Bacterial resistance to antibiotics constantly remodels the battlefront between infections and antibiotic therapy. Polymyxin B, a cationic peptide with an anti-Gram-negative spectrum of activity is ...re-entering use as a last resort measure and as an adjuvant. We use fluorescence dequenching to investigate the role of the rough chemotype bacterial lipopolysaccharide from
E. coli
BL21 as a molecular facilitator of membrane disruption by LPS. The minimal polymyxin B/lipid ratio required for leakage onset increased from 5.9 × 10
−4
to 1.9 × 10
−7
in the presence of rLPS. We confirm polymyxin B activity against
E. coli
BL21 by the agar diffusion method and determined a MIC of 291 μg ml
−1
. Changes in lipid membrane stability and dynamics in response to polymyxin and the role of LPS are investigated by
31
P NMR and high resolution
31
P MAS NMR relaxation is used to monitor selective molecular interactions between polymyxin B and rLPS within bilayer lipid membranes. We observe a strong facilitating effect from rLPS on the membrane lytic properties of polymyxin B and a specific, pyrophosphate-mediated process of molecular recognition of LPS by polymyxin B.
Polymyxin B uses bacterial LPS as docking receptor to cross the outer membrane.
Membranes of cells are active barriers, in which membrane proteins perform essential remodelling, transport and recognition functions that are vital to cells. Membrane proteins are key regulatory ...components of cells and represent essential targets for the modulation of cell function and pharmacological intervention. However, novel folds, low molarity and the need for lipid membrane support present serious challenges to the characterisation of their structure and interactions. We describe the use of solid state NMR as a versatile and informative approach for membrane and membrane protein studies, which uniquely provides information on structure, interactions and dynamics of membrane proteins. High resolution approaches are discussed in conjunction with applications of NMR methods to studies of membrane lipid and protein structure and interactions. Signal enhancement in high resolution NMR spectra through DNP is discussed as a tool for whole cell and interaction studies.
Many studies are uncovering functional roles for long noncoding RNAs (lncRNAs), yet few have been tested for in vivo relevance through genetic ablation in animal models. To investigate the functional ...relevance of lncRNAs in various physiological conditions, we have developed a collection of 18 lncRNA knockout strains in which the locus is maintained transcriptionally active. Initial characterization revealed peri- and postnatal lethal phenotypes in three mutant strains (Fendrr, Peril, and Mdgt), the latter two exhibiting incomplete penetrance and growth defects in survivors. We also report growth defects for two additional mutant strains (linc-Brn1b and linc-Pint). Further analysis revealed defects in lung, gastrointestinal tract, and heart in Fendrr(-/-) neonates, whereas linc-Brn1b(-/-) mutants displayed distinct abnormalities in the generation of upper layer II-IV neurons in the neocortex. This study demonstrates that lncRNAs play critical roles in vivo and provides a framework and impetus for future larger-scale functional investigation into the roles of lncRNA molecules. DOI: http://dx.doi.org/10.7554/eLife.01749.001.
Biofermentative production of styrene from renewable carbon sources is crucially dependent on strain tolerance and viability at elevated styrene concentrations. Solvent-driven collapse of bacterial ...plasma membranes limits yields and is technologically restrictive. Styrene is a hydrophobic solvent that readily partitions into the membrane interior and alters membrane-chain order and packing. We investigate styrene incorporation into model membranes and the role lipid chains play as determinants of membrane stability in the presence of styrene. MD simulations reveal styrene phase separation followed by irreversible segregation into the membrane interior. Solid state NMR shows committed partitioning of styrene into the membrane interior with persistence of the bilayer phase up to 67 mol % styrene. Saturated-chain lipid membranes were able to retain integrity even at 80 mol % styrene, whereas in unsaturated lipid membranes, we observe the onset of a non-bilayer phase of small lipid aggregates in coexistence with styrene-saturated membranes. Shorter-chain saturated lipid membranes were seen to tolerate styrene better, which is consistent with observed chain length reduction in bacteria grown in the presence of small molecule solvents. Unsaturation at mid-chain position appears to reduce the membrane tolerance to styrene and conversion from cis- to trans-chain unsaturation does not alter membrane phase stability but the lipid order in trans-chains is less affected than cis.
Neural progenitors self-renew and generate neurons throughout the central nervous system. Here, we uncover an unexpected regional specificity in the properties of neural progenitor cells, revealed by ...the function of a microRNA—miR-9. miR-9 is expressed in neural progenitors, and its knockdown results in an inhibition of neurogenesis along the anterior-posterior axis. However, the underlying mechanism differs—in the hindbrain, progenitors fail to exit the cell cycle, whereas in the forebrain they undergo apoptosis, counteracting the proliferative effect. Among several targets, we functionally identify hairy1 as a primary target of miR-9, regulated at the mRNA level. hairy1 mediates the effects of miR-9 on proliferation, through Fgf8 signaling in the forebrain and Wnt signaling in the hindbrain, but affects apoptosis only in the forebrain, via the p53 pathway. Our findings show a positional difference in the responsiveness of progenitors to miR-9 depletion, revealing an underlying divergence of their properties.
► miR-9 function in neural progenitors is context dependent ► miR-9 loss induces proliferation in the hindbrain and apoptosis in the forebrain ► The primary function of miR-9 in neurogenesis is to downregulate hairy1 mRNA levels ► miR-9/hairy1 affects proliferation via cyclinD/p27 and apoptosis via p53
Short-period (ultradian) oscillations of Hes1, a Notch signaling effector, are essential for maintaining neural progenitors in a proliferative state, while constitutive downregulation of Hes1 leads ...to neuronal differentiation. Hes1 oscillations are driven by autorepression, coupled with high instability of the protein and mRNA. It is unknown how Hes1 mRNA stability is controlled and furthermore, how cells exit oscillations in order to differentiate. Here, we identify a microRNA, miR-9, as a component of ultradian oscillations. We show that miR-9 controls the stability of Hes1 mRNA and that both miR-9 overexpression and lack of miR-9 dampens Hes1 oscillations. Reciprocally, Hes1 represses the transcription of miR-9, resulting in out-of-phase oscillations. However, unlike the primary transcript, mature miR-9 is very stable and thus accumulates over time. Given that raising miR-9 levels leads to dampening of oscillations, these findings provide support for a self-limiting mechanism whereby cells might terminate Hes1 oscillations and differentiate.
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► miR-9 controls Hes1 oscillations by regulating Hes1 mRNA stability in NPCs ► Hes1 represses miR-9 transcription, giving rise to antiphase oscillations ► The accumulation of mature miR-9 dampens Hes1 oscillations ► The double-negative feedback loop serves as a self-limiting oscillator
Hes1 short-period oscillations maintain neural progenitors in a proliferative state. How are oscillations controlled and how are they terminated? Papalopulu and colleagues report that a conserved microRNA, miR-9, controls Hes1 oscillations; overexpressing or knocking down miR-9 dampens Hes1 oscillations. Hes1 transcriptionally represses miR-9, resulting in antiphase oscillations of primary miR-9 transcripts. However, mature miR-9 accumulates over time, providing a mechanism to terminate Hes1 oscillations as its concentration increases. The authors propose that the miR-9/Hes1 regulatory feedback loop represents a “self-limiting oscillator.”
How multiple epigenetic layers and transcription factors (TFs) interact to facilitate brain development is largely unknown. Here, to systematically map the regulatory landscape of neural ...differentiation in the mouse neocortex, we profiled gene expression and chromatin accessibility in single cells and integrated these data with measurements of enhancer activity, DNA methylation and three-dimensional genome architecture in purified cell populations. This allowed us to identify thousands of new enhancers, their predicted target genes and the temporal relationships between enhancer activation, epigenome remodeling and gene expression. We characterize specific neuronal transcription factors associated with extensive and frequently coordinated changes across multiple epigenetic modalities. In addition, we functionally demonstrate a new role for Neurog2 in directly mediating enhancer activity, DNA demethylation, increasing chromatin accessibility and facilitating chromatin looping in vivo. Our work provides a global view of the gene regulatory logic of lineage specification in the cerebral cortex.
Biological membranes define the interface of life and its basic unit, the cell. Membrane proteins play key roles in membrane functions, yet their structure and mechanisms remain poorly understood. ...Breakthroughs in crystallography and electron microscopy have invigorated structural analysis while failing to characterise key functional interactions with lipids, small molecules and membrane modulators, as well as their conformational polymorphism and dynamics. NMR is uniquely suited to resolving atomic environments within complex molecular assemblies and reporting on membrane organisation, protein structure, lipid and polysaccharide composition, conformational variations and molecular interactions. The main challenge in membrane protein studies at the atomic level remains the need for a membrane environment to support their fold. NMR studies in membrane mimetics and membranes of increasing complexity offer close to native environments for structural and molecular studies of membrane proteins. Solution NMR inherits high resolution from small molecule analysis, providing insights from detergent solubilised proteins and small molecular assemblies. Solid-state NMR achieves high resolution in membrane samples through fast sample spinning or sample alignment. Recent developments in dynamic nuclear polarisation NMR allow signal enhancement by orders of magnitude opening new opportunities for expanding the applications of NMR to studies of native membranes and whole cells.
Mutations in the methyl-DNA-binding repressor protein MeCP2 cause the devastating neurodevelopmental disorder Rett syndrome. It has been challenging to understand how MeCP2 regulates transcription ...because MeCP2 binds broadly across the genome and MeCP2 mutations are associated with widespread small-magnitude changes in neuronal gene expression. We demonstrate here that MeCP2 represses nascent RNA transcription of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex. By measuring the rates of transcriptional initiation and elongation directly in the brain, we find that MeCP2 has no measurable effect on transcriptional elongation, but instead represses the rate at which Pol II initiates transcription of highly methylated long genes. These findings suggest a new model of MeCP2 function in which MeCP2 binds broadly across highly methylated regions of DNA, but acts at transcription start sites to attenuate transcriptional initiation.
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•MeCP2 represses transcription of highly methylated long genes through NCoR•Direct measurements of transcriptional initiation and elongation rates in the mouse brain•MeCP2 reduces transcriptional initiation, not elongation, of highly methylated long genes•Gene body-TSS contacts position distal MeCP2 molecules at the TSS
Mutations in MeCP2 cause the neurodevelopmental disorder Rett syndrome. Although MeCP2 binding is enriched in methylated gene bodies, Boxer et al. find that MeCP2 decreases the rate of transcriptional initiation, but not elongation, of highly methylated long genes in the brain through its interaction with the NCoR co-repressor complex.
Bioproduction of poly(methyl methacrylate) is a fast growing global industry that is limited by cellular toxicity of monomeric methacrylate intermediates to the producer strains. Maintaining high ...methacrylate concentrations during biofermentation, required by economically viable technologies, challenges bacterial membrane stability and cellular viability. Studying the stability of model lipid membranes in the presence of methacrylates offers unique molecular insights into the mechanisms of methacrylate toxicity, as well as into the fundamental structural bases of membrane assembly. We investigate the structure and stability of model membranes in the presence of high levels of methacrylate esters using solid-state nuclear magnetic resonance (NMR) and small-angle X-ray scattering (SAXS). Wide-line 31P NMR spectroscopy shows that butyl methacrylate (BMA) can be incorporated into the lipid bilayer at concentrations as high as 75 mol % without significantly disrupting membrane integrity and that lipid acyl chain composition can influence membrane tolerance and ability to accommodate BMA. Using high resolution 13C magic angle spinning (MAS) NMR, we show that the presence of 75 mol % BMA lowers the lipid main transition temperature by over 12 degrees, which suggests that BMA intercalates between the lipid chains, causing uncoupling of collective lipid motions that are typically dominated by chain trans–gauche isomerization. Potential uncoupling of the bilayer leaflets to accommodate a separate BMA subphase was not supported by the SAXS experiments, which showed that membrane thickness remained unchanged even at 80% BMA. Reduced X-ray scattering contrast at the polar/apolar interface suggests BMA localization in that region between the lipid molecules.
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