogenesis. Mig12-GFP localizes very strongly to the base of cilia and confocal imaging of this construct allows for simple visualization of the planar polarity of basal bodies that underlies polarized ciliary beating. Knockdown of Mig12 disrupts ciliogenesis. Finally, we show that ciliogenesis factors identified in the Xenopus epidermis are required in the midline to facilitate neural tube closure. These results provide further evidence of a requirement for cilia in neural tube morphogenesis and suggest that genes identified in the Xenopus epidermis play broad roles in ciliogenesis. The suites of genes identified here will provide a foundation for future studies, and may also contribute to our understanding of pathological changes in mucociliary epithelia that accompany diseases such as asthma.     

The orientation of ciliary basal bodies in quail oviduct is related to the ciliary beating cycle commencement

In mature ciliated cells, the basal feet associated to the basal bodies point out in the direction of the effective stroke of the ciliary beating. In contrast, during ciliogenesis, the basal feet of the newly anchored basal bodies are randomly oriented. The reorientation of basal bodies occurs during the beginning of the coordinated beating cycle of the cilia.     

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO(2)/HCO(3)(-) during exacerbations of airway diseases, but the role of CO(2)/HCO(3)(-)-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO(2)/HCO(3)(-)-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO(2)/HCO(3)(-)-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO(2)/HCO(3)(-) increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.