Lipid Analogues Research Articles

Probing the Hydrophobic Binding Pocket of G-Protein-Coupled Lysophosphatidylserine Receptor GPR34/LPS1 by Docking-Aided Structure-Activity Analysis.

The ligands of certain G-protein-coupled receptors (GPCRs) have been identified as endogenous lipids, such as lysophosphatidylserine (LysoPS). Here, we analyzed the molecular basis of the structure-activity relationship of ligands of GPR34, one of the LysoPS receptor subtypes, focusing on recognition of the long-chain fatty acid moiety by the hydrophobic pocket. By introducing benzene ring(s) into the fatty acid moiety of 2-deoxy-LysoPS, we explored the binding site's preference for the hydrophobic shape. A tribenzene-containing fatty acid surrogate with modifications of the terminal aromatic moiety showed potent agonistic activity toward GPR34. Computational docking of these derivatives with a homology modeling/molecular dynamics-based virtual binding site of GPR34 indicated that a kink in the benzene-based lipid surrogates matches the L-shaped hydrophobic pocket of GPR34. A tetrabenzene-based lipid analogue bearing a bulky tert-butyl group at the 4-position of the terminal benzene ring exhibited potent GPR34 agonistic activity, validating the present hydrophobic binding pocket model.

Abstract 116: Erufosine, a third-generation alkylphosphocholine with cytotoxic and cytostatic effects in breast and colorectal cancer cells

Abstract Introduction Breast and colorectal cancers are highly malignant diseases with low 5-year survival rate (<21%) in advanced stages, which highlights the need of finding new therapeutic options. Considering this, we are interested in exploring the anticancer potential of erufosine, a membrane targeting synthetic lipid analogue belonging to a class of antineoplastic agents known as alkylphosphocholines (APCs). Properties such as intravenous administration and significantly reduced hemolytic activity as compared to previous APC generations, and efficient cytotoxic effects against malignant cells make it an attractive anticancer compound. Materials and Methods In the present experiments, we studied the effects of erufosine against breast (MDA-MB-231 and MCF-7) and colorectal (SW480 and SW620) cancer cell lines. Following exposure to erufosine (0.78-100µM) for 24, 48 and 72h, cytotoxic effects were measured by MTT dye reduction assay and inhibitory concentrations (IC) were identified by GraphPad Prism software 6. Erufosine mediated cytostatic effects were highlighted by means of propidium iodide (PI) based labeling of the DNA followed by flow cytometry analysis. Furthermore, the cells were exposed to increasing concentrations of erufosine (IC25, 50, 75) and alterations in cell cycle relevant genes (CCNA1, CCND1, CCNE1, CCNE2, CDK1, CDK4) were investigated by qRT-PCR methodology. Results Erufosine was highly active against the selected cell lines (IC50 < 10µM, 24h) and induced significant arrest in the G2/M phase of the cell cycle. The cytostatic effects were more pronounced in the two metastatic cell lines of breast (MDA-MB-231) and colorectal (SW620) cancers. At molecular levels, erufosine induced expression of CCNA1 (≥ 2fold) in SW480, SW620, and MCF-7 cells, while the expression was inhibited in MDA-MB-231 cells at high concentrations (IC50, IC75). There were no significant alterations (≤ 2fold) in the expression levels of CCND1, CCNE1 and CDK4 in the four cell lines. CCNE2 was significantly downregulated in breast cancer cell lines only. Noteworthy, alterations in CDK1 were associated with cell lines from primary cancers, where the expression was upregulated in SW480 cells and downregulated in MCF-7 cells. Conclusion Antineoplastic effects of erufosine contribute to decreased proliferation of breast and colorectal cancer cell lines. This synthetic lipid induces arrest in G2/M phase and alterations in cell cycle relevant genes in the selected panel of breast and colorectal cancer cells. Characteristics like in vivo stability, minimal hemolytic activity, no toxicity to bone marrow and antineoplastic effects (cytotoxic/cytostatic) against cancer cells make it an attractive therapeutic compound. Further understanding at molecular levels supported by in vivo experimentation will pave the way to validate its therapeutic potential against cancers. Citation Format: Asim Pervaiz, Saqib Mahmood, Martin R. Berger. Erufosine, a third-generation alkylphosphocholine with cytotoxic and cytostatic effects in breast and colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 116. doi:10.1158/1538-7445.AM2017-116

Localization of Sphingolipid Enriched Plasma Membrane Regions and Long-Chain Base Composition during Mature-Fruit Abscission in Olive

Sphingolipids, found in membranes of eukaryotic cells, have been demonstrated to carry out functions in various processes in plant cells. However, the roles of these lipids in fruit abscission remain to be determined in plants. Biochemical and fluorescence microscopy imaging approach has been adopted to investigate the accumulation and distribution of sphingolipids during mature-fruit abscission in olive (Olea europaea L. cv. Picual). Here, a lipid-content analysis in live protoplasts of the olive abscission zone (AZ) was made with fluorescent dyes and lipid analogs, particularly plasma membrane sphingolipid-enriched domains, and their dynamics were investigated in relation to the timing of mature-fruit abscission. In olive AZ cells, the measured proportion of both polar lipids and sphingolipids increased as well as endocytosis was stimulated during mature-fruit abscission. Likewise, mature-fruit abscission resulted in quantitative and qualitative changes in sphingolipid long-chain bases (LCBs) in the olive AZ. The total LCB increase was due essentially to the increase of t18:1(8E) LCBs, suggesting that C-4 hydroxylation and Δ8 desaturation with a preference for (E)-isomer formation were quantitatively the most important sphingolipids in olive AZ during abscission. However, our results also showed a specific association between the dihydroxylated LCB sphinganine (d18:0) and the mature-fruit abscission. These results indicate a clear correlation between the sphingolipid composition and mature-fruit abscission. Moreover, measurements of endogenous sterol levels in the olive AZ revealed that it accumulated sitosterol and campesterol with a concomitant decrease in cycloartenol during abscission. In addition, underlying the distinct sterol composition of AZ during abscission, genes for key biosynthetic enzymes for sterol synthesis, for obtusifoliol 14α-demethylase (CYP51) and C-24 sterol methyltransferase2 (SMT2), were up-regulated during mature-fruit abscission, in parallel to the increase in sitosterol content. The differences found in AZ lipid content and the relationships established between LCB and sterol composition, offer new insights about sphingolipids and sterols in abscission.

Dendritic cell-based vaccine treated with polysorbitol transporter enhanced antigen-specific CD8+ T cell immune responses through cross-presentation

Abstract Cross-presentation of epitope from exogenous antigen onto MHC I molecule enhances CD8+ T cell activity is important in host protective immune responses against virus infections and cancer. We previously reported that polysorbitol transpoter (PST), prepared from sorbitol diacrylate and low-molecular-weight polyethylenimine, induces a long-term antigen specific B cell response and phagolysosomal escape of antigens in macrophages via mechanism called ‘proton-sponge effect’. Therefore, we hypothesize that PST could enhance the cross-presentation and antigen-specific CD8+ T cell immune responses. In the present study, we tried to determine whether PST could enhance cross-presentation of DC using model antigen Ovalbumin (OVA). PST formed a nano-sized complex with OVA (OVA-PST) enhanced SIINFEKL peptide loading onto MHC I molecules. The results further showed that it enhanced OVA-specific CD8+ T cell proliferation and activation. However, PST was not able to upregulate co-stimulatory molecules and pro-inflammatory cytokines, which are necessary to induce adequate protective immune response. Together with OVA-PST, Monophospholipid A (MPLA), a nontoxic TLR4 agonist which is analog of lipid A also used as co-delivery agent in nanoparticle peptide vaccine, upregulated the effects of OVA-PST complexes. Finally, when PST-OVA and MPLA applied to DC-based vaccine for tumor therapy, survival enhanced in mouse engrafted with tumor. Collectively, these results suggested that the PST enhances cross-presentation in DCs together with antigen-specific CD8+ T cell immune responses. Therefore PST with MPLA could act to improve DC-based vaccine.

Diffusion of lipids and GPI-anchored proteins in actin-free plasma membrane vesicles measured by STED-FCS.

Diffusion and interaction dynamics of molecules at the plasma membrane play an important role in cellular signaling and are suggested to be strongly associated with the actin cytoskeleton. Here we use superresolution STED microscopy combined with fluorescence correlation spectroscopy (STED-FCS) to access and compare the diffusion characteristics of fluorescent lipid analogues and GPI-anchored proteins (GPI-APs) in the live-cell plasma membrane and in actin cytoskeleton-free, cell-derived giant plasma membrane vesicles (GPMVs). Hindered diffusion of phospholipids and sphingolipids is abolished in the GPMVs, whereas transient nanodomain incorporation of ganglioside lipid GM1 is apparent in both the live-cell membrane and GPMVs. For GPI-APs, we detect two molecular pools in living cells; one pool shows high mobility with transient incorporation into nanodomains, and the other pool forms immobile clusters, both of which disappear in GPMVs. Our data underline the crucial role of the actin cortex in maintaining hindered diffusion modes of many but not all of the membrane molecules and highlight a powerful experimental approach to decipher specific influences on molecular plasma membrane dynamics.

Alkaline phosphatase binds tenaciously to titanium; implications for biological surface evaluation following bone implant retrieval

Enhancing the performance and longevity of titanium (Ti) implants continues to be a significant developmental theme in contemporary biomaterials design. Our specific focus pertains to the surface functionalisation of Ti using the bioactive lipid, lysophosphatidic acid (LPA) and certain phosphatase-resistant analogues of LPA. Coating survivorship to a plethora of testing regimens is required to align with due regulatory process before novel biomaterials can enter clinical trials. One of the key acceptance criteria is coating retention to the physical stresses experienced during implantation. In assessing coating stability to insertion into porcine bone we found that a subsequent in vitro assessment to confirm coating persistence was masked by abundant alkaline phosphatase (ALP) contamination adsorbed to the metal surface. Herein we report that ALP can bind to Ti in a matter of minutes by simply immersing Ti samples in aqueous solutions of the enzyme. We strongly discourage the in vitro monitoring of osteoblast and stromal cell ALP expression when assessing bioactive coating survivorship following Ti implant retrieval form native bone tissue.

Cell cycle dependent changes in the plasma membrane organization of mammalian cells

Lipid membranes are major structural elements of all eukaryotic and prokaryotic organisms. Although many aspects of their biology have been studied extensively, their dynamics and lateral heterogeneity are still not fully understood. Recently, we observed a cell-to-cell variability in the plasma membrane organization of CHO-K1 cells (Schwarzer et al., 2014). We surmised that cell cycle dependent changes of the individual cells from our unsynchronized cell population account for this phenomenon. In the present study, this hypothesis was tested. To this aim, CHO-K1 cells were arrested in different cell cycle phases by chemical treatments, and the order of their plasma membranes was determined by various fluorescent lipid analogues using fluorescence lifetime imaging microscopy. Our experiments exhibit significant differences in the membrane order of cells arrested in the G2/M or S phase compared to control cells. Our single-cell analysis also enabled the specific selection of mitotic cells, which displayed a significant increase of the membrane order compared to the control. In addition, the lipid raft marker GPImYFP was used to study the lateral organization of cell cycle arrested cells as well as mitotic cells and freely cycling samples. Again, significant differences were found between control and arrested cells and even more pronounced between control and mitotic cells.Our data demonstrate a direct correlation between cell cycle progression and plasma membrane organization, underlining that cell-to-cell heterogeneities of membrane properties have to be taken into account in cellular studies especially at the single-cell level.

Imidazolium-Based Lipid Analogues and Their Interaction with Phosphatidylcholine Membranes.

4,5-Dialkylated imidazolium lipid salts are a new class of lipid analogues showing distinct biological activities. The potential effects of the imidazolium lipids on artificial lipid membranes and the corresponding membrane interactions was analyzed. Therefore, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was employed to create an established lipid monolayer model and a bilayer membrane. Mixed monolayers of DPPC and 4,5-dialkylimidazolium lipids differing by their alkyl chain length (C7, C11, and C15) were characterized by surface pressure-area (π-A) isotherms using a Wilhelmy film balance in combination with epifluorescence microscopy. Monolayer hysteresis for binary mixtures was examined by recording triplicate consecutive compression-expansion cycles. The lipid miscibility and membrane stability of DPPC/imidazolium lipids were subsequently evaluated by the excess mean molecular area (ΔAex) and the excess Gibbs free energy (ΔGex) of mixing. Furthermore, the thermotropic behavior of mixed liposomes of DPPC/imidazolium lipids was investigated by differential scanning calorimetry (DSC). The C15-imidazolium lipid (C15-IMe·HI) forms a thermodynamically favored and kinetically reversible Langmuir monolayer with DPPC and exhibits a rigidification effect on both DPPC monolayer and bilayer structures at low molar fractions (X ≤ 0.3). However, the incorporation of the C11-imidazolium lipid (C11-IMe·HI) causes the formation of an unstable and irreversible Langmuir-Gibbs monolayer with DPPC and disordered DPPC liposomes. The C7-imidazolium lipid (C7-IMe·HI) displays negligible membrane activity. To better understand these results on a molecular level, all-atom molecular dynamics (MD) simulations were performed. The simulations yield two opposing molecular mechanisms governing the different behavior of the three imidazolium lipids: a lateral ordering effect and a free volume/stretching effect. Overall, our study provides the first evidence that the membrane interaction of the C15 and C11 derivatives modulates the structural organization of lipid membranes. On the contrary, for the C7 derivative its membrane activity is too low to contribute to its earlier reported potent cytotoxicity.

Diffusion of Single-Pass Transmembrane Receptors: From the Plasma Membrane into Giant Liposomes

To quantitatively examine the effect of membrane organization on lateral diffusion, we studied fluorescent carbocyanine lipid analogues and EGFP-tagged, single-pass transmembrane proteins in systems of decreasing complexity: (i) the plasma membrane (PM) of living cells, (ii) paraformaldehyde/dithiothreitol-induced giant plasma membrane vesicles (GPMVs), and (iii) giant unilamellar vesicles (GUVs) under physiological buffer conditions. A truncated, signaling-deficient interleukin-4 receptor subunit, showing efficient accumulation in the plasma membrane, served as a model transmembrane protein. Two-dimensional diffusion coefficients (D) were determined by fluorescence correlation spectroscopy (FCS) either at fixed positions (single-point, spFCS) or while scanning a circular orbit (circular scanning, csFCS). Consistent with a different inclusion sizes in the membrane, lipids diffuse slightly faster than the single-spanning membrane proteins in both membrane systems, GUVs and GPMVs. In GPMVs lipids and proteins consistently experienced a fivefold larger viscosity than in GUVs, reflecting the significant fraction of plasma membrane-derived proteins partitioning into GPMVs. Lipid and protein diffusion in the PM was, respectively, 2 times and 4–5 times slower in comparison to GPMVs. This discrepancy was quantitatively confirmed by csFCS. The similarity of diffusion of receptors and lipids in GPMVs and GUVs and its significant difference in the plasma membrane suggest that protein domains as small as EGFP convey sensitivity to the actin cortex on various length scales.

Tandem mass spectrometry of novel ether-linked phospholipid analogs of anionic pulmonary surfactant phospholipids.

Structural analogs of the bioactive lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol were synthesized with a xylitol polar head group and both diacyl and diether radyl groups. Mass spectral characterization of xylitol phospholipids (PX) was carried out using collisional activation and high-resolution mass measurements of positive molecular ion species and compared with the phosphatidylglycerol (PG) analogs. PX were synthesized using a transphosphatidylation reaction catalyzed by phospholipase D and purified by high-performance liquid chromatography (HPLC). Compounds were subjected to electrospray ionization and collision-induced dissociation (CID) was performed using a tandem quadrupole mass spectrometer to generate positive and negative molecular ions. Diether phospholipids were additionally analyzed by high-resolution mass spectrometry as protonated and sodiated molecular species in positive ion mode. Ester-linked PX analogs behaved similarly to PG after collisional activation of [M-H]- . The product ions formed by CID of the diether PG and PX negative ions only revealed information about the head group with no information about the aliphatic chains. In contrast, CID of protonated and sodiated diether phospholipid positive ions revealed reactions corresponding to cleavage of the ether chain, likely occurring by charge-driven reaction mechanisms. Novel PX analogs with diacyl and diether radyl substituents of the glycerol backbone were characterized by tandem mass spectrometry. These unique diether phospholipid analogs enabled exploration of ether cleavage reactions of the positive molecular ion species resulting from collision-induced decomposition. Copyright © 2016 John Wiley & Sons, Ltd.

High-fat Feeding Paradigm for Larval Zebrafish: Feeding, Live Imaging, and Quantification of Food Intake.

Zebrafish are emerging as a model of dietary lipid processing and metabolic disease. This protocol describes how to feed larval zebrafish a lipid-rich meal, which consists of an emulsion of chicken egg yolk liposomes created by sonicating egg yolk in embryo media. Detailed instructions are provided to screen larvae for egg yolk consumption so that larvae that fail to feed will not confound experimental results. The chicken egg yolk liposomes can be spiked with fluorescent lipid analogs, including fatty acids and cholesterol, enabling both systemic and subcellular visualization of dietary lipid processing. Several methods are described to mount larvae that are conducive to short- and long-term live imaging with both upright and inverted objectives at high and low magnification. Additionally presented is an assay to quantify larval food intake by extracting the lipids of larvae fed fluorescent lipid analogs, spotting the lipids on a thin layer chromatography plate, and quantifying the fluorescence. Finally, critical aspects of the procedures, important controls, options for modifying the protocols to address specific experimental questions, and potential limitations are discussed. These techniques can be applied not only to focused, hypothesis driven inquiries, but also to a variety of screens and live imaging techniques to study dietary lipid metabolism and the control of food intake.

Analytical Characterization of an Oil-in-Water Adjuvant Emulsion.

Adjuvants are typically used in subunit vaccine formulations to enhance immune responses elicited by individual antigens. Physical chemical characterization of novel adjuvants is an important step in ensuring their effective use in vaccine formulations. This paper reports application of a panel of quantitative assays developed to analyze and characterize an oil-in-water adjuvant emulsion, which contains glucopyranosyl lipid A (GLA) and is a squalene-based emulsion. GLA is a fully synthetic analogue of monophosphoryl lipid A, which is a Toll-like receptor type 4 agonist and an FDA-approved adjuvant. The GLA-stable emulsion (GLA-SE) is currently being used for a respiratory syncytial virus vaccine in a phase 2 clinical trial. GLA was quantitated using reverse-phased high-performance liquid chromatography (RP-HPLC) coupled to a mass spectrometric detector, achieving higher assay sensitivity than the charged aerosol detection routinely used. Quantitation of the excipients of GLA-SE, including squalene, egg phosphatidyl choline, and Poloxamer 188, was achieved using a simple and rapid RP-HPLC method with evaporative light scattering detection, eliminating chemical derivatization typically required for these chromophore-lacking compounds. DL-α-tocopherol, the antioxidant of the GLA-SE, was quantitated using a RP-HPLC method with conventional UV detection. The experimental results compared well with values expected for these compounds based on targeted composition of the adjuvant. The assays were applied to identify degradation of individual components in a GLA-SE sample that degraded into distinct aqueous and oil phases. The methods developed and reported here are effective tools in monitoring physicochemical integrity of the adjuvant, as well as in formulation studies.

Interaction of Human Chloride Intracellular Channel Protein 1 (CLIC1) with Lipid Bilayers: A Fluorescence Study.

Chloride intracellular channel protein 1 (CLIC1) is very unusual as it adopts a soluble glutathione S-transferase-like canonical fold but can also autoinsert into lipid bilayers to form an ion channel. The conversion between these forms involves a large, but reversible, structural rearrangement of the CLIC1 module. The only identified environmental triggers controlling the metamorphic transition of CLIC1 are pH and oxidation. Until now, there have been no high-resolution structural data available for the CLIC1 integral membrane state, and consequently, a limited understanding of how CLIC1 unfolds and refolds across the bilayer to form a membrane protein with ion channel activity exists. Here we show that fluorescence spectroscopy can be used to establish the interaction and position of CLIC1 in a lipid bilayer. Our method employs a fluorescence energy transfer (FRET) approach between CLIC1 and a dansyl-labeled lipid analogue to probe the CLIC1-lipid interface. Under oxidizing conditions, a strong FRET signal between the single tryptophan residue of CLIC1 (Trp35) and the dansyl-lipid analogue was detected. When considering the proportion of CLIC1 interacting with the lipid bilayer, as estimated by fluorescence quenching experiments, the FRET distance between Trp35 and the dansyl moiety on the membrane surface was determined to be ∼15 Å. This FRET-detected interaction provides direct structural evidence that CLIC1 associates with membranes. The results presented support the current model of an oxidation-driven interaction of CLIC1 with lipid bilayers and also propose a membrane anchoring role for Trp35.

Structural analogs of pulmonary surfactant phosphatidylglycerol inhibit toll-like receptor 2 and 4 signaling

The pulmonary surfactant phospholipid, 1-palmitoyl-2-oleoylphosphatidylglycerol (POPG), potently inhibits toll-like receptor (TLR)2 and TLR4 signaling from the cell surface of macrophages. Analogs of POPG that vary in polar head group length, hydroxylation, and alkyl branching were synthesized using a phospholipase D-catalyzed transphosphatidylation reaction and a 1-palmitoyl-2-oleoyl phosphatidylcholine substrate. Lipid analogs with C3 and C4 alkyl head group length (POP-propanol and POP-butanol) are less effective than POPG as TLR2 and TLR4 antagonists. However, adding a hydroxyl group at the alkyl chain 3- or 4-position (POP-propanediols or POP-butanediols) greatly increased their inhibitory effects against TLR2 and TLR4. POP-2',2'-dimethylpropanediol is a weak inhibitor of TLR2 and TLR4 activation that results in arachidonic acid release, but an effective inhibitor of TLR4 activation that results in TNF-α production. Addition of an amino group at the alkyl-2 position (POP-2'-aminopropanediol) completely abolished the antagonism of TLRs 2 and 4. Multiple analogs strongly bind to the TLR4 coreceptors, cluster of differentiation 14 (CD14) and myeloid differentiation 2, but competition for di[3-deoxy-D-manno-octulosonyl]-lipid A binding to CD14 is the best predictor of biological activity at the cellular level. Collectively, these findings identify new compounds for antagonizing TLR2 and TLR4 activation and define structural properties of POPG analogs for discriminating between two TLR systems.

Reorganization of Lipid Diffusion by Myelin Basic Protein as Revealed by STED Nanoscopy

Myelin is a multilayered membrane that ensheathes axonal fibers in the vertebrate nervous system, allowing fast propagation of nerve action potentials. It contains densely packed lipids, lacks an actin-based cytocortex, and requires myelin basic protein (MBP) as its major structural component. This protein is the basic constituent of the proteinaceous meshwork that is localized between adjacent cytoplasmic membranes of the myelin sheath. Yet, it is not clear how MBP influences the organization and dynamics of the lipid constituents of myelin. Here, we used optical stimulated emission depletion super-resolution microscopy in combination with fluorescence correlation spectroscopy to assess the characteristics of diffusion of different fluorescent lipid analogs in myelin membrane sheets of cultured oligodendrocytes and in micrometer-sized domains that were induced by MBP in live epithelial PtK2 cells. Lipid diffusion was significantly faster and less anomalous both in oligodendrocytes and inside the MBP-rich domains of PtK2 cells compared with undisturbed live PtK2 cells. Our data show that MBP reorganizes lipid diffusion, possibly by preventing the buildup of an actin-based cytocortex and by preventing most membrane proteins from entering the myelin sheath region. Yet, in contrast to myelin sheets in oligodendrocytes, the MBP-induced domains in epithelial PtK2 cells demonstrate no change in lipid order, indicating that segregation of long-chain lipids into myelin sheets is a process specific to oligodendrocytes.

Synthesis of a novel archaeal tetraether-type lipid containing a diorthoester group as a helper lipid for gene delivery

As an alternative to recombinant virus, synthetic carriers are widely developed for providing efficient and safe DNA delivery. To reach the nucleus where nucleic acid (NA) constructs are transcripted, chemical complexes have to overcome serious cellular traps such as endosomal escape and intracellular trafficking. Here, the design and the multi-step synthesis of a stabilizing tetraether lipid analogue of archaeal counterparts containing a diorthoester moiety in the middle of the bridging chain were described. The key step involved a double coupling reaction between two diether alcohols and a diketene acetal. Under aqueous acidic conditions, the diorthoester function of the tetraether lipid can be hydrolysed to yield two monopolar diether lipids. Applied to gene delivery in association with a cationic lipid, this new helper bipolar tetraether lipid led to lipid–DNA complexes with sizes and potential zeta values depending on the charge ratios (+/−). In addition, preliminary in vitro transfection assays supported the interest of these novel bipolar lipid-based lipoplex formulations compared to standard Lipofectamine-based formulations.

Interactions and Translational Dynamics of Phosphatidylinositol Bisphosphate (PIP2) Lipids in Asymmetric Lipid Bilayers.

Phosphatidylinositol phosphate (PIP) lipids are critical to many cell signaling pathways, in part by acting as molecular beacons that recruit peripheral membrane proteins to specific locations within the plasma membrane. Understanding the biophysics of PIP-protein interactions is critical to developing a chemically detailed model of cell communication. Resolving such interactions is challenging, even in model membrane systems, because of the difficulty in preparing PIP-containing membranes with high fluidity and integrity. Here we report on a simple, vesicle-based protocol for preparing asymmetric supported lipid bilayers in which fluorescent PIP lipid analogues are found only on the top leaflet of the supported membrane facing the bulk solution. With this asymmetric distribution of lipids between the leaflets, the fluorescent signal from the PIP lipid analogue reports directly on interactions between the peripheral molecules and the top leaflet of the membrane. Asymmetric PIP-containing bilayers are an ideal platform to investigate the interaction of PIP with peripheral membrane proteins using fluorescence-based imaging approaches. We demonstrate their usefulness here with a combined fluorescence correlation spectroscopy and single particle tracking study of the interaction between PIP2 lipids and a polycationic polymer, quaternized polyvinylpyridine (QPVP). With this approach we are able to quantify the microscopic features of the mobility coupling between PIP2 lipids and polybasic QPVP. With single particle tracking we observe individual PIP2 lipids switch from Brownian to intermittent motion as they become transiently trapped by QPVP.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.

Zebrafish Embryonic Lipidomic Analysis Reveals that the Yolk Cell Is Metabolically Active in Processing Lipid.

The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC)-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY) lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

Assessment of Nicotinic Acetylcholine Receptor Detergent Complexes Purity and Stability for Structural Studies

The torpedo californica (Tc) nicotinic acetylcholine receptor (nAChR) is a large (∼290 kDa) integral membrane protein composed of four homologous subunits with a stoichiometry α2βγδ. The main obstacle towards the achieving of high resolution structure of the nAChR is the preparation of milligram amounts of pure, functional and stable nAChR-detergent complex (nAChR-DC). A crucial aspect in the preparation nAChR-DCs is the functionality, purity and aggregation state of the solubilized protein. The main contaminants present in the crude Torpedo californica (Tc) membranes are the peripheral nAChR-associated proteins, mainly the 43-kDa rapsyn protein and the V-type ATPase. In the present study we performed an in-depth analysis of the purity of six nAChR-DCs prepared lipid analogue detergents: Foscholine (12,14,16) and Lysofoscholine (12,14,16) using microfluidic capillary gel electrophoresis (Angilent Bioanalizer 2100). This method provides a detailed analysis of the contaminants present in the nAChR-DCs. In addition, we evaluated the state of aggregation of these nAChR-DCs. The present study provides a roadmap for the preparation of high quality nAChR-DCs for structural studies. The preparation of functional and stable membrane protein-DCs from natural source will provide the groundwork to validate forthcoming developing technologies for the crystallization of large membrane protein complexes.