CHANGES IN THE ULTRASTRUCTURE OF SYMBIOTIC ZOOXANTHELLAE (SYMBIODINIUM SP., DINOPHYCEAE) IN FED AND STARVED SEA ANEMONES MAINTAINED UNDER HIGH AND LOW LIGHT1

The ultrastructure of symbiotic dinoflagellates (Symbiodinium sp., zooxanthellae) in the sea anemone Aiptasia pallida Verrill was examined in well-fed or starved (up to 120 days) anemones maintained under two light levels (5 and 50 μmol · m^?2· s^?1). Cell size of zooxanthellae was not affected by feeding history; however, both light and feeding history affected the relative cell volume of chloroplasts, lipids, and vacuoles. Stereological analysis of transmission electron micrographs showed that algae in low-light starved anemones had 10 times as much lipid (17.4% of cell volume) as those in well-fed anemones under the same light conditions (1.8%). The lipid content of algae from anemones in high light increased from 15.4% in well-fed anemones to 30.1% in starved anemones. The starch content of zooxanthellae in low-light anemones was law (4.1%) and not affected by feeding history, while the starch content of zooxanthellae in high-light anemones was greater (10.7%), with some differences among groups. Algal photoacclimation to low light included an increase in chloroplast relative volume from 17% (in well-fed high-light anemones) to 33% in well-fed low-light anemones. Starvation of the host resulted in a significant decrease in chloroplast volume in zooxanthellae in anemones at both light levels. Morphometry provides quantitative confirmation of biochemical and physiological data on zooxanthellae, because the changes in zooxanthellae with starvation of the host are consistent with other indicators of nutrient limitation of zooxanthellae of A. pallida held without food for long periods of time.     

Macronuclear DNA and total protein contents of mating types I and II of Paramecium primaurelia, during the phase of maturity and the transition to senescence. Preliminary observations]

Concerning the studies on mating type differentiation and life cycle development in Paramecium primaurelia stock 90, both macronuclear DNA and total protein contents have been measured cytofluorometrically in mating type I and mating type II isogenic cell lines growing in logarithmic phase, throughout their maturity period and transition to senescence. The target was to investigate whether the two mating types undergo clonal decline in different times, as the previous studies suggested. The results indicate that, throughout the maturity period, macronuclear DNA and total protein contents vary both in mating type I and mating type II cell lines; moreover, aged phenotypes as the dramatic decrease of both contents, firstly occur in mating type II which, therefore, appears to be submitted to clonal decline before mating type I.     

Measurement of the specific lipid content of attached cells in microtiter cultures

A method has been described to quantify intracellular neutral lipid content in attached cells in microtiter cultures. The procedure was based on oil red O staining of neutral lipid and Coomassie brilliant blue G-250 staining of total cellular protein. Results were expressed as the ratio of lipid to protein, the "specific lipid content" index. This measurement was shown to closely correspond to actual lipid per cell measurements under experimental conditions. The procedure was specific for neutral lipids and sensitive (greater than or equal to 50 ng triglyceride/well). Additionally, cell proliferation measurements could be made simultaneously, using protein staining data. Chromatic endpoints were measured using a spectrophotometer capable of reading individual wells of a microtiter plate. The procedure is recommended for applications in which the endpoint is neutral lipid droplet accumulation in attached cultured cells.     

Food ingestion and egestion in mating reactive populations of Paramecium primaurelia.

The study of food ingestion and egestion carried out on Paramecium primaurelia mating reactive cells shows that, after their transfer into a medium with suspended particles, the complementary mating type cells exhibit very significant differences in the food vacuole formation and egestion rate. Under the same external environmental conditions, the mating type II cells form and egest a higher number of food vacuoles when compared with mating type I cells. The higher rate of food vacuole formation shown by the mating type II cells is related to their faster growth rate.     

FRET multiphoton spectral imaging microscopy of 7-ketocholesterol and Nile Red in U937 monocytic cells loaded with 7-ketocholesterol

OBJECTIVE: To show the effect of 7-ketocholesterol (7KC) on cellular lipid content by means of flow cytometry and the interaction of 7KC with Nile Red (NR) via ultraviolet fluorescence resonance energy transfer (FRET) excitation of NR on U937 monocytic cells by means of 2-photon excitation confocal laser scanning microscopy (CLSM). STUDY DESIGN: Untreated and 7KC-treated U937 cells were stained with NR and analyzed by flow cytometry and CLSM. 3D sequences of images were obtained by spectral analysis in a 2-photon excitation CLSM and analyzed by the factor analysis of medical image sequences (FAMIS) algorithm, which provides factor curves and images. Factor images are the result of the FAMIS image processing method, which handles emission spectra. In FRET analysis, preparations are screened at selected UV wavelengths to avoid emission of NR in the absence of 7KC. RESULTS: During 7KC-induced cell death,flow cytometry and CLSM revealed a modification of the cellular lipid content. Factor images show FRET occurrence and subsequent colocalization of 7KC and NR. CONCLUSION: This investigation established the utility of 2-photon excitation CLSM to assess colocalization of 7KC with NR by FRET and to identify and distinguish polar and neutral lipids stained by NR that accumulate from the effect of 7KC.     

Lipids of human leukocytes: relation to celltype

Significant differences in lipid composition have been found between normal human lymphocytes and polymorphonuclear leukocytes (isolated from blood by means of glass-bead columns), abnormal leukocytes from patients with acute and chronic leukemia, and leukocytes from peritoneal exudates. Lipid extracts of isolated leukocytes were analyzed for total lipid, phosphorus, cholesterol, and plasmalogens. Individual phospholipids and neutral lipids were separated by thin-layer chromatography. The major phospholipids were phosphatidyl choline, ethanolamine glycerophosphatides, sphingomyelin, phosphatidyl serine, and phosphatidyl inositol. Plasmalogen was found mainly as phosphatidal ethanolamine. The neutral lipid fractions contained free cholesterol and various amounts of triglyceride, but little esterified cholesterol. Normal lymphocytes contained about half as much total lipid per cell as normal polymorphonuclear leukocytes, with a similar cholesterol:-lipid-P ratio but relatively more lecithin and less ethanolamine glycerophosphatide. Normal mature leukocytes, compared with immature cells of the same morphological series, had a higher total lipid content per cell, more cholesterol, and a higher ratio of cholesterol to lipid-P. Little difference was found in total lipid-P per cell, but mature cells contained relatively less lecithin and more sphingomyelin. These findings may reflect differences in the relative content of various intracellular organelles as well as possible differences in the quantity and composition of the plasma membrane.     

Effect of selection against deleterious mutations on the decline in heterozygosity at neutral loci in closely inbreeding populations.

Transition matrices for selfing and full-sib mating were derived to investigate the effect of selection against deleterious mutations on the process of inbreeding at a linked neutral locus. Selection was allowed to act within lines only (selection type I) or equally within and between lines (type II). For selfing lines under selection type I, inbreeding is always retarded, the retardation being determined by the recombination fraction between the neutral and selected loci and the inbreeding depression from the selected locus, irrespective of the selection coefficient (s) and dominance coefficient (h) of the mutant allele. For selfing under selection type II or full-sib mating under both selection types, inbreeding is delayed by weak selection (small s and sh), due to the associative overdominance created at the neutral locus, and accelerated by strong selection, due to the elevated differential contributions between alternative alleles at the neutral locus within individuals and between lines (for selection type II). For multiple fitness loci under selection, stochastic simulations were run for populations with selfing, full-sib mating, and random mating, using empirical estimates of mutation parameters and inbreeding load in Drosophila. The simulations results are in general compatible with empirical observations.     

Retinyl esters form lipid droplets independently of triacylglycerol and seipin

Lipid droplets store neutral lipids, primarily triacylglycerol and steryl esters. Seipin plays a role in lipid droplet biogenesis and is thought to determine the site of lipid droplet biogenesis and the size of newly formed lipid droplets. Here we show a seipin-independent pathway of lipid droplet biogenesis. In silico and in vitro experiments reveal that retinyl esters have the intrinsic propensity to sequester and nucleate in lipid bilayers. Production of retinyl esters in mammalian and yeast cells that do not normally produce retinyl esters causes the formation of lipid droplets, even in a yeast strain that produces only retinyl esters and no other neutral lipids. Seipin does not determine the size or biogenesis site of lipid droplets composed of only retinyl esters or steryl esters. These findings indicate that the role of seipin in lipid droplet biogenesis depends on the type of neutral lipid stored in forming droplets.     

Effects of caspase inhibitors (z-VAD-fmk, z-VDVAD-fmk) on Nile Red fluorescence pattern in 7-ketocholesterol-treated cells: investigation by flow cytometry and spectral imaging microscopy.

BACKGROUND: The 7-ketocholesterol (7KC)-induced cell death has some characteristics of apoptosis and is associated with polar lipid accumulation. So, we investigated the effects of the broad-spectrum caspase inhibitor z-VAD-fmk and of the caspase-2 inhibitor z-VDVAD-fmk on lipid profile evaluated by staining with Nile Red (NR). METHODS: The 7KC-treated human monocytic U937 cells were cultured in the absence or in the presence of the caspase inhibitors z-VAD-fmk or z-VDVAD-fmk. When staining with NR is performed, neutral and polar lipids have yellow and orange/red emission, respectively, and fluorescence was then analyzed by flow cytometry (FCM) and by confocal laser scanning microscopy (CLSM) combined with subsequent image processing. The 3D-image sequences were obtained by means of CLSM using spectral analysis, and were analyzed by the factor analysis of medical image sequences algorithm to differentiate spectra inside mixed fluorescence emission and get corresponding specific images. RESULTS: By FCM, comparatively to untreated cells, higher percentages of red fluorescent cells were identified in 7KC-treated cells. Factor curves and images reveal orange and red fluorescence emissions in 7KC-treated cells and show yellow, orange, and red fluorescence emissions in 7KC-treated cells cultured in the presence of z-VAD-fmk or z-VDVAD-fmk. CONCLUSIONS: Our data support that investigation by FCM and by spectral analysis in CLSM associated with subsequent image processing provides useful tools to determine the effect of caspase inhibitors on lipid content evaluated with NR. They also favor the hypothesis of relationships between caspase activity and polar lipid accumulation.     

Lipase-like 5 enzyme controls mitochondrial activity in response to starvation in Caenorhabditis elegans

The C. elegans lipase-like 5 (lipl-5) gene is predicted to code for a lipase homologous to the human gastric acid lipase. Its expression was previously shown to be modulated by nutritional or immune cues, but nothing is known about its impact on the lipid landscape and ensuing functional consequences. In the present work, we used mutants lacking LIPL-5 protein and found that lipl-5 is important for normal lipidome composition as well as its remodeling in response to food deprivation. Particularly, lipids with signaling functions such as ceramides and mitochondrial lipids were affected by lipl-5 silencing. In comparison with wild type worms, animals lacking LIPL-5 were enriched in cardiolipins linked to polyunsaturated C20 fatty acids and coenzyme Q-9. Differences in mitochondrial lipid composition were accompanied by differences in mitochondrial activity as mitochondria from well-fed lipl-5 mutants were significantly more able to oxidize respiratory substrates when compared with mitochondria from well-fed wild type worms. Strikingly, starvation elicited important changes in mitochondrial activity in wild type worms, but not in lipl-5 worms. This indicates that this lipase is a determinant of mitochondrial functional remodeling in response to food withdrawal.     

A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism

In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs) and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4), type 2A phosphatase and its related regulator (pph21 and sap185), type 2C protein phosphatases (ptc1, ptc4, ptc7) and dual phosphatases (pps1, msg5) were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190) were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive) in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.     

Perturbance Analysis of Nuclear Determination in Tetrahymena

Mating type frequencies were ascertained among the progeny of crosses of strains A × B, Tetrahymena thermophila under a number of different circumstances. The frequencies are different if the parents are severely starved than if they are well-fed at the time of conjugation; severe starvation of the progeny before the first post-zygotic division has an effect similar to that of starving the parents. Mating type frequencies may also be modified by isolating conjugating pairs into cell extracts before the new macronuclei begin to develop; the changes do not appear to be related in a meaningful way to the mating type of the cells used as a source of the cell extracts. A third means of changing the mating type frequencies involves the exposure of conjugating pairs to CaCl₂ solutions. Finally, changed frequency patterns may appear "spontaneously", and reflect either some as yet unsuspected environmental variable, or else an intrinsic metastable state that conditions the probabilities of mating type fixation. With the exception of the starvation effects, the pattern variations seem to fall into two groups. No satisfactory mechanism to account for these results is yet available.     

Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.     

Odd mating-type substances may work as precursor molecules of even mating-type substances in Paramecium caudatum

Mating-type substances are key molecules in the sexual recognition of the odd (O) and even (E) complementary mating-type cells in Paramecium caudatum. Indirect evidence suggested that the substances were proteins and were located on ventral surface cilia. Monoclonal antibodies inhibiting the mating reactivity of the O cells have been obtained. Using these antibodies, we tried to detect antigen molecules as dot-blot signals. Strong dot-blot signals of antigens were only detected from the mating reactive cells, but they were not detected from the well-fed and starved cells without mating reactivity. In addition to identifying the antigen on cilia and cytoplasm of the O cell, the antigen was detected from the cytoplasm of the E cells but never from their cilia. Furthermore, extracts of the E cells induced mating reaction with the living E cells but not with O cells. Thus, the O mating-type substances exist in the cytoplasm of the E mating-type cells, supporting strongly the hypothesis that O mating-type substances are precursor molecules of the E mating-type substances.     

Aluminum Tolerance Acquired during Phosphate Starvation in Cultured Tobacco Cells

Al toxicity in cultured tobacco cells (Nicotiana tabacum L. cv Samsun; nonchlorophyllic cell line SL) has been investigated in nutrient medium. In this system, Al and Fe(II) (ferrous ion) in the medium synergistically result in the accumulation of both Al and Fe, the peroxidation of lipids, and eventually death in cells at the logarithmic phase of growth (+P cells). A lipophilic antioxidant, N,N[prime]-diphenyl-p-phenylenediamine, protected +P cells from the peroxidation of lipids and from cell death, suggesting that a relationship exists between the two. Compared with +P cells, cells that had been starved of Pi (-P cells) were more tolerant to Al, accumulated 30 to 40% less Al and 70 to 90% less Fe, and did not show any evidence of the peroxidation of lipids during Al treatment. These results suggest that -P cells exhibit Al tolerance because their plasma membranes are protected from the peroxidation of lipids caused by the combination of Al and Fe(II). It seems likely that the exclusion of Fe from -P cells might suppress directly Fe-mediated peroxidation of lipids. Furthermore, since -P cells accumulated [beta]-carotene, it is proposed that this carotenoid pigment might function as a radical-trapping antioxidant in the plasma membrane of cells starved of Pi.     

Cell division induced by mechanical stimulation in starved Tetrahymena thermophila: cell cycle without synthesis of macronuclear DNA

Starved Tetrahymena thermophila cells underwent synchronous cell division 2 h after a mechanical stimulation. The macronucleus showed no obvious increase in DNA content before the cell division in the starvation medium, and the DNA content was decreased after the cell division. On the other hand, when the starved cells were given nutrient-supplied medium immediately after the mechanical stimulation, cell division was delayed for 3 h. This period was almost the same as that for G1 cells in the stationary culture to first division after transfer to fresh nutrient medium. These results suggest that the mechanical stimulation induces an early division of starved cells, skipping the macronuclear S-phase with the starved cells probably becoming trapped in G1. Starved cells that had finished division soon formed mating pairs with cells of the opposite type. These observations lead us to propose that cell division in starvation conditions may be necessary to reduce macronuclear DNA content prior to the mating of T. thermophila.     

Fat-specific protein 27, a novel lipid droplet protein that enhances triglyceride storage

Fat-specific protein (FSP)27/Cidec is most highly expressed in white and brown adipose tissues and increases in abundance by over 50-fold during adipogenesis. However, its function in adipocytes has remained elusive since its discovery over 15 years ago. Here we demonstrate that FSP27/Cidec localizes to lipid droplets in cultured adipocytes and functions to promote lipid accumulation. Ectopically expressed FSP27-GFP surrounds lipid droplets in 3T3-L1 adipocytes and colocalizes with the known lipid droplet protein perilipin. Immunostaining of endogenous FSP27 in 3T3-L1 adipocytes also confirmed its presence on lipid droplets. FSP27-GFP expression also markedly increases lipid droplet size and enhances accumulation of total neutral lipids in 3T3-L1 preadipocytes as well as other cell types such as COS cells. Conversely, RNA interference-based FSP27/Cidec depletion in mature adipocytes significantly stimulates lipolysis and reduces the size of lipid droplets. These data reveal FSP27/Cidec as a novel adipocyte lipid droplet protein that negatively regulates lipolysis and promotes triglyceride accumulation.     

Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, approximately 80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with (14)C-labeled OA or palmitic acid resulted in [(14)C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [(14)C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.     

The yeast lipin orthologue Pah1p is important for biogenesis of lipid droplets

Lipins are phosphatidate phosphatases that generate diacylglycerol (DAG). In this study, we report that yeast lipin, Pah1p, controls the formation of cytosolic lipid droplets. Disruption of PAH1 resulted in a 63% decrease in droplet number, although total neutral lipid levels did not change. This was accompanied by an accumulation of neutral lipids in the endoplasmic reticulum (ER). The droplet biogenesis defect was not a result of alterations in neutral lipid ratios. No droplets were visible in the absence of both PAH1 and steryl acyltransferases when grown in glucose medium, even though the strain produces as much triacylglycerol as wild type. The requirement of PAH1 for normal droplet formation can be bypassed by a knockout of DGK1. Nem1p, the activator of Pah1p, localizes to a single punctum per cell on the ER that is usually next to a droplet, suggesting that it is a site of droplet assembly. Overall, this study provides strong evidence that DAG generated by Pah1p is important for droplet biogenesis.