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At least 397 records · Page 22Linked to original sources
PubMed2003

Cellular and subcellular localization of the ARPKD protein; fibrocystin is expressed on primary cilia.

Autosomal recessive polycystic kidney disease (ARPKD) is an infantile form of PKD characterized by fusiform dilation of collecting ducts and congenital hepatic fibrosis. The ARPKD gene, PKHD1, is large (approximately 470 kb; 67 exons) with a 12222 bp longest open reading frame, although multiple different splice forms may be generated. The predicted full-length ARPKD protein, fibrocystin, is membrane bound with 4074 amino acids (447 kDa molecular weight). To characterize the pattern of fibrocystin expression we have generated four monoclonal antibodies (mAb) to the cytoplasmic tail of the protein. Western analysis of human kidney membrane protein showed an identical pattern with each mAb; a strongly expressing large product (>450 kDa), consistent with the predicted protein size, and a weaker approximately 220 kDa band. The same large product was detected in rat and mouse kidney with lower level expression in liver. To further show that these mAbs recognize fibrocystin, tissue from ARPKD patients was analyzed and no fibrocystin products were detected. Immunohistochemical analysis of the developing kidney showed expression in the branching ureteric bud and collecting ducts, expression that persisted into adulthood. Biliary duct staining was found in the liver, plus staining in the pancreas and developing testis. Immunofluorescence analysis of MDCK cells showed a major site of expression in the primary cilia. Recent studies have associated the disease protein in various human and animal forms of PKD with cilia. The localization of fibrocystin to cilia further strengthens that correlation and indicates that the primary defect in ARPKD may be linked to ciliary dysfunction.

Animals↗
PubMed2005

Characterization of the nephrocystin/nephrocystin-4 complex and subcellular localization of nephrocystin-4 to primary cilia and centrosomes.

Nephrocystin and nephrocystin-4 are newly identified proteins involved in familial juvenile nephronophthisis, an autosomal recessive nephropathy characterized by cyst formation and renal fibrosis. Nephrocystin is an adaptor protein that is able to associate with signaling molecules involved in cell adhesion and actin cytoskeleton organization, such as p130Cas, Pyk2, tensin and filamins. Nephrocystin was recently shown to interact and to co-localize with the microtubule component beta-tubulin to the primary cilia in renal epithelial cells, an organelle known to play a key role in the pathogenesis of cystic kidney diseases. In this study, we demonstrated that nephrocystin-4 also localizes to the primary cilia in polarized epithelial tubular cells, particularly at the basal bodies, and associates with microtubule component alpha-tubulin, suggesting a common role for the nephrocystin proteins in ciliary function. However, the co-localization of nephrocystin-4 with the microtubules is not restricted to the primary cilia, as nephrocystin-4 was also detected at the centrosomes of dividing cells and close to the cortical actin cytoskeleton in polarized cells. We also detected p130Cas and Pyk2 in the nephrocystin-4-containing complex, confirming the role of the nephrocystin proteins in cell-cell and cell-matrix adhesion signaling events. Finally, we refined the structural and functional regions involved in the interaction between nephrocystin and nephrocystin-4. These data suggest that nephrocystin and nephrocystin-4 belong to a multifunctional complex localized in actin- and microtubule-based structures involved in cell-cell and cell-matrix adhesion signaling as well as in cell division.

Adaptor Proteins, Signal Transducing↗
PubMed2025

The RNA splicing factor PRPF8 is required for left-right organiser cilia differentiation and determination of cardiac left-right asymmetry via regulation of Arl13b splicing.

Cilia function in the left-right organizer (LRO) is critical for determining internal organ asymmetry in vertebrates. To further understand the genetics of left-right asymmetry, we isolated a mouse mutant with laterality defects, l11Jus27, from a random mutagenesis screen. l11Jus27 mutants carry a missense mutation in the pre-mRNA processing factor, Prpf8. cephalophŏnus (cph) mutant zebrafish, carrying a protein truncating mutation in prpf8, phenocopy the laterality defects of l11Jus27 mutants. Prpf8 mutant mouse and fish embryos have increased expression of an alternative transcript encoding the cilium-associated protein, ARL13B, that lacks exon 9. In zebrafish, over-expression of the arl13b transcript lacking exon 9 perturbed cilium formation and caused laterality defects. The shorter ARL13B protein isoform lacked interactions with intraflagellar transport proteins. Our data suggest that PRPF8 plays a prominent role in LRO cilia by through the regulation of alternative splicing of ARL13B, thus uncovering a new mechanism for cilia-linked developmental defects.

ARL13B↗
PubMed1980

Structure and function of neutrophil leukocytes from patients with the immotile-cilia syndrome.

The various clinical manifestations of the recently characterized immotile-cilia syndrome can be traced to one cause--a structural defect of the cilia, making them immotile. It was regarded of interest to examine whether other aspects of cell motility may also be involved. For this reason the functions and structure of the neutrophil leukocytes were examined. Cells from eight patients with the immotile-cilia syndrome and healthy controls were investigated with regard to random and stimulated locomotion under agarose, orientation during migration, adherence, bactericidal capacity, and chemoluminescence. Four patients showed abnormally short migration distances of the leading front neutrophils after stimulation with serum and/or an E. coli bacterial factor (BF). Ascorbic acid did not restore the defective migration. Migrating neutrophils were significantly less oriented towards the serum-containing agarose well compared with the controls (p < 0.01). Adherence, bactericidal capacity for Staphylococcus aureus, chemoluminescence, random migration, and orientation during BF-induced migration were all normal. The number of microtubules in the pericentriolar region of the neutrophil granulocytes was unusually low in four of the eight patients. We conclude that the increased frequency of respiratory tract infections in patients with this syndrome is possibly due to defects in the granulocyte locomotory system, as well as to the defective mucociliary clearance of the airways.

Adult↗
PubMed1982

Giant cilia in the human organ of Corti.

Fusion of cilia, the growth of clumps of fused cilia and giant cilium formation have been studied in the normal human organ of Corti using the scanning electron microscope. These unusual forms are found mainly in the apical portions of the cochlea and appear to precede the loss of normal apical cilia which increases and extends in a basal direction with age. These changes may be due to low frequency noise damage or be a phenomenom of ageing. The mechanism of their formation is discussed in the light of recent experimental work on cell fusion.

Adult↗
PubMed1981

Single cilia formation in cells of the testicular interstitium in fertile men.

Testicular biopsies from 4 infertile men showing germinal cell aplasia with interstitial cell hyperplasia on light microscopy were studied by electron microscopy. Single cilia formation was observed in myoid cells, fibroblast-like cells and Leydig cells in the interstitium. Only 1 cilium was seen per cell. Transverse sections of most of cilia showed a 9 + 0 pattern of microtubule configuration. Cross striated rootlet fibers were attached to the basal bodies. The significance of the presence of cilia in the testicular interstitium is discussed.

Adult↗
PubMed2005

Lack of a laterality phenotype in Pkd1 knock-out embryos correlates with absence of polycystin-1 in nodal cilia.

The invariant asymmetric placement of thoracic and abdominal organs in the vertebrates is controlled by the left-asymmetric activity of the Nodal signaling cascade during embryogenesis. In the mouse embryo asymmetric induction of nodal is thought to be dependent on functional monocilia on the ventral node cells and on the Pkd2 gene, which encodes the calcium channel polycystin-2 (PC2). In humans mutations in PKD2 and PKD1 give rise to polycystic kidney disease. The PC1 and PC2 proteins are thought to function as part of a multifactorial complex. Localization of both proteins to the primary renal cilium suggested a function on cilia of the ventral node. Here we investigated Pkd1 knock-out embryos for laterality defects and found wild-type organ morphogenesis and normal expression of nodal and Pitx2. While PC2 localized to nodal cilia, no ciliary localization of PC1 was detected in mouse embryos. This finding was confirmed in an archetypical mammalian blastodisc, the rabbit embryo. Thus, absence of PC1 localization to cilia corresponded with a lack of laterality defects in Pkd1 knock-out embryos. Our results demonstrate a PC1-independent function of PC2 in left-right axis formation, and indirectly support a ciliary role of PC2 in this process.

Animals↗
PubMed1991

Multiple alpha-tubulin isoforms in cilia and cytoskeleton of Tetrahymena pyriformis generated by post-translational modifications. Studies during reciliation.

alpha-Tubulin microheterogeneity was studied in Tetrahymena pyriformis. Using two-dimensional electrophoretic analysis, we found between five and seven alpha-tubulin and four beta-tubulin isoforms in cilia and four or five alpha-tubulins and two beta-tubulins in cytoskeleton. Immunoblotting assay with anti-(acetyl alpha-tubulin) monoclonal antibody 6-11B-1 and [3H]acetate labelling revealed that the alpha-tubulin isoforms are post-translationally modified by acetylation. Our results also show that tubulins in the soluble cytoplasmic fraction are not acetylated. Nevertheless, a slight reaction with the antibody 6-11 B-1 can be observed in the taxol and vinblastine-treated cytoplasmic pool. Pulse/chase experiments using [35S]methionine during cell reciliation have demonstrated that the basic alpha-tubulin isoforms are converted into acidic isoforms in the absence of protein synthesis, suggesting that the basic alpha-tubulin is the precursor of the acidic forms which are found in cilia and cytoskeleton. In-vivo-translation selection demonstrated the existence of a single precursor molecule which corresponded to the most basic alpha-tubulin. Taken together, our results provide evidence for the existence of post-translational modifications, namely acetylation. Nevertheless, other post-translational mechanisms involved in the biosynthesis of microtubules of cilia and cytoskeleton are required to explain the whole alpha-tubulin heterogeneity.

Acetates↗
PubMed1983

Biochemical studies of olfaction: binding specificity of odorants to a cilia preparation from rainbow trout olfactory rosettes.

Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L-threonine, L-serine, and L-alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H-labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of L-[3H]lysine to site L was unaffected by addition of L-threonine, L-serine, or L-alanine, establishing its independence from site TSA. L-Arginine inhibited binding of L-[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.

Alanine↗
PubMedDate not supplied

Cilia-mediated oriented chemokinesis in Tetrahymena thermophila.

The role of the cilia in the locomotion ("gliding") of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination ("stop") of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the "stop." In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.

Animals↗
PubMedDate not supplied

Paramecium has two regulatory subunits of cyclic AMP-dependent protein kinase, one unique to cilia.

The subunit composition and intracellular location of the two forms of cAMP-dependent protein kinase of Paramecium cilia were determined using antibodies against the 40-kDa catalytic (C) and 44-kDa regulatory (R44) subunits of the 70-kDa cAMP-dependent protein kinase purified from deciliated cell bodies. Both C and R44 were present in soluble and particulate fractions of cilia and deciliated cells. Crude cilia and a soluble ciliary extract contained a 48-kDa protein (R48) weakly recognized by one of several monoclonal antibodies against R44, but not recognized by an anti-R44 polyclonal serum. Gel-filtration chromatography of a soluble ciliary extract resolved a 220-kDa form containing C and R48 and a 70-kDa form containing C and R44. In the large enzyme, R48 was the only protein to be autophosphorylated under conditions that allow autophosphorylation of R44. The subunits of the large enzyme subsequently were purified to homogeneity by cAMP-agarose chromatography. Both C and R48 were retained by the column and eluted with I M NaCl; no other proteins were purified in this step. These results confirm that the ciliary cAMP-dependent protein kinases have indistinguishable C subunits, but different R subunits. The small ciliary enzyme, like the cell-body enzyme, contains R44, whereas R48 is the R subunit of the large enzyme.

Animals↗
PubMedDate not supplied

Phagosome formation in Paramecium: roles of somatic and oral cilia and of solid particles as revealed by video microscopy.

The roles of somatic and oral cilia and solid particles during digestive vacuole (DV) formation in Paramecium multimicronucleatum were investigated using video-enhanced and immunofluorescence microscopy. Membrane incorporation into DVs was found to increase linearly with increasing particle concentration. The rate of discoidal vesicle transport to the cytopharynx was not affected by particles, showing that particles are not required for membrane trafficking to the cytopharynx. However, the presence of particles leads to an increased membrane fusion between the cytopharyngeal membrane and the discoidal vesicles. When live cells lost their somatic cilia on the left-ventral side anterior to the oral region due to deciliation, membrane incorporation into newly formed DVs was strongly inhibited. Using video-enhanced microscopy, latex beads were seen to be loaded along the quadrulus on the dorsal surface of the buccal cavity, but few beads were seen next to the dorsal and ventral peniculi. Particle sequestration into a pre-formed nascent digestive vacuole (NDV) was studied in Triton X-100-permeabilized cells whose ciliary beating was reactivated by the addition of Mg-ATP. Both beat frequency and the percentage of cells containing bead-labeled NDV were dependent on the Mg-ATP concentration: the higher the beat frequency, the higher the percentage of cells with a bead-labeled NDV. These results suggest that ciliary beating is probably the only mechanism required for particle accumulation in the NDV, while a coordinated beating of the somatic cilia on the left-ventral side anterior to the oral region as well as the quadrulus moves particles into the NDV. The beating of the peniculi may somehow prevent the backward flow of particles out of the NDV.

Animals↗
PubMed1981

Immotile cilia syndrome: radial spokes deficiency in a patient with Kartagener's triad.

Mucociliary transport and ultrastructure of nasal cilia in a 13 year old boy with Kartagener's triad, were investigated. Mucociliary transport was significantly delayed (greater than 30 minutes). Electron microscopy showed cilia lacking radial spokes, eccentric central tubules, and a dislocation of one the outer doublets. Dynein arms were present. We consider the radial spoke defect as a distinct congenital anomaly which contributes to the pathogenesis of the "immotile cilia syndrome".

Adolescent↗
PubMed1987

Characterization of metachronal wave of beating cilia on frog's palate epithelium in tissue culture.

1. A method is suggested to measure phase versus distance between beating cilia by means of a photoelectric device. A statistical method interpreting the results thus obtained is discussed. 2. It was found that: (a) an average phase exists between beating cilia, (b) despite strong fluctuations in phase on a short time scale, the average phase was kept constant over periods of 8 h, (c) the ciliary frequency and the length of the metachronal wave can be measured simultaneously. 3. The average phase differences are linearly dependent on distance. 4. The effective range of synchronization between cilia is of the order of 10 micron indicating that it occurs within one cell. 5. During the cycle of ciliary beating there are periods where coupling is stronger.

Animals↗
PubMed1981

Immotile cilia syndrome: a new cause of neonatal respiratory distress.

Kartagener's syndrome is a condition that consists of situs inversus, bronchiectasis, and sinusitis. Some patients have respiratory symptoms that date from early infancy, and electron microscopical examination has shown that adults with this condition lack dynein arms in ciliary microtubules. It has been suggested that an inherited defect in ciliary ultrastructure, the immotile cilia, is the basis for the syndrome. We report 6 patients who presented within the first 24 hours of life with tachypnoea, retraction, rales, and dextrocardia. Five of them had situs inversus. They all responded to physiotherapy after antibiotics had failed to improve their condition. Electron microscopical examination of the cilia in 2 of the infants confirmed deficiency of dynein arms. All 6 infants had recurrent respiratory infections. Four went on to develop recurrent otitis media, 2 had sinusitis, and 1 now has bronchiectasis. The immotile cilia syndrome should be added to the list of causes of respiratory distress in newborn infants. The condition is worth diagnosing because of the good response to physiotherapy, and because of the likelihood of recurrent respiratory problems.

Cilia↗
PubMed1976

Studies on ciliated epithelia of the human genital tract. I. Swelling of the cilia of Fallopian tube epithelium in organ cultures infected with Mycoplasma hominis.

Organ cultures of human Fallopian tubes were infected with Mycoplasma hominis. Scanning and transmission electron microscopy revealed swelling of the cilia of the tubal epithelial cells in infected cultures. In some, the entire cilia were swollen; in others, only the tips. Uninfected cultures kept for up to 7 days showed no structural changes in the cilia or other surface structures. M. hominis multiplied in organ cultures, but not in culture medium without tissue. A practical organ culture technique for the preparation of specimens for electron microscopy is described.

Cilia↗
PubMed2003

Functional analysis of cilia and ciliated epithelial ultrastructure in healthy children and young adults.

BACKGROUND: There are very few data on normal ciliary beat frequency, beat pattern, and ultrastructure in healthy children and adults. A study was undertaken to define ciliary structure, beat frequency and beat pattern in a healthy paediatric and young adult population. METHODS: Ciliated epithelial samples were obtained from 76 children and adult volunteers aged 6 months to 43 years by brushing the inferior nasal turbinate. Beating cilia were recorded using a digital high speed video camera which allowed analysis of ciliary beat pattern and beat frequency. Tissue was fixed for transmission electron microscopy. RESULTS: The mean ciliary beat frequency for the paediatric population (12.8 Hz (95% CI 12.3 to 13.3)) was higher than for the adult group (11.5 Hz (95% CI 10.3 to 12.7 Hz), p<0.01, t test); 10% (range 6-24%) of ciliated edges were found to have areas of dyskinetically beating cilia. All samples had evidence of mild epithelial damage. This reflected changes found in all measurements used for assessment of epithelial damage. Ciliary ultrastructural defects were found in less than 5% of cilia. CONCLUSION: Normal age related reference ranges have been established for ciliary structure and beat frequency. In a healthy population localised epithelial damage may be present causing areas of ciliary dyskinesia.

Adolescent↗
PubMed2006

Altered pH(i) regulation and Na(+)/HCO3(-) transporter activity in choroid plexus of cilia-defective Tg737(orpk) mutant mouse.

Tg737(orpk) mice have defects in cilia assembly and develop hydrocephalus in the perinatal period of life. Hydrocephalus is progressive and is thought to be initiated by abnormal ion and water transport across the choroid plexus epithelium. The pathology is further aggravated by the slow and disorganized beating of motile cilia on ependymal cells that contribute to decreased cerebrospinal fluid movement through the ventricles. Previously, we demonstrated that the hydrocephalus phenotype is associated with a marked increase in intracellular cAMP levels in choroid plexus epithelium, which is known to have regulatory effects on ion and fluid movement in many secretory epithelia. To evaluate whether the hydrocephalus in Tg737(orpk) mutants is associated with defects in ion transport, we compared the steady-state pH(i) and Na(+)-dependent transport activities of isolated choroid plexus epithelium tissue from Tg737(orpk) mutant and wild-type mice. The data indicate that Tg737(orpk) mutant choroid plexus epithelium have lower pH(i) and higher Na(+)-dependent HCO(3)(-) transport activity compared with wild-type choroid plexus epithelium. In addition, wild-type choroid plexus epithelium could be converted to a mutant phenotype with regard to the activity of Na(+)-dependent HCO(3)(-) transport by addition of dibutyryl-cAMP and mutant choroid plexus epithelium toward the wild-type phenotype by inhibiting PKA activity with H-89. Together, these data suggest that cilia have an important role in regulating normal physiology of choroid plexus epithelium and that ciliary dysfunction in Tg737(orpk) mutants disrupts a signaling pathway leading to elevated intracellular cAMP levels and aberrant regulation of pH(i) and ion transport activity.

Animals↗