An ovine hepatorenal fibrocystic model of a Meckel-like syndrome associated with dysmorphic primary cilia and TMEM67 mutations.

Meckel syndrome (MKS) is an inherited autosomal recessive hepatorenal fibrocystic syndrome, caused by mutations in TMEM67, characterized by occipital encephalocoele, renal cysts, hepatic fibrosis, and polydactyly. Here we describe an ovine model of MKS, with kidney and liver abnormalities, without polydactyly or occipital encephalocoele. Homozygous missense p.(Ile681Asn; Ile687Ser) mutations identified in ovine TMEM67 were pathogenic in zebrafish phenotype rescue assays. Meckelin protein was expressed in affected and unaffected kidney epithelial cells by immunoblotting, and in primary cilia of lamb kidney cyst epithelial cells by immunofluorescence. In contrast to primary cilia of relatively consistent length and morphology in unaffected kidney cells, those of affected cyst-lining cells displayed a range of short and extremely long cilia, as well as abnormal morphologies, such as bulbous regions along the axoneme. Putative cilia fragments were also consistently located within the cyst luminal contents. The abnormal ciliary phenotype was further confirmed in cultured interstitial fibroblasts from affected kidneys. These primary cilia dysmorphologies and length control defects were significantly greater in affected cells compared to unaffected controls. In conclusion, we describe abnormalities involving primary cilia length and morphology in the first reported example of a large animal model of MKS, in which we have identified TMEM67 mutations.

Does Harm Avoidance mediate effects of recollected parental bonding on mental distress in adulthood?

BACKGROUND: Adverse early life conditions such as perceived low quality of parental bonding increase vulnerability to stress and psychopathology in adulthood. However, the mechanisms by which perceptions of parental bonding translate into vulnerability are unclear and remain sparsely investigated in healthy populations. We proposed a model, in which the personality trait Harm Avoidance would mediate effects of recollected parental bonding during the first sixteen years of life on measures of perceived stress and mental distress severity in adulthood. METHOD: Five-hundred-eighteen adults (65.1 % women), aged 18-53years, completed questionnaires of parental bonding, perceived stress, trait Harm Avoidance, and severity of mental distress. Direct and indirect effects mediated through trait Harm Avoidance were examined in a structural equation model. RESULTS: Under the causal assumptions of our proposed model, indirect effects of trait Harm Avoidance mediated the relationship between parental overprotection and severity of mental distress, while significantly attenuating the direct effects of parental care on severity of mental distress. Moreover, indirect effects of trait Harm Avoidance significantly attenuated the direct effects of parental overprotection and care on perceived stress. CONCLUSION: In this large sample of mentally healthy adults, recollected parental bonding was significantly associated with levels of perceived stress and severity of mental distress. The results from our proposed model further suggest that trait Harm Avoidance may be a developmental link, by which the quality of recollected parental bonding in childhood translates into adult vulnerability to stress and mental distress.

Central 5-HT4 receptor binding as biomarker of serotonergic tonus in humans: a [11C]SB207145 PET study.

Identification of a biomarker that can inform on extracellular serotonin (5-HT) levels in the brains of living humans would enable greater understanding of the way brain circuits are modulated by serotonergic neurotransmission. Substantial evidence from studies in animals and humans indicates an inverse relationship between central 5-HT tonus and 5-HT type 4 receptor (5-HT4R) density, suggesting that 5-HT4R receptor density may be a biomarker marker for 5-HT tonus. Here, we investigated whether a 3-week administration of a selective serotonin reuptake inhibitor, expected to increase brain 5-HT levels, is associated with a decline in brain 5-HT4R binding. A total of 35 healthy men were studied in a placebo-controlled, randomized, double-blind study. Participants were assigned to receive 3 weeks of oral dosing with placebo or fluoxetine, 40 mg per day. Brain 5-HT4R binding was quantified at baseline and at follow-up with [(11)C]SB207145 positron emission tomography (PET). Three weeks of intervention with fluoxetine was associated with a 5.2% reduction in brain 5-HT4R binding (P=0.017), whereas placebo intervention did not change 5-HT4R binding (P=0.52). Our findings are consistent with a model, wherein the 5-HT4R density adjusts to changes in the extracellular 5-HT tonus. Our data demonstrate for the first time in humans that the imaging of central 5-HT4R binding may be used as an in vivo biomarker of the central 5-HT tonus.

Articular chondrocytes express connexin 43 hemichannels and P2 receptors - a putative mechanoreceptor complex involving the primary cilium?

Mechanical loading is essential for the health and homeostasis of articular cartilage, although the fundamental mechanotransduction pathways are unclear. Previous studies have demonstrated that cyclic compression up-regulates proteoglycan synthesis via an intracellular Ca(2+) signalling pathway, mediated by the release of ATP. However, the mechanism(s) of ATP release has not been elucidated. The present study examines expression of the putative mechanosensitive ATP-release channel, connexin 43 and whether it is expressed on the chondrocyte primary cilium, which acts as a mechanosensor in a variety of other cell types. In addition the study characterized the expression of a range of purine receptors through which ATP may activate downstream signalling events controlling cell function. Bovine articular chondrocytes were isolated by sequential enzyme digestion and seeded in agarose constructs. To verify the presence of functional hemichannels, Lucifer yellow (LY) uptake into viable cells was quantified following treatment with a hemichannel agonist (EGTA) and antagonist (flufenamic acid). LY uptake was observed in 45% of chondrocytes, increasing to 83% following EGTA treatment (P < 0.001). Treatment with the hemichannel blocker, flufenamic acid, significantly decreased LY uptake to less than 5% with and without EGTA. Immunofluorescence and confocal microscopy confirmed the presence of primary cilia and the expression of connexin 43. Approximately 50% of bovine chondrocyte primary cilia were decorated with connexin 43. Human chondrocytes in situ within cartilage explants also expressed connexin 43 hemichannels. However, expression was confined to the upper 200 microm of the tissue closest to the articular surface. Immunofluorescence revealed the expression of a range of P2X and P2Y receptor subtypes within human articular cartilage. In conclusion, the expression of functional hemichannels by articular chondrocytes may represent the mechanism through which mechanical loading activates ATP release as part of a purinergic mechanotransduction pathway. Furthermore, the expression of connexin 43 on the chondrocyte primary cilium suggests the possible involvement of the cilium in this pathway.

Primary cilia in osteoarthritic chondrocytes: from chondrons to clusters.

Osteoarthritis (OA) is a common joint disease characterized by articular cartilage degeneration. The etiology of OA is unknown. Because several previous studies have shown that primary cilia play critical roles in joint development, this study examined the incidence and morphology of primary cilia in chondrocytes during joint degeneration in a naturally occurring bovine model of OA. Primary cilia were detected using antibodies to acetylated alpha-tubulin in normal cartilage as well as in mild and severe OA tissue. In normal cartilage, cilia number and length were lowest in the superficial zone and increased with distance from the articular surface. In OA tissue, the incidence and length of cilia increased at the eroding articulating surface, resulting in an overall increased proportion of cilia. This is the first study to show that primary cilia are present on chondrocytes throughout OA progression and that the overall percentage of ciliated cells within the degenerating cartilage increases with OA severity.

Articular cartilage and growth plate defects are associated with chondrocyte cytoskeletal abnormalities in Tg737orpk mice lacking the primary cilia protein polaris.

Primary cilia are highly conserved organelles found on almost all eukaryotic cells. Tg737(orpk) (orpk) mice carry a hypomorphic mutation in the Tg737 gene resulting in the loss of polaris, a protein essential for ciliogenesis. Orpk mice have an array of skeletal patterning defects and show stunted growth after birth, suggesting defects in appositional and endochondral development. This study investigated the association between orpk tibial long bone growth and chondrocyte primary cilia expression using histomorphometric and immunohistochemical analysis. Wild-type chondrocytes throughout the developing epiphysis and growth plate expressed primary cilia, which showed a specific orientation away from the articular surface in the first 7-10 cell layers. In orpk mice, primary cilia were identified on very few cells and were significantly shorter. Orpk chondrocytes also showed significant increases in cytoplasmic tubulin, a likely result of failed ciliary assembly. The growth plates of orpk mice were significantly smaller in length and width, with marked changes in cellular organization in the presumptive articular cartilage, proliferative and hypertrophic zones. Cell density at the articular surface and in the hypertrophic zone was significantly altered, suggesting defects in both appositional and endochondral growth. In addition, orpk hypertrophic chondrocytes showed re-organization of the F-actin network into stress fibres and failed to fully undergo hypertrophy, while there was a marked reduction in type X collagen sequestration. These data suggest that failure to form a functional primary cilium affects chondrocyte differentiation and results in delayed chondrocyte hypertrophy within the orpk growth plate.

Nitric oxide levels and ciliary beat frequency in indigenous New Zealand children.

New Zealand children's morbidity from respiratory disease is high. This study examines whether subclinical ciliary abnormalities underlie the increased prevalence of respiratory disease in indigenous New Zealand children. A prospective study enrolled a group of healthy children who were screened for respiratory disease by questionnaire and lung function. Skin-prick tests were performed to control for atopy. Exhaled and nasal NO was measured online by a single-breath technique using chemiluminescence. Ciliary specimens were obtained by nasal brushings for assessment of structure and function. The ciliary beat frequency (CBF) (median CBF, 12.5 Hz; range, 10.4-16.8 Hz) and NO values (median exhaled NO, 5.6 ppb; range, 2.3-87.7 ppb; median nasal NO, 403 ppb; range, 34-1,120 ppb) for healthy New Zealand European (n=58), Pacific Island (n=61), and Maori (n=16) children were comparable with levels reported internationally. No ethnic differences in NO, atopy, or CBF were demonstrated. Despite an apparently normal ciliary beat, the percentage of ciliary structural defects was 3 times higher than reported controls (9%; range, 3.6-31.3%), with no difference across ethnic groups. In conclusion, it is unlikely that subclinical ciliary abnormalities underlie the increased prevalence of respiratory disease in indigenous New Zealand children. The high percentage of secondary ciliary defects suggests ongoing environmental or infective damage.

Ultrastructural, tomographic and confocal imaging of the chondrocyte primary cilium in situ.

Hyaline cartilage chondrocytes express one primary cilium per cell, but its function remains unknown. We examined the ultrastructure of chick embryo sternal chondrocyte cilia and their interaction with extracellular matrix molecules by transmission electron microscopy (TEM) and, for the first time, double-tilt electron tomography. Ciliary bending was also examined by confocal immunohistochemistry. Tomography and TEM showed the ciliary axoneme to interdigitate amongst collagen fibres and condensed proteoglycans. TEM also revealed the presence of electron-opaque particles in the proximal axoneme which may represent intraciliary-transport (ICT) particles. We observed a wide range of ciliary bending patterns. Some conformed to a heavy elastica model associated with shear stress. Others were acutely deformed, suggesting ciliary deflection by collagen fibres and proteoglycans with which the cilia make contact. We conclude that mechanical forces transmitted through these matrix macromolecules bend the primary cilium, identifying it as a potential mechanosensor involved in skeletal patterning and growth.

An integrated environmental perfusion chamber and heating system for long-term, high resolution imaging of living cells.

This communication presents the design and application of an integrated environmental perfusion chamber and stage heating blanket suitable for time-lapse video microscopy of living cells. The system consists of two independently regulated components: a perfusion chamber suitable for the maintenance of cell viability and the variable delivery of environmental factors, and a separate heating blanket to control the temperature of the microscope stage and limit thermal conduction from the perfusion chamber. Two contrasting experiments are presented to demonstrate the versatility of the system. One long-term sequence illustrates the behaviour of cells exposed to ceramic fibres. The other shows the shrinking response of cultured articular cartilage chondrons under dynamic hyper-osmotic conditions designed to simulate joint loading. The chamber is simple in design, economical to produce and permits long-term examination of dynamic cellular behaviour while satisfying the fundamental requirements for the maintenance of environmental factors that influence cell viability.

The 65-kDa carrot microtubule-associated protein forms regularly arranged filamentous cross-bridges between microtubules.

In plants, cortical microtubules (MTs) occur in characteristically parallel groups maintained up to one microtubule diameter apart by fine filamentous cross-bridges. However, none of the plant microtubule-associated proteins (MAPs) so far purified accounts for the observed separation between MTs in cells. We previously isolated from carrot cytoskeletons a MAP fraction including 120- and 65-kDa MAPs and have now separated the 65-kDa carrot MAP by sucrose density centrifugation. MAP65 does not induce tubulin polymerization but induces the formation of bundles of parallel MTs in a nucleotide-insensitive manner. The bundling effect is inhibited by porcine MAP2, but, unlike MAP2, MAP65 is heat-labile. In the electron microscope, MAP65 appears as filamentous cross-bridges, maintaining an intermicrotubule spacing of 25-30 nm. Microdensitometer-computer correlation analysis reveals that the cross-bridges are regularly spaced, showing a regular axial spacing that is compatible with a symmetrical helical superlattice for 13 protofilament MTs. Because MAP65 maintains in vitro the inter-MT spacing observed in plants and is shown to decorate cortical MTs, it is proposed that this MAP is important for the organization of the cortical array in vivo.

Inhibition of cytokinesis by asbestos and synthetic fibres.

Using high-resolution timelapse microscopy, we have followed individual phagocytized fibres through the later stages of division in MeT-5A human mesothelial cells and LLC-MK(2)monkey epithelial cells. The fibres used were crocidolite and chrysotile asbestos, fibrous glass (MMVF), and refractory ceramic fibres (RCF). Long fibres (15-80 microm) trapped within the cleavage furrow can partially or completely block cytokinesis. Cells proceed in one of three ways: (1) eventual completion of cytokinesis; (2) incomplete cytokinesis, resulting in two cells joined by a fibre-containing intercellular channel; or (3) failure of cytokinesis, resulting in a binucleate or trinucleate cell. Two factors associated with fibre-induced bi/trinucleation are: (1) an initial association between the fibre and the forming daughter nuclei, which is sometimes lost over time, and (2) disintegration of the midbody. The studies suggest that delay of cytokinesis by interzonal fibres can result in bi/trinucleation through the loss of midbody/intercellular bridge proteins that are required for completion of cytokinesis.

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Long crocidolite asbestos fibers cause polyploidy by sterically blocking cytokinesis.

Asbestos has been described as a physical carcinogen in that its carcinogenic effects appear to be related primarily to fiber dimensions. It has been hypothesized that long asbestos fibers may interfere with chromosome distribution during cell division, causing genomic changes that lead to cell transformation and neoplastic progression. Using high-resolution time-lapse light microscopy and serial-section electron microscopy, we have followed individual crocidolite asbestos fibers through the later stages of cell division in LLC-MK2 epithelial cells, and have detailed for the first time their effect on cytokinesis. We found that long fibers (15-55 microgram), trapped by the cleavage furrow, sterically blocked cytokinesis, sometimes resulting in the formation of a binucleated cell. The ends of blocking fibers were usually found within invaginations of the newly formed nuclei. Nuclear envelope-fiber attachment was evident when a chromatin strand ran with the fiber into the intercellular bridge. Such strands may break, causing chromosome structural rearrangements. Our data are the first to show that individual crocidolite fibers can cause genomic changes by sterically blocking cytokinesis and that fiber length and affinity for the nuclear envelope are important factors. Such genomic changes may be among the initial events leading to asbestos-induced cancers.

Behavior of crocidolite asbestos during mitosis in living vertebrate lung epithelial cells.

Asbestos has been described as a physical carcinogen in that long thin fibers are generally more carcinogenic than shorter thicker ones. It has been hypothesized that long thin fibers disrupt chromosome behavior during mitosis, causing chromosome abnormalities which lead to cell transformation and neoplastic progression. Using high-resolution time lapse video-enhanced light microscopy and the uniquely suited lung epithelial cells of the newt Taricha granulosa, we have characterized for the first time the behavior of crocidolite asbestos fibers, and their interactions with chromosomes, during mitosis in living cells. We found that the keratin cage surrounding the mitotic spindle inhibited fiber migration, resulting in spindles with few fibers. As in interphase, fibers displayed microtubule-mediated saltatory movements. Fiber position was only slightly affected by the ejection forces of the spindle asters. Physical interactions between crocidolite fibers and chromosomes occurred randomly within the spindle and along its edge. Crocidolite fibers showed no affinity toward chromatin and most encounters ended with the fiber passively yielding to the chromosome. In a few encounters along the spindle edge the chromosome yielded to the fiber, which remained stationary as if anchored to the keratin cage. We suggest that fibers thin enough to be caught in the keratin cage and long enough to protrude into the spindle are those fibers with the ability to snag or block moving chromosomes.

Concentrations of 4.5S RNA and Ffh protein in Escherichia coli: the stability of Ffh protein is dependent on the concentration of 4.5S RNA.

We measured the concentrations of both 4.5S RNA and Ffh protein under a variety of growth conditions and found that there were 400 molecules of 4.5S RNA per 10,000 ribosomes in wild-type cells and that the concentration of Ffh protein was one-fourth of that. This difference in concentration is 1 order of magnitude less than that previously reported but still significant. Pulse-chase labeling experiments indicated that Ffh protein is unstable in cells carrying ffh on high-copy-number plasmids and that simultaneous overproduction of 4.5S RNA stabilizes Ffh protein. Our analyses show that free Ffh protein is degraded with a half-life of approximately 20 min. We also tested whether three previously isolated suppressors of 4.5S RNA deficiency could reduce the requirement for Ffh protein. Since the two sffE suppressors do not suppress the Ffh requirement, we suggest that 4.5S RNA either acts in a sequential reaction with Ffh or has two functions.

Effect of 4.5S RNA depletion on Escherichia coli protein synthesis and secretion.

We examined the synthesis of individual proteins following depletion of 4.5S RNA by using a strain deficient in the induction of heat shock proteins. We found that initially the synthesis of all proteins was equally affected, and the peptide elongation rate was reduced by approximately 10%. For up to 1 generation time after the onset of inhibition of total protein synthesis, the processing of secreted proteins was unaffected. After further depletion of 4.5S RNA, accumulation of precursors of secreted proteins was observed under some growth conditions.

Analysis of microtubule arms and bridges in mitotic and interphase amebae of Dictyostelium discoideum.

We have analyzed transparencies of electron micrographs from ultrathin longitudinal sections through mitotic spindles of undifferentiated amebae of Dictyostelium discoideum for the presence of arms on microtubules (MTs) and bridges between them. We used the technique of microdensitometer scanning and computer-based model matching by cross-correlation and autocorrelation. We also determined that spindle MTs are composed of 13 protofilaments. Although regularly arranged lateral appendages are not a universal feature of MTs in these cells, both cross-correlation and autocorrelation analysis revealed that bridges between a kinetochore MT and its neighbor, and between MTs in the zone of overlap of the central spindle were significantly arranged on a 12-dimer superlattice. In addition, the autocorrelation analysis indicated a slight match with the 12-dimer model for neighboring non-kinetochore MTs. Although electron micrographs revealed putative arms on cytoplasmic and astral MTs, as well as bridges between central spindle MTs outside the zone of overlap, their arrangement did not match any of the models tested. Bridges between non-kinetochore MTs in the half-spindles possibly serve to reinforce the spindle scaffold. Bridges between kinetochore MTs and their neighbors may contribute to the mechanical stability of kinetochore fibers or they may be involved in poleward movements of the chromosomes. In the zone of overlap of the central spindle, the occurrence of frequent and regularly spaced bridges is consistent with models predicting that a sliding mechanism operates between MTs of opposite polarity in this region of the spindle to produce its elongation.

Microdensitometer-computer correlation analysis of two distinct, spatially segregated classes of microtubule bridges in Allogromia pseudopodia.

Previous video-light microscopic studies have shown that the microtubule bundles in the pseudopodia of foraminiferan protists display several types of movements in vivo, including active bending, zipping/splaying, and axial translocations. To gain insight into the types and arrangement of microtubule-associated proteins (e.g., mechanoenzymes, crosslinkers) in such a highly dynamic system, we employed microdensitometric-computer correlation methods to analyze, quantitatively, intermicrotubule bridges in thin-section electron micrographs of Allogromia laticollaris and Allogromia sp. (strain NF). Two distinct bridges occupying mutually exclusive zones between adjacent microtubules were identified. Type I bridges displayed a single axial repeat (34 nm for A. laticollaris and 28 nm for Allogromia sp.) and Type II bridges showed a typical 12-dimer helical superlattice pattern. In A. laticollaris, the two types of bridges were morphologically distinct: Type I bridges were aligned perpendicular to the microtubule wall and were 23-nm wide with an electron-lucent core; Type II bridges were irregular filaments projecting from the microtubules at various angles. When compared with the known distribution of microtubule-associated proteins in other systems, our findings indicate that, in vivo, Allogromia pseudopodial microtubules are decorated with MAP2-like bridges interrupted by discrete clusters of a dynein-like component.

Primary cilia cycle in PtK1 cells: effects of colcemid and taxol on cilia formation and resorption.

The effects of colcemid (0.16-1.0 microM) and taxol (10 microM) on the primary cilia cycle in PtK1 cells were studied by antitubulin immunofluorescence microscopy and by high-voltage electron microscopy of serial 0.25-micron sections. Although these drugs induce a fully characteristic rearrangement (taxol) or disassembly (colcemid) of cytoplasmic microtubules, neither affects the structure of primary cilia formed prior to the treatment or the resorption of primary cilia during the initial stages of mitosis. Cells arrested in mitosis by taxol or colcemid remain in mitosis for 5-7 h at 37 degrees C and then form 4N "micronucleated" restitution nuclei. Formation of primary cilia in these micronucleated cells is blocked by colcemid in a concentration-dependent fashion: normal cilia with expanded (ie, bulbed) distal ends form at the lower (0.16-0.25 microM) concentrations, while both cilia formation and centriole replication are inhibited at the higher (greater than or equal to 1.0 microM) concentrations. However, even in the presence of 1.0 microM colcemid, existing centrioles acquire the appendages characteristically associated with ciliating centrioles and attach to the dorsal cell surface. Continuous treatment with colcemid thus produces a population of cells enriched for the early stages of primary cilia formation. Micronucleated cells formed from a continuous taxol treatment contain two normal centriole pairs, and one or both parenting centrioles possess a primary cilium. Taxol, which has been reported to stabilize microtubules in vitro, does not inhibit the cell-cycle-dependent assembly and disassembly of axonemal microtubules in vivo.