A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.     

Ca2+/Calmodulin-dependent kinase II signaling causes skeletal overgrowth and premature chondrocyte maturation

The long bones of vertebrate limbs originate from cartilage templates and are formed by the process of endochondral ossification. This process requires that chondrocytes undergo a progressive maturation from proliferating to postmitotic prehypertrophic to mature, hypertrophic chondrocytes. Coordinated control of proliferation and maturation regulates growth of the skeletal elements. Various signals and pathways have been implicated in orchestrating these processes, but the underlying intracellular molecular mechanisms are often not entirely known. Here we demonstrated in the chick using replication-competent retroviruses that constitutive activation of Calcium/Calmodulin-dependent kinase II (CaMKII) in the developing wing resulted in elongation of skeletal elements associated with premature differentiation of chondrocytes. The premature maturation of chondrocytes was a cell-autonomous effect of constitutive CaMKII signaling associated with down-regulation of cell-cycle regulators and up-regulation of chondrocyte maturation markers. In contrast, the elongation of the skeletal elements resulted from a non-cell autonomous up-regulation of the Indian hedgehog responsive gene encoding Parathyroid-hormone-related peptide. Reduction of endogenous CaMKII activity by overexpressing an inhibitory peptide resulted in shortening of the skeletal elements associated with a delay in chondrocyte maturation. Thus, CaMKII is an essential component of intracellular signaling pathways regulating chondrocyte maturation.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

Ligand binding induces a conformational change in ifnar1 that is propagated to its membrane-proximal domain

The type I interferon (IFN) receptor plays a key role in innate immunity against viral and bacterial infections. Here, we show by intramolecular Förster resonance energy transfer spectroscopy that ligand binding induces substantial conformational changes in the ectodomain of ifnar1 (ifnar1-EC). Binding of IFNα2 and IFNβ induce very similar conformations of ifnar1, which were confirmed by single-particle electron microscopy analysis of the ternary complexes formed by IFNα2 or IFNβ with the two receptor subunits ifnar1-EC and ifnar2-EC. Photo-induced electron-transfer-based fluorescence quenching and single-molecule fluorescence lifetime measurements revealed that the ligand-induced conformational change in the membrane-distal domains of ifnar1-EC is propagated to its membrane-proximal domain, which is not involved in ligand recognition but is essential for signal activation. Temperature-dependent ligand binding studies as well as stopped-flow fluorescence experiments corroborated a multistep conformational change in ifnar1 upon ligand binding. Our results thus suggest that the relatively intricate architecture of the type I IFN receptor complex is designed to propagate the ligand binding event to and possibly even across the membrane by conformational changes.     

Tertiary structure and carbohydrate recognition by the chitin-binding domain of a hyperthermophilic chitinase from Pyrococcus furiosus

A chitinase is a hyperthermophilic glycosidase that effectively hydrolyzes both α and β crystalline chitins; that studied here was engineered from the genes PF1233 and PF1234 of Pyrococcus furiosus. This chitinase has unique structural features and contains two catalytic domains (AD1 and AD2) and two chitin-binding domains (ChBDs; ChBD1 and ChBD2). A partial enzyme carrying AD2 and ChBD2 also effectively hydrolyzes crystalline chitin. We determined the NMR and crystal structures of ChBD2, which significantly enhances the activity of the catalytic domain. There was no significant difference between the NMR and crystal structures. The overall structure of ChBD2, which consists of two four-stranded β-sheets, was composed of a typical β-sandwich architecture and was similar to that of other carbohydrate-binding module 2 family proteins, despite low sequence similarity. The chitin-binding surface identified by NMR was flat and contained a strip of three solvent-exposed Trp residues (Trp274, Trp308 and Trp326) flanked by acidic residues (Glu279 and Asp281). These acidic residues form a negatively charged patch and are a characteristic feature of ChBD2. Mutagenesis analysis indicated that hydrophobic interaction was dominant for the recognition of crystalline chitin and that the acidic residues were responsible for a higher substrate specificity of ChBD2 for chitin compared with that of cellulose. These results provide the first structure of a hyperthermostable ChBD and yield new insight into the mechanism of protein-carbohydrate recognition. This is important in the development of technology for the exploitation of biomass.     

Involvement of c-Src tyrosine kinase in SHP-1 phosphatase activation by Ang II AT2 receptors in rat fetal tissues

Angiotensin II (Ang II) AT(2) receptors are abundantly expressed in rat fetal tissues where they probably contribute to development. In the present study we examine the effects of Ang II type 2 receptor stimulation on SHP-1 activation. Ang II (10(-7) M) elicits a rapid and transient tyrosine phosphorylation of SHP-1, maximal at 1 min, in a dose-dependent form, blocked by the AT(2) antagonist, PD123319. SHP-1 phosphorylation is followed in time by tyrosine dephosphorylation of different proteins, suggesting a sequence of events. Ang II induces association of SHP-1 to AT(2) receptors as shown by co-immunoprecipitation, Western blot and binding assays. SHP-1 activity was determined in immunocomplexes obtained with either anti-AT(2) or anti-SHP-1 antibodies, after Ang II stimulation (1 min), in correlation with the maximal level of SHP-1 phosphorylation. Interestingly, following receptor stimulation (1 min) c-Src was associated to AT(2) or SHP-1 immunocomplexes. Preincubation with the c-Src inhibitor PP2 inhibited SHP-1 activation and c-Src association, thus confirming the participation of c-Src in this pathway. We demonstrated here for the first time the involvement of c-Src in SHP-1 activation via AT(2) receptors present in an ex vivo model expressing both receptor subtypes. In this model, AT(2) receptors are not constitutively associated to SHP-1 and SHP-1 is not constitutively activated. Thus, we clearly establish that SHP-1 activation, mediated by the AT(2) subtype, involves c-Src and precedes protein tyrosine dephosphorylation, in rat fetal membranes.     

Wnt5a induces homodimerization and activation of Ror2 receptor tyrosine kinase

Wnts are secreted glycoproteins that control vital biological processes, including embryogenesis, organogenesis and tumorigenesis. Wnts are classified into several subfamilies depending on the signaling pathways they activate, with the canonical subfamily activating the Wnt/beta-catenin pathway and the non-canonical subfamily activating a variety of other pathways, including the Wnt/calcium signaling and the small GTPase/c-Jun NH2-terminal kinase pathway. Wnts bind to a membrane receptor Frizzled and a co-receptor, the low-density lipoprotein receptor related protein. More recently, both canonical and non-canonical Wnts were shown to bind the Ror2 receptor tyrosine kinase. Ror2 is an orphan receptor that plays crucial roles in skeletal morphogenesis and promotes osteoblast differentiation and bone formation. Here we examine the effects of a canonical Wnt3a and a non-canonical Wnt5a on the signaling of the Ror2 receptor. We demonstrate that even though both Wnt5a and Wnt3a bound Ror2, only Wnt5a induced Ror2 homo-dimerization and tyrosine phosphorylation in U2OS human osteoblastic cells. Furthermore, Wnt5a treatment also resulted in increased phosphorylation of the Ror2 substrate, 14-3-3beta scaffold protein, indicating that Wnt5a binding causes activation of the Ror2 signaling cascade. Functionally, Wnt5a recapitulated the Ror2 activation phenotype, enhancing bone formation in the mouse calvarial bone explant cultures and potentiating osteoblastic differentiation of human mesenchymal stem cells. The effect of Wnt5a on osteoblastic differentiation was largely abolished upon Ror2 down-regulation. Thus we show that Wnt5a activates the classical receptor tyrosine kinase signaling cascade through the Ror2 receptor in cells of osteoblastic origin.