Modifying the m6A brain methylome by ALKBH5-mediated demethylation: a new contender for synaptic tagging.

Synaptic plasticity processes, which underlie learning and memory formation, require RNA to be translated local to synapses. The synaptic tagging hypothesis has previously been proposed to explain how mRNAs are available at specific activated synapses. However how RNA is regulated, and which transcripts are silenced or processed as part of the tagging process is still unknown. Modification of RNA by N6-methyladenosine (m6A/m) influences the cellular fate of mRNA. Here, by advanced microscopy, we showed that m6A demethylation by the eraser protein ALKBH5 occurs at active synaptic ribosomes and at synapses during short term plasticity. We demonstrated that at activated glutamatergic post-synaptic sites, both the YTHDF1 and YTHDF3 reader and the ALKBH5 eraser proteins increase in co-localisation to m6A-modified RNAs; but only the readers showed high co-localisation to modified RNAs during late-stage plasticity. The YTHDF1 and YTHFDF3 readers also exhibited differential roles during synaptic maturation suggesting that temporal and subcellular abundance may determine specific function. m6A-sequencing of human parahippocampus brain tissue revealed distinct white and grey matter m6A methylome profiles indicating that cellular context is a fundamental factor dictating regulated pathways. However, in both neuronal and glial cell-rich tissue, m6A effector proteins are themselves modified and m6A epitranscriptional and posttranslational modification processes coregulate protein cascades. We hypothesise that the availability m6A effector protein machinery in conjunction with RNA modification, may be important in the formation of condensed synaptic nanodomain assemblies through liquid-liquid phase separation. Our findings support that m6A demethylation by ALKBH5 is an intrinsic component of the synaptic tagging hypothesis and a molecular switch which leads to alterations in the RNA methylome, synaptic dysfunction and potentially reversible disease states.

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.

A novel O-linked glycan modulates Campylobacter jejuni major outer membrane protein-mediated adhesion to human histo-blood group antigens and chicken colonization.

Campylobacter jejuni is an important cause of human foodborne gastroenteritis; strategies to prevent infection are hampered by a poor understanding of the complex interactions between host and pathogen. Previous work showed that C. jejuni could bind human histo-blood group antigens (BgAgs) in vitro and that BgAgs could inhibit the binding of C. jejuni to human intestinal mucosa ex vivo. Here, the major flagella subunit protein (FlaA) and the major outer membrane protein (MOMP) were identified as BgAg-binding adhesins in C. jejuni NCTC11168. Significantly, the MOMP was shown to be O-glycosylated at Thr(268); previously only flagellin proteins were known to be O-glycosylated in C. jejuni. Substitution of MOMP Thr(268) led to significantly reduced binding to BgAgs. The O-glycan moiety was characterized as Gal(ß1-3)-GalNAc(ß1-4)-GalNAc(ß1-4)-GalNAcα1-Thr(268); modelling suggested that O-glycosylation has a notable effect on the conformation of MOMP and this modulates BgAg-binding capacity. Glycosylation of MOMP at Thr(268) promoted cell-to-cell binding, biofilm formation and adhesion to Caco-2 cells, and was required for the optimal colonization of chickens by C. jejuni, confirming the significance of this O-glycosylation in pathogenesis.

Pro-inflammatory cytokines can act as intracellular modulators of commensal bacterial virulence.

Interactions between commensal pathogens and hosts are critical for disease development but the underlying mechanisms for switching between the commensal and virulent states are unknown. We show that the human pathogen Neisseria meningitidis, the leading cause of pyogenic meningitis, can modulate gene expression via uptake of host pro-inflammatory cytokines leading to increased virulence. This uptake is mediated by type IV pili (Tfp) and reliant on the PilT ATPase activity. Two Tfp subunits, PilE and PilQ, are identified as the ligands for TNF-α and IL-8 in a glycan-dependent manner, and their deletion results in decreased virulence and increased survival in a mouse model. We propose a novel mechanism by which pathogens use the twitching motility mode of the Tfp machinery for sensing and importing host elicitors, aligning with the inflamed environment and switching to the virulent state.

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.

Localisation of a family of complex-forming ß-barrels in the T. vaginalis hydrogenosomal membrane.

Crucial to organellogenesis was the development of membrane translocases responsible for delivering proteins to new cellular compartments. This investigation examines the Trichomonas vaginalis hydrogenosome, a mitochondrially derived organelle. We identify an expanded family of putative ß-barrel proteins (THOM A-I) comprising nine related sequences. Sub-cellular localisation by immunofluorescence and biochemical fractionation is consistent with THOMs being localised to the hydrogenosomal membrane. Native gel electrophoresis and chemical cross-linking support the ability of THOM proteins to be components of membrane-bound oligomeric protein complexes, consistent with a role in protein translocation.

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.

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.

LIM-domain proteins, LIMD1, Ajuba, and WTIP are required for microRNA-mediated gene silencing.

In recent years there have been major advances with respect to the identification of the protein components and mechanisms of microRNA (miRNA) mediated silencing. However, the complete and precise repertoire of components and mechanism(s) of action remain to be fully elucidated. Herein we reveal the identification of a family of three LIM domain-containing proteins, LIMD1, Ajuba and WTIP (Ajuba LIM proteins) as novel mammalian processing body (P-body) components, which highlight a novel mechanism of miRNA-mediated gene silencing. Furthermore, we reveal that LIMD1, Ajuba, and WTIP bind to Ago1/2, RCK, Dcp2, and eIF4E in vivo, that they are required for miRNA-mediated, but not siRNA-mediated gene silencing and that all three proteins bind to the mRNA 5' m(7)GTP cap-protein complex. Mechanistically, we propose the Ajuba LIM proteins interact with the m(7)GTP cap structure via a specific interaction with eIF4E that prevents 4EBP1 and eIF4G interaction. In addition, these LIM-domain proteins facilitate miRNA-mediated gene silencing by acting as an essential molecular link between the translationally inhibited eIF4E-m(7)GTP-5(')cap and Ago1/2 within the miRISC complex attached to the 3'-UTR of mRNA, creating an inhibitory closed-loop complex.

The effect of allosteric modulators on the kinetics of agonist-G protein-coupled receptor interactions in single living cells.

Allosteric binding sites on adenosine -A(1) and -A(3) 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 A(1) and A(3) 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 A(1) and A(3) receptors were approximately 50 and 10 nM, respectively, suggesting that, even in live cells, low agonist concentrations predominantly detect high-affinity receptor states. At A(1) receptors, the dissociation of ABA-X-BY630 (30 nM) was significantly faster in the absence (k(off) = 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 microM; k(off) = 0.80 +/- 0.03 min(-1)) and allosteric inhibitor 4-methoxy-N-(7-methyl-3-(2-pyridinyl)-1-isoquinolinyl)benzamide (VUF5455; 1 microM; k(off) = 1.48 +/- 0.16 min(-1)). In contrast, ABA-X-BY630 dissociation from A(3) receptors was significantly slower in the absence (k(off) = 0.78 +/- 0.18 min(-1)) than in the presence of the allosteric inhibitors VUF5455 (1 microM; k(off) = 3.15 +/- 0.12 min(-1)) and PD81,723 (10 microM; k(off) = 2.46 +/- 0.18 min(-1)). An allosteric mechanism of action has previously not been identified for PD81,723 at the A(3) receptor or VUF5455 at the A(1) 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.

Laminin receptor initiates bacterial contact with the blood brain barrier in experimental meningitis models.

A diverse array of infectious agents, including prions and certain neurotropic viruses, bind to the laminin receptor (LR), and this determines tropism to the CNS. Bacterial meningitis in childhood is almost exclusively caused by the respiratory tract pathogens Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae, but the mechanism by which they initiate contact with the vascular endothelium of the blood brain barrier (BBB) is unknown. We hypothesized that an interaction with LR might underlie their CNS tropism. Using affinity chromatography, coimmunoprecipitation, retagging, and in vivo imaging approaches, we identified 37/67-kDa LR as a common receptor for all 3 bacteria on the surface of rodent and human brain microvascular endothelial cells. Mutagenesis studies indicated that the corresponding bacterial LR-binding adhesins were pneumococcal CbpA, meningococcal PilQ and PorA, and OmpP2 of H. influenzae. The results of competitive binding experiments suggest that a common adhesin recognition site is present in the carboxyl terminus of LR. Together, these findings suggest that disruption or modulation of the interaction of bacterial adhesins with LR might engender unexpectedly broad protection against bacterial meningitis and may provide a therapeutic target for the prevention and treatment of disease.

Physical interaction between TBX5 and MEF2C is required for early heart development.

TBX5 is a transcription factor which plays important roles in the development of the heart and upper limbs. Mutations in this gene produce the inherited disorder Holt-Oram syndrome. Here, we report a physical interaction between TBX5 and MEF2C leading to a synergistic activation of the alpha-cardiac myosin heavy chain (MYH6). Mutants of TBX5, TBX5G80R, and TBX5R279X that produce severe cardiac phenotypes impair the synergy. Using fluorescence resonance energy transfer, we demonstrate the interaction of TBX5 and MEF2C in living cells. We also show that they physically associate through their DNA-binding domains to form a complex on the MYH6 promoter. Morpholino-mediated knockdowns of Tbx5 and Mef2c in zebrafish suggest that the genetic interaction of these proteins is not only required for MYH6 expression but also essential for the early stages of heart development and survival. This is the first report of a functional interaction between a T-box protein and a MADS box factor that may be crucial in cardiomyocyte differentiation.

Transgenic enrichment of cardiomyocytes from human embryonic stem cells.

To realize the full scientific and clinical potential of human embryonic stem cell (hESC)-cardiomyocytes, strategies to overcome the high degree of heterogeneity of differentiated populations are required. Here we demonstrate the utility of two transgenic approaches in enrichment of cardiomyocytes derived from HUES-7 cells: (i) negative selection of proliferating cells with the herpes simplex virus thymidine kinase/ganciclovir (HSVtk/GCV) suicide gene system; and (ii) positive selection of cardiomyocytes expressing a bicistronic reporter [green fluorescent protein (GFP)-internal ribosome entry site (IRES)-puromycin-N-acetyltransferase (PAC)] from the human alphamyosin heavy chain promoter. Parental and transgenic HUES-7 cells were similar with regard to morphology, pluripotency marker expression, differentiation, and cardiomyocyte electrophysiology. Whereas immunostaining of dissociated cardiomyocyte preparations expressing HSVtk or PAC contained <7% cardiomyocytes, parallel cultures treated with GCV or puromycin, respectively, contained 33.4 +/- 2.1% or 91.5 +/- 4.3% cardiomyocytes corresponding to an enrichment factor of 6.7- or 14.5-fold. Drug-selected cardiomyocytes responded to chronotropic stimulation and displayed cardiac-specific action potentials, demonstrating that functionality was retained. Both transgenic strategies will be generically applicable and should readily translate to the enrichment of many other differentiated lineages derived from hESCs.

Common culture conditions for maintenance and cardiomyocyte differentiation of the human embryonic stem cell lines, BG01 and HUES-7.

Development of generic differentiation protocols that function in a range of independently-derived human embryonic stem cell (hESC) lines remains challenging due to considerable diversity in culture methods practiced between lines. Maintenance of BG01 and HUES-7 has routinely been on mouse embryonic fibroblast (MEF) feeder layers using manual- and trypsin-passaging, respectively. We adapted both lines to trypsin-passaging on feeders or on Matrigel in feeder-free conditions and assessed proliferation and cardiac differentiation. On feeders, undifferentiated proliferation of BG01 and HUES-7 was supported by all three media tested (BG-SK, HUES-C and HUES-nL), although incidence of karyotypic instability increased in both lines in BG-SK. On Matrigel, KSR-containing conditioned medium (CM) promoted undifferentiated cell proliferation, while differentiation occurred in CM containing Plasmanate or ES-screened Fetal Bovine Serum (FBS) and in unconditioned medium containing 100 ng/ml bFGF. Matrigel cultures were advantageous for transfection but detrimental to embryoid body (EB) formation. However, transfer of hESCs from Matrigel back to feeders and culturing to confluence was found to rescue EB formation. EBs formed efficiently when hESCs on feeders were treated with collagenase, harvested by scraping and then cultured in suspension in CM. Subsequent culture in FBS-containing medium produced spontaneously contracting EBs, for which the mean beat rate was 37.2 +/- 2.3 and 41.1 +/- 3.1 beats/min for BG01-EBs and HUES-7-EBs, respectively. Derived cardiomyocytes expressed cardiac genes and responded to pharmacological stimulation. Therefore the same culture and differentiation conditions functioned in two independently-derived hESC lines. Similar studies in other lines may facilitate development of universal protocols.

Molecular and phenotypic analyses of human embryonic stem cell-derived cardiomyocytes: opportunities and challenges for clinical translation.

Differentiation of human embryonic stem cells (hESCs) into cardiomyocytes in culture may offer unique opportunities for modeling genetic disorders, screening potentially cardiotoxic pharmaceutical agents or replacing cells of the diseased heart. However, before clinical utility can be realized, numerous hurdles must be overcome. Comprehensive molecular and phenotypic characterization is required but has so far been restricted to cardiomyocytes derived from a limited subset of hESC lines. Thus, we have initiated analysis of cardiomyocyte differentiation and function from a further two independently derived lines, BG01 and HUES-7. The challenge of improving cardiac cell induction, enrichment and maturation must also be addressed to meet the demands of high throughput pharmaceutical screening or to provide sufficient cells to repair an infarcted heart. Transplanted cells must functionally integrate without inducing arrhythmias, while survival and evasion of immune surveillance must be accomplished without tumorigenicity. This review evaluates the opportunities presented by hESC-derived cardiomyocytes and the progress towards surmounting the challenges of clinical translation.