Browse
Submit Data
Databases
API
Help

Dataset Information

14 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Metabolic phospholipid labeling of intact bacteria enables a fluorescence assay that detects compromised outer membranes.

ABSTRACT: Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.

SUBMITTER: Nilsson I

PROVIDER: S-EPMC7269758 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Nanoscale fluorescence correlation spectroscopy on intact living cell membranes with NSOM probes.

Project description:Characterization of molecular dynamics on living cell membranes at the nanoscale is fundamental to unravel the mechanisms of membrane organization and compartmentalization. Here we demonstrate the feasibility of fluorescence correlation spectroscopy (FCS) based on the nanometric illumination of near-field scanning optical microscopy (NSOM) probes on intact living cells. NSOM-FCS applied to fluorescent lipid analogs allowed us to reveal details of the diffusion hidden by larger illumination areas. Moreover, the technique offers the unique advantages of evanescent axial illumination and straightforward implementation of multiple color excitation. As such, NSOM-FCS represents a powerful tool to study a variety of dynamic processes occurring at the nanometer scale on cell membranes.

| S-EPMC3021660 | biostudies-literature

Gramicidin Lateral Distribution in Phospholipid Membranes: Fluorescence Phasor Plots and Statistical Mechanical Model.

Project description:When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content at 0.143 gramicidin mole fraction. To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries (critical gramicidin mole fractions, Xcr including 0.143). Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At Xcr, the size of cluster aggregates and regular distributions reach a local maximum. Before a similar model was developed for cholesterol/DMPC mixtures (Sugar and Chong (2012) J. Am. Chem. Soc. 134, 1164⁻1171) and here the similarities and differences are discussed between these two models.

| S-EPMC6274966 | biostudies-literature

Interaction of the neurotransmitter, neuropeptide Y, with phospholipid membranes: film balance and fluorescence microscopy studies.

Project description:The association of neuropeptide Y (NPY) with air-water interfaces and with phospholipid monolayers on water subphases and on physiological buffer has been investigated. Surface pressure (pi) versus molecular area (A) relations of the peptide at water surfaces depend on the concentration of the spreading solutions. Independent of that concentration, they show a transition from a low-density state to a high-density state at pi approximately 12 mN/m. Similar features are observed in the NPY adsorption to preformed monolayers (Deltapi(t --> infinity) as a function of pii = pi (t = 0) where t = 0 signifies the time of peptide injection). The transition is also observed in cospread lipid-NPY monolayers and is interpreted as the exclusion of the peptide from the surface layer. The reproducibility of the isotherms after expansion suggests that cospread lipid-peptide monolayers are thermodynamically stable and that the peptide remains associated with the monolayer after exclusion from the lipid surface. A comparison of NPY association with zwitterionic and with anionic lipids as well as a comparison of the interactions on pure water and on physiological buffer suggest that electrostatic attraction plays a major role in the energetics of peptide binding to the membrane surface. Dual label fluorescence microscopy demonstrates that the peptide associates preferentially with the disordered, liquid condensed monolayer phase and also suggests that it self-aggregates upon exceeding a critical surface concentration. A NPY variant with a distorted alpha-helix interacts with the surface as strongly as the natural NPY but expands the monolayers more. This suggests that the helix motif in the peptide is more important for the interaction with the receptor than for binding of the peptide to the membrane surface. In context, these observations attribute a specific role to the membrane in funneling the signal peptide to its membrane receptor.

| S-EPMC2553689 | biostudies-literature

Orientation of the Escherichia coli outer membrane protein OmpX in phospholipid bilayer membranes determined by solid-State NMR.

Project description:The solid-state NMR orientation-dependent frequencies measured for membrane proteins in macroscopically oriented lipid bilayers provide precise orientation restraints for structure determination in membranes. Here we show that this information can also be used to supplement crystallographic structural data to establish the orientation of a membrane protein in the membrane. This is achieved by incorporating a few orientation restraints, measured for the Escherichia coli outer membrane protein OmpX in magnetically oriented lipid bilayers (bicelles), in a simulated annealing calculation with the coordinates of the OmpX crystal structure. The (1)H-(15)N dipolar couplings measured for the seven Phe residues of OmpX in oriented bilayers can be assigned by back-calculation of the NMR spectrum from the crystal structure and are sufficient to establish the three-dimensional orientation of the protein in the membrane, while the (15)N chemical shifts provide a measure of cross-validation for the analysis. In C14 lipid bilayers, OmpX adopts a transmembrane orientation with a 7 degrees tilt of its beta-barrel axis relative to the membrane normal, matching the hydrophobic thickness of the barrel with that of the membrane.

| S-EPMC2899889 | biostudies-literature

Nanoparticle-induced surface reconstruction of phospholipid membranes.

Project description:The nonspecific adsorption of charged nanoparticles onto single-component phospholipid bilayers bearing phosphocholine headgroups is shown, from fluorescence and calorimetry experiments, to cause surface reconstruction at the points where nanoparticles adsorb. Nanoparticles of negative charge induce local gelation in otherwise fluid bilayers; nanoparticles of positive charge induce otherwise gelled membranes to fluidize locally. Through this mechanism, the phase state deviates from the nominal phase transition temperature by tens of degrees. This work generalizes the notions of environmentally induced surface reconstruction, prominent in metals and semiconductors. Bearing in mind that chemical composition in these single-component lipid bilayers is the same everywhere, this offers a mechanism to generate patchy functional properties in phospholipid membranes.

| S-EPMC2587577 | biostudies-literature

Bolaamphiphiles promote phospholipid translocation across vesicle membranes.

Project description:A series of membrane-spanning bolaamphiphiles (molecules with two hydrophilic end groups connected by a hydrophobic linker) were prepared by a modular synthetic method and evaluated for their abilities to affect the dynamics of a surrounding bilayer membrane. The goal was to determine if the bolaamphiphiles promote the translocation of phospholipids across vesicle membranes. The bolaamphiphiles were incorporated at low levels (up to 5 mol %) in vesicles composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). Inward translocation assays were performed using fluorescent, NBD-labeled phospholipid probes with phosphocholine (PC) or phosphoglycerol (PG) headgroups. The membrane-spanning bolaamphiphiles promote the translocation of both phospholipid probes in the order PG > PC, whereas shorter bolaamphiphiles (structures that must adopt a U-shape and keep both end groups in the same leaflet of the membrane), and regular amphiphiles with one hydrophilic end group, are inactive. These results are an exception to the rule-of-thumb that membrane-spanning bolaamphiphiles are inherently membrane-stabilizing molecules that inhibit all types of membrane transport.

| S-EPMC2531240 | biostudies-literature

Repeated photoporation with graphene quantum dots enables homogeneous labeling of live cells with extrinsic markers for fluorescence microscopy.

Project description:In the replacement of genetic probes, there is increasing interest in labeling living cells with high-quality extrinsic labels, which avoid over-expression artifacts and are available in a wide spectral range. This calls for a broadly applicable technology that can deliver such labels unambiguously to the cytosol of living cells. Here, we demonstrate that nanoparticle-sensitized photoporation can be used to this end as an emerging intracellular delivery technique. We replace the traditionally used gold nanoparticles with graphene nanoparticles as photothermal sensitizers to permeabilize the cell membrane upon laser irradiation. We demonstrate that the enhanced thermal stability of graphene quantum dots allows the formation of multiple vapor nanobubbles upon irradiation with short laser pulses, allowing the delivery of a variety of extrinsic cell labels efficiently and homogeneously into live cells. We demonstrate high-quality time-lapse imaging with confocal, total internal reflection fluorescence (TIRF), and Airyscan super-resolution microscopy. As the entire procedure is readily compatible with fluorescence (super resolution) microscopy, photoporation with graphene quantum dots has the potential to become the long-awaited generic platform for controlled intracellular delivery of fluorescent labels for live-cell imaging.

| S-EPMC6106998 | biostudies-literature

Compromised reactive but intact proactive inhibitory motor control in Tourette disorder.

Project description:Tourette disorder (TD) is characterized by tics, which are sudden repetitive involuntary movements or vocalizations. Deficits in inhibitory control in TD patients remain inconclusive from the traditional method of estimating the ability to stop an impending action, which requires careful interpretation of a metric derived from race model. One possible explanation for these inconsistencies is that race model's assumptions of independent and stochastic rise of GO and STOP process to a fixed threshold are often violated, making the classical metric to assess inhibitory control less robust. Here, we used a pair of metrics derived from a recent alternative model to address why stopping performance in TD is unaffected despite atypical neural circuitry. These new metrics distinguish between proactive and reactive inhibitory control and estimate them separately. When these metrics in adult TD group were contrasted with healthy controls (HC), we identified robust deficits in reactive control, but not in proactive control in TD. The TD group exhibited difficulty in slowing down the speed of movement preparation, which they rectified by their intact ability to postpone the movement.

| S-EPMC8828748 | biostudies-literature

Precise immuno-fluorescence canceling enables highly multiplexed imaging

Project description:This SuperSeries is composed of the SubSeries listed below.

2024-04-16 | GSE242887 | GEO

Cyanine Phototruncation Enables Spatiotemporal Cell Labeling.

Project description:Photoconvertible tracking strategies assess the dynamic migration of cell populations. Here we develop phototruncation-assisted cell tracking (PACT) and apply it to evaluate the migration of immune cells into tumor-draining lymphatics. This method is enabled by a recently discovered cyanine photoconversion reaction that leads to the two-carbon truncation and consequent blue-shift of these commonly used probes. By examining substituent effects on the heptamethine cyanine chromophore, we find that introduction of a single methoxy group increases the yield of the phototruncation reaction in neutral buffer by almost 8-fold. When converted to a membrane-bound cell-tracking variant, this probe can be applied in a series of in vitro and in vivo experiments. These include quantitative, time-dependent measurements of the migration of immune cells from tumors to tumor-draining lymph nodes. Unlike previously reported cellular photoconversion approaches, this method does not require genetic engineering and uses near-infrared (NIR) wavelengths. Overall, PACT provides a straightforward approach to label cell populations with spatiotemporal control.

| S-EPMC10523398 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data