Biogenesis and functions of lipid droplets in plants: Thematic Review Series: Lipid Droplet Synthesis and Metabolism: from Yeast to Man

The compartmentation of neutral lipids in plants is mostly associated with seed tissues, where triacylglycerols (TAGs) stored within lipid droplets (LDs) serve as an essential physiological energy and carbon reserve during postgerminative growth. However, some nonseed tissues, such as leaves, flowers and fruits, also synthesize and store TAGs, yet relatively little is known about the formation or function of LDs in these tissues. Characterization of LD-associated proteins, such as oleosins, caleosins, and sterol dehydrogenases (steroleosins), has revealed surprising features of LD function in plants, including stress responses, hormone signaling pathways, and various aspects of plant growth and development. Although oleosin and caleosin proteins are specific to plants, LD-associated sterol dehydrogenases also are present in mammals, and in both plants and mammals these enzymes have been shown to be important in (steroid) hormone metabolism and signaling. In addition, several other proteins known to be important in LD biogenesis in yeasts and mammals are conserved in plants, suggesting that at least some aspects of LD biogenesis and/or function are evolutionarily conserved.     

Transport of mercury compounds across bimolecular lipid membranes: effect of lipid composition, pH and chloride concentration

The use of bimolecular lipid membranes (BLM) as model membrane allows the analysis of the transport of mercury compounds across the lipidic barriers of biological membranes. The results of flux measurements show that two mercury compounds--HgCl2 and CH3HgCl--cross the BLM but the overall permeabilities are dependent on the pH of the aqueous media, and are not apparently influenced by the different phospholipid constituents of the bilayers. On the other hand, electrical measurements show that, function of the chemical speciation, the transport of this metal is done essentially in the neutral form.     

Inter-organelle lipid transfer: a channel model for Vps13 and chorein-N motif proteins

Membrane contact sites, where two organelles are in close proximity, are critical regulators of cellular membrane homeostasis, with roles in signaling, lipid metabolism, and ion dynamics. A growing catalog of specialized lipid transfer proteins carry out lipid exchange at these sites. Currently characterized eukaryotic lipid transport proteins are shuttles that typically extract a single lipid from the membrane of the donor organelle, solubilize it during transport through the cytosol, and deposit it in the acceptor organelle membrane. Here, we highlight the recently identified chorein_N family of lipid transporters, including the Vps13 proteins and the autophagy protein Atg2. These are elongated proteins that, distinct from previously characterized transport proteins, bind tens of lipids at once. They feature an extended channel, most likely lined with hydrophobic residues. We discuss the possibility that they are not shuttles but instead are bridges between membranes, with lipids traversing the cytosol via the hydrophobic channel.     

A versatile library of activity-based probes for fluorescence detection and/or affinity isolation of lipolytic enzymes

This work describes the synthesis of a library of fluorescent and/or biotinylated alkylphosphonate inhibitors being reactive towards serine hydrolases, especially lipases and esterases. Fluorescent inhibitors can be used for sensitive and rapid detection of active proteins by gel electrophoresis. Biotinylated inhibitors are applicable for the enrichment and isolation of active enzymes. Functionality as well as the different detection methods of the synthesized inhibitors were successfully tested with an enzyme preparation, namely cholesterol esterase from porcine pancreas (ppCE). Moreover, a biotinylated inhibitor was employed to enrich ppCE on avidin beads. Hence, our set of phosphonate inhibitors can be used for the detection and/or isolation of active serine hydrolases.     

Isolation and Chemical Characterization of Lipid A from Gram-negative Bacteria

Lipopolysaccharide (LPS) is the major cell surface molecule of gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three domains: the distal O-polysaccharide, a core oligosaccharide, and the lipid A domain consisting of a lipid A molecular species and 3-deoxy-D-manno-oct-2-ulosonic acid residues (Kdo). The lipid A domain is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In addition to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS, we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response, and have provided many new targets in the development of antibacterial compounds.     

The Lipid Droplet Knowledge Portal: A resource for systematic analyses of lipid droplet biology

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Drought acclimation and lipid composition in Folsomia candida: implications for cold shock,heat shock and acute desiccation stress

Many of the physiological adaptations evolved in terrestrial invertebrates to resist desiccation have also been shown to enhance the survival of low temperatures. In this study we have examined temporal changes in the physiology of the collembolan Folsomia candida during acclimation to mild desiccation stress (98.2% RH), and how physiological changes correlate with resistance to subsequent cold shock, heat shock and acute desiccation stress. Drought-acclimation increased the resistance to cold and acute drought but reduced the resistance to heat shock. The composition of membrane phospholipid fatty acids (PLFA) changed during acclimation resulting in a higher degree of unsaturation by the end of the 192-h acclimation period. This resembles typical membrane alterations seen in ectothermic animals exposed to cold. Only small changes were seen in the neutral lipid fraction. The temporal changes in cold resistance and drought resistance correlated well with changes in PLFA composition and accumulation of sugars and polyols (’cryoprotectives’). It is proposed that the drought-induced PLFA desaturation, combined with the membrane protecting accumulation of cryoprotectives, are important physiological adaptations providing tolerance to both desiccation and cold.     

Role for Phospholipid Flippase Complex of ATP8A1 and CDC50A Proteins in Cell Migration

Type IV P-type ATPases (P4-ATPases) and CDC50 family proteins form a putative phospholipid flippase complex that mediates the translocation of aminophospholipids such as phosphatidylserine (PS) and phosphatidylethanolamine (PE) from the outer to inner leaflets of the plasma membrane. In Chinese hamster ovary (CHO) cells, at least eight members of P4-ATPases were identified, but only a single CDC50 family protein, CDC50A, was expressed. We demonstrated that CDC50A associated with and recruited P4-ATPase ATP8A1 to the plasma membrane. Overexpression of CDC50A induced extensive cell spreading and greatly enhanced cell migration. Depletion of either CDC50A or ATP8A1 caused a severe defect in the formation of membrane ruffles, thereby inhibiting cell migration. Analyses of phospholipid translocation at the plasma membrane revealed that the depletion of CDC50A inhibited the inward translocation of both PS and PE, whereas the depletion of ATP8A1 inhibited the translocation of PE but not that of PS, suggesting that the inward translocation of cell-surface PE is involved in cell migration. This hypothesis was further examined by using a PE-binding peptide and a mutant cell line with defective PE synthesis; either cell-surface immobilization of PE by the PE-binding peptide or reduction in the cell-surface content of PE inhibited the formation of membrane ruffles, causing a severe defect in cell migration. These results indicate that the phospholipid flippase complex of ATP8A1 and CDC50A plays a major role in cell migration and suggest that the flippase-mediated translocation of PE at the plasma membrane is involved in the formation of membrane ruffles to promote cell migration.     

A new method for membrane reconstitution: Fusion of protein-containing vesicles with planar bilayer membranes below lipid phase transition temperature

A new method of membrane reconstitution was developed by fusion of channel protein containing vesicles with planar bilayer membranes. The fusion process only occurred below and near the phase transition temperature of the lipid used. We obtained the following results: 1. Our system is solvent-free and vesicles do not come into contact with the air/water interface. This obviates a possible denaturation of hydrophobic proteins. 2. Channel forming proteins and protein complexes, respectively, are active in a frozen lipid matrix. 3. We detected an unknown channel in cilia fragments. 4. Purified acetylcholine receptors form fluctuating channels in a membrane consisting of a pure synthetic lecithin (two component system).     

Oleaginous yeasts for biodiesel: Current and future trends in biology and production

Production of biodiesel from edible plant oils is quickly expanding worldwide to fill a need for renewable, environmentally-friendly liquid transportation fuels. Due to concerns over use of edible commodities for fuels, production of biodiesel from non-edible oils including microbial oils is being developed. Microalgae biodiesel is approaching commercial viability, but has some inherent limitations such as requirements for sunlight. While yeast oils have been studied for decades, recent years have seen significant developments including discovery of new oleaginous yeast species and strains, greater understanding of the metabolic pathways that determine oleaginicity, optimization of cultivation processes for conversion of various types of waste plant biomass to oil using oleaginous yeasts, and development of strains with enhanced oil production. This review examines aspects of oleaginous yeasts not covered in depth in other recent reviews. Topics include the history of oleaginous yeast research, especially advances in the early 20th century; the phylogenetic diversity of oleaginous species, beyond the few species commonly studied; and physiological characteristics that should be considered when choosing yeast species and strains to be utilized for conversion of a given type of plant biomass to oleochemicals. Standardized terms are proposed for units that describe yeast cell mass and lipid production.     

Chemo- and opto-genetic tools for dissecting the role of membrane contact sites in living cells: Recent advances and limitations

Membrane contact sites (MCSs) are morphologically defined intracellular structures where cellular membranes are closely apposed. Recent progress has significantly advanced our understanding of MCSs with the use of new tools and techniques. Visualization of MCSs in living cells by split fluorescence proteins or FRET-based techniques tells us the dynamic property of MCSs. Manipulation of MCSs by chemically-induced dimerization (CID) or light-induced dimerization (LID) greatly contributes to our understanding of their functional aspects including inter-organelle lipid transport mediated by lipid transfer proteins (LTPs). Here we highlight recent advances in these tools and techniques as applied to MCSs, and we discuss their advantages and limitations.     

Reconstitution of a Kv Channel into Lipid Membranes for Structural and Functional Studies

To study the lipid-protein interaction in a reductionistic fashion, it is necessary to incorporate the membrane proteins into membranes of well-defined lipid composition. We are studying the lipid-dependent gating effects in a prototype voltage-gated potassium (Kv) channel, and have worked out detailed procedures to reconstitute the channels into different membrane systems. Our reconstitution procedures take consideration of both detergent-induced fusion of vesicles and the fusion of protein/detergent micelles with the lipid/detergent mixed micelles as well as the importance of reaching an equilibrium distribution of lipids among the protein/detergent/lipid and the detergent/lipid mixed micelles. Our data suggested that the insertion of the channels in the lipid vesicles is relatively random in orientations, and the reconstitution efficiency is so high that no detectable protein aggregates were seen in fractionation experiments. We have utilized the reconstituted channels to determine the conformational states of the channels in different lipids, record electrical activities of a small number of channels incorporated in planar lipid bilayers, screen for conformation-specific ligands from a phage-displayed peptide library, and support the growth of 2D crystals of the channels in membranes. The reconstitution procedures described here may be adapted for studying other membrane proteins in lipid bilayers, especially for the investigation of the lipid effects on the eukaryotic voltage-gated ion channels.     

Mitochondrial membrane lipid remodeling in pathophysiology: A new target for diet and therapeutic interventions

Mitochondria are arbiters in the fragile balance between cell life and death. These organelles present an intricate membrane system, with a peculiar lipid composition and displaying transverse as well as lateral asymmetry. Some lipids are synthesized inside mitochondria, while others have to be imported or acquired in the form of precursors. Here, we review different processes, including external interventions (e.g., diet) and a range of biological events (apoptosis, disease and aging), which may result in alterations of mitochondrial membrane lipid content. Cardiolipin, the mitochondria lipid trademark, whose biosynthetic pathway is highly regulated, will deserve special attention in this review. The modulation of mitochondrial membrane lipid composition, especially by diet, as a therapeutic strategy for the treatment of some pathologies will be also addressed.     

Critical temperatures and a critical chain length in saturated diacylphosphatidylcholines: calorimetric, ultrasonic and Monte Carlo simulation study of chain-melting/ordering in aqueous lipid dispersions

Chain-ordering/melting transition in a series of saturated diacylphosphatidylcholines (PCs) in aqueous dispersions have been studied experimentally (calorimetric and ultrasonic techniques) and theoretically (an Ising-like lattice model). The shape of the calorimetric curves was compared with the theoretical data and interpreted in terms of the lateral interactions and critical temperatures determined for each lipid studied. A critical chain length has been found (between 16 and 17 C-atoms per chain) which subdivides PCs into two classes with different phase behavior. In shorter lipids, the transition takes place above their critical temperatures meaning that this is an intrinsically continuous transition. In longer lipids, the transition occurs below the critical temperatures of the lipids, meaning that the transition is intrinsically discontinuous (first-order). This conclusion was supported independently by the ultrasonic relaxation sensitive to density fluctuations. Interestingly, it is this length that is the most abundant among the saturated chains in biological membranes.     

Measuring molecular order for lipid membrane phase studies: Linear relationship between Laurdan generalized polarization and deuterium NMR order parameter

Two dimensional phase separation in lipid membranes and cell membranes is of interest to biology because of the idea of membrane rafts — compositionally heterogeneous liquid crystal domains with cellular functions. Few quantitative tools exist for characterizing and differentiating coexisting phases on a molecular scale. Lipid acyl chain order can be measured directly using deuterium nuclear magnetic resonance spectroscopy (²H NMR), or inferred using fluorescence microscopy along with the environment-sensitive probe Laurdan. We found a linear relationship between the ²H NMR order parameter and Laurdan generalized polarization. This observed correlation supports the idea that lipid chain order is tightly associated with the amount and dynamics of water molecules at the glycerol backbone level of the membrane.     

Bicelles: A natural ‘molecular goniometer’ for structural,dynamical and topological studies of molecules in membranes

Major biological processes occur at the biological membrane. One of the great challenges is to understand the function of chemical or biological molecules inside the membrane; as well of those involved in membrane trafficking. This requires obtaining a complete picture of the in situ structure and dynamics as well as the topology and orientation of these molecules in the membrane lipid bilayer. These led to the creation of several innovative models of biological membranes in order to investigate the structure and dynamics of amphiphilic molecules, as well as integral membrane proteins having single or multiple transmembrane segments. Because the determination of the structure, dynamics and topology of molecules in membranes requires a macroscopic alignment of the system, a new membrane model called ‘bicelles’ that represents a crossover between lipid vesicles and classical micelles has become very popular due to its property of spontaneous self-orientation in magnetic fields. In addition, crucial factors involved in mimicking natural membranes, such as sample hydration, pH and salinity limits, are easy to control in bicelle systems. Bicelles are composed of mixtures of long chain (14–18 carbons) and short chain phospholipids (6–8 carbons) hydrated up to 98% with buffers and may adopt various morphologies depending on lipid composition, temperature and hydration. We have been developing bicelle systems under the form of nano-discs made of lipids with saturated or biphenyl-containing fatty acyl chains. Depending on the lipid nature, these membranous nano-discs may be macroscopically oriented with their normal perpendicular or parallel to the magnetic field, providing a natural ‘molecular goniometer’ for structural and topological studies, especially in the field of NMR. Bicelles can also be spun at the magic angle and lead to the 3D structural determination of molecules in membranes.     

Complementary biophysical tools to investigate lipid specificity in the interaction between bioactive molecules and the plasma membrane: A review

Plasma membranes are complex entities common to all living cells. The basic principle of their organization appears very simple, but they are actually of high complexity and represent very dynamic structures. The interactions between bioactive molecules and lipids are important for numerous processes, from drug bioavailability to viral fusion. The cell membrane is a carefully balanced environment and any change inflicted upon its structure by a bioactive molecule must be considered in conjunction with the overall effect that this may have on the function and integrity of the membrane. Conceptually, understanding the molecular mechanisms by which bioactive molecules interact with cell membranes is of fundamental importance.     

Interaction of nonsteroidal anti-inflammatory drugs with membranes: In vitro assessment and relevance for their biological actions

Nonsteroidal anti-inflammatory drugs (NSAIDs) are among the most commonly used drugs in the world due to their anti-inflammatory, analgesic and antipyretic properties. Nevertheless, the consumption of these drugs is still associated with the occurrence of a wide spectrum of adverse effects. Regarding the major role of membranes in cellular events, the hypothesis that the biological actions of NSAIDs may be related to their effect at the membrane level has triggered the in vitro assessment of NSAIDs-membrane interactions. The use of membrane mimetic models, cell cultures, a wide range of experimental techniques and molecular dynamics simulations has been providing significant information about drugs partition and location within membranes and also about their effect on diverse membrane properties. These studies have indeed been providing evidences that the effect of NSAIDs at membrane level may be an additional mechanism of action and toxicity of NSAIDs. In fact, the pharmacokinetic properties of NSAIDs are closely related to the ability of these drugs to interact and overcome biological membranes. Moreover, the therapeutic actions of NSAIDs may also result from the indirect inhibition of cyclooxygenase due to the disturbing effect of NSAIDs on membrane properties. Furthermore, increasing evidences suggest that the disordering effects of these drugs on membranes may be in the basis of the NSAIDs-induced toxicity in diverse organ systems. Overall, the study of NSAIDs-membrane interactions has proved to be not only important for the better understanding of their pharmacological actions, but also for the rational development of new approaches to overcome NSAIDs adverse effects.