Gβ1 controls collective cell migration by regulating the protrusive activity of leader cells in the posterior lateral line primordium

Collective cell migration is critical for normal development, tissue repair and cancer metastasis. Migration of the posterior lateral line primordium (pLLP) generates the zebrafish sensory organs (neuromasts, NMs). This migration is promoted by the leader cells at the leading edge of the pLLP, which express the G protein-coupled chemokine receptor Cxcr4b and respond to the chemokine Cxcl12a. However, the mechanism by which Cxc112a/Cxcr4b signaling regulates pLLP migration remains unclear. Here we report that signal transduction by the heterotrimeric G protein subunit Gβ1 is essential for proper pLLP migration. Although both Gβ1 and Gβ4 are expressed in the pLLP and NMs, depletion of Gβ1 but not Gβ4 resulted in an arrest of pLLP migration. In embryos deficient for Gβ1, the pLLP cells migrated in an uncoordinated fashion and were unable to extend protrusions at the leading front, phenocopying those in embryos deficient for Cxcl12a or Cxcr4b. A transplantation assay showed that, like Cxcr4b, Gβ1 is required only in the leader cells of the pLLP. Analysis of F-actin dynamics in the pLLP revealed that whereas wild-type leader cells display extensive actin polymerization in the direction of pLLP migration, counterparts defective for Gβ1, Cxcr4b or Cxcl12a do not. Finally, synergy experiments revealed that Gβ1 and Cxcr4b interact genetically in regulating pLLP migration. Collectively, our data indicate that Gβ1 controls migration of the pLLP, likely by acting downstream of the Cxcl12a/Cxcr4b signaling. This study also provides compelling evidence for functional specificity among Gβ isoforms in vivo.     

Experimental evidence of the reciprocal oxidation of Bovine Serum Albumin and Linoleate in aqueous solution,initiated by HO free radicals

An investigation of radiation-induced oxidation of aqueous bovine serum albumin (BSA) in the presence of linoleate (LH) at pH 10.5 has been carried out in order to better understand the respective oxidative processes involved in both lipid and protein phases. Solutions containing BSA (15 μmol L⁻¹) and linoleate (15–600 μmol L⁻¹) below the critical micellar concentration (cmc = 2000 μmol L⁻¹), have been irradiated by γ-rays (¹³⁷Cs) at radiation doses ranging from 10 to 400 Gy (dose rate 9.5 Gy min⁻¹). It can be noticed that, in the absence of BSA, the main hydroperoxides formed from HO^•-induced linoleate oxidation below the cmc, do not exhibit a conjugated dienic structure. This was also verified in the presence of BSA. Selected chemical markers of oxidation have been monitored: non-conjugated dienic hydroperoxides and conjugated dienes (without hydroperoxide function) for linoleate oxidation, and carbonyl groups for BSA oxidation. We have shown that for the lowest linoleate concentration (15 μmol L⁻¹) in the presence of BSA (15 μmol L⁻¹), the formation of conjugated dienes was not observed, meaning that LH was not exposed to HO^• radicals attack. However, non-conjugated dienic lipid hydroperoxides were simultaneously detected, indicating that LH was secondarily oxidised by BSA oxidised species. Moreover, the oxidation of linoleate was found to be enhanced by the presence of BSA. For the highest linoleate concentration (600 μmol L⁻¹), the expected protection of BSA by LH was not observed, even if LH monomers were responsible for the total scavenging of HO^• radicals. In this latter case, the formation of non-conjugated dienic lipid hydroperoxides was lower than expected. Those results showed that BSA was not oxidised by the direct action of HO^• radicals but was undergoing a secondary oxidation by non-dienic lipid hydroperoxides and/or lipid radical intermediates, coming from the HO^•-induced linoleate oxidation.     

Very-Long-Chain Fatty Acids Are Involved in Polar Auxin Transport and Developmental Patterning in Arabidopsis

Very-long-chain fatty acids (VLCFAs) are essential for many aspects of plant development and necessary for the synthesis of seed storage triacylglycerols, epicuticular waxes, and sphingolipids. Identification of the acetyl-CoA carboxylase PASTICCINO3 and the 3-hydroxy acyl-CoA dehydratase PASTICCINO2 revealed that VLCFAs are important for cell proliferation and tissue patterning. Here, we show that the immunophilin PASTICCINO1 (PAS1) is also required for VLCFA synthesis. Impairment of PAS1 function results in reduction of VLCFA levels that particularly affects the composition of sphingolipids, known to be important for cell polarity in animals. Moreover, PAS1 associates with several enzymes of the VLCFA elongase complex in the endoplasmic reticulum. The pas1 mutants are deficient in lateral root formation and are characterized by an abnormal patterning of the embryo apex, which leads to defective cotyledon organogenesis. Our data indicate that in both tissues, defective organogenesis is associated with the mistargeting of the auxin efflux carrier PIN FORMED1 in specific cells, resulting in local alteration of polar auxin distribution. Furthermore, we show that exogenous VLCFAs rescue lateral root organogenesis and polar auxin distribution, indicating their direct involvement in these processes. Based on these data, we propose that PAS1 acts as a molecular scaffold for the fatty acid elongase complex in the endoplasmic reticulum and that the resulting VLCFAs are required for polar auxin transport and tissue patterning during plant development.     

A new regulation of IL-6 production in adult cardiomyocytes by β-adrenergic and IL-1β receptors and induction of cellular hypertrophy by IL-6 trans-signalling

The sympathetic nervous system and pro-inflammatory cytokines play key roles in numerous cardiovascular disorders. Chronic β-adrenergic receptor (β-AR) stimulation in myocardium induces expression of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and interleukin-6 (IL-6), which contribute to cardiac hypertrophy and failure. To evaluate the relationship between β-AR stimulation and pro-inflammatory cytokines, we studied the effects of the β-AR agonist isoprenaline (ISO) on IL-1-induced IL-6 production in adult rat ventricular myocytes (ARVMs). We report that ISO and IL-1 synergistically enhanced IL-6 gene expression and secretion. The synergistic effect of ISO was mimicked by cAMP elevating agents and involved the G_s protein/cAMP/PKA signalling pathway, but not the exchange factor EPAC. To evaluate the contribution of IL-6 to cellular hypertrophy, we examined the signalling pathways stimulated by the membrane-bound IL-6 receptor (IL-6R), and the IL-6 soluble receptor (sIL-6R) involved in the mechanism named IL-6 trans-signalling. The IL-6/sIL-6R complex promoted a rapid and persistent phosphorylation of STAT3^Tyr705 in ARVMs. Moreover, IL-6 trans-signalling increased protein synthesis, c-fos gene expression and B-type natriuretic peptide secretion, three markers of cardiac hypertrophy. IL-6 trans-signalling also increased cell size. In contrast, IL-6 alone had no significant effect on either cell size or STAT3 phosphorylation although it induced phosphorylation of ERK1/2, AKT and S6K, demonstrating the presence of a functional IL-6R in ARVMs. Taken together, these results demonstrate that β-AR stimulation synergises with IL-1 for IL-6 secretion in adult ventricular myocytes and indicate that IL-6 induces cardiac hypertrophy only via IL-6 trans-signalling. The IL-6 soluble receptor may thus serve as a switch for IL-6 to activate STAT3 phosphorylation and hypertrophy.     

Ethylene triggers needle abscission in root-detached balsam fir

Post-harvest needle abscission is a major challenge for Christmas tree and greenery industries. It was hypothesized that ethylene triggers abscission in balsam fir. Three experiments were conducted to test this hypothesis. In experiment 1, 70 balsam fir branches were collected, placed in water, and ethylene evolution was observed over time. In experiment 2, a 2 × 5 factorial experiment was designed to determine the effect of exogenous ethylene and an ethylene receptor blocker, 1-methylcyclopropene (1-MCP), on needle abscission. In experiment 3, a 2 × 6 factorial experiment was designed to determine the effect of exogenous ethylene and an ethylene inhibitor, aminoethoxyvinylglycine (AVG), on needle abscission. It was found that ethylene evolution was the highest 1–2 days prior to needle abscission, which was consistent in untreated branches and branches exposed to exogenous ethylene. Exposure to exogenous ethylene significantly decreased needle retention by 63%. When ethylene receptors were blocked by 1-MCP, needle retention increased by 147% despite the presence of ethylene and increased by 73% in the absence of ethylene when compared to the respective controls. When endogenous ethylene synthesis was inhibited by AVG, there was no improvement in needle retention in the presence of ethylene, but there was a 113% increase in needle retention in the absence of exogenous ethylene. Ethylene is strongly implicated as the signal triggering abscission in root-detached balsam fir.     

Septal and lateral wall localization of PBP5, the major D,D‐carboxypeptidase of Escherichia coli,requires substrate recognition and membrane attachment

The distribution of PBP5, the major D,D‐carboxypeptidase in Escherichia coli, was mapped by immunolabelling and by visualization of GFP fusion proteins in wild‐type cells and in mutants lacking one or more D,D‐carboxypeptidases. In addition to being scattered around the lateral envelope, PBP5 was also concentrated at nascent division sites prior to visible constriction. Inhibiting PBP2 activity (which eliminates wall elongation) shifted PBP5 to midcell, whereas inhibiting PBP3 (which aborts divisome invagination) led to the creation of PBP5 rings at positions of preseptal wall formation, implying that PBP5 localizes to areas of ongoing peptidoglycan synthesis. A PBP5(S44G) active site mutant was more evenly dispersed, indicating that localization required enzyme activity and the availability of pentapeptide substrates. Both the membrane bound and soluble forms of PBP5 converted pentapeptides to tetrapeptides in vitro and in vivo, and the enzymes accepted the same range of substrates, including sacculi, Lipid II, muropeptides and artificial substrates. However, only the membrane‐bound form localized to the developing septum and restored wild‐type rod morphology to shape defective mutants, suggesting that the two events are related. The results indicate that PBP5 localization to sites of ongoing peptidoglycan synthesis is substrate dependent and requires membrane attachment.     

Association of Hemolytic Activity of Pseudomonas entomophila,a Versatile Soil Bacterium,with Cyclic Lipopeptide Production

Pseudomonas entomophila is an entomopathogenic bacterium that is able to infect and kill Drosophila melanogaster upon ingestion. Its genome sequence suggests that it is a versatile soil bacterium closely related to Pseudomonas putida. The GacS/GacA two-component system plays a key role in P. entomophila pathogenicity, controlling many putative virulence factors and AprA, a secreted protease important to escape the fly immune response. P. entomophila secretes a strong diffusible hemolytic activity. Here, we showed that this activity is linked to the production of a new cyclic lipopeptide containing 14 amino acids and a 3-C₁₀OH fatty acid that we called entolysin. Three nonribosomal peptide synthetases (EtlA, EtlB, EtlC) were identified as responsible for entolysin biosynthesis. Two additional components (EtlR, MacAB) are necessary for its production and secretion. The P. entomophila GacS/GacA two-component system regulates entolysin production, and we demonstrated that its functioning requires two small RNAs and two RsmA-like proteins. Finally, entolysin is required for swarming motility, as described for other lipopeptides, but it does not participate in the virulence of P. entomophila for Drosophila. While investigating the physiological role of entolysin, we also uncovered new phenotypes associated with P. entomophila, including strong biocontrol abilities.Pseudomonas entomophila is a recently isolated Pseudomonas species that is closely related to the saprophytic soil bacterium Pseudomonas putida. It was initially characterized as a natural pathogen of Drosophila (63). Indeed, P. entomophila was first isolated from flies sampled in Guadeloupe, and it is highly pathogenic for Drosophila larvae and adults. P. entomophila can also effectively kill members of other insect orders (e.g., Bombyx mori, Anopheles gambiae), which makes it a new entomopathogenic bacterium. Its ability to infect and kill Drosophila melanogaster very efficiently after ingestion makes it an appropriate model for the study of host-pathogen interactions (38, 62, 63).In order to unravel features contributing to the entomopathogenic properties of P. entomophila, its genome was sequenced. The results suggest that this strain is a ubiquitous, metabolically versatile bacterium that may colonize diverse habitats, including soil, rhizosphere, and aquatic systems, as shown for P. putida KT2440 (62). However, in contrast to the P. putida genome, the P. entomophila genome contains many genes that are predicted to be important for virulence toward insects. Notably, P. entomophila could secrete many degradative enzymes (proteases and lipases), putative toxins, and secondary metabolites (62). Similar factors have been shown to play a key role in the virulence of other entomopathogenic bacteria like Photorhabdus and Xenorhabdus sp. (27, 29).Insertional mutagenesis allowed the identification of several P. entomophila genes required to infect and/or kill Drosophila. This analysis demonstrated that P. entomophila virulence is under the control of the GacS/GacA two-component system (62, 63), a global regulatory system which is known to control secondary metabolite production, protein secretion, and pathogenic abilities in gammaproteobacteria (37, 65). Another study indicates that P. entomophila can counteract the Drosophila gut immune response as a result of the secretion of an abundant protease, AprA, which degrades antimicrobial peptides produced by gut epithelia and thereby promotes bacterial persistence (38). However, an AprA-deficient mutant remains virulent to some extent, indicating that P. entomophila virulence is multifactorial, AprA being one virulence factor among others.The secretion of virulence factors is a common mechanism employed by pathogens to compromise host defenses. Several entomopathogenic bacteria (e.g., Photorhabdus luminescens) secrete toxins that allow them to impair host function (8). The starting point of this study was the observation that, in contrast to several other Pseudomonas strains, P. entomophila secretes a strong diffusible hemolytic activity (which is also controlled by the Gac system). This raises the possibility of a link between this hemolytic activity and the pathogenicity of P. entomophila for Drosophila. Indeed, bacterial hemolysins are exotoxins that attack blood cell membranes and cause cell rupture by poorly defined mechanisms. It was conceivable that this hemolytic activity could be a readout for the ability of P. entomophila to damage the epithelial cells of the Drosophila gut, which plays a crucial role in its virulence (10, 33, 63).In this study, the P. entomophila hemolytic factor was identified as a cyclic lipopeptide (CLP) whose structure was elucidated. CLPs are versatile molecules with antimicrobial, cytotoxic, and surfactant properties that are produced by members of the genera Bacillus, Serratia, Burkholderia, and Pseudomonas (31, 41, 43, 50). They are produced by a ribosome-independent mechanism that utilizes multifunctional enzymes called nonribosomal peptide synthetases (NRPSs) (42, 59). These NRPSs are composed of repeated amino acid activation modules containing domains for condensation, aminoacyl adenylation, and thiolation. Modules are responsible for activation and incorporation of amino acids into the growing peptide. A large number of prokaryotic and some eukaryotic organisms synthesize peptide metabolites via this nonribosomal mechanism of biosynthesis (42, 47).Several genes involved in P. entomophila lipopeptide production were identified, three of them encoding NRPSs. The physiological role of this lipopeptide was also investigated, and it does not seem to play a role in the process of virulence towards Drosophila and Dictyostelium or in the P. entomophila biocontrol activity that was uncovered by this study. This suggests that the lifestyle of this newly identified bacterium is probably quite versatile and that lipopeptide production could be required only under specific circumstances.     

A Major Determinant for Binding and Aminoacylation of tRNA in Cytoplasmic Alanyl-tRNA Synthetase Is Mutated in Dominant Axonal Charcot-Marie-Tooth Disease

Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at θ = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNA^Ala aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features.     

Adrenomedullin increases fibroblast-like synoviocyte adhesion to extracellular matrix proteins by upregulating integrin activation

Introduction  

Rheumatoid arthritis (RA) is characterized by bone and cartilage invasion by fibroblast-like synoviocytes (FLSs). Adrenomedullin, a peptide with anabolic and antiapoptotic properties, is secreted by rheumatoid FLSs. Adrenomedullin also increases the expression of adhesion molecules in endothelial cells and keratinocytes. Here, we investigated whether adrenomedullin mediated FLS adhesion to extracellular matrix (ECM) proteins.