LRRC6 Mutation Causes Primary Ciliary Dyskinesia with Dynein Arm Defects

Despite recent progress in defining the ciliome, the genetic basis for many cases of primary ciliary dyskinesia (PCD) remains elusive. We evaluated five children from two unrelated, consanguineous Palestinian families who had PCD with typical clinical features, reduced nasal nitric oxide concentrations, and absent dynein arms. Linkage analyses revealed a single common homozygous region on chromosome 8 and one candidate was conserved in organisms with motile cilia. Sequencing revealed a single novel mutation in LRRC6 (Leucine-rich repeat containing protein 6) that fit the model of autosomal recessive genetic transmission, leading to a change of a highly conserved amino acid from aspartic acid to histidine (Asp146His). LRRC6 was localized to the cytoplasm and was up-regulated during ciliogenesis in human airway epithelial cells in a Foxj1-dependent fashion. Nasal epithelial cells isolated from affected individuals and shRNA-mediated silencing in human airway epithelial cells, showed reduced LRRC6 expression, absent dynein arms, and slowed cilia beat frequency. Dynein arm proteins were either absent or mislocalized to the cytoplasm in airway epithelial cells from a primary ciliary dyskinesia subject. These findings suggest that LRRC6 plays a role in dynein arm assembly or trafficking and when mutated leads to primary ciliary dyskinesia with laterality defects.     

The functional expression and motile properties of recombinant outer arm dynein from Tetrahymena

Cilia and flagella are motile organelles that play various roles in eukaryotic cells. Ciliary movement is driven by axonemal dyneins (outer arm and inner arm dyneins) that bind to peripheral microtubule doublets. Elucidating the molecular mechanism of ciliary movement requires the genetic engineering of axonemal dyneins; however, no expression system for axonemal dyneins has been previously established. This study is the first to purify recombinant axonemal dynein with motile activity. In the ciliated protozoan Tetrahymena, recombinant outer arm dynein purified from ciliary extract was able to slide microtubules in a gliding assay. Furthermore, the recombinant dynein moved processively along microtubules in a single-molecule motility assay. This expression system will be useful for investigating the unique properties of diverse axonemal dyneins and will enable future molecular studies on ciliary movement.     

Defects in the cytoplasmic assembly of axonemal dynein arms cause morphological abnormalities and dysmotility in sperm cells leading to male infertility

Axonemal protein complexes, such as outer (ODA) and inner (IDA) dynein arms, are responsible for the generation and regulation of flagellar and ciliary beating. Studies in various ciliated model organisms have shown that axonemal dynein arms are first assembled in the cell cytoplasm and then delivered into axonemes during ciliogenesis. In humans, mutations in genes encoding for factors involved in this process cause structural and functional defects of motile cilia in various organs such as the airways and result in the hereditary disorder primary ciliary dyskinesia (PCD). Despite extensive knowledge about the cytoplasmic assembly of axonemal dynein arms in respiratory cilia, this process is still poorly understood in sperm flagella. To better define its clinical relevance on sperm structure and function, and thus male fertility, further investigations are required. Here we report the fertility status in different axonemal dynein preassembly mutant males (DNAAF2/ KTU, DNAAF4/ DYX1C1, DNAAF6/ PIH1D3, DNAAF7/ZMYND10, CFAP300/C11orf70 and LRRC6). Besides andrological examinations, we functionally and structurally analyzed sperm flagella of affected individuals by high-speed video- and transmission electron microscopy as well as systematically compared the composition of dynein arms in sperm flagella and respiratory cilia by immunofluorescence microscopy. Furthermore, we analyzed the flagellar length in dynein preassembly mutant sperm. We found that the process of axonemal dynein preassembly is also critical in sperm, by identifying defects of ODAs and IDAs in dysmotile sperm of these individuals. Interestingly, these mutant sperm consistently show a complete loss of ODAs, while some respiratory cilia from the same individual can retain ODAs in the proximal ciliary compartment. This agrees with reports of solely one distinct ODA type in sperm, compared to two different ODA types in proximal and distal respiratory ciliary axonemes. Consistent with observations in model organisms, we also determined a significant reduction of sperm flagellar length in these individuals. These findings are relevant to subsequent studies on the function and composition of sperm flagella in PCD patients and non-syndromic infertile males. Our study contributes to a better understanding of the fertility status in PCD-affected males and should help guide genetic and andrological counselling for affected males and their families.     

ARMC4 Mutations Cause Primary Ciliary Dyskinesia with Randomization of Left/Right Body Asymmetry

The motive forces for ciliary movement are generated by large multiprotein complexes referred to as outer dynein arms (ODAs), which are preassembled in the cytoplasm prior to transport to the ciliary axonemal compartment. In humans, defects in structural components, docking complexes, or cytoplasmic assembly factors can cause primary ciliary dyskinesia (PCD), a disorder characterized by chronic airway disease and defects in laterality. By using combined high resolution copy-number variant and mutation analysis, we identified ARMC4 mutations in twelve PCD individuals whose cells showed reduced numbers of ODAs and severely impaired ciliary beating. Transient suppression in zebrafish and analysis of an ENU mouse mutant confirmed in both model organisms that ARMC4 is critical for left-right patterning. We demonstrate that ARMC4 is an axonemal protein that is necessary for proper targeting and anchoring of ODAs.     

In vivo ciliogenesis in human fetal tracheal epithelium

Development of ciliated cells (CC) in the fetal human trachea was studied by light and electron microscopy in specimens obtained from 45 embryos or fetuses aged from 9 to 27 weeks of gestation (menstrual age). Four stages could be recognized during tracheal development. Up to 11 weeks (stage I), the trachea was covered with a columnar undifferentiated epithelium with abundant glycogen, apical microvilli, and primary cilia. From 12 to 18-19 weeks (stage II), centriologenesis and secondary ciliogenesis were very active, and the percentage of CC and secretory cells (SC) progressively increased. From 20 to 22-23 weeks, the density of CC was higher but, in parallel, the percentage of SC decreased (stage III). Throughout this period, the different steps of ciliogenesis could be identified in the same field, and the ciliated borders consisted of ciliary shafts with a disorderly arrangement. Megacilia were identified. Some of the preciliated cells had both cilia and secretory granules in their apical cytoplasm. After 24 weeks (stage IV), the ciliated border was apparently mature, the rootlets lengthened, and the cilia were correctly orientated. Whatever the fetal age, the density of CC was significantly higher (P less than .01) in the dorsal trachea compared to the ventral trachea. There are many similarities between animal and human ciliogenesis, but in human fetuses, most of the ciliary differentiation occurs early, during the first half of gestation. As demonstrated in experimental models, SC likely play a major role in genesis of CC during the fetal development of the human trachea.     

Loss-of-function mutations in a human gene related to Chlamydomonas reinhardtii dynein IC78 result in primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a group of heterogeneous disorders of unknown origin, usually inherited as an autosomal recessive trait. Its phenotype is characterized by axonemal abnormalities of respiratory cilia and sperm tails leading to bronchiectasis and sinusitis, which are sometimes associated with situs inversus (Kartagener syndrome) and male sterility. The main ciliary defect in PCD is an absence of dynein arms. We have isolated the first gene involved in PCD, using a candidate-gene approach developed on the basis of documented abnormalities of immotile strains of Chlamydomonas reinhardtii, which carry axonemal ultrastructural defects reminiscent of PCD. Taking advantage of the evolutionary conservation of genes encoding axonemal proteins, we have isolated a human sequence (DNAI1) related to IC78, a C. reinhardtii gene encoding a dynein intermediate chain in which mutations are associated with the absence of outer dynein arms. DNAI1 is highly expressed in trachea and testis and is composed of 20 exons located at 9p13-p21. Two loss-of-function mutations of DNAI1 have been identified in a patient with PCD characterized by immotile respiratory cilia lacking outer dynein arms. In addition, we excluded linkage between this gene and similar PCD phenotypes in five other affected families, providing a clear demonstration of locus heterogeneity. These data reveal the critical role of DNAI1 in the development of human axonemal structures and open up new means for identification of additional genes involved in related developmental defects.     

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.     

Development of human fetal lung in organ culture compared with in utero ontogeny

Summary In utero, at around 23 wk gestation, the progenitor epithelium of distal airway differentiates into type I and type II pneumatocytes. Human fetal lung organ cultures, as early as 12 wk gestation, have the competence to self-differentiate. Distal airway epithelial immunoreactivity to cytokeratins CK 7,8, and 18 decreases with differentiation both in utero and in organ culture, whereas reactivity to epithelial membrane antigen remains constant in both. As distal airways dilate, the mean percentage airspace of fetal lungs in organ culture increases to 58%, equivalent to lung of gestation 26.0±7.3 wk. In organ culture, capillary blood vessels, visualized by vimentin immunoreactivity, remodel and more closely approximate the epithelium but without direct invasion. In utero, at 23 wk gestation, elastin appears as condensation around airways and forms a basis for secondary crests which, by 29 wk gestation, evolve into alveolar septae. In organ culture, no elastin is deposited, no secondary or alveolar crests form, and the lung retains a simple saccular structure. Differentiation of the terminal airway epithelium and mesodermal maturational events to facilitate gas exchange, such as capillary invasion or secondary-alveolar crest formation, are almost synchronous in human lung in utero but clearly dissociate in organ culture.     

Regulation of ciliary motility: conserved protein kinases and phosphatases are targeted and anchored in the ciliary axoneme

Recent evidence has revealed that the dynein motors and highly conserved signaling proteins are localized within the ciliary 9 + 2 axoneme. One key mechanism for regulation of motility is phosphorylation. Here, we review diverse evidence, from multiple experimental organisms, that ciliary motility is regulated by phosphorylation/dephosphorylation of the dynein arms through kinases and phosphatases that are anchored immediately adjacent to their axonemal substrates.     

A Computational Model of Dynein Activation Patterns that Can Explain Nodal Cilia Rotation

Normal left-right patterning in vertebrates depends on the rotational movement of nodal cilia. In order to produce this ciliary motion, the activity of axonemal dyneins must be tightly regulated in a temporal and spatial manner; the specific activation pattern of the dynein motors in the nodal cilia has not been reported. Contemporary imaging techniques cannot directly assess dynein activity in a living cilium. In this study, we establish a three-dimensional model to mimic the ciliary ultrastructure and assume that the activation of dynein proteins is related to the interdoublet distance. By employing finite-element analysis and grid deformation techniques, we simulate the mechanical function of dyneins by pairs of point loads, investigate the time-variant interdoublet distance, and simulate the dynein-triggered ciliary motion. The computational results indicate that, to produce the rotational movement of nodal cilia, the dynein activity is transferred clockwise (looking from the tip) between the nine doublet microtubules, and along each microtubule, the dynein activation should occur faster at the basal region and slower when it is close to the ciliary tip. Moreover, the time cost by all the dyneins along one microtubule to be activated can be used to deduce the dynein activation pattern; it implies that, as an alternative method, measuring this time can indirectly reveal the dynein activity. The proposed protein-structure model can simulate the ciliary motion triggered by various dynein activation patterns explicitly and may contribute to furthering the studies on axonemal dynein activity.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Functional architecture of the outer arm dynein conformational switch

Dynein light chain 1 (LC1/DNAL1) is one of the most highly conserved components of ciliary axonemal outer arm dyneins, and it associates with both a heavy chain motor unit and tubulin located within the A-tubule of the axonemal outer doublet microtubules. In a variety of model systems, lack of LC1 or expression of mutant forms leads to profound defects in ciliary motility, including the failure of the hydrodynamic coupling needed for ciliary metachronal synchrony, random stalling during the power/recovery stroke transition, an aberrant response to imposed viscous load, and in some cases partial failure of motor assembly. These phenotypes have led to the proposal that LC1 acts as part of a mechanical switch to control motor function in response to alterations in axonemal curvature. Here we have used NMR chemical shift mapping to define the regions perturbed by a series of mutations in the C-terminal domain that yield a range of phenotypic effects on motility. In addition, we have identified the subdomain of LC1 involved in binding microtubules and characterized the consequences of an Asn → Ser alteration within the terminal leucine-rich repeat that in humans causes primary ciliary dyskinesia. Together, these data define a series of functional subdomains within LC1 and allow us to propose a structural model for the organization of the dynein heavy chain-LC1-microtubule ternary complex that is required for the coordinated activity of dynein motors in cilia.     

人胚鼻咽上皮发育分化与c—erbB—2的表达变化

应用免疫组织化学方法对人胚鼻咽的ｃ－ｅｒｂＢ－２表达情况进行了研究．结果表明,人胚鼻咽上皮的ｃ－ｅｒｂＢ－２表达没有发育阶段性与鼻咽部位置的差异,而与鼻咽上皮的种类密切相关．在假复层纤毛柱状上皮中,以纤毛层的ｃ－ｅｒｂＢ－２表达阳性信号最强;在典型的过渡型上皮中,ｃ－ｅｒｂＢ－２阳性反应细胞主要分布于上皮的下五分之四左右的区域,表层细胞无阳性信号出现;而在复层鳞状上皮中,ｃ－ｅｒｂＢ－２阳性细胞的位置进一步下移,主要分布于上皮的下三分之二左右的区域．这些结果提示,ｃ－ｅｒｂＢ－２在人胚鼻咽上皮中的表达随细胞分化程度的增加而降低直至完全没有表达．     

Lentivirus vector-mediated gene transfer to the developing bronchiolar airway epithelium in the fetal lamb

BACKGROUND: Development of effective and durable gene therapy for treatment of the respiratory manifestations of cystic fibrosis remains a formidable challenge. Obstacles include difficulty in achieving efficient gene transfer to mature airway epithelium and the need to stably transduce self-renewing epithelial progenitor cells in order to avoid loss of transgene expression through epithelial turnover. Targeting the developing airway epithelium during fetal life offers the prospect of circumventing these challenges. METHODS: In the current study we investigated vesicular stomatitis virus glycoprotein (VSVg)-pseudotyped HIV-1-derived lentivirus vector-mediated gene transfer to the airway epithelium of mid-gestation fetal lambs, both in vitro and in vivo. In the in vitro studies epithelial sheet explants and lung organ culture were used to examine transduction of the proximal and more distal airway epithelium, respectively. For the in vivo studies, vector was delivered directly into the proximal airway. RESULTS: We found that even during the early pseudoglandular and canalicular phases of lung development, occurring through mid-gestation, the proximal bronchial airway epithelium was relatively mature and highly resistant to lentivirus-mediated transduction. In contrast, the more distal bronchiolar airway epithelium was relatively permissive for transduction although the absolute levels achieved remained low. CONCLUSION: This result is promising as the bronchiolar airway epithelium is a major site of pathology in the cystic fibrosis airway, and much higher levels of transduction are likely to be achieved by developing strategies that increase the amount of vector reaching the more distal airway after intratracheal delivery.     

Ndel1 suppresses ciliogenesis in proliferating cells by regulating the trichoplein–Aurora A pathway

Primary cilia protrude from the surface of quiescent cells and disassemble at cell cycle reentry. We previously showed that ciliary reassembly is suppressed by trichoplein-mediated Aurora A activation pathway in growing cells. Here, we report that Ndel1, a well-known modulator of dynein activity, localizes at the subdistal appendage of the mother centriole, which nucleates a primary cilium. In the presence of serum, Ndel1 depletion reduces trichoplein at the mother centriole and induces unscheduled primary cilia formation, which is reverted by forced trichoplein expression or coknockdown of KCTD17 (an E3 ligase component protein for trichoplein). Serum starvation induced transient Ndel1 degradation, subsequent to the disappearance of trichoplein at the mother centriole. Forced expression of Ndel1 suppressed trichoplein degradation and axonemal microtubule extension during ciliogenesis, similar to trichoplein induction or KCTD17 knockdown. Most importantly, the proportion of ciliated and quiescent cells was increased in the kidney tubular epithelia of newborn Ndel1-hypomorphic mice. Thus, Ndel1 acts as a novel upstream regulator of the trichoplein–Aurora A pathway to inhibit primary cilia assembly.     

Ciliary motility: the components and cytoplasmic preassembly mechanisms of the axonemal dyneins

Motile cilia and flagella are organelles, which function in cell motility and in the transport of fluids over the surface of cells. Motility defects often result in a rare human disease, primary ciliary dyskinesia (PCD). Cell motility depends on axonemal dynein, a molecular motor that drives the beating of cilia and flagella. The dyneins are composed of multiple subunits, which are thought to be preassembled in the cytoplasm before they are transported into cilia and flagella. Axonemal dyneins have been extensively studied in Chlamydomonas. In addition, analyses of human PCDs over the past decade, together with studies in other model animals, have identified the conserved components required for dynein assembly. Recently also, the first cytoplasmic component of dynein assembly, kintoun (ktu), was elucidated through the analysis of a medaka mutant in combination with human genetics and cell biology and biochemical studies of Chlamydomonas. The components of dynein and the proteins involved in its cytoplasmic assembly process are discussed.     

Cobblestone HUVECs: a human model system for studying primary ciliogenesis

Primary cilia are microtubule based sensory organelles that play an important role in maintaining cellular homeostasis. Malfunctioning results in a number of abnormalities, diseases (ciliopathies) and certain types of cancer. Morphological and biochemical knowledge on cilia/flagella, (early) ciliogenesis and intraflagellar transport is often obtained from model systems (e.g. Chlamydomonas) or from multi ciliary cells like lung or kidney epithelium.In this study endothelial cells in isolated human umbilical veins (HUVs) and cultured human umbilical vein endothelial cells (HUVECs) are compared and used to study primary ciliogenesis. By combining fluorescence microscopy, SEM, 2D and 3D TEM techniques we found that under the tested culturing conditions 60% of cobblestone endothelial cells form a primary cilium. Only a few of these cilia are present (protruding) on the endothelial cell surface, meaning that most primary cilia are in the cytoplasm (non-protruding). This was also observed in situ in the endothelial cells in the umbilical vein. The exact function(s?) of these non-protruding cilia remains unclear.Ultra-structural analysis of cultured HUVECs and the endothelial layer of the human umbilical veins reveal that there are: vesicles inside the ciliary pocket during the early stages of ciliogenesis; tubules/vesicles from the cytoplasm fuse with the ciliary sheath; irregular axoneme patterns, and two round, membranous vesicles inside the basal body.We conclude that cobblestone cultured HUVECs are comparable to the in vivo epithelial lining of the umbilical veins and therefore provide a well defined, relatively simple human model system with a reproducible number of non-protruding primary cilia for studying ciliogenesis.     

Structural studies of ciliary components

Cilia are organelles found on most eukaryotic cells, where they serve important functions in motility, sensory reception, and signaling. Recent advances in electron tomography have facilitated a number of ultrastructural studies of ciliary components that have significantly improved our knowledge of cilium architecture. These studies have produced nanometer-resolution structures of axonemal dynein complexes, microtubule doublets and triplets, basal bodies, radial spokes, and nexin complexes. In addition to these electron tomography studies, several recently published crystal structures provide insights into the architecture and mechanism of dynein as well as the centriolar protein SAS-6, important for establishing the 9-fold symmetry of centrioles. Ciliary assembly requires intraflagellar transport (IFT), a process that moves macromolecules between the tip of the cilium and the cell body. IFT relies on a large 20-subunit protein complex that is thought to mediate the contacts between ciliary motor and cargo proteins. Structural investigations of IFT complexes are starting to emerge, including the first three-dimensional models of IFT material in situ, revealing how IFT particles organize into larger train-like arrays, and the high-resolution structure of the IFT25/27 subcomplex. In this review, we cover recent advances in the structural and mechanistic understanding of ciliary components and IFT complexes.