Adenosine-A3 receptors in neutrophil microdomains promote the formation of bacteria-tethering cytonemes

The A3‐adenosine receptor (A3AR) has recently emerged as a key regulator of neutrophil behaviour. Using a fluorescent A3AR ligand, we show that A3ARs aggregate in highly polarized immunomodulatory microdomains on human neutrophil membranes. In addition to regulating chemotaxis, A3ARs promote the formation of filipodia‐like projections (cytonemes) that can extend up to 100 μm to tether and ‘reel in’ pathogens. Exposure to bacteria or an A3AR agonist stimulates the formation of these projections and bacterial phagocytosis, whereas an A3AR‐selective antagonist inhibits cytoneme formation. Our results shed new light on the behaviour of neutrophils and identify the A3AR as a potential target for modulating their function

Dimerization of ABCG2 Analysed by Bimolecular Fluorescence Complementation

ABCG2 is one of three human ATP binding cassette transporters that are functionally capable of exporting a diverse range of substrates from cells. The physiological consequence of ABCG2 multidrug transport activity in leukaemia, and some solid tumours is the acquisition of cancer multidrug resistance. ABCG2 has a primary structure that infers that a minimal functional transporting unit would be a homodimer. Here we investigated the ability of a bimolecular fluorescence complementation approach to examine ABCG2 dimers, and to probe the role of individual amino acid substitutions in dimer formation. ABCG2 was tagged with fragments of venus fluorescent protein (vYFP), and this tagging did not perturb trafficking or function. Co-expression of two proteins bearing N-terminal and C-terminal fragments of YFP resulted in their association and detection of dimerization by fluorescence microscopy and flow cytometry. Point mutations in ABCG2 which may affect dimer formation were examined for alterations in the magnitude of fluorescence complementation signal. Bimolecular fluorescence complementation (BiFC) demonstrated specific ABCG2 dimer formation, but no changes in dimer formation, resulting from single amino acid substitutions, were detected by BiFC analysis

Nuclear trafficking, histone cleavage and induction of apoptosis by the meningococcal App and MspA autotransporters

Neisseria meningitidis, a major cause of bacterial meningitis and septicaemia, secretes multiple virulence factors, including the adhesion and penetration protein (App) and meningococcal serine protease A (MspA). Both are conserved, immunogenic, type Va autotransporters harbouring S6-family serine endopeptidase domains. Previous work suggested that both could mediate adherence to human cells, but their precise contribution to meningococcal pathogenesis was unclear. Here, we confirm that App and MspA are in vivo virulence factors since human CD46-expressing transgenic mice infected with meningococcal mutants lacking App, MspA or both had improved survival rates compared with mice infected with wild type. Confocal imaging showed that App and MspA were internalized by human cells and trafficked to the nucleus. Cross-linking and enzyme-linked immuno assay (ELISA) confirmed that mannose receptor (MR), transferrin receptor 1 (TfR1) and histones interact with MspA and App. Dendritic cell (DC) uptake could be blocked using mannan and transferrin, the specific physiological ligands for MR and TfR1, whereas in vitro clipping assays confirmed the ability of both proteins to proteolytically cleave the core histone H3. Finally, we show that App and MspA induce a dose-dependent increase in DC death via caspase-dependent apoptosis. Our data provide novel insights into the roles of App and MspA in meningococcal infection

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

Refining transcriptional programs in kidney development by integration of deep RNA-sequencing and array-based spatial profiling

<p>Abstract</p> <p>Background</p> <p>The developing mouse kidney is currently the best-characterized model of organogenesis at a transcriptional level. Detailed spatial maps have been generated for gene expression profiling combined with systematic <it>in situ </it>screening. These studies, however, fall short of capturing the transcriptional complexity arising from each locus due to the limited scope of microarray-based technology, which is largely based on "gene-centric" models.</p> <p>Results</p> <p>To address this, the polyadenylated RNA and microRNA transcriptomes of the 15.5 dpc mouse kidney were profiled using strand-specific RNA-sequencing (RNA-Seq) to a depth sufficient to complement spatial maps from pre-existing microarray datasets. The transcriptional complexity of RNAs arising from mouse RefSeq loci was catalogued; including 3568 alternatively spliced transcripts and 532 uncharacterized alternate 3' UTRs. Antisense expressions for 60% of RefSeq genes was also detected including uncharacterized non-coding transcripts overlapping kidney progenitor markers, Six2 and Sall1, and were validated by section <it>in situ </it>hybridization. Analysis of genes known to be involved in kidney development, particularly during mesenchymal-to-epithelial transition, showed an enrichment of non-coding antisense transcripts extended along protein-coding RNAs.</p> <p>Conclusion</p> <p>The resulting resource further refines the transcriptomic cartography of kidney organogenesis by integrating deep RNA sequencing data with locus-based information from previously published expression atlases. The added resolution of RNA-Seq has provided the basis for a transition from classical gene-centric models of kidney development towards more accurate and detailed "transcript-centric" representations, which highlights the extent of transcriptional complexity of genes that direct complex development events.</p

The Effect of Allosteric Modulators on the Kinetics of Agonist-G Protein-Coupled Receptor Interactions in Single Living Cells

Allosteric binding sites on adenosine -A1 and -A3 receptors represent attractive therapeutic targets for amplifying, in a spatially and temporally selective manner, the tissue protective actions of endogenous adenosine. This study has directly quantified the kinetics of agonist/G protein-coupled receptor interactions at the single-cell level, reflecting the physiological situation in which intracellular signaling proteins can exert major allosteric effects on agonist-receptor interactions. The association and dissociation rate constants at both A1 and A3 receptors, and therefore the affinity of the fluorescent adenosine derivative ABA-X-BY630 (structure appears in J Med Chem 50:782–793, 2007), were concentration-independent. The equilibrium dissociation constants of ABA-X-BY630 at A1 and A3 receptors were approximately 50 and 10 nM, respectively, suggesting that, even in live cells, low agonist concentrations predominantly detect high-affinity receptor states. At A1 receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (koff = 1.95 ± 0.09 min−1) compared with the presence of the allosteric enhancer (2-amino-4,5-dimethyl-3-thienyl)(3-(trifluoromethyl)phenyl)-methanone (PD81,723; 10 μM; koff = 0.80 ± 0.03 min−1) and allosteric inhibitor 4-methoxy-N-(7-methyl-3-(2-pyridinyl)-1-isoquinolinyl)benzamide (VUF5455; 1 μM; koff = 1.48 ± 0.16 min−1). In contrast, ABA-X-BY630 dissociation from A3 receptors was significantly slower in the absence (koff = 0.78 ± 0.18 min−1) than in the presence of the allosteric inhibitors VUF5455 (1 μM; koff = 3.15 ± 0.12 min−1) and PD81,723 (10 μM; koff = 2.46 ± 0.18 min−1). An allosteric mechanism of action has previously not been identified for PD81,723 at the A3 receptor or VUF5455 at the A1 receptor. Furthermore, the marked enhancement in fluorescent agonist dissociation by VUF5455 in living cells contrasts previous observations from broken cell preparations and emphasizes the need to study the allosteric regulation of agonist binding in living cells

Oligonucleotide primers employed.

<p>Oligonucleotide primers employed.</p

N-terminal tagging with ½ YFP molecules does not affect function of the ABCG2 protein.

<p>HEK293T cells were transiently transfected with YFP_ABCG2 constructs (C-E) or negative control (empty pcDNA vector; A) and positive control (His₁₂-ABCG2) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025818#s2" target="_blank">Methods</a> and Materials. Following transfection aliquots of cells were incubated with the ABCG2 substrate mitoxantrone in the presence or absence of the inhibitor fumitremorgin C (FTC) and cellular fluorescence determined by flow cytometry. Blue filled histograms represent the cellular fluorescence in the absence of FTC, and the rightward shift in the presence of the inhibitor (black lines) demonstrates functional competence at a level similar to those of the characterized control His₁₂-ABCG2 (B). The apparent large number of cells with zero fluorescence in the eYFP-ABCG2 panel (C) is a fluorescence artefact due to interference from the YFP fluorescence of this construct. The graphs are representative of >5 independent experiments.</p