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1.  Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection 
PLoS ONE  2013;8(3):e59925.
Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.
PMCID: PMC3602288  PMID: 23527286
2.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
PMCID: PMC3509786  PMID: 21131972
3.  Ktu/PF13 is required for cytoplasmic pre-assembly of axonemal dyneins 
Nature  2008;456(7222):611-616.
Cilia/flagella are highly conserved organelles that play diverse roles in cell motility and sensing extracellular signals. Motility defects in cilia/flagella often result in primary ciliary dyskinesia (PCD). However, the mechanisms underlying cilia formation and function, and in particular the cytoplasmic assembly of dyneins that power ciliary motility, are only poorly understood. Here we report a novel gene, kintoun (ktu), involved in this cytoplasmic process. This gene was first identified in a medaka mutant, and found to be mutated in PCD patients from two affected families as well as in the pf13 mutant of Chlamydomonas. In the absence of Ktu/PF13, both outer and inner dynein arms are missing or defective in the axoneme, leading to a loss of motility. Biochemical and immunohistochemical studies show that Ktu/PF13 is one of the long-sought proteins involved in pre-assembly of dynein arm complexes in the cytoplasm before intraflagellar transport loads them for the ciliary compartment.
PMCID: PMC3279746  PMID: 19052621
4.  The coiled-coil domain containing protein CCDC40 is essential for motile cilia function and left-right axis formation 
Nature genetics  2010;43(1):79-84.
Primary ciliary dyskinesia (PCD) is a genetically heterogeneous autosomal recessive disorder characterized by recurrent infections of the respiratory tract associated with abnormal function of motile cilia. Approximately half of PCD patients also have alterations in the left-right organization of internal organ positioning including situs inversus and situs ambiguous (Kartagener’s Syndrome, KS). Here we identify an uncharacterized coiled-coil domain containing protein (CCDC40) essential for correct left-right patterning in mouse, zebrafish and humans. Ccdc40 is expressed in tissues that contain motile cilia and mutation of Ccdc40 results in cilia with reduced ranges of motility. Importantly, we demonstrate that CCDC40 deficiency causes a novel PCD variant characterized by misplacement of central pair microtubules and defective axonemal assembly of inner dynein arms (IDAs) and dynein regulator complexes (DRCs). CCDC40 localizes to motile cilia and the apical cytoplasm and is responsible for axonemal recruitment of CCDC39, which is also mutated in a similar PCD variant.
PMCID: PMC3132183  PMID: 21131974
5.  DNAH5 Mutations Are a Common Cause of Primary Ciliary Dyskinesia with Outer Dynein Arm Defects 
Rationale: Primary ciliary dyskinesia (PCD) is characterized by recurrent airway infections and randomization of left–right body asymmetry. To date, autosomal recessive mutations have only been identified in a small number of patients involving DNAI1 and DNAH5, which encode outer dynein arm components.
Methods: We screened 109 white PCD families originating from Europe and North America for presence of DNAH5 mutations by haplotype analyses and/or sequencing.
Results: Haplotype analyses excluded linkage in 26 families. In 30 PCD families, we identified 33 novel (12 nonsense, 8 frameshift, 5 splicing, and 8 missense mutations) and two known DNAH5 mutations. We observed clustering of mutations within five exons harboring 27 mutant alleles (52%) of the 52 detected mutant alleles. Interestingly, 6 (32%) of 19 PCD families with DNAH5 mutations from North America carry the novel founder mutation 10815delT. Electron microscopic analyses in 22 patients with PCD with mutations invariably detected outer dynein arm ciliary defects. High-resolution immunofluorescence imaging of respiratory epithelial cells from eight patients with DNAH5 mutations showed mislocalization of mutant DNAH5 and accumulation at the microtubule organizing centers. Mutant DNAH5 was absent throughout the ciliary axoneme in seven patients and remained detectable in the proximal ciliary axoneme in one patient carrying compound heterozygous splicing mutations at the 3′-end (IVS75-2A>T, IVS76+5G>A). In a preselected subpopulation with documented outer dynein arm defects (n = 47), DNAH5 mutations were identified in 53% of patients.
Conclusions: DNAH5 is frequently mutated in patients with PCD exhibiting outer dynein arm defects and mutations cluster in five exons.
PMCID: PMC2662904  PMID: 16627867
cilia; DNAH5; outer dynein arm; primary ciliary dyskinesia
6.  The von Hippel-Lindau tumor suppressor protein controls ciliogenesis by orienting microtubule growth 
The Journal of Cell Biology  2006;175(4):547-554.
Cilia are specialized organelles that play an important role in several biological processes, including mechanosensation, photoperception, and osmosignaling. Mutations in proteins localized to cilia have been implicated in a growing number of human diseases. In this study, we demonstrate that the von Hippel-Lindau (VHL) protein (pVHL) is a ciliary protein that controls ciliogenesis in kidney cells. Knockdown of pVHL impeded the formation of cilia in mouse inner medullary collecting duct 3 kidney cells, whereas the expression of pVHL in VHL-negative renal cancer cells rescued the ciliogenesis defect. Using green fluorescent protein–tagged end-binding protein 1 to label microtubule plus ends, we found that pVHL does not affect the microtubule growth rate but is needed to orient the growth of microtubules toward the cell periphery, a prerequisite for the formation of cilia. Furthermore, pVHL interacts with the Par3–Par6–atypical PKC complex, suggesting a mechanism for linking polarity pathways to microtubule capture and ciliogenesis.
PMCID: PMC2064591  PMID: 17101696
7.  Mislocalization of DNAH5 and DNAH9 in Respiratory Cells from Patients with Primary Ciliary Dyskinesia 
Rationale: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by recurrent infections of the airways and situs inversus in half of the affected offspring. The most frequent genetic defects comprise recessive mutations of DNAH5 and DNAI1, which encode outer dynein arm (ODA) components. Diagnosis of PCD usually relies on electron microscopy, which is technically demanding and sometimes difficult to interpret. Methods: Using specific antibodies, we determined the subcellular localization of the ODA heavy chains DNAH5 and DNAH9 in human respiratory epithelial and sperm cells of patients with PCD and control subjects by high-resolution immunofluorescence imaging. We also assessed cilia and sperm tail function by high-speed video microscopy. Results: In normal ciliated airway epithelium, DNAH5 and DNAH9 show a specific regional distribution along the ciliary axoneme, indicating the existence of at least two distinct ODA types. DNAH5 was completely or only distally absent from the respiratory ciliary axoneme in patients with PCD with DNAH5− (n = 3) or DNAI1− (n = 1) mutations, respectively, and instead accumulated at the microtubule-organizing centers. In contrast to respiratory cilia, sperm tails from a patient with DNAH5 mutations had normal ODA heavy chain distribution, suggesting different modes of ODA generation in these cell types. Blinded investigation of a large cohort of patients with PCD and control subjects identified DNAH5 mislocalization in all patients diagnosed with ODA defects by electron microscopy (n = 16). Cilia with complete axonemal DNAH5 deficiency were immotile, whereas cilia with distal DNAH5 deficiency showed residual motility. Conclusions: Immunofluorescence staining can detect ODA defects, which will possibly aid PCD diagnosis.
PMCID: PMC2718478  PMID: 15750039
fluorescent antibody technique; genetics; respiratory tract diseases

Results 1-7 (7)