Lipid Dye Research Articles

Nonequilibrium Collective Dynamics in Photoexcited Lipid Multilayers by Time Resolved Diffuse X-Ray Scattering

We study the nonequilibrium shape fluctuations in fluorescence labeled phospholipid multibilayers composed of the model lipid DOPC and the well-known lipid dye Texas red, driven out of equilibrium by short laser pulses. The temporal evolution of the lipid bilayer undulations after excitation was recorded by time resolved x-ray diffraction. Already at moderate peak intensities (Pp≤10(5) W/cm2), pulsed laser illumination leads to significant changes of the undulation modes in a well-defined lateral wavelength band. The observed phenomena evolve on nano- to microsecond time scales after optical excitation, and can be described in terms of a modulation instability in the lipid multilamellar stack.

Imaging the Effects of Prostaglandin Analogues on Cultured Trabecular Meshwork Cells by Coherent Anti-Stokes Raman Scattering

The aim of this study was to nondestructively monitor morphological changes to the lipid membranes of primary cultures of living human trabecular meshwork cells (hTMC) without the application of exogenous label. Live hTMC were imaged using two nonlinear optical techniques: coherent anti-Stokes Raman scattering (CARS) and two-photon autofluorescence (TPAF). The hTMC were treated with a commercial formulation of latanoprost (0.5 μg/mL) for 24 hours before imaging. Untreated cells and cells treated with vehicle containing the preservative benzalkonium chloride (BAK; 2 μg/mL) were imaged as controls. After CARS/TPAF imaging, hTMC were fixed, stained with the fluorescent lipid dye Nile Red, and imaged by conventional confocal microscopy to verify lipid membrane structures. Analysis of CARS/TPAF images of hTMC treated with latanoprost revealed multiple intracellular lipid membranes absent from untreated or BAK-treated hTMC. Treatment of hTMC with sodium fluoride or ouabain, agents shown to cause morphological changes to hTMC, also did not induce formation of intracellular lipid membranes. CARS microscopy detected changes in living hTMC morphology that were validated by subsequent histological stain. Prostaglandin-induced changes to hTMC involved rearrangement of lipid membranes within these cells. These in vitro results identify a novel biological response to a class of antiglaucoma drugs, and further experiments are needed to establish how this effect is involved in the hypotensive action of prostaglandin analogues in vivo.

DNA-Based Patterning of Tethered Membrane Patches

Solid-supported lipid bilayers are useful model systems for mimicking cellular membranes; however, the interaction of the bilayer with the surface can disrupt the function of integral membrane proteins. As a result, many groups have introduced tethered lipid bilayers, which retain the proximity to the surface, enabling surface-sensitive techniques, but physically distance the bilayer from the surface. We have recently developed a method for spatially separating a lipid bilayer from a solid support using DNA lipids (Chung and Boxer et al., J. Struct. Biol., 2009). In this system, a DNA strand is covalently attached to a silane-modified glass slide or SiO2 wafer. The complementary DNA strand conjugated to a lipid moiety is inserted into giant unilamellar vesicles (GUVs), and the DNA-modified GUVs hybridize to the strands on the surface, inducing flattening and rupture of the GUV to a planar tethered lipid bilayer. However, the location of the patch is random, determined by where the DNA-GUV initially binds with its complement. To allow greater versatility and control, we sought a way to pattern tethered membrane patches. We present a method for creating spatially distinct tethered membrane patches on a glass slide using microarray printing. Surface-reactive DNA sequences are spotted onto the slide, incubated to covalently link the DNA to the surface, and DNA-GUVs patches are formed selectively on the printed DNA. Different DNA sequences can be printed on the same slide, creating a unique handle on each GUV patch. This handle enables the creation of patches of different lipid compositions, dyes, and/or DNA-lipid sequences in adjacent but distinct areas, and the control over the placement of the tethered lipid bilayer potentially allows interfacing with devices. This approach would also enable rapid screening of different patches in protein binding assays and as targets for membrane fusion.

Temperature-Dependent Phase Behavior of the Synaptosomal Membranes from Mammalian and Marine Invertebrate Synaptosomes

Given reports of raft domains in rodent brain synaptosomes, we probed for their conservation in a poikilotherm, the Woods Hole squid Loligo. Because rafts are described as liquid-ordered phase-separated domains, we expected phase transition temperatures above body temperature if raft function is important to neuronal activity. We tested this hypothesis by comparing synaptosomes, intact nerve endings, from animals that live at two very different temperatures: mouse (body temperature-37°C) and squid (body temperature-20°C). We measured the temperature-dependence of the lipid phase of intact synaptosomes in the absence of exogenous probes by using line-width and spinning sideband intensities of lipid hydrocarbon chain resonances, using proton magic angle spinning NMR spectra as a function of temperature between 0 and 40°C. We also determined the phase behavior of total synaptosomal lipids by repeating the NMR protocol on extracted lipids. To our surprise, there was no detectable signal for liquid-ordered lipid phases at the body temperature of either species. However, there was a difference in the temperature of the onset of order as synaptosomes were cooled below body temperature: the phase state of mouse synaptosomal membranes changes drastically below 24°C, whereas this change occurs below 8°C for the squid synaptosomal membranes. We then measured the composition of synaptosomes in terms of total lipid heads and tails, and the main difference arises from high concentrations of omega-3 poly-unsaturated fatty acids and cholesterol in the squid. Fluorescence microscopy images of squid synaptosomes stained with lipid dyes confirmed the formation of domains below, but not above the phase transition temperatures obtained from NMR measurements. Thus although the membranes of synaptosomes contain lipids that can phase-separate, these lipids remain in the liquid-disordered state at the usual physiological temperatures for squid and mouse.

Impact of PEGylation on biodistribution and tumor accumulation of Lipid-Mu peptide-DNA

For gene-based therapeutic approaches that require transfection of specific cell targets in vivo, it is important to design the delivery system to have optimized tissue distribution. Surface PEGylation of liposomes and polymer nanoparticles has been shown to lead to prolonged blood circulation and also preferential accumulation in tumor sites resulting from the enhanced permeability and retention (EPR) effect. The aim of this study was to investigate the effect of different surface PEGylation densities on resulted biodistribution profiles of Lipid-Mu peptide-DNA (LMD) nanoparticles. LMD particles containing the near infrared fluorescent lipid dye, Cy5.5-DSPE, were injected intravenously, and whole-body fluorescence images of the live animal were recorded. Analysis of these time series of images indicated that LMDs with different surface PEG2000 densities had distinctively different biodistribution and tumor accumulation profiles. LMDs containing approximately 15–25% of surface PEG2000 were shown to have the highest accumulation and longest residence time in tumor. LMD distribution and pharmacokinetic profiles in other organs were also observed to be different and were analyzed.

Parallel Dip‐Pen Nanolithography using Spore‐ and Colloid‐Terminated Cantilevers

Parallel dip-pen nanolithography is used to generate micrometer-scale patterns with protein and lipid dyes on both a glass surface and spore layer. Spore- and colloid-based tips are used to facilitate parallel patterning.

One-step synthesis of dye-incorporated porous silica particles

Fluorescent nanoparticles have a variety of biomedical applications as diagnostics and traceable drug delivery agents. Highly fluorescent porous silica nanoparticles were synthesized in a water/oil phase by a microemulsion method. What is unique about the resulting porous silica nanoparticles is the combination of a single-step, efficient synthesis and the high stability of its fluorescence emission in the resulting materials. The key of the success of this approach is the choice of a lipid dye that functions as a surrogate surfactant in the preparation. The surfactant dye was incorporated at the interface of the inorganic silica matrix and organic environment (pore template), and thus insures the stability of the dye–silica hybrid structure. The resulting fluorescent silica materials have a number of properties that make them attractive for biomedical applications: the availability of various color of the resulting nanoparticle from among a broad spectrum of commercially dyes, the controllablity of pore size (diameters of ~5 nm) and particle size (diameters of ~40 nm) by adjusting template monomer concentration and the water/oil ratio, and the stability and durability of particle fluorescence because of the deep insertion of surfactant’s tail into the silica matrix.

Permeation of Amphipathic Drug through a Lipid Bilayer: Curcumin

Curcumin is a natural compound extracted from rhizomes of turmeric and is an example of amphipathic drug that bind to cell membranes. Extensive research over the last 50 years indicated that curcumin has antiinfammatory, antiangiogenic, antioxidant, wound-healing, and anticancer effects. The ability of drugs to permeate through membrane is very important to drug delivery. Passive transmembrane permeability is a major mechanism for drug absorption. In this study we used the aspirated GUV method to study how curcumin molecules permeate through a lipid membrane. Previous equilibrium studies showed that curcumin has two bound states on a lipid bilayer: one on the headgroup-chain interface (S) and another in the hydrocarbon chain region (I). The I state energy is significantly higher than the S state, so that initially as curcumin diffuse to the bilayer binds almost exclusively in the S state. Because of the existence of the I state, curcumin in the S state can translocate through the hydrocarbon barrier. We proved by the quenching ability of curcumin on lipid dye that, when the concentration is lower than the critical concentration, curcumin molecules which initial binding to the outer-layer can translocate into the inner-layer. The translocated molecules will then desorb from the inner layer thus permeate through membrane into the vesicle. We proved by content dye that below the critical concentration curcumin permeate through the membrane without making leakage. Surprisingly we found that when the concentration is higher than the critical concentration, the bilayer leaks dye molecules, thus further increases the permeation rate. Thus our results suggest that when a lipid bilayer is stretched beyond a certain limit by molecular binding, it might become leaky to small molecules. We believe that this complicated concentration-dependent permeation rate could be a common pattern for amphipathic drugs.

The MARVEL transmembrane motif of occludin mediates oligomerization and targeting to the basolateral surface in epithelia

Occludin (Ocln), a MARVEL-motif-containing protein, is found in all tight junctions. MARVEL motifs are comprised of four transmembrane helices associated with the localization to or formation of diverse membrane subdomains by interacting with the proximal lipid environment. The functions of the Ocln MARVEL motif are unknown. Bioinformatics sequence- and structure-based analyses demonstrated that the MARVEL domain of Ocln family proteins has distinct evolutionarily conserved sequence features that are consistent with its basolateral membrane localization. Live-cell microscopy, fluorescence resonance energy transfer (FRET) and bimolecular fluorescence complementation (BiFC) were used to analyze the intracellular distribution and self-association of fluorescent-protein-tagged full-length human Ocln or the Ocln MARVEL motif excluding the cytosolic C- and N-termini (amino acids 60-269, FP-MARVEL-Ocln). FP-MARVEL-Ocln efficiently arrived at the plasma membrane (PM) and was sorted to the basolateral PM in filter-grown polarized MDCK cells. A series of conserved aromatic amino acids within the MARVEL domain were found to be associated with Ocln dimerization using BiFC. FP-MARVEL-Ocln inhibited membrane pore growth during Triton-X-100-induced solubilization and was shown to increase the membrane-ordered state using Laurdan, a lipid dye. These data demonstrate that the Ocln MARVEL domain mediates self-association and correct sorting to the basolateral membrane.

Molecular Convergence of Bacterial and Eukaryotic Surface Order

The conservation of fluidity is a theme common to all cell membranes. In this study, an analysis of lipid packing was conducted via C-laurdan spectroscopy of cell surface membranes prepared from representative species of Bacteria and Eukarya. We found that despite their radical differences in composition (namely the presence and absence of membrane-rigidifying sterol) the membrane order of all taxa converges on a remarkably similar level. To understand how this similarity is constructed, we reconstituted membranes with either bacterial or eukaryotic components. We found that transmembrane segments of proteins have an important role in buffering lipid-mediated packing. This buffering ensures that sterol-free and sterol-containing membranes exhibit similar barrier properties.

Imaging and analysis of Pseudomonas aeruginosa swarming and rhamnolipid production.

Many bacteria spread over surfaces by "swarming" in groups. A problem for scientists who study swarming is the acquisition of statistically significant data that distinguish two observations or detail the temporal patterns and two-dimensional heterogeneities that occur. It is currently difficult to quantify differences between observed swarm phenotypes. Here, we present a method for acquisition of temporal surface motility data using time-lapse fluorescence and bioluminescence imaging. We specifically demonstrate three applications of our technique with the bacterium Pseudomonas aeruginosa. First, we quantify the temporal distribution of P. aeruginosa cells tagged with green fluorescent protein (GFP) and the surfactant rhamnolipid stained with the lipid dye Nile red. Second, we distinguish swarming of P. aeruginosa and Salmonella enterica serovar Typhimurium in a coswarming experiment. Lastly, we quantify differences in swarming and rhamnolipid production of several P. aeruginosa strains. While the best swarming strains produced the most rhamnolipid on surfaces, planktonic culture rhamnolipid production did not correlate with surface growth rhamnolipid production.

Transfer of cell membrane components via trogocytosis occurs in CD4+Foxp3+CD25+ regulatory T-cell contact-dependent suppression

A key component of the immune system is its ability to establish and maintain peripheral tolerance. Naturally occurring CD4+CD25+Foxp3+ regulatory T (nTreg) cells represent an important means by which this is accomplished, through their potent ability to suppress the actions of both CD4+ and CD8+ effector (Teff) cells in vitro and in vivo. We hypothesized that direct contact between nTreg and Teff cells is sufficient for nTreg cell-contact suppression. We first show that nTreg cell suppression is independent of APCs and their derived co-stimulatory signals. We then used a two-colour, lipid dye labelling and quantification approach to formally demonstrate that nTreg cells specifically form cell conjugates with responding T (Tresp) cells only under TCR activating conditions. Strikingly, activated CD4+ nTreg cells undergo progressive trogocytosis, a process by which membrane fragments are transferred from one cell subset to another, with Tresp cells more readily than Teff cells. These results are the first to show that nTreg cell cognate interactions with Tresp cells leads to trogocytosis between the cells, and the first to relate the degree of trogocytosis with the level of nTreg-mediated suppression.

Reversible solid-state mechanochromic fluorescence from a boron lipid dye

Difluoroboron β-diketone complexes are versatile light-emitting molecules that exhibit tunable emission in both solution and the solid state. Among this class of dyes, difluoroboron dibenzoylmethane-polylactide (BF2dbmPLA) polymers have been investigated for their molecular weight dependent fluorescence where the polymer chain plays an important role in BF2dbm solid-state emission. Here the substituent effects were further examined with a lipid chain replacing polylactide. Surprising process dependent and reversible mechanochromic fluorescence was discovered for the boron dodecane complex (BF2dbmOC12H25). A thermally annealed spin-cast film of the lipid dye on glass exhibited blue fluorescence under UV light but after shearing or scratching, the mechanically perturbed region turned yellow-green. The blue coloration could be rapidly recovered by thermal treatment of the film. The phenomena were investigated by steady-state fluorescence spectroscopy at room, low, and high temperatures, in situfluorescence microscopy, fluorescence lifetime measurements, and X-ray diffraction. Consistent with previous findings, the ordered-to-amorphous structural change that occurs upon mechanical perturbation may increase molecular rotational freedom, allowing for more efficient excimer emission, which typically occurs at longer wavelengths.

Negative Potentials Across Biological Membranes Promote Fusion by Class II and Class III Viral Proteins

Voltage was investigated as a factor in the fusion of virions. Virions, pseudotyped with a class II, SFV E1 or VEEV E, or a class III protein, VSV G, were prepared with GFP within the core and a fluorescent lipid. This allowed both hemifusion and fusion to be monitored. Voltage clamping the target cell showed that fusion is promoted by a negative potential and hindered by a positive potential. Hemifusion occurred independent of polarity. Lipid dye movement, in the absence of content mixing, ceased before complete transfer for positive potentials, indicating that reversion of hemifused membranes into two distinct membranes is responsible for voltage dependence and inhibition of fusion. Content mixing quickly followed lipid dye transfer for a negative potential, providing a direct demonstration that hemifusion induced by class II and class III viral proteins is a functional intermediate of fusion. In the hemifused state, virions that fused exhibited slower lipid transfer than did nonfusing virions. All viruses with class II or III fusion proteins may utilize voltage to achieve infection.

Observation of Beta-Amyloid Formation Via Membrane Binding

Alzheimer's Aβ-40 and penetratin exhibited the same conformation changes upon binding to membranes. Recently we have studied the thermodynamics of membrane-mediated β-aggregate formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the α-helical conformation at low peptide-to-lipid (P/L) ratios. As P/L exceeds a lipid dependent critical value P/L∗, small β-aggregates were formed, which served as the nuclei for large β-aggregates. We tested this free energy description in a kinetic experiment using GUVs. A GUV made of 7:3 DOPC/DOPG and 0.2% lipid dye was aspirated by a micropipette and transferred to a solution of containing penetratin in various concentrations. As the peptides began to bind to the GUV, the membrane area initially expanded till it reached a maximum (this corresponds to P/L->P/L∗). Then the area began to decrease from the maximum expanded value (corresponding to P/L exceeding P/L∗ where the membrane thinning decreases with increasing P/L). Concomitant with the area decrease, lipid aggregates began to appear on the surface of GUV and some of them came off the GUV surface.

Voltage Across the Target Cell Membrane is a Strong Regulator of Fusion of Virus Containing Class II or Class III Proteins

Simultaneous electrophysiological voltage clamp and confocal microscopy were used to investigate the dependence of fusion on the voltage across the membrane of the target cell for class II and class III fusion proteins. Cells expressing the fusion protein of Semliki Forest Virus or Venezuelan Equine Encephalitis Virus, both class II, or Vesicular Stomatitis Virus, class III, fused to target cells that were voltage-clamped to a trans-negative potential. Fusion did not occur for positive potentials. This pattern also held for fusion of pseudotyped virions to cells. These virions contained one of the three types of fusion proteins and a fluorescent lipid in the envelopes, and GFP within their core, allowing both hemifusion and fusion to be monitored after acidification of the external medium. Fusion of the pseudovirions exhibited the same voltage-dependence as found for cell-cell fusion. Hemifusion occurred for either voltage polarity at physiological temperature, but the kinetics of hemifusion depended on polarity and lipid dye transfer from virus to cell was aborted for positive, but not for negative potentials. The latter strongly indicates that hemifused membranes separate back into two distinct membranes for positive potentials, accounting for the block of fusion for this polarity. Creating hemifusion at low temperature allowed the hemifusion stage to be captured and studied. Fluorescent lipid dye clearly left the virus and content mixing was subsequently observed for trans-negative potentials, showing that hemifusion is a functional intermediate of fusion in this system. Voltage may be an important driving force for infection by all viruses that utilize class II and III proteins. Supported by NIH GM 27367.

A new model system swims into focus: using the zebrafish to visualize intestinal lipid metabolism in vivo

Many fundamental questions remain regarding the cellular and molecular mechanisms of digestive lipid metabolism. One major impediment to answering important questions in the field has been the lack of a tractable and sufficiently complex model system. Until recently, most studies of lipid metabolism have been performed in vitro or in mice, yet each approach possesses certain limitations. The zebrafish (Danio rerio) offers an excellent model system in which to study lipid metabolism in vivo, owing to its small size, genetic tractability and optical clarity. Fluorescent lipid dyes and optical reporters of lipid-modifying enzymes are now being used in live zebrafish to generate visible readouts of digestive physiology. Here we review recent advances in visualizing intestinal lipid metabolism in live larval zebrafish.

Histopathological changes in brain arteriovenous malformations after embolization using Onyx or N-butyl cyanoacrylate

The aim of this study was to analyze the histopathological changes in a consecutive series of 32 patients with brain arteriovenous malformations that were resected after undergoing endovascular embolization (22 using Onyx and 10 using N-butyl cyanoacrylate [NBCA]). Selections from fixed paraffin-embedded specimens were stained for histological examination with H & E and Verhoeff-van Gieson stain. Lipid dye Oil Red O was used to stain vessel specimens that were embolized using NBCA. Specimens were evaluated for the presence of embolic agent, inflammation, angionecrosis, and evidence of recanalization. These results were correlated with the time interval between the bleeding, embolization, and resection. The smallest vessel occluded by the embolic agent was 5 microm in the Onyx group and 20 microm in the NBCA group. There was evidence of vascular or perivascular inflammation in 20 (90.9%) of 22 and 9 (90%) of 10 specimens after Onyx and NBCA embolization, respectively. Chronic foreign-body giant cells were observed in 12 (54.5%) of 22 specimens after Onyx embolization, but were absent in specimens after NBCA embolization. Angionecrosis of the embolized vessel was observed in 13 (59.1%) of 22 specimens and in 4 (40%) of 10 specimens after Onyx and NBCA embolization, respectively. There was evidence of recanalization in Onyx embolized vessels in 4 (18.2%) of 22 specimens, and there was no evidence of recanalization after NBCA embolization. Onyx penetrates much smaller vessels than NBCA. Inflammation occurs with both embolic agents at equal frequency. Evidence of chronic foreign-body giant cells and recanalization after Onyx embolization shows a long-standing reaction to Onyx and raises questions about the permanence of occlusion after Onyx embolization.

Esterified Cholesterol Is Highly Localized to Bruch's Membrane, as Revealed by Lipid Histochemistry in Wholemounts of Human Choroid

Accumulation of neutral lipids in Bruch's membrane (BrM) is a major age change in human retina and contributes to the formation of extracellular lesions associated with age-related macular degeneration. We developed a BrM-choroid wholemounting technique suitable for reliable staining and evaluated different fluorescent lipid dyes for topographic semiquantitative analysis of BrM lipids. Thin BrM-choroid complexes with partially stripped choroid from 10 aged donor eyes were prepared with an optimized wholemounting technique. Preparation quality was monitored by examining 1-mum-thick sections of representative samples. The staining patterns of Nile Red, BODIPY 493/503, filipin for unesterified cholesterol (UC-F), filipin for esterified cholesterol (EC-F), and Oil Red O in wholemounts were compared with their staining patterns in chorioretinal sections, using wide-field epi-fluorescence microscopy. Wholemounts exhibited optimal flatness on the BrM side. Reduced tissue thickness allowed reliable dye penetration and staining of BrM. Only EC-F was with high specificity localized to BrM and demonstrated an intense and distinct granular staining pattern not previously appreciated in chorioretinal sections. All other lipid dyes also stained choroidal or retinal tissue intensely. No dye provided perfect characteristics in regard to representing all neutral lipid classes present in BrM or to fluorescence intensity. Nevertheless, only EC-F was highly localized to BrM with a specific granular pattern. Because direct assays indicate that esterified cholesterol is abundantly present in BrM, we consider EC-F the most valuable choice for analyzing neutral lipid deposits in human BrM.

Imaging Single Virus Fusion Reveals the HIV-1 Entry Pathway

Viruses whose fusion proteins are activated by interactions with cellular receptors at neutral pH, including HIV-1, are assumed to fuse directly with the plasma membrane. However, direct virus fusion at the cell surface has not been explicitly demonstrated. To differentiate between HIV-1 fusion with the plasma membrane (PM) and with endosomal membrane (EM), we performed time-resolved single virus imaging. Pseudoviruses bearing HIV-1 Env glycoprotein were generated and co-labeled with a content marker (Gag-GFP) and a red lipophilic membrane dye. Upon virus maturation, the Gag-GFP is cleaved by viral protease yielding a smaller GFP-tagged fragment that is readily released from virions permeabilized with saponin. This marker thus provided a convenient means to detect small pore formation during virus-cell fusion. Double-labeled viruses were adhered in the cold to HeLa-derived cells expressing CD4 and coreceptors. Fusion was triggered by shifting to 37°C and monitored by laser scanning confocal microscopy. Imaging of single HIV-cell fusion revealed the occurrence of both PM and EM fusion events. Fluorescent viruses undergoing PM fusion transferred their lipid marker (hemifusion), but not content marker (full fusion). By contrast, full fusion with an endosome was consistently observed. These EM fusion events were manifested in disappearance of a content marker, but not the lipid dye due to its limited dilution in an endosomal membrane. These results demonstrate that, contrary to a common perception, HIV-1 enters HeLa-derived target cells by receptor/coreceptor-mediated endocytosis followed by fusion with endosomes and delivery of viral nucleocapsid into the cytosol.