Lipid Analogues Research Articles

Proteome-wide detection of phospholipid–protein interactions in mitochondria by photocrosslinking and click chemistry

Photoactivatable lipid analogues are uniquely suited for the detection of lipid-protein interactions in biological membranes. Based on photocrosslinking, new methodology has been developed for the proteome-wide detection of lipid-protein interactions. Bifunctional lipid analogues containing a tag for click chemistry in addition to the photoactivatable moiety enable the enrichment of the crosslinked proteins that is required for subsequent identification by mass spectrometry. In principle the phospholipid interaction-based membrane protein proteomics approach is applicable to any biomembrane and any lipid. Here, we review the background and the development of the new methodology. Results obtained with photocrosslinking in purified mitochondrial membranes from the yeast Saccharomyces cerevisiae are summarized and future perspectives discussed.

Single-Molecule Study of the Dynamics of Lipid-Like Molecules in the E. Coli Outer Membrane

While there have been many studies on the diffusion of membrane lipids in eukaryotic cells, which have given insight into the structure and organization of these membranes, little is known to date of their mobility in bacterial membranes, specifically the Gram negative bacteria, Escherichia coli. The E. coli outer envelope consists of inner and outer lipid membranes that are separated by a periplasmic space containing the cell wall. The outer membrane is unique in that it is thinner than mammalian plasma membranes and consists of a phospholipid inner leaflet with a predominantly lipopolysaccharide (LPS) outer leaflet. Here we look at the diffusion of the fluorescent lipid analog 3,3′-dioctadecylindocyanine iodide (DiI(C18)) and Alexa488-LPS in the outer membrane of live E. coli cells using single molecule imaging/tracking techniques. The diffusion coefficient of DiI(C18) was found to be (5.2 ± 0.2) × 10−11 cm2/sat time scales of 0.33 s. By contrast, the diffusion coefficient of DiI(C18) in human epithelial cancer cells of the nasopharanyx (KB) is found to be (1.94 ± 0.2) × 10−8 cm2/s, in good agreement with previously measured diffusion coefficients of DiI(C18) in other mammalian cells. The mobility of LPS in the outer membrane and the implications of the slow diffusion of DiI(C18) on the structure of the outer membrane of E. coli will be discussed.

Imaging of Mobile Stable Lipid Rafts in the Live Cell Plasma Membrane and their Involvement in Cellular Signaling During Heat Shock

The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, also termed lipid or membrane rafts. Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition and heterogeneity. We present here a method which allows for the first time the demonstration that single rafts diffuse as stable platforms in the live cell plasma membrane. Our method senses rafts by their property to assemble a characteristic set of fluorescent marker-proteins or lipids on a time-scale of seconds. The special photobleaching protocol TOCCSL (Thinning Out Clusters while Conserving Stoichiometry of Labeling1) was used to reduce the surface density of labeled mobile rafts down to the level of well-isolated diffraction-limited spots, without altering the single spot brightness. The statistical distribution of probe molecules per raft was determined by single molecule brightness analysis. For demonstration, we used the consensus markers Bodipy-GM1, a fluorescent lipid analogue, and glycosylphosphatidylinositol-anchored monomeric GFP. For both markers we found cholesterol-dependent association in the plasma membrane of living CHO and Jurkat T cells in the resting state, indicating the presence of small, mobile, stable rafts hosting these probes. We further applied the technology to address structural changes in the plasma membrane during fever-type heat shock: at elevated temperatures mGFP-GPI homo-association disappeared, accompanied by an increase in the expression of the small heat shock protein Hsp27.1. Moertelmaier, M., Brameshuber, M., Linimeier, M., Schutz, G. J. & Stockinger, H. Thinning out clusters while conserving stoichiometry of labeling. Appl Phys Lett 87, 263903 (2005).

Fluorescence Lifetime Tuning—A Novel Approach to Study Flip-Flop Kinetics in Supported Phospholipid Bilayers

In the present work we introduce a straightforward fluorescent assay that can be applied in studies of the transbilayer movement (flip-flop) of fluorescent lipid analogues across supported phospholipid bilayers (SPBs). The assay is based on the distance dependent fluorescence quenching by light absorbing surfaces. Applied to SPBs this effect leads to strong differences in fluorescence lifetimes when the dye moves from the outer lipid leaflet to the leaflet in contact with the support. Herein, we present the basic principles of this novel approach, and comment on its advantages over the commonly used methods for investigating flip-flop dynamics across lipid bilayers. We test the assay on the fluorescent lipid analog Atto633-DOPE and the 3-hydroxyflavone F2N12S probe in SPBs composed of DOPC/ DOPS lipids. Moreover, we compare and discuss the flip-flop rates of the probes with respect to their lateral diffusion coefficients.

New Generation of Liposomes Called Archaeosomes Based on Natural or Synthetic Archaeal Lipids as Innovative Formulations for Drug Delivery

Archaeosomes made from natural archaeal membrane lipids and/or synthetic lipid analogues have been extensively studied for potential applications in drug and vaccine delivery over the past decade only. Archaeal-type lipids consist of archaeol (diether) and/or caldarchaeol (tetraether) core structures wherein regularly branched and usually fully saturated phytanyl chains (20-40 carbons in lengths), are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone. Archaeosomes constitute a novel generation of liposomes that exhibit high stabilities to low or high temperatures, acidic or alkaline pH, oxidative conditions, high pressure, action of phospholipases, bile salts and serum proteins. These properties associated with a good safety profile are beneficial for nanotechnological applications in drug and gene delivery. Additionally, archaeosome formulations could be used as efficient carriers of antigens and/or adjuvants promoting antigen-specific, humoral and cell-mediated immune responses, in addition to antigen-specific mucosal immune responses in the vaccinated hosts. The immune responses are well sustained over time, and are subject to strong memory responses. Nanodelivery-based vaccinations using archaeosomes could then represent a promising approach for treating and preventing infections, allergies, and neoplastic or cancer diseases. In this review, the few recent US, World and European patents developing archaeosomes for these biotechnological applications in Health are discussed.

Determination of Lipid Degradation by Marine Lipase‐Producing Bacteria: Critical Evaluation of Lipase Activity Assays

With the aim of obtaining a better understanding of lipids-lipases interactions in bacterioplankton communities in oceans, we used different methods for measuring lipase activities in pure cultures of the marine strain Alteromonas macleodii. The decay of tripalmitate added to cultures was followed chemically over time. In an enzymatic approach, lipase activities were measured using the fluorogenic lipid analogs MUF-palmitate and ELF-palmitate. When hydrolyzed by lipase, the non-fluorescent substrates release MUF and ELF Alcohol (ELFA) which are fluorescent. As shown by spectrofluorometry, ELF-palmitate was an efficient competitor for MUF-palmitate. However, the activities reached using these two fluorogenic substrates were different, but still much higher than the tripalmitate hydrolysis rate, measured chemically. MUF- and ELF-palmitate would not be hydrolyzed by lipase sensu stricto (defined as triacylglycerol acylesterase E.C. 3.1.1.3) but rather reflects lipolytic activities in a broad sense. ELFA is also water-insoluble and theoretically precipitates in the external membrane of bacteria causing its hydrolysis, which would allow microscopic identification of active cells. By epifluorescence microscopy, the accumulation of ELFA fluorescence over time was detected (as large, diffuse halos), but no precipitates were clearly associated with bacteria on slide preparations, neither for pure cultures of Alteromonas macleodii nor for natural samples from the Bay of Marseille, France. Among possible biases, those related to the hydrophobic/hydrophilic conditions required for precipitation are discussed.

New GM1 Ganglioside Derivatives for Selective Single and Double Labelling of the Natural Glycosphingolipid Skeleton

AbstractSelective single and double labelling of the natural ganglioside GM1 enables one to introduce various markers into different parts of the glycosphingolipid molecule without changing the natural skeleton. To that end, N‐Fmoc‐2‐amino‐, N‐Fmoc‐18‐amino‐ and S‐(ethoxythiocarbonyl)‐18‐mercaptostearic acids have been prepared and coupled with the primary amino group in the sphingosine part of lyso‐GM1 and deAc‐deAcyl‐GM1 gangliosides. The products of these coupling reactions – building blocks 16a, 16b, 16c, 26 and 27 – may be used for the synthesis of GM1 derivatives with one or two fluorescent dye moieties or other labels of various polarities. Examples of various labelling strategies, using hydrophilic and lipophilic photostable fluorescent dyes, have been made available. The GM1 derivatives 17a, 22a and 23c labelled with the fluorescent dye ATTO 647N or the doubly labelled derivative 25b can be used as probes in fluorescence correlation spectroscopy (in conventional microscopy or stimulated emission depletion nanoscopy) to study the diffusion of lipid analogues in model or live cell membranes.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Microbial activities accompanying decomposition of cladoceran and copepod carcasses under different environmental conditions

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 57:89-100 (2009) - DOI: https://doi.org/10.3354/ame01331 Microbial activities accompanying decomposition of cladoceran and copepod carcasses under different environmental conditions Kam W. Tang1,*, Samantha L. Bickel1, Claudia Dziallas2, Hans-Peter Grossart2 1Virginia Institute of Marine Science, 1208 Greate Road, Gloucester Point, Virginia 23062, USA 2Leibniz Institute of Freshwater Ecology and Inland Fisheries, Alte Fischerhuette 2, 16775 Stechlin, Germany *Email: kamtang@vims.edu ABSTRACT: Cladoceran and copepod carcasses in both marine and freshwater environments represent concentrated reservoirs of organic substrates for water column bacteria. We studied the microbial abundance, activities, and diversity associated with decomposing carcasses of different zooplankton species over short and long time scales, and in oligotrophic vs. eutrophic environments. Fresh carcasses of Daphnia cucullata, Diaphanosoma brachyurum, and Eudiaptomus gracilis were rapidly colonized by bacteria, which reached peak abundances within 1.5 d at 20°C and then decreased. Cell-specific exoenzymatic activity on protein and lipid analogs and production rate of bacteria associated with the carcasses were all higher than in the ambient water. ANOSIM analyses of DGGE banding patterns revealed that bacterial communities associated with both cladoceran and copepod carcasses rapidly diverged from the initial bacterial community in the ambient water. The high similarity of bacteria on both types of carcasses indicates that the carcasses were decomposed by similar bacterial groups. Estimated carcass decomposition rate was lower at 6°C, with an estimated Q10 of 2.4. Carcasses suspended in the eutrophic Lake Dagow had a higher average carbon loss rate than those suspended in the oligotrophic Lake Stechlin. Cladoceran carcasses were initially colonized by bacteria faster than copepod carcasses in both laboratory and field experiments, suggesting that cladoceran carcasses were more prone to exploitation by bacteria, yet copepod carcasses lost carbon at higher rates. Overall, our results suggest that pelagic zooplankton production can be directly converted to water column bacterial production via carcass decomposition, especially during the mid-summer zooplankton decline commonly observed in lakes. KEY WORDS: Decomposition · Exoenzymes · Bacterial production · Bacterial community composition · Zooplankton carcasses Full text in pdf format PreviousNextCite this article as: Tang KW, Bickel SL, Dziallas C, Grossart HP (2009) Microbial activities accompanying decomposition of cladoceran and copepod carcasses under different environmental conditions. Aquat Microb Ecol 57:89-100. https://doi.org/10.3354/ame01331 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 57, No. 1. Online publication date: September 18, 2009 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2009 Inter-Research.

Probing HIV-1 Membrane Liquid Order by Laurdan Staining Reveals Producer Cell-dependent Differences

Viruses acquire their envelope by budding from a host cell membrane, but viral lipid composition may differ from that of the budding membrane. We have previously reported that the HIV-1 membrane is highly enriched in cholesterol, sphingolipids, and other raft lipids, suggesting that the virus may bud from pre-existing or virus-induced lipid rafts. Here, we employed the environmentally sensitive fluorescent dye Laurdan to study the membrane lateral structure of HIV-1 derived from different cell lines. Differences in viral membrane order detected by Laurdan staining were shown by mass spectrometry to be due to differences in lipid composition. Isogenic viruses from two different cell lines were both strongly enriched in raft lipids and displayed a liquid-ordered membrane, but these effects were significantly more pronounced for HIV-1 from the T-cell line MT-4 compared with virus from 293T cells. Host-dependent differences in the lipidomes predominantly affected the ratio of sphingomyelins (including dihydrosphingomyelin) to phosphatidylcholine, whereas cholesterol contents were similar. Accordingly, treatment of infectious HIV-1 with the sphingomyelin-binding toxins Equinatoxin-II or lysenin showed differential inhibition of infectivity. Liposomes consisting of lipids that had been extracted from viral particles exhibited slightly less liquid order than the respective viral membranes, which is likely to be due to absence of membrane proteins and to loss of lipid asymmetry. Synthetic liposomes consisting of a quaternary lipid mixture emulating the viral lipids showed a liquid order similar to liposomes derived from virion lipids. Thus, Laurdan staining represents a rapid and quantitative method to probe viral membrane liquid order and may prove useful in the search for lipid active drugs.

Exploring the Effects of Sterols in Model Lipid Membranes Using Single-Molecule Orientations

Single-molecule orientations of the fluorescent lipid analogue BODIPY-PC doped into lipid monolayers and bilayers of DPPC are used to characterize the structure present in the films as a function of sterol content. Out-of-focus polarized total internal reflection fluorescence microscopy (PTIRF-M) measurements are used to characterize the single-molecule tilt angles with respect to the membrane normal. Tilt angle histograms for Langmuir-Blodgett monolayers of DPPC reveal bimodal distributions at all surface pressures studied. A linear dependence in the dye population oriented normal to the membrane plane with surface pressure is found and used to characterize the equivalent surface pressure of supported bilayers formed through vesicle fusion. These measurements reveal an equivalent surface pressure of approximately 23 mN/m, which is somewhat lower than the currently accepted value of approximately 30-35 mN/m.(1-7) The effect of cholesterol, ergosterol, and lanosterol on membrane structure is also compared between DPPC bilayers and monolayers transferred at approximately 23 mN/m. The addition of cholesterol leads to dramatic changes in the tilt angle histograms while lanosterol has essentially no effect. The addition of ergosterol has a slight influence at higher concentrations. Using the average tilt angle calculated from the single-molecule histograms, the order parameter S is calculated as a function of cholesterol and compared with previous studies.(8-10).

Lipid Lateral Mobility in Cochlear Outer Hair Cells: Regional Differences and Regulation by Cholesterol

The outer hair cell (OHC) lateral plasma membrane houses the transmembrane protein prestin, a necessary component of the yet unknown molecular mechanism(s) underlying electromotility and the exquisite sensitivity and frequency selectivity of mammalian hearing. The importance of the plasma membrane environment in modulating OHC electromotility has been substantiated by recent studies demonstrating that membrane cholesterol alters prestin activity in a manner consistent with cholesterol-induced changes in auditory function. Cholesterol is known to affect membrane material properties, and measurements of lipid lateral mobility provide a method to asses these changes in living OHCs. Using fluorescence recovery after photobleaching (FRAP), we characterized regional differences in the lateral diffusion of the lipid analog di-8-ANEPPS in OHCs and investigated whether lipid mobility, which reflects membrane fluidity, is sensitive to membrane cholesterol. FRAP experiments revealed quantitative differences in lipid lateral mobility among the apical, lateral, and basal regions of the OHC and demonstrated that diffusion in individual regions is uniquely sensitive to cholesterol manipulations. Interestingly, in the lateral region, both cholesterol depletion and loading significantly reduced the effective diffusion coefficient from control values. Thus, the fluidity of the OHC lateral plasma membrane is regulated by cholesterol levels in a non-monotonic manner, suggesting that the overall material properties of the lateral plasma membrane are optimally tuned for OHC function in the native state. These results support the idea that the cholesterol-dependent regulation of prestin function and electromotility correlates with changes in the properties of the lipid environment that surrounds and supports prestin.

Combined structural and biological activities for new polyunsaturated fatty derivatives obtained by biotechnological process

The objective of this study is to demonstrate the use of new polyunsaturated fatty derivatives for cutaneous applications. These new compounds present an analogue structure of cutaneous lipid, stabilize the polyunsaturated fatty acids (face to oxidation) and demonstrate specific biological activities. Three molecules described are Omega 6 fatty acid stabilized compound (O6FASC), the O3FASC and the O9FASC. The derivatives are synthesized via the same biotechnological process. This work describes the choice of final structure, the design of the biotechnological process and the free solvent enzymatic synthesis used for the synthesis of these three cutaneous lipid analogues. The restructuring effect of such analogues has been demonstrated with an in vivo study on volunteers. The stabilization of the O3FASC and O6FASC, and the biological activities of these three compounds are presented. The O6FASC shows very good results in anti-inflammatory effects; the O3FASC has anti-stress activities, whereas the O9FASC presents interesting results in improving elasticity and firmness. All these activity tests are presented in this work.

Synthesis and evaluation of a novel liposome containing BPA–peptide conjugate for BNCT

We aimed at securing sufficient concentrations of 10B in boron neutron capture therapy (BNCT) by developing a new drug delivery system. We have designed and developed a novel lipid analog and succeeded in using it to develop the new boron component liposome. It consisted of three different kinds of amino acid derivatives and two fatty acids, and could react directly with the peptide synthesized first on resin by Fmoc solid-phase synthesis. In this study, lipid analog conjugated with HIV-TAT peptide (domain of human immunodeficiency virus TAT protein) and boronophenylalanine (BPA) was synthesized and successfully incorporated into liposomes.

Regulation of plasma endocannabinoids differs in pregnant versus non‐pregnant women

Endogenous cannabinoids, endocannabinoids, are lipid messengers and analogs of polyunsaturated fatty acids. The endocannabinoid metabolome is comprised of over a dozen metabolites associated with a variety of functions in central and peripheral physiology, reproduction and development. We quantified 15 plasma analytes of the endocannabinoid metabolome in pregnant women (n=34) at 20‐22 weeks versus non‐pregnant women (n=9) using liquid chromotography‐mass spectrometry. Ethanolamides were higher in pregnant versus non‐pregnant women (mean±SD): palmitoylethanolamine,; (5.32± 4.07 pg/ μl versus 1.98 ± 0.40 pg/ μl, p<0.01), oleoylethanolamine (3.08 ± 1.72 pg/ μl versus 1.37 ± 0.39 pg/ μl, p<0.01) and arachidonoylethanolamine (0.68 ± 0.22 pg/ μl versus 0.24 ± 0.10 pg/ μl, p < 0.01). The glycerols: 2‐arachidonolglyerol was significantly higher during pregnancy (133.17 ± 71.06 pg/ μl versus 9.96 ± 5.80 pg/ μl, p<0.01) while 2‐ oleoyglycerol was lower in pregnancy (472.01 ± 299.50 pg/ μl versus 1217.64 ± 466.35 pg/ μl, p<0.01); 2‐palmitoyglycerol was detected only in pregnancy (1850 ± 1254 pg/µl). In conclusion, pregnancy altered specific metabolites in the plasma endocannibinoid system.

Sorting of Lipids and Proteins in Membrane Curvature Gradients

The sorting of lipids and proteins in cellular trafficking pathways is a process of central importance in maintaining compartmentalization in eukaryotic cells. However, the mechanisms behind these sorting phenomena are currently far from being understood. Among several mechanistic suggestions, membrane curvature has been invoked as a means to segregate lipids and proteins in cellular sorting centers. To assess this hypothesis, we investigate the sorting of lipid analog dye trace components between highly curved tubular membranes and essentially flat membranes of giant unilamellar vesicles. Our experimental findings indicate that intracellular lipid sorting, contrary to frequent assumptions, is unlikely to occur by lipids fitting into membrane regions of appropriate curvature. This observation is explained in the framework of statistical mechanical lattice models that show that entropy, rather than curvature energy, dominates lipid distribution in the absence of strongly preferential lateral intermolecular interactions. Combined with previous findings of curvature induced phase segregation, we conclude that lipid cooperativity is required to enable efficient sorting. In contrast to lipid analog dyes, the peripheral membrane binding protein Cholera toxin subunit B is effectively curvature-sorted. The sorting of Cholera toxin subunit B is rationalized by statistical models. We discuss the implications of our findings for intracellular sorting mechanisms.

Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative

Cholesterol-rich, liquid-ordered (L o) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (L d) domains, the number of such analogs with a high affinity for biologically relevant L o domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The L d phase domains were also labeled with 1,1′-didodecyl-3,3,3′,3′-tetramethylindocarbocyanine (DiI-C 12) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 ± 0.08 ns, compared with 4.1 ± 0.4 ns in the presence of DiI-C 12. The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C 12 (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 ± 0.2 nm. These results also indicate preferential partitioning ( K p = 1.88) of Bdp-Chol into the L o phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence correlation spectroscopy, is (7.4 ± 0.3) × 10 −8 cm 2/s and (5.0 ± 0.2) × 10 −8 cm 2/s in the L d and L o phases, respectively. Experimental translational/rotational diffusion coefficient ratios are compared with theoretical predictions using the hydrodynamic model (Saffman-Delbrück). The results suggest that Bdp-Chol is likely to form a complex with other lipid molecules during its macroscopic diffusion in GUV lipid bilayers at room temperature. Our integrated, multiscale results demonstrate the potential of this cholesterol analog for studying lipid-lipid interactions, lipid order, and membrane fluidity of biologically relevant L o domains.

Measuring Diffusion of Lipid-like Probes in Artificial and Natural Membranes by Raster Image Correlation Spectroscopy (RICS): Use of a Commercial Laser-Scanning Microscope with Analog Detection

The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial CLSM (Zeiss LSM 510 META) in the study of the diffusion of the fluorescent lipid analog 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indodicarbocyanine perchlorate (DiI-C(18)(5)) both in giant unilamellar vesicles and in the plasma membrane of living oligodendrocytes, i.e., the myelin-producing cells of the central nervous system. It is shown that RICS on a commercial CLSM with analog detection allows for reliable results in the study of membrane diffusion by removal of unwanted correlations introduced by the analog detection system. The results obtained compare well with those collected by FRAP and FCS.

In vitro assay for drug‐induced hepatosteatosis using rat primary hepatocytes, a fluorescent lipid analog and gene expression analysis

To evaluate new drugs' potential for hepatosteatosis, we developed a cell-based assay using a fluorescent fatty acid analog: BODIPY558/568 C12. Rat primary hepatocytes were exposed to positive reference compounds [cyclosporine A (CsA), clofibrate (CFR), tetracycline (TC), valproic acid (VPA), carbon tetrachloride (CCl4), tamoxifen (TMX)] in the presence of BODIPY558/568 C12. The formation of fluorscecent particles or lipid droplets in the cytoplasm was confirmed by confocal laser scanning microscopy and electron microscopy respectively. The accumulation of BODIPY558/568 C12 was measured by fluorometry and high content imaging method. All positive reference compounds increased fluorescent particles in number and fluorescence intensity. High content imaging was more sensitive and selective method than fluorometry to detect fluorescent particles. Gene expression analysis of the hepatocytes showed two patterns: genes related to lipid metabolism/synthesis were down-regulated by oxidative stress inducing compounds: CsA, TC and TMX, and up-regulated by peroxisome proliferator-activated receptor-alpha agonists: CFR and VPA. From these findings, we concluded that the cell-based assay developed in this study is an appropriate method to predict drugs' potential for hepatosteatosis, and gene expression analysis is useful to profile the mechanism of the hepatosteatosis.

Detection of Lipid Domains in Model and Plasma Membranes by Fluorescence Lifetime Imaging Microscopy of Fluorescent Lipid Analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate that lipid domains in plasma membranes of mammalian cells are short lived and in the submicron range. To unravel the lateral heterogeneity of cellular membranes, in particular of mammalian plasma membranes, at this scale various techniques of fluorescence spectroscopy and microscopy have been applied. Measuring fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of 1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in Giant Unilamellar Vesicles (GUVs) and the plasma membrane of mammalian cells by Fluorescence Lifetime Imaging (FLIM). For GUVs forming microscopically visible lipid domains the fluorescence lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct being about 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM. Application of this approach to mammalian cells revealed that while the fluorescence lifetime is sensitive to the composition of the plasma membrane, distinct lipid domains as found for GUVs were not detected. A broad distribution of the long lifetime was found for C6-NBD-PC inserted in the plasma membrane of these cells centred around 11 ns. However, FLIM studies on lipid domain forming giant vesicles derived from the plasma membrane of HeLa-cells rather support a recent hypothesis that an ensemble of lipid domains being of submicroscopic size exist in the plasma membrane.

The Influence of Sterol Composition on Transbilayer Diffusion Rates

Cholesterol is a uniquely important sterol to mammalian cell membranes and has been shown to suppress the translocation of lipids between leaflets of a bilayer and therefore plays an important role in maintaining lipid asymmetry. Leaflet composition is regulated by the active transport of lipids by membrane proteins, while thermal diffusion across a membrane tries to randomize the leaflet composition. We have measured the transbilayer diffusion rates for three different sterols over a wide range of compositions. The sterols studied were all cholesterol analogs; including dihydrocholesterol, ergosterol, a component of fungal cell membranes, and stigmasterol, an unsaturated plant sterol. Temperature was varied to determine its influence on transbilayer diffusion rates. We find that sterol structure does have an influence on the rate at which lipids move between bilayer leaflets. Transbilayer diffusion measurements were made using a sodium dithionite assay to monitor the location of lipid analogues within DMPC/sterol liposomes.