A.I.P.M. de Kroon

Lipid map of the mammalian cell

Technological developments, especially in mass spectrometry and bioinformatics, have revealed that living cells contain thousands rather than dozens of different lipids [for classification and nomenclature, see Fahy et al. (Fahy et al., 2009)]. Now, the resulting questions are what is the relevance of each of these unique molecules for the cell and how do cells use lipids for their vital functions? The answer requires an integrative approach – cellular lipidomics – which addresses first the distribution of all lipids between the various organelle membranes and then their local organization within each membrane. To understand lipid homeostasis and its dynamics, one has to study the localized metabolism of lipids, their transport within and between the various membranes, and the sensors and effectors that govern these processes. In terms of function, above all, we need to understand the physical behavior of complex lipid mixtures and their effect on local protein structure, organization and function. Finally, in the course of evolution, many lipids and lipid metabolites have acquired key functions in the signaling networks that wire the cell, by binding to cognate receptors and by recruiting proteins to specific membranes. The accompanying poster describes the lipid content of the various organelle membranes, illustrates lipid localization and dynamics in various subcellular locations, and explains the structure of lipids and their biosynthetic pathways. Below, we highlight additional issues that are important in lipid cell biology, and aim to provide a framework and a timely update for lipid systems biology.

Glycoalkaloids selectively permeabilize cholesterol containing biomembranes

The effects of the glycoalkaloids alpha-solanine, alpha-chaconine and alpha-tomatine on different cell types were studied in order to investigate the membrane action of these compounds. Hemolysis of erythrocytes was compared to 6-carboxyfluorescein leakage from both ghosts and erythrocyte lipid vesicles, whereas leakage of enzymes from mitochondria and the apical and baso-lateral side of Caco-2 cells was determined. Furthermore, the effects of glycoalkaloids on the gap-junctional communication between Caco-2 cells was studied. From these experiments, it was found that glycoalkaloids specifically induced membrane disruptive effects of cholesterol containing membranes as was previously reported in model membrane studies. In addition, alpha-chaconine was found to selectively decrease gap-junctional intercellular communication. Furthermore, the glycoalkaloids were more potent in permeabilizing the outer membrane of mitochondria compared to digitonin at the low concentrations used.

High Cytotoxicity of Cisplatin Nanocapsules in Ovarian Carcinoma Cells Depends on Uptake by Caveolae-Mediated Endocytosis

Purpose: Cisplatin nanocapsules, nanoprecipitates of cisplatin encapsulated in phospholipid bilayers, exhibit increased in vitro toxicity compared with the free drug toward a panel of human ovarian carcinoma cell lines. To elucidate the mechanism of cell killing by nanocapsules and to understand the cell line dependence of nanocapsule efficacy, the route of uptake and the intracellular fate of the nanocapsules were investigated. Experimental Design: Intracellular platinum accumulation and cisplatin-DNA-adduct formation were measured in cell lines that differ in sensitivity to cisplatin nanocapsules. Confocal fluorescence microscopy in combination with down-regulation with small interfering RNA was used to map the route of cellular uptake of nanocapsules containing fluorescein-labeled cisplatin. Results: In sensitive cell lines, cisplatin from nanocapsules is taken up much more efficiently than the free compound. In IGROV-1 cells, the increased platinum accumulation results in augmented cisplatin-DNA-adduct formation. Confocal fluorescence microscopy revealed that the uptake of nanocapsules is energy dependent. Colocalization with markers of early and late endosomes indicated uptake via endocytosis. Down-regulation of caveolin-1 with small interfering RNA inhibited the uptake and cytotoxic effect of nanocapsules in IGROV-1 cells. Ovarian carcinoma cells, in which the nanocapsules are less effective than in IGROV-1 cells, do not internalize the nanocapsules (OVCAR-3) or accumulate them in an endocytic compartment after clathrin-mediated endocytosis (A2780). Conclusions: The high cytotoxicity of cisplatin nanocapsules requires caveolin-1-dependent endocytosis that is followed by release of the drug from a late endosomal/lysosomal compartment and cisplatin-DNA-adduct formation. The findings may be applied in predicting the efficacy of nanoparticulate anticancer drug delivery systems in treating different tumor types.

The pH dependence of headgroup and acyl chain structure and dynamics of phosphatidylserine, studied by 2H-NMR

By varying the pH, the influence of the ionization degree on the structure and dynamics of aqueous dispersions of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS) was studied, using 2H-NMR methods. For this purpose DOPS was synthesized with deuterium labels incorporated either stereospecifically at the beta-position of the serine headgroup ([2-2H]DOPS) or at the 11-position of both acyl chains ([11,11-2H2]DOPS), allowing the effects of pH on headgroup and acyl chains to be measured in parallel. A large scale synthesis procedure of stereospecific 1,2-dioleoyl-sn-glycero-3-phospho-[2-2H]-L- serine is described. The quadrupolar splitting (delta nu q) of [2-2H]DOPS is shown to be a sensitive sensor for the degree of protonation of the molecule. Whereas the delta nu q of [2-2H]DOPS decreases upon lowering the pH, that of [11,11-2H2]DOPS gradually increases, indicating an increase in acyl chain ordering. In the pH range below the pKa value, DOPS exhibits a temperature-dependent bilayer to hexagonal HII phase transition, apparent from the 31P-NMR spectra and the occurrence of a second component in the [11,11-2H2]DOPS 2H-NMR spectrum, with a much smaller delta nu q. The HII phase component in spectra from [2-2H]DOPS coincides with the isotropic position and has no defined delta nu q. In the bilayer organization delta nu q and spin-lattice relaxation time (T1) values for the acyl chain deuterated DOPS are similar to those obtained for other lipid systems. In contrast the PS headgroup region displays a relatively rigid structure as evidenced by a large delta nu q and very small T1 values. Upon adopting the HII phase the T1 values of the acyl chain deuterons are hardly affected. The uniqueness of the PS headgroup with respect to structure and motional properties is reinforced by the occurrence of a T1 minimum at 45 degrees C in the measurement of the temperature dependence of T1 for [2-2H]DOPS in the hexagonal HII configuration. Quantitative analysis yields a correlation time (tau c) for the motions determining T1 under these conditions, of 3.45 ns.

Transbilayer movement of phosphatidylcholine in the mitochondrial outer membrane of Saccharomyces cerevisiae is rapid and bidirectional.

The process of transmembrane movement of phosphatidylcholine (PC) across the outer membrane of mitochondria was investigated in vitro in mitochondrial outer membrane vesicles (OMV) from the yeast Saccharomyces cerevisiae. Phosphatidylcholine-transfer protein (PC-TP) was used to extract radiolabeled PC from OMV, with small unilamellar vesicles serving as acceptor system. Endogenously radiolabeled PC synthesized either via the CDP-choline pathway or via methylation of phosphatidylethanolamine can be extracted completely from the OMV with a t(1/2) of 1 min or less at 30 degrees C. The size of the pool of PC in OMV available for exchange by PC-TP is not affected by pretreatment of the OMV with proteinase K or sulfhydryl reagents. In the reverse experiment where radiolabeled PC was introduced into the OMV, similar characteristics for the exchange were found. The accessibility of labeled PC to externally added phospholipase A(2) was used as a measure for its transmembrane distribution. It was found that PC is not exclusively located in the outer leaflet of the OMV. Only 30-35% can be degraded in intact OMV by phospholipase A(2), irrespective of whether the PC is introduced by PC-TP or endogenously synthesized via either of the pathways of biosynthesis. The results demonstrate the occurrence of rapid bidirectional transbilayer movement of both endogenous and in vitro introduced PC in OMV. Furthermore, there appears to be no preference for mitochondrial import of PC synthesized by either of the pathways in vivo.

Anionic phospholipids can mediate membrane insertion of the anionic part of a bound peptide

The effect of anionic lipids on the membrane insertion of a carboxyl group on a specially designed palmitoylated peptide was studied, using tryptophan fluorescence. It is demonstrated that the negatively charged membrane surface of mixed phosphatidylcholine/phosphatidylglycerol small unilamellar vesicles enhances the protonation of the C‐terminal carboxyl group, and the subsequent insertion of that part of the peptide.

Association of synthetic model peptides with phospholipid vesicles induced by a membrane potential

Hydrophobic model peptides, consisting of 5 or 6 amino acids and carrying a nett positive charge at the amino terminus, exhibit a dramatically increased association with large unilamellar egg-PC vesicles upon application of a valinomycin-induced K+ diffusion potential, negative inside. The association of the peptides is largely reversible, apparent from a release of peptide upon dissipation of the membrane potential.

Phosphatidylcholine is essential for efficient functioning of the mitochondrial glycerol-3-phosphate dehydrogenase Gut2 in Saccharomyces cerevisiae

Gut2, the mitochondrial glycerol-3-phosphate dehydrogenase, was previously shown to become preferentially labelled with photoactivatable phosphatidylcholine (PC), pointing to a functional relation between these molecules. In the present study we analyzed whether Gut2 functioning depends on the PC content of yeast cells, using PC biosynthetic mutants in which the PC content was lowered. PC depletion was found to reduce growth on glycerol and to increase glycerol excretion, both indicating that PC is needed for optimal Gut2 functioning in vivo. Using several in vitro approaches the nature of the dependence of Gut2 functioning on cellular PC contents was investigated. The results of these experiments suggest that it is unlikely that the effects observed in vivo are due to changes in cellular Gut2 content, in specific activity of Gut2 in isolated mitochondria, or in the membrane association of Gut2, upon lowering the PC level. The in vivo effects are more likely an indirect result of PC depletion-induced changes in the cellular context in which Gut2 functions, that are not manifested in the in vitro systems used.

Chapter 5 Lipid-peptide interactions in model systems: Membrane insertion and translocation of peptides

Publisher Summary Lipid–peptide/protein interactions determine the structure and function of biomembranes. These interactions play an important role in both the activity of membrane-bound enzymes and the transport of solutes across membranes. There is growing experimental evidence that the interaction of peptides or proteins with the lipid phase of biological membranes plays a role in processes such as membrane insertion and the translocation of precursor proteins. The study of peptide–lipid model systems provides information on the possibilities and limitations of the putative function of peptide–lipid interactions in various biological processes. This chapter presents an overview of the results that have emerged from the integrated approach that is chosen to study the molecular details of membrane insertion and the translocation of peptides. The main parameters affecting peptide–lipid interactions have been investigated in well-defined systems consisting of synthetic model peptides and phospholipid vesicles. This enabled a coherent analysis of the influence of the peptides charge and hydrophobicity, of the membrane surface charge, and of the presence of transmembrane ion gradients that give rise to pH gradients and membrane potentials on lipid–peptide interaction.

The contributions of biosynthesis and acyl chain remodelling to the molecular species profile of phosphatidylcholine in yeast

Phosphatidylcholine (PC) is a very abundant membrane lipid in most eukaryotes, including yeast. The molecular species profile of PC, i.e. the ensemble of PC molecules with acyl chains differing in number of carbon atoms and double bonds, is important for membrane function. Pathways of PC synthesis and turnover maintain PC homoeostasis and determine the molecular species profile of PC. Studies addressing the processes involved in establishing the molecular species composition of PC in yeast using stable isotope labelling combined with detection by MS are reviewed.