The intercentriolar linkage is critical for the ability of heterologous centrosomes to induce parthenogenesis in Xenopus.

Centrosomes isolated from various sources, including human cells, have the capacity to induce parthenogenetic development when injected into unfertilized amphibian eggs. We recently isolated calf thymus centrosomes and showed that they differ structurally and functionally from previously isolated centrosomes of KE37 cells, in that the two centrioles in calf thymocytes are linearly associated by their proximal ends through a mass of electron dense material and nucleate few microtubules from their distal ends (Komesli, S., F. Tournier, M. Paintrand, R. Margolis, D. Job, and M. Bornens. 1989. J. Cell Biol. 109:2869-2878). We report here that these centrosomes are also unable to induce egg cleavage and examine the various possibilities which could account for this lack of competence. The results show that: (a) the kinetics of microtubule assembly on calf thymus centrosomes in Xenopus extracts are comparable to those of KE37 centrosomes; (b) centrosomes isolated from thymus of calves raised under controlled conditions (without anabolic agents) also lack competence; (c) centrosomes isolated from bovine cells of other tissues are competent; (d) centrosomes isolated from thymus of three other species (rat, mouse, and human) are competent. Since the lack of activity of calf thymus centrosomes apparently was not linked to species or tissue differences, we compared the ultrastructure of the centrosomes in the various centrosome preparations. The results show a strict correlation between the linear arrangement of centrioles and the lack of activity of the centrosomes. They suggest that the centrosome cycle can be blocked when the centrioles are prevented from separating into a nonlinear configuration, a step which might be critical for the initiation of procentriole budding. They also indicate that the centrosome may be involved in the G0-G1 transition.

Mass isolation of calf thymus centrosomes: identification of a specific configuration.

Centrosomes from calf thymocytes were isolated using a simple preparative procedure that provides large yields of free organelles. A comparative study with centrosomes isolated from human cultured lymphoblasts has led to the discovery of important differences in the structure of the two isolates and in their capacity to nucleate microtubules from purified tubulin. The possibility that the centrosomal structure depends upon the growth state of cells is discussed.

IL-13 alters mucociliary differentiation and ciliary beating of human respiratory epithelial cells.

In animal models of asthma, interleukin-13 (IL-13) induces goblet cell metaplasia, eosinophil infiltration of the bronchial mucosa, and bronchial hyperreactivity, but the basis of its effects on airway epithelia remain unknown. Lesions of the epithelial barrier, frequently observed in asthma and other chronic lung inflammatory diseases, are repaired through proliferation, migration, and differentiation of epithelial cells. An inflammatory process may then, therefore, influence epithelial regeneration. We have thus investigated the effect of IL-13 on mucociliary differentiation of human nasal epithelial cells in primary culture. We show that IL-13 alters ciliated cell differentiation and increases the proportion of secretory cells. IL-13 downregulates the actin-binding protein ezrin and other cytoskeletal components. IL-13 also impairs lateral cell contacts and interferes with the apical localization of ezrin seen in differentiated ciliated cells. In addition, an IL-4 antagonistic mutant protein (Y124D), which binds to the IL-4 receptor alpha subunit, a common chain of IL-4 and IL-13 receptors, inhibits IL-13's effects. IL-13 also decreases ciliary beat frequency in a time- and dose-dependent manner. These results suggest that, in human allergic asthmatic responses, IL-13 affects both ciliated and secretory cell differentiation, leading to airway damage and obstruction.

The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment.

Suppressor of fused, Su(fu), was identified as a semi-dominant suppressor of the putative serine/threonine kinase encoded by the segment polarity gene fused in Drosophila melanogaster. The amorphic Su(fu) mutation is viable, shows a maternal effect and displays no phenotype by itself. Su(fu) mutations are often found associated to karmoisin (kar) mutations but two complementation groups can be clearly identified. By using a differential hybridization screening method, we have cloned the Su(fu) region and identified chromosomal rearrangements associated with Su(fu) mutations. Two classes of cDNAs with similar developmental patterns, including a maternal contribution, are detectable in the region. Transformation experiments clearly assigned the Su(fu)+ function to one of these transcription units while the other one can be most likely assigned to the kar+ function. Surprisingly the 5' end of the kar RNA mapped within the 3' untranslated region of the Su(fu) transcribed sequence. The Su(fu) gene encodes a 53-kD protein, which contains a PEST sequence and shows no significant homologies with known proteins. Genetic analysis shows that proper development requires a fine tuning of the genetic doses of fu and Su(fu) both maternally and zygotically. These results, together with previous genetic and molecular data, suggest that fused and Suppressor of fused could act through a competitive posttraductionnal modification of a common target in the hedgehog signaling pathway.

Ciliated differentiation of rabbit tracheal epithelial cells in vitro.

Primary cultures of rabbit tracheal epithelial (RbTE) cells have been performed in two different ways. Quantitative analysis of both proliferative capacities and ciliated differentiation process were carried out using epithelial cell cultures from tracheal explants and from dissociated tracheal epithelial cells in air-liquid interface conditions. We show that both alpha- and beta-tubulins from RbTE cells are polyglutamylated and that this posttranslational modification is restricted to cilia axonemes and centrioles of non-ciliated cells. A monoclonal antibody raised against polyglutamylated tubulins was used to quantify the proportion of ciliated cells. Even though epithelial cells from outgrowths obtained by the explant technique highly proliferated during the first days of culture, no ciliated differentiation occurred. On the other hand, using air-liquid interface conditions after proliferation of dissociated cells, we could observe and quantify a ciliated cell differentiation in vitro by both Western blot and flow cytometric analysis. The specific detection and quantification of ciliated cells open the way for the biochemical and molecular characterization of centriolar components during ciliated differentiation.

Thermodynamic Origin of the Vitreous Transition.

The vitreous transition is characterized by a freezing of atomic degrees of freedom at a temperature Tg depending on the heating and cooling rates. A kinetic origin is generally attributed to this phenomenon instead of a thermodynamic one which we develop here. Completed homogeneous nucleation laws reflecting the energy saving due to Fermi energy equalization of nascent crystals and their melt are used. They are applied to bulk metallic glasses and extended to inorganic glasses and polymers. A transition T*g among various Tg corresponds to a crystal homogeneous nucleation temperature, leading to a preliminary formation of a cluster distribution during the relaxation time preceding the long steady-state nucleation time of crystals in small samples. The thermally-activated energy barrier ΔG*2ls/kBT at T*g for homogeneous nucleation is nearly the same in all glass-forming melts and determined by similar values of viscosity and a thermally-activated diffusion barrier from melt to cluster. The glass transition T*g is a material constant and a linear function of the energy saving associated with charge transfers from nascent clusters to the melt. The vitreous transition and the melting temperatures alone are used to predict the free-volume disappearance temperature equal to the Vogel-Fulcher-Tammann temperature of fragile glass-forming melts, in agreement with many viscosity measurements. The reversible thermodynamic vitreous transition is determined by the disappearance temperature T*g of the fully-relaxed enthalpy Hr that is not time dependent; the observed specific heat jump at T*g is equal to the proportionality coefficient of Hr with (T*g - Ta) for T ≤ T*g as expected from the enthalpy excess stored by a quenched undercooled melt at the annealing temperature Ta and relaxed towards an equilibrium vitreous state. However, the heat flux measurements found in literature over the last 50 years only gave an out-of-equilibrium Tg since the enthalpy is continuous at T*g without visible heat jump.

[Production of partial blastulas by parthenogenesis in Xenopus]. / Production de blastulas partielles par parthénogenèse chez le xénope.

In mature Xenopus eggs, the cell cycle can be triggered by pricking the egg or by an electric shock. However, no cleavage occurs unless centriole-containing fractions or isolated centrosomes are injected at the time of egg activation. We have obtained for an average of one heterologous centrosome injected per oocyte a complete parthenogenetic development. We also observed that the success rate of blastula formation declined linearly with the time elapsing between oocyte activation and centrosome injection. Moreover, in most cases, large areas of the blastulas remained uncleaved, interfering with gastrulation and blocking further development.

Parthenogenesis in Xenopus eggs injected with centrosomes from synchronized human lymphoid cells.

In Xenopus eggs, normal development requires the participation of the centrosome provided by the sperm. Injection of foreign centrosomes purified from exponentially growing mammalian cells enables the eggs to undertake parthenogenesis. In order to know whether such a complementation required centrosomes already committed to duplication, we have prepared centrosomes from human cells synchronized at different stages of the cell cycle (G0, G1, G2). We show that the three types of centrosome possess a similar parthenogenetic activity and conclude that duplication of heterologous centrosome can be triggered in Xenopus eggs.

Centrosomes competent for parthenogenesis in Xenopus eggs support procentriole budding in cell-free extracts.

Heterologous centrosomes from diversed species including humans promote egg cleavage when injected into metaphase-arrested Xenopus eggs. We have recently isolated centrosomes from calf thymocytes and shown that they were unable to induce egg cleavage, an inability that was apparently correlated with the peculiar structure of these centrosomes rather than with a lack of microtubule-nucleating activity: the two centrioles were associated in a colinear orientation by their proximal ends. To promote cleavage, a heterologous centrosome probably is required to duplicate, although this has not yet been demonstrated. Therefore, we designed an in vitro assay that would enable us to directly observe the duplication process. We show that competent centrosomes from KE37 cells synchronized in G1 phase initiate procentriole budding in interphasic extracts from Xenopus eggs in the absence of protein synthesis, whereas calf thymocyte centrosomes do not. Since calf thymocyte centrosomes do not support parthenogenesis, the present results suggest that duplication of the foreign centrosome is required for centrosome-induced parthenogenesis. Furthermore, procentriole budding takes place in the absence of protein synthesis in egg extracts arrested in S phase. This in vitro assay should contribute to the identification of molecular mechanisms involved in the initiation of centrosome duplication.

Interleukin-13 induces proliferation of human airway epithelial cells in vitro via a mechanism mediated by transforming growth factor-alpha.

Remodeling of the airways, as occurs in asthmatic patients, is associated with the continual presence of inflammatory mediators and Th2 cytokines, especially interleukin (IL)-13, during cycles of epithelial injury and repair. In this study, we examined the effect of IL-13 on well-differentiated normal human bronchial epithelial (NHBE) cells maintained in air-liquid interface culture. IL-13 induced proliferation of NHBE cells after 24 h exposure, as reflected by [(3)H]thymidine uptake and cell counts. The effects of IL-13 were mediated through the epidermal growth factor receptor (EGFR), as proliferation was attenuated by AG1478, an EGFR tyrosine kinase inhibitor. Proliferation appeared to be mediated by transforming growth factor (TGF)-alpha, a potent ligand for EGFR, which was released rapidly from NHBE cells in response to IL-13. Neutralizing antibody to TGF-alpha, but not antibodies against other potentially important growth factors (EGF, heparin binding epidermal growth factor-like growth factor [HB-EGF], platelet-derived growth factor [PDGF]), inhibited the mitogenic response to IL-13. This study provides the first experimental evidence that IL-13 can initiate a proliferative response of human airway epithelium in the absence of inflammatory cells or other cell types. The results are consistent with a mechanism whereby IL-13 induces release of TGF-alpha from the epithelial cells, which in turn binds via an autocrine/paracrine-type action to the EGFR, initiating proliferation. IL-13-induced airway remodeling in vivo may involve this epithelium-driven response.

Effects of retinoic acid receptor-selective agonists on human nasal epithelial cell differentiation.

Retinoids play a critical role in the maintenance of the mucociliary phenotype of epithelial cells in the upper respiratory tract. To determine the role of retinoic acid receptors (RARs) in the regulation of epithelial differentiation, we tested the effect of the synthetic retinoids CD336, CD2019, and CD666, selective agonists for RARalpha, RARbeta, and RARgamma, respectively, during differentiation of human nasal epithelial (HNE) cells in vitro. Using glutamylated tubulin and transglutaminase I (Tg I) as markers of ciliated cell and squamous cell differentiation, respectively, we showed that retinoic acid (RA) stimulated mucociliary differentiation and, in parallel, inhibited squamous cell differentiation. The agonists of the three RARs independently induced ciliogenesis and inhibited squamous cell differentiation by downregulating Tg I expression in a dose- and time-dependent manner. Antagonists specific for the three RARs abolished the effects of the corresponding agonists, demonstrating an RAR-specific mediated effect. Moreover, treatment of retinoid-deficient cultures with RAR agonists induced conversion of the squamous-like phenotype into a ciliated phenotype. In conclusion, all three RARs are potentially involved in the differentiating effects of RA in respiratory epithelial cells.

Sinefungin and taxol effects on cell cycle and cytoskeleton of Leishmania donovani promastigotes.

Sinefungin is an antibiotic possessing a strong anti-leishmanial activity. Among the most important effects of this molecule on Leishmania donovani promastigotes are morphological modifications and a very rapid and effective inhibition of DNA synthesis. These cells contain a single DNA-rich mitochondrion whose division cycle is coordinated with the nuclear division cycle. We have developed a flow-cytometric procedure based upon mithramycin as fluorochrome that can perform quantitative cell cycle analysis on the nuclear DNA. Cell cycle progression was analyzed to establish that sinefungin irreversibly blocks the promastigotes in early S phase. Sinefungin did not react with stationary cells as they were arrested in G1. Surprisingly, taxol, a microtubule-stabilizing drug, induced the same morphological modifications as sinefungin although it interfered with the G2/M progression. According to immunofluorescence studies, the stable microtubular network is apparently affected neither by taxol nor by sinefungin.

Drosophila centrosomes are unable to trigger parthenogenetic development of Xenopus eggs.

Centrosomes are powerful and exclusive parthenogenetic agents in the Xenopus egg. We have previously shown that heterologous centrosomes from various vertebrate species were able to promote egg cleavage in Xenopus and that human centrosome activity was associated with an insoluble proteinacious structure that is not significantly simpler than the native centrosome. In this work, we have investigated the parthenogenetic capacity of more evolutionary distant centrosomes. We show that centrosomes devoid of centrioles, such as SPBs isolated from Saccharomyces cerevisiae, do not form asters of microtubules in cytoplasmic extracts from Xenopus eggs, and are inactive in the parthenogenetic test. We further show that Drosophila centrosomes which possess a typical centriole architecture, and are quite active to nucleate microtubules in Xenopus cytoplasmic extracts, are unable to trigger egg cleavage. This was observed both with centrosomes isolated from Drosophila syncytial embryos and nucleus-centrosome complexes from the Drosophila Kc23 cell line. We demonstrate that this inability could not be restored after pre-incubation of Drosophila centrosomes in the egg cytoplasm before injection. We conclude that the parthenogenetic activity of a centrosome is not directly linked to its capacity to nucleate microtubules from the egg tubulin, and that the evolutionary conserved nine-fold symmetrical structure of the centriole cannot be considered as sufficient for triggering procentriole assembly.

Polyglutamylation and polyglycylation of alpha- and beta-tubulins during in vitro ciliated cell differentiation of human respiratory epithelial cells.

Tubulins are the major proteins within centriolar and axonemal structures. In all cell types studied so far, numerous alpha- and beta-tubulin isoforms are generated both by expression of a multigenic family and various post-translational modifications. We have developed a primary culture of human nasal epithelial cells where the ciliated cell differentiation process has been observed and quantified. We have used this system to study several properties concerning polyglutamylation and polyglycylation of tubulin. GT335, a monoclonal antibody directed against glutamylated tubulins, stained the centriole/basal bodies and the axonemes of ciliated cells, and the centrioles of non-ciliated cells. By contrast, axonemal but not centriolar tubulins were polyglycylated. Several polyglutamylated and polyglycylated tubulin isotypes were detected by two-dimensional electrophoresis, using GT335 and a specific monoclonal antibody (TAP952) directed against short polyglycyl chains. Immunoelectron microscopy experiments revealed that polyglycylation only affected axonemal tubulin. Using the same technical approach, polyglutamylation was shown to be an early event in the centriole assembly process, as gold particles were detected in fibrogranular material corresponding to the first cytoplasmic structures involved in centriologenesis. In a functional assay, GT335 and TAP952 had a dose-dependent inhibitory effect on ciliary beat frequency. TAP952 had only a weak effect while GT335 treatment led to a total arrest of beating. These results strongly suggest that in human ciliated epithelial cells, tubulin polyglycylation has only a structural role in cilia axonemes, while polyglutamylation may have a function both in centriole assembly and in cilia activity.

Differential expression and cellular distribution of centrin isoforms during human ciliated cell differentiation in vitro.

Centrin protein is an ubiquitously expressed cytoskeletal component and is a member of the EF-hand superfamily of calcium-binding proteins. It was first discovered in the flagellar apparatus of unicellular green algae where it is involved in contraction of Ca(2+)-sensitive structures. Centrin protein is associated with centrosome-related structures such as spindle pole body in yeast, and centriole/basal bodies in flagellar and ciliated cells. Three centrin genes have been cloned in human cells. In this work, we have performed a comparative biochemical and functional analysis of centrin isoforms using a primary culture of human nasal epithelial cells which provides an efficient way to obtain a complete ciliated cell differentiation process. RT-PCR experiments show that the expression of the three human centrin genes increases during cell differentiation, and that only centrin 2 and 3 are expressed during cell proliferation. Using polyclonal antibodies raised against recombinant human centrin 2 and 3, we show a specific pattern of protein expression. Ultrastructural immunolocalization suggests that centrin proteins are involved in the early process of centriole assembly, as they are concentrated within the precursor structures of centriole/basal bodies. It also shows a differential localisation of centrin proteins in mature centriole/basal bodies, suggesting different functions for centrins 1/2 and centrin 3. This is also supported by functional analyses showing that centrin 1 and/or centrin 2 are involved in ciliary beating.

Experimental implementation of a Wiener filter in a hybrid digital--optical correlator.

We present the implementation of a clutter-tolerant filter in a hybrid correlator system. Wiener filters were mapped with a complex encoding technique onto a smectic A(*) liquid-crystal spatial light modulator (SLM). The technique overcomes the problem of representing high-dynamic-range data on SLM's that have limited modulation capabilities. It also provides a compact image recognition system that is robust enough for many real-world applications. Experimental results are presented.

Microfabrication by use of a spatial light modulator in the ultraviolet: experimental results.

We report the development of a new microstereophotolithography technique for creation of three-dimensional microcomponents by use of a planar, layer-by-layer process of exposure, in which a spatial light modulator is used as a dynamic lithographic mask. The system operates in the UV to take advantage of the wide supply of commercially available photopolymers designed for conventional stereolithography. With this novel procedure it is possible to build components with feature sizes as small as a few micrometers. The experimental setup is briefly described, and the first microcomponent fabricated by this system is shown.