CCTα and CCTδ chaperonin subunits are essential and required for cilia assembly and maintenance in Tetrahymena

Background - The eukaryotic cytosolic chaperonin CCT is a hetero-oligomeric complex formed by two rings connected back-to-back, each composed of eight distinct subunits (CCTalpha to CCTzeta). CCT complex mediates the folding, of a wide range of newly synthesised proteins including tubulin (alpha, beta and gamma) and actin, as quantitatively major substrates. Methodology/Principal findings - We disrupted the genes encoding CCTalpha and CCTdelta subunits in the ciliate Tetrahymena. Cells lacking the zygotic expression of either CCTalpha or CCTdelta showed a loss of cell body microtubules, failed to assemble new cilia and died within 2 cell cycles. We also show that loss of CCT subunit activity leads to axoneme shortening and splaying of tips of axonemal microtubules. An epitope-tagged CCTalpha rescued the gene knockout phenotype and localized primarily to the tips of cilia. A mutation in CCTalpha, G346E, at a residue also present in the related protein implicated in the Bardet Biedel Syndrome, BBS6, also caused defects in cilia and impaired CCTalpha localization in cilia. Conclusions/Significance - Our results demonstrate that the CCT subunits are essential and required for ciliary assembly and maintenance of axoneme structure, especially at the tips of cilia.

Improving a Cluster Based Directional Channel Model in Realistic Macro-Cell Environment

In this paper a realistic directional channel model that is an extension of the COST 273 channel model is presented. The model uses a cluster of scatterers and visibility region generation based strategy with increased realism, due to the introduction of terrain and clutter information. New approaches for path-loss prediction and line of sight modeling are considered, affecting the cluster path gain model implementation. The new model was implemented using terrain, clutter, street and user mobility information for the city of Lisbon, Portugal. Some of the model's outputs are presented, mainly path loss and small/large-scale fading statistics.

Equilibrium self-assembly of colloids with distinct interaction sites: Thermodynamics, percolation, and cluster distribution functions

We calculate the equilibrium thermodynamic properties, percolation threshold, and cluster distribution functions for a model of associating colloids, which consists of hard spherical particles having on their surfaces three short-ranged attractive sites (sticky spots) of two different types, A and B. The thermodynamic properties are calculated using Wertheim's perturbation theory of associating fluids. This also allows us to find the onset of self-assembly, which can be quantified by the maxima of the specific heat at constant volume. The percolation threshold is derived, under the no-loop assumption, for the correlated bond model: In all cases it is two percolated phases that become identical at a critical point, when one exists. Finally, the cluster size distributions are calculated by mapping the model onto an effective model, characterized by a-state-dependent-functionality (f) over bar and unique bonding probability (p) over bar. The mapping is based on the asymptotic limit of the cluster distributions functions of the generic model and the effective parameters are defined through the requirement that the equilibrium cluster distributions of the true and effective models have the same number-averaged and weight-averaged sizes at all densities and temperatures. We also study the model numerically in the case where BB interactions are missing. In this limit, AB bonds either provide branching between A-chains (Y-junctions) if epsilon(AB)/epsilon(AA) is small, or drive the formation of a hyperbranched polymer if epsilon(AB)/epsilon(AA) is large. We find that the theoretical predictions describe quite accurately the numerical data, especially in the region where Y-junctions are present. There is fairly good agreement between theoretical and numerical results both for the thermodynamic (number of bonds and phase coexistence) and the connectivity properties of the model (cluster size distributions and percolation locus).

DRhoGEF2 regulates cellular tension and cell pulsations in the Amnioserosa during Drosophila dorsal closure

Coordination of apical constriction in epithelial sheets is a fundamental process during embryogenesis. Here, we show that DRhoGEF2 is a key regulator of apical pulsation and constriction of amnioserosal cells during Drosophila dorsal closure. Amnioserosal cells mutant for DRhoGEF2 exhibit a consistent decrease in amnioserosa pulsations whereas overexpression of DRhoGEF2 in this tissue leads to an increase in the contraction time of pulsations. We probed the physical properties of the amnioserosa to show that the average tension in DRhoGEF2 mutant cells is lower than wild-type and that overexpression of DRhoGEF2 results in a tissue that is more solid-like than wild-type. We also observe that in the DRhoGEF2 overexpressing cells there is a dramatic increase of apical actomyosin coalescence that can contribute to the generation of more contractile forces, leading to amnioserosal cells with smaller apical surface than wild-type. Conversely, in DRhoGEF2 mutants, the apical actomyosin coalescence is impaired. These results identify DRhoGEF2 as an upstream regulator of the actomyosin contractile machinery that drives amnioserosa cells pulsations and apical constriction.

The expression of tubulin cofactor A (TBCA) is regulated by a noncoding antisense Tbca RNA during testis maturation

Abstract - Recently, long noncoding RNAs have emerged as pivotal molecules for the regulation of coding genes' expression. These molecules might result from antisense transcription of functional genes originating natural antisense transcripts (NATs) or from transcriptional active pseudogenes. TBCA interacts with β-tubulin and is involved in the folding and dimerization of new tubulin heterodimers, the building blocks of microtubules. Methodology/Principal findings: We found that the mouse genome contains two structurally distinct Tbca genes located in chromosomes 13 (Tbca13) and 16 (Tbca16). Interestingly, the two Tbca genes albeit ubiquitously expressed, present differential expression during mouse testis maturation. In fact, as testis maturation progresses Tbca13 mRNA levels increase progressively, while Tbca16 mRNA levels decrease. This suggests a regulatory mechanism between the two genes and prompted us to investigate the presence of the two proteins. However, using tandem mass spectrometry we were unable to identify the TBCA16 protein in testis extracts even in those corresponding to the maturation step with the highest levels of Tbca16 transcripts. These puzzling results led us to re-analyze the expression of Tbca16. We then detected that Tbca16 transcription produces sense and natural antisense transcripts. Strikingly, the specific depletion by RNAi of these transcripts leads to an increase of Tbca13 transcript levels in a mouse spermatocyte cell line. Conclusions/Significance: Our results demonstrate that Tbca13 mRNA levels are post-transcriptionally regulated by the sense and natural antisense Tbca16 mRNA levels. We propose that this regulatory mechanism operates during spermatogenesis, a process that involves microtubule rearrangements, the assembly of specific microtubule structures and requires critical TBCA levels.

Mob1: defining cell polarity for proper cell division

Mob1 is a component of both the mitotic exit network and Hippo pathway, being required for cytokinesis, control of cell proliferation and apoptosis. Cell division accuracy is crucial in maintaining cell ploidy and genomic stability and relies on the correct establishment of the cell division axis, which is under the control of the cell's environment and its intrinsic polarity. The ciliate Tetrahymena thermophila possesses a permanent anterior-posterior axis, left-right asymmetry and divides symmetrically. These unique features of Tetrahymena prompted us to investigate the role of Tetrahymena Mob1. Unexpectedly, we found that Mob1 accumulated in basal bodies at the posterior pole of the cell, and is the first molecular polarity marker so far described in Tetrahymena. In addition, Mob1 depletion caused the abnormal establishment of the cell division plane, providing clear evidence that Mob1 is important for its definition. Furthermore, cytokinesis was arrested and ciliogenesis delayed in Tetrahymena cells depleted of Mob1. This is the first evidence for an involvement of Mob1 in cilia biology. In conclusion, we show that Mob1 is an important cell polarity marker that is crucial for correct division plane placement, for cytokinesis completion and for normal cilia growth rates.

A new cloning system based on the OprI lipoprotein for the production of recombinant bacterial cell wall-derived immunogenic formulations

The conjugation of antigens with ligands of pattern recognition receptors (PRR) is emerging as a promising strategy for the modulation of specific immunity. Here, we describe a new Escherichia coli system for the cloning and expression of heterologous antigens in fusion with the OprI lipoprotein, a TLR ligand from the Pseudomonas aeruginosa outer membrane (OM). Analysis of the OprI expressed by this system reveals a triacylated lipid moiety mainly composed by palmitic acid residues. By offering a tight regulation of expression and allowing for antigen purification by metal affinity chromatography, the new system circumvents the major drawbacks of former versions. In addition, the anchoring of OprI to the OM of the host cell is further explored for the production of novel recombinant bacterial cell wall-derived formulations (OM fragments and OM vesicles) with distinct potential for PRR activation. As an example, the African swine fever virus ORF A104R was cloned and the recombinant antigen was obtained in the three formulations. Overall, our results validate a new system suitable for the production of immunogenic formulations that can be used for the development of experimental vaccines and for studies on the modulation of acquired immunity.

Determination of chitin content in fungal cell wall: an alternative flow cytometric method

The conventional methods used to evaluate chitin content in fungi, such as biochemical assessment of glucosamine release after acid hydrolysis or epifluorescence microscopy, are low throughput, laborious, time-consuming, and cannot evaluate a large number of cells. We developed a flow cytometric assay, efficient, and fast, based on Calcofluor White staining to measure chitin content in yeast cells. A staining index was defined, its value was directly related to chitin amount and taking into consideration the different levels of autofluorecence. Twenty-two Candida spp. and four Cryptococcus neoformans clinical isolates with distinct susceptibility profiles to caspofungin were evaluated. Candida albicans clinical isolate SC5314, and isogenic strains with deletions in chitin synthase 3 (chs3Δ/chs3Δ) and genes encoding predicted Glycosyl Phosphatidyl Inositol (GPI)-anchored proteins (pga31Δ/Δ and pga62Δ/Δ), were used as controls. As expected, the wild-type strain displayed a significant higher chitin content (P < 0.001) than chs3Δ/chs3Δ and pga31Δ/Δ especially in the presence of caspofungin. Ca. parapsilosis, Ca. tropicalis, and Ca. albicans showed higher cell wall chitin content. Although no relationship between chitin content and antifungal drug susceptibility phenotype was found, an association was established between the paradoxical growth effect in the presence of high caspofungin concentrations and the chitin content. This novel flow cytometry protocol revealed to be a simple and reliable assay to estimate cell wall chitin content of fungi.

A novel flow cytometric protocol for assessment of yeast cell adhesion

Microbial adhesion is a field of recognized relevance and, as such, an impressive array of tools has been developed to understand its molecular mechanisms and ultimately for its quantification. Some of the major limitations found within these methodologies concern the incubation time, the small number of cells analyzed, and the operator's subjectivity. To overcome these aspects, we have developed a quantitative method to measure yeast cells' adhesion through flow cytometry. In this methodology, a suspension of yeast cells is mixed with green fluorescent polystyrene microspheres (uncoated or coated with host proteins). Within 2 h, an adhesion profile is obtained based on two parameters: percentage and cells-microsphere population's distribution pattern. This flow cytometry protocol represents a useful tool to quantify yeast adhesion to different substrata in a large scale, providing manifold data in a speedy and informative manner.

TBCCD1, a new centrosomal protein, is required for centrosome and Golgi apparatus positioning

In animal cells the centrosome is positioned at the cell centre in close association with the nucleus. The mechanisms responsible for this are not completely understood. Here, we report the first characterization of human TBCC-domain containing 1 (TBCCD1), a protein related to tubulin cofactor C. TBCCD1 localizes at the centrosome and at the spindle midzone, midbody and basal bodies of primary and motile cilia. Knockdown of TBCCD1 in RPE-1 cells caused the dissociation of the centrosome from the nucleus and disorganization of the Golgi apparatus. TBCCD1-depleted cells are larger, less efficient in primary cilia assembly and their migration is slower in wound-healing assays. However, the major microtubule-nucleating activity of the centrosome is not affected by TBCCD1 silencing. We propose that TBCCD1 is a key regulator of centrosome positioning and consequently of internal cell organization.

Cell-specific regulation of acetylcholinesterase expression under inflammatory conditions

Acetylcholine (ACh) has been shown to exert an anti-inflammatory function by down-modulating the expression of pro-inflammatory cytokines. Its availability can be regulated at different levels, namely at its synthesis and degradation steps. Accordingly, the expression of acetylcholinesterase (AChE), the enzyme responsible for ACh hydrolysis, has been observed to be modulated in inflammation. To further address the mechanisms underlying this effect, we aimed here at characterizing AChE expression in distinct cellular types pivotal to the inflammatory response. This study was performed in the human acute leukaemia monocytyc cell line, THP-1, in human monocyte-derived primary macrophages and in human umbilical cord vein endothelial cells (HUVEC). In order to subject these cells to inflammatory conditions, THP-1 and macrophage were treated with lipopolysaccharide (LPS) from E.coli and HUVEC were stimulated with the tumour necrosis factor α (TNF-α). Our results showed that although AChE expression was generally up-regulated at the mRNA level under inflammatory conditions, distinct AChE protein expression profiles were aurprisingly observed among the distinct cellular types studied. Altogether, these results argue for the existence of cell specific mechanisms that regulate the expression of acetylcholinesterase in inflammation.

Cobalt and Zinc Compounds Bearing 1,10-Phenanthroline-5,6-dione or 1,3,5-Triaza-7-phosphaadamantane Derivatives - Synthesis, Characterization, Cytotoxicity, and Cell Selectivity Studies

The compounds [mPTA][CoCl4] (1, mPTA = N-methyl-1,3,5-triaza-7-phosphaadamantane cation), [CoCl(H2O)(DION)(2)][BF4] (2, DION = 1,10-phenanthroline-5,6-dione), [Zn(DION)(2)]Cl-2 (3) and [ZnCl(O-PTA=O)(DION)][BF4] (4) were synthesized by reaction of CoCl2 with [mPTA]I or DION and ZnCl2 with DION or 1,3,5-triaza-7-phosphaadamantane-7-oxide (PTA=O) and DION, respectively. All complexes are water soluble and have been characterized by IR, far-IR, H-1, C-13 and P-31{H-1} NMR spectroscopy, ESI-MS, elemental analyses and single-crystal X-ray diffraction structural analysis (for 1). They were screened against the human tumour cell lines HCT116, HepG2 and MCF7. Complexes 2 and 3 exhibit the highest in vitro cytotoxicity and show lower cytotoxic activities in normal human fibroblast cell line than in HCT116 tumour cell line, which demonstrates their slight specificity for this type of tumour cell.

CCTα

In the cell, the correct folding of many proteins depends on the function of preexisting ones known as Molecular Chaperones (for a review see Hartl and Hayer-Hartl 2009). These, were defined as proteins that bind to and stabilize an otherwise unstable conformation of another protein, and by controlling binding and release, facilitate its correct fate in vivo, be it folding, oligomeric assembly, transport to a particular subcellular compartment, or disposal by degradation. Molecular chaperones do not convey steric information specifying correct folding: instead, they prevent incorrect interactions within and between nonnative peptides, thus typically increasing the yield but not the rate of folding reactions. Molecular chaperones are ubiquitous and comprise several protein families that are structurally unrelated (Hartl and Hayer-Hartl 2009). The Hsp70s and the Chaperonin families have been extensively studied.

Non-coding RNAs: multi-tasking molecules in the cell

In the last years it has become increasingly clear that the mammalian transcriptome is highly complex and includes a large number of small non-coding RNAs (sncRNAs) and long noncoding RNAs (lncRNAs). Here we review the biogenesis pathways of the three classes of sncRNAs, namely short interfering RNAs (siRNAs), microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs). These ncRNAs have been extensively studied and are involved in pathways leading to specific gene silencing and the protection of genomes against virus and transposons, for example. Also, lncRNAs have emerged as pivotal molecules for the transcriptional and post-transcriptional regulation of gene expression which is supported by their tissue-specific expression patterns, subcellular distribution, and developmental regulation. Therefore, we also focus our attention on their role in differentiation and development. SncRNAs and lncRNAs play critical roles in defining DNA methylation patterns, as well as chromatin remodeling thus having a substantial effect in epigenetics. The identification of some overlaps in their biogenesis pathways and functional roles raises the hypothesis that these molecules play concerted functions in vivo, creating complex regulatory networks where cooperation with regulatory proteins is necessary. We also highlighted the implications of biogenesis and gene expression deregulation of sncRNAs and lncRNAs in human diseases like cancer.

Translation termination and protein folding pathway genes are not correlated in gastric cancer

Background: The eukaryotic release factor 3 (eRF3) has been shown to affect both tubulin and actin cytoskeleton, suggesting a role in cytoskeleton assembly, mitotic spindle formation and chromosome segregation. Also, direct interactions between eRF3 and subunits of the cytosolic chaperonin CCT have been described. Moreover, both eRF3a and CCT subunits have been described to be up-regulated in cancer tissues. Our aim was to evaluate the hypothesis that eRF3 expression levels are correlated with the expression of genes encoding proteins involved in the tubulin folding pathways. Methods: Relative expression levels of eRF1, eRF3a/GSPT1, PFDN4, CCT2, CCT4, and TBCA genes in tumour samples relative to their adjacent normal tissues were investigated using real time-polymerase chain reaction in 20 gastric cancer patients. Results: The expression levels of eRF3a/GSPT1 were not correlated with the expression levels of the other genes studied. However, significant correlations were detected between the other genes, both within intestinal and diffuse type tumours. Conclusions: eRF3a/GSPT1 expression at the mRNA level is independent from both cell translation rates and from the expression of the genes involved in tubulin-folding pathways. The differences in the patterns of expression of the genes studied support the hypothesis of genetically independent pathways in the origin of intestinal and diffuse type gastric tumours.