Transformation of Lipid Bodies Related to Hydrocarbon Accumulation in a Green Alga,Botryococcus braunii (Race B)

The colonial microalga Botryococcus braunii accumulates large quantities of hydrocarbons mainly in the extracellular space; most other oleaginous microalgae store lipids in the cytoplasm. Botryococcus braunii is classified into three principal races (A, B, and L) based on the types of hydrocarbons. Race B has attracted the most attention as an alternative to petroleum by its higher hydrocarbon contents than the other races and its hydrocarbon components, botryococcenes and methylsqualenes, both can be readily converted into biofuels. We studied race B using fluorescence and electron microscopy, and clarify the stage when extracellular hydrocarbon accumulation occurs during the cell cycle, in a correlation with the behavior and structural changes of the lipid bodies and discussed development of the algal colony. New accumulation of lipids on the cell surface occurred after cell division in the basolateral region of daughter cells. While lipid bodies were observed throughout the cell cycle, their size and inclusions were dynamically changing. When cells began dividing, the lipid bodies increased in size and inclusions until the extracellular accumulation of lipids started. Most of the lipids disappeared from the cytoplasm concomitant with the extracellular accumulation, and then reformed. We therefore hypothesize that lipid bodies produced during the growth of B. braunii are related to lipid secretion. New lipids secreted at the cell surface formed layers of oil droplets, to a maximum depth of six layers, and fused to form flattened, continuous sheets. The sheets that combined a pair of daughter cells remained during successive cellular divisions and the colony increased in size with increasing number of cells.     

Docosahexaenoic Acid Production and Lipid-Body Formation in Schizochytrium limacinum SR21

Schizochytrium limacinum SR21, a thraustochytrid (Labyrinturomycota), is a heterotrophic marine microorganism. SR21 has attracted recent attention because of the production of docosahexaenoic acid (DHA). We obtained highly concentrated SR21 zoospores and successfully observed synchronous growth. We investigated changes of lipid content and fatty acid composition during the growth. The morphological features of the lipid bodies were also described via fluorescent and electron microscopy. The cells developed quickly after zoospore settlement. Lipid bodies developed in accordance with an increase in lipid content during the 8-h synchronous growth. The total lipid was composed mainly of triacylglycerol, sterol esters, and phosphatidylcholine. The proportion of neutral lipids (triacylglycerol and sterol esters) in the total lipid was fairly constant during growth. The fatty acid composition of neutral lipids, primary components of the lipid body, and phospholipids, primary components of the cell membranes, was nearly unchanged during the synchronous growth. However, the DHA content of the phospholipids decreased drastically after a 10-day culture. Electron micrographs prepared using a high-pressure freeze substitution technique revealed a fine structure of light- and dark-staining bands inside the lipid bodies in many stages of the cells.     

Active Hydrocarbon Biosynthesis and Accumulation in a Green Alga,Botryococcus braunii (Race A)

Among oleaginous microalgae, the colonial green alga Botryococcus braunii accumulates especially large quantities of hydrocarbons. This accumulation may be achieved more by storage of lipids in the extracellular space rather than in the cytoplasm, as is the case for all other examined oleaginous microalgae. The stage of hydrocarbon synthesis during the cell cycle was determined by autoradiography. The cell cycle of B. braunii race A was synchronized by aminouracil treatment, and cells were taken at various stages in the cell cycle and cultured in a medium containing [¹⁴C]acetate. Incorporation of ¹⁴C into hydrocarbons was detected. The highest labeling occurred just after septum formation, when it was about 2.6 times the rate during interphase. Fluorescent and electron microscopy revealed that new lipid accumulation on the cell surface occurred during at least two different growth stages and sites of cells. Lipid bodies in the cytoplasm were not prominent in interphase cells. These lipid bodies then increased in number, size, and inclusions, reaching maximum values just before the first lipid accumulation on the cell surface at the cell apex. Most of them disappeared from the cytoplasm concomitant with the second new accumulation at the basolateral region, where extracellular lipids continuously accumulated. The rough endoplasmic reticulum near the plasma membrane is prominent in B. braunii, and the endoplasmic reticulum was often in contact with both a chloroplast and lipid bodies in cells with increasing numbers of lipid bodies. We discuss the transport pathway of precursors of extracellular hydrocarbons in race A.     

Cytoplasmic lipid bodies of neutrophils: formation induced by cis- unsaturated fatty acids and mediated by protein kinase C

Lipid bodies, nonmembrane-bound cytoplasmic inclusions, serve as repositories of esterified arachidonate and are increased in cells associated with inflammatory reactions. We have evaluated stimuli and mechanisms responsible for lipid body formation within human polymorphonuclear leukocytes (PMNs). Arachidonic acid and oleic acid stimulated dose-dependent formation of lipid bodies over 0.5-1 h. Other C20 and C18 fatty acids were less active and demonstrated rank orders as follows: cis-unsaturated fatty acids were much more active than trans-fatty acids, and activity diminished with decreasing numbers of double bonds. Lipid bodies elicited in vitro with cis-fatty acids were ultrastructurally identical to lipid bodies present in PMNs in vivo. Lipid body induction was not because of fatty acid-elicited oxidants or fatty acid-induced ATP depletion. Cis-fatty acid-induced activation of protein kinase C (PKC) was involved in lipid body formation as evidenced by the capacity of other PKC activators, 1-oleoyl-2-acetyl-glycerol and two active phorbol esters, phorbol myristate acetate, and phorbol 12,13 dibutyrate, but not an inactive phorbol, to induce lipid body formation. The PKC inhibitor, 1-O-hexadecyl-2-O-methyl-glycerol, inhibited PMN lipid body formation induced by oleic and arachidonic acids and by 1-oleoyl-2-acetyl-glycerol and phorbol myristate acetate. Other PKC inhibitors (staurosporine, H-7) also inhibited lipid body formation. Formation of lipid bodies in PMNs is a specific cellular response, stimulated by cis-fatty acids and diglycerides and apparently mediated by PKC, which results in the mobilization and deposition of lipids within discrete, ultrastructurally defined cytoplasmic domains.     

Morphological and spectrometric analyses of lipids accumulation in a novel oleaginous microalga, Eustigmatos cf. polyphem (Eustigmatophyceae)

Eustigmatos cf. polyphem is a yellow-green unicellular edaphic microalga belonging to the eustigmatophyte. The characteristics of lipids accumulation of E. cf. polyphem grown in a bubble cylindrical photobioreactor under nitrogen-limited conditions was dissected by morphological and spectrometric analyses. Total lipids accumulation rate increased rapidly at early growth phase, with the emergence of many small lipid bodies. Afterwards, lipid bodies became abundant and enlarged primarily because of the progressive accumulation of lipids and the fusion of nearby lipid bodies. Maximum total lipids and neutral lipids content reached up to 60.59 % and 53.08 % of cell dry weight, accompanied with a biomass dry weight 7.9 g/l. E. cf. polyphem is thus referred to as an oleaginous microalga for biodiesel production due to its high biomass and considerable production of oils.     

Acquisition of membrane lipids by differentiating glyoxysomes: role of lipid bodies

Glyoxysomes in cotyledons of cotton (Gossypium hirsutum, L.) seedlings enlarge dramatically within 48 h after seed imbibition (Kunce, C.M., R.N. Trelease, and D.C. Doman. 1984. Planta (Berl.). 161:156-164) to effect mobilization of stored cotton-seed oil. We discovered that the membranes of enlarging glyoxysomes at all stages examined contained a large percentage (36-62% by weight) of nonpolar lipid, nearly all of which were triacylglycerols (TAGs) and TAG metabolites. Free fatty acids comprised the largest percentage of these nonpolar lipids. Six uncommon (and as yet unidentified) fatty acids constituted the majority (51%) of both the free fatty acids and the fatty acids in TAGs of glyoxysome membranes; the same six uncommon fatty acids were less than 7% of the acyl constituents in TAGs extracted from cotton-seed storage lipid bodies. TAGs of lipid bodies primarily were composed of palmitic, oleic, and linoleic acids (together 70%). Together, these three major storage fatty acids were less than 10% of both the free fatty acids and fatty acids in TAGs of glyoxysome membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) constituted a major portion of glyoxysome membrane phospholipids (together 61% by weight). Pulse-chase radiolabeling experiments in vivo clearly demonstrated that 14C-PC and 14C-PE were synthesized from 14C-choline and 14C-ethanolamine, respectively, in ER of cotyledons, and then transported to mitochondria; however, these lipids were not transported to enlarging glyoxysomes. The lack of ER involvement in glyoxysome membrane phospholipid synthesis, and the similarities in lipid compositions between lipid bodies and membranes of glyoxysomes, led us to formulate and test a new hypothesis whereby lipid bodies serve as the dynamic source of nonpolar lipids and phospholipids for membrane expansion of enlarging glyoxysomes. In a cell-free system, 3H-triolein (TO) and 3H-PC were indeed transferred from lipid bodies to glyoxysomes. 3H-PC, but not 3H-TO, also was transferred to mitochondria in vitro. The amount of lipid transferred increased linearly with respect to time and amount of acceptor organelle protein, and transfer occurred only when lipid body membrane proteins were associated with the donor lipid bodies. 3H-TO was transferred to and incorporated into glyoxysome membranes, and then hydrolyzed to free fatty acids. 3H-PC was transferred to and incorporated into glyoxysome and mitochondria membranes without subsequent hydrolysis. Our data are inconsistent with the hypothesis that ER contributes membrane lipids to glyoxysomes during postgerminative seedling growth.(ABSTRACT TRUNCATED AT 400 WORDS)     

The lipoxygenase-dependent oxygenation of lipid body membranes is promoted by a patatin-type phospholipase in cucumber cotyledons

Oilseed germination is characterized by the mobilization of storage lipids as a carbon and energy source for embryonic growth. In addition to storage lipid degradation in germinating oilseeds via the direct action of a triacylglycerol lipase (TGL) on the storage lipids, a second degradation pathway that is dependent on a specific lipid body trilinoleate 13-lipoxygenase (13-LOX) has been proposed in several plant species. The activity of this specific 13-LOX leads first to the formation of ester lipid hydroperoxides. These hydroperoxy fatty acids are then preferentially cleaved off by a TGL and serve as a substrate for glyoxysomal β-oxidation. As a prerequisite for triacylglycerol (TAG) mobilization, a partial degradation of the phospholipid monolayer and/or membrane proteins of the oil body has been discussed. Evidence has now been found for both processes: partial degradation of the proteins caleosin and oleosin was observed and simultaneously a patatin-like protein together with transient phospholipase (PLase) activity could be detected at the oil body membranes during germination. Moreover, in vitro experiments with isolated oil bodies from mature seeds revealed that the formation of 13-LOX-derived lipid peroxides in lipid body membranes is increased after incubation with the purified recombinant patatin-like protein. These experiments suggest that in vivo the degradation of storage lipids in cucumber cotyledons is promoted by the activity of a specific oil body PLase, which leads to an increased decomposition of the oil body membrane by the 13-LOX and thereby TAGs may be better accessible to LOX and TGL.     

The synthesis and transport of lipids for axonal growth and nerve regeneration

Neurons are unique polarized cells in which the growing axon is often located up to a meter or more from the cell body. Consequently, the intracellular movement of membrane lipids and proteins between cell bodies and axons poses a special challenge. The mechanisms of lipid transport within neurons are, for the most part, unknown although lipid transport via vesicles and via cholesterol- and sphingolipid-rich 'rafts' are considered likely mechanisms. Very active anterograde and retrograde transport of lipid-containing vesicles occurs between the cell body and distal axons. However, it is becoming clear that the axon need not obtain all of its membrane constituents from the cell body. For example, the synthesis of phosphatidylcholine, the major membrane phospholipid, occurs in axons, and its synthesis at this location is required for axonal elongation. In contrast, cholesterol synthesis appears to occur only in cell bodies, and cholesterol is efficiently delivered from cell bodies to axons by anterograde transport. Cholesterol that is required for axonal growth can also be exogenously supplied from lipoproteins to axons of cultured neurons. Several studies have suggested a role for apolipoprotein E in lipid delivery for growth and regeneration of axons after a nerve injury. Alternatively, or in addition, apolipoprotein E has been proposed to be a ligand for receptors that mediate signal transduction cascades. Lipids are also transported from axons to myelin, although the importance of this process for myelination is not clear.     

Cytosolic phospholipase A2-driven PGE2 synthesis within unsaturated fatty acids-induced lipid bodies of epithelial cells

Cytoplasmic lipid bodies (also known as lipid droplets) are intracellular deposits of arachidonic acid (AA), which can be metabolized for eicosanoid generation. PGE₂ is a major AA metabolite produced by epithelial cells and can modulate restoration of epithelium homeostasis after injury. We studied lipid body biogenesis and their role in AA metabolic pathway in an epithelial cell line derived from normal rat intestinal epithelium, IEC-6 cells. Lipid bodies were virtually absent in confluent IEC-6 cells. Stimulation of confluent IEC-6 cells with unsaturated fatty acids, including AA or oleic acid (OA), induced rapid lipid body assembly that was independent on its metabolism to PGE₂, but dependent on G-coupled receptor-driven signaling through p38, PKC, and PI3K. Newly formed lipid bodies compartmentalized cytosolic phospholipase (cPL)A₂-α, while facilitated AA mobilization and synthesis of PGE₂ within epithelial cells. Thus, both lipid body-related events, including highly regulated biogenesis and functional assembly of cPLA₂-α-driven enhanced AA mobilization and PGE₂ production, may have key roles in epithelial cell-driven inflammatory functions, and may represent relevant therapeutic targets of epithelial pathologies.     

Lipid bodies and lipid body formation in an oleaginous fungus, Mortierella ramanniana var. angulispora

Mortierella ramanniana var. angulispora accumulates triacylglycerol (TG) in lipid bodies. Studies on lipid transport into lipid bodies are essential for elucidating mechanisms of lipid body formation. We used fluorescent dyes and fluorescent lipid analogs to visualize lipid body formation with a confocal laser scanning microscope. Different sizes of lipid bodies were stained by Nile red, a lipid body marker – one with a diameter of about 1 μm and the other with a diameter of about 2–3 μm. Lipid bodies matured into larger ones with culture. To metabolically monitor lipid bodies, we used 1-palmitoyl, 2-[5-(5,7-dimethyl boron dipyrromethene difluoride)-1-pentanoyl]-phosphatidic acid (C₅-DMB-PA), and C₅-DMB-phosphatidylcholine (C₅-DMB-PC). These were taken up into fungal cells and incorporated into intracellular organelles at 30°C. C₅-DMB-PA was quickly incorporated into lipid bodies while C₅-DMB-PC was initially incorporated into internal membranes, presumably endoplasmic reticulum membranes, and fluorescence was then gradually transported into lipid bodies. The transport of fluorescent lipids accompanied their metabolism into diacylglycerol (DG) and TG, which, taken together with the fluorescence distribution, suggested that conversion to TG was not necessary for transport into lipid bodies. It is likely that the synthesized DG was mainly located in lipid bodies and the conversion to TG took place in lipid bodies. C₅-DMB-PA and C₅-DMB-PC were converted to DG and TG in the membrane and lipid body fractions of this fungus, which agreed with in vivo metabolism of these fluorescent lipids and in vitro enzyme activity related to PA and PC metabolism. These results indicate that transport and metabolism of C₅-DMB-PA and C₅-DMB-PC represent two different routes for lipid body formation in this fungus.     

Conversion of membrane lipid acyl groups to triacylglycerol and formation of lipid bodies upon nitrogen starvation in biofuel green algae Chlorella UTEX29

Algal lipids are ideal biofuel sources. Our objective was to determine the contributors to triacylglycerol (TAG) accumulation and lipid body formation in Chlorella UTEX29 under nitrogen (N) deprivation. A fivefold increase in intracellular lipids following N starvation for 24 h confirmed the oleaginous characteristics of UTEX29. Ultrastructural studies revealed increased number of lipid bodies and decreased starch granules in N-starved cells compared to N-replete cells. Lipid bodies were observed as early as 3 h after N removal and plastids collapsed after 48 h of stress. Moreover, the identification of intracellular pyrenoids and differences in the expected nutritional requirements for Chlorella protothecoides (as UTEX29 is currently classified) led us to conduct a phylogenetic study using 18S and actin cDNA sequences. This indicated UTEX29 to be more phylogenetically related to Chlorella vulgaris. To investigate the fate of different lipids after N starvation, radiolabeling using ¹⁴C-acetate was used. A significant decrease in ¹⁴C-galactolipids and phospholipids matched the increase in ¹⁴C-TAG starting at 3 h of N starvation, consistent with acyl groups from structural lipids as sources for TAG under N starvation. These results have important implications for the identification of key steps controlling oil accumulation in N-starved biofuel algae and demonstrate membrane recycling during lipid body formation.     

Leukocyte lipid bodies — Biogenesis and functions in inflammation

Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infectious, neoplasic and other inflammatory conditions. Here, we will review the contemporary evidence related to the biogenesis and structure of leukocyte lipid bodies (also known as lipid droplets) as inflammatory organelles. Studies of leukocyte lipid bodies are providing functional, ultrastructural and protein compositional evidences that lipid bodies are not solely storage depots of neutral lipid. Over the past years substantial progresses have been made to demonstrate that lipid body biogenesis is a highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place leukocyte lipid bodies as inducible cytoplasmic organelles with roles in cell signaling and activation, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Pertinent to the roles of lipid bodies in inflammation and cell signaling, enzymes involved in eicosanoid synthesis are localized at lipid bodies and lipid bodies are sites for eicosanoid generation. Collectively, lipid bodies in leukocytes are emerging as critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.     

Heterologous expression of AtClo1, a plant oil body protein, induces lipid accumulation in yeast

Proteomic approaches on lipid bodies have led to the identification of proteins associated with this compartment, showing that, rather than the inert fat depot, lipid droplets appear as complex dynamic organelles with roles in metabolism control and cell signaling. We focused our investigations on caleosin [ Arabidopsis thaliana caleosin 1 (AtClo1)], a minor protein of the Arabidopsis thaliana seed lipid body. AtClo1 shares an original triblock structure, which confers to the protein the capacity to insert at the lipid body surface. In addition, AtClo1 possesses a calcium-binding domain. The study of plants deficient in caleosin revealed its involvement in storage lipid degradation during seed germination. Using Saccharomyces cerevisiae as a heterologous expression system, we investigated the potential role of AtClo1 in lipid body biogenesis and filling. The green fluorescent protein-tagged protein was correctly targeted to lipid bodies. We observed an increase in the number and size of lipid bodies. Moreover, transformed yeasts accumulated more fatty acids (+46.6%). We confirmed that this excess of fatty acids was due to overaccumulation of lipid body neutral lipids, triacylglycerols and steryl esters. We showed that the original intrinsic properties of AtClo1 protein were sufficient to generate a functional lipid body membrane and to promote overaccumulation of storage lipids in yeast oil bodies.     

Seasonal Dynamics in the Chemistry and Structure of the Fat Bodies of Bumblebee Queens

Insects’ fat bodies are responsible for nutrient storage and for a significant part of intermediary metabolism. Thus, it can be expected that the structure and content of the fat body will adaptively change, if an insect is going through different life stages. Bumblebee queens belong to such insects as they dramatically change their physiology several times over their lives in relation to their solitary overwintering, independent colony foundation stage, and during the colony life-cycle ending in the senescent stage. Here, we report on changes in the ultrastructure and lipid composition of the peripheral fat body of Bombus terrestris queens in relation to seasonal changes in the queens’ activity. Six life stages are defined and evaluated in particular: pharate, callow, before and after hibernation, egg-laying, and senescence. Transmission electron microscopy revealed that the fat body contained two main cell types–adipocytes and oenocytes. Only adipocytes reveal important changes related to the life phase, and mostly the ration between inclusion and cytoplasm volume varies among particular stages. Both electron microscopy and chemical analyses of lipids highlighted seasonal variability in the quantity of the stored lipids, which peaked prior to hibernation. Triacylglycerols appeared to be the main energy source during hibernation, while the amount of glycogen before and after hibernation remained unchanged. In addition, we observed that the representation of some fatty acids within the triacylglycerols change during the queen’s life. Last but not least, we show that fat body cell membranes do not undergo substantial changes concerning phospholipid composition in relation to overwintering. This finding supports the hypothesis that the cold-adaptation strategy of bumblebee queens is more likely to be based on polyol accumulation than on the restructuring of lipid membranes.     

Triacylglycerol lipases of the yeast

All eukaryotes including the yeast contain a lipid storage compartment which is named lipid particle, lipid droplet or oil body. Lipids accumulating in this subcellular fraction serve as a depot of energy and building blocks for membrane lipid synthesis. In the yeast, the major storage lipids are triacylglycerols (TGs) and steryl esters (SEs). An important step in the life cycle of these non-polar lipids is their mobilization from their site of storage and channeling of their degradation components to the appropriate metabolic pathways. A key step in this mobilization process is hydrolysis of TG and SE which is accomplished by lipases and hydrolases. In this review, we describe our recent knowledge of TG lipases from the yeast based on biochemical, molecular biological and cell biological information. We report about recent findings addressing the versatile role of TG lipases in lipid metabolism, and discuss non-polar lipid homeostasis and its newly discovered links to various cell biological processes in the yeast.     

MEMBRANE SYNTHESIS IN BACILLUS SUBTILIS : III. The Morphological Localization of the Sites of Membrane Synthesis

The results of several lines of investigation indicate that membrane growth in Bacillus subtilis does not occur at one or a small number of discrete zones. No indications of large regions of membrane conservation were observed. Kinetic labeling experiments of mesosomal and plasma membrane lipids indicate that the mesosomal lipids are not precursors of the plasma membrane lipids. Density shift experiments, in which the changes in buoyant density of membranes were studied after growth in deuterated media, showed no indication of large zones of conservation during membrane growth. Radioautography of thin sections of cells pulse labeled with tritiated glycerol showed no indication of specific zones of lipid synthesis. The consequences of these results for models of cell growth and division are discussed.     

CDP-Diacylglycerol Synthetase Coordinates Cell Growth and Fat Storage through Phosphatidylinositol Metabolism and the Insulin Pathway

During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.     

Storage lipid synthesis is non-essential in yeast.

Steryl esters and triacylglycerol (TAG) are the main storage lipids in eukaryotic cells. In the yeast Saccharomyces cerevisiae, these storage lipids accumulate during stationary growth phase within organelles known as lipid bodies. We have used single and multiple gene disruptions to study storage lipid synthesis in yeast. Four genes, ARE1, ARE2, DGA1, and LRO1, were found to contribute to TAG synthesis. The most significant contribution is made by DGA1, which encodes a novel acyl-CoA:diacylglycerol acyltransferase. Two of the genes, ARE1 and ARE2, are also involved in steryl ester synthesis. A yeast strain that lacks all four genes is viable and has no apparent growth defects under standard conditions. The strain is devoid of both TAG and steryl esters, and fluorescence microscopy revealed that it also lacks lipid bodies. We conclude that neither storage lipids nor lipid bodies are essential for growth in yeast.