Separable mechanisms drive local and global polarity establishment in the Caenorhabditiselegans intestinal epithelium.

Apico-basolateral polarization is essential for epithelial cells to function as selective barriers and transporters, and to provide mechanical resilience to organs. Epithelial polarity is established locally, within individual cells to establish distinct apical, junctional and basolateral domains, and globally, within a tissue where cells coordinately orient their apico-basolateral axes. Using live imaging of endogenously tagged proteins and tissue-specific protein depletion in the Caenorhabditiselegans embryonic intestine, we found that local and global polarity establishment are temporally and genetically separable. Local polarity is initiated prior to global polarity and is robust to perturbation. PAR-3 is required for global polarization across the intestine but local polarity can arise in its absence, as small groups of cells eventually established polarized domains in PAR-3-depleted intestines in a HMR-1 (E-cadherin)-dependent manner. Despite the role of PAR-3 in localizing PKC-3 to the apical surface, we additionally found that PAR-3 and PKC-3/aPKC have distinct roles in the establishment and maintenance of local and global polarity. Taken together, our results indicate that different mechanisms are required for local and global polarity establishment in vivo.

Versatile strategy for isolating transcription activator-like effector nuclease-mediated knockout mutants in Caenorhabditis elegans.

Targeted genome editing using transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 systems has recently emerged as a potentially powerful method for creating locus-specific mutations in Caenorhabditis elegans. Due to the low mutation frequencies, one of the crucial steps in using these technologies is screening animals that harbor a targeted mutation. In previous studies, identifying targeted mutations in C. elegans usually depended on observations of fluorescent markers such as a green fluorescent protein or visible phenotypes such as dumpy and uncoordinated phenotypes. However, this strategy is limited in practice because the phenotypes caused by targeted mutations such as defects in sensory behaviors are often apparently invisible. Here, we describe a versatile strategy for isolating C. elegans knockout mutants by TALEN-mediated genome editing and a heteroduplex mobility assay. We applied TALENs to engineer the locus of the neural gene glr-1, which is a C. elegans AMPA-type receptor orthologue that is known to have crucial roles in various sensory behaviors. Knockout mutations in the glr-1 locus, which caused defective mechanosensory behaviors, were efficiently identified by the heteroduplex mobility assay. Thus, we demonstrated the utility of a TALEN-based knockout strategy for creating C. elegans with mutations that cause invisible phenotypes.

Caenorhabditis elegans-based in vivo screening of bioactives from marine sponge-associated bacteria against Vibrio alginolyticus.

AIM: To establish Caenorhabditis elegans based in vivo method for screening bioactives from marine sponge associated bacteria (SAB) against Vibrio species. METHODS AND RESULTS: About 256 SAB isolates were screened for their ability to rescue C. elegans infected with Vibrio species. The chloroform extract of the positive isolate was subjected to column fractionation and purity of the active fraction was analysed using HPLC. Further, the components were elucidated using GC/MS. The active fraction was tested for its in vivo rescue activity, antibacterial and anti-QS activity. In vivo colonization reduction and biofilm inhibition efficiency were assessed using GFP-tagged V. alginolyticus using confocal laser scanning microscopy (CLSM). The ability of the active fraction in modulating expression of V. alginolyticus quorum sensing (QS) regulators luxT and lafK was measured using real-time PCR. The results indicated that the chloroform extract of SAB4.2 displayed significant rescue activity against V. alginolyticus by inhibiting the QS pathway. HPLC analysis of the active fraction revealed a single major peak and GC/MS analysis suggested Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl) as the major constituent. The potent bacterial isolate was identified as Alcaligenes faecalis. CONCLUSIONS: In vivo screening using C. elegans identified a marine isolate that inhibits the virulence of V. alginolyticus by interrupting the QS pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: The study provides a C. elegans based in vivo screening method for identifying bioactives from natural resources by overcoming the disadvantages of traditional in vitro plate assays.

Leuconostoc mesenteroides Strains Isolated from Carrots Show Probiotic Features.

Lactic acid bacteria (LAB) share several beneficial effects on human organisms, such as bioactive metabolites' release, pathogens' competition and immune stimulation. This study aimed at determining the probiotic potential of autochthonous lactic acid bacteria isolated from carrots. In particular, the work reported the characterization at the species level of four LAB strains deriving from carrots harvested in Fucino highland, Abruzzo (Italy). Ribosomal 16S DNA analysis allowed identification of three strains belonging to Leuconostoc mesenteroides and a Weissella soli strain. In vitro and in vivo assays were performed to investigate the probiotic potential of the different isolates. Among them, L. mesenteroides C2 and L. mesenteroides C7 showed high survival percentages under in vitro simulated gastro-intestinal conditions, antibiotic susceptibly and the ability to inhibit in vitro growth against Salmonella enterica serovar Typhimurium, Listeria monocytogenes, Pseudomonas aeruginosa and Staphylococcus aureus pathogens. In parallel, the simple model Caenorhabditis elegans was used for in vivo screenings. L. mesenteroides C2 and L. mesenteroides C7 strains significantly induced pro-longevity effects, protection from pathogens' infection and innate immunity stimulation. Overall, these results showed that some autochthonous LAB from vegetables such as carrots have functional features to be considered as novel probiotic candidates.

Kettin, the large actin-binding protein with multiple immunoglobulin domains, is essential for sarcomeric actin assembly and larval development in Caenorhabditis elegans.

Among many essential genes in the nematode Caenorhabditis elegans, let-330 is located on the left arm of chromosome V and was identified as the largest target of a mutagen in this region. However, let-330 gene has not been characterized at the molecular level. Here, we report that two sequenced let-330 alleles are nonsense mutations of ketn-1, a previously characterized gene encoding kettin. Kettin is a large actin-binding protein of 472 kDa with 31 immunoglobulin domains and is expressed in muscle cells in C. elegans. let-330/ketn-1 mutants are homozygous lethal at the first larval stage with mild defects in body elongation. These mutants have severe defects in sarcomeric actin and myosin assembly in striated muscle. However, α-actinin and vinculin, which are components of the dense bodies anchoring actin to the membranes, were not significantly disorganized by let-330/ketn-1 mutation. Kettin localizes to embryonic myofibrils before α-actinin is expressed, and α-actinin deficiency does not affect kettin localization in larval muscle. Depletion of vinculin minimally affects kettin localization but significantly reduces colocalization of actin with kettin in embryonic muscle cells. These results indicate that kettin is an essential protein for sarcomeric assembly of actin filaments in muscle cells.

Amyloid-beta (Aß1-42)-induced paralysis in Caenorhabditis elegans is reduced through NHR-49/PPARalpha.

Alzheimer´s disease is a neurodegenerative disorder characterized by the misfolding and aggregation of amyloid ß (Aß). Agonists of peroxisomal proliferator-activated receptors (PPARs) are discussed as anti-amyloidogenic compounds, e.g. due to their cholesterol-lowering activities. In a previous study we have shown in Caenorhabditis elegans expressing human Aß in muscle cells, that inhibition of steroid-signaling, by RNAi of respective members of the signaling pathway or by reducing cellular cholesterol uptake, both increases the nuclear translocation of the foxo transcription factor DAF-16 and concomitantly reduces Aß-induced paralysis. Using RNAi in the present study we show that NHR-49/PPARalpha inhibits steroidal-signaling upstream of DAF-9, a cytochrome P450-dependent enzyme which generates dafachronic acids as ligands for the nuclear hormone receptor DAF-12, and upstream of DAF-12 itself. The NHR-49/PPARalpha agonist fenofibrate reduces Aß-induced paralysis in dependence on nhr-49 and nuclear translocation of DAF-16. In conclusion, activation of NHR-49/PPARalpha inhibits the steroidal-signaling pathway which increases the nuclear translocation of DAF-16 and inhibits the Aß-induced phenotype in an Alzheimer model of C. elegans.

Transcription-independent TFIIIC-bound sites cluster near heterochromatin boundaries within lamina-associated domains in C. elegans.

BACKGROUND: Chromatin organization is central to precise control of gene expression. In various eukaryotic species, domains of pervasive cis-chromatin interactions demarcate functional domains of the genomes. In nematode Caenorhabditis elegans, however, pervasive chromatin contact domains are limited to the dosage-compensated sex chromosome, leaving the principle of C. elegans chromatin organization unclear. Transcription factor III C (TFIIIC) is a basal transcription factor complex for RNA polymerase III, and is implicated in chromatin organization. TFIIIC binding without RNA polymerase III co-occupancy, referred to as extra-TFIIIC binding, has been implicated in insulating active and inactive chromatin domains in yeasts, flies, and mammalian cells. Whether extra-TFIIIC sites are present and contribute to chromatin organization in C. elegans remains unknown. RESULTS: We identified 504 TFIIIC-bound sites absent of RNA polymerase III and TATA-binding protein co-occupancy characteristic of extra-TFIIIC sites in C. elegans embryos. Extra-TFIIIC sites constituted half of all identified TFIIIC binding sites in the genome. Extra-TFIIIC sites formed dense clusters in cis. The clusters of extra-TFIIIC sites were highly over-represented within the distal arm domains of the autosomes that presented a high level of heterochromatin-associated histone H3K9 trimethylation (H3K9me3). Furthermore, extra-TFIIIC clusters were embedded in the lamina-associated domains. Despite the heterochromatin environment of extra-TFIIIC sites, the individual clusters of extra-TFIIIC sites were devoid of and resided near the individual H3K9me3-marked regions. CONCLUSION: Clusters of extra-TFIIIC sites were pervasive in the arm domains of C. elegans autosomes, near the outer boundaries of H3K9me3-marked regions. Given the reported activity of extra-TFIIIC sites in heterochromatin insulation in yeasts, our observation raised the possibility that TFIIIC may also demarcate heterochromatin in C. elegans.

Depletion of cdc-25.3, a Caenorhabditis elegans orthologue of cdc25, increases physiological germline apoptosis.

In Caenorhabditis elegans hermaphrodites, physiological germline apoptosis is higher in cdc-25.3 mutants than in wild-type. The elevated germline apoptosis in cdc-25.3 mutants seems to be induced by accumulation of double-stranded DNA breaks (DSBs). Both DNA damage and synapsis checkpoint genes are required to increase the germline apoptosis. Notably, the number of germ cells that lose P-granule components, PGL-1 and PGL-3, increase in cdc-25.3 mutants, and the increase in germline apoptosis requires the activity of SIR-2.1, a Sirtuin orthologue. These results suggest that elevation of germline apoptosis in cdc-25.3 mutants is induced by accumulation of DSBs, leading to a loss of PGL-1 and PGL-3 in germ cells, which promotes cytoplasmic translocation of SIR-2.1, and finally activates the core apoptotic machinery.

Acute blockade of the Caenorhabditis elegans dopamine transporter DAT-1 by the mammalian norepinephrine transporter inhibitor nisoxetine reveals the influence of genetic modifications of dopamine signaling in vivo.

Modulation of neurotransmission by the catecholamine dopamine (DA) is conserved across phylogeny. In the nematode Caenorhabditis elegans, excess DA signaling triggers Swimming-Induced Paralysis (Swip), a phenotype first described in animals with loss of function mutations in the presynaptic DA transporter (dat-1). Swip has proven to be a phenotype suitable for the identification of novel dat-1 mutations as well as the identification of novel genes that impact DA signaling. Pharmacological manipulations can also induce Swip, though the reagents employed to date lack specificity and potency, limiting their use in evaluation of dat-1 expression and function. Our lab previously established the mammalian norepinephrine transporter (NET) inhibitor nisoxetine to be a potent antagonist of DA uptake conferred by DAT-1 following heterologous expression. Here we demonstrate the ability of low (µM) concentrations of nisoxetine to trigger Swip within minutes of incubation, with paralysis dependent on DA release and signaling, and non-additive with Swip triggered by dat-1 deletion. Using nisoxetine in combination with genetic mutations that impact DA release, we further demonstrate the utility of the drug for demonstrating contributions of presynaptic DA receptors and ion channels to Swip. Together, these findings reveal nisoxetine as a powerful reagent for monitoring multiple dimensions of DA signaling in vivo, thus providing a new resource that can be used to evaluate contributions of dat-1 and other genes linked to DA signaling without the potential for compensations that attend constitutive genetic mutations.

ß-Phenylethylamine requires the dopamine transporter to increase extracellular dopamine in Caenorhabditis elegans dopaminergic neurons.

ß-Phenylethylamine (ßPEA) is an endogenous amine that has been shown to increase the synaptic levels of dopamine (DA). A number of in vitro and behavioral studies suggest the dopamine transporter (DAT) plays a role in the effects generated by ßPEA, however the mechanism through which ßPEA affects DAT has not yet been elucidated. Here, we used Caenorhabditis (C.) elegans DAT (DAT-1) expressing LLC-pk1 cells and neuronal cultures to investigate whether the ßPEA-induced increase of extracellular DA required DAT-1. Our data show that ßPEA increases extracellular dopamine both in DAT-1 transfected cells and cultures of differentiated neurons. RTI-55, a cocaine homologue and DAT inhibitor, completely blocked the ßPEA-induced effect in transfected cells. However in neuronal cultures, RTI-55 only partly inhibited the increase of extracellular DA generated by ßPEA. These results suggest that ßPEA requires DAT-1 and other, not yet identified proteins, to increase extracellular DA when tested in a native system. Furthermore, our results suggest that ßPEA-induced increase of extracellular DA does not require functional monoamine vesicles as genetic ablation of the C. elegans homologue vesicular monoamine transporter, cat-1, did not compromise the ability of ßPEA to increase extracellular DA. Finally, our electrophysiology data show that ßPEA caused fast-rising and self-inactivating amperometric currents in a subset of wild-type DA neurons but not in neurons isolated from dat-1 knockout animals. Taken together, these data demonstrate that in both DA neurons and heterogeneous cultures of differentiated C. elegans neurons, ßPEA releases cytoplasmic DA through DAT-1 to ultimately increase the extracellular concentration of DA.