Fluorescent Lipid Probes Research Articles

Lateral stress profile and fluorescent lipid probes. FRET pair of probes that introduces minimal distortions into lipid packing

The modern theory of lipid membrane structure incorporates the concept of lateral stress profile. The latter represents the forces that act on any solute inside the membrane. We used this concept to propose two lipid probes that introduce minimal distortions into the lipid bilayer packing: the surface pressure isotherms and volt-potentials of the pure and mixed (probe-containing) lipid monolayers are equal. The probes represent a FRET pair. They are applicable in lipid transfer and vesicle fusion experiments.

Influence of stabilizing components on the integrity of antitumor liposomes loaded with lipophilic prodrug in the bilayer.

Previously, we proposed a liposomal formulation of melphalan (Mlph)-a chemotherapeutic alkylating agent-incorporated in a fluid lipid bilayer in the form of dioleoylglyceride ester. In this work, we compared the stabilizing effect of different amphiphiles included in the Mlph-liposomes, such as phosphatidylinositol (PI), ganglioside GM1, a conjugate of N-carboxymethyl-modified oligoglycine with dioleoylphosphatidylethanolamine (acidic lipopeptide), and polyethylene glycol (2000 Da) conjugated with dipalmitoylphosphatidylethanolamine (PEG-lipid), upon incubation in human serum. Mean hydrodynamic diameter values (86-90 nm) were similar among different liposome samples, while zeta potential values considerably varied. The formulations were incubated in human serum at 37 °C for different time intervals up to 24 h. Liposome integrity was evaluated by changes in fluorescence upon leakage of calcein or disruption of Förster resonance energy transfer between donor and acceptor fluorescent lipid probes in the bilayer. The best stabilization of liposomes was achieved upon the addition of ganglioside GM1 or the acidic lipopeptide. Inclusion of 10 mol% PI improved liposome stability only for the first 4 h of incubation. Pegylated liposomal formulations of melphalan lipophilic prodrug with fluid phase bilayer were the least stable, which is probably due to the propensity of the PEG-lipid to exit liposome membranes. Cholesterol-containing bilayers of liquid ordered phase, supplemented with sufficient amounts of the PEG-lipid, showed good stability in serum.

The Blomia tropicalis allergen Blo t 7 stimulates innate immune signalling pathways through TLR2.

Although the house dust mite species Blomia tropicalis is a leading cause of allergic diseases in tropical and subtropical regions, the identification and characterization of the allergenic proteins remain incomplete. We aimed to characterize a recombinant form of Blo t 7 (rBlo t 7) in terms of IgE reactivity, lipid-binding activity and ability to stimulate innate immunity. The mature Blo t 7 cDNA was cloned by PCR methods for the expression of a secreted form of the allergen in P. pastoris. The IgE reactivity to purified rBlo t 7 as well as the potential cross-reactivity with Der p 7 was determined by ELISA. The lipid-binding capacity of rBlo t 7 was assayed using fluorescent lipid probes. The stimulation of TLR2 signalling pathway by rBlo t 7 was examined in cell activation and reporter assays. The amplified mature Blo t 7 cDNA revealed the presence of a 60 base pair insertion compared with the reference sequence registered in the GenBank database. Multiple protein sequence alignments of group 7 mite allergens confirmed that this longer deduced amino acid sequence was the authentic Blo t 7 polypeptide chain. Analysis of IgE reactivity can classify rBlo t 7 as an intermediate B. tropicalis allergen which displayed weak cross-reactivity with Der p 7. Purified rBlo t 7 was shown to bind selectively the naturally fluorescent lipid probe cis-parinaric (cPNA) with a dissociation constant of 2 μmol/L. The group 7 Blomia allergen stimulated the TLR2-, NF-kB- and MAPK-dependent production of IL-8 and GM-CSF in respiratory epithelial cells. Through its propensity to transport fatty acids/lipids and to stimulate TLR2 signalling pathways in airway epithelial cells, Blo t 7 can represent a key allergen for the initiation of the B. tropicalis-induced airway inflammation.

Identification and characterization of the major pseudocoelomic proteins of the giant kidney worm, Dioctophyme renale

BackgroundThe giant kidney worm, Dioctophyme renale, is a debilitating and potentially lethal parasite that inhabits and destroys, typically host’s right kidney, and may also be found in ectopic sites. It is circumglobally distributed, mainly in dogs, and is increasingly regarded as a threat to other domestic animals and humans. There is little information on the parasite’s true incidence, or immune responses to it, and none on its biochemistry and molecular biology.ResultsWe characterised the soluble proteins of body wall, intestine, gonads and pseudocelomic fluid (PCF) of adult parasites. Two proteins, P17 and P44, dominate the PCF of both male and females. P17 is of 16,622 Da by mass spectrometry, and accounts for the intense red colour of the adult parasites. It may function to carry or scavenge oxygen and be related to the ‘nemoglobins’ found in other nematode clades. P44 is of 44,460 Da and was found to associate with fatty acids by thin layer chromatography. Using environment-sensitive fluorescent lipid probes, P44 proved to be a hydrophobic ligand-binding protein with a binding site that is highly apolar, and competitive displacement experiments showed that P44 binds fatty acids. It may therefore have a role in distributing lipids within the parasites and, if also secreted, might influence local inflammatory and tissue responses. N-terminal and internal peptide amino-acid sequences of P44 indicate a relationship with a cysteine- and histidine-rich protein of unknown function from Trichinella spiralis.ConclusionsThe dominant proteins of D. renale PCF are, like those of large ascaridids, likely to be involved in lipid and oxygen handling, although there is evidence of strong divergence between the two groups.

Influence of metal cations and cholesterol on lipid-amphotericin membrane

Amphotericin B(AmB) has been widely used in antifungal therapy. AmB molecules combine with cholesterol to form pores that can be toxic to human cells, thus greatly limiting its clinical application. The interaction between AmB and the cell membrane may be influenced by potassium, sodium and calcium ions. In this study, the bilayer in large unilamellar lipid-drug liposomes with or without cholesterol was employed as a model membrane. N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine(NBD-PE) and 1-palmitoyl-2-[6-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)aminodoclecanoyl]-sn-glysero-3-phosphocholine(6-NBD-PC) are two kinds of fluorescent lipid probes, and the NBD group is attached to the polar lipid headgroup in the former, but to the sn-2 fatty acyl chain in the latter. The effect of these metal cations on the lipid-drug membrane was monitored by red edge excitation shift(REES), fluorescence polarization, and the fluorescence lifetime of lipid probes in hydrophilic and hydrophobic areas of the membrane. These ions have different effects on the lipid-AmB membrane. Cholesterol can strengthen the packing ability of the membrane, which is influenced differently by potassium, sodium and calcium ions. Moreover, the influence of these ions on the membrane may be relative to the method of ion transportation through the membrane. This study is significant to understand the reduction of AmB’s cellular toxicity.

Abstract 206: Intestinal LXR Directs Absorbed Lipids to Storage

Post-prandial hypertriglyceridemia has emerged as a cardiovascular risk factor with limited therapeutic options. We have taken genetic and physiological approaches to identify and characertize novel factors that regulate intestinal lipid handling in the hope of revealing cellular pathways that dampen delivery of dietary lipids, opening the therapeutic window to blunting post-prandial lipid excursions. Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. These nuclear hormone receptors are activated by oxysterol, ligands that accumulate when excess cholesterol is present. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets, as visualized with histological stains, live fluorescent lipid probes, and ultrastructural analyses. This delay in transport of ingested neutral lipids protects animals over-expressing nr1h3 in the intestine from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a , which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. We have identified a gene regulatory network in enterocytes whereby the pace of delivery of dietary lipids is gated by Lxrs. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists’ induction of hepatic lipogenesis and hypertriglyceridemia. The development of intestine-limited Lxr agonists could be of therapeutic value in blunting post-prandial hypertriglyceridemia.

Far-Red Fluorescent Lipid-Polymer Probes for an Efficient Labeling of Enveloped Viruses.

Far‐red emitting fluorescent lipid probes are desirable to label enveloped viruses, for their efficient tracking by optical microscopy inside autofluorescent cells. Most used probes are rapidly released from membranes, leading to fluorescence signal decay and loss of contrast. Here, water‐soluble lipid‐polymer probes are synthesized harboring hydrophilic or hydrophobic far‐red emitting dyes, and exhibiting enhanced brightness. They efficiently label Hepatitis C Virus pseudotyped particles (HCVpp), more stably and reproducibly than commercial probes, and a strong fluorescence signal is observed with a high contrast. Labeling with such probes do not alter virion morphology, integrity, nor infectivity. Finally, it is shown by fluorescence microscopy that these probes enable efficient tracking of labeled HCVpp inside hepatocarcinoma cells used as model hepatocytes, in spite of their autofluorescence up to 700 nm. These novel fluorescent lipid‐polymer probes should therefore enable a better characterization of early stages of infection of autofluorescent cells by enveloped viruses.

Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 2. QSAR models that predict localization of fluorescent probes used to identify (“specifically stain”) various biomembranes and membranous organelles

We discuss a variety of biological targets including generic biomembranes and the membranes of the endoplasmic reticulum, endosomes/lysosomes, Golgi body, mitochondria (outer and inner membranes) and the plasma membrane of usual fluidity. For each target, we discuss the access of probes to the target membrane, probe uptake into the membrane and the mechanism of selectivity of the probe uptake. A statement of the QSAR decision rule that describes the required physicochemical features of probes that enable selective staining also is provided, followed by comments on exceptions and limits. Examples of probes typically used to demonstrate each target structure are noted and decision rule tabulations are provided for probes that localize in particular targets; these tabulations show distribution of probes in the conceptual space defined by the relevant structure parameters (“parameter space”). Some general implications and limitations of the QSAR models for probe targeting are discussed including the roles of certain cell and protocol factors that play significant roles in lipid staining. A case example illustrates the predictive ability of QSAR models. Key limiting values of the head group hydrophilicity parameter associated with membrane-probe interactions are discussed in an appendix.

Uptake and localization mechanisms of fluorescent and colored lipid probes. Part 3. Protocols for predicting intracellular localization of lipid probes using QSAR models

We provide detailed protocols for applying the QSAR decision-rule models described in Part 2 of this paper. These procedures permit prediction of the intracellular localization of fluorescent probes or of any small molecular xenobiotic whether fluorescent or not. Also included is a set of notes that give practical advice on various possible problems and limitations of the methods, together with a flow chart that provides a graphical algorithmic summary of the QSAR models.

Ceramide and Cholesterol Effects on Phospholipid Bilayers under the AFM: Characterization of Complex Lipid Phases

Atomic force microscopy (AFM) has been applied to the characterization of palmitoylceramide (pCer) and cholesterol (Chol) incorporation into phospholipid-based supported planar bilayers (SPBs) at 22°C. Phospholipids were dipalmitoyl phosphatidylcholine (DPPC) or palmitoyl sphingomyelin (pSM). Membranes of different compositions were prepared by either the vesicle adsorption or the spin-coating procedures (the latter strictly for pCer-containing mixtures) and analyzed with a combination of AFM imaging (domain segregation, bilayer thickness and roughness) and force spectroscopy (mechanical resistance to indentation). The mixtures under study were pure phospholipids (pSM, DPPC), phospholipid:Chol (70:30), phospholipid:Chol:pCer (2:1:1) and phospholipid:pCer (90:10, 80:20 and 70:30). Binary phospholipid:pCer mixtures at increasing ceramide ratios gave rise to highly-resistant segregated domains with increasing extension but similar properties in terms of breakthrough forces, thicknesses and roughnesses. These ceramide-enriched domains are able to exclude a fluorescent lipid probe (DiIC18) due to their high intermolecular packing. Interestingly, these domains have been reported to disappear when model membranes become highly enriched in cholesterol in fluid membranes or in the absence of a fluid phase (our case). Indeed, the ternary mixtures (2:1:1) gave rise to a homogenous lamellar gel phase with significantly different properties when compared to all of the other mixtures under study: ternary mixtures showed a reduced thickness and an intermediate roughness and mechanical resistance when compared to phospholipid:Chol (70:30) and phospholipid:pCer. These differences were statistically significant. More importantly, at those relatively high pCer and Chol concentrations in ternary mixtures, no mutual displacement of these molecules was observed. The data become relevant in the context of sphingolipid signaling and membrane platform formation.

Brain-SM/SDPC/Cholesterol Phase Diagram

We report the solved 3-component phase diagram for the biologically relevant mixture of brain-SM/ SDPC (18:0,22:6)-PC/cholesterol. Polyunsaturated acyl chains can have special influences on the mixing and phase behaviors of lipid mixtures. Their high degree of unsaturation affects the physical properties of biomembranes in ways that are still not fully understood, and their high concentration in some membranes makes them key players in membrane structure. FRET techniques and fluorescence microscopy imaging of giant unilamellar vesicles (GUVs) were employed for phase boundary visualization and phase identification of this 3-component mixture. Fluorescent lipid probes having complementary partitioning behavior were used to distinguish between phases. Of particular interest is the “raft” region of Ld + Lo phase coexistence, which shows phase separation by FRET methods, yet GUVs appear uniform under microscopy. Taken together these results indicate nanoscopic Ld + Lo phase separation in brain-SM/ SDPC (18:0,22:6)-PC/cholesterol mixture.

Synthesis of multifunctional lipid–polymer conjugates: application to the elaboration of bright far-red fluorescent lipid probes

Well-defined multifunctional lipid-polymer conjugates as new tools for the functionalization of lipid assemblies.

Changes in Order Parameters Associated with Ceramide-Mediated Membrane Reorganization Measured Using pTIRFM

The enzymatic generation of ceramide has significant effects on the biophysical properties of lipid bilayers and can lead to the extensive reorganization of cell membranes. We have synthesized and characterized a headgroup-labeled fluorescent lipid probe (NBD-ceramide, NBD-Cer) and demonstrated that it can be used for polarized total internal reflection fluorescence microscopy experiments to probe changes in membrane order that result from ceramide incorporation. NBD-Cer measures significantly higher order parameters for the liquid-ordered (Lo) domains ([P2] = 0.40 ± 0.03) than for the liquid-disordered phase (Ld, fluid, [P2] = 0.22 ± 0.02) of phase-separated bilayers prepared from egg sphingomyelin, dioleolyphosphatidylcholine, and cholesterol mixtures. The probe also responds to changes in packing induced by the direct incorporation of ceramide or the variation in the ionic strength of the aqueous medium. Order parameter maps obtained after enzyme treatment of bilayers with coexisting Lo and Ld phases show two distinct types of behavior. In regions of high enzyme activity, the initial Lo/Ld domains are replaced by large, dark features that have high membrane order corroborating previous hypotheses that these are ceramide-enriched regions of the membrane. In areas of low enzyme activity, the size and shape of the Lo domains are conserved, but there is an increase in the order parameter for the initial Ld phase ([P2] = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the Lo domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.

Ganglioside Influence on Phospholipid Films Investigated with Single Molecule Fluorescence Measurements

Single molecule fluorescence measurements are used to probe the effects of GM1 in DPPC monolayers. Langmuir-Blodgett films of GM1 and DPPC were doped with ~10(-8) mol % of the fluorescent lipid probe, BODIPY-PC, and transferred onto glass substrates at 23 mN/m. As shown previously, the individual orientation of each BODIPY-PC probe in the membrane can be measured using defocused polarized total internal reflection fluorescence microscopy, revealing changes in film properties at the molecular level. Here, BODIPY-PC tilt angle histograms are used to characterize the effects of GM1 in DPPC films from 0.05 to 100 mol % GM1. At high GM1 levels (>5 mol % GM1), trends in the single molecule measurements agree with previous bulk measurements showing the turnover from condensing to expanding influence of GM1 at 15-20 mol %, thus validating the single molecule approach. At biologically relevant, low concentrations of GM1 (<5 mol % GM1), where bulk fluorescence measurements are less informative, the single molecule measurements reveal a marked influence of GM1 on film properties. The addition of trace amounts of GM1 to DPPC films leads to an expansion of the film which continues to 0.10 mol % GM1, above which the trend reverses and the condensing effect previously noted is observed.

High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy

Angle-resolved linear dichroism is a recent technique that exploits images recorded using an illumination field whose polarization angle is sequentially rotated during acquisition. It allows to retrieve orientation information of the fluorescent molecules, namely the average orientation angle and the amplitude of the fluctuations around this average. In order to boost up the acquisition speed without sacrificing the axial sectioning, we propose to combine a spinning disk confocal excitation scheme together with an electrooptical polarization switching and a camera acquisition. The polarization distortions induced when passing through the spinning disk system have been quantified and effectively compensated. The signal to noise features of the camera have been analyzed in detail so that the precision of the method can be quantified. The technique has been successfully tested on giant unilamellar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC18 and di-8-ANEPPQ. It was able to acquire precise orientation images at full frame rates in the range of a second, ultimately limited by the fluorophore brightness and the camera sensitivity.

Limited Perturbation of a DPPC Bilayer by Fluorescent Lipid Probes: A Molecular Dynamics Study

The properties of lipid bilayer nanometer-scale domains could be crucial for understanding cell membranes. Fluorescent probes are often used to study bilayers, yet their effects on host lipids are not well understood. We used molecular dynamics simulations to investigate perturbations in a fluid DPPC bilayer upon incorporation of three indocarbocyanine probes: DiI-C18:0, DiI-C18:2, or DiI-C12:0. We find a 10-12% decrease in chain order for DPPC in the solvation shell nearest the probe but smaller effects in subsequent shells, indicating that the probes significantly alter only their local environment. We also observe order perturbations of lipids directly across from the probe in the opposite leaflet. Additionally, the DPPC headgroup phosphorus-to-nitrogen vector of lipids nearest the probe exhibits preferential orientation pointing away from the DiI. We show that, while DiI probes perturb their local environment, they do not strongly influence the average properties of "nanoscopic" domains containing a few hundred lipids.

A Novel Fluorescent Lipid Probe for Dry Eye: Retrieval by Tear Lipocalin in Humans

A fluorescent probe was used to identify mucin-depleted areas on the ocular surface and to test the hypothesis that tear lipocalin retrieves lipids from the eyes of normal and dry eye subjects. Fluorescein-labeled octadecyl ester, FODE, was characterized by mass spectrometry and absorbance spectrophotometry. The use of FODE to define mucin defects was studied with impression membranes under conditions that selectively deplete mucin. The kinetics of FODE removal from the ocular surface were analyzed by sampling tears from control and dry eye patients at various times. The tear protein-FODE complexes were isolated by gel filtration and ion exchange chromatographies, monitored with absorption and fluorescent spectroscopies, and analyzed by gel electrophoresis. Immunoprecipitation verified FODE complexed to tear lipocalin in tears. FODE exhibits an isosbestic point at 473 nm, pKa of 7.5, and red shift relative to fluorescein. The low solubility of FODE in buffer is enhanced with 1% Tween 80 and ethanol. FODE adheres to the ocular surface of dry eye patients. FODE produces visible staining at the contact sites of membranes, which correlates with removal of mucin. Despite the fact that tear lipocalin is reduced in dry eye patients, FODE removal follows similar rapid exponential decay functions for all subjects. FODE is bound to tear lipocalin in tears. Tear lipocalin retrieves lipid rapidly from the human ocular surface in mild to moderate dry eye disease and controls. With improvements in solubility, FODE may have potential as a fluorescent probe to identify mucin-depleted areas.

Golgi Apparatus and Endoplasmic Reticulum Dynamic during Meiotic Development in Canine Oocytes

The Golgi apparatus (GA) and endoplasmic reticulum (ER) play a central role in the events related to intracellular trafficking distribution. This work evaluated the dynamics and localization of the GA and ER in canine oocytes during meiotic development in vitro. Cumulus-oocytes complexes (COCs) from ovaries of adult bitches were incubated for IVM for 0, 48, 72 and 96 h. At each time, the nuclear status was determined using DAPI staining, and the GA was evaluated by immunofluorescence using two antibodies against Golgi proteins: GM130 and Giantin. ER was analysed with fluorescent lipid probes (ER-Tracker), for living cells. Golgi structures were homogeneous in the cytoplasm in non-matured oocytes, mainly in those GV-arrested oocytes. In contrast, at 48 h and from GVBD stage, the immunolocalization began to be subcortical, increasing at 72 h and 96 h. Meiotic development increased with time and the majority of oocytes at MI-MII stages showed cortical distribution of Golgi structure. Living ZP intact non-matured oocytes showed a reticular pattern of ER that covered oocyte cortex. Confocal microscopy showed that, in all levels cuts the fluorescence marks were located in the cortical region, irrespective of culture time. The changes and localization in these organelles during IVM might be related to meiotic development, but in a non-synchronous manner.

Effects of chemical modification of sphingomyelin ammonium group on formation of liquid-ordered phase

Sphingomyelin (SM) and cholesterol form microdomains called lipid rafts in cellular membranes. To develop a versatile fluorescent lipid probe, chemical modifications to both the hydrophobic and hydrophilic portions of SM are essential. Few reports describing SM probes with a fluorophore at the polar head group have been published. This study examined the effect of substitution on an ammonium moiety of SM on the membrane properties of SM. Two SM analogs with small propargyl and allyl groups on the quaternary nitrogen atom were synthesized and subjected to analysis using differential scanning calorimetry, fluorescent anisotropy, detergent solubilization, surface pressure, and density measurements. Results demonstrated that the two SM analogs retained the membrane properties of SM, including formation of an ordered phase and the ability to interact with cholesterol. A dansyl-substituted SM was prepared for fluorescent measurements. Dansyl-SM showed less of a propensity to form microdomains. These findings imply the potential application of N-substituted SMs as a raft-specific molecular probe.