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Sample records for abundant lipid droplets

  1. Lipid droplets go nuclear.

    PubMed

    Farese, Robert V; Walther, Tobias C

    2016-01-01

    Lipid droplets (LDs) are sometimes found in the nucleus of some cells. In this issue, Ohsaki et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201507122) show that the nuclear membrane, promyelocytic leukemia bodies, and the protein PML-II play a role in nuclear LD formation, suggesting functional relationships between these structures. PMID:26728852

  2. Expanding roles for lipid droplets

    PubMed Central

    Welte, Michael A.

    2015-01-01

    Summary Lipid droplets are the intracellular sites for neutral lipid storage. They are critical for lipid metabolism and energy homeostasis, and their dysfunction has been linked to many diseases. Accumulating evidence suggests that the roles lipid droplets play in biology are significantly broader than previously anticipated. Lipid droplets are the source of molecules important in the nucleus: they can sequester transcription factors and chromatin components and generate the lipid ligands for certain nuclear receptors. Lipid droplets have also emerged as important nodes for fatty acid trafficking, both inside the cell and between cells. In immunity, new roles for droplets, not directly linked to lipid metabolism, have been uncovered, as assembly platforms for specific viruses and as reservoirs for proteins that fight intracellular pathogens. Until recently, knowledge about droplets in the nervous system has been minimal, but now there are multiple links between lipid droplets and neurodegeneration: Many candidate genes for Hereditary Spastic Paraplegia also have central roles in lipid-droplet formation and maintenance, and mitochondrial dysfunction in neurons can lead to transient accumulating of lipid droplets in neighboring glial cells, an event that may, in turn, contribute to neuronal damage. As the cell biology and biochemistry of lipid droplets are increasingly well understood, the next few years should yield many new mechanistic insights into these novel functions of lipid droplets. PMID:26035793

  3. Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein.

    PubMed

    Vrablik, Tracy L; Petyuk, Vladislav A; Larson, Emily M; Smith, Richard D; Watts, Jennifer L

    2015-10-01

    Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified Caenorhabditis elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols is rich in fatty acid species obtained from the dietary Escherichia coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a very similar proteome in both strains, except that the most abundant protein in the C. elegans lipid droplet proteome, MDT-28, is relatively less abundant in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans. Finally, we confirmed the localization of one of the newly identified lipid droplet proteins, ACS-4. We found that ACS-4 localizes to the surface of lipid droplets in the C. elegans intestine and skin. This study bolsters C. elegans as a model to study the dynamics and functions of lipid droplets in a multicellular organism.

  4. Lipidomic and proteomic analysis of Caenorhabditis elegans lipid droplets and identification of ACS-4 as a lipid droplet-associated protein

    SciTech Connect

    Vrablik, Tracy L.; Petyuk, Vladislav A.; Larson, Emily M.; Smith, Richard D.; Watts, Jennifer

    2015-06-27

    Lipid droplets are cytoplasmic organelles that store neutral lipids for membrane synthesis and energy reserves. In this study, we characterized the lipid and protein composition of purified C. elegans lipid droplets. These lipid droplets are composed mainly of triacylglycerols, surrounded by a phospholipid monolayer composed primarily of phosphatidylcholine and phosphatidylethanolamine. The fatty acid composition of the triacylglycerols was rich in fatty acid species obtained from the dietary E. coli, including cyclopropane fatty acids and cis-vaccenic acid. Unlike other organisms, C. elegans lipid droplets contain very little cholesterol or cholesterol esters. Comparison of the lipid droplet proteomes of wild type and high-fat daf-2 mutant strains shows a relative decrease of MDT-28 abundance in lipid droplets isolated from daf-2 mutants. Functional analysis of lipid droplet proteins identified in our proteomic studies indicated an enrichment of proteins required for growth and fat homeostasis in C. elegans.

  5. Lipid Droplets And Cellular Lipid Metabolism

    PubMed Central

    Walther, Tobias C.; Farese, Robert V.

    2013-01-01

    Among organelles, lipid droplets (LDs) uniquely constitute a hydrophobic phase in the aqueous environment of the cytosol. Their hydrophobic core of neutral lipids stores metabolic energy and membrane components, making LDs hubs for lipid metabolism. In addition, LDs are implicated in a number of other cellular functions, ranging from protein storage and degradation to viral replication. These processes are functionally linked to many physiological and pathological conditions, including obesity and related metabolic diseases. Despite their important functions and nearly ubiquitous presence in cells, many aspects of LD biology are unknown. In the past few years, the pace of LD investigation has increased, providing new insights. Here, we review the current knowledge of LD cell biology and its translation to physiology. PMID:22524315

  6. HEPATIC STELLATE CELL LIPID DROPLETS: A SPECIALIZED LIPID DROPLET FOR RETINOID STORAGE

    PubMed Central

    Blaner, William S.; O’Byrne, Sheila M.; Wongsiriroj, Nuttaporn; Kluwe, Johannes; D’Ambrosio, Diana; Jiang, Hongfeng; Schwabe, Robert F.; Hillman, Elizabeth M.C.; Piantedosi, Roseann; Libien, Jenny

    2009-01-01

    The majority of retinoid (vitamin A and its metabolites) present in the body of a healthy vertebrate is contained within lipid droplets present in the cytoplasm of hepatic stellate cells (HSCs). Two types of lipid droplets have been identified through histological analysis of HSCs within the liver: smaller droplets bounded by a unit membrane and larger membrane-free droplets. Dietary retinoid intake but not triglyceride intake markedly influences the number and size of HSC lipid droplets. The lipids present in rat HSC lipid droplets include retinyl ester, triglyceride, cholesteryl ester, cholesterol, phospholipids and free fatty acids. Retinyl ester and triglyceride are present at similar concentrations, and together these two classes of lipid account for approximately three-quarters of the total lipid in HSC lipid droplets. Both adipocyte-differentiation related protein and TIP47 have been identified by immunohistochemical analysis to be present in HSC lipid droplets. Lecithin:retinol acyltransferase (LRAT), an enzyme responsible for all retinyl ester synthesis within the liver, is required for HSC lipid droplet formation, since Lrat-deficient mice completely lack HSC lipid droplets. When HSCs become activated in response to hepatic injury, the lipid droplets and their retinoid contents are rapidly lost. Although loss of HSC lipid droplets is a hallmark of developing liver disease, it is not known whether this contributes to disease development or occurs simply as a consequence of disease progression. Collectively, the available information suggests that HSC lipid droplets are specialized organelles for hepatic retinoid storage and that loss of HSC lipid droplets may contribute to the development of hepatic disease. PMID:19071229

  7. Identification of a Major Lipid Droplet Protein in a Marine Diatom Phaeodactylum tricornutum.

    PubMed

    Yoneda, Kohei; Yoshida, Masaki; Suzuki, Iwane; Watanabe, Makoto M

    2016-02-01

    Various kinds of organisms, including microalgae, accumulate neutral lipids in distinct intracellular compartments called lipid droplets. Generally, lipid droplets are generated from the endoplasmic reticulum, and particular proteins localize on their surface. Some of these proteins function as structural proteins to prevent fusion between the lipid droplets, and the others could have an enzymatic role or might be involved in intracellular membrane trafficking. However, information about lipid droplet proteins in microalgae is scarce as compared with that in animals and land plants. We focused on the oil-producing, marine, pennate diatom Phaeodactylum tricornutum that forms lipid droplets during nitrogen deprivation and we investigated the proteins located on the lipid droplets. After 6 d of cultivation in a nitrate-deficient medium, the mature lipid droplets were isolated by sucrose density gradient centrifugation. Proteomic analyses revealed five proteins, with Stramenopile-type lipid droplet protein (StLDP) being the most abundant protein in the lipid droplet fraction. Although the primary sequence of StLDP did not have homology to any known lipid droplet proteins, StLDP had a central hydrophobic domain. This structural feature is also detected in oleosin of land plants and in lipid droplet surface protein (LDSP) of Nannochloropsis species. As a proline knot motif of oleosin, conservative proline residues existed in the hydrophobic domain. StLDP was up-regulated during nitrate deprivation, and fluctuations of StLDP expression levels corresponded to the size of the lipid droplets. PMID:26738549

  8. Droplet Microfluidics for Artificial Lipid Bilayers

    NASA Astrophysics Data System (ADS)

    Punnamaraju, Srikoundinya; Steckl, Andrew

    2012-02-01

    Droplet interface bilayer is a versatile approach that allows formation of artificial lipid bilayer membrane at the interface of two lipid monolayer coated aqueous droplets in a lipid filled oil medium. Versatility exists in the form of voltage control of DIB area, ability of forming networks of DIBs, volume control of droplets and lipid-oil, and ease of reformation. Significant effect of voltage on the area and capacitance of DIB as well as DIB networks are characterized using simultaneous optical and electrical recordings. Mechanisms behind voltage-induced effects on DIBs are investigated. Photo induced effect on the DIB membrane porosity is obtained by incorporating UVC-sensitive photo-polymerizable lipids in DIB. Photo-induced effects can be extended for in-vitro studies of triggered release of encapsulated contents across membranes. A droplet based low voltage digital microfluidic platform is developed to automate DIB formation, which could potentially be used for forming arrays of lipid bilayer membranes.

  9. Dictyostelium Lipid Droplets Host Novel Proteins

    PubMed Central

    Du, Xiaoli; Barisch, Caroline; Paschke, Peggy; Herrfurth, Cornelia; Bertinetti, Oliver; Pawolleck, Nadine; Otto, Heike; Rühling, Harald; Feussner, Ivo; Herberg, Friedrich W.

    2013-01-01

    Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane. PMID:24036346

  10. Nuclear lipid droplets: a novel nuclear domain.

    PubMed

    Layerenza, J P; González, P; García de Bravo, M M; Polo, M P; Sisti, M S; Ves-Losada, A

    2013-02-01

    We investigated nuclear neutral-lipid (NL) composition and organization, as NL may represent an alternative source for providing fatty acids and cholesterol (C) to membranes, signaling paths, and transcription factors in the nucleus. We show here that nuclear NL were organized into nonpolar domains in the form of nuclear-lipid droplets (nLD). By fluorescent confocal microscopy, representative nLD were observed in situ within the nuclei of rat hepatocytes in vivo and HepG2 cells, maintained under standard conditions in culture, and within nuclei isolated from rat liver. nLD were resistant to Triton X-100 and became stained with Sudan Red, OsO4, and BODIPY493/503. nLD and control cytosolic-lipid droplets (cLD) were isolated from rat-liver nuclei and from homogenates, respectively, by sucrose-gradient sedimentation. Lipids were extracted, separated by thin-layer chromatography, and quantified. nLD were composed of 37% lipids and 63% proteins. The nLD lipid composition was as follows: 19% triacylglycerols (TAG), 39% cholesteryl esters, 27% C, and 15% polar lipids; whereas the cLD composition contained different proportions of these same lipid classes, in particular 91% TAG. The TAG fatty acids from both lipid droplets were enriched in oleic, linoleic, and palmitic acids. The TAG from the nLD corresponded to a small pool, whereas the TAG from the cLD constituted the main cellular pool (at about 100% yield from the total homogenate). In conclusion, nLD are a domain within the nucleus where NL are stored and organized and may be involved in nuclear lipid homeostasis. PMID:23098923

  11. Proteomic analysis of murine testes lipid droplets

    PubMed Central

    Wang, Weiyi; Wei, Suning; Li, Linghai; Su, Xueying; Du, Congkuo; Li, Fengjuan; Geng, Bin; Liu, Pingsheng; Xu, Guoheng

    2015-01-01

    Testicular Leydig cells contain abundant cytoplasmic lipid droplets (LDs) as a cholesteryl-ester store for releasing cholesterols as the precursor substrate for testosterone biosynthesis. Here, we identified the protein composition of testicular LDs purified from adult mice by using mass spectrometry and immunodetection. Among 337 proteins identified, 144 were previously detected in LD proteomes; 44 were confirmed by microscopy. Testicular LDs contained multiple Rab GTPases, chaperones, and proteins involved in glucuronidation, ubiquination and transport, many known to modulate LD formation and LD-related cellular functions. In particular, testicular LDs contained many members of both the perilipin family and classical lipase/esterase superfamily assembled predominately in adipocyte LDs. Thus, testicular LDs might be regulated similar to adipocyte LDs. Remarkably, testicular LDs contained a large number of classical enzymes for biosynthesis and metabolism of cholesterol and hormonal steroids, so steroidogenic reactions might occur on testicular LDs or the steroidogenic enzymes and products could be transferred through testicular LDs. These characteristics differ from the LDs in most other types of cells, so testicular LDs could be an active organelle functionally involved in steroidogenesis. PMID:26159641

  12. Keeping FIT, storing fat: Lipid droplet biogenesis.

    PubMed

    Choudhary, Vineet; Golden, Andy; Prinz, William A

    2016-01-01

    All eukaryotes store excess lipids in organelles known as lipid droplets (LDs), which play central roles in lipid metabolism. Understanding LD biogenesis and metabolism is critical for understanding the pathophysiology of lipid metabolic disorders like obesity and atherosclerosis. LDs are composed of a core of neutral lipids surrounded by a monolayer of phospholipids that often contains coat proteins. Nascent LDs bud from the endoplasmic reticulum (ER) but the mechanism is not known. In this commentary we discuss our recent finding that a conserved family of proteins called fat storage-inducing transmembrane (FIT) proteins is necessary for LDs budding from the ER. In cells lacking FIT proteins, LDs remain in the ER membrane. C. elegans has a single FIT protein (FITM-2), which we found is essential; almost all homozygous fitm-2 animals die as larvae and those that survive to adulthood give rise to embryos that die as L1 and L2 larvae. Homozygous fitm-2 animals have a number of abnormalities including a significant decrease in intestinal LDs and dramatic defects in muscle development. Understanding how FIT proteins mediate LD biogenesis and what roles they play in lipid metabolism and development are fascinating challenges for the future. PMID:27383728

  13. ARFGAP1 Is Dynamically Associated with Lipid Droplets in Hepatocytes

    PubMed Central

    Alamri, Hussam; Feng, Shi Bo; Kalantari, Fariba; Negi, Sarita; Wong, Amy H. Y.; Mazur, Alexander; Asp, Lennart; Fazel, Ali; Salman, Ayat; Lazaris, Anthoula; Metrakos, Peter; Bergeron, John J. M.; Nilsson, Tommy

    2014-01-01

    The ARF GTPase Activating Protein 1 (ARFGAP1) associates mainly with the cytosolic side of Golgi cisternal membranes where it participates in the formation of both COPI and clathrin-coated vesicles. In this study, we show that ARFGAP1 associates transiently with lipid droplets upon addition of oleate in cultured cells. Also, that addition of cyclic AMP shifts ARFGAP1 from lipid droplets to the Golgi apparatus and that overexpression and knockdown of ARFGAP1 affect lipid droplet formation. Examination of human liver tissue reveals that ARFGAP1 is found associated with lipid droplets at steady state in some but not all hepatocytes. PMID:25397679

  14. The Lipid-Droplet Proteome Reveals that Droplets Are a Protein-Storage Depot

    SciTech Connect

    Cermelli, Silvia; Guo, Yi; Gross, Steven P.; Welte, Michael

    2006-09-19

    Lipid droplets are ubiquitous organelles that are among the basic building blocks of eukaryotic cells. Despite central roles for cholesterol homeostasis and lipid metabolism, their function and protein composition are poorly understood. Results: We purified lipid droplets from Drosophila embryos and analyzed the associated proteins by capillary LC-MS-MS. Important functional groups include enzymes involved in lipid metabolism, signaling molecules, and proteins related to membrane trafficking. Unexpectedly, histones H2A, H2Av, and H2B were present. Using biochemistry, genetics, real-time imaging, and cell biology, we confirm that roughly 50% of certain embryonic histones are physically attached to lipid droplets, a localization conserved in other fly species. Histone association with droplets starts during oogenesis and is prominent in early embryos, but it is undetectable in later stages or in cultured cells. Histones on droplets are not irreversibly trapped; quantitation of droplet histone levels and transplantation experiments suggest that histones are transferred from droplets to nuclei as development proceeds. When this maternal store of histones is unavailable because lipid droplets are mislocalized, zygotic histone production starts prematurely. Conclusions: Because we uncover a striking proteomic similarity of Drosophila droplets to mammalian lipid droplets, Drosophila likely provides a good model for understanding droplet function in general. Our analysis also reveals a new function for these organelles; the massive nature of histone association with droplets and its developmental time-course suggest that droplets sequester maternally provided proteins until they are needed. We propose that lipid droplets can serve as transient storage depots for proteins that lack appropriate binding partners in the cell. Such sequestration may provide a general cellular strategy for handling excess proteins.

  15. Microorganism lipid droplets and biofuel development.

    PubMed

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-12-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech 'factories' to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development.

  16. Morphologically and Functionally Distinct Lipid Droplet Subpopulations

    PubMed Central

    Zhang, Shuyan; Wang, Yang; Cui, Liujuan; Deng, Yaqin; Xu, Shimeng; Yu, Jinhai; Cichello, Simon; Serrero, Ginette; Ying, Yunshu; Liu, Pingsheng

    2016-01-01

    Lipid droplet (LD), a multi-functional organelle, is often found to associate with other cellular membranous structures and vary in size in a given cell, which may be related to their functional diversity. Here we established a method to separate LD subpopulations from isolated CHO K2 LDs into three different size categories. The subpopulation with smallest LDs was nearly free of ER and other membranous structures while those with larger LDs contained intact ER. These distinct subpopulations of LDs differed in their protein composition and ability to recruit proteins. This method was also applicable to LDs obtained from other sources, such as Huh7 cells, mouse liver and brown adipose tissue, et al. We developed an in vitro assay requiring only isolated LDs, Coenzyme A, and ATP to drive lipid synthesis. The LD subpopulation nearly depleted of ER was able to incorporate fatty acids into triacylglycerol and phospholipids. Together, our data demonstrate that LDs in a given cell are heterogeneous in size and function, and suggest that LDs are one of cellular lipid synthetic organelles. PMID:27386790

  17. Microorganism lipid droplets and biofuel development

    PubMed Central

    Liu, Yingmei; Zhang, Congyan; Shen, Xipeng; Zhang, Xuelin; Cichello, Simon; Guan, Hongbin; Liu, Pingsheng

    2013-01-01

    Lipid droplet (LD) is a cellular organelle that stores neutral lipids as a source of energy and carbon. However, recent research has emerged that the organelle is involved in lipid synthesis, transportation, and metabolism, as well as mediating cellular protein storage and degradation. With the exception of multi-cellular organisms, some unicellular microorganisms have been observed to contain LDs. The organelle has been isolated and characterized from numerous organisms. Triacylglycerol (TAG) accumulation in LDs can be in excess of 50% of the dry weight in some microorganisms, and a maximum of 87% in some instances. These microorganisms include eukaryotes such as yeast and green algae as well as prokaryotes such as bacteria. Some organisms obtain carbon from CO2 via photosynthesis, while the majority utilizes carbon from various types of biomass. Therefore, high TAG content generated by utilizing waste or cheap biomass, coupled with an efficient conversion rate, present these organisms as bio-tech ‘factories’ to produce biodiesel. This review summarizes LD research in these organisms and provides useful information for further LD biological research and microorganism biodiesel development. [BMB Reports 2013; 46(12): 575-581] PMID:24355300

  18. Isolation of Lipid Droplets from Cells by Density Gradient Centrifugation.

    PubMed

    Brasaemle, Dawn L; Wolins, Nathan E

    2016-01-01

    Lipid droplets are organelles found in most mammalian cells, as well as in various plant tissues and yeast. They are composed of a core of neutral lipids surrounded by a membrane monolayer of phospholipids and cholesterol in which specific proteins are embedded. This unit provides protocols for isolating lipid droplets from mammalian cells by discontinuous density gradient centrifugation. © 2016 by John Wiley & Sons, Inc. PMID:27580706

  19. Microalgal lipid droplets: composition, diversity, biogenesis and functions.

    PubMed

    Goold, Hugh; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2015-04-01

    Lipid droplet is the major site of neutral lipid storage in eukaryotic cells, and increasing evidence show its involvement in numerous cellular processes such as lipid homeostasis, signaling, trafficking and inter-organelle communications. Although the biogenesis, structure, and functions of lipid droplets have been well documented for seeds of vascular plants, mammalian adipose tissues, insects and yeasts, relative little is known about lipid droplets in microalgae. Over the past 5 years, the growing interest of microalgae as a platform for biofuel, green chemicals or value-added polyunsaturated fatty acid production has brought algal lipid droplets into spotlight. Studies conducted on the green microalga Chlamydomonas reinhardtii and other model microalgae such as Haematococcus and Nannochloropsis species have led to the identification of proteins associated with lipid droplets, which include putative structural proteins different from plant oleosins and animal perilipins, as well as candidate proteins for lipid biosynthesis, mobilization, trafficking and homeostasis. Biochemical and microscopy studies have also started to shed light on the role of chloroplasts in the biogenesis of lipid droplets in Chlamydomonas. PMID:25433857

  20. Spatial distribution of lipid droplets during starvation: Implications for lipophagy.

    PubMed

    Barbosa, Antonio Daniel; Siniossoglou, Symeon

    2016-01-01

    Survival during starvation depends largely on metabolic energy, which is stored in the form of neutral lipids in specialized organelles known as lipid droplets. The precursors for the synthesis of neutral lipids are also used for membrane biogenesis, which is required for cell growth and proliferation. Therefore cells must possess mechanisms to preferentially channel lipid precursors toward either membrane synthesis or lipid droplet storage, in response to nutrient status. How this partitioning is spatially regulated within the endoplasmic reticulum (ER) where lipid droplets co-localize, remains poorly understood. We have recently shown that at the onset of starvation lipid droplets concentrate at a perinuclear ER subdomain flanking the nucleus-vacuole junction (NVJ) and that this is crucial for maintaining proper nuclear shape and ER membrane organization. Here we show that disruption of the NVJ does not block the translocation and internalization of lipid droplets into the vacuole for their degradation, which takes place at later stages of starvation. We propose that alternative pathways of lipid droplet translocation from the ER to the vacuole may exist to enable stationary phase-induced lipophagy. PMID:27574533

  1. Increased lipid droplet accumulation associated with a peripheral sensory neuropathy.

    PubMed

    Marshall, Lee L; Stimpson, Scott E; Hyland, Ryan; Coorssen, Jens R; Myers, Simon J

    2014-04-01

    Hereditary sensory neuropathy type 1 (HSN-1) is an autosomal dominant neurodegenerative disease caused by missense mutations in the SPTLC1 gene. The SPTLC1 protein is part of the SPT enzyme which is a ubiquitously expressed, critical and thus highly regulated endoplasmic reticulum bound membrane enzyme that maintains sphingolipid concentrations and thus contributes to lipid metabolism, signalling, and membrane structural functions. Lipid droplets are dynamic organelles containing sphingolipids and membrane bound proteins surrounding a core of neutral lipids, and thus mediate the intracellular transport of these specific molecules. Current literature suggests that there are increased numbers of lipid droplets and alterations of lipid metabolism in a variety of other autosomal dominant neurodegenerative diseases, including Alzheimer's and Parkinson's disease. This study establishes for the first time, a significant increase in the presence of lipid droplets in HSN-1 patient-derived lymphoblasts, indicating a potential connection between lipid droplets and the pathomechanism of HSN-1. However, the expression of adipophilin (ADFP), which has been implicated in the regulation of lipid metabolism, was not altered in lipid droplets from the HSN-1 patient-derived lymphoblasts. This appears to be the first report of increased lipid body accumulation in a peripheral neuropathy, suggesting a fundamental molecular linkage between a number of neurodegenerative diseases. PMID:24711860

  2. Dropping in on the lipid droplet- tumor protein D52 (TPD52) as a new regulator and resident protein.

    PubMed

    Chen, Yuyan; Frost, Sarah; Byrne, Jennifer A

    2016-01-01

    Lipid droplets are essential for both the storage and retrieval of excess cellular nutrients, and their biology is regulated by a diverse range of cellular proteins, some of which function at the lipid droplet. Numerous studies have characterized lipid droplet proteomes in different organisms and cell types, and RNAi whole genome screening studies have examined the genetic regulation of lipid storage in C. elegans and D. melanogaster. While tumor protein D52 (TPD52) did not emerge from earlier studies as a strong candidate, exogenous expression of human TPD52 in cultured cells resulted in significantly increased numbers of lipid droplets, and oleic acid supplementation increased TPD52 detection at both lipid droplets and the Golgi apparatus. These results suggest that direct testing of proteins that are infrequently but recurrently identified in proteomic and RNAi screening studies may identify novel lipid droplet regulators. While the analysis of these possibly lower-abundance or itinerant lipid droplet proteins may be more technically challenging, such proteins could facilitate a more detailed interrogation of emerging aspects of lipid droplet biology. PMID:27617178

  3. Adipocyte size fluctuation, mechano-active lipid droplets and caveolae.

    PubMed

    Le Lay, Soazig; Briand, Nolwenn; Dugail, Isabelle

    2015-01-01

    Recent data indicate that cell size fluctuation, a key property in adipocyte pathophysiology primarily dependent on lipid storage, is linked to a novel function of lipid droplet organelles acting as mechano-active organelles to regulate cell membrane remodeling and caveolae dynamics. PMID:26167412

  4. Lipid droplet analysis using in vitro bovine oocytes and embryos.

    PubMed

    Ordoñez-Leon, E A; Merchant, H; Medrano, A; Kjelland, M; Romo, S

    2014-04-01

    The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos. PMID:24467659

  5. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores

    PubMed Central

    Bickel, Perry E.; Tansey, John T.; Welte, Michael A.

    2009-01-01

    Summary The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kiloDaltons (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  6. PAT proteins, an ancient family of lipid droplet proteins that regulate cellular lipid stores.

    PubMed

    Bickel, Perry E; Tansey, John T; Welte, Michael A

    2009-06-01

    The PAT family of lipid droplet proteins includes 5 members in mammals: perilipin, adipose differentiation-related protein (ADRP), tail-interacting protein of 47 kDa (TIP47), S3-12, and OXPAT. Members of this family are also present in evolutionarily distant organisms, including insects, slime molds and fungi. All PAT proteins share sequence similarity and the ability to bind intracellular lipid droplets, either constitutively or in response to metabolic stimuli, such as increased lipid flux into or out of lipid droplets. Positioned at the lipid droplet surface, PAT proteins manage access of other proteins (lipases) to the lipid esters within the lipid droplet core and can interact with cellular machinery important for lipid droplet biogenesis. Genetic variations in the gene for the best-characterized of the mammalian PAT proteins, perilipin, have been associated with metabolic phenotypes, including type 2 diabetes mellitus and obesity. In this review, we discuss how the PAT proteins regulate cellular lipid metabolism both in mammals and in model organisms. PMID:19375517

  7. Spastin binds to lipid droplets and affects lipid metabolism.

    PubMed

    Papadopoulos, Chrisovalantis; Orso, Genny; Mancuso, Giuseppe; Herholz, Marija; Gumeni, Sentiljana; Tadepalle, Nimesha; Jüngst, Christian; Tzschichholz, Anne; Schauss, Astrid; Höning, Stefan; Trifunovic, Aleksandra; Daga, Andrea; Rugarli, Elena I

    2015-04-01

    Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP.

  8. Lipid droplet detection by the cavity perturbation method

    NASA Astrophysics Data System (ADS)

    Blakey, R. T.; Mason, A.; Rolph, C. E.; Bond, G.; Al-Shamma'a, A. I.

    2011-08-01

    There are currently no point-of-care diagnosis strategies available to indicate the presence of neoplasmic growth. This research aims to develop a novel diagnostic strategy based on detecting TAG accumulation in cells. This element of the research is a preliminary experiment to prove the concept of detecting TAG lipid droplets in YEPD media. It was found that a change in mono-unsaturated concentration can be detected by the frequency shift in a resonant cavity. The dielectric constant of TAG vegetable oils was calculated at 2.34-2.39. It was also found that concentrations of lipid droplet can be differentiated up to 5% (v/v).

  9. Lipoprotein lipase activity is required for cardiac lipid droplet production.

    PubMed

    Trent, Chad M; Yu, Shuiqing; Hu, Yunying; Skoller, Nathan; Huggins, Lesley A; Homma, Shunichi; Goldberg, Ira J

    2014-04-01

    The rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice.

  10. Lipoprotein lipase activity is required for cardiac lipid droplet production.

    PubMed

    Trent, Chad M; Yu, Shuiqing; Hu, Yunying; Skoller, Nathan; Huggins, Lesley A; Homma, Shunichi; Goldberg, Ira J

    2014-04-01

    The rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice. PMID:24493834

  11. Lipid droplet meets a mitochondrial protein to regulate adipocyte lipolysis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In response to adrenergic stimulation, adipocytes undergo protein kinase A (PKA)-stimulated lipolysis. A key PKA target in this context is perilipin 1, a major regulator of lipolysis on lipid droplets (LDs). A study published in this issue of The EMBO Journal (Pidoux et al, 2011) identifies optic at...

  12. Multivalent niacin nanoconjugates for delivery to cytoplasmic lipid droplets.

    PubMed

    Sharma, Anjali; Khatchadourian, Armen; Khanna, Kunal; Sharma, Rishi; Kakkar, Ashok; Maysinger, Dusica

    2011-02-01

    We report here the design, synthesis, and properties, of multifunctional niacin nanoconjugates based on dendritic, miktoarm and linear backbone nanocarriers, using "click" chemistry. The conjugates were in this instance used to deliver the therapeutic agent niacin to lipid droplets. The desired combination of niacin, a lipophilic fluorescent dye (BODIPY), and polyethylene glycol (PEG), was achieved by covalently linking the desired agents to the selected carrier. The nanocarriers containing niacin and BODIPY were found almost exclusively within cytoplasmic lipid droplets in the cells used in this study (living hepatocytes and microglia), whereas the trifunctional carrier containing niacin, BODIPY and PEG was partially localized within these organelles but also elsewhere in the cytoplasmic compartment. Spectrofluorometric analyses, confocal microscopy and fluorescence cell sorting revealed different rates and extent of multifunctional conjugate(s) internalization in the two cell types. Even micromolar concentrations of the internalized multifunctional conjugates did not cause significant cell death or mitochondrial functional impairment, suggesting that they are suitable candidate nanostructures for lipid droplet imaging and for targeting drugs to these cellular organelles. These studies provide an efficient and easy way to synthesize multifunctional nanocarriers by click chemistry, applicable to the synthesis of related multifunctional nanostructures and to their use in the targeting of cellular organelles, including lipid droplets.

  13. The Causative Gene in Chanarian Dorfman Syndrome Regulates Lipid Droplet Homeostasis in C. elegans

    PubMed Central

    Xie, Meng; Roy, Richard

    2015-01-01

    AMP-activated kinase (AMPK) is a key regulator of many cellular mechanisms required for adjustment to various stresses induced by the changing environment. In C. elegans dauer larvae AMPK-null mutants expire prematurely due to hyperactive Adipose Triglyceride Lipase (ATGL-1) followed by rapid depletion of triglyceride stores. We found that the compromise of one of the three C. elegans orthologues of human cgi-58 significantly improves the survival of AMPK-deficient dauers. We also provide evidence that C. elegans CGI-58 acts as a co-activator of ATGL-1, while it also functions cooperatively to maintain regular lipid droplet structure. Surprisingly, we show that it also acts independently of ATGL-1 to restrict lipid droplet coalescence by altering the surface abundance and composition of long chain (C20) polyunsaturated fatty acids (PUFAs). Our data reveal a novel structural role of CGI-58 in maintaining lipid droplet homeostasis through its effects on droplet composition, morphology and lipid hydrolysis; a conserved function that may account for some of the ATGL-1-independent features unique to Chanarin-Dorfman Syndrome. PMID:26083785

  14. Apoptosis-induced mitochondrial dysfunction causes cytoplasmic lipid droplet formation.

    PubMed

    Boren, J; Brindle, K M

    2012-09-01

    A characteristic of apoptosis is the rapid accumulation of cytoplasmic lipid droplets, which are composed largely of neutral lipids. The proton signals from these lipids have been used for the non-invasive detection of cell death using magnetic resonance spectroscopy. We show here that despite an apoptosis-induced decrease in the levels and activities of enzymes involved in lipogenesis, which occurs downstream of p53 activation and inhibition of the mTOR signaling pathway, the increase in lipid accumulation is due to increased de novo lipid synthesis. This results from inhibition of mitochondrial fatty acid β-oxidation, which coupled with an increase in acyl-CoA synthetase activity, diverts fatty acids away from oxidation and into lipid synthesis. The inhibition of fatty acid oxidation can be explained by a rapid rise in mitochondrial membrane potential and an attendant increase in the levels of reactive oxygen species. PMID:22460322

  15. Lipid droplet mobilization: The different ways to loosen the purse strings.

    PubMed

    D'Andrea, Sabine

    2016-01-01

    Cytosolic lipid droplets are dynamic lipid-storage organelles that play a crucial role as reservoirs of metabolic energy and membrane precursors. These organelles are present in virtually all cell types, from unicellular to pluricellular organisms. Despite similar structural organization, lipid droplets are heterogeneous in morphology, distribution and composition. The protein repertoire associated to lipid droplet controls the organelle dynamics. Distinct structural lipid droplet proteins are associated to specific lipolytic pathways. The role of these structural lipid droplet-associated proteins in the control of lipid droplet degradation and lipid store mobilization is discussed. The control of the strictly-regulated lipolysis in lipid-storing tissues is compared between mammals and plants. Differences in the cellular regulation of lipolysis between lipid-storing tissues and other cell types are also discussed.

  16. Models of lipid droplets growth and fission in adipocyte cells

    SciTech Connect

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2015-08-15

    Lipid droplets (LD) are spherical cellular inclusion devoted to lipids storage. It is well known that excessive accumulation of lipids leads to several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis and atherosclerosis. LDs' size range from fraction to one hundred of micrometers in adipocytes and is related to the lipid content, but their growth is still a puzzling question. It has been suggested that LDs can grow in size due to the fusion process by which a larger LD is obtained by the merging of two smaller LDs, but these events seems to be rare and difficult to be observed. Many other processes are thought to be involved in the number and growth of LDs, like the de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets. Moreover the number and size of LDs are influenced by the catabolism and the absorption or interaction with other organelles. The comprehension of these processes could help in the confinement of the pathologies related to lipid accumulation. In this study the LDs' size distribution, number and the total volume of immature (n=12), mature (n=12, 10-days differentiated) and lipolytic (n=12) 3T3-L1 adipocytes were considered. More than 11,000 LDs were measured in the 36 cells after Oil Red O staining. In a previous work Monte Carlo simulations were used to mimic the fusion process alone between LDs. We found that, considering the fusion as the only process acting on the LDs, the size distribution in mature adipocytes can be obtained with numerical simulation starting from the size distribution in immature cells provided a very high rate of fusion events. In this paper Monte Carlo simulations were developed to mimic the interaction between LDs taking into account many other processes in addition to fusion (de novo formation and the growth through additional neutral lipid deposition in pre-existing droplets) in order to reproduce the LDs growth and we also simulated the catabolism

  17. Perilipins: Lipid Droplet Coat Proteins Adapted for Tissue-Specific Energy Storage and Utilization, and Lipid Cytoprotection

    PubMed Central

    Sztalryd, Carole; Kimmel, Alan R.

    2014-01-01

    Cytosolic lipid storage droplets are primary functional organelles that regulate cellular lipid metabolism and homeostasis. Paradoxically, excess lipid stores are linked to both adaptive (fasting and chronic exercise) and mal-adaptive (obesity and related health complications) conditions. Thus, collective metabolic and physiological processes must balance lipid storage and utilization with prevention of lipocytotoxicity and compounding tissue dysfunctions, urging the need to further define the connection of mammalian lipid droplet function and lipid homeostasis. The perilipins are a multi-protein family that targets lipid droplet surfaces and regulates lipid storage and hydrolysis. Study of perilipin functions has provided insight into the physiological roles of cytosolic lipid droplets and their relationship with obesity-related pathologies. Here, we review the current knowledge of the multiple perilipin proteins in regulating tissue-specific lipid droplets and associations with tissue and systemic energetics. PMID:24036367

  18. Lxr-driven enterocyte lipid droplet formation delays transport of ingested lipids[S

    PubMed Central

    Cruz-Garcia, Lourdes; Schlegel, Amnon

    2014-01-01

    Liver X receptors (Lxrs) are master regulators of cholesterol catabolism, driving the elimination of cholesterol from the periphery to the lumen of the intestine. Development of pharmacological agents to activate Lxrs has been hindered by synthetic Lxr agonists’ induction of hepatic lipogenesis and hypertriglyceridemia. Elucidating the function of Lxrs in regulating enterocyte lipid handling might identify novel aspects of lipid metabolism that are pharmacologically amenable. We took a genetic approach centered on the single Lxr gene nr1h3 in zebrafish to study the role of Lxr in enterocyte lipid metabolism. Loss of nr1h3 function causes anticipated gene regulatory changes and cholesterol intolerance, collectively reflecting high evolutionary conservation of zebrafish Lxra function. Intestinal nr1h3 activation delays transport of absorbed neutral lipids, with accumulation of neutral lipids in enterocyte cytoplasmic droplets. This delay in transport of ingested neutral lipids protects animals from hypercholesterolemia and hepatic steatosis induced by a high-fat diet. On a gene regulatory level, Lxra induces expression of acsl3a, which encodes acyl-CoA synthetase long-chain family member 3a, a lipid droplet-anchored protein that directs fatty acyl chains into lipids. Forced overexpression of acls3a in enterocytes delays, in part, the appearance of neutral lipids in the vasculature of zebrafish larvae. Activation of Lxr in the intestine cell-autonomously regulates the rate of delivery of absorbed lipids by inducting a temporary lipid intestinal droplet storage depot. PMID:25030662

  19. Triacylglycerol Storage in Lipid Droplets in Procyclic Trypanosoma brucei

    PubMed Central

    Allmann, Stefan; Mazet, Muriel; Ziebart, Nicole; Bouyssou, Guillaume; Fouillen, Laetitia; Dupuy, Jean-William; Bonneu, Marc; Moreau, Patrick; Bringaud, Frédéric; Boshart, Michael

    2014-01-01

    Carbon storage is likely to enable adaptation of trypanosomes to nutritional challenges or bottlenecks during their stage development and migration in the tsetse. Lipid droplets are candidates for this function. This report shows that feeding of T. brucei with oleate results in a 4–5 fold increase in the number of lipid droplets, as quantified by confocal fluorescence microscopy and by flow cytometry of BODIPY 493/503-stained cells. The triacylglycerol (TAG) content also increased 4–5 fold, and labeled oleate is incorporated into TAG. Fatty acid carbon can thus be stored as TAG in lipid droplets under physiological growth conditions in procyclic T. brucei. β-oxidation has been suggested as a possible catabolic pathway for lipids in T. brucei. A single candidate gene, TFEα1 with coding capacity for a subunit of the trifunctional enzyme complex was identified. TFEα1 is expressed in procyclic T. brucei and present in glycosomal proteomes, Unexpectedly, a TFEα1 gene knock-out mutant still expressed wild-type levels of previously reported NADP-dependent 3-hydroxyacyl-CoA dehydrogenase activity, and therefore, another gene encodes this enzymatic activity. Homozygous Δtfeα1/Δtfeα1 null mutant cells show a normal growth rate and an unchanged glycosomal proteome in procyclic T. brucei. The decay kinetics of accumulated lipid droplets upon oleate withdrawal can be fully accounted for by the dilution effect of cell division in wild-type and Δtfeα1/Δtfeα1 cells. The absence of net catabolism of stored TAG in procyclic T. brucei, even under strictly glucose-free conditions, does not formally exclude a flux through TAG, in which biosynthesis equals catabolism. Also, the possibility remains that TAG catabolism is completely repressed by other carbon sources in culture media or developmentally activated in post-procyclic stages in the tsetse. PMID:25493940

  20. N-terminus of seed caleosins is essential for lipid droplet sorting but not for lipid accumulation.

    PubMed

    Purkrtová, Zita; Chardot, Thierry; Froissard, Marine

    2015-08-01

    Caleosin, a calcium-binding protein associated with plant lipid droplets, stimulates lipid accumulation when heterologously expressed in Saccharomyces cerevisiae. Accumulated lipids are stored in cytoplasmic lipid droplets that are stabilised by incorporated caleosin. We designed a set of mutants affecting putative crucial sites for caleosin function and association with lipid droplets, i.e. the N-terminus, the EF-hand motif and the proline-knot motif. We investigated the effect of introduced mutations on caleosin capacity to initiate lipid accumulation and on caleosin sorting within cell as well as on its association with lipid droplets. Our results strongly suggest that the N-terminal domain is essential for proper protein sorting and targeting to lipid droplets but not for enhancing lipid accumulation. PMID:26032334

  1. The physics of lipid droplet nucleation, growth and budding.

    PubMed

    Thiam, Abdou Rachid; Forêt, Lionel

    2016-08-01

    Lipid droplets (LDs) are intracellular oil-in-water emulsion droplets, covered by a phospholipid monolayer and mainly present in the cytosol. Despite their important role in cellular metabolism and growing number of newly identified functions, LD formation mechanism from the endoplasmic reticulum remains poorly understood. To form a LD, the oil molecules synthesized in the ER accumulate between the monolayer leaflets and induce deformation of the membrane. This formation process works through three steps: nucleation, growth and budding, exactly as in phase separation and dewetting phenomena. These steps involve sequential biophysical membrane remodeling mechanisms for which we present basic tools of statistical physics, membrane biophysics, and soft matter science underlying them. We aim to highlight relevant factors that could control LD formation size, site and number through this physics description. An emphasis will be given to a currently underestimated contribution of the molecular interactions between lipids to favor an energetically costless mechanism of LD formation.

  2. Protein Crowding Is a Determinant of Lipid Droplet Protein Composition.

    PubMed

    Kory, Nora; Thiam, Abdou-Rachid; Farese, Robert V; Walther, Tobias C

    2015-08-10

    Lipid droplets (LDs) are lipid storage organelles that grow or shrink, depending on the availability of metabolic energy. Proteins recruited to LDs mediate many metabolic functions, including phosphatidylcholine and triglyceride synthesis. How the LD protein composition is tuned to the supply and demand for lipids remains unclear. We show that LDs, in contrast to other organelles, have limited capacity for protein binding. Consequently, macromolecular crowding plays a major role in determining LD protein composition. During lipolysis, when LDs and their surfaces shrink, some, but not all, proteins become displaced. In vitro studies show that macromolecular crowding, rather than changes in monolayer lipid composition, causes proteins to fall off the LD surface. As predicted by a crowding model, proteins compete for binding to the surfaces of LDs. Moreover, the LD binding affinity determines protein localization during lipolysis. Our findings identify protein crowding as an important principle in determining LD protein composition. PMID:26212136

  3. Dynamics of the lipid droplet proteome of the Oleaginous yeast rhodosporidium toruloides.

    PubMed

    Zhu, Zhiwei; Ding, Yunfeng; Gong, Zhiwei; Yang, Li; Zhang, Sufang; Zhang, Congyan; Lin, Xinping; Shen, Hongwei; Zou, Hanfa; Xie, Zhensheng; Yang, Fuquan; Zhao, Xudong; Liu, Pingsheng; Zhao, Zongbao K

    2015-03-01

    Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

  4. Dynamics of the Lipid Droplet Proteome of the Oleaginous Yeast Rhodosporidium toruloides

    PubMed Central

    Zhu, Zhiwei; Ding, Yunfeng; Gong, Zhiwei; Yang, Li; Zhang, Sufang; Zhang, Congyan; Lin, Xinping; Shen, Hongwei; Zou, Hanfa; Xie, Zhensheng; Yang, Fuquan; Zhao, Xudong

    2015-01-01

    Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae, Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers. PMID:25576482

  5. Epithelial thickness and lipid droplets in the hepatopancreas of Porcellio scaber (Crustacea: Isopoda) in different physiological conditions.

    PubMed

    Leser, Vladka; Drobne, Damjana; Vilhar, Barbara; Kladnik, Ales; Znidarsic, Nada; Strus, Jasna

    2008-01-01

    We investigated the morphometric characteristics of the hepatopancreatic epithelium in the terrestrial isopod Porcellio scaber during acclimatization to laboratory conditions, during the daily cycle, the molt cycle, and fasting. The hepatopancreatic epithelium was analyzed using computer-assisted microscopy of serial sections of the hepatopancreatic tubes. In addition, the abundance, the distribution, and the size of lipid droplets in the hepatopancreatic epithelium were recorded. The experimental animals were collected in the field and transferred to the laboratory. The hepatopancreatic epithelium was thinner and lipid droplets reduced after 2 months of acclimatization to laboratory conditions. The daily cycle and the molt cycle affected neither the epithelial thickness nor the abundance of lipid droplets. But in animals fasted for 2 weeks, these two parameters were significantly reduced. Based on both the epithelial thickness and the abundance of lipid droplets in B cells, we propose criteria for estimating the stress status of the animals. With the possibility to determine the stress status, many studies on isopods gain in relevance.

  6. Role of adipose specific lipid droplet proteins in maintaining whole body energy homeostasis☆

    PubMed Central

    Konige, Manige; Wang, Hong; Sztalryd, Carole

    2015-01-01

    Excess or insufficient lipid storage in white adipose tissue lipid droplets is associated with dyslipidemia, insulin resistance and increased risk for diabetes type 2. Thus, maintenance of adipose lipid droplet growth and function is critical to preserve whole body insulin sensitivity and energy homeostasis. Progress in understanding biology of lipid droplets has underscored the role of proteins that interact with lipid droplets. Here, we review the current knowledge of adipose specific lipid droplet proteins, which share unique functions controlling adipocyte lipid storage, limiting lipid spill-over and lipotoxic effects thought to contribute to disease. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease. PMID:23688782

  7. Lipid Droplet-Associated Proteins (LDAPs) Are Required for the Dynamic Regulation of Neutral Lipid Compartmentation in Plant Cells.

    PubMed

    Gidda, Satinder K; Park, Sunjung; Pyc, Michal; Yurchenko, Olga; Cai, Yingqi; Wu, Peng; Andrews, David W; Chapman, Kent D; Dyer, John M; Mullen, Robert T

    2016-04-01

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols in seeds, their biogenesis and function in nonseed tissues are poorly understood. Recently, we identified a class of plant-specific, lipid droplet-associated proteins (LDAPs) that are abundant components of LDs in nonseed cell types. Here, we characterized the three LDAPs in Arabidopsis (Arabidopsis thaliana) to gain insight to their targeting, assembly, and influence on LD function and dynamics. While all three LDAPs targeted specifically to the LD surface, truncation analysis of LDAP3 revealed that essentially the entire protein was required for LD localization. The association of LDAP3 with LDs was detergent sensitive, but the protein bound with similar affinity to synthetic liposomes of various phospholipid compositions, suggesting that other factors contributed to targeting specificity. Investigation of LD dynamics in leaves revealed that LD abundance was modulated during the diurnal cycle, and characterization of LDAP misexpression mutants indicated that all three LDAPs were important for this process. LD abundance was increased significantly during abiotic stress, and characterization of mutant lines revealed that LDAP1 and LDAP3 were required for the proper induction of LDs during heat and cold temperature stress, respectively. Furthermore, LDAP1 was required for proper neutral lipid compartmentalization and triacylglycerol degradation during postgerminative growth. Taken together, these studies reveal that LDAPs are required for the maintenance and regulation of LDs in plant cells and perform nonredundant functions in various physiological contexts, including stress response and postgerminative growth. PMID:26896396

  8. Identification of major proteins in the lipid droplet-enriched fraction isolated from the human hepatocyte cell line HuH7.

    PubMed

    Fujimoto, Yasuyuki; Itabe, Hiroyuki; Sakai, Jun; Makita, Minoru; Noda, Junich; Mori, Masahiro; Higashi, Yusuke; Kojima, Shinichi; Takano, Tatsuya

    2004-02-01

    Recent studies have revealed the presence of intracellular lipid droplets in wide variety of species. In mammalian cells, there exist proteins specifically localize in lipid droplets. However, the protein profile in the droplet remains yet to be clarified. In this study, a fraction enriched with lipid droplets was isolated from a human hepatocyte cell line HuH7 using sucrose density gradient centrifugation, and 17 major proteins in the fraction were identified using nano LC-MS/MS techniques. Adipose differentiation-related protein (ADRP) was the most abundant protein in the fraction. The secondary abundant proteins were identified to be acyl-CoA synthetase 3 (ACS3) and 17beta-hydroxysteroid dehydrogenase 11 (17betaHSD11). Included in the identified proteins were five lipid-metabolizing enzymes as well as two lipid droplet-specific proteins. When HuH7 cell lysate was fractionated by a density gradient, most of 17betaHSD11 was found in the droplet-enriched fraction. In immunocytochemical analysis, 17betaHSD11 showed ring-shaped images which overlapped with those for ADRP. These results suggest that a specific set of proteins is enriched in the lipid droplet-enriched fraction and that 17betaHSD11 localizes specifically in the fraction. PMID:14741744

  9. GRAF1a is a brain-specific protein that promotes lipid droplet clustering and growth, and is enriched at lipid droplet junctions

    PubMed Central

    Lucken-Ardjomande Häsler, Safa; Vallis, Yvonne; Jolin, Helen E.; McKenzie, Andrew N.; McMahon, Harvey T.

    2014-01-01

    ABSTRACT Lipid droplets are found in all cell types. Normally present at low levels in the brain, they accumulate in tumours and are associated with neurodegenerative diseases. However, little is known about the mechanisms controlling their homeostasis in the brain. We found that GRAF1a, the longest GRAF1 isoform (GRAF1 is also known as ARHGAP26), was enriched in the brains of neonates. Endogenous GRAF1a was found on lipid droplets in oleic-acid-fed primary glial cells. Exclusive localization required a GRAF1a-specific hydrophobic segment and two membrane-binding regions, a BAR and a PH domain. Overexpression of GRAF1a promoted lipid droplet clustering, inhibited droplet mobility and severely perturbed lipolysis following the chase of cells overloaded with fatty acids. Under these conditions, GRAF1a concentrated at the interface between lipid droplets. Although GRAF1-knockout mice did not show any gross abnormal phenotype, the total lipid droplet volume that accumulated in GRAF1−/− primary glia upon incubation with fatty acids was reduced compared to GRAF1+/+ cells. These results provide additional insights into the mechanisms contributing to lipid droplet growth in non-adipocyte cells, and suggest that proteins with membrane sculpting BAR domains play a role in droplet homeostasis. PMID:25189622

  10. Cidea controls lipid droplet fusion and lipid storage in brown and white adipose tissue.

    PubMed

    Wu, Lizhen; Zhou, Linkang; Chen, Cheng; Gong, Jingyi; Xu, Li; Ye, Jing; Li, De; Li, Peng

    2014-01-01

    Excess lipid storage in adipose tissue results in the development of obesity and other metabolic disorders including diabetes, fatty liver and cardiovascular diseases. The lipid droplet (LD) is an important subcellular organelle responsible for lipid storage. We previously observed that Fsp27, a member of the CIDE family proteins, is localized to LD-contact sites and promotes atypical LD fusion and growth. Cidea, a close homolog of Fsp27, is expressed at high levels in brown adipose tissue. However, the exact role of Cidea in promoting LD fusion and lipid storage in adipose tissue remains unknown. Here, we expressed Cidea in Fsp27-knockdown adipocytes and observed that Cidea has similar activity to Fsp27 in promoting lipid storage and LD fusion and growth. Next, we generated Cidea and Fsp27 double-deficient mice and observed that these animals had drastically reduced adipose tissue mass and a strong lean phenotype. In addition, Cidea/Fsp27 double-deficient mice had improved insulin sensitivity and were intolerant to cold. Furthermore, we observed that the brown and white adipose tissues of Cidea/Fsp27 double-deficient mice had significantly reduced lipid storage and contained smaller LDs compared to those of Cidea or Fsp27 single deficient mice. Overall, these data reveal an important role of Cidea in controlling lipid droplet fusion, lipid storage in brown and white adipose tissue, and the development of obesity.

  11. Lipid droplets form complexes with viroplasms and are crucial for rotavirus replication.

    PubMed

    Crawford, Sue E; Desselberger, Ulrich

    2016-08-01

    Recent evidence has demonstrated that a variety of pathogens target cellular lipid metabolism for their replication. Lipid droplets are a major contributor to lipid homeostasis and contain neutral fats but are also recognized as dynamic organelles involved in signal transduction, membrane trafficking and modulation of immune and inflammatory responses. Rotaviruses co-opt lipid droplets for their replication. Rotavirus viroplasms, sites of viral RNA replication and immature particle assembly, form complexes with cellular lipid droplets early in infection. Chemical compounds blocking fatty acid synthesis or interfering with lipid droplet homeostasis decrease viroplasm formation and the yield of infectious viral progeny. Lipid droplets are vital for the replication of rotaviruses as well as various members of the Flaviviridae family and several intracellular bacteria. Chemical compounds decreasing intracellular triglyceride content reduced rotavirus replication in an animal model and should be considered as potential therapeutic agents against disease caused by rotaviruses, flaviviruses and intracellular bacteria. PMID:27341619

  12. Proteome Analysis of Cytoplasmatic and Plastidic β-Carotene Lipid Droplets in Dunaliella bardawil1[OPEN

    PubMed Central

    Davidi, Lital; Levin, Yishai; Ben-Dor, Shifra; Pick, Uri

    2015-01-01

    The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets. PMID:25404729

  13. Proteome analysis of cytoplasmatic and plastidic β-carotene lipid droplets in Dunaliella bardawil.

    PubMed

    Davidi, Lital; Levin, Yishai; Ben-Dor, Shifra; Pick, Uri

    2015-01-01

    The halotolerant green alga Dunaliella bardawil is unique in that it accumulates under stress two types of lipid droplets: cytoplasmatic lipid droplets (CLD) and β-carotene-rich (βC) plastoglobuli. Recently, we isolated and analyzed the lipid and pigment compositions of these lipid droplets. Here, we describe their proteome analysis. A contamination filter and an enrichment filter were utilized to define core proteins. A proteome database of Dunaliella salina/D. bardawil was constructed to aid the identification of lipid droplet proteins. A total of 124 and 42 core proteins were identified in βC-plastoglobuli and CLD, respectively, with only eight common proteins. Dunaliella spp. CLD resemble cytoplasmic droplets from Chlamydomonas reinhardtii and contain major lipid droplet-associated protein and enzymes involved in lipid and sterol metabolism. The βC-plastoglobuli proteome resembles the C. reinhardtii eyespot and Arabidopsis (Arabidopsis thaliana) plastoglobule proteomes and contains carotene-globule-associated protein, plastid-lipid-associated protein-fibrillins, SOUL heme-binding proteins, phytyl ester synthases, β-carotene biosynthesis enzymes, and proteins involved in membrane remodeling/lipid droplet biogenesis: VESICLE-INDUCING PLASTID PROTEIN1, synaptotagmin, and the eyespot assembly proteins EYE3 and SOUL3. Based on these and previous results, we propose models for the biogenesis of βC-plastoglobuli and the biosynthesis of β-carotene within βC-plastoglobuli and hypothesize that βC-plastoglobuli evolved from eyespot lipid droplets.

  14. TIP47 functions in the biogenesis of lipid droplets.

    PubMed

    Bulankina, Anna V; Deggerich, Anke; Wenzel, Dirk; Mutenda, Kudzai; Wittmann, Julia G; Rudolph, Markus G; Burger, Koert N J; Höning, Stefan

    2009-05-18

    TIP47 (tail-interacting protein of 47 kD) was characterized as a cargo selection device for mannose 6-phosphate receptors (MPRs), directing their transport from endosomes to the trans-Golgi network. In contrast, our current analysis shows that cytosolic TIP47 is not recruited to organelles of the biosynthetic and endocytic pathways. Knockdown of TIP47 expression had no effect on MPR distribution or trafficking and did not affect lysosomal enzyme sorting. Therefore, our data argue against a function of TIP47 as a sorting device. Instead, TIP47 is recruited to lipid droplets (LDs) by an amino-terminal sequence comprising 11-mer repeats. We show that TIP47 has apolipoprotein-like properties and reorganizes liposomes into small lipid discs. Suppression of TIP47 blocked LD maturation and decreased the incorporation of triacylglycerol into LDs. We conclude that TIP47 functions in the biogenesis of LDs.

  15. Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin.

    PubMed

    Gross, Danielle N; Miyoshi, Hideaki; Hosaka, Toshio; Zhang, Hui-Hong; Pino, Elizabeth C; Souza, Sandra; Obin, Martin; Greenberg, Andrew S; Pilch, Paul F

    2006-02-01

    In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure. PMID:16239256

  16. Lipid droplet-associated proteins in atherosclerosis (Review).

    PubMed

    Plakkal Ayyappan, Janeesh; Paul, Antoni; Goo, Young-Hwa

    2016-06-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low‑density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL‑derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD‑associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell‑targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed. PMID:27082419

  17. Hydrophobic and Basic Domains Target Proteins to Lipid Droplets

    PubMed Central

    Ingelmo-Torres, Mercedes; González-Moreno, Elena; Kassan, Adam; Hanzal-Bayer, Michael; Tebar, Francesc; Herms, Albert; Grewal, Thomas; Hancock, John F.; Enrich, Carlos; Bosch, Marta; Gross, Steven P.; Parton, Robert G.; Pol, Albert

    2010-01-01

    In recent years, progress in the study of the lateral organization of the plasma membrane has led to the proposal that mammalian cells use two different organelles to store lipids: intracellular lipid droplets (LDs) and plasma membrane caveolae. Experimental evidence suggests that caveolin (CAV) may act as a sensitive lipid-organizing molecule that physically connects these two lipid-storing organelles. Here, we determine the sequences necessary for efficient sorting of CAV to LDs. We show that targeting is a process cooperatively mediated by two motifs. CAV's central hydrophobic domain (Hyd) anchors CAV to the endoplasmic reticulum (ER). Next, positively charged sequences (Pos-Seqs) mediate sorting of CAVs into LDs. Our findings were confirmed by identifying an equivalent, non-conserved but functionally interchangeable Pos-Seq in ALDI, a bona fide LD-resident protein. Using this information, we were able to retarget a cytosolic protein and convert it to an LD-resident protein. Further studies suggest three requirements for targeting via this mechanism: the positive charge of the Pos-Seq, physical proximity between Pos-Seq and Hyd and a precise spatial orientation between both motifs. The study uncovers remarkable similarities with the signals that target proteins to the membrane of mitochondria and peroxisomes PMID:19874557

  18. Lipid droplet-associated proteins in atherosclerosis (Review)

    PubMed Central

    AYYAPPAN, JANEESH PLAKKAL; PAUL, ANTONI; GOO, YOUNG-HWA

    2016-01-01

    Accumulation of atherosclerotic plaques in arterial walls leads to major cardiovascular diseases and stroke. Macrophages/foam cells are central components of atherosclerotic plaques, which populate the arterial wall in order to remove harmful modified low-density lipoprotein (LDL) particles, resulting in the accumulation of lipids, mostly LDL-derived cholesterol ester, in cytosolic lipid droplets (LDs). At present, LDs are recognized as dynamic organelles that govern cellular metabolic processes. LDs consist of an inner core of neutral lipids surrounded by a monolayer of phospholipids and free cholesterol, and contain LD-associated proteins (LDAPs) that regulate LD functions. Foam cells are characterized by an aberrant accumulation of cytosolic LDs, and are considered a hallmark of atherosclerotic lesions through all stages of development. Previous studies have investigated the mechanisms underlying foam cell formation, aiming to discover therapeutic strategies that target foam cells and intervene against atherosclerosis. It is well established that LDAPs have a major role in the pathogenesis of metabolic diseases caused by dysfunction of lipid metabolism, and several studies have linked LDAPs to the development of atherosclerosis. In this review, several foam cell-targeting pathways have been described, with an emphasis on the role of LDAPs in cholesterol mobilization from macrophages. In addition, the potential of LDAPs as therapeutic targets to prevent the progression and/or facilitate the regression of the disease has been discussed. PMID:27082419

  19. Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

    PubMed Central

    Robenek, Horst; Buers, Insa; Robenek, Mirko J.; Hofnagel, Oliver; Ruebel, Anneke; Troyer, David; Severs, Nicholas J.

    2011-01-01

    Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies. PMID:21490801

  20. Developmental changes in the protein composition of Manduca sexta lipid droplets

    PubMed Central

    Soulages, Jose L; Firdaus, Sarah J; Hartson, Steve; Chen, Xiao; Howard, Alisha D.; Arrese, Estela L

    2012-01-01

    The lipid droplets (LDs) are intracellular organelles mainly dedicated to the storage and provision of fatty acids. To accomplish these functions the LDs interact with other organelles and cytosolic proteins. In order to explore possible correlations between the physiological states of cells and the protein composition of LDs we have determined and compared the proteomic profiles of lipid droplets isolated from the fat bodies of 5th-instar larvae and adult Manduca sexta insects and from ovaries. These LD-rich tissues represent three clearly distinct metabolic states in regard to lipid metabolism: 1) Larval fat body synthesizes fatty acids (FA) and accumulates large amounts as triglyceride (TG); 2) Fat body from adult insects provides FA to support reproduction and flight; 3) Ovaries do not synthesize FA, but accumulate considerable amounts of TG in LDs. Major qualitative and semi-quantitative variations in the protein compositions of the LDs isolated from these three tissues were observed by MS/MS and partially validated by immuno-blotting. The differences observed included changes in the abundance of lipid droplet specific proteins, cytosolic proteins, mitochondrial proteins and also proteins associated with the machinery of protein synthesis. These results suggest that changes in the interaction of LDs with other organelles and cytosolic proteins are tightly related to the physiological state of cells. Herein, we summarize and compare the protein compositions of three subtypes of LDs and also describe for the first time the proteomic profile of LDs from an insect ovary. The compositions and compositional differences found among the LDs are discussed to provide a platform for future studies on the role of LDs, and their associated proteins, in cellular metabolism. PMID:22245367

  1. The fusion of lipid droplets is involved in fat loss during cooking of duck "foie gras".

    PubMed

    Théron, L; Astruc, T; Bouillier-Oudot, M; Molette, C; Vénien, A; Peyrin, F; Vitezica, Z G; Fernandez, X

    2011-12-01

    Fat loss during cooking of duck "foie gras" is the main quality issue in processing plants. To better understand this phenomenon, a histological and ultrastructural study was conducted. The aim was to characterize changes in lipid droplets of duck "foie gras" related to fat loss during cooking. Ten fatty livers were sampled before and after cooking and prepared for optical and transmission electron microscopy. In raw livers, the lipid droplets were nearly spherical while after cooking, they were larger and lost their spherical shape. We also observed a decrease in the number of droplets after cooking, probably due to droplet fusion caused by the heat treatment. Before cooking, there were fewer lipid droplets and a higher osmium tetroxyde staining intensity in the fatty liver, which later gave a lower technological yield. Fat loss during cooking was higher when there was more fusion of lipid droplets before cooking.

  2. Dynamic Regulation of Hepatic Lipid Droplet Properties by Diet

    PubMed Central

    Crunk, Amanda E.; Monks, Jenifer; Murakami, Aya; Jackman, Matthew; MacLean, Paul S.; Ladinsky, Mark; Bales, Elise S.; Cain, Shannon; Orlicky, David J.; McManaman, James L.

    2013-01-01

    Cytoplasmic lipid droplets (CLD) are organelle-like structures that function in neutral lipid storage, transport and metabolism through the actions of specific surface-associated proteins. Although diet and metabolism influence hepatic CLD levels, how they affect CLD protein composition is largely unknown. We used non-biased, shotgun, proteomics in combination with metabolic analysis, quantitative immunoblotting, electron microscopy and confocal imaging to define the effects of low- and high-fat diets on CLD properties in fasted-refed mice. We found that the hepatic CLD proteome is distinct from that of CLD from other mammalian tissues, containing enzymes from multiple metabolic pathways. The hepatic CLD proteome is also differentially affected by dietary fat content and hepatic metabolic status. High fat feeding markedly increased the CLD surface density of perilipin-2, a critical regulator of hepatic neutral lipid storage, whereas it reduced CLD levels of betaine-homocysteine S-methyltransferase, an enzyme regulator of homocysteine levels linked to fatty liver disease and hepatocellular carcinoma. Collectively our data demonstrate that the hepatic CLD proteome is enriched in metabolic enzymes, and that it is qualitatively and quantitatively regulated by diet and metabolism. These findings implicate CLD in the regulation of hepatic metabolic processes, and suggest that their properties undergo reorganization in response to hepatic metabolic demands. PMID:23874434

  3. Seipin is required for converting nascent to mature lipid droplets

    PubMed Central

    Wang, Huajin; Becuwe, Michel; Housden, Benjamin E; Chitraju, Chandramohan; Porras, Ashley J; Graham, Morven M; Liu, Xinran N; Thiam, Abdou Rachid; Savage, David B; Agarwal, Anil K; Garg, Abhimanyu; Olarte, Maria-Jesus; Lin, Qingqing; Fröhlich, Florian; Hannibal-Bach, Hans Kristian; Upadhyayula, Srigokul; Perrimon, Norbert; Kirchhausen, Tomas; Ejsing, Christer S; Walther, Tobias C; Farese, Robert V

    2016-01-01

    How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using Drosophila and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete step in LD formation—the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs. DOI: http://dx.doi.org/10.7554/eLife.16582.001 PMID:27564575

  4. Lipid droplets form from distinct regions of the cell in the fission yeast Schizosaccharomyces pombe

    DOE PAGES

    Meyers, Alex; del Rio, Zuania P.; Beaver, Rachael A.; Morris, Ryan M.; Weiskittel, Taylor M.; Alshibli, Amany K.; Mannik, Jaana; Morrell-Falvey, Jennifer; Dalhaimer, Paul

    2016-04-29

    Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation aroundmore » the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. Lastly, the results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell.« less

  5. Lipid Droplets Form from Distinct Regions of the Cell in the Fission Yeast Schizosaccharomyces pombe.

    PubMed

    Meyers, Alex; Del Rio, Zuania P; Beaver, Rachael A; Morris, Ryan M; Weiskittel, Taylor M; Alshibli, Amany K; Mannik, Jaana; Morrell-Falvey, Jennifer; Dalhaimer, Paul

    2016-06-01

    Eukaryotic cells store cholesterol/sterol esters (SEs) and triacylglycerols (TAGs) in lipid droplets, which form from the contiguous endoplasmic reticulum (ER) network. However, it is not known if droplets preferentially form from certain regions of the ER over others. Here, we used fission yeast Schizosaccharomyces pombe cells where the nuclear and cortical/peripheral ER domains are distinguishable by light microscopy to show that SE-enriched lipid droplets form away from the nucleus at the cell tips, whereas TAG-enriched lipid droplets form around the nucleus. Sterols localize to the regions of the cells where droplets enriched in SEs are observed. TAG droplet formation around the nucleus appears to be a strong function of diacylglycerol (DAG) homeostasis with Cpt1p, which coverts DAG into phosphatidylcholine and phosphatidylethanolamine localized exclusively to the nuclear ER. Also, Dgk1p, which converts DAG into phosphatidic acid localized strongly to the nuclear ER over the cortical/peripheral ER. We also show that TAG more readily translocates from the ER to lipid droplets than do SEs. The results augment the standard lipid droplet formation model, which has SEs and TAGs flowing into the same nascent lipid droplet regardless of its biogenesis point in the cell.

  6. Oleosin of subcellular lipid droplets evolved in green algae.

    PubMed

    Huang, Nan-Lan; Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-04-01

    In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants. PMID:23391579

  7. Oleosin of subcellular lipid droplets evolved in green algae.

    PubMed

    Huang, Nan-Lan; Huang, Ming-Der; Chen, Tung-Ling L; Huang, Anthony H C

    2013-04-01

    In primitive and higher plants, intracellular storage lipid droplets (LDs) of triacylglycerols are stabilized with a surface layer of phospholipids and oleosin. In chlorophytes (green algae), a protein termed major lipid-droplet protein (MLDP) rather than oleosin on LDs was recently reported. We explored whether MLDP was present directly on algal LDs and whether algae had oleosin genes and oleosins. Immunofluorescence microscopy revealed that MLDP in the chlorophyte Chlamydomonas reinhardtii was associated with endoplasmic reticulum subdomains adjacent to but not directly on LDs. In C. reinhardtii, low levels of a transcript encoding an oleosin-like protein (oleolike) in zygotes-tetrads and a transcript encoding oleosin in vegetative cells transferred to an acetate-enriched medium were found in transcriptomes and by reverse transcription-polymerase chain reaction. The C. reinhardtii LD fraction contained minimal proteins with no detectable oleolike or oleosin. Several charophytes (advanced green algae) possessed low levels of transcripts encoding oleosin but not oleolike. In the charophyte Spirogyra grevilleana, levels of oleosin transcripts increased greatly in cells undergoing conjugation for zygote formation, and the LD fraction from these cells contained minimal proteins, two of which were oleosins identified via proteomics. Because the minimal oleolike and oleosins in algae were difficult to detect, we tested their subcellular locations in Physcomitrella patens transformed with the respective algal genes tagged with a Green Fluorescent Protein gene and localized the algal proteins on P. patens LDs. Overall, oleosin genes having weak and cell/development-specific expression were present in green algae. We present a hypothesis for the evolution of oleosins from algae to plants.

  8. Multiplex coherent anti-Stokes Raman scattering microscopy on lipid droplets in HeLa cells

    NASA Astrophysics Data System (ADS)

    Rinia, Hilde A.; Burger, Koert N. J.; Bonn, Mischa; Müller, Michiel

    2007-07-01

    Lipid droplets have become a major research topic recently, as they are found to be involved in obesity related diseases. Most of this research has been focused on the localization of the proteins playing a role in lipid droplet formation or breakdown. The role of different lipid species however remains unclear because it is difficult to distinguish different fatty acids with the present microscopy techniques. Coherent Anti-Stokes Raman scattering (CARS) is the non-linear analogue of spontaneous Raman scattering. Multiplex CARS microscopy can provide quantitative, chemical and physical information, making it an excellent tool to study the composition and thermodynamic phase of lipid droplets. To investigate the potential of CARS in this field, we have incubated HeLa cells with four different fatty acids, varying in saturation. The fatty acids were internalized by the cells and stored as lipid droplets, which we imaged with multiplex CARS microscopy. We were able to distinguish either of the fatty acids as such in lipid droplets inside the cells. Furthermore, we found that solid phase fatty acids were fluidized when present in lipid droplets. This illustrates the potential of CARS microscopy to elucidate the possible role of the chemistry of fatty acids in lipid droplet regulation.

  9. Direct comparison of fatty acid ratios in single cellular lipid droplets as determined by Raman spectroscopy and gas chromatography

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cellular lipid droplets are the least studied and least understood cellular organelles in eukaryotic and prokaryotic cells. Despite a broad research trying to understand lipid droplets it has not been possible to determine the composition of individual cellular lipid droplets. In this paper we prese...

  10. Autophagy regulation depends on ER homeostasis controlled by lipid droplets.

    PubMed

    Velázquez, Ariadna P; Graef, Martin

    2016-08-01

    Macroautophagy (hereafter autophagy) is a highly conserved homeostasis and quality control process critically linked to neurodegeneration, metabolic diseases, cancer, and aging. A key feature of autophagy is the de novo formation of autophagosomes, double-membrane vesicular structures encapsulating cytoplasmic cargo for vacuolar turnover and recycling. The membrane rearrangements underlying nucleation, expansion, closure, and vacuolar fusion of autophagosomes are driven by multicomponent core autophagy machinery in cooperation with numerous factors involved in a variety of cellular processes. Our current understanding of the origin and contribution of diverse membrane sources to autophagosome biogenesis and of cellular functions enabling stress-appropriate autophagy responses critical for cell health and survival remains limited. Here, we summarize and discuss our recent findings analyzing the role of lipid droplets (LDs), conserved intracellular storage compartments for neutral lipids, for autophagy regulation. Our data indicate that LDs are dispensable as membrane sources, but fulfill critical functions for maintaining endoplasmic reticulum (ER) homeostasis, including buffering of newly synthesized fatty acids and maintenance of phospholipid composition, required for intact autophagy regulation and cell survival during nutrient stress.

  11. Biogenesis and functions of lipid droplets in plants

    PubMed Central

    Chapman, Kent D.; Dyer, John M.; Mullen, Robert T.

    2012-01-01

    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. PMID:22045929

  12. Nitrogen-Deprivation Elevates Lipid Levels in Symbiodinium spp. by Lipid Droplet Accumulation: Morphological and Compositional Analyses

    PubMed Central

    Jiang, Pei-Luen; Pasaribu, Buntora; Chen, Chii-Shiarng

    2014-01-01

    Stable cnidarian-dinoflagellate (genus Symbiodinium) endosymbioses depend on the regulation of nutrient transport between Symbiodinium populations and their hosts. It has been previously shown that the host cytosol is a nitrogen-deficient environment for the intracellular Symbiodinium and may act to limit growth rates of symbionts during the symbiotic association. This study aimed to investigate the cell proliferation, as well as ultrastructural and lipid compositional changes, in free-living Symbiodinium spp. (clade B) upon nitrogen (N)-deprivation. The cell proliferation of the N-deprived cells decreased significantly. Furthermore, staining with a fluorescent probe, boron dipyrromethane 493/503 (BODIPY 493/503), indicated that lipid contents progressively accumulated in the N-deprived cells. Lipid analyses further showed that both triacylglycerol (TAG) and cholesterol ester (CE) were drastically enriched, with polyunsaturated fatty acids (PUFA; i.e., docosahexaenoic acid, heneicosapentaenoic acid, and oleic acid) became more abundant. Ultrastructural examinations showed that the increase in concentration of these lipid species was due to the accumulation of lipid droplets (LDs), a cellular feature that have previously shown to be pivotal in the maintenance of intact endosymbioses. Integrity of these stable LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Proteomic analyses of these LDs identified proteins putatively involved in lipid metabolism, signaling, stress response and energy metabolism. These results suggest that LDs production may be an adaptive response that enables Symbiodinium to maintain sufficient cellular energy stores for survival under the N-deprived conditions in the host cytoplasm. PMID:24475285

  13. Repressive effects of oat extracts on intracellular lipid-droplet formation in adipocytes and a three-dimensional subcutaneous adipose tissue model.

    PubMed

    Kato, Shinya; Kato, Yuko; Shibata, Hiroki; Saitoh, Yasukazu; Miwa, Nobuhiko

    2015-04-01

    We assessed the repression of lipid-droplet formation in mouse mesenchymal stromal preadipocytes OP9 by specified oat extracts (Hatomugi, Coix lacryma-jobi var. ma-yuen) named "SPH" which were proteolytically and glucosyl-transferredly prepared from finely-milled oat whole-grain. Stimulation of OP9 preadipocytes with insulin-containing serum-replacement promoted differentiation to adipocytes, concurrently with an increase in the intracellular lipid droplets by 51.5%, which were repressed by SPH-bulk or SPH-water-extract at 840ppm, to 33.5% or 46.9%, respectively, but not by SPH-ethanol-extract at the same dose, showing the hydrophilic property of the anti-adipogenetic ingredients. The intracellular lipid droplets were scanty for intact preadipocytes, small-sized but abundant for the SPH-unadministered adipocytes, and large-sized but few for SPH-bulk-administered adipocytes being coexistent with many lipid-droplet-lacking viable cells, suggesting "the all-or-none rule" for lipid-droplet generation in cell-to-cell. Hydrogen-peroxide-induced cell death in human epidermal keratinocytes HaCaT was prevented by SPH-bulk at 100 or 150ppm by 5.6-8.1%, being consistent with higher viabilities of SPH-bulk-administered OP9 cells, together with repressions of both cell shrinkage and cell detachment from the culture substratum. In three-dimensional subcutaneous adipose tissue models reconstructed with HaCaT-keratinocytes and OP9-preadipocytes, lipid droplets were accumulated in dermal OP9-cell-parts, and repressed to 43.5% by SPH-bulk at 840ppm concurrently with marked diminishment of huge aggregates of lipid droplets. Thus SPH-bulk suppresses adipogenesis-associated lipid-droplet accumulation during differentiation of OP9 preadipocytes together with lowered cytotoxicity to either HaCaT keratinocytes or the preadipocytes. PMID:25686949

  14. Lipid droplets maintain lipid homeostasis during anaphase for efficient cell separation in budding yeast

    PubMed Central

    Yang, Po-Lin; Hsu, Tzu-Han; Wang, Chao-Wen; Chen, Rey-Huei

    2016-01-01

    The neutral lipids steryl ester and triacylglycerol (TAG) are stored in the membrane-bound organelle lipid droplet (LD) in essentially all eukaryotic cells. It is unclear what physiological conditions require the mobilization or storage of these lipids. Here, we study the budding yeast mutant are1Δ are2Δ dga1Δ lro1Δ, which cannot synthesize the neutral lipids and therefore lacks LDs. This quadruple mutant is delayed at cell separation upon release from mitotic arrest. The cells have abnormal septa, unstable septin assembly during cytokinesis, and prolonged exocytosis at the division site at the end of cytokinesis. Lipidomic analysis shows a marked increase of diacylglycerol (DAG) and phosphatidic acid, the precursors for TAG, in the mutant during mitotic exit. The cytokinesis and separation defects are rescued by adding phospholipid precursors or inhibiting fatty acid synthesis, which both reduce DAG levels. Our results suggest that converting excess lipids to neutral lipids for storage during mitotic exit is important for proper execution of cytokinesis and efficient cell separation. PMID:27307588

  15. Isolation of Cellular Lipid Droplets: Two Purification Techniques Starting from Yeast Cells and Human Placentas

    PubMed Central

    Dalhaimer, Paul

    2014-01-01

    Lipid droplets are dynamic organelles that can be found in most eukaryotic and certain prokaryotic cells. Structurally, the droplets consist of a core of neutral lipids surrounded by a phospholipid monolayer. One of the most useful techniques in determining the cellular roles of droplets has been proteomic identification of bound proteins, which can be isolated along with the droplets. Here, two methods are described to isolate lipid droplets and their bound proteins from two wide-ranging eukaryotes: fission yeast and human placental villous cells. Although both techniques have differences, the main method - density gradient centrifugation - is shared by both preparations. This shows the wide applicability of the presented droplet isolation techniques. In the first protocol, yeast cells are converted into spheroplasts by enzymatic digestion of their cell walls. The resulting spheroplasts are then gently lysed in a loose-fitting homogenizer. Ficoll is added to the lysate to provide a density gradient, and the mixture is centrifuged three times. After the first spin, the lipid droplets are localized to the white-colored floating layer of the centrifuge tubes along with the endoplasmic reticulum (ER), the plasma membrane, and vacuoles. Two subsequent spins are used to remove these other three organelles. The result is a layer that has only droplets and bound proteins. In the second protocol, placental villous cells are isolated from human term placentas by enzymatic digestion with trypsin and DNase I. The cells are homogenized in a loose-fitting homogenizer. Low-speed and medium-speed centrifugation steps are used to remove unbroken cells, cellular debris, nuclei, and mitochondria. Sucrose is added to the homogenate to provide a density gradient and the mixture is centrifuged to separate the lipid droplets from the other cellular fractions. The purity of the lipid droplets in both protocols is confirmed by Western Blot analysis. The droplet fractions from both preps

  16. Identification of a new class of lipid droplet-associated proteins in plants.

    PubMed

    Horn, Patrick J; James, Christopher N; Gidda, Satinder K; Kilaru, Aruna; Dyer, John M; Mullen, Robert T; Ohlrogge, John B; Chapman, Kent D

    2013-08-01

    Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652

  17. Identification of a new class of lipid droplet-associated proteins in plants.

    PubMed

    Horn, Patrick J; James, Christopher N; Gidda, Satinder K; Kilaru, Aruna; Dyer, John M; Mullen, Robert T; Ohlrogge, John B; Chapman, Kent D

    2013-08-01

    Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues.

  18. Conserved Amphipathic Helices Mediate Lipid Droplet Targeting of Perilipins 1–3*

    PubMed Central

    Rowe, Emily R.; Mimmack, Michael L.; Barbosa, Antonio D.; Haider, Afreen; Isaac, Iona; Ouberai, Myriam M.; Thiam, Abdou Rachid; Patel, Satish; Saudek, Vladimir; Siniossoglou, Symeon; Savage, David B.

    2016-01-01

    Perilipins (PLINs) play a key role in energy storage by orchestrating the activity of lipases on the surface of lipid droplets. Failure of this activity results in severe metabolic disease in humans. Unlike all other lipid droplet-associated proteins, PLINs localize almost exclusively to the phospholipid monolayer surrounding the droplet. To understand how they sense and associate with the unique topology of the droplet surface, we studied the localization of human PLINs in Saccharomyces cerevisiae, demonstrating that the targeting mechanism is highly conserved and that 11-mer repeat regions are sufficient for droplet targeting. Mutations designed to disrupt folding of this region into amphipathic helices (AHs) significantly decreased lipid droplet targeting in vivo and in vitro. Finally, we demonstrated a substantial increase in the helicity of this region in the presence of detergent micelles, which was prevented by an AH-disrupting missense mutation. We conclude that highly conserved 11-mer repeat regions of PLINs target lipid droplets by folding into AHs on the droplet surface, thus enabling PLINs to regulate the interface between the hydrophobic lipid core and its surrounding hydrophilic environment. PMID:26742848

  19. The FATP1–DGAT2 complex facilitates lipid droplet expansion at the ER–lipid droplet interface

    PubMed Central

    Xu, Ningyi; Zhang, Shaobing O.; Cole, Ronald A.; McKinney, Sean A.; Guo, Fengli; Haas, Joel T.; Bobba, Sudheer; Farese, Robert V.

    2012-01-01

    At the subcellular level, fat storage is confined to the evolutionarily conserved compartments termed lipid droplets (LDs), which are closely associated with the endoplasmic reticulum (ER). However, the molecular mechanisms that enable ER–LD interaction and facilitate neutral lipid loading into LDs are poorly understood. In this paper, we present evidence that FATP1/acyl-CoA synthetase and DGAT2/diacylglycerol acyltransferase are components of a triglyceride synthesis complex that facilitates LD expansion. A loss of FATP1 or DGAT2 function blocked LD expansion in Caenorhabditis elegans. FATP1 preferentially associated with DGAT2, and they acted synergistically to promote LD expansion in mammalian cells. Live imaging indicated that FATP1 and DGAT2 are ER and LD resident proteins, respectively, and electron microscopy revealed FATP1 and DGAT2 foci close to the LD surface. Furthermore, DGAT2 that was retained in the ER failed to support LD expansion. We propose that the evolutionarily conserved FATP1–DGAT2 complex acts at the ER–LD interface and couples the synthesis and deposition of triglycerides into LDs both physically and functionally. PMID:22927462

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

    PubMed

    Chapman, Kent D; Dyer, John M; Mullen, Robert T

    2012-02-01

    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. PMID:22045929

  1. Adsorption of protein-coated lipid droplets to mixed biopolymer hydrogel surfaces: role of biopolymer diffusion.

    PubMed

    Vargas, Maria; Weiss, Jochen; McClements, D Julian

    2007-12-18

    The adsorption of charged particles to hydrogel surfaces is important in a number of natural and industrial processes. In this study, the adsorption of cationic lipid droplets to the surfaces of anionic hydrogels was examined. An oil-in-water emulsion containing cationic beta-lactoglobulin-coated lipid droplets was prepared (d32=0.24 microm, zeta=+74 mV, pH 3.0). An anionic hydrogel containing 0.1 wt % beet pectin and 1.5 wt % agar (pH 3.0) was prepared. Emulsions containing different lipid droplet concentrations (0.3-5 wt %) were brought into contact with the hydrogel surfaces for different times (0-24 h). The adsorption of lipid droplets to the hydrogel surfaces could not be explained by a typical adsorption isotherm. We found that the electrical charge on the nonadsorbed lipid droplets became less positive or even became negative in the presence of the hydrogel and that extensive droplet aggregation occurred, which was attributed to the ability of pectin molecules to diffuse through the hydrogels and interact with the lipid droplets. These results may have important consequences for understanding certain industrial and biological processes, as well as for the design of controlled or triggered release systems.

  2. The phosphorylation of serine 492 of perilipin a directs lipid droplet fragmentation and dispersion.

    PubMed

    Marcinkiewicz, Amy; Gauthier, Denise; Garcia, Anne; Brasaemle, Dawn L

    2006-04-28

    Perilipin A is a key regulator of triacylglycerol storage and hydrolysis in adipocytes; phosphorylation of perilipin A by protein kinase A facilitates maximal lipolysis. Chronic stimulation of lipolysis in 3T3-L1 adipocytes causes large perinuclear lipid droplets to fragment into myriad dispersed perilipin A-covered microlipid droplets. In cultured fibroblasts stably expressing ectopic perilipin A, clustered lipid droplets disperse throughout the cytoplasm upon incubation of the cells with forskolin and isobutylmethylxanthine (IBMX) to elevate levels of cAMP and activate protein kinase A, mirroring events observed in adipocytes. Furthermore, diethylum-belliferyl phosphate inhibits stimulated lipolysis but not the dispersion of lipid droplets, suggesting that products of lipolysis are not required for this remodeling process. We hypothesized that protein kinase A-mediated phosphorylation of perilipin A triggers the remodeling of lipid droplets. The mutation of serine 492 of perilipin A to alanine prevented the dispersion of clustered lipid droplets in fibroblasts stably expressing the mutated perilipin upon incubation with forskolin and IBMX. In contrast, the substitution of serines 81, 222, 276, or 433 with alanine, either singly or in combinations, did not affect the protein kinase A-mediated remodeling of lipid droplets. Interestingly, substitution of serines 433, 492, and 517 of perilipin A with glutamic acid residues blocked the dispersion of clustered lipid droplets in cells incubated with forskolin and IBMX, indicating that the addition of a negative charge does not mimic a phosphate group. We conclude that protein kinase A-mediated phosphorylation of serine 492 of perilipin A drives the fragmentation and dispersion of lipid droplets. PMID:16488886

  3. Low abundances of synthetics lipids in phantoms

    NASA Astrophysics Data System (ADS)

    Villanueva-Luna, A. E.; Santiago-Alvarado, A.; Castro-Ramos, J.; Vazquez-Montiel, S.; Flores-Gil, A.; Aguilar-Soto, J.; Delgado-Atencio, J. A.

    2012-03-01

    Phantoms simulate optical characteristics of tissues. Phantoms use to mimic light distributions in living tissue. Several Phantoms compositions made of silicone, polyester, polyurethane, and epoxy resin have been described in the literature. These kinds of phantoms have the problem of long time preservation. In this work, we describe the fabrication and characterization of phantoms with low concentrations of synthetic lipid using Raman spectroscopy. We fabricate four phantoms made of Polydimethylsiloxane (PDMS). These phantoms have synthetic lipid content of cholesterol and triglycerides. The size of our phantoms is 1 x 1 cm and 5 mm of thickness.We used the point-to-point mapping technique. Finally, we compared advantages and performance of made PDMS and gelatin phantoms.

  4. Regulation of lipid droplet size in mammary epithelial cells by remodeling of membrane lipid composition-a potential mechanism.

    PubMed

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors-intracellular lipid droplets-and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  5. Activation of the lipid droplet controls the rate of lipolysis of triglycerides in the insect fat body.

    PubMed

    Patel, Rajesh T; Soulages, Jose L; Hariharasundaram, Balaji; Arrese, Estela L

    2005-06-17

    The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.

  6. The polyomavirus BK agnoprotein co-localizes with lipid droplets

    SciTech Connect

    Unterstab, Gunhild; Gosert, Rainer; Leuenberger, David; Lorentz, Pascal; Rinaldo, Christine H.; Hirsch, Hans H.

    2010-04-10

    Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function.

  7. The polyomavirus BK agnoprotein co-localizes with lipid droplets.

    PubMed

    Unterstab, Gunhild; Gosert, Rainer; Leuenberger, David; Lorentz, Pascal; Rinaldo, Christine H; Hirsch, Hans H

    2010-04-10

    Agnoprotein encoded by human polyomavirus BK (BKV) is a late cytoplasmic protein of 66 amino acids (aa) of unknown function. Immunofluorescence microscopy revealed a fine granular and a vesicular distribution in donut-like structures. Using BKV(Dunlop)-infected or agnoprotein-transfected cells, we investigated agnoprotein co-localization with subcellular structures. We found that agnoprotein co-localizes with lipid droplets (LD) in primary human renal tubular epithelial cells as well as in other cells supporting BKV replication in vitro (UTA, Vero cells). Using agnoprotein-enhanced green fluorescent protein (EGFP) fusion constructs, we demonstrate that agnoprotein aa 20-42 are required for targeting LD, whereas aa 1-20 or aa 42-66 were not. Agnoprotein aa 22-40 are predicted to form an amphipathic helix, and mutations A25D and F39E, disrupting its hydrophobic domain, prevented LD targeting. However, changing the phosphorylation site serine-11 to alanine or aspartic acid did not alter LD co-localization. Our findings provide new clues to unravel agnoprotein function. PMID:20138326

  8. A novel role for lipid droplets in the organismal antibacterial response

    PubMed Central

    Anand, Preetha; Cermelli, Silvia; Li, Zhihuan; Kassan, Adam; Bosch, Marta; Sigua, Robilyn; Huang, Lan; Ouellette, Andre J; Pol, Albert; Welte, Michael A; Gross, Steven P

    2012-01-01

    We previously discovered histones bound to cytosolic lipid droplets (LDs); here we show that this forms a cellular antibacterial defense system. Sequestered on droplets under normal conditions, in the presence of bacterial lipopolysaccharide (LPS) or lipoteichoic acid (LTA), histones are released from the droplets and kill bacteria efficiently in vitro. Droplet-bound histones also function in vivo: when injected into Drosophila embryos lacking droplet-bound histones, bacteria grow rapidly. In contrast, bacteria injected into embryos with droplet-bound histones die. Embryos with droplet-bound histones displayed more than a fourfold survival advantage when challenged with four different bacterial species. Our data suggests that this intracellular antibacterial defense system may function in adult flies, and also potentially in mice. DOI: http://dx.doi.org/10.7554/eLife.00003.001 PMID:23150794

  9. Lipid droplet-associated proteins (LDAPs) are required for the dynamic regulation of neutral lipid compartmentation in plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Eukaryotic cells compartmentalize neutral lipids into organelles called lipid droplets (LDs), and while much is known about the role of LDs in storing triacylglycerols (TAGs) in seeds, their biogenesis and function in non-seed tissues is poorly understood. Recently, we identified a class of plant-sp...

  10. Pharmacological inhibition of lipid droplet formation enhances the effectiveness of curcumin in glioblastoma.

    PubMed

    Zhang, Issan; Cui, Yiming; Amiri, Abdolali; Ding, Yidan; Campbell, Robert E; Maysinger, Dusica

    2016-03-01

    Increased lipid droplet number and fatty acid synthesis allow glioblastoma multiforme, the most common and aggressive type of brain cancer, to withstand accelerated metabolic rates and resist therapeutic treatments. Lipid droplets are postulated to sequester hydrophobic therapeutic agents, thereby reducing drug effectiveness. We hypothesized that the inhibition of lipid droplet accumulation in glioblastoma cells using pyrrolidine-2, a cytoplasmic phospholipase A2 alpha inhibitor, can sensitize cancer cells to the killing effect of curcumin, a promising anticancer agent isolated from the turmeric spice. We observed that curcumin localized in the lipid droplets of human U251N glioblastoma cells. Reduction of lipid droplet number using pyrrolidine-2 drastically enhanced the therapeutic effect of curcumin in both 2D and 3D glioblastoma cell models. The mode of cell death involved was found to be mediated by caspase-3. Comparatively, the current clinical chemotherapeutic standard, temozolomide, was significantly less effective in inducing glioblastoma cell death. Together, our results suggest that the inhibition of lipid droplet accumulation is an effective way to enhance the chemotherapeutic effect of curcumin against glioblastoma multiforme.

  11. Lipid droplets may lay a spacial foundation for vasculogenic mimicry formation in hepatocellular carcinoma.

    PubMed

    Li, Yue; Cai, Weiwei; Yi, Qingqing; Xie, Fengshan; Liu, Yanling; Du, Bin; Feng, Lei; Qiu, Liying

    2014-07-01

    Vasculogenic mimicry is a highly patterned vascular channel distinguished from the endothelium-dependent blood vessel. Vasculogenic mimicry is lined by highly aggressive tumor cells, and is associated with tumor grade, invasion and metastasis, and poor clinical prognosis. Much attention has been focused on the signaling pathways and the tumor microenvironment needed for vasculogenic mimicry formation, however, the studies on the spacial foundation for vasculogenic mimicry formation are limited. There are many lipid droplets in hepatocellular carcinoma due to steatosis, while increased numbers of lipid droplets also have been reported in many other neoplastic processes. The role of lipid droplets in tumor is still unclear. Based on the similar structural and morphological characteristics between vasculogenic mimicry and lipid droplet, we speculate that the lipid droplets may lay a spacial foundation for vasculogenic mimicry formation by a way of "space placeholder" in HCC. Experimental data and limited clinical literatures support the hypothesis to a certain degree. This hypothesis may provide a new idea for the study of vasculogenic mimicry and also provide a new direction for the functional study of lipid droplets in tumor.

  12. The Perilipins: Major Cytosolic Lipid Droplet-Associated Proteins and Their Roles in Cellular Lipid Storage, Mobilization, and Systemic Homeostasis.

    PubMed

    Kimmel, Alan R; Sztalryd, Carole

    2016-07-17

    The discovery by Dr. Constantine Londos of perilipin 1, the major scaffold protein at the surface of cytosolic lipid droplets in adipocytes, marked a fundamental conceptual change in the understanding of lipolytic regulation. Focus then shifted from the enzymatic activation of lipases to substrate accessibility, mediated by perilipin-dependent protein sequestration and recruitment. Consequently, the lipid droplet became recognized as a unique, metabolically active cellular organelle and its surface as the active site for novel protein-protein interactions. A new area of investigation emerged, centered on lipid droplets' biology and their role in energy homeostasis. The perilipin family is of ancient origin and has expanded to include five mammalian genes and a growing list of evolutionarily conserved members. Universally, the perilipins modulate cellular lipid storage. This review provides a summary that connects the perilipins to both cellular and whole-body homeostasis. PMID:27431369

  13. Identification of lipid droplet structure-like/resident proteins in Caenorhabditis elegans.

    PubMed

    Na, Huimin; Zhang, Peng; Chen, Yong; Zhu, Xiaotong; Liu, Yi; Liu, Yangli; Xie, Kang; Xu, Ningyi; Yang, Fuquan; Yu, Yong; Cichello, Simon; Mak, Ho Yi; Wang, Meng C; Zhang, Hong; Liu, Pingsheng

    2015-10-01

    The lipid droplet (LD) is a cellular organelle that stores neutral lipids in cells and has been linked with metabolic disorders. Caenorhabditis elegans has many characteristics which make it an excellent animal model for studying LDs. However, unlike in mammalian cells, no LD structure-like/resident proteins have been identified in C. elegans, which has limited the utility of this model for the study of lipid storage and metabolism. Herein based on three lines of evidence, we identified that MDT-28 and DHS-3 previously identified in C. elegans LD proteome were two LD structure-like/resident proteins. First, MDT-28 and DHS-3 were found to be the two most abundant LD proteins in the worm. Second, the proteins were specifically localized to LDs and we identified the domains responsible for this targeting in both proteins. Third and most importantly, the depletion of MDT-28 induced LD clustering while DHS-3 deletion reduced triacylglycerol content (TAG). We further characterized the proteins finding that MDT-28 was ubiquitously expressed in the intestine, muscle, hypodermis, and embryos, whereas DHS-3 was expressed mainly in intestinal cells. Together, these two LD structure-like/resident proteins provide a basis for future mechanistic studies into the dynamics and functions of LDs in C. elegans.

  14. Regulation of Lipid Droplet Size in Mammary Epithelial Cells by Remodeling of Membrane Lipid Composition—A Potential Mechanism

    PubMed Central

    Cohen, Bat-Chen; Shamay, Avi; Argov-Argaman, Nurit

    2015-01-01

    Milk fat globule size is determined by the size of its precursors—intracellular lipid droplets—and is tightly associated with its composition. We examined the relationship between phospholipid composition of mammary epithelial cells and the size of both intracellular and secreted milk fat globules. Primary culture of mammary epithelial cells was cultured in medium without free fatty acids (control) or with 0.1 mM free capric, palmitic or oleic acid for 24 h. The amount and composition of the cellular lipids and the size of the lipid droplets were determined in the cells and medium. Mitochondrial quantity and expression levels of genes associated with mitochondrial biogenesis and polar lipid composition were determined. Cells cultured with oleic and palmitic acids contained similar quantities of triglycerides, 3.1- and 3.8-fold higher than in controls, respectively (P < 0.0001). When cultured with oleic acid, 22% of the cells contained large lipid droplets (>3 μm) and phosphatidylethanolamine concentration was higher by 23 and 63% compared with that in the control and palmitic acid treatments, respectively (P < 0.0001). In the presence of palmitic acid, only 4% of the cells contained large lipid droplets and the membrane phosphatidylcholine concentration was 22% and 16% higher than that in the control and oleic acid treatments, respectively (P < 0.0001). In the oleic acid treatment, approximately 40% of the lipid droplets were larger than 5 μm whereas in that of the palmitic acid treatment, only 16% of the droplets were in this size range. Triglyceride secretion in the oleic acid treatment was 2- and 12-fold higher compared with that in the palmitic acid and control treatments, respectively. Results imply that membrane composition of bovine mammary epithelial cells plays a role in controlling intracellular and secreted lipid droplets size, and that this process is not associated with cellular triglyceride content. PMID:25756421

  15. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes.

    PubMed

    Love, Joseph D; Suzuki, Takashi; Robinson, Delia B; Harris, Carla M; Johnson, Joyce E; Mohler, Peter J; Jerome, W Gray; Swift, Larry L

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology.

  16. Microsomal Triglyceride Transfer Protein (MTP) Associates with Cytosolic Lipid Droplets in 3T3-L1 Adipocytes

    PubMed Central

    Robinson, Delia B.; Harris, Carla M.; Johnson, Joyce E.; Mohler, Peter J.; Jerome, W. Gray; Swift, Larry L.

    2015-01-01

    Lipid droplets are intracellular energy storage organelles composed of a hydrophobic core of neutral lipid, surrounded by a monolayer of phospholipid and a diverse array of proteins. The function of the vast majority of these proteins with regard to the formation and/or turnover of lipid droplets is unknown. Our laboratory was the first to report that microsomal triglyceride transfer protein (MTP), a lipid transfer protein essential for the assembly of triglyceride-rich lipoproteins, was expressed in adipose tissue of humans and mice. In addition, our studies suggested that MTP was associated with lipid droplets in both brown and white fat. Our observations led us to hypothesize that MTP plays a key role in lipid droplet formation and/or turnover. The objective of these studies was to gain insight into the function of MTP in adipocytes. Using molecular, biochemical, and morphologic approaches we have shown: 1) MTP protein levels increase nearly five-fold as 3T3-L1 cells differentiate into adipocytes. 2) As 3T3-L1 cells undergo differentiation, MTP moves from the juxtanuclear region of the cell to the surface of lipid droplets. MTP and perilipin 2, a major lipid droplet surface protein, are found on the same droplets; however, MTP does not co-localize with perilipin 2. 3) Inhibition of MTP activity has no effect on the movement of triglyceride out of the cell either as a lipid complex or via lipolysis. 4) MTP is found associated with lipid droplets within hepatocytes from human fatty livers, suggesting that association of MTP with lipid droplets is not restricted to adipocytes. In summary, our data demonstrate that MTP is a lipid droplet-associated protein. Its location on the surface of the droplet in adipocytes and hepatocytes, coupled with its known function as a lipid transfer protein and its increased expression during adipocyte differentiation suggest a role in lipid droplet biology. PMID:26267806

  17. Antioxidant Role for Lipid Droplets in a Stem Cell Niche of Drosophila

    PubMed Central

    Bailey, Andrew P.; Koster, Grielof; Guillermier, Christelle; Hirst, Elizabeth M.A.; MacRae, James I.; Lechene, Claude P.; Postle, Anthony D.; Gould, Alex P.

    2015-01-01

    Summary Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. We now find that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. This study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts. PMID:26451484

  18. Lipid droplet-associated proteins in alcoholic liver disease: a potential linkage with hepatocellular damage

    PubMed Central

    Ikura, Yoshihiro; Caldwell, Stephen H

    2015-01-01

    Steatosis is a characteristic morphological change of alcoholic liver disease, but its pathologic significance is still obscure. Regardless of cell types, intracellular lipid droplets are coated with a phospholipid monolayer, on which many kinds of lipid droplet-associated proteins are present. These proteins, such as the perilipin family of proteins and the cell death inducing DNA fragmentation factor (DFF) 45-like effectors, are recognized to play important roles in lipid metabolism in the physiological settings. In addition, recent lipidology studies have revealed that expression of the lipid droplet-associated proteins possibly participate in the pathologic processes of many metabolic disorders, including fatty liver and insulin resistance. Hence, controlling protein expressions is expected to offer novel therapeutic options. In this review, we summarize collected data concerning the potential contribution of the lipid droplet-associated proteins to the development of alcoholic fatty liver. Without exception, existing data indicates that the lipid droplet-associated proteins, especially the perilipin family proteins, are important factors in alcoholic fatty liver. These proteins exert a prosteatotic effect, and their expression is closely associated with lipotoxicity based on endoplasmic reticulum stress and oxidative injury. Although suppression of their expression may be beneficial, careful consideration is required because these proteins simultaneously function as protective factors against lipotoxicity. PMID:26464614

  19. A test of current models for the mechanism of milk-lipid droplet secretion

    PubMed Central

    Jeong, Jaekwang; Lisinski, Ivonne; Kadegowda, Anil K.G.; Shin, Hyunsu; Wooding, F.B. Peter; Daniels, Brian R.; Schaack, Jerome; Mather, Ian H.

    2013-01-01

    Milk lipid is secreted by a unique process, during which triacylglycerol droplets bud from mammary cells coated with an outer bilayer of apical membrane. In all current schemes, the integral protein butyrophilin 1A1 (BTN) is postulated to serve as a transmembrane scaffold, which interacts, either with itself, or with the peripheral proteins, xanthine oxidoreductase (XOR) and possibly perilipin-2 (PLIN2), to form an immobile bridging complex between the droplet and apical surface. In one such scheme, BTN on the surface of cytoplasmic lipid droplets interacts directly with BTN in the apical membrane without binding to either XOR or PLIN2. We tested these models using both biochemical and morphological approaches. BTN was concentrated in the apical membrane in all species examined and contained mature N-linked glycans. We found no evidence for the association of unprocessed BTN with intracellular lipid droplets. BTN-enhanced-green-fluorescent-protein was highly mobile in areas of mouse milk-lipid droplets that had not undergone post-secretion changes, and endogenous mouse BTN comprised only 0.5–0.7%, (w/w) of the total protein, i.e., over fifty-fold less than in the milk-lipid droplets of cow and other species. These data are incompatible with models of milk-lipid secretion in which BTN is the major component of an immobile global adhesive complex and suggest that interactions between BTN and other proteins at the time of secretion are more transient than previously predicted. The high mobility of BTN in lipid droplets, mark it as a potential mobile signaling molecule in milk. PMID:23738536

  20. Direct comparison of fatty acid ratios in single cellular lipid droplets as determined by comparative Raman spectroscopy and gas chromatography.

    PubMed

    Schie, Iwan W; Nolte, Lena; Pedersen, Theresa L; Smith, Zach; Wu, Jian; Yahiatène, Idir; Newman, John W; Huser, Thomas

    2013-11-01

    Cellular lipid droplets are the least studied and least understood cellular organelles in eukaryotic and prokaryotic cells. Despite a significant body of research studying the physiology of lipid droplets it has not yet been possible to fully determine the composition of individual cellular lipid droplets. In this paper we use Raman spectroscopy on single cellular lipid droplets and least-squares fitting of pure fatty acid spectra to determine the composition of individual lipid droplets in cells after treatment with different ratios of oleic and palmitic acid. We validate the results of the Raman spectroscopy-based single lipid droplet analysis with results obtained by gas chromatography analysis of millions of cells, and find that our approach can accurately predict the relative amount of a specific fatty acid in the lipid droplet. Based on these results we show that the fatty acid composition in individual lipid droplets is on average similar to that of all lipid droplets found in the sample. Furthermore, we expand this approach to the investigation of the lipid composition in single cellular peroxisomes. We determine the location of cellular peroxisomes based on two-photon excitation fluorescence (TPEF) imaging of peroxisomes labeled with the green fluorescent protein, and successive Raman spectroscopy of peroxisomes. We find that in some cases peroxisomes can produce a detectable CARS signal, and that the peroxisomal Raman spectra exhibit an oleic acid-like signature.

  1. COPI buds 60-nm lipid droplets from reconstituted water-phospholipid-triacylglyceride interfaces, suggesting a tension clamp function.

    PubMed

    Thiam, Abdou Rachid; Antonny, Bruno; Wang, Jing; Delacotte, Jérôme; Wilfling, Florian; Walther, Tobias C; Beck, Rainer; Rothman, James E; Pincet, Frédéric

    2013-08-13

    Intracellular trafficking between organelles is achieved by coat protein complexes, coat protomers, that bud vesicles from bilayer membranes. Lipid droplets are protected by a monolayer and thus seem unsuitable targets for coatomers. Unexpectedly, coat protein complex I (COPI) is required for lipid droplet targeting of some proteins, suggesting a possible direct interaction between COPI and lipid droplets. Here, we find that COPI coat components can bud 60-nm triacylglycerol nanodroplets from artificial lipid droplet (LD) interfaces. This budding decreases phospholipid packing of the monolayer decorating the mother LD. As a result, hydrophobic triacylglycerol molecules become more exposed to the aqueous environment, increasing LD surface tension. In vivo, this surface tension increase may prime lipid droplets for reactions with neighboring proteins or membranes. It provides a mechanism fundamentally different from transport vesicle formation by COPI, likely responsible for the diverse lipid droplet phenotypes associated with depletion of COPI subunits.

  2. Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro.

    PubMed

    Cao, Yi; Jantzen, Kim; Gouveia, Ana Cecilia Damiao; Skovmand, Astrid; Roursgaard, Martin; Loft, Steffen; Møller, Peter

    2015-07-01

    Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3h, but not 24h. Intracellular level of reduced glutathione was increased after 24h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction.

  3. Automobile diesel exhaust particles induce lipid droplet formation in macrophages in vitro.

    PubMed

    Cao, Yi; Jantzen, Kim; Gouveia, Ana Cecilia Damiao; Skovmand, Astrid; Roursgaard, Martin; Loft, Steffen; Møller, Peter

    2015-07-01

    Exposure to diesel exhaust particles (DEP) has been associated with adverse cardiopulmonary health effects, which may be related to dysregulation of lipid metabolism and formation of macrophage foam cells. In this study, THP-1 derived macrophages were exposed to an automobile generated DEP (A-DEP) for 24h to study lipid droplet formation and possible mechanisms. The results show that A-DEP did not induce cytotoxicity. The production of reactive oxygen species was only significantly increased after exposure for 3h, but not 24h. Intracellular level of reduced glutathione was increased after 24h exposure. These results combined indicate an adaptive response to oxidative stress. Exposure to A-DEP was associated with significantly increased formation of lipid droplets, as well as changes in lysosomal function, assessed as reduced LysoTracker staining. In conclusion, these results indicated that exposure to A-DEP may induce formation of lipid droplets in macrophages in vitro possibly via lysosomal dysfunction. PMID:26122084

  4. Influence of apolipoprotein A-V on hepatocyte lipid droplet formation.

    PubMed

    Gao, Xuan; Forte, Trudy M; Ryan, Robert O

    2012-10-19

    Apolipoprotein A-V (apoA-V) is postulated to modulate intra-hepatic triglyceride (TG) trafficking. Stably transfected McA-RH7777 hepatocarcinoma cells expressing human apoA-V displayed enhanced neutral lipid staining while conditioned media from these cells had 40±8% less TG than cells transfected with a control vector. To obtain homogeneous cell lines expressing different amounts of apoA-V, a strategy of clonal selection was pursued. Immunoblot analysis of two distinct apoA-V stable cell lines yielded one that expresses low amounts of apoA-V and another that expresses higher amounts. Confocal fluorescence microscopy of control cells and cells expressing low levels of apoA-V had similar numbers of lipid droplets while cells expressing higher amounts of apoA-V had twice as many lipid droplets, on average. Thus, apoA-V expression promotes lipid droplet accumulation in these cells.

  5. A Lipid Droplet Protein of Nannochloropsis with Functions Partially Analogous to Plant Oleosins1[W][OA

    PubMed Central

    Vieler, Astrid; Brubaker, Shane B.; Vick, Bertrand; Benning, Christoph

    2012-01-01

    As our understanding of the dynamics of lipid droplets (LDs) in animal, plant, and fungal cells is rapidly evolving, still little is known about the formation and turnover of these organelles in microalgae. Yet with the growing importance of algal feedstock for the production of biofuels and high-value lipids, there is a need to understand the mechanisms of LD dynamics in microalgae. Thus, we investigated the proteins associated with LDs of the emerging heterokont model alga Nannochloropsis sp. and discovered an abundant hydrophobic lipid droplet surface protein (LDSP) with unique primary sequence but structural similarities to other LD proteins. LDSP abundance in Nannochloropsis cells closely tracked the amount of triacylglycerols during conditions of oil accumulation and degradation. Functional characterization of LDSP in an Arabidopsis (Arabidopsis thaliana) OLEOSIN1-deficient mutant allowed a separation of its physical and structural properties in its interaction with LDs from its physiological or biochemical activities. Although LDSP presence in Arabidopsis predictably affected LD size, it could not reverse the physiological impact of OLEOSIN deficiency on triacylglycerol hydrolysis during germination. PMID:22307965

  6. Acute effects of ACTH on dissociated adrenocortical cells: quantitative changes in mitochondria and lipid droplets.

    PubMed

    Zoller, L C; Malamed, S

    1975-08-01

    To study the role of certain organelles in steroidogenesis, dissociated rat adrenocortical cells were incubated for two hours with ACTH at a concentration that induces a high level of steroid production. Sections of ACTH treated and untreated cells were photographed in the electron microscope, and morphometric analysis was undertaken to assess possible ACTH-induced changes in total cell volume, volume density and numerical denisty of lipid droplets and mitochondria. There was no change in total cell volume. Lipid droplet volume density and numerical density decreased. Mitochondrial volume density did not change, but numerical density increased. The decrease in lipid droplet volume density indicates a rapid depletion of cholesterol for steroid production. This depletion is almost entirely due to the disappearance of lipid droplets, rather than to an overall diminution in their size, as shown by the decrease in lipid droplet numerical density. The mitochondrial data suggest that the adrenocortical cell has an adedquate mitochondrial apparatus to respond to acute ACTH stimulation with increased steroid output without an increase inmitochondrial volume.

  7. Storable droplet interface lipid bilayers for cell-free ion channel studies.

    PubMed

    Jung, Sung-Ho; Choi, Sangbaek; Kim, Young-Rok; Jeon, Tae-Joon

    2012-01-01

    An artificially created lipid bilayer is an important platform in studying ion channels and engineered biosensor applications. However, a lipid bilayer created using conventional techniques is fragile and short-lived, and the measurement of ion channels requires expertise and laborious procedures, precluding practical applications. Here, we demonstrate a storable droplet lipid bilayer precursor frozen with ion channels, resulting in a droplet interface bilayer upon thawing. A small vial with an aqueous droplet in organic solution was flash frozen in -80 °C methanol immediately after an aqueous droplet was introduced into the organic solution and gravity draws the droplet down to the interface upon thawing. A lipid bilayer created along the interface using this method had giga-ohm resistance and typical specific capacitance values. The noise level of this system is favorably comparable to the conventional system. The subsequent incorporation of ion channels, alpha-hemolysin and gramicidin A, showed typical conductance values consistent with those in previous literatures. This novel system to create a lipid bilayer as a whole can be automated from its manufacture to use and indefinitely stored when frozen. As a result, ion channel measurements can be carried out in any place, increasing the accessibility of ion channel studies as well as a number of applications, such as biosensors, ion channel drug screening, and biophysical studies. PMID:21909672

  8. An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER

    PubMed Central

    Markgraf, Daniel F.; Klemm, Robin W.; Junker, Mirco; Hannibal-Bach, Hans K.; Ejsing, Christer S.; Rapoport, Tom A.

    2014-01-01

    Eukaryotic cells store neutral lipids, such as triacylglycerol (TAG), in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show in S. cerevisiae that LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein, Ice2p, facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG-degradation and -synthesis, promoting the rapid re-localization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER, and explain how cells switch neutral lipid metabolism from storage to consumption. PMID:24373967

  9. An ER protein functionally couples neutral lipid metabolism on lipid droplets to membrane lipid synthesis in the ER.

    PubMed

    Markgraf, Daniel F; Klemm, Robin W; Junker, Mirco; Hannibal-Bach, Hans K; Ejsing, Christer S; Rapoport, Tom A

    2014-01-16

    Eukaryotic cells store neutral lipids such as triacylglycerol (TAG) in lipid droplets (LDs). Here, we have addressed how LDs are functionally linked to the endoplasmic reticulum (ER). We show that, in S. cerevisiae, LD growth is sustained by LD-localized enzymes. When LDs grow in early stationary phase, the diacylglycerol acyl-transferase Dga1p moves from the ER to LDs and is responsible for all TAG synthesis from diacylglycerol (DAG). During LD breakdown in early exponential phase, an ER membrane protein (Ice2p) facilitates TAG utilization for membrane-lipid synthesis. Ice2p has a cytosolic domain with affinity for LDs and is required for the efficient utilization of LD-derived DAG in the ER. Ice2p breaks a futile cycle on LDs between TAG degradation and synthesis, promoting the rapid relocalization of Dga1p to the ER. Our results show that Ice2p functionally links LDs with the ER and explain how cells switch neutral lipid metabolism from storage to consumption.

  10. SLDP: a novel protein related to caleosin is associated with the endosymbiotic Symbiodinium lipid droplets from Euphyllia glabrescens.

    PubMed

    Pasaribu, Buntora; Lin, I-Ping; Tzen, Jason T C; Jauh, Guang-Yuh; Fan, Tung-Yung; Ju, Yu-Min; Cheng, Jing-O; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2014-10-01

    Intracellular lipid droplets (LDs) have been proposed to play a key role in the mutualistic endosymbiosis between reef-building corals and the dinoflagellate endosymbiont Symbiodinium spp. This study investigates and identifies LD proteins in Symbiodinium from Euphyllia glabrescens. Discontinuous Percoll gradient centrifugation was used to separate Symbiodinium cells from E. glabrescens tentacles. Furthermore, staining with a fluorescent probe, Nile red, indicated that lipids accumulated in that freshly isolated Symbiodinium cells and lipid analyses further showed polyunsaturated fatty acids (PUFA) was abundant. The stable LDs were purified from endosymbiotic Symbiodinium cells. The structural integrity of the Symbiodinium LDs was maintained via electronegative repulsion and steric hindrance possibly provided by their surface proteins. Protein extracts from the purified LDs revealed a major protein band with a molecular weight of 20 kDa, which was termed Symbiodinium lipid droplet protein (SLDP). Interestingly, immunological cross-recognition analysis revealed that SLDP was detected strongly by the anti-sesame and anti-cycad caleosin antibodies. It was suggested that the stable Symbiodinium LDs were sheltered by this unique structural protein and was suggested that SLDP might be homologous to caleosin to a certain extent.

  11. Quantitative Lipid Droplet Proteome Analysis Identifies Annexin A3 as a Cofactor for HCV Particle Production.

    PubMed

    Rösch, Kathrin; Kwiatkowski, Marcel; Hofmann, Sarah; Schöbel, Anja; Grüttner, Cordula; Wurlitzer, Marcus; Schlüter, Hartmut; Herker, Eva

    2016-09-20

    Lipid droplets are vital to hepatitis C virus (HCV) infection as the putative sites of virion assembly, but morphogenesis and egress of virions remain ill defined. We performed quantitative lipid droplet proteome analysis of HCV-infected cells to identify co-factors of that process. Our results demonstrate that HCV disconnects lipid droplets from their metabolic function. Annexin A3 (ANXA3), a protein enriched in lipid droplet fractions, strongly impacted HCV replication and was characterized further: ANXA3 is recruited to lipid-rich fractions in HCV-infected cells by the viral core and NS5A proteins. ANXA3 knockdown does not affect HCV RNA replication but severely impairs virion production with lower specific infectivity and higher density of secreted virions. ANXA3 is essential for the interaction of viral envelope E2 with apolipoprotein E (ApoE) and for trafficking, but not lipidation, of ApoE in HCV-infected cells. Thus, we identified ANXA3 as a regulator of HCV maturation and egress. PMID:27653686

  12. The emergence of lipid droplets in yeast: current status and experimental approaches.

    PubMed

    Radulovic, Maja; Knittelfelder, Oskar; Cristobal-Sarramian, Alvaro; Kolb, Dagmar; Wolinski, Heimo; Kohlwein, Sepp D

    2013-11-01

    The 'discovery' of lipid droplets as a metabolically highly active subcellular organelle has sparked great scientific interest in its research in recent years. The previous view of a rather inert storage pool of neutral lipids--triacylglycerol and sterols or steryl esters--has markedly changed. Driven by the endemic dimensions of lipid-associated disorders on the one hand, and the promising biotechnological application to generate oils ('biodiesel') from single-celled organisms on the other, multiple model organisms are exploited in basic and applied research to develop a better understanding of biogenesis and metabolism of this organelle. This article summarizes the current status of LD research in yeast and experimental approaches to obtain insight into the regulatory and structural components driving lipid droplet formation and their physiological and pathophysiological roles in lipid homeostasis.

  13. Lipid droplet organelle distribution in populations of dividing cells studied by simulation

    NASA Astrophysics Data System (ADS)

    Dalhaimer, Paul

    2013-06-01

    One of the key questions in cell biology is how organelles are passed from parent to daughter cells. To help address this question, I used Brownian dynamics to simulate lipid droplets as model organelles in populations of dividing cells. Lipid droplets are dynamic bodies that can form both de novo and by fission, they can also be depleted. The quantitative interplay among these three events is unknown but would seem crucial for controlling droplet distribution in populations of dividing cells. Surprisingly, of the three main events studied: biogenesis, fission, and depletion, the third played the key role in maintaining droplet organelle number—and to a lesser extent volume—in populations of dividing cells where formation events would have seemed paramount. In the case of lipid droplets, this provides computational evidence that they must be sustained, most likely through contacts with the endoplasmic reticulum. The findings also agree with video microscopy experiments over much shorter timescales where droplet depletion in fission yeast cells was not observed. In general, this work shows that organelle maintenance is invaluable and lack thereof cannot necessarily be compensated for by organelle formation. This study provides a time-accurate, physical-based template for long-term cell division studies.

  14. Chains, Sheets and Droplets: Assemblies of Hydrophobic Gold Nanocrystals with Saturated Phosphatidylcholine Lipid and Squalene

    PubMed Central

    Rasch, Michael R.; Bosoy, Christian; Yu, Yixuan; Korgel, Brian A.

    2012-01-01

    Assemblies of saturated 1,2-diacyl-phosphatidylcholine lipid and hydrophobic dodecanethiol-capped 1.8 nm diameter gold nanocrystals were studied as a function of lipid chain length and the addition of the naturally-occurring oil, squalene. The gold nanocrystals formed various lipid-stabilized agglomerates, sometimes fusing with lipid vesicle bilayers. The nanocrystal assembly structure depended on the hydrocarbon chain length of the lipid fatty acids. Lipid with the shortest fatty acid length studied, dilauroyl-phosphatidylcholine, created extended chains of gold nanocrystals. Lipid with slightly longer fatty acid chains created planar sheets of nanocrystals. Further increases of the fatty acid chain length led to spherical agglomerates. The inclusion of squalene led to lipid- and nanocrystal-coated oil droplets. PMID:23033891

  15. Entangled in a membranous web: ER and lipid droplet reorganization during hepatitis C virus infection.

    PubMed

    Meyers, Nathan L; Fontaine, Krystal A; Kumar, G Renuka; Ott, Melanie

    2016-08-01

    Hepatitis C virus (HCV) is a major cause of liver disease worldwide. To establish and maintain chronic infection, HCV extensively rearranges cellular organelles to generate distinct compartments for viral RNA replication and virion assembly. Here, we review our current knowledge of how HCV proliferates and remodels ER-derived membranes while preserving and expanding associated lipid droplets during viral infection. Unraveling the molecular mechanisms responsible for HCV-induced membrane reorganization will enhance our understanding of the HCV life-cycle, the associated liver pathology, and the biology of the ER:lipid droplet interface in general. PMID:27240021

  16. Response of pigeon guillemots to variable abundance of high-lipid and low-lipid prey

    USGS Publications Warehouse

    Litzow, M.A.; Piatt, J.F.; Prichard, A.K.; Roby, D.D.

    2002-01-01

    Populations of the pigeon guillemot (Cepphus columba) and other piscivores have been in decline for several decades in the Gulf of Alaska and Bering Sea, and a decline in abundance of lipid-rich schooling fishes is hypothesized as the major cause. We tested this hypothesis by studying the breeding biology of pigeon guillemots during 1995-1999 while simultaneously measuring prey abundance with beach seines and bottom trawls. Our study area (Kachemak Bay, Alaska) comprises two oceanographically distinct areas. Populations of a lipid-rich schooling fish, Pacific sand lance (Ammodytes hexapterus), were higher in the warmer Inner Bay than in the colder Outer Bay, and sand lance abundance was higher during warm years. Populations of low-lipid content demersal fishes were similar between areas. Chick survival to age 15 days was 47% higher in the Inner Bay (high-lipid diet) than in the Outer Bay (low-lipid diet), and estimated reproductive success (chicks fledged nest-1) was 62% higher in the Inner Bay than in the Outer Bay. Chick provisioning rate (kJ chick-1 h-1) increased with the proportion of sand lance in the diet (r2=0.21), as did growth rate (g day-1) of younger (beta) chicks in two-chick broods (r2=0.14). Pigeon guillemots in the Inner Bay switched to demersal prey during years of below-average sand lance abundance, and these birds reacted to 38-fold interannual changes in sand lance abundance with reductions in beta chick growth rates, with no decline in beta chick survival. In contrast, the proportion of nests experiencing brood reduction in the Outer Bay (demersal diet) increased >300% during years of below-average demersal abundance, although demersal fish abundance varied only 4-fold among years. Our results support the hypothesis that recovery of pigeon guillemot populations from the effects of the Exxon Valdez oil spill is limited by availability of lipid-rich prey.

  17. Unexpected roles of plastoglobules (plastid lipid droplets) in vitamin K1 and E metabolism.

    PubMed

    Spicher, Livia; Kessler, Felix

    2015-06-01

    Tocopherol (vitamin E) and phylloquinone (vitamin K1) are lipid-soluble antioxidants that can only be synthesized by photosynthetic organisms. These compounds function primarily at the thylakoid membrane but are also present in chloroplast lipid droplets, also known as plastoglobules (PG). Depending on environmental conditions and stage of plant development, changes in the content, number and size of PG occur. PG are directly connected to the thylakoid membrane via the outer lipid leaflet. Apart from storage, PG are active in metabolism and likely trafficking of diverse lipid species. This review presents recent advances on how plastoglobules are implicated in the biosynthesis and metabolism of vitamin E and K.

  18. Assessment of lipidomic species in hepatocyte lipid droplets from stressed mouse models.

    PubMed

    Hartler, Jürgen; Köfeler, Harald C; Trötzmüller, Martin; Thallinger, Gerhard G; Spener, Friedrich

    2014-01-01

    Lipid droplets are considered to be the hub for storage and metabolism of cellular lipids. In previous work we have phenotyped the lipidome of murine hepatocyte lipid droplets using liquid chromatography-mass spectrometry (UHPLC-MS) plus integrated MS/MS, followed by automatic analysis of the MS data. The organelles were isolated after intervention studies involving nutritional stress (extended feeding of a high fat diet or short term fasting), genetic stress due to knock-out of adipocyte triglyceride lipase, or by combined application of nutritional and genetic stress together ('super stress'). Lipidomics at the level of lipid species (profiling of lipid classes) and lipid molecular species (structural analysis in parallel) has unraveled clear lipid droplet phenotypes as judged by patterns seen best in triacylglycerol (TG) lipidomes, but also in diacylglycerol and phosphatidylcholine lipidomes. The combined view of these data presented here validates the methods used and provides high quality lipidomic data for further bioinformatic inspections. Examples are given for identification of TG species subsets considered surrogates for whole TG lipidomes. PMID:25977802

  19. A Seoul-Fluor-based bioprobe for lipid droplets and its application in image-based high throughput screening.

    PubMed

    Kim, Eunha; Lee, Sanghee; Park, Seung Bum

    2012-02-25

    We developed a novel fluorescent bioprobe (SF44) that can specifically visualize the cellular lipid droplets in in vitro and in vivo systems and illustrated the mechanistic rationale of its fluorogenic property. Its application to image-based high throughput screening led us to the identification of a new small-molecule modulator of lipid droplet formation.

  20. Adipose triglyceride lipase acts on neutrophil lipid droplets to regulate substrate availability for lipid mediator synthesis

    PubMed Central

    Schlager, Stefanie; Goeritzer, Madeleine; Jandl, Katharina; Frei, Robert; Vujic, Nemanja; Kolb, Dagmar; Strohmaier, Heimo; Dorow, Juliane; Eichmann, Thomas O.; Rosenberger, Angelika; Wölfler, Albert; Lass, Achim; Kershaw, Erin E.; Ceglarek, Uta; Dichlberger, Andrea; Heinemann, Akos; Kratky, Dagmar

    2015-01-01

    In humans, mutations in ATGL lead to TG accumulation in LDs of most tissues and cells, including peripheral blood leukocytes. This pathologic condition is called Jordans’ anomaly, in which functional consequences have not been investigated. In the present study, we tested the hypothesis that ATGL plays a role in leukocyte LD metabolism and immune cell function. Similar to humans with loss-of-function mutations in ATGL, we found that global and myeloid-specific Atgl−/− mice exhibit Jordans’ anomaly with increased abundance of intracellular TG-rich LDs in neutrophil granulocytes. In a model of inflammatory peritonitis, lipid accumulation was also observed in monocytes and macrophages but not in eosinophils or lymphocytes. Neutrophils from Atgl−/− mice showed enhanced immune responses in vitro, which were more prominent in cells from global compared with myeloid-specific Atgl−/− mice. Mechanistically, ATGL−/− as well as pharmacological inhibition of ATGL led to an impaired release of lipid mediators from neutrophils. These findings demonstrate that the release of lipid mediators is dependent on the liberation of precursor molecules from the TG-rich pool of LDs by ATGL. Our data provide mechanistic insights into Jordans’ anomaly in neutrophils and suggest that ATGL is a potent regulator of immune cell function and inflammatory diseases. PMID:26109679

  1. Chromatic aberration correction and deconvolution for UV sensitive imaging of fluorescent sterols in cytoplasmic lipid droplets.

    PubMed

    Wüstner, Daniel; Faergeman, Nils J

    2008-08-01

    Intrinsically fluorescent sterols, like dehydroergosterol (DHE), mimic cholesterol closely and are therefore suitable to determine cholesterol transport by fluorescence microscopy. Disadvantages of DHE are its low quantum yield, rapid bleaching, and the fact that its excitation and emission is in the UV region of the spectrum. Thus, one has to deal with chromatic aberration and low signal-to-noise ratio. We developed a method to correct for chromatic aberration between the UV channel and the red/green channel in multicolor imaging of DHE compared with the lipid droplet marker Nile Red in living macrophage foam cells and in adipocytes. We used deconvolution microscopy and developed image segmentation techniques to assess the DHE content of lipid droplets in both cell types in an automated manner. Pulse-chase studies and colocalization analysis were performed to monitor the redistribution of DHE upon adipocyte differentiation. DHE is targeted to transferrin-positive recycling endosomes in preadipocytes but associates with droplets in mature adipocytes. Only in adipocytes but not in foam cells fluorescent sterol was confined to the droplet-limiting membrane. We developed an approach to visualize and quantify sterol content of lipid droplets in living cells with potential for automated high content screening of cellular sterol transport.

  2. Ordering and stability in lipid droplets with applications to low-density lipoproteins.

    PubMed

    Lancaster, Jarrett L; Antonijevic, Todor; Starobin, Joseph M

    2014-06-01

    In this article, we present a framework for investigating the order-disorder transition in lipid droplets using the standard Ising model. While a single lipid droplet is itself a complex system whose constituent cholesteryl esters each possesses many degrees of freedom, we present justification for using this effective approach to isolate the underlying physics. It is argued that the behavior of the esters confined within lipid droplets is significantly different from that of a bulk system of similar esters, which is adequately described by continuum mean-field theory in the thermodynamic limit. When the droplet's shell is modeled as an elastic membrane, a simple picture emerges for a transition between two ordered phases within the core which is tuned by the strength of interactions between the esters. Triglyceride concentration is proposed as a variable which strongly influences the strength of interactions between cholesteryl esters within droplets. The possible relevance of this mechanism to the well known atherogenic nature of small low-density lipoprotein particles is discussed in detail.

  3. Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells.

    PubMed

    Gidda, Satinder K; Watt, Samantha; Collins-Silva, Jillian; Kilaru, Aruna; Arondel, Vincent; Yurchenko, Olga; Horn, Patrick J; James, Christopher N; Shintani, David; Ohlrogge, John B; Chapman, Kent D; Mullen, Robert T; Dyer, John M

    2013-11-01

    While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed. PMID:24305619

  4. Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells.

    PubMed

    Gidda, Satinder K; Watt, Samantha; Collins-Silva, Jillian; Kilaru, Aruna; Arondel, Vincent; Yurchenko, Olga; Horn, Patrick J; James, Christopher N; Shintani, David; Ohlrogge, John B; Chapman, Kent D; Mullen, Robert T; Dyer, John M

    2013-11-01

    While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, lipid droplets are delineated by a half-unit membrane whose protein constituents are poorly understood, except in the specialized case of oleosins, which are associated with seed lipid droplets. Recently, we identified a new class of lipid-droplet associated proteins called LDAPs that localize specifically to the lipid droplet surface within plant cells and share extensive sequence similarity with the small rubber particle proteins (SRPPs) found in rubber-accumulating plants. Here, we provide additional evidence for a role of LDAPs in lipid accumulation in oil-rich fruit tissues, and further explore the functional relationships between LDAPs and SRPPs. In addition, we propose that the larger LDAP/SRPP protein family plays important roles in the compartmentalization of lipophilic compounds, including triacylglycerols and polyisoprenoids, into lipid droplets within plant cells. Potential roles in lipid droplet biogenesis and function of these proteins also are discussed.

  5. A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.

    PubMed

    Czekalska, Magdalena A; Kaminski, Tomasz S; Jakiela, Slawomir; Tanuj Sapra, K; Bayley, Hagan; Garstecki, Piotr

    2015-01-21

    This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated. PMID:25412368

  6. Arabidopsis SEIPIN proteins modulate triacylglycerol accumulation and influence lipid droplet proliferation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. By contrast to the single SEIPIN genes in humans and yeast, there are three SEIPIN homologues in Arabidopsis thaliana, designated At-SEIPIN1, At-SEIPIN2 and At-SEIPIN3. Here, a yeast (Saccharomy...

  7. Lipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells

    PubMed Central

    Astanina, Ksenia; Koch, Marcus; Jüngst, Christian; Zumbusch, Andreas; Kiemer, Alexandra K.

    2015-01-01

    Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research. PMID:26095213

  8. Lipid droplets as a novel cargo of tunnelling nanotubes in endothelial cells.

    PubMed

    Astanina, Ksenia; Koch, Marcus; Jüngst, Christian; Zumbusch, Andreas; Kiemer, Alexandra K

    2015-01-01

    Intercellular communication is a fundamental process in the development and functioning of multicellular organisms. Recently, an essentially new type of intercellular communication, based on thin membrane channels between cells, has been reported. These structures, termed intercellular or tunnelling nanotubes (TNTs), permit the direct exchange of various components or signals (e.g., ions, proteins, or organelles) between non-adjacent cells at distances over 100 μm. Our studies revealed the presence of tunnelling nanotubes in microvascular endothelial cells (HMEC-1). The TNTs were studied with live cell imaging, environmental scanning electron microscopy (ESEM), and coherent anti-Stokes Raman scattering spectroscopy (CARS). Tunneling nanotubes showed marked persistence: the TNTs could connect cells over long distances (up to 150 μm) for several hours. Several cellular organelles were present in TNTs, such as lysosomes and mitochondria. Moreover, we could identify lipid droplets as a novel type of cargo in the TNTs. Under angiogenic conditions (VEGF treatment) the number of lipid droplets increased significantly. Arachidonic acid application not only increased the number of lipid droplets but also tripled the extent of TNT formation. Taken together, our results provide the first demonstration of lipid droplets as a cargo of TNTs and thereby open a new field in intercellular communication research.

  9. Liver X Receptors Balance Lipid Stores in Hepatic Stellate Cells via Rab18, a Retinoid Responsive Lipid Droplet Protein

    PubMed Central

    O’Mahony, Fiona; Wroblewski, Kevin; O’Byrne, Sheila M.; Jiang, Hongfeng; Clerkin, Kara; Benhammou, Jihane; Blaner, William S.; Beaven, Simon W.

    2014-01-01

    Liver X receptors (LXRs) are determinants of hepatic stellate cell (HSC) activation and liver fibrosis. Freshly isolated HSCs from Lxrαβ−/− mice have increased lipid droplet (LD) size but the functional consequences of this are unknown. Our aim was to determine whether LXRs link cholesterol to retinoid storage in HSCs and how this impacts activation. Primary HSCs from Lxrαβ−/− and wild-type (WT) mice were profiled by gene array during in vitro activation. Lipid content was quantified by HPLC and mass spectroscopy. Primary HSCs were treated with nuclear receptor ligands, transfected with siRNA and plasmid constructs, and analyzed by immunocytochemistry. Lxrαβ−/− HSCs have increased cholesterol and retinyl esters (CEs & REs). The retinoid increase drives intrinsic retinoic acid receptor (RAR) signaling and activation occurs more rapidly in Lxrαβ−/− HSCs. We identify Rab18 as a novel retinoic acid responsive, lipid droplet associated protein that helps mediate stellate cell activation. Rab18 mRNA, protein, and membrane insertion increase during activation. Both Rab18 GTPase activity and isoprenylation are required for stellate cell lipid droplet loss and induction of activation markers. These phenomena are accelerated in the Lxrαβ−/− HSCs, where there is greater retinoic acid flux. Conversely, Rab18 knockdown retards lipid droplet loss in culture and blocks activation, just like the functional mutants. Rab18 is also induced with acute liver injury in vivo. Conclusion Retinoid and cholesterol metabolism are linked in stellate cells by the LD associated protein, Rab18. Retinoid overload helps explain the pro-fibrotic phenotype of Lxrαβ−/− mice and we establish a pivotal role for Rab18 GTPase activity and membrane insertion in wild-type stellate cell activation. Interference with Rab18 may have significant therapeutic benefit in ameliorating liver fibrosis. PMID:25482505

  10. Membrane attachment and structure models of lipid storage droplet protein 1.

    PubMed

    Lin, Penghui; Chen, Xiao; Moktan, Hem; Arrese, Estela L; Duan, Lian; Wang, Liying; Soulages, Jose L; Zhou, Donghua H

    2014-03-01

    Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center. PMID:24333382

  11. Membrane attachment and structure models of lipid storage droplet protein 1

    PubMed Central

    Lin, Penghui; Chen, Xiao; Moktan, Hem; Arrese, Estela L.; Duan, Lian; Wang, Liying; Soulages, Jose L.; Zhou, Donghua H.

    2014-01-01

    Neutral lipid triglycerides, a main reserve for fat and energy, are stored in organelles called lipid droplets. The storage and release of triglycerides are actively regulated by several proteins specific to the droplet surface, one of which in insects is PLIN1. PLIN1 plays a key role in the activation of triglyceride hydrolysis upon phosphorylation. However, the structure of PLIN1 and its relation to functions remain elusive due to its insolubility and crystallization difficulty. Here we report the first solid-state NMR study on the Drosophila melanogaster PLIN1 in combination with molecular dynamics simulation to show the structural basis for its lipid droplet attachment. NMR spin diffusion experiments were consistent with the predicted membrane attachment motif of PLIN1. The data indicated that PLIN1 has close contact with the terminal methyl groups of the phospholipid acyl chains. Structure models for the membrane attachment motif were generated based on hydrophobicity analysis and NMR membrane insertion depth information. Simulated NMR spectra from a trans-model agreed with experimental spectra. In this model, lipids from the bottom leaflet were very close to the surface in the region enclosed by membrane attachment motif. This may imply that in real lipid droplet, triglyceride molecules might be brought close to the surface by the same mechanism, ready to leave the droplet in the event of lipolysis. Juxtaposition of triglyceride lipase structure to the trans-model suggested a possible interaction of a conserved segment with the lipase by electrostatic interactions, opening the lipase lid to expose the catalytic center. PMID:24333382

  12. The conserved metalloprotease invadolysin localizes to the surface of lipid droplets

    PubMed Central

    Cobbe, Neville; Marshall, Kathryn M.; Rao, Shubha Gururaja; Chang, Ching-Wen; Di Cara, Francesca; Duca, Edward; Vass, Sharron; Kassan, Adam; Heck, Margarete M. S.

    2009-01-01

    Summary Invadolysin is a metalloprotease conserved in many different organisms, previously shown to be essential in Drosophila with roles in cell division and cell migration. The gene seems to be ubiquitously expressed and four distinct splice variants have been identified in human cells but not in most other species examined. Immunofluorescent detection of human invadolysin in cultured cells reveals the protein to be associated with the surface of lipid droplets. By means of subcellular fractionation, we have independently confirmed the association of invadolysin with lipid droplets. We thus identify invadolysin as the first metalloprotease located on these dynamic organelles. In addition, analysis of larval fat-body morphological appearance and triglyceride levels in the Drosophila invadolysin mutant suggests that invadolysin plays a role in lipid storage or metabolism. PMID:19706689

  13. Droplet immobilization within a polymeric organogel improves lipid bilayer durability and portability.

    PubMed

    Venkatesan, Guru A; Sarles, Stephen A

    2016-05-24

    The droplet interface bilayer (DIB) is a promising technique for assembling lipid membrane-based materials and devices using water droplets in oil, but it has largely been limited to laboratory environments due to its liquid construction. With a vision to transform this lab-based technique into a more-durable embodiment, we investigate the use of a polymer-based organogel to encapsulate DIBs within a more-solid material matrix to improve their handling and portability. Specifically, a temperature-sensitive organogel formed from hexadecane and poly[styrene-b-(ethylene-co-butylene)-b-styrene] (SEBS) triblock copolymer is used to replace the liquid solvent that surrounds the lipid-coated droplets to establish a novel liquid-in-gel DIB system. Through specific capacitance measurements and single-channel recordings of the pore forming peptide alamethicin, we verify that the structural and functional membrane properties are retained when DIBs are assembled within SEBS organogel. In addition, we demonstrate that organogel encapsulation offers improved handling of droplets and yields DIBs with a near 3× higher bilayer durability, as quantified by the lateral acceleration required to rupture the membrane, compared to liquid-in-liquid DIBs in oil. This encapsulated DIB system provides a barrier against contamination from the environment and offers a new material platform for supporting multilayered DIB-based devices as well as other digital microfluidic systems that feature water droplets in oil. PMID:27164314

  14. Motor-mediated cortical versus astral microtubule organization in lipid-monolayered droplets.

    PubMed

    Baumann, Hella; Surrey, Thomas

    2014-08-01

    The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we studied the effect of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from 5-100 μm, which covers the size range of typical cell bodies. We found that droplet size alone had a major organizing influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organizations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. Our results provide insights into the combined organizing influence of spatial constraints and cross-linking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid-monolayered droplet system characterized here can be expanded readily to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organization inside liposomes surrounded by a deformable lipid bilayer. PMID:24966327

  15. Motor-mediated Cortical versus Astral Microtubule Organization in Lipid-monolayered Droplets

    PubMed Central

    Baumann, Hella; Surrey, Thomas

    2014-01-01

    The correct spatial organization of microtubules is of crucial importance for determining the internal architecture of eukaryotic cells. Microtubules are arranged in space by a multitude of biochemical activities and by spatial constraints imposed by the cell boundary. The principles underlying the establishment of distinct intracellular architectures are only poorly understood. Here, we studied the effect of spatial confinement on the self-organization of purified motors and microtubules that are encapsulated in lipid-monolayered droplets in oil, varying in diameter from 5–100 μm, which covers the size range of typical cell bodies. We found that droplet size alone had a major organizing influence. The presence of a microtubule-crosslinking motor protein decreased the number of accessible types of microtubule organizations. Depending on the degree of spatial confinement, the presence of the motor caused either the formation of a cortical array of bent microtubule bundles or the generation of single microtubule asters in the droplets. These are two of the most prominent forms of microtubule arrangements in plant and metazoan cells. Our results provide insights into the combined organizing influence of spatial constraints and cross-linking motor activities determining distinct microtubule architectures in a minimal biomimetic system. In the future, this simple lipid-monolayered droplet system characterized here can be expanded readily to include further biochemical activities or used as the starting point for the investigation of motor-mediated microtubule organization inside liposomes surrounded by a deformable lipid bilayer. PMID:24966327

  16. Identification of a New Class of Lipid Droplet-Associated Proteins in Plants1[C][W][OPEN

    PubMed Central

    Horn, Patrick J.; James, Christopher N.; Gidda, Satinder K.; Kilaru, Aruna; Dyer, John M.; Mullen, Robert T.; Ohlrogge, John B.; Chapman, Kent D.

    2013-01-01

    Lipid droplets in plants (also known as oil bodies, lipid bodies, or oleosomes) are well characterized in seeds, and oleosins, the major proteins associated with their surface, were shown to be important for stabilizing lipid droplets during seed desiccation and rehydration. However, lipid droplets occur in essentially all plant cell types, many of which may not require oleosin-mediated stabilization. The proteins associated with the surface of nonseed lipid droplets, which are likely to influence the formation, stability, and turnover of this compartment, remain to be elucidated. Here, we have combined lipidomic, proteomic, and transcriptomic studies of avocado (Persea americana) mesocarp to identify two new lipid droplet-associated proteins, which we named LDAP1 and LDAP2. These proteins are highly similar to each other and also to the small rubber particle proteins that accumulate in rubber-producing plants. An Arabidopsis (Arabidopsis thaliana) homolog to LDAP1 and LDAP2, At3g05500, was localized to the surface of lipid droplets after transient expression in tobacco (Nicotiana tabacum) cells that were induced to accumulate triacylglycerols. We propose that small rubber particle protein-like proteins are involved in the general process of binding and perhaps the stabilization of lipid-rich particles in the cytosol of plant cells and that the avocado and Arabidopsis protein members reveal a new aspect of the cellular machinery that is involved in the packaging of triacylglycerols in plant tissues. PMID:23821652

  17. Lipid droplets fusion in adipocyte differentiated 3T3-L1 cells: A Monte Carlo simulation

    SciTech Connect

    Boschi, Federico; Rizzatti, Vanni; Zamboni, Mauro; Sbarbati, Andrea

    2014-02-15

    Several human worldwide diseases like obesity, type 2 diabetes, hepatic steatosis, atherosclerosis and other metabolic pathologies are related to the excessive accumulation of lipids in cells. Lipids accumulate in spherical cellular inclusions called lipid droplets (LDs) whose sizes range from fraction to one hundred of micrometers in adipocytes. It has been suggested that LDs can grow in size due to a fusion process by which a larger LD is obtained with spherical shape and volume equal to the sum of the progenitors’ ones. In this study, the size distribution of two populations of LDs was analyzed in immature and mature (5-days differentiated) 3T3-L1 adipocytes (first and second populations, respectively) after Oil Red O staining. A Monte Carlo simulation of interaction between LDs has been developed in order to quantify the size distribution and the number of fusion events needed to obtain the distribution of the second population size starting from the first one. Four models are presented here based on different kinds of interaction: a surface weighted interaction (R2 Model), a volume weighted interaction (R3 Model), a random interaction (Random model) and an interaction related to the place where the LDs are born (Nearest Model). The last two models mimic quite well the behavior found in the experimental data. This work represents a first step in developing numerical simulations of the LDs growth process. Due to the complex phenomena involving LDs (absorption, growth through additional neutral lipid deposition in existing droplets, de novo formation and catabolism) the study focuses on the fusion process. The results suggest that, to obtain the observed size distribution, a number of fusion events comparable with the number of LDs themselves is needed. Moreover the MC approach results a powerful tool for investigating the LDs growth process. Highlights: • We evaluated the role of the fusion process in the synthesis of the lipid droplets. • We compared the

  18. Olanzapine promotes the accumulation of lipid droplets and the expression of multiple perilipins in human adipocytes.

    PubMed

    Nimura, Satomi; Yamaguchi, Tomohiro; Ueda, Koki; Kadokura, Karin; Aiuchi, Toshihiro; Kato, Rina; Obama, Takashi; Itabe, Hiroyuki

    2015-11-27

    Second generation antipsychotics are useful for the treatment of schizophrenia, but concerns have been raised about the side effects of diabetes mellitus and obesity. Olanzapine, especially, is associated with more weight gain than the others. It has been reported that olanzapine promotes adipocyte-differentiation in rodents both in vivo and in vitro. In this study the effects of antipsychotics on human adipocytes were investigated by using human mesenchymal stem cells (hMSCs). When hMSCs were differentiated and treated with various antipsychotics, olanzapine and clozapine increased intracellular lipids. Olanzapine induced lipid accumulation in a dose-dependent manner. Proteomic analysis revealed that PLIN4 and several enzymes for lipid metabolism were increased in the hMSCs after olanzapine treatment. During adipocyte differentiation, olanzapine increased the protein expression of PLIN1, PLIN2 and PLIN4. These proteins are known to be associated with the initial stage of lipid droplet formation. Immunocytochemistry showed that olanzapine increased and enlarged the lipid droplets coated with PLIN1 and PLIN2 while PLIN4 was largely distributed in the cytosol. mRNA expression of PLIN2, but not PLIN1 or PLIN4, was increased by olanzapine. On the other hand, olanzapine did not alter the mRNA level of transcription regulators involved in adipocyte-differentiation or adipokines. The present study shows that olanzapine induced transient PLIN2 expression in hMSCs that could result in an accumulation of lipid droplets and overexpression of PLIN1 and PLIN4, providing information of possible interest for olanzapine-induced weight gain. PMID:26471304

  19. 17{beta}-Hydroxysteroid dehydrogenase type 13 is a liver-specific lipid droplet-associated protein

    SciTech Connect

    Horiguchi, Yuka; Araki, Makoto; Motojima, Kiyoto

    2008-05-30

    17{beta}-Hydroxysteroid dehydrogenase (17{beta}HSD) type 13 is identified as a new lipid droplet-associated protein. 17{beta}HSD type 13 has an N-terminal sequence similar to that of 17{beta}HSD type 11, and both sequences function as an endoplasmic reticulum and lipid droplet-targeting signal. Localization of native 17{beta}HSD type 13 on the lipid droplets was confirmed by subcellular fractionation and Western blotting. In contrast to 17{beta}HSD type 11, however, expression of 17{beta}HSD type 13 is largely restricted to the liver and is not enhanced by peroxisome proliferator-activated receptor {alpha} and its ligand. Instead the expression level of 17{beta}HSD type 13 in the receptor-null mice was increased several-fold. 17{beta}HSD type 13 may have a distinct physiological role as a lipid droplet-associated protein in the liver.

  20. Characterization of the interaction of diacylglycerol acyltransferase-2 with the endoplasmic reticulum and lipid droplets.

    PubMed

    McFie, Pamela J; Jin, Youzhi; Banman, Shanna L; Beauchamp, Erwan; Berthiaume, Luc G; Stone, Scot J

    2014-09-01

    Acyl CoA:diacylglycerol acyltransferase-2 (DGAT2) is an integral membrane protein that catalyzes the synthesis of triacylglycerol (TG). DGAT2 is present in the endoplasmic reticulum (ER) and also localizes to lipid droplets when cells are stimulated with oleate. Previous studies have shown that DGAT2 can interact with membranes and lipid droplets independently of its two transmembrane domains, suggesting the presence of an additional membrane binding domain. In order to identify additional membrane binding regions, we confirmed that DGAT2 has only two transmembrane domains and demonstrated that the loop connecting them is present in the ER lumen. Increasing the length of this short loop from 5 to 27 amino acids impaired the ability of DGAT2 to localize to lipid droplets. Using a mutagenesis approach, we were able to identify a stretch of amino acids that appears to have a role in binding DGAT2 to the ER membrane. Our results confirm that murine DGAT2 has only two transmembrane domains but also can interact with membranes via a previously unidentified helical domain containing its active site.

  1. Targeting of Nir2 to lipid droplets is regulated by a specific threonine residue within its PI-transfer domain.

    PubMed

    Litvak, Vladimir; Shaul, Yoav D; Shulewitz, Mark; Amarilio, Roy; Carmon, Shari; Lev, Sima

    2002-09-01

    Nir2, like its Drosophila homolog retinal degeneration B (RdgB), contains an N-terminal phosphatidylinositol-transfer protein (PI-TP)-like domain. Previous studies have suggested that RdgB plays an important role in the fly phototransduction cascade and that its PI-transfer domain is critical for this function. In this domain, a specific mutation, T59E, induces a dominant retinal degeneration phenotype. Here we show that a similar mutation, T59E in the human Nir2 protein, targets Nir2 to spherical cytosolic structures identified as lipid droplets by the lipophilic dye Nile red. A truncated Nir2T59E mutant consisting of only the PI-transfer domain was also targeted to lipid droplets, whereas neither the wild-type Nir2 nor the Nir2T59A mutant was associated with lipid droplets under regular growth conditions. However, oleic-acid treatment caused translocation of wild-type Nir2, but not translocation of the T59A mutant, to lipid droplets. This treatment also induced partial targeting of endogenous Nir2, which is mainly associated with the Golgi apparatus, to lipid droplets. Targeting of Nir2 to lipid droplets was attributed to its enhanced threonine phosphorylation. These results suggest that a specific threonine within the PI-transfer domain of Nir2 provides a regulatory site for targeting to lipid droplets. In conjunction with the role of PI-TPs in lipid transport, this targeting may affect intracellular lipid trafficking and distribution and may provide the molecular basis underlying the dominant effect of the RdgB-T59E mutant on retinal degeneration. PMID:12225667

  2. Do cytoplasmic lipid droplets accumulate in immature oocytes from over-conditioned repeat breeder dairy heifers?

    PubMed

    Awasthi, H; Saravia, F; Rodríguez-Martínez, H; Båge, R

    2010-10-01

    One of the main sources of repeat breeding in dairy cattle, caused by fertilization failure or early embryonic death, is metabolic stress during lactation. Nutrition seems also to play a role when the condition is seen in heifers, where oocyte cytoplasmic maturation is impaired. To determine whether over conditioning affects oocyte morphology, immature oocytes were collected by ovum pick-up (OPU) twice weekly during 5 weeks from three over-conditioned repeat breeder dairy heifers (RBH) and two normal virgin heifers (VH, controls) of the Swedish Red breed, monitored by body weight and condition. Oocyte quality was assessed under stereomicroscope and further examined by transmission electron microscope for accumulation of cytoplasmic lipid deposits. After OPU, the RBH yielded more low quality oocytes (60% vs 52% for VH, p = 0.14). The relative occupancy of osmophilic lipid droplets in the cytoplasm was higher in oocytes of bad quality compared with good ones, especially in RBH (p = 0.08) but also in VH (p = 0.11). Moreover, the oocytes from over-conditioned RBH showed higher amounts of cytoplasmic lipid deposits both in good (p = 0.14) and, even more prominent, in bad quality oocytes (p = 0.06). Such accumulation of lipid droplets may imply increased sensitivity to oxidative stress, hinder cytoplasmic maturation and lead to subfertility, as accounted in over-conditioned repeat breeders of the Swedish Red breed. PMID:19735437

  3. Glial Lipid Droplets and ROS Induced by Mitochondrial Defects Promote Neurodegeneration

    PubMed Central

    Liu, Lucy; Zhang, Ke; Sandoval, Hector; Yamamoto, Shinya; Jaiswal, Manish; Sanz, Elisenda; Li, Zhihong; Hui, Jessica; Graham, Brett H.; Quintana, Albert; Bellen, Hugo J.

    2014-01-01

    Reactive oxygen species (ROS) and mitochondrial defects in neurons are implicated in neurodegenerative disease. Here we find that a key consequence of ROS and neuronal mitochondrial dysfunction is the accumulation of lipid droplets (LD) in glia. In Drosophila, ROS triggers c-Jun-N-terminal Kinase (JNK) and Sterol Regulatory Element Binding Protein (SREBP) activity in neurons leading to LD accumulation in glia prior to or at the onset of neurodegeneration. The accumulated lipids are peroxidated in the presence of ROS. Reducing LD accumulation in glia and lipid peroxidation via targeted lipase overexpression and/or lowering ROS significantly delays the onset of neurodegeneration. Furthermore, a similar pathway leads to glial LD accumulation in Ndufs4 mutant mice with neuronal mitochondrial defects, suggesting that LD accumulation following mitochondrial dysfunction is an evolutionarily conserved phenomenon, and represents an early, transient indicator and promoter of neurodegenerative disease. PMID:25594180

  4. The seipin complex Fld1/Ldb16 stabilizes ER–lipid droplet contact sites

    PubMed Central

    Grippa, Alexandra; Buxó, Laura; Mora, Gabriel; Funaya, Charlotta; Idrissi, Fatima-Zahra; Mancuso, Francesco; Gomez, Raul; Muntanyà, Júlia; Sabidó, Eduard

    2015-01-01

    Lipid droplets (LDs) are storage organelles consisting of a neutral lipid core surrounded by a phospholipid monolayer and a set of LD-specific proteins. Most LD components are synthesized in the endoplasmic reticulum (ER), an organelle that is often physically connected with LDs. How LD identity is established while maintaining biochemical and physical connections with the ER is not known. Here, we show that the yeast seipin Fld1, in complex with the ER membrane protein Ldb16, prevents equilibration of ER and LD surface components by stabilizing the contact sites between the two organelles. In the absence of the Fld1/Ldb16 complex, assembly of LDs results in phospholipid packing defects leading to aberrant distribution of lipid-binding proteins and abnormal LDs. We propose that the Fld1/Ldb16 complex facilitates the establishment of LD identity by acting as a diffusion barrier at the ER–LD contact sites. PMID:26572621

  5. Specific Accumulation of Lipid Droplets in Hepatocyte Nuclei of PFOA-exposed BALB/c Mice

    NASA Astrophysics Data System (ADS)

    Wang, Ling; Wang, Yu; Liang, Yong; Li, Jia; Liu, Yuchen; Zhang, Jie; Zhang, Aiqian; Fu, Jianjie; Jiang, Guibin

    2013-07-01

    Lipid droplets (LDs), which are important storage structures for neutral lipids and organelles of diverse functions, participate in various cellular activities. In this study, BALB/c mice, fed a regular or a high-fat diet, were exposed to the synthetic perfluorinated compound, perfluorooctanoic acid (PFOA). PFOA-exposed mice had altered serum lipid and lipoprotein levels, and hydropic degeneration or ballooning degeneration of hepatocytes. Moreover, we report for the first time that LDs accumulate in hepatic nuclei after PFOA exposure. As PFOA resembles fatty acids (FA) in its structure, this chemical may interfere with the transportation and metabolism of FA as well as LDs in the cell. This abnormal localization of LDs in the nucleus may be related to the cause of PFOA toxicity.

  6. Arabidopsis SEIPIN Proteins Modulate Triacylglycerol Accumulation and Influence Lipid Droplet Proliferation[OPEN

    PubMed Central

    2015-01-01

    The lipodystrophy protein SEIPIN is important for lipid droplet (LD) biogenesis in human and yeast cells. In contrast with the single SEIPIN genes in humans and yeast, there are three SEIPIN homologs in Arabidopsis thaliana, designated SEIPIN1, SEIPIN2, and SEIPIN3. Essentially nothing is known about the functions of SEIPIN homologs in plants. Here, a yeast (Saccharomyces cerevisiae) SEIPIN deletion mutant strain and a plant (Nicotiana benthamiana) transient expression system were used to test the ability of Arabidopsis SEIPINs to influence LD morphology. In both species, expression of SEIPIN1 promoted accumulation of large-sized lipid droplets, while expression of SEIPIN2 and especially SEIPIN3 promoted small LDs. Arabidopsis SEIPINs increased triacylglycerol levels and altered composition. In tobacco, endoplasmic reticulum (ER)-localized SEIPINs reorganized the normal, reticulated ER structure into discrete ER domains that colocalized with LDs. N-terminal deletions and swapping experiments of SEIPIN1 and 3 revealed that this region of SEIPIN determines LD size. Ectopic overexpression of SEIPIN1 in Arabidopsis resulted in increased numbers of large LDs in leaves, as well as in seeds, and increased seed oil content by up to 10% over wild-type seeds. By contrast, RNAi suppression of SEIPIN1 resulted in smaller seeds and, as a consequence, a reduction in the amount of oil per seed compared with the wild type. Overall, our results indicate that Arabidopsis SEIPINs are part of a conserved LD biogenesis machinery in eukaryotes and that in plants these proteins may have evolved specialized roles in the storage of neutral lipids by differentially modulating the number and sizes of lipid droplets. PMID:26362606

  7. Proteomic Analysis of Lipid Droplets from Caco-2/TC7 Enterocytes Identifies Novel Modulators of Lipid Secretion

    PubMed Central

    Beilstein, Frauke; Bouchoux, Julien; Rousset, Monique; Demignot, Sylvie

    2013-01-01

    In enterocytes, the dynamic accumulation and depletion of triacylglycerol (TAG) in lipid droplets (LD) during fat absorption suggests that cytosolic LD-associated TAG contribute to TAG-rich lipoprotein (TRL) production. To get insight into the mechanisms controlling the storage/secretion balance of TAG, we used as a tool hepatitis C virus core protein, which localizes onto LDs, and thus may modify their protein coat and decrease TRL secretion. We compared the proteome of LD fractions isolated from Caco-2/TC7 enterocytes expressing or not hepatitis C virus core protein by a differential proteomic approach (isobaric tag for relative and absolute quantitation (iTRAQ) labeling coupled with liquid chromatography and tandem mass spectrometry). We identified 42 proteins, 21 being involved in lipid metabolism. Perilipin-2/ADRP, which is suggested to stabilize long term-stored TAG, was enriched in LD fractions isolated from Caco-2/TC7 expressing core protein while perilipin-3/TIP47, which is involved in LD synthesis from newly synthesized TAG, was decreased. Endoplasmic reticulum-associated proteins were strongly decreased, suggesting reduced interactions between LD and endoplasmic reticulum, where TRL assembly occurs. For the first time, we show that 17β-hydroxysteroid dehydrogenase 2 (DHB2), which catalyzes the conversion of 17-keto to 17 β-hydroxysteroids and which was the most highly enriched protein in core expressing cells, is localized to LD and interferes with TAG secretion, probably through its capacity to inactivate testosterone. Overall, we identified potential new players of lipid droplet dynamics, which may be involved in the balance between lipid storage and secretion, and may be altered in enterocytes in pathological conditions such as insulin resistance, type II diabetes and obesity. PMID:23301014

  8. Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of lipophilic compounds in plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    While lipid droplets have traditionally been considered as inert sites for the storage of triacylglycerols and sterol esters, they are now recognized as dynamic and functionally diverse organelles involved in energy homeostasis, lipid signaling, and stress responses. Unlike most other organelles, li...

  9. Disruption of the human CGI-58 homologue in Arabidopsis results in lipid droplet accumulation in the cytosol of plant cells

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CGI-58 has been identified as the causative gene in the human neutral lipid storage disease called Chanarin-Dorfman Syndrome. This disorder results in accumulation of intracellular lipid droplets in non-adipose tissues. Here we show that disruption of the homologous CGI-58 gene in Arabidopsis thal...

  10. Bioactive Hybrid Particles from Poly(D,L-lactide-co-glycolide) Nanoparticle Stabilized Lipid Droplets.

    PubMed

    Joyce, Paul; Whitby, Catherine P; Prestidge, Clive A

    2015-08-12

    Biodegradable and bioactive hybrid particles composed of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles and medium-chain triglycerides were prepared by spray drying lipid-in-water emulsions stabilized by PLGA nanoparticles, to form PLGA-lipid hybrid (PLH) microparticles approximately 5 μm in mean diameter. The nanoparticle stabilizer was varied and mannitol was also incorporated during the preparation to investigate the effect of stabilizer charge and cryoprotectant content on the particle microstructure. An in vitro lipolysis model was used to demonstrate the particles' bioactivity by manipulating the digestion kinetics of encapsulated lipid by pancreatic lipase in simulated gastrointestinal fluid. Lipid digestion kinetics were enhanced in PLH and PLGA-lipid-mannitol hybrid (PLMH) microparticles for both stabilizers, compared to a coarse emulsion, in biorelevant media. An optimal digestion rate was observed for the negatively charged PLMH system, evidenced by a 2-fold increase in the pseudo-first-order rate constant compared to a coarse emulsion. Improved microparticle redispersion, probed by dual dye confocal fluorescence microscopy, increased the available surface area of lipid for lipase adsorption, enhancing digestion kinetics. Thereby, lipase action was controlled in hybrid microparticles by altering the surface charge and carbohydrate content. Our results demonstrate that bioactive microparticles composed of versatile and biodegradable polymeric particles and oil droplets have great potential for use in smart food and nutrient delivery, as well as safer and more efficacious oral delivery of drugs and drug combinations. PMID:26181279

  11. RORα and 25-Hydroxycholesterol Crosstalk Regulates Lipid Droplet Homeostasis in Macrophages

    PubMed Central

    Tuong, Zewen Kelvin; Lau, Patrick; Du, Ximing; Condon, Nicholas D.; Goode, Joel M.; Oh, Tae Gyu; Yeo, Jeremy C.; Muscat, George E. O.; Stow, Jennifer L.

    2016-01-01

    Nuclear hormone receptors have important roles in the regulation of metabolic and inflammatory pathways. The retinoid-related orphan receptor alpha (Rorα)-deficient staggerer (sg/sg) mice display several phenotypes indicative of aberrant lipid metabolism, including dyslipidemia, and increased susceptibility to atherosclerosis. In this study we demonstrate that macrophages from sg/sg mice have increased ability to accumulate lipids and accordingly exhibit larger lipid droplets (LD). We have previously shown that BMMs from sg/sg mice have significantly decreased expression of cholesterol 25-hydroxylase (Ch25h) mRNA, the enzyme that produces the oxysterol, 25-hydroxycholesterol (25HC), and now confirm this at the protein level. 25HC functions as an inverse agonist for RORα. siRNA knockdown of Ch25h in macrophages up-regulates Vldlr mRNA expression and causes increased accumulation of LDs. Treatment with physiological concentrations of 25HC in sg/sg macrophages restored lipid accumulation back to normal levels. Thus, 25HC and RORα signify a new pathway involved in the regulation of lipid homeostasis in macrophages, potentially via increased uptake of lipid which is suggested by mRNA expression changes in Vldlr and other related genes. PMID:26812621

  12. Biology and pathobiology of lipid droplets and their potential role in the protection of the organ of Corti.

    PubMed

    Urrutia, Raul A; Kalinec, Federico

    2015-12-01

    The current review article seeks to extend our understanding on the role of lipid droplets within the organ of Corti. In addition to presenting an overview of the current information about the origin, structure and function of lipid droplets we draw inferences from the collective body of knowledge about this cellular organelle to build a conceptual framework to better understanding their role in auditory function. This conceptual model considers that lipid droplets play a significant role in the synthesis, storage, and release of lipids and proteins for energetic use and/or modulating cell signaling pathways. We describe the role and mechanism by which LD play a role in human diseases, and we also review emerging data from our laboratory revealing the potential role of lipid droplets from Hensen cells in the auditory organ. We suggest that lipid droplets might help to develop rapidly and efficiently the resolution phase of inflammatory responses in the mammalian cochlea, preventing inflammatory damage of the delicate inner ear structures and, consequently, sensorineural hearing loss.

  13. Visualization and Analysis of Hepatitis C Virus Structural Proteins at Lipid Droplets by Super-Resolution Microscopy

    PubMed Central

    Reimer, Rudolph; Herker, Eva

    2014-01-01

    Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet–associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites. PMID:25019511

  14. Rotavirus replication and the role of cellular lipid droplets: New therapeutic targets?

    PubMed

    Lever, Andrew; Desselberger, Ulrich

    2016-06-01

    Rotaviruses (RVs) are a major cause of acute gastroenteritis in infants and young children worldwide. These viruses infect the villous epithelium of the small intestine. Part of their replication occurs in cytoplasmic inclusion bodies termed viroplasms. Viroplasms and the lipid droplets (LDs) of cellular organelles are known to interact both physically and functionally. Compounds interfering with the homoeostasis of LDs significantly decrease the production of infectious RV progeny. There is considerable scope for more detailed exploration of such compounds as potential antiviral agents for a disease for which at present no specific therapy exists.

  15. The Phospholipid:Diacylglycerol Acyltransferase Lro1 Is Responsible for Hepatitis C Virus Core-Induced Lipid Droplet Formation in a Yeast Model System

    PubMed Central

    Wang, Chao-Wen; Cheng, Yun-Hsin; Irokawa, Hayato; Hwang, Gi-Wook; Naganuma, Akira; Kuge, Shusuke

    2016-01-01

    Chronic infection with the hepatitis C virus frequently induces steatosis, which is a significant risk factor for liver pathogenesis. Steatosis is characterized by the accumulation of lipid droplets in hepatocytes. The structural protein core of the virus induces lipid droplet formation and localizes on the surface of the lipid droplets. However, the precise molecular mechanisms for the core-induced formation of lipid droplets remain elusive. Recently, we showed that the expression of the core protein in yeast as a model system could induce lipid droplet formation. In this study, we probed the cellular factors responsible for the formation of core-induced lipid-droplets in yeast cells. We demonstrated that one of the enzymes responsible for triglyceride synthesis, a phospholipid:diacylglycerol acyltransferase (Lro1), is required for the core-induced lipid droplet formation. While core proteins inhibit Lro1 degradation and alter Lro1 localization, the characteristic localization of Lro1 adjacent to the lipid droplets appeared to be responsible for the core-induced lipid droplet formation. RNA virus genomes have evolved using high mutation rates to maintain their ability to replicate. Our observations suggest a functional relationship between the core protein with hepatocytes and yeast cells. The possible interactions between core proteins and the endoplasmic reticulum membrane affect the mobilization of specific proteins. PMID:27459103

  16. Long-term live cell microscopy studies of lipid droplet fusion dynamics in adipocytes[S

    PubMed Central

    Jüngst, Christian; Klein, Matthias; Zumbusch, Andreas

    2013-01-01

    During the adipogenic differentiation process of mesenchymal stem cells, lipid droplets (LDs) grow slowly by transferring lipids between each other. Recent findings hint at the possibility that a fusion pore is involved. In this study, we analyze lipid transfer data obtained in long-term label-free microscopy studies in the framework of a Hagen-Poiseuille model. The data obtained show a LD fusion process in which the lipid transfer directionality depends on the size difference between LDs, whereas the respective rates depend on the size difference and additionally on the diameter of the smaller LDs. For the data analysis, the viscosity of the transferred material has to be known. We demonstrate that a viscosity-dependent molecular rotor dye can be used to measure LD viscosities in live cells. On this basis, we calculate the diameter of a putative lipid transfer channel which appears to have a direct dependence on the diameter of the smaller of the two participating LDs. PMID:24103784

  17. Cholesterol mobilization from hepatic lipid droplets during endotoxemia is altered in obese ob/ob mice.

    PubMed

    Arisqueta, Lino; Navarro-Imaz, Hiart; Rueda, Yuri; Fresnedo, Olatz

    2015-10-01

    The innate immune response to pathogens during the acute phase response includes lipid metabolism adaptations. Hepatic triacylglycerol (TG) and cholesteryl ester (CE) storage in and mobilization from lipid droplets (LDs) respond to metabolic changes under the control of liver X receptor (LXR) transactivation and cytokine transduction. To evaluate whether alterations of these mechanisms have an impact in the adaptive response to endotoxemia, we analysed liver metabolism changes in lipopolysaccharide (LPS)-treated ob/ob mice, which show altered metabolic and innate responses and a higher sensitivity to sepsis. Lipid composition of serum lipoproteins and hepatic LDs was determined in wild type and ob/ob mice 24 h after LPS treatment. Liver metabolic profiling was done by measuring enzyme activities and mRNA levels. Increased CE hydrolase activity in LDs from endotoxemic mice was accompanied by a lower content of CE and low or no induction of LXR-mediated expression of genes involved in HDL secretion. The attenuated response in liver lipid mobilization accompanied by the strain-specific cholesterol enrichment of secreted VLDL might lead to accumulation of LDL cholesterol. According to our findings, obese leptin-deficient mice present an altered control of hepatic lipid metabolism responses to LPS, which might be, in part at least, a consequence of impaired LXR. PMID:25953914

  18. Adsorption kinetics dictate monolayer self-assembly for both lipid-in and lipid-out approaches to droplet interface bilayer formation

    SciTech Connect

    Venkatesan, Guru A.; Lee, Joonho; Farimani, Amir Barati; Heiranian, Mohammad; Collier, C. Patrick; Narayana, Aluru; Sarles, Stephen A.

    2015-11-10

    The droplet interface bilayer (DIB) is a method to assemble planar lipid bilayer membranes between lipid-coated aqueous droplets and has gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous droplet–oil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: “lipid-in”, in which phospholipids in the form of liposomes are placed in water, and “lipid-out”, in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods and exhibit different success rates for bilayer formation. In our study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. Also, we clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid placement and

  19. Adsorption kinetics dictate monolayer self-assembly for both lipid-in and lipid-out approaches to droplet interface bilayer formation

    DOE PAGES

    Venkatesan, Guru A.; Lee, Joonho; Farimani, Amir Barati; Heiranian, Mohammad; Collier, C. Patrick; Narayana, Aluru; Sarles, Stephen A.

    2015-11-10

    The droplet interface bilayer (DIB) is a method to assemble planar lipid bilayer membranes between lipid-coated aqueous droplets and has gained popularity among researchers in many fields. Well-packed lipid monolayer on aqueous droplet–oil interfaces is a prerequisite for successfully assembling DIBs. Such monolayers can be achieved by two different techniques: “lipid-in”, in which phospholipids in the form of liposomes are placed in water, and “lipid-out”, in which phospholipids are placed in oil as inverse micelles. While both approaches are capable of monolayer assembly needed for bilayer formation, droplet pairs assembled with these two techniques require significantly different incubation periods andmore » exhibit different success rates for bilayer formation. In our study, we combine experimental interfacial tension measurements with molecular dynamics simulations of phospholipids (DPhPC and DOPC) assembled from water and oil origins to understand the differences in kinetics of monolayer formation. With the results from simulations and by using a simplified model to analyze dynamic interfacial tensions, we conclude that, at high lipid concentrations common to DIBs, monolayer formation is simple adsorption controlled for lipid-in technique, whereas it is predominantly adsorption-barrier controlled for the lipid-out technique due to the interaction of interface-bound lipids with lipid structures in the subsurface. The adsorption barrier established in lipid-out technique leads to a prolonged incubation time and lower bilayer formation success rate, proving a good correlation between interfacial tension measurements and bilayer formation. Also, we clarify that advective flow expedites monolayer formation and improves bilayer formation success rate by disrupting lipid structures, rather than enhancing diffusion, in the subsurface and at the interface for lipid-out technique. Additionally, electrical properties of DIBs formed with varying lipid

  20. Conditions of endoplasmic reticulum stress stimulate lipid droplet formation in Saccharomyces cerevisiae.

    PubMed

    Fei, Weihua; Wang, Han; Fu, Xin; Bielby, Christopher; Yang, Hongyuan

    2009-11-15

    LDs (lipid droplets) are cellular organelles which can be found in nearly all eukaryotic cells. Despite their importance in cell biology, the mechanism underlying LD biogenesis remains largely unknown. In the present study we report that conditions of ER (endoplasmic reticulum) stress stimulate LD formation in Saccharomyces cerevisiae. We found that LDs accumulated in yeast mutants with compromised protein glycosylation or ER-associated protein degradation. Moreover, tunicamycin and Brefeldin A, agents which induce ER stress, were found to stimulate LD formation. In contrast, the restoration of protein glycosylation reduced LD accumulation. Interestingly, enhanced neutral lipids synthesis and LD formation under conditions of ER stress was not dependent on Ire1p. Lastly, we demonstrated that the absence of LDs did not compromise cell viability under ER stress. Our results suggest that although more LDs are produced, LDs are not essential to cell survival under ER stress. PMID:19708857

  1. Lipid droplets: a new player in colorectal cancer stem cells unveiled by spectroscopic imaging.

    PubMed

    Tirinato, Luca; Liberale, Carlo; Di Franco, Simone; Candeloro, Patrizio; Benfante, Antonina; La Rocca, Rosanna; Potze, Lisette; Marotta, Roberto; Ruffilli, Roberta; Rajamanickam, Vijayakumar P; Malerba, Mario; De Angelis, Francesco; Falqui, Andrea; Carbone, Ennio; Todaro, Matilde; Medema, Jan Paul; Stassi, Giorgio; Di Fabrizio, Enzo

    2015-01-01

    The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs.

  2. Lipid Droplets: A New Player in Colorectal Cancer Stem Cells Unveiled by Spectroscopic Imaging

    PubMed Central

    Tirinato, Luca; Liberale, Carlo; Di Franco, Simone; Candeloro, Patrizio; Benfante, Antonina; La Rocca, Rosanna; Potze, Lisette; Marotta, Roberto; Ruffilli, Roberta; Rajamanickam, Vijayakumar P; Malerba, Mario; De Angelis, Francesco; Falqui, Andrea; Carbone, Ennio; Todaro, Matilde; Medema, Jan Paul; Stassi, Giorgio; Di Fabrizio, Enzo

    2015-01-01

    The cancer stem cell (CSC) model is describing tumors as a hierarchical organized system and CSCs are suggested to be responsible for cancer recurrence after therapy. The identification of specific markers of CSCs is therefore of paramount importance. Here, we show that high levels of lipid droplets (LDs) are a distinctive mark of CSCs in colorectal (CR) cancer. This increased lipid content was clearly revealed by label-free Raman spectroscopy and it directly correlates with well-accepted CR-CSC markers as CD133 and Wnt pathway activity. By xenotransplantation experiments, we have finally demonstrated that CR-CSCs overexpressing LDs retain most tumorigenic potential. A relevant conceptual advance in this work is the demonstration that a cellular organelle, the LD, is a signature of CSCs, in addition to molecular markers. A further functional characterization of LDs could lead soon to design new target therapies against CR-CSCs. Stem Cells 2015;33:35–44 PMID:25186497

  3. Composition and occurrence of lipid droplets in the cyanobacterium Nostoc punctiforme

    PubMed Central

    Peramuna, Anantha; Summers, Michael L.

    2014-01-01

    Inclusions of neutral lipids termed lipid droplets (LDs) located throughout the cell were identified in the cyanobacterium Nostoc punctiforme by staining with lipophyllic fluorescent dyes. LDs increased in number upon entry into stationary phase and addition of exogenous fructose indicating a role for carbon storage, whereas high-light stress did not increase LD numbers. LD accumulation increased when nitrate was used as the nitrogen source during exponential growth as compared to added ammonia or nitrogen–fixing conditions. Analysis of isolated LDs revealed enrichment of triacylglycerol (TAG), - tochopherol, and C17 alkanes. LD TAG from exponential phase growth contained mainly saturated C16 and C18 fatty acids whereas stationary phase LD TAG had additional unsaturated fatty acids characteristic of whole cells. This is the first characterization of cyanobacterial LD composition and conditions leading to their production. Based upon their abnormally large size and atypical location these structures represent a novel sub-organelle in cyanobacteria. PMID:25135835

  4. A conserved family of proteins facilitates nascent lipid droplet budding from the ER

    PubMed Central

    Choudhary, Vineet; Ojha, Namrata; Golden, Andy

    2015-01-01

    Lipid droplets (LDs) are found in all cells and play critical roles in lipid metabolism. De novo LD biogenesis occurs in the endoplasmic reticulum (ER) but is not well understood. We imaged early stages of LD biogenesis using electron microscopy and found that nascent LDs form lens-like structures that are in the ER membrane, raising the question of how these nascent LDs bud from the ER as they grow. We found that a conserved family of proteins, fat storage-inducing transmembrane (FIT) proteins, is required for proper budding of LDs from the ER. Elimination or reduction of FIT proteins in yeast and higher eukaryotes causes LDs to remain in the ER membrane. Deletion of the single FIT protein in Caenorhabditis elegans is lethal, suggesting that LD budding is an essential process in this organism. Our findings indicated that FIT proteins are necessary to promote budding of nascent LDs from the ER. PMID:26504167

  5. Lipid Droplets Purified from Drosophila Embryos as an Endogenous Handle for Precise Motor Transport Measurements

    PubMed Central

    Bartsch, Tobias F.; Longoria, Rafael A.; Florin, Ernst-Ludwig; Shubeita, George T.

    2013-01-01

    Molecular motor proteins are responsible for long-range transport of vesicles and organelles. Recent works have elucidated the richness of the transport complex, with multiple teams of similar and dissimilar motors and their cofactors attached to individual cargoes. The interaction among these different proteins, and with the microtubules along which they translocate, results in the intricate patterns of cargo transport observed in cells. High-precision and high-bandwidth measurements are required to capture the dynamics of these interactions, yet the crowdedness in the cell necessitates performing such measurements in vitro. Here, we show that endogenous cargoes, lipid droplets purified from Drosophila embryos, can be used to perform high-precision and high-bandwidth optical trapping experiments to study motor regulation in vitro. Purified droplets have constituents of the endogenous transport complex attached to them and exhibit long-range motility. A novel method to determine the quality of the droplets for high-resolution measurements in an optical trap showed that they compare well with plastic beads in terms of roundness, homogeneity, position sensitivity, and trapping stiffness. Using high-resolution and high-bandwidth position measurements, we demonstrate that we can follow the series of binding and unbinding events that lead to the onset of active transport. PMID:24010661

  6. Droplet-Shooting and Size-Filtration (DSSF) Method for Synthesis of Cell-Sized Liposomes with Controlled Lipid Compositions.

    PubMed

    Morita, Masamune; Onoe, Hiroaki; Yanagisawa, Miho; Ito, Hiroaki; Ichikawa, Masatoshi; Fujiwara, Kei; Saito, Hirohide; Takinoue, Masahiro

    2015-09-21

    We report a centrifugal microfluidic method, droplet-shooting and size-filtration (DSSF), for the production of cell-sized liposomes with controlled lipid compositions. This involves the generation of large and small droplets from the tip of a glass capillary and the selective transfer of small droplets through an oil-water interface, thus resulting in the generation of cell-sized liposomes. We demonstrate control of the microdomain formation as well as the formation of asymmetric lipid bilayer liposomes of uniform size by the control of lipid composition. The DSSF method involves simple microfluidics and is easy to use. In addition, only a small volume (0.5-2 μL) of sample solution is required for the formation of hundreds of cell-sized liposomes. We believe that this method can be applied to generate cell-sized liposomes for a wide variety of uses, such as the construction of artificial cell-like systems.

  7. A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans

    PubMed Central

    Li, Shiwei; Xu, Shibin; Ma, Yanli; Wu, Shuang; Feng, Yu; Cui, Qingpo; Chen, Lifeng; Zhou, Shuang; Kong, Yuanyuan; Zhang, Xiaoyu; Yu, Jialei; Wu, Mengdi; Zhang, Shaobing O.

    2016-01-01

    To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 × 105 mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion. PMID:27261001

  8. Lipid droplets and their component triglycerides and steryl esters regulate autophagosome biogenesis

    PubMed Central

    Shpilka, Tomer; Welter, Evelyn; Borovsky, Noam; Amar, Nira; Mari, Muriel; Reggiori, Fulvio; Elazar, Zvulun

    2015-01-01

    Autophagy is a major catabolic process responsible for the delivery of proteins and organelles to the lysosome/vacuole for degradation. Malfunction of this pathway has been implicated in numerous pathological conditions. Different organelles have been found to contribute to the formation of autophagosomes, but the exact mechanism mediating this process remains obscure. Here, we show that lipid droplets (LDs) are important for the regulation of starvation-induced autophagy. Deletion of Dga1 and Lro1 enzymes responsible for triacylglycerol (TAG) synthesis, or of Are1 and Are2 enzymes responsible for the synthesis of steryl esters (STE), results in the inhibition of autophagy. Moreover, we identified the STE hydrolase Yeh1 and the TAG lipase Ayr1 as well as the lipase/hydrolase Ldh1 as essential for autophagy. Finally, we provide evidence that the ER-LD contact-site proteins Ice2 and Ldb16 regulate autophagy. Our study thus highlights the importance of lipid droplet dynamics for the autophagic process under nitrogen starvation. PMID:26162625

  9. A microfluidic device to study the digestion of trapped lipid droplets.

    PubMed

    Marze, Sébastien; Algaba, Hugo; Marquis, Mélanie

    2014-07-25

    At the junction of chemistry, physics and biology, digestion involves many processes. Studying the mechanisms in such a complex system is challenging because numerous interactions coexist. Even in an apparently simple system such as an emulsion, many physicochemical characteristics affect lipid digestion. Moreover, these characteristics are difficult to control using conventional in vitro techniques. The goal of this work was to design a microfluidic device allowing the study of well-controlled individual oil droplets under gastrointestinal digestion conditions. Different parameters were investigated in order to validate the relevance of this device compared to conventional in vitro techniques using emulsions. Various triglycerides and digestion conditions were tested with droplets of the same initial diameter generated by a flow focusing device, then placed in individual traps of a microfluidic chamber for digestion with continuous digestive juice renewal. The results are in good agreement with those obtained with conventional in vitro techniques and open the way to screening of lipid digestion, in particular, bioaccessibility of lipophilic molecules, a prerequisite for bioavailability studied in nutrition, pharmacology, and toxicology.

  10. A Genetic Screen for Mutants with Supersized Lipid Droplets in Caenorhabditis elegans.

    PubMed

    Li, Shiwei; Xu, Shibin; Ma, Yanli; Wu, Shuang; Feng, Yu; Cui, Qingpo; Chen, Lifeng; Zhou, Shuang; Kong, Yuanyuan; Zhang, Xiaoyu; Yu, Jialei; Wu, Mengdi; Zhang, Shaobing O

    2016-01-01

    To identify genes that regulate the dynamics of lipid droplet (LD) size, we have used the genetically tractable model organism Caenorhabditis elegans, whose wild-type LD population displays a steady state of size with an upper limit of 3 μm in diameter. From a saturated forward genetic screen of 6.7 × 10(5) mutagenized haploid genomes, we isolated 118 mutants with supersized intestinal LDs often reaching 10 μm. These mutants define nine novel complementation groups, in addition to four known genes (maoc-1, dhs-28, daf-22, and prx-10). The nine groups are named drop (lipid droplet abnormal) and categorized into four classes. Class I mutants drop-5 and drop-9, similar to prx-10, are up-regulated in ACS-22-DGAT-2-dependent LD growth, resistant to LD hydrolysis, and defective in peroxisome import. Class II mutants drop-2, drop-3, drop-6, and drop-7 are up-regulated in LD growth, are resistant to LD hydrolysis, but are not defective in peroxisome import. Class III mutants drop-1 and drop-8 are neither up-regulated in LD growth nor resistant to LD hydrolysis, but seemingly up-regulated in LD fusion. Class IV mutant drop-4 is cloned as sams-1 and, different to the other three classes, is ACS-22-independent and hydrolysis-resistant. These four classes of supersized LD mutants should be valuable for mechanistic studies of LD cellular processes including growth, hydrolysis, and fusion. PMID:27261001

  11. Function of Lipid Storage Droplet 1 (Lsd1) in Wing Development of Drosophila melanogaster

    PubMed Central

    Men, Tran Thanh; Binh, Tran Duy; Yamaguchi, Masamitsu; Huy, Nguyen Tien; Kamei, Kaeko

    2016-01-01

    Perilipins are evolutionarily conserved from Drosophila to humans, the lipid storage droplet 1 (Lsd1) is a Drosophila homolog of human perilipin 1. The function of Lsd1 as a regulator of lipolysis in Drosophila has been demonstrated, as the Lsd1 mutant causes an increase of lipid droplet size. However, the functions of this gene during development are still under investigation. In order to determine the function of Lsd1 during development, Lsd1 was knocked down in Drosophila using the GAL4-UAS system. Selective knockdown of Lsd1 in the dorsal wing disc caused an atrophied wing phenotype. The generation of reactive oxygen species in the wing pouch compartment of the Lsd1-knockdown flies was significantly higher than in the control. Immunostaining with caspase-3 antibody revealed a greater number of apoptotic cells in Lsd1-knockdown wing discs than in the control. Cell death by autophagy was also induced in the knockdown flies. Moreover, cells deprived of Lsd1 showed mitochondrial expansion and decreased ATP levels. These results strongly suggest that knockdown of Lsd1 induces mitochondrial stress and the production of reactive oxygen species that result in cell death, via apoptosis and the autophagy pathway. These results highlight the roles of Drosophila Lsd1 during wing development. PMID:27136547

  12. Dynamics of lipid droplets induced by the hepatitis C virus core protein

    SciTech Connect

    Lyn, Rodney K.; Kennedy, David C.; Stolow, Albert; Ridsdale, Andrew; Pezacki, John Paul

    2010-09-03

    Research highlights: {yields} Hepatitis C virus uses lipid droplets (LD) onto which HCV core proteins bind. {yields} HCV core proteins on LDs facilitate viral particle assembly. {yields} We used a novel combination of CARS, two-photon fluorescence, and DIC microscopies. {yields} Particle tracking experiments show that core slowly affects LD localization. {yields} Particle tracking measured the change in speed and directionality of LD movement. -- Abstract: The hepatitis C virus (HCV) is a global health problem, with limited treatment options and no vaccine available. HCV uses components of the host cell to proliferate, including lipid droplets (LD) onto which HCV core proteins bind and facilitate viral particle assembly. We have measured the dynamics of HCV core protein-mediated changes in LDs and rates of LD movement on microtubules using a combination of coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF), and differential interference contrast (DIC) microscopies. Results show that the HCV core protein induces rapid increases in LD size. Particle tracking experiments show that HCV core protein slowly affects LD localization by controlling the directionality of LD movement on microtubules. These dynamic processes ultimately aid HCV in propagating and the molecules and interactions involved represent novel targets for potential therapeutic intervention.

  13. Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes.

    PubMed

    Arrese, Estela L; Rivera, Laticia; Hamada, Masakazu; Mirza, Saima; Hartson, Steve D; Weintraub, Susan; Soulages, Jose L

    2008-05-01

    Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately alpha-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect. PMID:18342616

  14. Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus

    PubMed Central

    Barletta, Ana Beatriz Ferreira; Alves, Liliane Rosa; Nascimento Silva, Maria Clara L.; Sim, Shuzhen; Dimopoulos, George; Liechocki, Sally; Maya-Monteiro, Clarissa M.; Sorgine, Marcos H. Ferreira

    2016-01-01

    In mammals, lipid droplets (LDs) are ubiquitous organelles that modulate immune and inflammatory responses through the production of lipid mediators. In insects, it is unknown whether LDs play any role during the development of immune responses. We show that Aedes aegypti Aag2 cells – an immune responsive cell lineage – accumulates LDs when challenged with Enterobacter cloacae, Sindbis, and Dengue viruses. Microarray analysis of Aag2 challenged with E.cloacae or infected with Dengue virus revealed high transcripts levels of genes associated with lipid storage and LDs biogenesis, correlating with the increased LDs numbers in those conditions. Similarly, in mosquitoes, LDs accumulate in midgut cells in response to Serratia marcescens and Sindbis virus or when the native microbiota proliferates, following a blood meal. Also, constitutive activation of Toll and IMD pathways by knocking-down their respective negative modulators (Cactus and Caspar) increases LDs numbers in the midgut. Our results show for the first time an infection-induced LDs accumulation in response to both bacterial and viral infections in Ae. Aegypti, and we propose a role for LDs in mosquito immunity. These findings open new venues for further studies in insect immune responses associated with lipid metabolism. PMID:26887863

  15. Lipid droplet-mediated ER homeostasis regulates autophagy and cell survival during starvation.

    PubMed

    Velázquez, Ariadna P; Tatsuta, Takashi; Ghillebert, Ruben; Drescher, Ingmar; Graef, Martin

    2016-03-14

    Lipid droplets (LDs) are conserved organelles for intracellular neutral lipid storage. Recent studies suggest that LDs function as direct lipid sources for autophagy, a central catabolic process in homeostasis and stress response. Here, we demonstrate that LDs are dispensable as a membrane source for autophagy, but fulfill critical functions for endoplasmic reticulum (ER) homeostasis linked to autophagy regulation. In the absence of LDs, yeast cells display alterations in their phospholipid composition and fail to buffer de novo fatty acid (FA) synthesis causing chronic stress and morphologic changes in the ER. These defects compromise regulation of autophagy, including formation of multiple aberrant Atg8 puncta and drastically impaired autophagosome biogenesis, leading to severe defects in nutrient stress survival. Importantly, metabolically corrected phospholipid composition and improved FA resistance of LD-deficient cells cure autophagy and cell survival. Together, our findings provide novel insight into the complex interrelation between LD-mediated lipid homeostasis and the regulation of autophagy potentially relevant for neurodegenerative and metabolic diseases.

  16. Emerging role of lipid droplets in Aedes aegypti immune response against bacteria and Dengue virus.

    PubMed

    Barletta, Ana Beatriz Ferreira; Alves, Liliane Rosa; Silva, Maria Clara L Nascimento; Sim, Shuzhen; Dimopoulos, George; Liechocki, Sally; Maya-Monteiro, Clarissa M; Sorgine, Marcos H Ferreira

    2016-01-01

    In mammals, lipid droplets (LDs) are ubiquitous organelles that modulate immune and inflammatory responses through the production of lipid mediators. In insects, it is unknown whether LDs play any role during the development of immune responses. We show that Aedes aegypti Aag2 cells - an immune responsive cell lineage - accumulates LDs when challenged with Enterobacter cloacae, Sindbis, and Dengue viruses. Microarray analysis of Aag2 challenged with E.cloacae or infected with Dengue virus revealed high transcripts levels of genes associated with lipid storage and LDs biogenesis, correlating with the increased LDs numbers in those conditions. Similarly, in mosquitoes, LDs accumulate in midgut cells in response to Serratia marcescens and Sindbis virus or when the native microbiota proliferates, following a blood meal. Also, constitutive activation of Toll and IMD pathways by knocking-down their respective negative modulators (Cactus and Caspar) increases LDs numbers in the midgut. Our results show for the first time an infection-induced LDs accumulation in response to both bacterial and viral infections in Ae. Aegypti, and we propose a role for LDs in mosquito immunity. These findings open new venues for further studies in insect immune responses associated with lipid metabolism. PMID:26887863

  17. Chlamydia trachomatis Infection Leads to Defined Alterations to the Lipid Droplet Proteome in Epithelial Cells

    PubMed Central

    Saka, Hector Alex; Thompson, J. Will; Chen, Yi-Shan; Dubois, Laura G.; Haas, Joel T.; Moseley, Arthur; Valdivia, Raphael H.

    2015-01-01

    The obligate intracellular bacterium Chlamydia trachomatis is a major human pathogen and a main cause of genital and ocular diseases. During its intracellular cycle, C. trachomatis replicates inside a membrane-bound vacuole termed an “inclusion”. Acquisition of lipids (and other nutrients) from the host cell is a critical step in chlamydial replication. Lipid droplets (LD) are ubiquitous, ER-derived neutral lipid-rich storage organelles surrounded by a phospholipids monolayer and associated proteins. Previous studies have shown that LDs accumulate at the periphery of, and eventually translocate into, the chlamydial inclusion. These observations point out to Chlamydia-mediated manipulation of LDs in infected cells, which may impact the function and thereby the protein composition of these organelles. By means of a label-free quantitative mass spectrometry approach we found that the LD proteome is modified in the context of C. trachomatis infection. We determined that LDs isolated from C. trachomatis-infected cells were enriched in proteins related to lipid metabolism, biosynthesis and LD-specific functions. Interestingly, consistent with the observation that LDs intimately associate with the inclusion, a subset of inclusion membrane proteins co-purified with LD protein extracts. Finally, genetic ablation of LDs negatively affected generation of C. trachomatis infectious progeny, consistent with a role for LD biogenesis in optimal chlamydial growth. PMID:25909443

  18. Saturating Light Induces Sustained Accumulation of Oil in Plastidal Lipid Droplets in Chlamydomonas reinhardtii.

    PubMed

    Goold, Hugh Douglas; Cuiné, Stéphan; Légeret, Bertrand; Liang, Yuanxue; Brugière, Sabine; Auroy, Pascaline; Javot, Hélène; Tardif, Marianne; Jones, Brian; Beisson, Fred; Peltier, Gilles; Li-Beisson, Yonghua

    2016-08-01

    Enriching algal biomass in energy density is an important goal in algal biotechnology. Nitrogen (N) starvation is considered the most potent trigger of oil accumulation in microalgae and has been thoroughly investigated. However, N starvation causes the slow down and eventually the arrest of biomass growth. In this study, we show that exposing a Chlamydomonas reinhardtii culture to saturating light (SL) under a nonlimiting CO2 concentration in turbidostatic photobioreactors induces a sustained accumulation of lipid droplets (LDs) without compromising growth, which results in much higher oil productivity than N starvation. We also show that the polar membrane lipid fraction of SL-induced LDs is rich in plastidial lipids (approximately 70%), in contrast to N starvation-induced LDs, which contain approximately 60% lipids of endoplasmic reticulum origin. Proteomic analysis of LDs isolated from SL-exposed cells identified more than 200 proteins, including known proteins of lipid metabolism, as well as 74 proteins uniquely present in SL-induced LDs. LDs induced by SL and N depletion thus differ in protein and lipid contents. Taken together, lipidomic and proteomic data thus show that a large part of the sustained oil accumulation occurring under SL is likely due to the formation of plastidial LDs. We discuss our data in relation to the different metabolic routes used by microalgae to accumulate oil reserves depending on cultivation conditions. Finally, we propose a model in which oil accumulation is governed by an imbalance between photosynthesis and growth, which can be achieved by impairing growth or by boosting photosynthetic carbon fixation, with the latter resulting in higher oil productivity. PMID:27297678

  19. Influence of squalene on lipid particle/droplet and membrane organization in the yeast Saccharomyces cerevisiae

    PubMed Central

    Spanova, Miroslava; Zweytick, Dagmar; Lohner, Karl; Klug, Lisa; Leitner, Erich; Hermetter, Albin; Daum, Günther

    2012-01-01

    In a previous study (Spanova et al., 2010, J. Biol. Chem., 285, 6127–6133) we demonstrated that squalene, an intermediate of sterol biosynthesis, accumulates in yeast strains bearing a deletion of the HEM1 gene. In such strains, the vast majority of squalene is stored in lipid particles/droplets together with triacylglycerols and steryl esters. In mutants lacking the ability to form lipid particles, however, substantial amounts of squalene accumulate in organelle membranes. In the present study, we investigated the effect of squalene on biophysical properties of lipid particles and biological membranes and compared these results to artificial membranes. Our experiments showed that squalene together with triacylglycerols forms the fluid core of lipid particles surrounded by only a few steryl ester shells which transform into a fluid phase below growth temperature. In the hem1∆ deletion mutant a slight disordering effect on steryl esters was observed indicated by loss of the high temperature transition. Also in biological membranes from the hem1∆ mutant strain the effect of squalene per se is difficult to pinpoint because multiple effects such as levels of sterols and unsaturated fatty acids contribute to physical membrane properties. Fluorescence spectroscopic studies using endoplasmic reticulum, plasma membrane and artificial membranes revealed that it is not the absolute squalene level in membranes but rather the squalene to sterol ratio which mainly affects membrane fluidity/rigidity. In a fluid membrane environment squalene induces rigidity of the membrane, whereas in rigid membranes there is almost no additive effect of squalene. In summary, our results demonstrate that squalene (i) can be well accommodated in yeast lipid particles and organelle membranes without causing deleterious effects; and (ii) although not being a typical membrane lipid may be regarded as a mild modulator of biophysical membrane properties. PMID:22342273

  20. Human serum activates CIDEB-mediated lipid droplet enlargement in hepatoma cells

    SciTech Connect

    Singaravelu, Ragunath; Lyn, Rodney K.; Srinivasan, Prashanth; Delcorde, Julie; Steenbergen, Rineke H.; Tyrrell, D. Lorne; Pezacki, John P.

    2013-11-15

    Highlights: •Human serum induced differentiation of hepatoma cells increases cellular lipid droplet (LD) size. •The observed increase in LD size correlates with increased PGC-1α and CIDEB expression. •Induction of CIDEB expression correlates with rescue of VLDL secretion and loss of ADRP. •siRNA knockdown of CIDEB impairs the human serum mediated increase in LD size. •This system represents a cost-efficient model to study CIDEB’s role in lipid biology. -- Abstract: Human hepatocytes constitutively express the lipid droplet (LD) associated protein cell death-inducing DFFA-like effector B (CIDEB). CIDEB mediates LD fusion, as well as very-low-density lipoprotein (VLDL) maturation. However, there are limited cell culture models readily available to study CIDEB’s role in these biological processes, as hepatoma cell lines express negligible levels of CIDEB. Recent work has highlighted the ability of human serum to differentiate hepatoma cells. Herein, we demonstrate that culturing Huh7.5 cells in media supplemented with human serum activates CIDEB expression. This activation occurs through the induced expression of PGC-1α, a positive transcriptional regulator of CIDEB. Coherent anti-Stokes Raman scattering (CARS) microscopy revealed a correlation between CIDEB levels and LD size in human serum treated Huh7.5 cells. Human serum treatment also resulted in a rapid decrease in the levels of adipose differentiation-related protein (ADRP). Furthermore, individual overexpression of CIDEB was sufficient to down-regulate ADRP protein levels. siRNA knockdown of CIDEB revealed that the human serum mediated increase in LD size was CIDEB-dependent. Overall, our work highlights CIDEB’s role in LD fusion, and presents a new model system to study the PGC-1α/CIDEB pathway’s role in LD dynamics and the VLDL pathway.

  1. Group X secreted phospholipase A2 induces lipid droplet formation and prolongs breast cancer cell survival

    PubMed Central

    2013-01-01

    Background Alterations in lipid metabolism are inherent to the metabolic transformations that support tumorigenesis. The relationship between the synthesis, storage and use of lipids and their importance in cancer is poorly understood. The human group X secreted phospholipase A2 (hGX sPLA2) releases fatty acids (FAs) from cell membranes and lipoproteins, but its involvement in the regulation of cellular FA metabolism and cancer is not known. Results Here we demonstrate that hGX sPLA2 induces lipid droplet (LD) formation in invasive breast cancer cells, stimulates their proliferation and prevents their death on serum deprivation. The effects of hGX sPLA2 are shown to be dependent on its enzymatic activity, are mimicked by oleic acid and include activation of protein kinase B/Akt, a cell survival signaling kinase. The hGX sPLA2-stimulated LD biogenesis is accompanied by AMP-activated protein kinase (AMPK) activation, up-regulation of FA oxidation enzymes and the LD-coating protein perilipin 2, and suppression of lipogenic gene expression. Prolonged activation of AMPK inhibited hGX sPLA2-induced LD formation, while etomoxir, an inhibitor of FA oxidation, abrogated both LD formation and cell survival. The hGX sPLA2-induced changes in lipid metabolism provide a minimal immediate proliferative advantage during growth under optimal conditions, but they confer to the breast cancer cells a sustained ability to resist apoptosis during nutrient and growth factor limitation. Conclusion Our results identify hGX sPLA2 as a novel modulator of lipid metabolism that promotes breast cancer cell growth and survival by stimulating LD formation and FA oxidation. PMID:24070020

  2. Lipid Droplets: A Key Cellular Organelle Associated with Cancer Cell Survival under Normoxia and Hypoxia

    PubMed Central

    Koizume, Shiro; Miyagi, Yohei

    2016-01-01

    The Warburg effect describes the phenomenon by which cancer cells obtain energy from glycolysis even under normoxic (O2-sufficient) conditions. Tumor tissues are generally exposed to hypoxia owing to inefficient and aberrant vasculature. Cancer cells have multiple molecular mechanisms to adapt to such stress conditions by reprogramming the cellular metabolism. Hypoxia-inducible factors are major transcription factors induced in cancer cells in response to hypoxia that contribute to the metabolic changes. In addition, cancer cells within hypoxic tumor areas have reduced access to serum components such as nutrients and lipids. However, the effect of such serum factor deprivation on cancer cell biology in the context of tumor hypoxia is not fully understood. Cancer cells are lipid-rich under normoxia and hypoxia, leading to the increased generation of a cellular organelle, the lipid droplet (LD). In recent years, the LD-mediated stress response mechanisms of cancer cells have been revealed. This review focuses on the production and functions of LDs in various types of cancer cells in relation to the associated cellular environment factors including tissue oxygenation status and metabolic mechanisms. This information will contribute to the current understanding of how cancer cells adapt to diverse tumor environments to promote their survival. PMID:27589734

  3. Lipid Droplet Accumulation and Impaired Fat Efflux in Polarized Hepatic Cells: Consequences of Ethanol Metabolism

    PubMed Central

    McVicker, Benita L.; Rasineni, Karuna; Tuma, Dean J.; McNiven, Mark A.; Casey, Carol A.

    2012-01-01

    Steatosis, an early manifestation in alcoholic liver disease, is associated with the accumulation of hepatocellular lipid droplets (LDs). However, the role ethanol metabolism has in LD formation and turnover remains undefined. Here, we assessed LD dynamics following ethanol and oleic acid treatment to ethanol-metabolizing WIF-B cells (a hybrid of human fibroblasts (WI 38) and Fao rat hepatoma cells). An OA dose-dependent increase in triglyceride and stained lipids was identified which doubled (P < 0.05) in the presence of ethanol. This effect was blunted with the inclusion of an alcohol metabolism inhibitor. The ethanol/ OA combination also induced adipophilin, LD coat protein involved in the attenuation of lipolysis. Additionally, ethanol treatment resulted in a significant reduction in lipid efflux. These data demonstrate that the metabolism of ethanol in hepatic cells is related to LD accumulation, impaired fat efflux, and enhancements in LD-associated proteins. These alterations in LD dynamics may contribute to ethanol-mediated defects in hepatocellular LD regulation and the formation of steatosis. PMID:22506128

  4. Lipid Droplets: A Key Cellular Organelle Associated with Cancer Cell Survival under Normoxia and Hypoxia.

    PubMed

    Koizume, Shiro; Miyagi, Yohei

    2016-01-01

    The Warburg effect describes the phenomenon by which cancer cells obtain energy from glycolysis even under normoxic (O₂-sufficient) conditions. Tumor tissues are generally exposed to hypoxia owing to inefficient and aberrant vasculature. Cancer cells have multiple molecular mechanisms to adapt to such stress conditions by reprogramming the cellular metabolism. Hypoxia-inducible factors are major transcription factors induced in cancer cells in response to hypoxia that contribute to the metabolic changes. In addition, cancer cells within hypoxic tumor areas have reduced access to serum components such as nutrients and lipids. However, the effect of such serum factor deprivation on cancer cell biology in the context of tumor hypoxia is not fully understood. Cancer cells are lipid-rich under normoxia and hypoxia, leading to the increased generation of a cellular organelle, the lipid droplet (LD). In recent years, the LD-mediated stress response mechanisms of cancer cells have been revealed. This review focuses on the production and functions of LDs in various types of cancer cells in relation to the associated cellular environment factors including tissue oxygenation status and metabolic mechanisms. This information will contribute to the current understanding of how cancer cells adapt to diverse tumor environments to promote their survival. PMID:27589734

  5. Electron spin resonance studies on intact cells and isolated lipid droplets from fatty acid-modified L1210 murine leukemia.

    PubMed

    Simon, I; Burns, C P; Spector, A A

    1982-07-01

    It has been suggested that the formation of cytoplasmic lipid droplets may produce an artifact and be responsible for the differences in membrane physical properties detected in lipid-modified cells using fluorescence polarization or spin label probes. To investigate this, the electron spin resonance spectra of lipid droplets isolated from the cytoplasm of L1210 leukemia cells were compared with spectra obtained from the intact cell. Mice bearing the L1210 leukemia were fed diets containing either 16% sunflower oil or 16% coconut oil in order to modify the fatty acid composition of the tumor. A microsome-rich fraction prepared from L1210 cells grown in animals fed the sunflower oil-rich diet contained more polyenoic fatty acids (52 versus 29%), while microsomes from L1210 cells grown in animals fed the coconut oil-rich diets contained more monoenoic fatty acids (37 versus 12%). The order parameter calculated for lipid droplets labeled with the 5-nitroxystearic acid spin probe was only about one-half that of intact cells, whereas it was similar to that obtained for pure triolein droplets suspended in buffer. Order parameters of the inner hyperfine splittings calculated from the spectra of cells grown in the sunflower oil-fed animals [0.543 +/- 0.001 (S.E.)] were lower than those from the cells grown in animals fed the coconut oil diets (0.555 +/- 0.002) (p less than 0.005). In contrast, the order parameters of the lipid droplets isolated from the cells grown in animals fed sunflower oil (0.303 +/- 0.029) or coconut oil (0.295 +/- 0.021) were not significantly different, indicating that motion of a spin label probe in the highly fluid cytoplasmic lipid droplets is not affected by these types of modifications in cellular fatty acid composition. Therefore, the electron spin resonance changes that are observed in the intact cells cannot be due to localization of the probe in cytoplasmic lipid droplets. These results support the conclusion that the electron spin

  6. Intramembrane proteolysis promotes trafficking of hepatitis C virus core protein to lipid droplets.

    PubMed

    McLauchlan, John; Lemberg, Marius K; Hope, Graham; Martoglio, Bruno

    2002-08-01

    Hepatitis C virus (HCV) is the major causative pathogen associated with liver cirrhosis and hepatocellular carcinoma. The virus has a positive-sense RNA genome encoding a single polyprotein with the virion components located in the N-terminal portion. During biosynthesis of the polyprotein, an internal signal sequence between the core protein and the envelope protein E1 targets the nascent polypeptide to the endoplasmic reticulum (ER) membrane for translocation of E1 into the ER. Following membrane insertion, the signal sequence is cleaved from E1 by signal peptidase. Here we provide evidence that after cleavage by signal peptidase, the signal peptide is further processed by the intramembrane-cleaving protease SPP that promotes the release of core protein from the ER membrane. Core protein is then free for subsequent trafficking to lipid droplets. This study represents an example of a potential role for intramembrane proteolysis in the maturation of a viral protein. PMID:12145199

  7. Role of the hydrophobic domain in targeting caveolin-1 to lipid droplets

    PubMed Central

    Ostermeyer, Anne G.; Ramcharan, Lynne T.; Zeng, Youchun; Lublin, Douglas M.; Brown, Deborah A.

    2004-01-01

    Although caveolins normally reside in caveolae, they can accumulate on the surface of cytoplasmic lipid droplets (LDs). Here, we first provided support for our model that overaccumulation of caveolins in the endoplasmic reticulum (ER) diverts the proteins to nascent LDs budding from the ER. Next, we found that a mutant H-Ras, present on the cytoplasmic surface of the ER but lacking a hydrophobic peptide domain, did not accumulate on LDs. We used the fact that wild-type caveolin-1 accumulates in LDs after brefeldin A treatment or when linked to an ER retrieval motif to search for mutants defective in LD targeting. The hydrophobic domain, but no specific sequence therein, was required for LD targeting of caveolin-1. Certain Leu insertions blocked LD targeting, independently of hydrophobic domain length, but dependent on their position in the domain. We propose that proper packing of putative hydrophobic helices may be required for LD targeting of caveolin-1. PMID:14709541

  8. Lipid Droplets Are Essential for Efficient Clearance of Cytosolic Inclusion Bodies.

    PubMed

    Moldavski, Ofer; Amen, Triana; Levin-Zaidman, Smadar; Eisenstein, Miriam; Rogachev, Ilana; Brandis, Alexander; Kaganovich, Daniel; Schuldiner, Maya

    2015-06-01

    Exposing cells to folding stress causes a subset of their proteins to misfold and accumulate in inclusion bodies (IBs). IB formation and clearance are both active processes, but little is known about their mechanism. To shed light on this issue, we performed a screen with over 4,000 fluorescently tagged yeast proteins for co-localization with a model misfolded protein that marks IBs during folding stress. We identified 13 proteins that co-localize to IBs. Remarkably, one of these IB proteins, the uncharacterized and conserved protein Iml2, exhibited strong physical interactions with lipid droplet (LD) proteins. Indeed, we here show that IBs and LDs are spatially and functionally linked. We further demonstrate a mechanism for IB clearance via a sterol-based metabolite emanating from LDs. Our findings therefore uncover a function for Iml2 and LDs in regulating a critical stage of cellular proteostasis.

  9. Pioglitazone reduces lipid droplets in cholesterolosis of the gallbladder by increasing ABCA1 and NCEH1 expression.

    PubMed

    Wang, Jing-Min; Wang, Dong; Tan, Yu-Yan; Zhao, Gang; Ji, Zhen-Ling

    2015-01-01

    As a cholesterol-induced metabolic disease, cholesterolosis of the gallbladder is often resected clinically, which could lead to many complications. The histopathology of cholesterolosis is due to excessive lipid droplet accumulation in epithelial and subcutaneous tissues. The main components of lipid droplets are cholesterol esters (CEs). Removal of CEs from gallbladder epithelial cells (GBECs) is very important for maintaining intracellular cholesterol homeostasis and for treating cholesterol-related diseases. In this study, pioglitazone was used to reduce intracellular CEs. To further elucidate the mechanism, cholesterolosis GBECs were treated with pioglitazone, 22-(R)-hydroxycholesterol (a liver X receptor α (LXRα) agonist), or peroxisome proliferator-activated receptor gamma (PPARγ) siRNA. Western blotting for PPARγ, LXRα, ATP-binding cassette transporter A1 (ABCA1), and neutral cholesteryl ester hydrolase 1 (NCEH1) was performed. At length, cholesterol efflux to apoA-I was measured, and oil red O staining was used to visualize lipid droplet variations in cells. In conclusion, we observed that pioglitazone increased ABCA1 expression in an LXR-dependent manner and NCEH1 expression in an LXRα-independent manner, which mobilized CE hydrolysis and cholesterol efflux to reduce lipid droplet content in cholesterolosis GBECs. Our data provide a plausible alternative to human gallbladder cholesterolosis.

  10. Fluorescent phosphocholine--a specific marker for the endoplasmic reticulum and for lipid droplets in Chara internodal cells.

    PubMed

    Foissner, Ilse

    2009-12-01

    The staining pattern of 1,2-bis(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (Bodipy PC) was investigated in internodal cells of the green alga Chara corallina. Ten minutes after dye addition, Bodipy-PC-derived fluorescence appeared in lipid droplets and after 1 h in the cortical endoplasmic reticulum (ER) and in the inner ER tubes. Staining of the ER required energy but was independent of an intact actin or microtubule cytoskeleton and independent of vesicular endocytosis. The size of the lipid droplets varied between 0.25 microm in elongating cells and 3.2 microm in senescent internodes. They moved together with or along the cortical ER cisternae in a cytoskeleton-independent manner or remained immobile up to several minutes. Detachment of lipid droplets from the cortical ER or fusion of lipid droplets was never observed. The results of this study suggest that Bodipy PC is a valuable, less toxic alternative to 3,3'-dihexyloxacarbocyanine iodide (DiOC6) staining of the ER in Chara. They confirm an earlier report about microtubule-dependent cortical ER morphology and dynamics in elongating internodes and offer new perspectives for the study of organelle interactions.

  11. Antiatherogenic activity of fungal beauveriolides, inhibitors of lipid droplet accumulation in macrophages

    PubMed Central

    Namatame, Ichiji; Tomoda, Hiroshi; Ishibashi, Shun; Ōmura, Satoshi

    2004-01-01

    Beauveriolides I and III, isolated from the culture broth of fungal Beauveria sp. FO-6979, showed potent inhibitory activity of lipid droplet accumulation in primary mouse peritoneal macrophages. The cellular molecular target of this inhibitory activity was studied in macrophages. Beauveriolides I and III strongly inhibited the cholesteryl ester (CE) synthesis with IC50 values of 0.78 and 0.41 μM, respectively, without showing significant effects on the triacylglycerol and phospholipid synthesis. Furthermore, lysosomal cholesterol metabolism to CE in macrophages was inhibited by the compounds, indicating that the inhibition site lies within steps between cholesterol departure from the lysosome and CE synthesis in the endoplasmic reticulum. Therefore, acyl-CoA:cholesterol acyltransferase (ACAT) activity in the membrane fractions prepared from mouse macrophages was studied, resulting in a dose-dependent inhibition by beauveriolides I and III with IC50 values of 6.0 and 5.5 μM, respectively. Thus, we showed that the beauveriolides inhibit macrophage ACAT activity specifically, resulting in blockage of the CE synthesis, leading to a reduction of lipid droplets in macrophages. ACAT activity in the membrane fractions prepared from mouse liver and Caco-2 cells was also inhibited, indicating that the beauveriolides block both ACAT-1 and -2. Moreover, beauveriolides I and III exert antiatherogenic activity in both low-density lipoprotein receptor- and apolipoprotein E-knockout mice without any side effects such as diarrhea or cytotoxicity to adrenal tissues as observed for many synthetic ACAT inhibitors. Beauveriolides I and III are the first microbial cyclodepsipeptides having an in vivo antiatherosclerotic effect and show promise as potential lead compounds for antiatherosclerotic agents. PMID:14718664

  12. Lipid Droplets, Perilipins and Cytokeratins – Unravelled Liaisons in Epithelium-Derived Cells

    PubMed Central

    Heid, Hans; Rickelt, Steffen; Zimbelmann, Ralf; Winter, Stefanie; Schumacher, Heiderose; Dörflinger, Yvette

    2013-01-01

    Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron– in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins. PMID:23704888

  13. Lipid droplets, perilipins and cytokeratins--unravelled liaisons in epithelium-derived cells.

    PubMed

    Heid, Hans; Rickelt, Steffen; Zimbelmann, Ralf; Winter, Stefanie; Schumacher, Heiderose; Dörflinger, Yvette

    2013-01-01

    Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5). Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron- in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.

  14. Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria.

    PubMed

    Strauss, Juliette A; Shaw, Christopher S; Bradley, Helen; Wilson, Oliver J; Dorval, Thierry; Pilling, James; Wagenmakers, Anton J M

    2016-01-01

    Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD-derived fatty acids (FAs) into neighboring mitochondria for β-oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase (r = 0.50 ± 0.012) and to a lesser extent with LDs (r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles. PMID:26733245

  15. Apoptosis inhibitor of macrophage (AIM) diminishes lipid droplet-coating proteins leading to lipolysis in adipocytes

    SciTech Connect

    Iwamura, Yoshihiro; Mori, Mayumi; Nakashima, Katsuhiko; Mikami, Toshiyuki; Murayama, Katsuhisa; Arai, Satoko; Miyazaki, Toru

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer AIM induces lipolysis in a distinct manner from that of hormone-dependent lipolysis. Black-Right-Pointing-Pointer AIM ablates activity of peroxisome proliferator-activated receptor in adipocytes. Black-Right-Pointing-Pointer AIM reduces mRNA levels of lipid-droplet coating proteins leading to lipolysis. -- Abstract: Under fasting conditions, triacylglycerol in adipose tissue undergoes lipolysis to supply fatty acids as energy substrates. Such lipolysis is regulated by hormones, which activate lipases via stimulation of specific signalling cascades. We previously showed that macrophage-derived soluble protein, AIM induces obesity-associated lipolysis, triggering chronic inflammation in fat tissue which causes insulin resistance. However, the mechanism of how AIM mediates lipolysis remains unknown. Here we show that AIM induces lipolysis in a manner distinct from that of hormone-dependent lipolysis, without activation or augmentation of lipases. In vivo and in vitro, AIM did not enhance phosphorylation of hormone-sensitive lipase (HSL) in adipocytes, a hallmark of hormone-dependent lipolysis activation. Similarly, adipose tissue from obese AIM-deficient and wild-type mice showed comparable HSL phosphorylation. Consistent with the suppressive effect of AIM on fatty acid synthase activity, the amount of saturated and unsaturated fatty acids was reduced in adipocytes treated with AIM. This response ablated transcriptional activity of peroxisome proliferator-activated receptor (PPAR{gamma}), leading to diminished gene expression of lipid-droplet coating proteins including fat-specific protein 27 (FSP27) and Perilipin, which are indispensable for triacylglycerol storage in adipocytes. Accordingly, the lipolytic effect of AIM was overcome by a PPAR{gamma}-agonist or forced expression of FSP27, while it was synergized by a PPAR{gamma}-antagonist. Overall, distinct modes of lipolysis appear to take place in different physiological

  16. ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment.

    PubMed

    Suzuki, Michitaka; Murakami, Tatsuro; Cheng, Jinglei; Kano, Hiroyuki; Fukata, Masaki; Fujimoto, Toyoshi

    2015-06-15

    Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs.

  17. Translation inhibitors induce formation of cholesterol ester-rich lipid droplets.

    PubMed

    Suzuki, Michitaka; Ohsaki, Yuki; Tatematsu, Tsuyako; Shinohara, Yuki; Maeda, Takashi; Cheng, Jinglei; Fujimoto, Toyoshi

    2012-01-01

    Lipid droplets (LDs) in non-adipocytes contain triglycerides (TG) and cholesterol esters (CE) in variable ratios. TG-rich LDs are generated when unsaturated fatty acids are administered, but the conditions that induce CE-rich LD formation are less well characterized. In the present study, we found that protein translation inhibitors such as cycloheximide (CHX) induced generation of CE-rich LDs and that TIP47 (perilipin 3) was recruited to the LDs, although the expression of this protein was reduced drastically. Electron microscopy revealed that LDs formed in CHX-treated cells possess a distinct electron-dense rim that is not found in TG-rich LDs, whose formation is induced by oleic acid. CHX treatment caused upregulation of mTORC1, but the CHX-induced increase in CE-rich LDs occurred even when rapamycin or Torin1 was given along with CHX. Moreover, the increase in CE was seen in both wild-type and autophagy-deficient Atg5-null mouse embryonic fibroblasts, indicating that mTORC1 activation and suppression of autophagy are not necessary to induce the observed phenomenon. The results showed that translation inhibitors cause a significant change in the lipid ester composition of LDs by a mechanism independent of mTORC1 signaling and autophagy. PMID:22879956

  18. Trapping toxins within lipid droplets is a resistance mechanism in fungi

    PubMed Central

    Chang, Wenqiang; Zhang, Ming; Zheng, Sha; Li, Ying; Li, Xiaobin; Li, Wei; Li, Gang; Lin, Zhaomin; Xie, Zhiyu; Zhao, Zuntian; Lou, Hongxiang

    2015-01-01

    Lipid droplets (LDs) act as intracellular storage organelles in most types of cells and are principally involved in energy homeostasis and lipid metabolism. However, the role of LDs in resistance to toxins in fungi remains largely unknown. Here, we show that the trapping of endogenous toxins by LDs is a self-resistance mechanism in the toxin producer, while absorbing external lipophilic toxins is a resistance mechanism in the toxin recipient that acts to quench the production of reactive oxygen species. We found that an endolichenic fungus that generates phototoxic perylenequinones (PQs) trapped the PQs inside LDs. Using a model that incorporates the fungicidal action of hypocrellin A (HA), a PQ derivative, we showed that yeast cells escaped killing by trapping toxins inside LDs. Furthermore, LD-deficient mutants were hypersusceptible to HA-mediated phototoxins and other fungicides. Our study identified a previously unrecognised function of LDs in fungi that has implications for our understanding of environmental adaptation strategies for fungi and antifungal drug discovery. PMID:26463663

  19. ELMOD2 is anchored to lipid droplets by palmitoylation and regulates adipocyte triglyceride lipase recruitment.

    PubMed

    Suzuki, Michitaka; Murakami, Tatsuro; Cheng, Jinglei; Kano, Hiroyuki; Fukata, Masaki; Fujimoto, Toyoshi

    2015-06-15

    Adipocyte triglyceride lipase (ATGL) is the major enzyme involved in the hydrolysis of triglycerides. The Arf1-coat protein complex I (COPI) machinery is known to be engaged in the recruitment of ATGL to lipid droplets (LDs), but the regulatory mechanism has not been clarified. In the present study, we found that ELMOD2, a putative noncanonical Arf-GTPase activating protein (GAP) localizing in LDs, plays an important role in controlling ATGL transport to LDs. We showed that knockdown of ELMOD2 by RNA interference induced an increase in the amount of ATGL existing in LDs and decreased the total cellular triglycerides. These effects of ELMOD2 knockdown were canceled by transfection of small interfering RNA-resistant cDNA of wild-type ELMOD2 but not by that of mutated ELMOD2 lacking the Arf-GAP activity. ELMOD2 was distributed in the endoplasmic reticulum and mitochondria as well as in LDs, but palmitoylation was required only for distribution to LDs. An ELMOD2 mutant deficient in palmitoylation failed to reconstitute the ATGL transport after the ELMOD2 knockdown, indicating that distribution in LDs is indispensable to the functionality of ELMOD2. These results indicate that ELMOD2 regulates ATGL transport and cellular lipid metabolism by modulating the Arf1-COPI activity in LDs. PMID:25904333

  20. Characterization of the Proteome of Cytoplasmic Lipid Droplets in Mouse Enterocytes after a Dietary Fat Challenge

    PubMed Central

    D’Aquila, Theresa; Sirohi, Devika; Grabowski, Jeffrey M.; Hedrick, Victoria E.; Paul, Lake N.; Greenberg, Andrew S.; Kuhn, Richard J.; Buhman, Kimberly K.

    2015-01-01

    Dietary fat absorption by the small intestine is a multistep process that regulates the uptake and delivery of essential nutrients and energy. One step of this process is the temporary storage of dietary fat in cytoplasmic lipid droplets (CLDs). The storage and mobilization of dietary fat is thought to be regulated by proteins that associate with the CLD; however, mechanistic details of this process are currently unknown. In this study we analyzed the proteome of CLDs isolated from enterocytes harvested from the small intestine of mice following a dietary fat challenge. In this analysis we identified 181 proteins associated with the CLD fraction, of which 37 are associated with known lipid related metabolic pathways. We confirmed the localization of several of these proteins on or around the CLD through confocal and electron microscopy, including perilipin 3, apolipoprotein A-IV, and acyl-CoA synthetase long-chain family member 5. The identification of the enterocyte CLD proteome provides new insight into potential regulators of CLD metabolism and the process of dietary fat absorption. PMID:25992653

  1. PML isoform II plays a critical role in nuclear lipid droplet formation

    PubMed Central

    Ohsaki, Yuki; Kawai, Takeshi; Yoshikawa, Yukichika; Cheng, Jinglei; Jokitalo, Eija

    2016-01-01

    Lipid droplets (LDs) in the nucleus of hepatocyte-derived cell lines were found to be associated with premyelocytic leukemia (PML) nuclear bodies (NBs) and type I nucleoplasmic reticulum (NR) or the extension of the inner nuclear membrane. Knockdown of PML isoform II (PML-II) caused a significant decrease in both nuclear LDs and type I NR, whereas overexpression of PML-II increased both. Notably, these effects were evident only in limited types of cells, in which a moderate number of nuclear LDs exist intrinsically, and PML-II was targeted not only at PML NBs, but also at the nuclear envelope, excluding lamins and SUN proteins. Knockdown of SUN proteins induced a significant increase in the type I NR and nuclear LDs, but these effects were cancelled by simultaneous knockdown of PML-II. Nuclear LDs harbored diacylglycerol O-acyltransferase 2 and CTP:phosphocholine cytidylyltransferase α and incorporated newly synthesized lipid esters. These results corroborated that PML-II plays a critical role in generating nuclear LDs in specific cell types. PMID:26728854

  2. Fenofibrate insulates diacylglycerol in lipid droplet/ER and preserves insulin signaling transduction in the liver of high fat fed mice.

    PubMed

    Chan, Stanley M H; Zeng, Xiao-Yi; Sun, Ruo-Qiong; Jo, Eunjung; Zhou, Xiu; Wang, Hao; Li, Songpei; Xu, Aimin; Watt, Matthew J; Ye, Ji-Ming

    2015-07-01

    Hepatic steatosis is often associated with insulin resistance as a hallmark of the metabolic syndrome in the liver. The present study investigated the effects of PPARα activation induced by fenofibrate (FB) on the relationship of insulin resistance and hepatic steatosis in mice fed a high-fat (HF) diet, which increases lipid influx into the liver. Mice were fed HF diet to induce insulin resistance and hepatic steatosis with or without FB. FB activated PPARα and ameliorated HF diet-induced glucose intolerance and hepatic insulin resistance without altering either hepatic steatosis or inflammation signaling (JNK or IKK). Interestingly, FB treatment simultaneously increased fatty acid (FA) synthesis (50%) and oxidation (66%, both p<0.01) into intermediate lipid metabolites, suggesting a FA oxidation-synthesis cycling in operation. Associated with these effects, diacylglycerols (DAGs) were sequestered within the lipid droplet/ER compartment, thus reducing their deposition in the cellular membrane, which is known to impair insulin signal transduction. These findings suggest that the reduction in membrane DAGs (rather than total hepatic steatosis) may be critical for the protection by fenofibrate-induced PPARα activation against hepatic insulin resistance induced by dietary fat. PMID:25906681

  3. Enhancement of carotenoid bioaccessibility from carrots using excipient emulsions: influence of particle size of digestible lipid droplets.

    PubMed

    Zhang, Ruojie; Zhang, Zipei; Zou, Liqiang; Xiao, Hang; Zhang, Guodong; Decker, Eric Andrew; McClements, David Julian

    2016-01-01

    The influence of initial lipid droplet size on the ability of excipient emulsions to increase carotenoid bioaccessibility from carrots was investigated using a simulated gastrointestinal tract (GIT). Corn oil-in-water excipient emulsions were fabricated with different surface-weighted mean droplet diameters: d32 = 0.17 μm (fine), 0.46 μm (medium), and, 10 μm (large). Bulk oil containing a similar quantity of lipids as the emulsions was used as a control. The excipient emulsions and control were mixed with pureed carrots, and then passed through a simulated GIT (mouth, stomach, and small intestine), and changes in particle size, charge, microstructure, lipid digestion, and carotenoid bioaccessibility were measured. Carotenoid bioaccessibility significantly increased with decreasing lipid droplet size in the excipient emulsions, which was attributed to the rapid formation of mixed micelles that could solubilize the carotenoids in the intestinal fluids. These results have important implications for designing excipient foods, such as dressings, dips, creams, and sauces, to increase the bioavailability of health-promoting nutraceuticals in foods. PMID:26583923

  4. Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition.

    PubMed

    Khanthusaeng, Vilaivan; Thammasiri, Jiratti; Bass, Casie S; Navanukraw, Chainarong; Borowicz, Pawel; Redmer, Dale A; Grazul-Bilska, Anna T

    2016-07-01

    Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n=9), overfed (O; 2×C, n=12), or underfed (U; 0.6×C, n=10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P<0.05) at the mid than early-luteal phase, and were not affected by nutritional plane. LD total volume and number were greater (P<0.001) in LLC than SLC; however, mean volume of individual LD was greater (P<0.02) in SLC than LLC. In LLC, total LD volume was greater (P<0.02) in O than C and U ewes. In SLC, total LD volume and number was greater (P<0.003) at the mid than early-luteal phase, and percentage of cell volume occupied by LD was greater (P<0.002) in U than C and O ewes. These data demonstrate that both stage of luteal development and nutritional plane affect selected LD measurements and thus may affect luteal functions

  5. Lipid droplets in cultured luteal cells in non-pregnant sheep fed different planes of nutrition.

    PubMed

    Khanthusaeng, Vilaivan; Thammasiri, Jiratti; Bass, Casie S; Navanukraw, Chainarong; Borowicz, Pawel; Redmer, Dale A; Grazul-Bilska, Anna T

    2016-07-01

    Accumulation of lipid droplets (LD) in luteal cells likely is important for energy storage and steroidogenesis in the highly metabolically active corpus luteum (CL). The objective of this study was to determine the effect of plane of nutrition on progesterone (P4) secretion, and lipid droplet number and size in cultured ovine luteal cells. Ewes were randomly assigned to one of three nutritional groups: control (C; 100% NRC requirements, n=9), overfed (O; 2×C, n=12), or underfed (U; 0.6×C, n=10). Superovulation was induced by follicle stimulating hormone injections. At the early and mid-luteal phases of the estrous cycle, CL were dissected from ovaries, and luteal cells isolated enzymatically. Luteal cells were incubated overnight in medium containing serum in chamber slides. Media were then changed to serum-free and after 24h incubation, media were collected for P4 analysis, and cells were fixed in formalin and stained with BODIPY followed by DAPI staining. Z-stacks of optical sections of large and small luteal cells (LLC and SLC, respectively) were obtained using a laser-scanning microscope. Rendered 3D images of individual LLC and SLC were analyzed for cell volume, and total and individual LD volume, number and percentage of cellular volume occupied by LD by using Imaris software. Concentrations of P4 in serum and media were greater (P<0.05) at the mid than early-luteal phase, and were not affected by nutritional plane. LD total volume and number were greater (P<0.001) in LLC than SLC; however, mean volume of individual LD was greater (P<0.02) in SLC than LLC. In LLC, total LD volume was greater (P<0.02) in O than C and U ewes. In SLC, total LD volume and number was greater (P<0.003) at the mid than early-luteal phase, and percentage of cell volume occupied by LD was greater (P<0.002) in U than C and O ewes. These data demonstrate that both stage of luteal development and nutritional plane affect selected LD measurements and thus may affect luteal functions

  6. Structure of a CGI-58 motif provides the molecular basis of lipid droplet anchoring.

    PubMed

    Boeszoermenyi, Andras; Nagy, Harald Manuel; Arthanari, Haribabu; Pillip, Christoph Jens; Lindermuth, Hanna; Luna, Rafael Eulogio; Wagner, Gerhard; Zechner, Rudolf; Zangger, Klaus; Oberer, Monika

    2015-10-30

    Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp(21) and Trp(25)) and right (harboring Trp(29)) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp(21) and Trp(25) and two adjacent leucines. Trp(29) serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm.

  7. Structure of a CGI-58 Motif Provides the Molecular Basis of Lipid Droplet Anchoring*

    PubMed Central

    Boeszoermenyi, Andras; Nagy, Harald Manuel; Arthanari, Haribabu; Pillip, Christoph Jens; Lindermuth, Hanna; Luna, Rafael Eulogio; Wagner, Gerhard; Zechner, Rudolf; Zangger, Klaus; Oberer, Monika

    2015-01-01

    Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10–31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp21 and Trp25) and right (harboring Trp29) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp21 and Trp25 and two adjacent leucines. Trp29 serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm. PMID:26350461

  8. Structure of a CGI-58 motif provides the molecular basis of lipid droplet anchoring.

    PubMed

    Boeszoermenyi, Andras; Nagy, Harald Manuel; Arthanari, Haribabu; Pillip, Christoph Jens; Lindermuth, Hanna; Luna, Rafael Eulogio; Wagner, Gerhard; Zechner, Rudolf; Zangger, Klaus; Oberer, Monika

    2015-10-30

    Triacylglycerols (TGs) stored in lipid droplets (LDs) are hydrolyzed in a highly regulated metabolic process called lipolysis to free fatty acids that serve as energy substrates for β-oxidation, precursors for membrane lipids and signaling molecules. Comparative gene identification-58 (CGI-58) stimulates the enzymatic activity of adipose triglyceride lipase (ATGL), which catalyzes the hydrolysis of TGs to diacylglycerols and free fatty acids. In adipose tissue, protein-protein interactions between CGI-58 and the LD coating protein perilipin 1 restrain the ability of CGI-58 to activate ATGL under basal conditions. Phosphorylation of perilipin 1 disrupts these interactions and mobilizes CGI-58 for the activation of ATGL. We have previously demonstrated that the removal of a peptide at the N terminus (residues 10-31) of CGI-58 abrogates CGI-58 localization to LDs and CGI-58-mediated activation of ATGL. Here, we show that this tryptophan-rich N-terminal peptide serves as an independent LD anchor, with its three tryptophans serving as focal points of the left (harboring Trp(21) and Trp(25)) and right (harboring Trp(29)) anchor arms. The solution state NMR structure of a peptide comprising the LD anchor bound to dodecylphosphocholine micelles as LD mimic reveals that the left arm forms a concise hydrophobic core comprising tryptophans Trp(21) and Trp(25) and two adjacent leucines. Trp(29) serves as the core of a functionally independent anchor arm. Consequently, simultaneous tryptophan alanine permutations in both arms abolish localization and activity of CGI-58 as opposed to tryptophan substitutions that occur in only one arm. PMID:26350461

  9. Unique Regulation of Adipose Triglyceride Lipase (ATGL) by Perilipin 5, a Lipid Droplet-associated Protein*

    PubMed Central

    Wang, Hong; Bell, Ming; Sreenevasan, Urmilla; Hu, Hong; Liu, Jun; Dalen, Knut; Londos, Constantine; Yamaguchi, Tomohiro; Rizzo, Mark A.; Coleman, Rosalind; Gong, Dawei; Brasaemle, Dawn; Sztalryd, Carole

    2011-01-01

    Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [32P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [32P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL. PMID:21393244

  10. Simple and Stable Lipid Bilayer Formation: A Droplets Contacting Method using Parylene Micro-pores for Multiple Ion Channel Recordings

    NASA Astrophysics Data System (ADS)

    Tsuji, Yutaro; Kawano, Ryuji; Osaki, Toshihisa; Sasaki, Hirotaka; Miki, Norihisa; Takeuchi, Shoji

    This paper describes multiple ion-channel recordings through membrane proteins reconstituted in bilayer lipid membranes (BLMs) array. The BLMs array can be prepared by “Droplets Contacting Method” which forms BLMs at the interface of two lipid monolayers. Since this method does not require skilled techniques, it is highly reproducible and can be applied to automated system. We used a double well chip (DWC) for the droplets contacting method. We attempted to confine the BLMs forming areas with parylene micro-pore (parylene double well chip, PDWC) to augment the mechanical stability of BLMs. Subsequently, we arrayed the PDWC with electrodes for multiple recordings of channel proteins. We successfully demonstrated 14 channels simultaneous ion channel recordings through α-hemolysin.

  11. Lipid droplets hypertrophy: a crucial determining factor in insulin regulation by adipocytes

    NASA Astrophysics Data System (ADS)

    Sanjabi, Bahram; Dashty, Monireh; Özcan, Behiye; Akbarkhanzadeh, Vishtaseb; Rahimi, Mehran; Vinciguerra, Manlio; van Rooij, Felix; Al-Lahham, Saad; Sheedfar, Fareeba; van Kooten, Theo G.; Spek, C. Arnold; Rowshani, Ajda T.; van der Want, Johannes; Klaassen, Rene; Sijbrands, Eric; Peppelenbosch, Maikel P.; Rezaee, Farhad

    2015-03-01

    Lipid droplets (LDs) hypertrophy in adipocytes is the main cause of energy metabolic system dysfunction, obesity and its afflictions such as T2D. However, the role of adipocytes in linking energy metabolic disorders with insulin regulation is unknown in humans. Human adipocytes constitutively synthesize and secrete insulin, which is biologically functional. Insulin concentrations and release are fat mass- and LDs-dependent respectively. Fat reduction mediated by bariatric surgery repairs obesity-associated T2D. The expression of genes, like PCSK1 (proinsulin conversion enzyme), GCG (Glucagon), GPLD1, CD38 and NNAT, involved in insulin regulation/release were differentially expressed in pancreas and adipose tissue (AT). INS (insulin) and GCG expression reduced in human AT-T2D as compared to AT-control, but remained unchanged in pancreas in either state. Insulin levels (mRNA/protein) were higher in AT derived from prediabetes BB rats with destructed pancreatic β-cells and controls than pancreas derived from the same rats respectively. Insulin expression in 10 human primary cell types including adipocytes and macrophages is an evidence for extrapancreatic insulin-producing cells. The data suggest a crosstalk between AT and pancreas to fine-tune energy metabolic system or may minimize the metabolic damage during diabetes. This study opens new avenues towards T2D therapy with a great impact on public health.

  12. Modulation Effect of Peroxisome Proliferator-Activated Receptor Agonists on Lipid Droplet Proteins in Liver.

    PubMed

    Zhu, Yun-Xia; Zhang, Ming-Liang; Zhong, Yuan; Wang, Chen; Jia, Wei-Ping

    2016-01-01

    Peroxisome proliferator-activated receptor (PPAR) agonists are used for treating hyperglycemia and type 2 diabetes. However, the mechanism of action of these agonists is still under investigation. The lipid droplet-associated proteins FSP27/CIDEC and LSDP5, regulated directly by PPARγ and PPARα, are associated with hepatic steatosis and insulin sensitivity. Here, we evaluated the expression levels of FSP27/CIDEC and LSDP5 and the regulation of these proteins by consumption of a high-fat diet (HFD) or administration of PPAR agonists. Mice with diet-induced obesity were treated with the PPARγ or PPARα agonist, pioglitazone or fenofibrate, respectively. Liver tissues from db/db diabetic mice and human were also collected. Interestingly, FSP27/CIEDC was expressed in mouse and human livers and was upregulated in obese C57BL/6J mice. Fenofibrate treatment decreased hepatic triglyceride (TG) content and FSP27/CIDEC protein expression in mice fed an HFD diet. In mice, LSDP5 was not detected, even in the context of insulin resistance or treatment with PPAR agonists. However, LSDP5 was highly expressed in humans, with elevated expression observed in the fatty liver. We concluded that fenofibrate greatly decreased hepatic TG content and FSP27/CIDEC protein expression in mice fed an HFD, suggesting a potential regulatory role for fenofibrate in the amelioration of hepatic steatosis.

  13. Peroxisomes, lipid droplets, and endoplasmic reticulum “hitchhike” on motile early endosomes

    PubMed Central

    Guimaraes, Sofia C.; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R.A.; Schrader, Michael

    2015-01-01

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. PMID:26620910

  14. Nuclear-localized CTP:phosphocholine cytidylyltransferase α regulates phosphatidylcholine synthesis required for lipid droplet biogenesis

    PubMed Central

    Aitchison, Adam J.; Arsenault, Daniel J.; Ridgway, Neale D.

    2015-01-01

    The reversible association of CTP:phosphocholine cytidylyltransferase α (CCTα) with membranes regulates the synthesis of phosphatidylcholine (PC) by the CDP-choline (Kennedy) pathway. Based on results with insect CCT homologues, translocation of nuclear CCTα onto cytoplasmic lipid droplets (LDs) is proposed to stimulate the synthesis of PC that is required for LD biogenesis and triacylglycerol (TAG) storage. We examined whether this regulatory mechanism applied to LD biogenesis in mammalian cells. During 3T3-L1 and human preadipocyte differentiation, CCTα expression and PC synthesis was induced. In 3T3-L1 cells, CCTα translocated from the nucleoplasm to the nuclear envelope and cytosol but did not associate with LDs. The enzyme also remained in the nucleus during human adipocyte differentiation. RNAi silencing in 3T3-L1 cells showed that CCTα regulated LD size but did not affect TAG storage or adipogenesis. LD biogenesis in nonadipocyte cell lines treated with oleate also promoted CCTα translocation to the nuclear envelope and/or cytoplasm but not LDs. In rat intestinal epithelial cells, CCTα silencing increased LD size, but LD number and TAG deposition were decreased due to oleate-induced cytotoxicity. We conclude that CCTα increases PC synthesis for LD biogenesis by translocation to the nuclear envelope and not cytoplasmic LDs. PMID:26108622

  15. Optimization of Seoul-Fluor-based lipid droplet bioprobes and their application in microalgae for bio-fuel study.

    PubMed

    Lee, Youngjun; Na, Sangcheol; Lee, Sanghee; Jeon, Noo Li; Park, Seung Bum

    2013-05-01

    We synthesized a series of Seoul-Fluor-based lipid droplet bioprobes with a linear range of lipophilicity and identified SF44 and SF58 as SF-based LD bioprobes in microalgae for biofuel research as well as in mammalian cells. Unlike Nile Red, SF-based bioprobes can stain algal LDs with excellent efficiency under the non-invasive and non-cytotoxic conditions.

  16. Recent discoveries on absorption of dietary fat: Presence, synthesis, and metabolism of cytoplasmic lipid droplets within enterocytes.

    PubMed

    D'Aquila, Theresa; Hung, Yu-Han; Carreiro, Alicia; Buhman, Kimberly K

    2016-08-01

    Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, cardiovascular disease, and cancer. Within enterocytes, the digestive products of dietary fat are re-synthesized into triacylglycerol, which is either secreted on chylomicrons or stored within cytoplasmic lipid droplets (CLDs). CLDs were originally thought to be inert stores of neutral lipids, but are now recognized as dynamic organelles that function in multiple cellular processes in addition to lipid metabolism. This review will highlight recent discoveries related to dietary fat absorption with an emphasis on the presence, synthesis, and metabolism of CLDs within this process. PMID:27108063

  17. Recent discoveries on absorption of dietary fat: Presence, synthesis, and metabolism of cytoplasmic lipid droplets within enterocytes.

    PubMed

    D'Aquila, Theresa; Hung, Yu-Han; Carreiro, Alicia; Buhman, Kimberly K

    2016-08-01

    Dietary fat provides essential nutrients, contributes to energy balance, and regulates blood lipid concentrations. These functions are important to health, but can also become dysregulated and contribute to diseases such as obesity, diabetes, cardiovascular disease, and cancer. Within enterocytes, the digestive products of dietary fat are re-synthesized into triacylglycerol, which is either secreted on chylomicrons or stored within cytoplasmic lipid droplets (CLDs). CLDs were originally thought to be inert stores of neutral lipids, but are now recognized as dynamic organelles that function in multiple cellular processes in addition to lipid metabolism. This review will highlight recent discoveries related to dietary fat absorption with an emphasis on the presence, synthesis, and metabolism of CLDs within this process.

  18. The deubiquitinase activity of the Salmonella pathogenicity island 2 effector, SseL, prevents accumulation of cellular lipid droplets.

    PubMed

    Arena, Ellen T; Auweter, Sigrid D; Antunes, L Caetano M; Vogl, A Wayne; Han, Jun; Guttman, Julian A; Croxen, Matthew A; Menendez, Alfredo; Covey, Scott D; Borchers, Christoph H; Finlay, B Brett

    2011-11-01

    To cause disease, Salmonella enterica serovar Typhimurium requires two type III secretion systems that are encoded by Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2). These secretion systems serve to deliver specialized proteins (effectors) into the host cell cytosol. While the importance of these effectors to promote colonization and replication within the host has been established, the specific roles of individual secreted effectors in the disease process are not well understood. In this study, we used an in vivo gallbladder epithelial cell infection model to study the function of the SPI-2-encoded type III effector, SseL. The deletion of the sseL gene resulted in bacterial filamentation and elongation and the unusual localization of Salmonella within infected epithelial cells. Infection with the ΔsseL strain also caused dramatic changes in host cell lipid metabolism and led to the massive accumulation of lipid droplets in infected cells. This phenotype was directly attributable to the deubiquitinase activity of SseL, as a Salmonella strain carrying a single point mutation in the catalytic cysteine also resulted in extensive lipid droplet accumulation. The excessive buildup of lipids due to the absence of a functional sseL gene also was observed in murine livers during S. Typhimurium infection. These results suggest that SseL alters host lipid metabolism in infected epithelial cells by modifying the ubiquitination patterns of cellular targets.

  19. Label-Free Analysis of Cellular Lipid Droplet Formation by Non-Linear Microscopy

    NASA Astrophysics Data System (ADS)

    Schie, Iwan W.

    Cellular lipid droplets (LD) are cellular organelles that can be found in every cell type. Recent research indicates that cellular LD are involved in a large number of cellular metabolic functions, such as lipid metabolism, protection from lipotoxicity, protein storage and degradation, and many more. LD formation is frequently associated with adverse health effects, i.e. alcoholic and non-alcoholic fatty liver disease, diabetes type-2, as well as many cardiovascular disorders. Despite their wide presence, LDs are the least studied and most poorly understood cellular organelles. Typically, LDs are investigated using fluorescence-based techniques that require staining with exogenous fluorophores. Other techniques, e.g. biochemical assays, require the destruction of cells that prohibit the analysis of living cells. Therefore, in my thesis research I developed a novel compound fast-scanning nonlinear optical microscope equipped with the ability to also acquire Raman spectra at specific image locations. This system allows us to image label-free cellular LD formation in living cells and analyze the composition of single cellular LDs. Images can be acquired at near video-rate (˜16 frames/s). Furthermore, the system has the ability to acquire very large images of tissue of up to 7.5x15 cm2 total area by stitching together scans with dimensions of 1x1 mm2 in less than 1 minute. The system also enables the user to acquire Raman spectra from points of interest in the multiphoton images and provides chemically-specific data from sample volumes as small as 1 femtoliter. In my thesis I used this setup to determine the effects of VLDL lipolysis products on primary rat hepatocytes. By analyzing the Raman spectra and comparing the peak ratios for saturated and unsaturated fatty acid it was determined that the small cellular LD are highly saturated, while large cellular LDs contain mostly unsaturated lipids. Furthermore, I established a method to determine the specific contribution

  20. The distribution and abundance of archaeal tetraether lipids in U.S. Great Basin hot springs.

    PubMed

    Paraiso, Julienne J; Williams, Amanda J; Huang, Qiuyuan; Wei, Yuli; Dijkstra, Paul; Hungate, Bruce A; Dong, Hailiang; Hedlund, Brian P; Zhang, Chuanlun L

    2013-01-01

    Isoprenoidal glycerol dialkyl glycerol tetraethers (iGDGTs) are core membrane lipids of many archaea that enhance the integrity of cytoplasmic membranes in extreme environments. We examined the iGDGT profiles and corresponding aqueous geochemistry in 40 hot spring sediment and microbial mat samples from the U.S. Great Basin with temperatures ranging from 31 to 95°C and pH ranging from 6.8 to 10.7. The absolute abundance of iGDGTs correlated negatively with pH and positively with temperature. High lipid concentrations, distinct lipid profiles, and a strong relationship between polar and core lipids in hot spring samples suggested in situ production of most iGDGTs rather than contamination from local soils. Two-way cluster analysis and non-metric multidimensional scaling (NMS) of polar iGDGTs indicated that the relative abundance of individual lipids was most strongly related to temperature (r (2) = 0.546), with moderate correlations with pH (r (2) = 0.359), nitrite (r (2) = 0.286), oxygen (r (2) = 0.259), and nitrate (r (2) = 0.215). Relative abundance profiles of individual polar iGDGTs indicated potential temperature optima for iGDGT-0 (≤70°C), iGDGT-3 (≥55°C), and iGDGT-4 (≥60°C). These relationships likely reflect both physiological adaptations and community-level population shifts in response to temperature differences, such as a shift from cooler samples with more abundant methanogens to higher-temperature samples with more abundant Crenarchaeota. Crenarchaeol was widely distributed across the temperature gradient, which is consistent with other reports of abundant crenarchaeol in Great Basin hot springs and suggests a wide distribution for thermophilic ammonia-oxidizing archaea (AOA).

  1. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

    PubMed

    Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-10-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs.

  2. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6[S

    PubMed Central

    Eichmann, Thomas O.; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G.; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-01-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. PMID:26330055

  3. ATGL and CGI-58 are lipid droplet proteins of the hepatic stellate cell line HSC-T6.

    PubMed

    Eichmann, Thomas O; Grumet, Lukas; Taschler, Ulrike; Hartler, Jürgen; Heier, Christoph; Woblistin, Aaron; Pajed, Laura; Kollroser, Manfred; Rechberger, Gerald; Thallinger, Gerhard G; Zechner, Rudolf; Haemmerle, Günter; Zimmermann, Robert; Lass, Achim

    2015-10-01

    Lipid droplets (LDs) of hepatic stellate cells (HSCs) contain large amounts of vitamin A [in the form of retinyl esters (REs)] as well as other neutral lipids such as TGs. During times of insufficient vitamin A availability, RE stores are mobilized to ensure a constant supply to the body. To date, little is known about the enzymes responsible for the hydrolysis of neutral lipid esters, in particular of REs, in HSCs. In this study, we aimed to identify LD-associated neutral lipid hydrolases by a proteomic approach using the rat stellate cell line HSC-T6. First, we loaded cells with retinol and FAs to promote lipid synthesis and deposition within LDs. Then, LDs were isolated and lipid composition and the LD proteome were analyzed. Among other proteins, we found perilipin 2, adipose TG lipase (ATGL), and comparative gene identification-58 (CGI-58), known and established LD proteins. Bioinformatic search of the LD proteome for α/β-hydrolase fold-containing proteins revealed no yet uncharacterized neutral lipid hydrolases. In in vitro activity assays, we show that rat (r)ATGL, coactivated by rat (r)CGI-58, efficiently hydrolyzes TGs and REs. These findings suggest that rATGL and rCGI-58 are LD-resident proteins in HSCs and participate in the mobilization of both REs and TGs. PMID:26330055

  4. Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function.

    PubMed

    Gallardo-Montejano, Violeta I; Saxena, Geetu; Kusminski, Christine M; Yang, Chaofeng; McAfee, John L; Hahner, Lisa; Hoch, Kathleen; Dubinsky, William; Narkar, Vihang A; Bickel, Perry E

    2016-01-01

    Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction. PMID:27554864

  5. Nuclear Perilipin 5 integrates lipid droplet lipolysis with PGC-1α/SIRT1-dependent transcriptional regulation of mitochondrial function

    PubMed Central

    Gallardo-Montejano, Violeta I.; Saxena, Geetu; Kusminski, Christine M.; Yang, Chaofeng; McAfee, John L.; Hahner, Lisa; Hoch, Kathleen; Dubinsky, William; Narkar, Vihang A.; Bickel, Perry E.

    2016-01-01

    Dysfunctional cellular lipid metabolism contributes to common chronic human diseases, including type 2 diabetes, obesity, fatty liver disease and diabetic cardiomyopathy. How cells balance lipid storage and mitochondrial oxidative capacity is poorly understood. Here we identify the lipid droplet protein Perilipin 5 as a catecholamine-triggered interaction partner of PGC-1α. We report that during catecholamine-stimulated lipolysis, Perilipin 5 is phosphorylated by protein kinase A and forms transcriptional complexes with PGC-1α and SIRT1 in the nucleus. Perilipin 5 promotes PGC-1α co-activator function by disinhibiting SIRT1 deacetylase activity. We show by gain-and-loss of function studies in cells that nuclear Perilipin 5 promotes transcription of genes that mediate mitochondrial biogenesis and oxidative function. We propose that Perilipin 5 is an important molecular link that couples the coordinated catecholamine activation of the PKA pathway and of lipid droplet lipolysis with transcriptional regulation to promote efficient fatty acid catabolism and prevent mitochondrial dysfunction. PMID:27554864

  6. Milk fat globule membrane coating of large lipid droplets in the diet of young mice prevents body fat accumulation in adulthood.

    PubMed

    Baars, Annemarie; Oosting, Annemarie; Engels, Eefje; Kegler, Diane; Kodde, Andrea; Schipper, Lidewij; Verkade, Henkjan J; van der Beek, Eline M

    2016-06-01

    Epidemiological studies have demonstrated protective effects of breast-feeding on childhood obesity. Differences between human milk and infant milk formula (IMF) in dietary lipid structure may contribute to this effect. In our mouse model, feeding a diet containing large lipid droplets coated with phospholipids (PL) (Nuturis®; PL of milk fat globule membrane (MFGM) fraction origin) in early life protected against excessive body fat accumulation following a diet challenge in adult life. We now set out to determine the relevance of increased droplet size and/or MFGM lipid droplet coating to the observed anti-obesogenic effects in adult life. From day 16 to 42, male mouse pups were exposed to diets with small (S) or large (L) lipid droplets (0·3 v. 2·9 µm average mode diameter, respectively), either without MFGM or with MFGM coating around the lipid droplet, resulting in four groups: S (control diet), L, Scoating and Lcoating (Nuturis® IMF diet). Mice were subsequently challenged with a Western-style diet until dissection at postnatal day 98. A non-challenged group served as reference (REF). We repeatedly determined body composition between postnatal day 42 and 98. At day 98 plasma and gene expression measurements were performed. Only the Nuturis® IMF diet (Lcoating) in early life containing MFGM-coated large lipid droplets reduced body fat mass to a level comparable with the REF group. These data support the notion that the structural aspects of lipids in human milk, for example, both lipid droplet size as well as the MFGM coating, may contribute to its reported protective effect against obesity in later life. PMID:27040581

  7. Milk fat globule membrane coating of large lipid droplets in the diet of young mice prevents body fat accumulation in adulthood.

    PubMed

    Baars, Annemarie; Oosting, Annemarie; Engels, Eefje; Kegler, Diane; Kodde, Andrea; Schipper, Lidewij; Verkade, Henkjan J; van der Beek, Eline M

    2016-06-01

    Epidemiological studies have demonstrated protective effects of breast-feeding on childhood obesity. Differences between human milk and infant milk formula (IMF) in dietary lipid structure may contribute to this effect. In our mouse model, feeding a diet containing large lipid droplets coated with phospholipids (PL) (Nuturis®; PL of milk fat globule membrane (MFGM) fraction origin) in early life protected against excessive body fat accumulation following a diet challenge in adult life. We now set out to determine the relevance of increased droplet size and/or MFGM lipid droplet coating to the observed anti-obesogenic effects in adult life. From day 16 to 42, male mouse pups were exposed to diets with small (S) or large (L) lipid droplets (0·3 v. 2·9 µm average mode diameter, respectively), either without MFGM or with MFGM coating around the lipid droplet, resulting in four groups: S (control diet), L, Scoating and Lcoating (Nuturis® IMF diet). Mice were subsequently challenged with a Western-style diet until dissection at postnatal day 98. A non-challenged group served as reference (REF). We repeatedly determined body composition between postnatal day 42 and 98. At day 98 plasma and gene expression measurements were performed. Only the Nuturis® IMF diet (Lcoating) in early life containing MFGM-coated large lipid droplets reduced body fat mass to a level comparable with the REF group. These data support the notion that the structural aspects of lipids in human milk, for example, both lipid droplet size as well as the MFGM coating, may contribute to its reported protective effect against obesity in later life.

  8. Lipid Droplet Formation, Their Localization and Dynamics during Leishmania major Macrophage Infection

    PubMed Central

    Rabhi, Sameh; Rabhi, Imen; Trentin, Bernadette; Piquemal, David; Regnault, Béatrice; Goyard, Sophie; Lang, Thierry; Descoteaux, Albert; Enninga, Jost; Guizani-Tabbane, Lamia

    2016-01-01

    Leishmania, the causative agent of vector-borne diseases, known as leishmaniases, is an obligate intracellular parasite within mammalian hosts. The outcome of infection depends largely on the activation status of macrophages, the first line of mammalian defense and the major target cells for parasite replication. Understanding the strategies developed by the parasite to circumvent macrophage defense mechanisms and to survive within those cells help defining novel therapeutic approaches for leishmaniasis. We previously showed the formation of lipid droplets (LDs) in L. major infected macrophages. Here, we provide novel insights on the origin of the formed LDs by determining their cellular distribution and to what extent these high-energy sources are directed to the proximity of Leishmania parasites. We show that the ability of L. major to trigger macrophage LD accumulation is independent of parasite viability and uptake and can also be observed in non-infected cells through paracrine stimuli suggesting that LD formation is from cellular origin. The accumulation of LDs is demonstrated using confocal microscopy and live-cell imagin in parasite-free cytoplasmic region of the host cell, but also promptly recruited to the proximity of Leishmania parasites. Indeed LDs are observed inside parasitophorous vacuole and in parasite cytoplasm suggesting that Leishmania parasites besides producing their own LDs, may take advantage of these high energy sources. Otherwise, these LDs may help cells defending against parasitic infection. These metabolic changes, rising as common features during the last years, occur in host cells infected by a large number of pathogens and seem to play an important role in pathogenesis. Understanding how Leishmania parasites and different pathogens exploit this LD accumulation will help us define the common mechanism used by these different pathogens to manipulate and/or take advantage of this high-energy source. PMID:26871576

  9. Lipid droplet remodeling and interaction with mitochondria in mouse brown adipose tissue during cold treatment.

    PubMed

    Yu, Jinhai; Zhang, Shuyan; Cui, Liujuan; Wang, Weiyi; Na, Huimin; Zhu, Xiaotong; Li, Linghai; Xu, Guoheng; Yang, Fuquan; Christian, Mark; Liu, Pingsheng

    2015-05-01

    Brown adipose tissue (BAT) maintains animal body temperature by non-shivering thermogenesis, which is through uncoupling protein 1 (UCP1) that uncouples oxidative phosphorylation and utilizes β-oxidation of fatty acids released from triacylglycerol (TAG) in lipid droplets (LDs). Increasing BAT activity and "browning" other tissues such as white adipose tissue (WAT) can enhance the expenditure of excess stored energy, and in turn reduce prevalence of metabolic diseases. Although many studies have characterized the biology of BAT and brown adipocytes, BAT LDs especially their activation induced by cold exposure remain to be explored. We have isolated LDs from mouse interscapular BAT and characterized the full proteome using mass spectrometry. Both morphological and biochemical experiments showed that the LDs could tightly associate with mitochondria. Under cold treatment mouse BAT started expressing LD structure protein PLIN-2/ADRP and increased expression of PLIN1. Both hormone sensitive lipase (HSL) and adipose TAG lipase (ATGL) were increased in LDs. In addition, isolated BAT LDs showed increased levels of the mitochondrial protein UCP1, and prolonged cold exposure could stimulate BAT mitochondrial cristae biogenesis. These changes were in agreement with the data from transcriptional analysis. Our results provide the BAT LD proteome for the first time and show that BAT LDs facilitate heat production by coupling increasing TAG hydrolysis through recruitment of ATGL and HSL to the organelle and expression of another LD resident protein PLIN2/ADRP, as well as by tightly associating with activated mitochondria. These findings will benefit the study of BAT activation and the interaction between LDs and mitochondria.

  10. Dynamic changes in lipid droplet-associated proteins in the "browning" of white adipose tissues.

    PubMed

    Barneda, David; Frontini, Andrea; Cinti, Saverio; Christian, Mark

    2013-05-01

    The morphological and functional differences between lipid droplets (LDs) in brown (BAT) and white (WAT) adipose tissues will largely be determined by their associated proteins. Analysing mRNA expression in mice fat depots we have found that most LD protein genes are expressed at higher levels in BAT, with the greatest differences observed for Cidea and Plin5. Prolonged cold exposure, which induces the appearance of brown-like adipocytes in mice WAT depots, was accompanied with the potentiation of the lipolytic machinery, with changes in ATGL, CGI-58 and G0S2 gene expression. However the major change detected in WAT was the enhancement of Cidea mRNA. Together with the increase in Cidec, it indicates that LD enlargement through LD-LD transference of fat is an important process during WAT browning. To study the dynamics of this phenotypic change, we have applied 4D confocal microscopy in differentiated 3T3-L1 cells under sustained β-adrenergic stimulation. Under these conditions the cells experienced a LD remodelling cycle, with progressive reduction on the LD size by lipolysis, followed by the formation of new LDs, which were subjected to an enlargement process, likely to be CIDE-triggered, until the cell returned to the basal state. This transformation would be triggered by the activation of a thermogenic futile cycle of lipolysis/lipogenesis and could facilitate the molecular mechanism for the unilocular to multilocular transformation during WAT browning. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.

  11. Increase in cellular triacylglycerol content and emergence of large ER-associated lipid droplets in the absence of CDP-DG synthase function.

    PubMed

    He, Yue; Yam, Candice; Pomraning, Kyle; Chin, Jacqueline S R; Yew, Joanne Y; Freitag, Michael; Oliferenko, Snezhana

    2014-12-15

    Excess fatty acids and sterols are stored as triacylglycerols and sterol esters in specialized cellular organelles, called lipid droplets. Understanding what determines the cellular amount of neutral lipids and their packaging into lipid droplets is of fundamental and applied interest. Using two species of fission yeast, we show that cycling cells deficient in the function of the ER-resident CDP-DG synthase Cds1 exhibit markedly increased triacylglycerol content and assemble large lipid droplets closely associated with the ER membranes. We demonstrate that these unusual structures recruit the triacylglycerol synthesis machinery and grow by expansion rather than by fusion. Our results suggest that interfering with the CDP-DG route of phosphatidic acid utilization rewires cellular metabolism to adopt a triacylglycerol-rich lifestyle reliant on the Kennedy pathway. PMID:25318672

  12. AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae.

    PubMed

    Xie, Meng; Roy, Richard

    2015-01-01

    Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress. PMID:26098762

  13. Nitrogen Deprivation Induces Lipid Droplet Accumulation and Alters Fatty Acid Metabolism in Symbiotic Dinoflagellates Isolated from Aiptasia pulchella

    NASA Astrophysics Data System (ADS)

    Weng, Li-Chi; Pasaribu, Buntora; -Ping Lin, I.; Tsai, Ching-Hsiu; Chen, Chii-Shiarng; Jiang, Pei-Luen

    2014-07-01

    The stability of cnidarian-dinoflagellate (genus Symbiodinium spp.) endosymbioses depends on the regulation of nutrient transport between Symbiodinium populations and their hosts. Previously, we successfully induced the production of lipid droplets in the free-living cultured Symbiodinium (clade B) under the nitrogen-deprivation condition for 5 days. Therefore, the present study aimed at understanding the disruption of the endosymbiotic relationship between the cnidarians and dinoflagellates by nitrogen deprivation using Aiptasia pulchella as an example. Transmission electron micrographs revealed the formation of lipid droplets induced by nitrogen deprivation, and the lipid analyses further showed that polyunsaturated fatty acids were drastically enriched in Symbiodinium after 30 days of nitrogen deprivation, although these were unaffected after 5 days of nitrogen starvation. The present study also suggested that the host provided nitrogen to the symbiotic cells during short-term environmental stress. However, the relationship started to deteriorate after 30 days. These findings provide a more detailed understanding of the mechanisms of the symbiotic relationship between the symbiotic dinoflagellates in terms of the nitrogen source, which might provide more information for the explanation of the regulatory mechanism underlying endosymbiotic associations.

  14. AMP-Activated Kinase Regulates Lipid Droplet Localization and Stability of Adipose Triglyceride Lipase in C. elegans Dauer Larvae

    PubMed Central

    Xie, Meng; Roy, Richard

    2015-01-01

    Animals have developed diverse mechanisms to adapt to their changing environment. Like many organisms the free-living nematode C. elegans can alternate between a reproductive mode or a diapause-like "dauer" stage during larval development to circumvent harsh environmental conditions. The master metabolic regulator AMP-activated protein kinase (AMPK) is critical for survival during the dauer stage, where it phosphorylates adipose triglyceride lipase (ATGL-1) at multiple sites to block lipid hydrolysis and ultimately protect the cellular triglyceride-based energy depot from rapid depletion. However, how the AMPK-mediated phosphorylation affects the function of ATGL-1 has not been characterised at the molecular level. Here we show that AMPK phosphorylation leads to the generation of 14-3-3 binding sites on ATGL-1, which are recognized by the C. elegans 14-3-3 protein orthologue PAR-5. Physical interaction of ATGL-1 with PAR-5 results in sequestration of ATGL-1 away from the lipid droplets and eventual proteasome-mediated degradation. In addition, we also show that the major AMPK phosphorylation site on ATGL-1, Ser 303, is required for both modification of its lipid droplet localization and its degradation. Our data provide mechanistic insight as to how AMPK functions to enhance survival through its ability to protect the accumulated triglyceride deposits from rapid hydrolysis to preserve the energy stores during periods of extended environmental duress. PMID:26098762

  15. Impact of dietary fiber coatings on behavior of protein-stabilized lipid droplets under simulated gastrointestinal conditions.

    PubMed

    Tokle, Tanushree; Lesmes, Uri; Decker, Eric Andrew; McClements, David Julian

    2012-01-01

    Multilayer emulsions containing lipid droplets coated by lactoferrin (LF) - anionic polysaccharide layers have improved resistance to environmental stresses (such as pH, salt, and temperature), but their behavior within the gastrointestinal tract (GIT) is currently unknown. The objective of this research was therefore to monitor changes in the physicochemical properties and digestibility of these systems under simulated GIT conditions. Primary emulsions (5% corn oil, 0.5% LF) were prepared using a high-pressure homogenizer. Secondary emulsions (5% corn oil, 0.5% LF, 0.5% polysaccharide) were prepared by incorporating alginate, low methoxyl pectin (LMP) or high methoxyl pectin (HMP) into primary emulsions. Emulsions were then subjected to simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions in sequence. LF, LF-LMP and LF-HMP emulsions were stable to droplet aggregation in the stomach but aggregated in the small intestine, whereas LF-alginate emulsions aggregated in both the stomach and small intestine. The presence of a dietary fiber coating around the initial lipid droplets had little influence on the total extent of lipid digestion in SIF, but LF-alginate emulsions had a slower initial digestion rate than the other emulsions. These results suggest that the dietary fiber coatings may become detached in the small intestine, or that they were permeable to digestive enzymes. Pepsin was found to have little influence on the physical stability or digestibility of the emulsions. The knowledge obtained from this study is important for the design of delivery systems for encapsulation and release of lipophilic bioactive ingredients.

  16. Lipid Droplets and Peroxisomes: Key Players in Cellular Lipid Homeostasis or A Matter of Fat—Store ’em Up or Burn ’em Down

    PubMed Central

    Kohlwein, Sepp D.; Veenhuis, Marten; van der Klei, Ida J.

    2013-01-01

    Lipid droplets (LDs) and peroxisomes are central players in cellular lipid homeostasis: some of their main functions are to control the metabolic flux and availability of fatty acids (LDs and peroxisomes) as well as of sterols (LDs). Both fatty acids and sterols serve multiple functions in the cell—as membrane stabilizers affecting membrane fluidity, as crucial structural elements of membrane-forming phospholipids and sphingolipids, as protein modifiers and signaling molecules, and last but not least, as a rich carbon and energy source. In addition, peroxisomes harbor enzymes of the malic acid shunt, which is indispensable to regenerate oxaloacetate for gluconeogenesis, thus allowing yeast cells to generate sugars from fatty acids or nonfermentable carbon sources. Therefore, failure of LD and peroxisome biogenesis and function are likely to lead to deregulated lipid fluxes and disrupted energy homeostasis with detrimental consequences for the cell. These pathological consequences of LD and peroxisome failure have indeed sparked great biomedical interest in understanding the biogenesis of these organelles, their functional roles in lipid homeostasis, interaction with cellular metabolism and other organelles, as well as their regulation, turnover, and inheritance. These questions are particularly burning in view of the pandemic development of lipid-associated disorders worldwide. PMID:23275493

  17. Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy

    PubMed Central

    Jaeger, Daniel; Pilger, Christian; Hachmeister, Henning; Oberländer, Elina; Wördenweber, Robin; Wichmann, Julian; Mussgnug, Jan H.; Huser, Thomas; Kruse, Olaf

    2016-01-01

    Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation. PMID:27767024

  18. Negatively-charged residues in the polar carboxy-terminal region in FSP27 are indispensable for expanding lipid droplets.

    PubMed

    Tamori, Yoshikazu; Tateya, Sanshiro; Ijuin, Takeshi; Nishimoto, Yuki; Nakajima, Shinsuke; Ogawa, Wataru

    2016-03-01

    FSP27 has an important role in large lipid droplet (LD) formation because it exchanges lipids at the contact site between LDs. In the present study, we clarify that the amino-terminal domain of FSP27 (amino acids 1-130) is dispensable for LD enlargement, although it accelerates LD growth. LD expansion depends on the carboxy-terminal domain of FSP27 (amino acids 131-239). Especially, the negative charge of the acidic residues (D215, E218, E219 and E220) in the polar carboxy-terminal region (amino acids 202-239) is essential for the enlargement of LD. We propose that the carboxy-terminal domain of FSP27 has a crucial role in LD expansion, whereas the amino-terminal domain only has a supportive role. PMID:26921608

  19. Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis

    PubMed Central

    Smagris, Eriks; BasuRay, Soumik; Li, John; Huang, Yongcheng; Lai, Ka-man V; Gromada, Jesper; Cohen, Jonathan C; Hobbs, Helen H

    2015-01-01

    A sequence polymorphism (rs738409, I148M) in patatin-like phospholipid domain containing protein 3 (PNPLA3) is strongly associated with nonalcoholic fatty liver disease (NAFLD), but the mechanistic basis for this association remains enigmatic. Neither ablation nor overexpression of wild-type PNPLA3 affects liver fat content in mice, whereas hepatic overexpression of the human 148M transgene causes steatosis. To determine whether the 148M allele causes fat accumulation in the liver when expressed at physiological levels, we introduced a methionine codon at position 148 of the mouse Pnpla3 gene. Knockin mice had normal levels of hepatic fat on a chow diet, but when challenged with a high-sucrose diet their liver fat levels increased 2 to 3-fold compared to wild-type littermates without any associated changes in glucose homeostasis. The increased liver fat in the knockin mice was accompanied by a 40-fold increase in PNPLA3 on hepatic lipid droplets, with no increase in hepatic PNPLA3 messenger RNA (mRNA). Similar results were obtained when the catalytic dyad of PNPLA3 was inactivated by substituting the catalytic serine with alanine (S47A). Conclusion: These data provide the first direct evidence that physiological expression of PNPLA3 148M variant causes NAFLD, and that the accumulation of catalytically inactive PNPLA3 on the surfaces of lipid droplets is associated with the accumulation of TG in the liver. (Hepatology 2015;61:108–118) PMID:24917523

  20. Label-Free Digital Quantification of Lipid Droplets in Single Cells by Stimulated Raman Microscopy on a Microfluidic Platform.

    PubMed

    Cao, Chen; Zhou, Dong; Chen, Tao; Streets, Aaron M; Huang, Yanyi

    2016-05-01

    Quantitative characterization of a single-cell phenotype remains challenging. We combined a scalable microfluidic array of parallel cell culture chambers and stimulated Raman scattering (SRS) microscopy to quantitatively characterize the response of lipid droplet (LD) formation to free-fatty-acid stimuli with single-LD resolution at the single-cell level. By enabling the systematic live-cell imaging with SRS microscopy in a microfluidic device, we were able to quantify the morphology of over a thousand live cells in 10 different chemical environments and with 8 replicates for each culture condition, in a single experiment, and without relying on fluorescent labeling. We developed an image processing pipeline for cell segmentation and LD morphology quantification using dual-channel SRS images. This allows us to construct distributions of the morphological parameters of LDs in the cellular population and expose the vast phenotypic heterogeneity among genetically similar cells. Specifically, this approach provides an analytical tool for quantitatively investigating LD morphology in live cells in situ. With this high-throughput, high-resolution, and label-free method, we found that LD growth dynamics showed considerable cell to cell variation. Lipid accumulation in nonadipocyte cells is mainly reflected in the increase of LD number, as opposed to an increase in their size or lipid concentration. Our method allows statistical single-cell quantification of the LD distribution for further investigation of lipid metabolism and dynamic behavior, and also extends the possibility to couple with other "omics" technologies in the future.

  1. Modest hypoxia significantly reduces triglyceride content and lipid droplet size in 3T3-L1 adipocytes

    SciTech Connect

    Hashimoto, Takeshi; Yokokawa, Takumi; Endo, Yuriko; Iwanaka, Nobumasa; Higashida, Kazuhiko; Taguchi, Sadayoshi

    2013-10-11

    Highlights: •Long-term hypoxia decreased the size of LDs and lipid storage in 3T3-L1 adipocytes. •Long-term hypoxia increased basal lipolysis in 3T3-L1 adipocytes. •Hypoxia decreased lipid-associated proteins in 3T3-L1 adipocytes. •Hypoxia decreased basal glucose uptake and lipogenic proteins in 3T3-L1 adipocytes. •Hypoxia-mediated lipogenesis may be an attractive therapeutic target against obesity. -- Abstract: Background: A previous study has demonstrated that endurance training under hypoxia results in a greater reduction in body fat mass compared to exercise under normoxia. However, the cellular and molecular mechanisms that underlie this hypoxia-mediated reduction in fat mass remain uncertain. Here, we examine the effects of modest hypoxia on adipocyte function. Methods: Differentiated 3T3-L1 adipocytes were incubated at 5% O{sub 2} for 1 week (long-term hypoxia, HL) or one day (short-term hypoxia, HS) and compared with a normoxia control (NC). Results: HL, but not HS, resulted in a significant reduction in lipid droplet size and triglyceride content (by 50%) compared to NC (p < 0.01). As estimated by glycerol release, isoproterenol-induced lipolysis was significantly lowered by hypoxia, whereas the release of free fatty acids under the basal condition was prominently enhanced with HL compared to NC or HS (p < 0.01). Lipolysis-associated proteins, such as perilipin 1 and hormone-sensitive lipase, were unchanged, whereas adipose triglyceride lipase and its activator protein CGI-58 were decreased with HL in comparison to NC. Interestingly, such lipogenic proteins as fatty acid synthase, lipin-1, and peroxisome proliferator-activated receptor gamma were decreased. Furthermore, the uptake of glucose, the major precursor of 3-glycerol phosphate for triglyceride synthesis, was significantly reduced in HL compared to NC or HS (p < 0.01). Conclusion: We conclude that hypoxia has a direct impact on reducing the triglyceride content and lipid droplet size via

  2. The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix

    PubMed Central

    Barneda, David; Planas-Iglesias, Joan; Gaspar, Maria L; Mohammadyani, Dariush; Prasannan, Sunil; Dormann, Dirk; Han, Gil-Soo; Jesch, Stephen A; Carman, George M; Kagan, Valerian; Parker, Malcolm G; Ktistakis, Nicholas T; Klein-Seetharaman, Judith; Dixon, Ann M; Henry, Susan A; Christian, Mark

    2015-01-01

    Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue, and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs, which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD–LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat. DOI: http://dx.doi.org/10.7554/eLife.07485.001 PMID:26609809

  3. Transcript Abundance of Putative Lipid Phosphate Phosphatases During Development of Trypanosoma brucei in the Tsetse Fly.

    PubMed

    Alves e Silva, Thiago Luiz; Savage, Amy F; Aksoy, Serap

    2016-04-01

    African trypanosomes (Trypanosoma brucei spp.) cause devastating diseases in sub-Saharan Africa. Trypanosomes differentiate repeatedly during development in tsetse flies before gaining mammalian infectivity in fly salivary glands. Lipid phosphate phosphatases (LPPs) are involved in diverse biological processes, such as cell differentiation and cell migration. Gene sequences encoding two putative T. brucei LPP proteins were used to search the T. brucei genome, revealing two additional putative family members. Putative structural features and transcript abundance during parasite development in tsetse fly were characterized. Three of the four LPP proteins are predicted to have six transmembrane domains, while the fourth shows only one. Semiquantitative gene expression revealed differential regulation of LPPs during parasite development. Transcript abundance for three of the four putative LPP genes was elevated in parasites infecting salivary glands, but not mammalian-infective metacyclic cells in fly saliva, indicating a potential role of this family in parasite establishment in tsetse salivary glands.

  4. Trans-10,cis-12 CLA increases adipocyte lipolysis and alters lipid droplet-associated proteins: role of mTOR and ERK signaling.

    PubMed

    Chung, Soonkyu; Brown, Jonathan Mark; Sandberg, Maria Boysen; McIntosh, Michael

    2005-05-01

    Lipid droplet-associated proteins play an important role in adipocyte triglyceride (TG) metabolism. Here, we show that trans-10,cis-12 conjugated linoleic acid (CLA), but not cis-9,trans-11 CLA, increased lipolysis and altered human adipocyte lipid droplet morphology. Before this change in morphology, there was a rapid trans-10,cis-12 CLA-induced increase in the accumulation of perilipin A in the cytosol, followed by the disappearance of perilipin A protein. In contrast, protein levels of adipose differentiation-related protein (ADRP) were increased in cultures treated with trans-10,cis-12 CLA. Immunostaining revealed that ADRP localized to the surface of small lipid droplets, displacing perilipin. Intriguingly, trans-10,cis-12 CLA increased ADRP protein expression to a much greater extent than ADRP mRNA without affecting stability, suggesting translational control of ADRP. To this end, we found that trans-10,cis-12 CLA increased activation of the mammalian target of rapamycin/p70 S6 ribosomal protein kinase/S6 ribosomal protein (mTOR/p70S6K/S6) pathway. Collectively, these data demonstrate that the trans-10,cis-12 CLA-mediated reduction of human adipocyte TG content is associated with the differential localization and expression of lipid droplet-associated proteins. This process involves both the translational control of ADRP through the activation of mTOR/p70S6K/S6 signaling and transcriptional control of perilipin A. PMID:15716587

  5. Postprandial VLDL lipolysis products increase monocyte adhesion and lipid droplet formation via activation of ERK2 and NFκB

    PubMed Central

    Altman, Robin; Norman, Jennifer E.; Rutledge, John C.

    2013-01-01

    Postprandial lipemia is characterized by a transient increase in circulating triglyceride-rich lipoproteins such as very low-density lipoprotein (VLDL) and has been shown to activate monocytes in vivo. Lipolysis of VLDL releases remnant particles, phospholipids, monoglycerides, diglycerides, and fatty acids in close proximity to endothelial cells and monocytes. We hypothesized that postprandial VLDL lipolysis products could activate and recruit monocytes by increasing monocyte expression of proinflammatory cytokines and adhesion molecules, and that such activation is related to the development of lipid droplets. Freshly isolated human monocytes were treated with VLDL lipolysis products (2.28 mmol/l triglycerides + 2 U/ml lipoprotein lipase), and monocyte adhesion to a primed endothelial monolayer was observed using a parallel plate flow chamber coupled with a CCD camera. Treated monocytes showed more rolling and adhesion than controls, and an increase in transmigration between endothelial cells. The increased adhesive events were related to elevated expression of key integrin complexes including Mac-1 [αm-integrin (CD11b)/β2-integrin (CD18)], CR4 [αx-integrin (CD11c)/CD18] and VLA-4 [α4-integrin (CD49d)/β1-integrin (CD29)] on treated monocytes. Treatment of peripheral blood mononuclear cells (PBMCs) and THP-1 monocytes with VLDL lipolysis products increased expression of TNFα, IL-1β, and IL-8 over controls, with concurrent activation of NFkB and AP-1. NFκB and AP-1-induced cytokine and integrin expression was dependent on ERK and Akt phosphorylation. Additionally, fatty acids from VLDL lipolysis products induced ERK2-dependent lipid droplet formation in monocytes, suggesting a link to inflammatory signaling pathways. These results provide novel mechanisms for postprandial monocyte activation by VLDL lipolysis products, suggesting new pathways and biomarkers for chronic, intermittent vascular injury. PMID:24163071

  6. Autophagy-like processes are involved in lipid droplet degradation in Auxenochlorella protothecoides during the heterotrophy-autotrophy transition

    PubMed Central

    Zhao, Li; Dai, Junbiao; Wu, Qingyu

    2014-01-01

    Autophagy is a cellular degradation process that recycles cytoplasmic components in eukaryotes. Although intensively studied in yeast, plants, and mammals, autophagy in microalgae is not well understood. Auxenochlorella protothecoides is a green microalga that has the ability to grow either autotrophically when under light or heterotrophically when in media containing glucose. The two growth modes are inter-convertible and transition between them is accompanied by drastic changes in morphology and cellular composition; however, the mechanisms underlying these changes are unknown. In this study, we identified autophagy-related genes and characterized their roles in the degradation of lipid droplets during the heterotrophy-to-autotrophy (HA) transition in A. protothecoides. Most of the proteins constituting the eukaryotic “core machinery” were conserved in A. protothecoides. Two proteins, Atg4 and Atg8, were further investigated. A. protothecoides ATG4 was cloned from a cDNA library and expressed within yeast, and was able to functionally restore the autophagy pathway in atg4Δ yeast during nitrogen starvation. Furthermore, Atg8, which displayed high sequence identity with its yeast homolog, was able to conjugate to phosphatidylethanolamine (PE) in vitro and was recruited to the phagophore assembly site in yeast. We also identified a C-terminal glycine residue, G118, that was the cleavage site for Atg4. Finally, we used confocal and transmission electron microscopy to reveal that autophagic-like vacuoles were detectable in algal cells during the HA transition. Our data suggested that the lipid droplets in heterotrophic cells were engulfed directly by the autophagic-like vacuole instead of via autophagosomes. PMID:25177326

  7. Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice.

    PubMed

    Baumeier, Christian; Kaiser, Daniel; Heeren, Jörg; Scheja, Ludger; John, Clara; Weise, Christoph; Eravci, Murat; Lagerpusch, Merit; Schulze, Gunnar; Joost, Hans-Georg; Schwenk, Robert Wolfgang; Schürmann, Annette

    2015-05-01

    Caloric restriction and intermittent fasting are known to improve glucose homeostasis and insulin resistance in several species including humans. The aim of this study was to unravel potential mechanisms by which these interventions improve insulin sensitivity and protect from type 2 diabetes. Diabetes-susceptible New Zealand Obese mice were either 10% calorie restricted (CR) or fasted every other day (IF), and compared to ad libitum (AL) fed control mice. AL mice showed a diabetes prevalence of 43%, whereas mice under CR and IF were completely protected against hyperglycemia. Proteomic analysis of hepatic lipid droplets revealed significantly higher levels of PSMD9 (co-activator Bridge-1), MIF (macrophage migration inhibitor factor), TCEB2 (transcription elongation factor B (SIII), polypeptide 2), ACY1 (aminoacylase 1) and FABP5 (fatty acid binding protein 5), and a marked reduction of GSTA3 (glutathione S-transferase alpha 3) in samples of CR and IF mice. In addition, accumulation of diacylglycerols (DAGs) was significantly reduced in livers of IF mice (P=0.045) while CR mice showed a similar tendency (P=0.062). In particular, 9 DAG species were significantly reduced in response to IF, of which DAG-40:4 and DAG-40:7 also showed significant effects after CR. This was associated with a decreased PKCε activation and might explain the improved insulin sensitivity. In conclusion, our data indicate that protection against diabetes upon caloric restriction and intermittent fasting associates with a modulation of lipid droplet protein composition and reduction of intracellular DAG species. PMID:25645620

  8. [Deposition of exogenous and endogenously generated unsaturated fatty acids in lipid droplets triacylglycerol as a mechanism of its sequestration in epithelial cells].

    PubMed

    Fedorova, E V; Fok, E M; Bakhteeva, V T; Lavrova, E A; Parnova, R G

    2014-08-01

    Neutral lipids are deposited in intracellular compartments called lipid droplets, which are known to be critically implicated in regulation of cellular lipid metabolism. These organelles consist of a core of neutral lipids, mainly triacylglycerol (TAG) and cholesteryl esters, surrounded by phospholipid monolayer. Using Nile red lipid staining and [3H]-arachidonic and [3H]-oleic acids as precursors for lipid biosynthesis, we have evaluated the mechanisms of lipid body induction elicited by exogenous fatty acids within primary cultured epithelial cells from the frog urinary bladder. It was found that arachidonic and oleic acids at concentrations 10-50 tM stimulated lipid droplets formation accompanied by accumulation of TAG and by the significant increase of incorporation of fatty acids into TAG indicating an enhanced TAG biosynthesis. No changes of cholesteryl esters content were observed under these conditions. In cells, prelabelled with [3H]-oleic acids, etomoxir, an inhibitor of O-carnitine palmitroyltansferase 1, decreased oxidation of oleic acid and increased its incorporation into TAG leading to intracellular TAG accumulation. In cells, prelabelled with [3H]-arachidonic acid, diclofenac, an inhibitor of cyclooxygenase 1 and 2, led to significant decrease in cellular PGE2 production and to reesterification of free arachidonic acid to TAG but not to phospholipids. Taking together, these data evidence that in isolated frog urinary bladder epithelial cells, reacylation of unsaturated free fatty acids into TAG is a main route of their metabolic conversion under the conditions of the increased cytosolic level of free fatty acids.

  9. Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation

    PubMed Central

    Kessler, Sonja M.; Laggai, Stephan; Van Wonterghem, Elien; Gemperlein, Katja; Müller, Rolf; Haybaeck, Johannes; Vandenbroucke, Roosmarijn E.; Ogris, Manfred; Libert, Claude; Kiemer, Alexandra K.

    2016-01-01

    Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Hydrodynamic gene delivery of an Igf2 plasmid used for transient Igf2 overexpression employing codon-optimized plasmid DNA resulted in a strong induction of hepatic Igf2 expression. The exogenously delivered Igf2 had no influence on endogenous Igf2 expression. The downstream kinase AKT was activated in Igf2 animals. Decreased ALT levels mirrored the cytoprotective effect of IGF2. Serum cholesterol was increased and sulfo-phospho-vanillin colorimetric assay confirmed lipid accumulation in Igf2-livers while no signs of inflammation were observed. Interestingly, hepatic cholesterol and phospholipids, determined by thin layer chromatography, and free cholesterol by filipin staining, were specifically increased. Lipid droplet (LD) size was not changed, but their number was significantly elevated. Furthermore, free cholesterol, which can be stored in LDs and has been reported to be critical for steatosis progression, was elevated in Igf2 overexpressing mice. Accordingly, Hmgcr/HmgCoAR was upregulated. To have a closer look at de novo lipid synthesis we investigated expression of the lipogenic transcription factor SREBF1 and its target genes. SREBF1 was induced and also SREBF1 target genes were slightly upregulated. Interestingly, the expression of Cpt1a, which is responsible for mitochondrial fatty acid oxidation, was induced. Hepatic IGF2 expression induces a fatty liver, characterized by increased cholesterol and phospholipids leading to accumulation of LDs. We therefore suggest a causal role for IGF2 in hepatic lipid accumulation. PMID:27199763

  10. Transient Hepatic Overexpression of Insulin-Like Growth Factor 2 Induces Free Cholesterol and Lipid Droplet Formation.

    PubMed

    Kessler, Sonja M; Laggai, Stephan; Van Wonterg, Elien; Gemperlein, Katja; Müller, Rolf; Haybaeck, Johannes; Vandenbroucke, Roosmarijn E; Ogris, Manfred; Libert, Claude; Kiemer, Alexandra K

    2016-01-01

    Although insulin-like growth factor 2 (IGF2) has been reported to be overexpressed in steatosis and steatohepatitis, a causal role of IGF2 in steatosis development remains elusive. Aim of our study was to decipher the role of IGF2 in steatosis development. Hydrodynamic gene delivery of an Igf2 plasmid used for transient Igf2 overexpression employing codon-optimized plasmid DNA resulted in a strong induction of hepatic Igf2 expression. The exogenously delivered Igf2 had no influence on endogenous Igf2 expression. The downstream kinase AKT was activated in Igf2 animals. Decreased ALT levels mirrored the cytoprotective effect of IGF2. Serum cholesterol was increased and sulfo-phospho-vanillin colorimetric assay confirmed lipid accumulation in Igf2-livers while no signs of inflammation were observed. Interestingly, hepatic cholesterol and phospholipids, determined by thin layer chromatography, and free cholesterol by filipin staining, were specifically increased. Lipid droplet (LD) size was not changed, but their number was significantly elevated. Furthermore, free cholesterol, which can be stored in LDs and has been reported to be critical for steatosis progression, was elevated in Igf2 overexpressing mice. Accordingly, Hmgcr/HmgCoAR was upregulated. To have a closer look at de novo lipid synthesis we investigated expression of the lipogenic transcription factor SREBF1 and its target genes. SREBF1 was induced and also SREBF1 target genes were slightly upregulated. Interestingly, the expression of Cpt1a, which is responsible for mitochondrial fatty acid oxidation, was induced. Hepatic IGF2 expression induces a fatty liver, characterized by increased cholesterol and phospholipids leading to accumulation of LDs. We therefore suggest a causal role for IGF2 in hepatic lipid accumulation. PMID:27199763

  11. Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy.

    PubMed

    Yi, Yung-Hsiang; Chien, Cheng-Hao; Chen, Wei-Wen; Ma, Tian-Hsiang; Liu, Kuan-Yu; Chang, Yu-Sun; Chang, Ta-Chau; Lo, Szecheng J

    2014-01-01

    Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals. PMID:23979461

  12. Lipid droplet pattern and nondroplet-like structure in two fat mutants of Caenorhabditis elegans revealed by coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Yi, Yung-Hsiang; Chien, Cheng-Hao; Chen, Wei-Wen; Ma, Tian-Hsiang; Liu, Kuan-Yu; Chang, Yu-Sun; Chang, Ta-Chau; Lo, Szecheng J.

    2014-01-01

    Lipid is an important energy source and essential component for plasma and organelle membranes in all kinds of cells. Coherent anti-Stokes Raman scattering (CARS) microscopy is a label-free and nonlinear optical technique that can be used to monitor the lipid distribution in live organisms. Here, we utilize CARS microscopy to investigate the pattern of lipid droplets in two live Caenorhabditis elegans mutants (fat-2 and fat-3). The CARS images showed a striking decrease in the size, number, and content of lipid droplets in the fat-2 mutant but a slight difference in the fat-3 mutant as compared with the wild-type worm. Moreover, a nondroplet-like structure with enhanced CARS signal was detected for the first time in the uterus of fat-2 and fat-3 mutants. In addition, transgenic fat-2 mutant expressing a GFP fusion protein of vitellogenin-2 (a yolk lipoprotein) revealed that the enhanced CARS signal colocalized with the GFP signal, which suggests that the nondroplet-like structure is primarily due to the accumulation of yolk lipoproteins. Together, this study implies that CARS microscopy is a potential tool to study the distribution of yolk lipoproteins, in addition to lipid droplets, in live animals.

  13. Enhancing Alkane Production in Cyanobacterial Lipid Droplets: A ModeFl Platform for Industrially Relevant Compound Production

    PubMed Central

    Peramuna, Anantha; Morton, Ray; Summers, Michael L.

    2015-01-01

    Cyanobacterial lipid droplets (LDs) are packed with hydrophobic energy-dense compounds and have great potential for biotechnological expression and the compartmentalization of high value compounds. Nostoc punctiforme normally accumulates LDs containing neutral lipids, and small amounts of heptadecane, during the stationary phase of growth. In this study, we further enhanced heptadecane production in N. punctiforme by introducing extrachromosomal copies of aar/adc genes, and report the discovery of a putative novel lipase encoded by Npun_F5141, which further enhanced alkane production. Extra copies of all three genes in high light conditions resulted in a 16-fold higher accumulation of heptadecane compared to the wild type strain in the exponential phase. LD accumulation during exponential phase also increased massively to accommodate the heptadecane production. A large number of small, less fluorescent LDs were observed at the cell periphery in exponential growth phase, whereas fewer number of highly fluorescent, much larger LDs were localized towards the center of the cell in the stationary phase. These advances demonstrate that cyanobacterial LDs are an ideal model platform to make industrially relevant compounds, such as alkanes, during exponential growth, and provide insight into LD formation in cyanobacteria. PMID:25821934

  14. Comparative proteomics reveals abnormal binding of ATGL and dysferlin on lipid droplets from pressure overload-induced dysfunctional rat hearts.

    PubMed

    Li, Linghai; Zhang, Huina; Wang, Weiyi; Hong, Yun; Wang, Jifeng; Zhang, Shuyan; Xu, Shimeng; Shu, Qingbo; Li, Juanfen; Yang, Fuquan; Zheng, Min; Qian, Zongjie; Liu, Pingsheng

    2016-01-01

    Excessive retention of neutral lipids in cardiac lipid droplets (LDs) is a common observation in cardiomyopathy. Thus, the systematic investigation of the cardiac LD proteome will help to dissect the underlying mechanisms linking cardiac steatosis and myocardial dysfunction. Here, after isolation of LDs from normal and dysfunctional Sprague-Dawley rat hearts, we identified 752 heart-associated LD proteins using iTRAQ quantitative proteomic method, including 451 proteins previously unreported on LDs. The most noteworthy finding was the identification of the membrane resealing protein, dysferlin. An analysis of dysferlin truncation mutants indicated that its C2 domain was responsible for its LD localization. Quantitative proteomic results further determined that 27 proteins were increased and 16 proteins were decreased in LDs from post pressure overload-induced dysfunctional hearts, compared with normal hearts. Notably, adipose triacylglycerol lipase (ATGL) was dramatically decreased and dysferlin was substantially increased on dysfunctional cardiac LDs. This study for the first time reveals the dataset of the heart LD proteome in healthy tissue and the variation of it under cardiac dysfunction. These findings highlight an association between the altered LD protein localization of dysferlin and ATGL and myocardial dysfunction. PMID:26795240

  15. Comparative proteomics reveals abnormal binding of ATGL and dysferlin on lipid droplets from pressure overload-induced dysfunctional rat hearts

    PubMed Central

    Li, Linghai; Zhang, Huina; Wang, Weiyi; Hong, Yun; Wang, Jifeng; Zhang, Shuyan; Xu, Shimeng; Shu, Qingbo; Li, Juanfen; Yang, Fuquan; Zheng, Min; Qian, Zongjie; Liu, Pingsheng

    2016-01-01

    Excessive retention of neutral lipids in cardiac lipid droplets (LDs) is a common observation in cardiomyopathy. Thus, the systematic investigation of the cardiac LD proteome will help to dissect the underlying mechanisms linking cardiac steatosis and myocardial dysfunction. Here, after isolation of LDs from normal and dysfunctional Sprague-Dawley rat hearts, we identified 752 heart-associated LD proteins using iTRAQ quantitative proteomic method, including 451 proteins previously unreported on LDs. The most noteworthy finding was the identification of the membrane resealing protein, dysferlin. An analysis of dysferlin truncation mutants indicated that its C2 domain was responsible for its LD localization. Quantitative proteomic results further determined that 27 proteins were increased and 16 proteins were decreased in LDs from post pressure overload-induced dysfunctional hearts, compared with normal hearts. Notably, adipose triacylglycerol lipase (ATGL) was dramatically decreased and dysferlin was substantially increased on dysfunctional cardiac LDs. This study for the first time reveals the dataset of the heart LD proteome in healthy tissue and the variation of it under cardiac dysfunction. These findings highlight an association between the altered LD protein localization of dysferlin and ATGL and myocardial dysfunction. PMID:26795240

  16. The lipid droplet enzyme Tgl1p hydrolyzes both steryl esters and triglycerides in the yeast, Saccharomyces cerevisiae.

    PubMed

    Jandrositz, Anita; Petschnigg, Julia; Zimmermann, Robert; Natter, Klaus; Scholze, Hubert; Hermetter, Albin; Kohlwein, Sepp D; Leber, Regina

    2005-06-15

    Based on sequence homology to mammalian acid lipases, yeast reading frame YKL140w was predicted to encode a triacylglycerol (TAG) lipase in yeast and was hence named as TGL1, triglyceride lipase 1. A deletion of TGL1, however, resulted in an increase of the cellular steryl ester content. Fluorescently labeled lipid analogs that become covalently linked to the enzyme active site upon catalysis were used to discriminate between the lipase and esterase activities of Tgl1p. Tgl1p preferred single-chain esterase inhibitors over lipase inhibitors in vitro. Under assay conditions optimal for acid lipases, Tgl1p exhibited steryl esterase activity only and lacked any triglyceride lipase activity. In contrast, at pH 7.4, Tgl1p also exhibited TAG lipase activity; however, steryl ester hydrolase activity was still predominant. Tgl1p localized exclusively to lipid droplets which are the intracellular storage compartment of steryl esters and triacylglycerols in the yeast S. cerevisiae. In a tgl1 deletion mutant, the mobilization of steryl esters in vivo was delayed, but not abolished, suggesting the existence of additional enzymes involved in steryl ester mobilization.

  17. The Perilipin Homologue, Lipid Storage Droplet 2, Regulates Sleep Homeostasis and Prevents Learning Impairments Following Sleep Loss

    PubMed Central

    Thimgan, Matthew S.; Suzuki, Yasuko; Seugnet, Laurent; Gottschalk, Laura; Shaw, Paul J.

    2010-01-01

    Extended periods of waking result in physiological impairments in humans, rats, and flies. Sleep homeostasis, the increase in sleep observed following sleep loss, is believed to counter the negative effects of prolonged waking by restoring vital biological processes that are degraded during sleep deprivation. Sleep homeostasis, as with other behaviors, is influenced by both genes and environment. We report here that during periods of starvation, flies remain spontaneously awake but, in contrast to sleep deprivation, do not accrue any of the negative consequences of prolonged waking. Specifically, the homeostatic response and learning impairments that are a characteristic of sleep loss are not observed following prolonged waking induced by starvation. Recently, two genes, brummer (bmm) and Lipid storage droplet 2 (Lsd2), have been shown to modulate the response to starvation. bmm mutants have excess fat and are resistant to starvation, whereas Lsd2 mutants are lean and sensitive to starvation. Thus, we hypothesized that bmm and Lsd2 may play a role in sleep regulation. Indeed, bmm mutant flies display a large homeostatic response following sleep deprivation. In contrast, Lsd2 mutant flies, which phenocopy aspects of starvation as measured by low triglyceride stores, do not exhibit a homeostatic response following sleep loss. Importantly, Lsd2 mutant flies are not learning impaired after sleep deprivation. These results provide the first genetic evidence, to our knowledge, that lipid metabolism plays an important role in regulating the homeostatic response and can protect against neuronal impairments induced by prolonged waking. PMID:20824166

  18. Turnover of the actomyosin complex in zebrafish embryos directs geometric remodelling and the recruitment of lipid droplets

    PubMed Central

    Dutta, Asmita; Kumar Sinha, Deepak

    2015-01-01

    Lipid droplets (LDs), reservoirs of cholesterols and fats, are organelles that hydrolyse lipids in the cell. In zebrafish embryos, the actomyosin complex and filamentous microtubules control the periodic regulation of the LD geometry. Contrary to the existing hypothesis that LD transport involves the kinesin-microtubule system, we find that their recruitment to the blastodisc depends on the actomyosin turnover and is independent of the microtubules. For the first time we report the existence of two distinct states of LDs, an inactive and an active state, that occur periodically, coupled weakly to the cleavage cycles. LDs are bigger, more circular and more stable in the inactive state in which the geometry of the LDs is maintained by actomyosin as well as microtubules. The active state has smaller and irregularly shaped LDs that show shape fluctuations that are linked to actin depolymerization. Because most functions of LDs employ surface interactions, our findings on the LD geometry and its regulation bring new insights to the mechanisms associated with specific functions of LDs, such as their storage capacity for fats or proteins, lipolysis etc. PMID:26355567

  19. Postprandial triglyceride-rich lipoproteins regulate perilipin-2 and perilipin-3 lipid-droplet-associated proteins in macrophages.

    PubMed

    Varela, Lourdes M; López, Sergio; Ortega-Gómez, Almudena; Bermúdez, Beatriz; Buers, Insa; Robenek, Horst; Muriana, Francisco J G; Abia, Rocío

    2015-04-01

    Lipid accumulation in macrophages contributes to atherosclerosis. Within macrophages, lipids are stored in lipid droplets (LDs); perilipin-2 and perilipin-3 are the main LD-associated proteins. Postprandial triglyceride (TG)-rich lipoproteins induce LD accumulation in macrophages. The role of postprandial lipoproteins in perilipin-2 and perilipin-3 regulation was studied. TG-rich lipoproteins (TRLs) induced the levels of intracellular TGs, LDs and perilipin-2 protein expression in THP-1 macrophages and in Apoe(-/-) mice bone-marrow-derived macrophages with low and high basal levels of TGs. Perilipin-3 was only synthesized in mice macrophages with low basal levels of TGs. The regulation was dependent on the fatty acid composition of the lipoproteins; monounsaturated and polyunsaturated fatty acids (PUFAs) more strongly attenuated these effects compared with saturated fatty acids. In THP-1 macrophages, immunofluorescence microscopy and freeze-fracture immunogold labeling indicated that the lipoproteins translocated perilipin-3 from the cytoplasm to the LD surface; only the lipoproteins that were rich in PUFAs suppressed this effect. Chemical inhibition showed that lipoproteins induced perilipin-2 protein expression through the peroxisome proliferator-activated nuclear receptor (PPAR) PPARα and PPARγ pathways. Overall, our data indicate that postprandial TRLs may be involved in atherosclerotic plaque formation through the regulation of perilipin-2 and perilipin-3 proteins in macrophages. Because the fatty acid composition of the lipoproteins is dependent on the type of fat consumed, the ingestion of olive oil, which is rich in monounsaturated fatty acids, and fish oil, which is rich in omega-3 fatty acids, can be considered a good nutritional strategy to reduce the risk of atherosclerosis by LD-associated proteins decrease. PMID:25595097

  20. Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages1[OPEN

    PubMed Central

    2016-01-01

    Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 μm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species. PMID:27208281

  1. Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages.

    PubMed

    Huang, Ming-Der; Huang, Anthony H C

    2016-07-01

    Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 μm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species. PMID:27208281

  2. Subcellular Lipid Droplets in Vanilla Leaf Epidermis and Avocado Mesocarp Are Coated with Oleosins of Distinct Phylogenic Lineages.

    PubMed

    Huang, Ming-Der; Huang, Anthony H C

    2016-07-01

    Subcellular lipid droplets (LDs) in diverse plant cells and species are coated with stabilizing oleosins of at least five phylogenic lineages and perform different functions. We examined two types of inadequately studied LDs for coated oleosins and their characteristics. The epidermis but not mesophyll of leaves of vanilla (Vanilla planifolia) and most other Asparagales species contained solitary and clustered LDs (<0.5 μm), some previously studied by electron microscopy and speculated to be for cuticle formation. In vanilla leaves, transcripts of oleosins of the U lineage were present in both epidermis and mesophyll, but oleosin occurred only in epidermis. Immuno-confocal laser scanning microscopy revealed that the LDs were coated with oleosins. LDs in isolated fractions did not coalesce, and the fractions contained heterogeneous proteins including oleosins and diverse lipids. These findings reflect the in situ structure and possible functions of the LDs. Fruit mesocarp of avocado (Persea americana) and other Lauraceae species possessed large LDs, which likely function in attracting animals for seed dispersal. They contained transcripts of oleosin of a novel M phylogenic lineage. Each avocado mesocarp fatty cell possessed one to several large LDs (5 to 20 μm) and at their periphery, numerous small LDs (<0.5 μm). Immuno-confocal laser scanning microscopy revealed that oleosin was present mostly on the small LDs. LDs in isolated fractions coalesced rapidly, and the fraction contained oleosin and several other proteins and triacylglycerols as the main lipids. These two new types of oleosin-LDs exemplify the evolutionary plasticity of oleosins-LDs in generating novel functions in diverse cell types and species.

  3. The proteome of cytosolic lipid droplets isolated from differentiated Caco-2/TC7 enterocytes reveals cell-specific characteristics

    PubMed Central

    Bouchoux, Julien; Beilstein, Frauke; Pauquai, Thomas; Guerrera, I. Chiara; Chateau, Danielle; Ly, Nathalie; Alqub, Malik; Klein, Christophe; Chambaz, Jean; Rousset, Monique; Lacorte, Jean-Marc; Morel, Etienne; Demignot, Sylvie

    2011-01-01

    Background information. Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization. Results. Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has

  4. Masking apertures enabling automation and solution exchange in sessile droplet lipid bilayers.

    PubMed

    Portonovo, Shiva A; Schmidt, Jacob

    2012-02-01

    Reconstitution of ion channels and transmembrane proteins in planar lipid bilayer membranes allow for their scientific study in highly controlled environments. Recent work with lipid bilayers formed from mechanically joined monolayers has shown their potential for wider technological application, including automation and parallelization. However, bilayer areas are highly sensitive to variations in mechanical position and the bilayers themselves cannot withstand significant perfusion of adjacent solutions. Toward this end, here we describe use of an aperture that masks the monolayer contact area, enabling formation of highly consistent bilayer areas and significantly reducing their variation with changes in relative position of the monolayers. Further, use of the aperture enables flow of solution adjacent to the bilayer without rupture or significant change in bilayer area. The device design is scalable and compatible with SBS standard instrumentation and automation technology, potentially enabling its use for rapid, parallel automated measurements of ion channels for large scale scientific studies and pharmaceutical screening.

  5. Cell proliferation, apoptosis and accumulation of lipid droplets in U937-1 cells incubated with eicosapentaenoic acid.

    PubMed Central

    Finstad, H S; Drevon, C A; Kulseth, M A; Synstad, A V; Knudsen, E; Kolset, S O

    1998-01-01

    The monocytic cell line U937-1 was cultured in the presence of eicosapentaenoic acid (20:5, n-3) (EPA) or oleic acid (18:1, n-9) (OA). EPA caused a dose-dependent inhibition of cell proliferation, whereas OA had no effect. At the highest EPA concentrations, 120 and 240 microM, inhibition of cell proliferation was accompanied by initiation of apoptosis. A concentration of 60 microM EPA caused a 35% reduction in cell proliferation without inducing apoptosis, and was therefore used for further studies. Addition of antioxidants or inhibitors of eicosanoid synthesis had no influence on the reduced cell proliferation after EPA treatment. The inhibition required continuous presence of EPA in the incubation medium as the cells resumed a normal proliferation rate when they were placed in EPA-free medium. The inhibition of proliferation was not accompanied by differentiation into macrophage-like cells, as expression of serglycin and the ability to perform respiratory burst was unaffected by EPA. Expression of CD23 mRNA increased when the cells were incubated with EPA, but to a smaller extent than after retinoic acid (RA) or PMA treatment. Furthermore, expression of the monocytic differentiation markers CD36 and CD68 was lower in cells treated with EPA or OA when compared with untreated cells. The cell cycle distribution of U937-1 cells was similar in cells incubated with EPA or PMA, whereas RA-treated cells accumulated in the G1 phase. Side scatter increased in cells incubated with EPA and OA, which was ascribed to an accumulation of lipid droplets after examination of the cells by electron microscopy. The number of droplets per cell was higher in cells exposed to EPA than OA. The cellular triacylglycerol (TAG) increased 5.5- and 15.5-fold after incubation with OA and EPA respectively. No difference in the cellular content of cholesterol compared with untreated cells was observed. The TAG fraction in EPA-treated cells contained high amounts of EPA and docosapentaenoic acid

  6. Regulation of lipid droplet-associated proteins following growth hormone administration and feed restriction in lactating Holstein cows.

    PubMed

    Faylon, M P; Koltes, D E; Spurlock, D M

    2014-05-01

    Lipid metabolism plays a crucial role in the adaptation of dairy cows to periods of energy insufficiency. The objective of the current study was to determine if lipolytic proteins are consistently regulated when energy mobilization is stimulated by different factors. We evaluated 2 models of altered energy balance in mid-lactation Holstein cows, including feed restriction (FR) and administration of bovine growth hormone (GH), by quantifying the abundance and (or) phosphorylation of hormone-sensitive lipase (HSL), perilipin (PLIN), and adipose triglyceride lipase (ATGL). For GH administration, adipose tissue and blood samples were collected 4d before and 3 and 7d after administration of GH (n=20 cows). Similarly, adipose and blood samples were obtained 6d before and 1 and 4d after initiation of FR (n=18 cows). Estimated net energy balance decreased and nonesterified fatty acid concentration increased in both experimental models. Decreased ATGL and PLIN protein abundance was observed with GH administration and FR. Additionally, the abundance of phosphorylated HSLSer565 decreased in both models. Decreased abundance of phosphorylated PLIN was observed with GH administration, but not FR. Decreased ATGL protein abundance appears to be a consistent response to energy insufficiency in lactating cows, as this response was also described with negative energy balance at the onset of lactation. In contrast, the abundance of PLIN protein and phosphorylation of HSL using antibodies targeting serine residue 565 of HSL (HSLSer565) were altered in the current research, but not at the onset of lactation. Our findings demonstrate that lipolysis is altered through the regulation of multiple proteins, and that this regulation differs according to physiological state in lactating cows. PMID:24630665

  7. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions. PMID:26733203

  8. Differential Roles of Cell Death-inducing DNA Fragmentation Factor-α-like Effector (CIDE) Proteins in Promoting Lipid Droplet Fusion and Growth in Subpopulations of Hepatocytes.

    PubMed

    Xu, Wenyi; Wu, Lizhen; Yu, Miao; Chen, Feng-Jung; Arshad, Muhammad; Xia, Xiayu; Ren, Hao; Yu, Jinhai; Xu, Li; Xu, Dijin; Li, John Zhong; Li, Peng; Zhou, Linkang

    2016-02-26

    Lipid droplets (LDs) are dynamic subcellular organelles whose growth is closely linked to obesity and hepatic steatosis. Cell death-inducing DNA fragmentation factor-α-like effector (CIDE) proteins, including Cidea, Cideb, and Cidec (also called Fsp27), play important roles in lipid metabolism. Cidea and Cidec are LD-associated proteins that promote atypical LD fusion in adipocytes. Here, we find that CIDE proteins are all localized to LD-LD contact sites (LDCSs) and promote lipid transfer, LD fusion, and growth in hepatocytes. We have identified two types of hepatocytes, one with small LDs (small LD-containing hepatocytes, SLHs) and one with large LDs (large LD-containing hepatocytes, LLHs) in the liver. Cideb is localized to LDCSs and promotes lipid exchange and LD fusion in both SLHs and LLHs, whereas Cidea and Cidec are specifically localized to the LDCSs and promote lipid exchange and LD fusion in LLHs. Cideb-deficient SLHs have reduced LD sizes and lower lipid exchange activities. Fasting dramatically induces the expression of Cidea/Cidec and increases the percentage of LLHs in the liver. The majority of the hepatocytes from the liver of obese mice are Cidea/Cidec-positive LLHs. Knocking down Cidea or Cidec significantly reduced lipid storage in the livers of obese animals. Our data reveal that CIDE proteins play differential roles in promoting LD fusion and lipid storage; Cideb promotes lipid storage under normal diet conditions, whereas Cidea and Cidec are responsible for liver steatosis under fasting and obese conditions.

  9. Dietary supplementation with soy isoflavones or replacement with soy proteins prevents hepatic lipid droplet accumulation and alters expression of genes involved in lipid metabolism in rats.

    PubMed

    Xiao, Chao Wu; Wood, Carla M; Weber, Dorcas; Aziz, Syed A; Mehta, Rekha; Griffin, Philip; Cockell, Kevin A

    2014-01-01

    Accumulation of hepatic lipid droplet (HLD) is the hallmark pathology of non-alcoholic fatty liver disease (NAFLD). This study examined the effects of soy isoflavones (ISF) and different amounts of soy proteins on the accumulation of HLD, lipid metabolism and related gene expression in rats. Weanling Sprague-Dawley rats were fed diets containing either 20 % casein protein without (D1) or with (D2) supplemental ISF (50 mg/kg diet) or substitution of casein with increasing amounts of alcohol-washed soy protein isolate (SPI, 5, 10, and 20 %; D3, D4, D5) for 90 days. Dietary casein (20 %) induced accumulation of HLD in female, but not in male rats. Both soy proteins and ISF remarkably prevented the formation of HLD. Soy proteins lowered hepatic total cholesterol and triglyceride in a dose-dependent manner. Interestingly, soy proteins but not ISF significantly increased free fatty acids in the liver of the female rats compared to D1. Proteomic analysis showed that at least 3 enzymes involved in lipogenesis were down-regulated and 7 proteins related to fatty acid β-oxidation or lipolysis were up-regulated by soy protein over D1. Additionally, 9 differentially expressed proteins identified were related to amino acid metabolism, 5 to glycolysis and 2 to cholesterol metabolism. Dietary ISF and SPI markedly reduced hepatic-peroxisome-proliferator-activated receptor γ2 (PPARγ2) and fat-specific protein 27 (FSP27) in female rats. Overall, this study has shown that partial or full replacement of dietary casein by soy protein or supplementation with soy ISF can effectively prevent the accumulation of HLD. The potential molecular mechanism(s) involved might be due to suppression of lipogenesis and stimulation of lipolysis and down-regulation of PPARγ2 and FSP27. This suggests that consumption of soy foods or supplements might be a useful strategy for the prevention or treatment of fatty liver diseases.

  10. Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.

    PubMed

    Xie, Xitao; Langlais, Paul; Zhang, Xiaodong; Heckmann, Bradlee L; Saarinen, Alicia M; Mandarino, Lawrence J; Liu, Jun

    2014-06-15

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL.

  11. Septin 9 induces lipid droplets growth by a phosphatidylinositol-5-phosphate and microtubule-dependent mechanism hijacked by HCV

    PubMed Central

    Akil, Abdellah; Peng, Juan; Omrane, Mohyeddine; Gondeau, Claire; Desterke, Christophe; Marin, Mickaël; Tronchère, Hélène; Taveneau, Cyntia; Sar, Sokhavuth; Briolotti, Philippe; Benjelloun, Soumaya; Benjouad, Abdelaziz; Maurel, Patrick; Thiers, Valérie; Bressanelli, Stéphane; Samuel, Didier; Bréchot, Christian; Gassama-Diagne, Ama

    2016-01-01

    The accumulation of lipid droplets (LD) is frequently observed in hepatitis C virus (HCV) infection and represents an important risk factor for the development of liver steatosis and cirrhosis. The mechanisms of LD biogenesis and growth remain open questions. Here, transcriptome analysis reveals a significant upregulation of septin 9 in HCV-induced cirrhosis compared with the normal liver. HCV infection increases septin 9 expression and induces its assembly into filaments. Septin 9 regulates LD growth and perinuclear accumulation in a manner dependent on dynamic microtubules. The effects of septin 9 on LDs are also dependent on binding to PtdIns5P, which, in turn, controls the formation of septin 9 filaments and its interaction with microtubules. This previously undescribed cooperation between PtdIns5P and septin 9 regulates oleate-induced accumulation of LDs. Overall, our data offer a novel route for LD growth through the involvement of a septin 9/PtdIns5P signalling pathway. PMID:27417143

  12. Adenovirus RIDα uncovers a novel pathway requiring ORP1L for lipid droplet formation independent of NPC1.

    PubMed

    Cianciola, Nicholas L; Greene, Diane J; Morton, Richard E; Carlin, Cathleen R

    2013-11-01

    Niemann-Pick disease type C (NPC) is caused by mutations in NPC1 or NPC2, which coordinate egress of low-density-lipoprotein (LDL)-cholesterol from late endosomes. We previously reported that the adenovirus-encoded protein RIDα rescues the cholesterol storage phenotype in NPC1-mutant fibroblasts. We show here that RIDα reconstitutes deficient endosome-to-endoplasmic reticulum (ER) transport, allowing excess LDL-cholesterol to be esterified by acyl-CoA:cholesterol acyltransferase and stored in lipid droplets (LDs) in NPC1-deficient cells. Furthermore, the RIDα pathway is regulated by the oxysterol-binding protein ORP1L. Studies have classified ORP1L as a sterol sensor involved in LE positioning downstream of GTP-Rab7. Our data, however, suggest that ORP1L may play a role in transport of LDL-cholesterol to a specific ER pool designated for LD formation. In contrast to NPC1, which is dispensable, the RIDα/ORP1L-dependent route requires functional NPC2. Although NPC1/NPC2 constitutes the major pathway, therapies that amplify minor egress routes for LDL-cholesterol could significantly improve clinical management of patients with loss-of-function NPC1 mutations. The molecular identity of putative alternative pathways, however, is poorly characterized. We propose RIDα as a model system for understanding physiological egress routes that use ORP1L to activate ER feedback responses involved in LD formation.

  13. Identification of a novel phosphorylation site in adipose triglyceride lipase as a regulator of lipid droplet localization.

    PubMed

    Xie, Xitao; Langlais, Paul; Zhang, Xiaodong; Heckmann, Bradlee L; Saarinen, Alicia M; Mandarino, Lawrence J; Liu, Jun

    2014-06-15

    Adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triacylglycerol (TG) hydrolysis, has long been known to be a phosphoprotein. However, the potential phosphorylation events that are involved in the regulation of ATGL function remain incompletely defined. Here, using a combinatorial proteomics approach, we obtained evidence that at least eight different sites of ATGL can be phosphorylated in adipocytes. Among them, Thr³⁷² resides within the hydrophobic region known to mediate lipid droplet (LD) targeting. Although it had no impact on the TG hydrolase activity, substitution of phosphorylation-mimic Asp for Thr³⁷² eliminated LD localization and LD-degrading capacity of ATGL expressed in HeLa cells. In contrast, mutation of Thr³⁷² to Ala gave a protein that bound LDs and functioned the same as the wild-type protein. In nonstimulated adipocytes, the Asp mutation led to decreased LD association and basal lipolytic activity of ATGL, whereas the Ala mutation produced opposite effects. Moreover, the LD translocation of ATGL upon β-adrenergic stimulation was also compromised by the Asp mutation. In accord with these findings, the Ala mutation promoted and the Asp mutation attenuated the capacity of ATGL to mediate lipolysis in adipocytes under both basal and stimulated conditions. Collectively, these studies identified Thr³⁷² as a novel phosphorylation site that may play a critical role in determining subcellular distribution as well as lipolytic action of ATGL. PMID:24801391

  14. Cell survival during complete nutrient deprivation depends on lipid droplet-fueled β-oxidation of fatty acids.

    PubMed

    Cabodevilla, Ainara G; Sánchez-Caballero, Laura; Nintou, Eleni; Boiadjieva, Violeta G; Picatoste, Fernando; Gubern, Albert; Claro, Enrique

    2013-09-27

    Cells exposed to stress of different origins synthesize triacylglycerols and generate lipid droplets (LD), but the physiological relevance of this response is uncertain. Using complete nutrient deprivation of cells in culture as a simple model of stress, we have addressed whether LD biogenesis has a protective role in cells committed to die. Complete nutrient deprivation induced the biogenesis of LD in human LN18 glioblastoma and HeLa cells and also in CHO and rat primary astrocytes. In all cell types, death was associated with LD depletion and was accelerated by blocking LD biogenesis after pharmacological inhibition of Group IVA phospholipase A2 (cPLA2α) or down-regulation of ceramide kinase. Nutrient deprivation also induced β-oxidation of fatty acids that was sensitive to cPLA2α inhibition, and cell survival in these conditions became strictly dependent on fatty acid catabolism. These results show that, during nutrient deprivation, cell viability is sustained by β-oxidation of fatty acids that requires biogenesis and mobilization of LD.

  15. Activation of hormone-sensitive lipase requires two steps, protein phosphorylation and binding to the PAT-1 domain of lipid droplet coat proteins.

    PubMed

    Wang, Hong; Hu, Liping; Dalen, Knut; Dorward, Heidi; Marcinkiewicz, Amy; Russell, Deanna; Gong, Dawei; Londos, Constantine; Yamaguchi, Tomohiro; Holm, Cecilia; Rizzo, Mark A; Brasaemle, Dawn; Sztalryd, Carole

    2009-11-13

    Lipolysis is an important metabolic pathway controlling energy homeostasis through degradation of triglycerides stored in lipid droplets and release of fatty acids. Lipid droplets of mammalian cells are coated with one or more members of the PAT protein family, which serve important functions in regulating lipolysis. In this study, we investigate the mechanisms by which PAT family members, perilipin A, adipose differentiation-related protein (ADFP), and LSDP5, control lipolysis catalyzed by hormone-sensitive lipase (HSL), a major lipase in adipocytes and several non-adipose cells. We applied fluorescence microscopic tools to analyze proteins in situ in cultured Chinese hamster ovary cells using fluorescence recovery after photobleaching and anisotropy Forster resonance energy transfer. Fluorescence recovery after photobleaching data show that ADFP and LSDP5 exchange between lipid droplet and cytoplasmic pools, whereas perilipin A does not. Differences in protein mobility do not correlate with PAT protein-mediated control of lipolysis catalyzed by HSL or endogenous lipases. Forster resonance energy transfer and co-immunoprecipitation experiments reveal that each of the three PAT proteins bind HSL through interaction of the lipase with amino acids within the highly conserved amino-terminal PAT-1 domain. ADFP and LSDP5 bind HSL under basal conditions, whereas phosphorylation of serine residues within three amino-terminal protein kinase A consensus sequences of perilipin A is required for HSL binding and maximal lipolysis. Finally, protein kinase A-mediated phosphorylation of HSL increases lipolysis in cells expressing ADFP or LSDP5; in contrast, phosphorylation of perilipin A exerts the major control over HSL-mediated lipolysis when perilipin is the main lipid droplet protein. PMID:19717842

  16. Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine.

    PubMed

    Prats, Clara; Donsmark, Morten; Qvortrup, Klaus; Londos, Constantine; Sztalryd, Carole; Holm, Cecilia; Galbo, Henrik; Ploug, Thorkil

    2006-11-01

    A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

  17. Induction of liver steatosis and lipid droplet formation in ATF6alpha-knockout mice burdened with pharmacological endoplasmic reticulum stress.

    PubMed

    Yamamoto, Keisuke; Takahara, Kazuna; Oyadomari, Seiichi; Okada, Tetsuya; Sato, Takashi; Harada, Akihiro; Mori, Kazutoshi

    2010-09-01

    Accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates homeostatic responses collectively termed the unfolded protein response. Among the three principal signaling pathways operating in mammals, activating transcription factor (ATF)6alpha plays a pivotal role in transcriptional induction of ER-localized molecular chaperones and folding enzymes as well as components of ER-associated degradation, and thereby mouse embryonic fibroblasts deficient in ATF6alpha are sensitive to ER stress. However, ATF6alpha-knockout mice show no apparent phenotype under normal growing conditions. In this report, we burdened mice with intraperitoneal injection of the ER stress-inducing reagent tunicamycin and found that wild-type mice were able to recover from the insult, whereas ATF6alpha-knockout mice exhibited liver dysfunction and steatosis. Thus, ATF6alpha-knockout mice accumulated neutral lipids in the liver such as triacylglycerol and cholesterol, which was ascribable to blockage of beta-oxidation of fatty acids caused by decreased mRNA levels of the enzymes involved in the process, suppression of very-low-density lipoprotein formation due to destabilized apolipoprotein B-100, and stimulation of lipid droplet formation resulting from transcriptional induction of adipose differentiation-related protein. Accordingly, the hepatocytes of tunicamycin-injected knockout mice were filled with many lipid droplets. These results establish links among ER stress, lipid metabolism, and steatosis.

  18. Distribution of vitamin A-storing lipid droplets in hepatic stellate cells in liver lobules--a comparative study.

    PubMed

    Higashi, Nobuyo; Senoo, Haruki

    2003-03-01

    To investigate the storage mechanisms of vitamin A, we examined the liver of adult polar bears and arctic foxes, which physiologically store a large amount of vitamin A, by high-performance liquid chromatography (HPLC), transmission electron microscopy (TEM) morphometry, gold chloride staining, fluorescence microscopy for the detection of autofluorescence of vitamin A, staining with hematoxylin-eosin (H&E), Masson's trichrome, and Ishii and Ishii's silver impregnation. HPLC revealed that the polar bears and arctic foxes contained 1.8-1.9 x 10(4) nmol total retinol (retinol plus retinyl esters) per gram liver. In the arctic foxes, the composition of the retinyl esters was found to be 51.1% palmitate, 26.6% oleate, 15.4% stearate, and 7% linoleate. The hepatic stellate cells of the arctic animals were demonstrated by TEM to contain the bulk of the vitamin A-lipid droplets in their cytoplasm. The liver lobules of the arctic animals showed a zonal gradient in the storage of vitamin A. The gradient was expressed as a symmetric crescendo-decrescendo profile starting at the periportal zone, peaking at the middle zone, and sloping down toward the central zone in the liver lobule. The density (i.e., cell number per area) of hepatic stellate cells was essentially the same among the zones. The gradient and the composition of the retinyl esters in storing vitamin A were not changed by differences in the vitamin A amount in the livers. These results indicate that the heterogeneity of vitamin A-storage capacity in hepatic stellate cells of arctic foxes and polar bears is genetically determined. PMID:12552640

  19. Lipid droplet binding thalidomide analogs activate endoplasmic reticulum stress and suppress hepatocellular carcinoma in a chemically induced transgenic mouse model

    PubMed Central

    2013-01-01

    Background Hepatocellular carcinoma (HCC) is the most frequent and aggressive primary tumor of the liver and it has limited treatment options. Results In this study, we report the in vitro and in vivo effects of two novel amino-trifluoro-phtalimide analogs, Ac-915 and Ac-2010. Both compounds bind lipid droplets and endoplasmic reticulum membrane, and interact with several proteins with chaperone functions (HSP60, HSP70, HSP90, and protein disulfide isomerase) as determined by affinity chromatography and resonant waveguide optical biosensor technology. Both compounds inhibited protein disulfide isomerase activity and induced cell death of different HCC cells at sub or low micromolar ranges detected by classical biochemical end-point assay as well as with real-time label-free measurements. Besides cell proliferation inhibiton, analogs also inhibited cell migration even at 250 nM. Relative biodistribution of the analogs was analysed in native tissue sections of different organs after administration of drugs, and by using fluorescent confocal microscopy based on the inherent blue fluorescence of the compounds. The analogs mainly accumulated in the liver. The effects of Ac-915 and Ac-2010 were also demonstrated on the advanced stages of hepatocarcinogenesis in a transgenic mouse model of N-nitrosodiethylamine (DEN)-induced HCC. Significantly less tumor development was found in the livers of the Ac-915- or Ac-2010-treated groups compared with control mice, characterized by less liver tumor incidence, fewer tumors and smaller tumor size. Conclusion These results imply that these amino-trifluoro-phthalimide analogs could serve potent clinical candidates against HCC alone or in combination with dietary polyunsaturated fatty acids. PMID:24268070

  20. Picoliter droplet-based digital peptide nucleic acid clamp PCR and dielectric sorting for low abundant K-ras mutations

    NASA Astrophysics Data System (ADS)

    Zhang, Huidan; Sperling, Ralph; Rotem, Assaf; Shan, Lianfeng; Heyman, John; Zhang, Yizhe; Weitz, David

    2012-02-01

    Colorectal cancer (CRC) remains the second leading cause of cancer-related mortality in the US, and the 5-year survival of metastatic CRC (mCRC) is less than 10%. Although monoclonal antibodies against epidermal growth factor receptor (EGFR) provide incremental improvements in survival, approximately 40% of mCRC patients with activating KRAS mutations won't benefit from this therapy. Peptide nucleic acid (PNA), a synthetic non-extendable oligonucleotides, can bind strongly to completely complementary wild-type KRAS by Watson-Crick base pairing and suppress its amplification during PCR, while any mutant allele will show unhindered amplification. The method is particularly suitable for the simultaneously detection of several adjoining mutant sites, just as mutations of codons 12 and 13 of KRAS gene where there are totally 12 possible mutation types. In this work, we describe the development and validation of this method, based on the droplet-based digital PCR. Using a microfluidic system, single target DNA molecule is compartmentalized in microdroplets together with PNA specific for wild-type KRAS, thermocycled and the fluorescence of each droplet was detected, followed by sorting and sequencing. It enables the precise determination of all possible mutant KRAS simultaneously, and the precise quantification of a single mutated KRAS in excess background unmutated KRAS.

  1. Small organic solutes in sticky droplets from orb webs of the spider Zygiella atrica (Araneae; Araneidae): β-alaninamide is a novel and abundant component.

    PubMed

    Townley, Mark A; Pu, Qinglin; Zercher, Charles K; Neefus, Christopher D; Tillinghast, Edward K

    2012-10-01

    In northeastern North America, Zygiella atrica often build their orb webs near the ocean. We analyzed individual field-built Z. atrica webs to determine if organic low-molecular-mass solutes (LMM) in their sticky droplets showed any unusual features not previously seen in orb webs of other species living in less salty environments. While two of the three most abundant organic LMM (putrescine (butane-1,4-diamine) and GABamide (4-aminobutanamide)) are already well-known from webs of inland spiders, the third major LMM, β-alaninamide (3-aminopropanamide), a homolog of GABamide, has not been detected in sticky droplets from any other araneoid spiders (27 species). It remains to be established, however, whether or not use of β-alaninamide is related to proximity to saltwater. We observed variability in organic LMM composition in Z. atrica webs that appeared to be influenced more by an undetermined factor associated with different collecting locations and/or collection dates than by different genders or instars. Shifts in composition when adult females were transferred from the field to the laboratory were also observed. Structural similarities and inverse correlations among β-alaninamide, GABamide, and N-acetylputrescine suggest that they may form a series of LMM fulfilling essentially the same, as yet unknown, role in the webs of those species in which they occur.

  2. Small organic solutes in sticky droplets from orb webs of the spider Zygiella atrica (Araneae; Araneidae): β-alaninamide is a novel and abundant component.

    PubMed

    Townley, Mark A; Pu, Qinglin; Zercher, Charles K; Neefus, Christopher D; Tillinghast, Edward K

    2012-10-01

    In northeastern North America, Zygiella atrica often build their orb webs near the ocean. We analyzed individual field-built Z. atrica webs to determine if organic low-molecular-mass solutes (LMM) in their sticky droplets showed any unusual features not previously seen in orb webs of other species living in less salty environments. While two of the three most abundant organic LMM (putrescine (butane-1,4-diamine) and GABamide (4-aminobutanamide)) are already well-known from webs of inland spiders, the third major LMM, β-alaninamide (3-aminopropanamide), a homolog of GABamide, has not been detected in sticky droplets from any other araneoid spiders (27 species). It remains to be established, however, whether or not use of β-alaninamide is related to proximity to saltwater. We observed variability in organic LMM composition in Z. atrica webs that appeared to be influenced more by an undetermined factor associated with different collecting locations and/or collection dates than by different genders or instars. Shifts in composition when adult females were transferred from the field to the laboratory were also observed. Structural similarities and inverse correlations among β-alaninamide, GABamide, and N-acetylputrescine suggest that they may form a series of LMM fulfilling essentially the same, as yet unknown, role in the webs of those species in which they occur. PMID:23081916

  3. Effects of dietary plant meal and soya-saponin supplementation on intestinal and hepatic lipid droplet accumulation and lipoprotein and sterol metabolism in Atlantic salmon (Salmo salar L.).

    PubMed

    Gu, Min; Kortner, Trond M; Penn, Michael; Hansen, Anne Kristine; Krogdahl, Åshild

    2014-02-01

    Altered lipid metabolism has been shown in fish fed plant protein sources. The present study aimed to gain further insights into how intestinal and hepatic lipid absorption and metabolism are modulated by plant meal (PM) and soya-saponin (SA) inclusion in salmon feed. Post-smolt Atlantic salmon were fed for 10 weeks one of four diets based on fishmeal or PM, with or without 10 g/kg SA. PM inclusion resulted in decreased growth performance, excessive lipid droplet accumulation in the pyloric caeca and liver, and reduced plasma cholesterol levels. Intestinal and hepatic gene expression profiling revealed an up-regulation of the expression of genes involved in lipid absorption and lipoprotein (LP) synthesis (apo, fatty acid transporters, microsomal TAG transfer protein, acyl-CoA cholesterol acyltransferase, choline kinase and choline-phosphate cytidylyltransferase A), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase) and associated transcription factors (sterol regulatory element-binding protein 2 and PPARγ). SA inclusion resulted in reduced body pools of cholesterol and bile salts. The hepatic gene expression of the rate-limiting enzyme in bile acid biosynthesis (cytochrome P450 7A1 (cyp7a1)) as well as the transcription factor liver X receptor and the bile acid transporter abcb11 (ATP-binding cassette B11) was down-regulated by SA inclusion. A significant interaction was observed between PM inclusion and SA inclusion for plasma cholesterol levels. In conclusion, gene expression profiling suggested that the capacity for LP assembly and cholesterol synthesis was up-regulated by PM exposure, probably as a compensatory mechanism for excessive lipid droplet accumulation and reduced plasma cholesterol levels. SA inclusion had hypocholesterolaemic effects on Atlantic salmon, accompanied by decreased bile salt metabolism.

  4. Droplet organelles?

    PubMed

    Courchaine, Edward M; Lu, Alice; Neugebauer, Karla M

    2016-08-01

    Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology.

  5. Droplet organelles?

    PubMed

    Courchaine, Edward M; Lu, Alice; Neugebauer, Karla M

    2016-08-01

    Cells contain numerous, molecularly distinct cellular compartments that are not enclosed by lipid bilayers. These compartments are implicated in a wide range of cellular activities, and they have been variously described as bodies, granules, or organelles. Recent evidence suggests that a liquid-liquid phase separation (LLPS) process may drive their formation, possibly justifying the unifying term "droplet organelle". A veritable deluge of recent publications points to the importance of low-complexity proteins and RNA in determining the physical properties of phase-separated structures. Many of the proteins linked to such structures are implicated in human diseases, such as amyotrophic lateral sclerosis (ALS). We provide an overview of the organizational principles that characterize putative "droplet organelles" in healthy and diseased cells, connecting protein biochemistry with cell physiology. PMID:27357569

  6. Abundant genetic overlap between blood lipids and immune-mediated diseases indicates shared molecular genetic mechanisms.

    PubMed

    Andreassen, Ole A; Desikan, Rahul S; Wang, Yunpeng; Thompson, Wesley K; Schork, Andrew J; Zuber, Verena; Doncheva, Nadezhda T; Ellinghaus, Eva; Albrecht, Mario; Mattingsdal, Morten; Franke, Andre; Lie, Benedicte A; Mills, Ian G; Mills, Ian; Aukrust, Pål; McEvoy, Linda K; Djurovic, Srdjan; Karlsen, Tom H; Dale, Anders M

    2015-01-01

    Epidemiological studies suggest a relationship between blood lipids and immune-mediated diseases, but the nature of these associations is not well understood. We used genome-wide association studies (GWAS) to investigate shared single nucleotide polymorphisms (SNPs) between blood lipids and immune-mediated diseases. We analyzed data from GWAS (n~200,000 individuals), applying new False Discovery Rate (FDR) methods, to investigate genetic overlap between blood lipid levels [triglycerides (TG), low density lipoproteins (LDL), high density lipoproteins (HDL)] and a selection of archetypal immune-mediated diseases (Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, psoriasis and sarcoidosis). We found significant polygenic pleiotropy between the blood lipids and all the investigated immune-mediated diseases. We discovered several shared risk loci between the immune-mediated diseases and TG (n = 88), LDL (n = 87) and HDL (n = 52). Three-way analyses differentiated the pattern of pleiotropy among the immune-mediated diseases. The new pleiotropic loci increased the number of functional gene network nodes representing blood lipid loci by 40%. Pathway analyses implicated several novel shared mechanisms for immune pathogenesis and lipid biology, including glycosphingolipid synthesis (e.g. FUT2) and intestinal host-microbe interactions (e.g. ATG16L1). We demonstrate a shared genetic basis for blood lipids and immune-mediated diseases independent of environmental factors. Our findings provide novel mechanistic insights into dyslipidemia and immune-mediated diseases and may have implications for therapeutic trials involving lipid-lowering and anti-inflammatory agents. PMID:25853426

  7. Abundant genetic overlap between blood lipids and immune-mediated diseases indicates shared molecular genetic mechanisms.

    PubMed

    Andreassen, Ole A; Desikan, Rahul S; Wang, Yunpeng; Thompson, Wesley K; Schork, Andrew J; Zuber, Verena; Doncheva, Nadezhda T; Ellinghaus, Eva; Albrecht, Mario; Mattingsdal, Morten; Franke, Andre; Lie, Benedicte A; Mills, Ian G; Mills, Ian; Aukrust, Pål; McEvoy, Linda K; Djurovic, Srdjan; Karlsen, Tom H; Dale, Anders M

    2015-01-01

    Epidemiological studies suggest a relationship between blood lipids and immune-mediated diseases, but the nature of these associations is not well understood. We used genome-wide association studies (GWAS) to investigate shared single nucleotide polymorphisms (SNPs) between blood lipids and immune-mediated diseases. We analyzed data from GWAS (n~200,000 individuals), applying new False Discovery Rate (FDR) methods, to investigate genetic overlap between blood lipid levels [triglycerides (TG), low density lipoproteins (LDL), high density lipoproteins (HDL)] and a selection of archetypal immune-mediated diseases (Crohn's disease, ulcerative colitis, rheumatoid arthritis, type 1 diabetes, celiac disease, psoriasis and sarcoidosis). We found significant polygenic pleiotropy between the blood lipids and all the investigated immune-mediated diseases. We discovered several shared risk loci between the immune-mediated diseases and TG (n = 88), LDL (n = 87) and HDL (n = 52). Three-way analyses differentiated the pattern of pleiotropy among the immune-mediated diseases. The new pleiotropic loci increased the number of functional gene network nodes representing blood lipid loci by 40%. Pathway analyses implicated several novel shared mechanisms for immune pathogenesis and lipid biology, including glycosphingolipid synthesis (e.g. FUT2) and intestinal host-microbe interactions (e.g. ATG16L1). We demonstrate a shared genetic basis for blood lipids and immune-mediated diseases independent of environmental factors. Our findings provide novel mechanistic insights into dyslipidemia and immune-mediated diseases and may have implications for therapeutic trials involving lipid-lowering and anti-inflammatory agents.

  8. Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule

    PubMed Central

    Gaida, Matthias M.; Mayer, Christine; Dapunt, Ulrike; Stegmaier, Sabine; Schirmacher, Peter; Wabnitz, Guido H.; Hänsch, G. Maria

    2016-01-01

    T2R38 belongs to the family of bitter receptors and was initially detected in cells of the oral cavity. We now describe expression of T2R38 in tumor cells in patients with pancreatic cancer and in tumor-derived cell lines. T2R38 is localized predominantly intracellular in association with lipid droplets, particularly with the lipid droplet membrane. The receptor can be activated by the bona fide ligand for T2R38, phenylthiourea (PTU), and by N-acetyl-dodecanoyl homoserine (AHL-12), a quorum sensing molecule of Pseudomonas aeruginosa, the latter is the only known natural ligand for T2R38. In response to PTU or AHL-12, key transcription factors are activated including phosphorylation of the MAP kinases p38 and ERK1/2, and upregulation of NFATc1. Moreover, we found increased expression of the multi-drug resistance protein 1 (also known as ABCB1), a transmembrane transporter molecule, participating in shuttling of a plethora of drugs, such as chemotherapeutics or antibiotics. In conclusion, our data indicate a new, additional function of the taste receptor T2R38 beyond sensing ‘bitter’. Moreover, because T2R38 can be stimulated by a bacteria-derived signaling molecule the receptor could link microbiota and cancer. PMID:26862855

  9. The broad-spectrum antiviral compound ST-669 restricts chlamydial inclusion development and bacterial growth and localizes to host cell lipid droplets within treated cells.

    PubMed

    Sandoz, Kelsi M; Valiant, William G; Eriksen, Steven G; Hruby, Dennis E; Allen, Robert D; Rockey, Daniel D

    2014-07-01

    Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a lipid droplet-dependent process. PMID:24777097

  10. Expression of the bitter receptor T2R38 in pancreatic cancer: localization in lipid droplets and activation by a bacteria-derived quorum-sensing molecule.

    PubMed

    Gaida, Matthias M; Mayer, Christine; Dapunt, Ulrike; Stegmaier, Sabine; Schirmacher, Peter; Wabnitz, Guido H; Hänsch, G Maria

    2016-03-15

    T2R38 belongs to the family of bitter receptors and was initially detected in cells of the oral cavity. We now describe expression of T2R38 in tumor cells in patients with pancreatic cancer and in tumor-derived cell lines. T2R38 is localized predominantly intracellular in association with lipid droplets, particularly with the lipid droplet membrane. The receptor can be activated by the bona fide ligand for T2R38, phenylthiourea (PTU), and by N-acetyl-dodecanoyl homoserine (AHL-12), a quorum sensing molecule of Pseudomonas aeruginosa, the latter is the only known natural ligand for T2R38. In response to PTU or AHL-12, key transcription factors are activated including phosphorylation of the MAP kinases p38 and ERK1/2, and upregulation of NFATc1. Moreover, we found increased expression of the multi-drug resistance protein 1 (also known as ABCB1), a transmembrane transporter molecule, participating in shuttling of a plethora of drugs, such as chemotherapeutics or antibiotics. In conclusion, our data indicate a new, additional function of the taste receptor T2R38 beyond sensing "bitter". Moreover, because T2R38 can be stimulated by a bacteria-derived signaling molecule the receptor could link microbiota and cancer. PMID:26862855

  11. The Broad-Spectrum Antiviral Compound ST-669 Restricts Chlamydial Inclusion Development and Bacterial Growth and Localizes to Host Cell Lipid Droplets within Treated Cells

    PubMed Central

    Sandoz, Kelsi M.; Valiant, William G.; Eriksen, Steven G.; Hruby, Dennis E.; Allen, Robert D.

    2014-01-01

    Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization. Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a lipid droplet-dependent process. PMID:24777097

  12. A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis.

    PubMed

    Ren, Jihui; Pei-Chen Lin, Coney; Pathak, Manish C; Temple, Brenda R S; Nile, Aaron H; Mousley, Carl J; Duncan, Mara C; Eckert, Debra M; Leiker, Thomas J; Ivanova, Pavlina T; Myers, David S; Murphy, Robert C; Brown, H Alex; Verdaasdonk, Jolien; Bloom, Kerry S; Ortlund, Eric A; Neiman, Aaron M; Bankaitis, Vytas A

    2014-03-01

    Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches. PMID:24403601

  13. A phosphatidylinositol transfer protein integrates phosphoinositide signaling with lipid droplet metabolism to regulate a developmental program of nutrient stress-induced membrane biogenesis

    SciTech Connect

    Ren, Jihui; Lin, Coney Pei-Chen; Pathak, Manish C.; Temple, Brenda R.S.; Nile, Aaron H.; Mousley, Carl J.; Duncan, Mara C.; Eckert, Debra M.; Leiker, Thomas J.; Ivanova, Pavlina T.; Myers, David S.; Murphy, Robert C.; Brown, H. Alex; Verdaasdonk, Jolien; Bloom, Kerry S.; Ortlund, Eric A.; Neiman, Aaron M.; Bankaitis, Vytas A.

    2014-07-11

    Lipid droplet (LD) utilization is an important cellular activity that regulates energy balance and release of lipid second messengers. Because fatty acids exhibit both beneficial and toxic properties, their release from LDs must be controlled. Here we demonstrate that yeast Sfh3, an unusual Sec14-like phosphatidylinositol transfer protein, is an LD-associated protein that inhibits lipid mobilization from these particles. We further document a complex biochemical diversification of LDs during sporulation in which Sfh3 and select other LD proteins redistribute into discrete LD subpopulations. The data show that Sfh3 modulates the efficiency with which a neutral lipid hydrolase-rich LD subclass is consumed during biogenesis of specialized membrane envelopes that package replicated haploid meiotic genomes. These results present novel insights into the interface between phosphoinositide signaling and developmental regulation of LD metabolism and unveil meiosis-specific aspects of Sfh3 (and phosphoinositide) biology that are invisible to contemporary haploid-centric cell biological, proteomic, and functional genomics approaches.

  14. Remodeling of host phosphatidylcholine by Chlamydia acyltransferase is regulated by acyl-CoA binding protein ACBD6 associated with lipid droplets

    PubMed Central

    Soupene, Eric; Wang, Derek; Kuypers, Frans A

    2015-01-01

    The bacterial human pathogen Chlamydia trachomatis invades cells as an infectious elementary body (EB). The EB is internalized into a vacuole that is hidden from the host defense mechanism, and is modified to sustain the development of the replicative reticulate body (RB). Inside this parasitophorous compartment, called the inclusion, the pathogen survives supported by an active exchange of nutrients and proteins with the host cell. We show that host lipids are scavenged and modified into bacterial-specific lipids by the action of a shared human-bacterial acylation mechanism. The bacterial acylating enzymes for the essential lipids 1-acyl-sn-glycerol 3-phosphate and 1-acyl-sn-phosphatidylcholine were identified as CT453 and CT775, respectively. Bacterial CT775 was found to be associated with lipid droplets (LDs). During the development of C. trachomatis, the human acyl-CoA carrier hACBD6 was recruited to cytosolic LDs and translocated into the inclusion. hACBD6 protein modulated the activity of CT775 in an acyl-CoA dependent fashion and sustained the activity of the bacterial acyltransferase by buffering the concentration of acyl-CoAs. We propose that disruption of the binding activity of the acyl-CoA carrier might represent a new drug-target to prevent growth of C. trachomatis. PMID:25604091

  15. RNAi screens reveal novel metabolic regulators: RIP140, MAP4k4 and the lipid droplet associated fat specific protein (FSP) 27.

    PubMed

    Puri, V; Virbasius, J V; Guilherme, A; Czech, M P

    2008-01-01

    Adipose tissue modulates whole body metabolism and insulin sensitivity by controlling circulating lipid levels and producing molecules that can regulate fatty acid metabolism in such tissues as muscle and liver. We have developed RNA interference (RNAi) screens to identify genes in cultured adipocytes that regulate insulin signalling and key metabolic pathways. These short interfering RNA (siRNA)-based screens identified the transcriptional corepressor receptor interacting protein 140 (RIP140) (J Clin Invest 116: 125, 2006) and the mitogen-activated protein kinase (MAP4k4) (Proc Natl Acad Sci USA 103: 2087, 2006) as negative regulators of insulin-responsive hexose uptake and oxidative metabolism. Gene expression profiling revealed that RIP140 depletion upregulates the expression of clusters of genes in the pathways of glucose uptake, glycolysis, tricarboxylic acid cycle, fatty acid oxidation, mitochondrial biogenesis and oxidative phosphorylation. RIP140-null mice resist weight gain on a high-fat diet and display enhanced glucose tolerance. MAP4k4 depletion in adipocytes increases many of the RIP140-sensitive genes, increases adipogenesis and mediates some actions of tumour necrosis factor-alpha (TNF-alpha). Remarkably, another hit in our RNAi screens was fat specific protein 27 (FSP27), a highly expressed isoform of Cidea. We discovered that FSP27 unexpectedly associates specifically with lipid droplets and regulates fat storage. We conclude that RIP140, MAP4k4 and the novel lipid droplet protein FSP27 are powerful regulators of adipose tissue metabolism and are potential therapeutic targets for controlling metabolic disease. The discovery of these novel proteins validates the power of RNAi screening for discovery of new therapeutic approaches to type 2 diabetes and obesity. PMID:18171433

  16. Lipid droplet and early autophagosomal membrane targeting of Atg2A and Atg14L in human tumor cells[S

    PubMed Central

    Pfisterer, Simon G.; Bakula, Daniela; Frickey, Tancred; Cezanne, Alice; Brigger, Daniel; Tschan, Mario P.; Robenek, Horst; Proikas-Cezanne, Tassula

    2014-01-01

    Autophagy is a lysosomal bulk degradation pathway for cytoplasmic cargo, such as long-lived proteins, lipids, and organelles. Induced upon nutrient starvation, autophagic degradation is accomplished by the concerted actions of autophagy-related (ATG) proteins. Here we demonstrate that two ATGs, human Atg2A and Atg14L, colocalize at cytoplasmic lipid droplets (LDs) and are functionally involved in controlling the number and size of LDs in human tumor cell lines. We show that Atg2A is targeted to cytoplasmic ADRP-positive LDs that migrate bidirectionally along microtubules. The LD localization of Atg2A was found to be independent of the autophagic status. Further, Atg2A colocalized with Atg14L under nutrient-rich conditions when autophagy was not induced. Upon nutrient starvation and dependent on phosphatidylinositol 3-phosphate [PtdIns(3)P] generation, both Atg2A and Atg14L were also specifically targeted to endoplasmic reticulum-associated early autophagosomal membranes, marked by the PtdIns(3)P effectors double-FYVE containing protein 1 (DFCP1) and WD-repeat protein interacting with phosphoinositides 1 (WIPI-1), both of which function at the onset of autophagy. These data provide evidence for additional roles of Atg2A and Atg14L in the formation of early autophagosomal membranes and also in lipid metabolism. PMID:24776541

  17. Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

    PubMed

    Senoo, Haruki; Mezaki, Yoshihiro; Morii, Mayako; Hebiguchi, Taku; Miura, Mitsutaka; Imai, Katsuyuki

    2013-11-01

    Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A. PMID:23765517

  18. Uptake and storage of vitamin A as lipid droplets in the cytoplasm of cells in the lamina propria mucosae of the rat intestine.

    PubMed

    Senoo, Haruki; Mezaki, Yoshihiro; Morii, Mayako; Hebiguchi, Taku; Miura, Mitsutaka; Imai, Katsuyuki

    2013-11-01

    Vitamin A (retinyl palmitate) was injected subcutaneously or administered to rats by tube feeding. After subcutaneous injection, vitamin A was taken up and stored in cells of the lamina propria mucosae of the rat intestine. After oral administration, vitamin A was absorbed by the intestinal absorptive epithelial cells and transferred to cells of the lamina propria mucosae, where cells took up and stored the transferred vitamin A. The morphology of these cells was similar to that of hepatic stellate cells (also called vitamin A-storing cells, lipocytes, interstitial cells, fat-storing cells or Ito cells). Thus, these cells in the intestine could take up vitamin A from the systemic circulation and as well as by intestinal absorption, and store the vitamin in the lipid droplets in their cytoplasm. The data suggest that these cells are extrahepatic stellate cells of the digestive tract that may play roles in both the absorption and homeostasis of vitamin A.

  19. CaMKII-MEDIATED PHOSPHORYLATION OF THE BOMBYX MORI LIPID STORAGE DROPLET PROTEIN-1 (BmLsd1), AN INSECT PAT FAMILY PROTEIN, IS ESSENTIAL FOR SILKMOTH SEX PHEROMONE BIOSYNTHESIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

  20. Hormone signaling linked to silkmoth sex pheromone biosynthesis involves Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of the insect PAT family protein Bombyx mori lipid storage droplet protein-1(BmLsd)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The structurally-related members of the PAT family of proteins, which are so name based on similarity amongst perilipin, adipophilin/adipocyte differentiation-related protein (ADRP), and tail-interacting protein of 47 kilodaltons (TIP47), are cytoplasmic lipid droplet (LD)-associated proteins charac...

  1. Leptin activation of mTOR pathway in intestinal epithelial cell triggers lipid droplet formation, cytokine production and increased cell proliferation.

    PubMed

    Fazolini, Narayana P B; Cruz, André L S; Werneck, Miriam B F; Viola, João P B; Maya-Monteiro, Clarissa M; Bozza, Patrícia T

    2015-01-01

    Accumulating evidence suggests that obesity and enhanced inflammatory reactions are predisposing conditions for developing colon cancer. Obesity is associated with high levels of circulating leptin. Leptin is an adipocytokine that is secreted by adipose tissue and modulates immune response and inflammation. Lipid droplets (LD) are organelles involved in lipid metabolism and production of inflammatory mediators, and increased numbers of LD were observed in human colon cancer. Leptin induces the formation of LD in macrophages in a PI3K/mTOR pathway-dependent manner. Moreover, the mTOR is a serine/threonine kinase that plays a key role in cellular growth and is frequently altered in tumors. We therefore investigated the role of leptin in the modulation of mTOR pathway and regulation of lipid metabolism and inflammatory phenotype in intestinal epithelial cells (IEC-6 cells). We show that leptin promotes a dose- and time-dependent enhancement of LD formation. The biogenesis of LD was accompanied by enhanced CXCL1/CINC-1, CCL2/MCP-1 and TGF-β production and increased COX-2 expression in these cells. We demonstrated that leptin-induced increased phosphorylation of STAT3 and AKT and a dose and time-dependent mTORC activation with enhanced phosphorilation of the downstream protein P70S6K protein. Pre-treatment with rapamycin significantly inhibited leptin effects in LD formation, COX-2 and TGF-β production in IEC-6 cells. Moreover, leptin was able to stimulate the proliferation of epithelial cells on a mTOR-dependent manner. We conclude that leptin regulates lipid metabolism, cytokine production and proliferation of intestinal cells through a mechanism largely dependent on activation of the mTOR pathway, thus suggesting that leptin-induced mTOR activation may contribute to the obesity-related enhanced susceptibility to colon carcinoma. PMID:26017929

  2. Effects on Transcriptional Regulation and Lipid Droplet Characteristics in the Liver of Female Juvenile Pigs after Early Postnatal Feed Restriction and Refeeding Are Dependent on Birth Weight

    PubMed Central

    Nebendahl, Constance; Krüger, Ricarda; Görs, Solvig; Albrecht, Elke; Martens, Karen; Hennig, Steffen; Storm, Niels; Höppner, Wolfgang; Pfuhl, Ralf; Metzler-Zebeli, Barbara U.; Hammon, Harald M.; Metges, Cornelia C.

    2013-01-01

    Epidemiological and experimental data indicate that caloric restriction in early postnatal life may improve liver lipid metabolism in low birth weight individuals. The present study investigated transcriptional and metabolic responses to low (U) and normal (N) birth weight (d 75, T1) and postnatal feed restriction (R, 60% of controls, d 98, T2) followed by subsequent refeeding until d 131 of age (T3). Liver tissue studies were performed with a total of 42 female pigs which were born by multiparous German landrace sows. Overall, 194 genes were differentially expressed in the liver of U vs. N (T1) animals with roles in lipid metabolism. The total mean area and number of lipid droplets (LD) was about 4.6- and 3.7 times higher in U compared to N. In U, the mean LD size (µm2) was 24.9% higher. 3-week feed restriction reduced total mean area of LDs by 58.3 and 72.7% in U and N, respectively. A functional role of the affected genes in amino acid metabolism was additionally indicated. This was reflected by a 17.0% higher arginine concentration in the liver of UR animals (vs. NR). To evaluate persistency of effects, analyses were also done after refeeding period at T3. Overall, 4 and 22 genes show persistent regulation in U and N animals after 5 weeks of refeeding, respectively. These genes are involved in e.g. processes of lipid and protein metabolism and glucose homeostasis. Moreover, the recovery of total mean LD area in U and N animals back to the previous T1 level was observed. However, when compared to controls, the mean LD size was still reduced by 23.3% in UR, whereas it was increased in NR (+24.7%). The present results suggest that short-term postnatal feed restriction period programmed juvenile U animals for an increased rate of hepatic lipolysis in later life. PMID:24260100

  3. Inhibition of HCV Replication by Oxysterol-Binding Protein-Related Protein 4 (ORP4) through Interaction with HCV NS5B and Alteration of Lipid Droplet Formation

    PubMed Central

    Wen, Yahong; Liu, Ziqing; Ridgway, Neale D.; Kao, C. Cheng; He, Johnny J.

    2013-01-01

    Hepatitis C virus (HCV) RNA replication involves complex interactions among the 3’x RNA element within the HCV 3’ untranslated region, viral and host proteins. However, many of the host proteins remain unknown. In this study, we devised an RNA affinity chromatography /2D/MASS proteomics strategy and identified nine putative 3’ X-associated host proteins; among them is oxysterol-binding protein-related protein 4 (ORP4), a cytoplasmic receptor for oxysterols. We determined the relationship between ORP4 expression and HCV replication. A very low level of constitutive ORP4 expression was detected in hepatocytes. Ectopically expressed ORP4 was detected in the endoplasmic reticulum and inhibited luciferase reporter gene expression in HCV subgenomic replicon cells and HCV core expression in JFH-1-infected cells. Expression of ORP4S, an ORP4 variant that lacked the N-terminal pleckstrin-homology domain but contained the C-terminal oxysterol-binding domain also inhibited HCV replication, pointing to an important role of the oxysterol-binding domain in ORP4-mediated inhibition of HCV replication. ORP4 was found to associate with HCV NS5B and its expression led to inhibition of the NS5B activity. ORP4 expression had little effect on intracellular lipid synthesis and secretion, but it induced lipid droplet formation in the context of HCV replication. Taken together, these results demonstrate that ORP4 is a negative regulator of HCV replication, likely via interaction with HCV NS5B in the replication complex and regulation of intracellular lipid homeostasis. This work supports the important role of lipids and their metabolism in HCV replication and pathogenesis. PMID:24069433

  4. 6-gingerol prevents adipogenesis and the accumulation of cytoplasmic lipid droplets in 3T3-L1 cells.

    PubMed

    Tzeng, Thing-Fong; Liu, I-Min

    2013-04-15

    6-Gingerol ((S)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-3-decanone) is one of the pungent constituents of Zingiber zerumbet (L) Smith (Zingiberaceae family). In this study, we investigated the effects of 6-gingerol on the inhibition of adipogenesis in 3T3-L1 cells. After treatment with 6-gingerol in differentiation medium for 4 or 8 days, the 3T3-L1 cells were lysed for experimental analysis. Cells were stained with Oil-Red-O to detect oil droplets in adipocytes. The 3T3-L1 cells were lysed and measured for triglyceride contents. The protein expression of adipogenesis-related transcription factor was evaluated by Western blot analysis. 6-Gingerol suppressed oil droplet accumulation and reduced the droplet size in a concentration (5-15 μg/ml)- and time-dependent manner. Treatment of 3T3-L1 cells with 6-gingerol reduced the protein levels of peroxisome proliferator-activated receptor (PPAR)γ and CCAAT/enhancer-binding protein (C/EBP)α. Additionally, the protein levels of fatty acid synthase (FAS) and adipocyte-specific fatty acid binding protein (aP2) decreased upon treatment with 6-gingerol. Meanwhile, 6-gingerol diminished the insulin-stimulated serine phosphorylation of Akt (Ser473) and GSK3β (Ser9). These results suggest that 6-gingerol effectively suppresses adipogenesis and that it exerts its role mainly through the significant down-regulation of PPARγ and C/EBPα and subsequently inhibits FAS and aP2 expression. 6-Gingerol also inhibited differentiation in 3T3-L1 cells by attenuating the Akt/GSK3β pathway. Our findings provide important insights into the mechanisms underlying the anti-adipogenic activity of 6-gingerol. PMID:23369342

  5. Apolipoproteins C-I and C-III Inhibit Lipoprotein Lipase Activity by Displacement of the Enzyme from Lipid Droplets*

    PubMed Central

    Larsson, Mikael; Vorrsjö, Evelina; Talmud, Philippa; Lookene, Aivar; Olivecrona, Gunilla

    2013-01-01

    Apolipoproteins (apo) C-I and C-III are known to inhibit lipoprotein lipase (LPL) activity, but the molecular mechanisms for this remain obscure. We present evidence that either apoC-I or apoC-III, when bound to triglyceride-rich lipoproteins, prevent binding of LPL to the lipid/water interface. This results in decreased lipolytic activity of the enzyme. Site-directed mutagenesis revealed that hydrophobic amino acid residues centrally located in the apoC-III molecule are critical for attachment to lipid emulsion particles and consequently inhibition of LPL activity. Triglyceride-rich lipoproteins stabilize LPL and protect the enzyme from inactivating factors such as angiopoietin-like protein 4 (angptl4). The addition of either apoC-I or apoC-III to triglyceride-rich particles severely diminished their protective effect on LPL and rendered the enzyme more susceptible to inactivation by angptl4. These observations were seen using chylomicrons as well as the synthetic lipid emulsion Intralipid. In the presence of the LPL activator protein apoC-II, more of apoC-I or apoC-III was needed for displacement of LPL from the lipid/water interface. In conclusion, we show that apoC-I and apoC-III inhibit lipolysis by displacing LPL from lipid emulsion particles. We also propose a role for these apolipoproteins in the irreversible inactivation of LPL by factors such as angptl4. PMID:24121499

  6. Microbial Diversity and Lipid Abundance in Microbial Mats from a Sulfidic, Saline, Warm Spring in Utah, USA

    NASA Astrophysics Data System (ADS)

    Gong, J.; Edwardson, C.; Mackey, T. J.; Dzaugis, M.; Ibarra, Y.; Course 2012, G.; Frantz, C. M.; Osburn, M. R.; Hirst, M.; Williamson, C.; Hanselmann, K.; Caporaso, J.; Sessions, A. L.; Spear, J. R.

    2012-12-01

    The microbial diversity of Stinking Springs, a sulfidic, saline, warm spring northeast of the Great Salt Lake was investigated. The measured pH, temperature, salinity, and sulfide concentration along the flow path ranged from 6.64-7.77, 40-28° C, 2.9-2.2%, and 250 μM to negligible, respectively. Five sites were selected along the flow path and within each site microbial mats were dissected into depth profiles based on the color and texture of the mat layers. Genomic DNA was extracted from each layer, and the 16S rRNA gene was amplified and sequenced on the Roche 454 Titanium platform. Fatty acids were also extracted from the mat layers and analyzed by liquid chromatography and mass spectrometry. The mats at Stinking Springs were classified into roughly two morphologies with respect to their spatial distribution: loose, sometimes floating mats proximal to the spring source; and thicker, well-laminated mats distal to the spring source. Loosely-laminated mats were found in turbulent stream flow environments, whereas well-laminated mats were common in less turbulent sheet flows. Phototrophs, sulfur oxidizers, sulfate reducers, methanogens, other bacteria and archaea were identified by 16S rRNA gene sequences. Diatoms, identified by microscopy and lipid analysis were found to increase in abundance with distance from the source. Methanogens were generally more abundant in deeper mat laminae. Photoheterotrophs were found in all mat layers. Microbial diversity increased significantly with depth at most sites. In addition, two distinct microbial streamers were identified and characterized at the two fast flowing sites. These two streamer varieties were dominated by either cyanobacteria or flavobacteria. Overall, our genomic and lipid analysis suggest that the physical and chemical environment is more predictive of the community composition than mat morphology. Site Map

  7. Amber Light (590 nm) Induces the Breakdown of Lipid Droplets through Autophagy-Related Lysosomal Degradation in Differentiated Adipocytes.

    PubMed

    Choi, Min Sik; Kim, Hyoung-June; Ham, Mira; Choi, Dong-Hwa; Lee, Tae Ryong; Shin, Dong Wook

    2016-01-01

    Lipolysis in the adipocytes provides free fatty acids for other tissues in response to the energy demand. With the rapid increase in obesity-related diseases, finding novel stimuli or mechanisms that regulate lipid metabolism becomes important. We examined the effects of visible light (410, 457, 505, 530, 590, and 660 nm) irradiation on lipolysis regulation in adipocytes differentiated from human adipose-derived stem cells (ADSCs). Interestingly, 590 nm (amber) light irradiation significantly reduced the concentration of lipid droplets (LDs). We further investigated the lipolytic signaling pathways that are involved in 590 nm light irradiation-induced breakdown of LDs. Immunoblot analysis revealed that 590 nm light irradiation-induced phosphorylation of hormone-sensitive lipase (HSL) was insufficient to promote reduction of LDs. We observed that 590 nm light irradiation decreased the expression of perilipin 1. We found that 590 nm light irradiation, but not 505 nm, induced conversion of LC3 I to LC3 II, a representative autophagic marker. We further demonstrated that the lysosomal inhibitors leupeptin/NH4Cl inhibited 590 nm light irradiation-induced reduction of LDs in differentiated adipocytes. Our data suggest that 590 nm light irradiation-induced LD breakdown is partially mediated by autophagy-related lysosomal degradation, and can be applied in clinical settings to reduce obesity. PMID:27346059

  8. Amber Light (590 nm) Induces the Breakdown of Lipid Droplets through Autophagy-Related Lysosomal Degradation in Differentiated Adipocytes

    PubMed Central

    Choi, Min Sik; Kim, Hyoung-June; Ham, Mira; Choi, Dong-Hwa; Lee, Tae Ryong; Shin, Dong Wook

    2016-01-01

    Lipolysis in the adipocytes provides free fatty acids for other tissues in response to the energy demand. With the rapid increase in obesity-related diseases, finding novel stimuli or mechanisms that regulate lipid metabolism becomes important. We examined the effects of visible light (410, 457, 505, 530, 590, and 660 nm) irradiation on lipolysis regulation in adipocytes differentiated from human adipose-derived stem cells (ADSCs). Interestingly, 590 nm (amber) light irradiation significantly reduced the concentration of lipid droplets (LDs). We further investigated the lipolytic signaling pathways that are involved in 590 nm light irradiation-induced breakdown of LDs. Immunoblot analysis revealed that 590 nm light irradiation-induced phosphorylation of hormone-sensitive lipase (HSL) was insufficient to promote reduction of LDs. We observed that 590 nm light irradiation decreased the expression of perilipin 1. We found that 590 nm light irradiation, but not 505 nm, induced conversion of LC3 I to LC3 II, a representative autophagic marker. We further demonstrated that the lysosomal inhibitors leupeptin/NH4Cl inhibited 590 nm light irradiation-induced reduction of LDs in differentiated adipocytes. Our data suggest that 590 nm light irradiation-induced LD breakdown is partially mediated by autophagy-related lysosomal degradation, and can be applied in clinical settings to reduce obesity. PMID:27346059

  9. Shwachman-Diamond syndrome presenting with early ichthyosis, associated dermal and epidermal intracellular lipid droplets, hypoglycemia, and later distinctive clinical SDS phenotype.

    PubMed

    Scalais, Emmanuel; Connerotte, Anne-Catherine; Despontin, Karine; Biver, Armand; Ceuterick-de Groote, Chantal; Alders, Marielle; Kolivras, Athanassios; Hachem, Jean-Pierre; De Meirleir, Linda

    2016-07-01

    Shwachman-Diamond syndrome (SDS) is a recessive ribosomopathy, characterized by bone marrow failure and exocrine pancreatic insufficiency (ePI) often associated with neurodevelopmental and skeletal abnormalities. The aim of this report is to describe a SDS patient with early ichthyosis associated with dermal and epidermal intracellular lipid droplets (iLDs), hypoglycemia and later a distinctive clinical SDS phenotype. At 3 months of age, she had ichthyosis, growth retardation, and failure to thrive. She had not cytopenia. Ultrasonography (US) showed pancreatic diffuse high echogenicity. Subsequently fasting hypoketotic hypoglycemia occurred without permanent hepatomegaly or hyperlipidemia. Continuous gavage feeding was followed by clinical improvement including ichthyosis and hypoglycemia. After 14 months of age, she developed persistent neutropenia and ePI consistent with SDS. The ichthyotic skin biopsy, performed at 5 months of age, disclosed iLDs in all epidermal layers, in melanocytes, eccrine sweat glands, Schwann cells and dermal fibroblasts. These iLDs were reminiscent of those described in Dorfman-Chanarin syndrome (DCS) or Wolman's disease. Both LIPA and CGI-58 analysis did not revealed pathogenic mutation. By sequencing SBDS, a compound heterozygous for a previously reported gene mutation (c.258 + 2T>C) and a novel mutation (c.284T>G) were found. Defective SBDS may hypothetically interfere as in DCS, with neutral lipid metabolism and play a role in the SDS phenotype such as ichthyosis with dermal and epidermal iLDs and hypoglycemia. This interference with neutral lipid metabolism must most likely occur in the cytoplasm compartment as in DCS and not in the lysosomal compartment as in Wolman's disease. © 2016 Wiley Periodicals, Inc. PMID:27127007

  10. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

    PubMed

    McDonough, Patrick M; Ingermanson, Randall S; Loy, Patricia A; Koon, Erick D; Whittaker, Ross; Laris, Casey A; Hilton, Jeffrey M; Nicoll, James B; Buehrer, Benjamin M; Price, Jeffrey H

    2011-06-01

    Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes. PMID:21186937

  11. Quantification of hormone sensitive lipase phosphorylation and colocalization with lipid droplets in murine 3T3L1 and human subcutaneous adipocytes via automated digital microscopy and high-content analysis.

    PubMed

    McDonough, Patrick M; Ingermanson, Randall S; Loy, Patricia A; Koon, Erick D; Whittaker, Ross; Laris, Casey A; Hilton, Jeffrey M; Nicoll, James B; Buehrer, Benjamin M; Price, Jeffrey H

    2011-06-01

    Lipolysis in adipocytes is associated with phosphorylation of hormone sensitive lipase (HSL) and translocation of HSL to lipid droplets. In this study, adipocytes were cultured in a high-throughput format (96-well dishes), exposed to lipolytic agents, and then fixed and labeled for nuclei, lipid droplets, and HSL (or HSL phosphorylated on serine 660 [pHSLser660]). The cells were imaged via automated digital fluorescence microscopy, and high-content analysis (HCA) methods were used to quantify HSL phosphorylation and the degree to which HSL (or pHSLser660) colocalizes with the lipid droplets. HSL:lipid droplet colocalization was quantified through use of Pearson's correlation, Mander's M1 Colocalization, and the Tanimoto coefficient. For murine 3T3L1 adipocytes, isoproterenol, Lys-γ3-melanocyte stimulating hormone, and forskolin elicited the appearance and colocalization of pHSLser660, whereas atrial natriuretic peptide (ANP) did not. For human subcutaneous adipocytes, isoproterenol, forskolin, and ANP activated HSL phosphorylation/colocalization, but Lys-γ3-melanocyte stimulating hormone had little or no effect. Since ANP activates guanosine 3',5'-cyclic monophosphate (cGMP)-dependent protein kinase, HSL serine 660 is likely a substrate for cGMP-dependent protein kinase in human adipocytes. For both adipocyte model systems, adipocytes with the greatest lipid content displayed the greatest lipolytic responses. The results for pHSLser660 were consistent with release of glycerol by the cells, a well-established assay of lipolysis, and the HCA methods yielded Z' values >0.50. The results illustrate several key differences between human and murine adipocytes and demonstrate advantages of utilizing HCA techniques to study lipolysis in cultured adipocytes.

  12. The Hepatitis C Virus Core Protein Inhibits Adipose Triglyceride Lipase (ATGL)-mediated Lipid Mobilization and Enhances the ATGL Interaction with Comparative Gene Identification 58 (CGI-58) and Lipid Droplets*

    PubMed Central

    Camus, Gregory; Schweiger, Martina; Herker, Eva; Harris, Charles; Kondratowicz, Andrew S.; Tsou, Chia-Lin; Farese, Robert V.; Herath, Kithsiri; Previs, Stephen F.; Roddy, Thomas P.; Pinto, Shirly; Zechner, Rudolf; Ott, Melanie

    2014-01-01

    Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL−/− mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs. PMID:25381252

  13. The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets.

    PubMed

    Camus, Gregory; Schweiger, Martina; Herker, Eva; Harris, Charles; Kondratowicz, Andrew S; Tsou, Chia-Lin; Farese, Robert V; Herath, Kithsiri; Previs, Stephen F; Roddy, Thomas P; Pinto, Shirly; Zechner, Rudolf; Ott, Melanie

    2014-12-26

    Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs. PMID:25381252

  14. The hepatitis C virus core protein inhibits adipose triglyceride lipase (ATGL)-mediated lipid mobilization and enhances the ATGL interaction with comparative gene identification 58 (CGI-58) and lipid droplets.

    PubMed

    Camus, Gregory; Schweiger, Martina; Herker, Eva; Harris, Charles; Kondratowicz, Andrew S; Tsou, Chia-Lin; Farese, Robert V; Herath, Kithsiri; Previs, Stephen F; Roddy, Thomas P; Pinto, Shirly; Zechner, Rudolf; Ott, Melanie

    2014-12-26

    Liver steatosis is a common health problem associated with hepatitis C virus (HCV) and an important risk factor for the development of liver fibrosis and cancer. Steatosis is caused by triglycerides (TG) accumulating in lipid droplets (LDs), cellular organelles composed of neutral lipids surrounded by a monolayer of phospholipids. The HCV nucleocapsid core localizes to the surface of LDs and induces steatosis in cultured cells and mouse livers by decreasing intracellular TG degradation (lipolysis). Here we report that core at the surface of LDs interferes with the activity of adipose triglyceride lipase (ATGL), the key lipolytic enzyme in the first step of TG breakdown. Expressing core in livers or mouse embryonic fibroblasts of ATGL(-/-) mice no longer decreases TG degradation as observed in LDs from wild-type mice, supporting the model that core reduces lipolysis by engaging ATGL. Core must localize at LDs to inhibit lipolysis, as ex vivo TG hydrolysis is impaired in purified LDs coated with core but not when free core is added to LDs. Coimmunoprecipitation experiments revealed that core does not directly interact with the ATGL complex but, unexpectedly, increased the interaction between ATGL and its activator CGI-58 as well as the recruitment of both proteins to LDs. These data link the anti-lipolytic activity of the HCV core protein with altered ATGL binding to CGI-58 and the enhanced association of both proteins with LDs.

  15. Ultra-large scale AFM of lipid droplet arrays: investigating the ink transfer volume in dip pen nanolithography

    NASA Astrophysics Data System (ADS)

    Förste, Alexander; Pfirrmann, Marco; Sachs, Johannes; Gröger, Roland; Walheim, Stefan; Brinkmann, Falko; Hirtz, Michael; Fuchs, Harald; Schimmel, Thomas

    2015-05-01

    There are only few quantitative studies commenting on the writing process in dip-pen nanolithography with lipids. Lipids are important carrier ink molecules for the delivery of bio-functional patters in bio-nanotechnology. In order to better understand and control the writing process, more information on the transfer of lipid material from the tip to the substrate is needed. The dependence of the transferred ink volume on the dwell time of the tip on the substrate was investigated by topography measurements with an atomic force microscope (AFM) that is characterized by an ultra-large scan range of 800 × 800 μm2. For this purpose arrays of dots of the phospholipid1,2-dioleoyl-sn-glycero-3-phosphocholine were written onto planar glass substrates and the resulting pattern was imaged by large scan area AFM. Two writing regimes were identified, characterized of either a steady decline or a constant ink volume transfer per dot feature. For the steady state ink transfer, a linear relationship between the dwell time and the dot volume was determined, which is characterized by a flow rate of about 16 femtoliters per second. A dependence of the ink transport from the length of pauses before and in between writing the structures was observed and should be taken into account during pattern design when aiming at best writing homogeneity. The ultra-large scan range of the utilized AFM allowed for a simultaneous study of the entire preparation area of almost 1 mm2, yielding good statistic results.

  16. Ultra-large scale AFM of lipid droplet arrays: investigating the ink transfer volume in dip pen nanolithography.

    PubMed

    Förste, Alexander; Pfirrmann, Marco; Sachs, Johannes; Gröger, Roland; Walheim, Stefan; Brinkmann, Falko; Hirtz, Michael; Fuchs, Harald; Schimmel, Thomas

    2015-05-01

    There are only few quantitative studies commenting on the writing process in dip-pen nanolithography with lipids. Lipids are important carrier ink molecules for the delivery of bio-functional patters in bio-nanotechnology. In order to better understand and control the writing process, more information on the transfer of lipid material from the tip to the substrate is needed. The dependence of the transferred ink volume on the dwell time of the tip on the substrate was investigated by topography measurements with an atomic force microscope (AFM) that is characterized by an ultra-large scan range of 800 × 800 μm(2). For this purpose arrays of dots of the phospholipid1,2-dioleoyl-sn-glycero-3-phosphocholine were written onto planar glass substrates and the resulting pattern was imaged by large scan area AFM. Two writing regimes were identified, characterized of either a steady decline or a constant ink volume transfer per dot feature. For the steady state ink transfer, a linear relationship between the dwell time and the dot volume was determined, which is characterized by a flow rate of about 16 femtoliters per second. A dependence of the ink transport from the length of pauses before and in between writing the structures was observed and should be taken into account during pattern design when aiming at best writing homogeneity. The ultra-large scan range of the utilized AFM allowed for a simultaneous study of the entire preparation area of almost 1 mm(2), yielding good statistic results. PMID:25854547

  17. Draft Genome Sequence of the Basidiomycetous Yeast-Like Fungus Pseudozyma hubeiensis SY62, Which Produces an Abundant Amount of the Biosurfactant Mannosylerythritol Lipids.

    PubMed

    Konishi, Masaaki; Hatada, Yuji; Horiuchi, Jun-Ichi

    2013-01-01

    The basidiomycetous yeast-like fungus Pseudozyma hubeiensis strain SY62 is capable of producing an abundant amount of the glycolipid biosurfactant mannosylerythritol lipids (MELs), which are a major component of monoacetylated MEL (MEL-C). To reveal the synthetic pathway of the MELs of strain SY62, we present the 18.44-Mb draft genome sequence. PMID:23814110

  18. Draft Genome Sequence of the Basidiomycetous Yeast-Like Fungus Pseudozyma hubeiensis SY62, Which Produces an Abundant Amount of the Biosurfactant Mannosylerythritol Lipids

    PubMed Central

    Hatada, Yuji; Horiuchi, Jun-ichi

    2013-01-01

    The basidiomycetous yeast-like fungus Pseudozyma hubeiensis strain SY62 is capable of producing an abundant amount of the glycolipid biosurfactant mannosylerythritol lipids (MELs), which are a major component of monoacetylated MEL (MEL-C). To reveal the synthetic pathway of the MELs of strain SY62, we present the 18.44-Mb draft genome sequence. PMID:23814110

  19. Toxoplasma gondii-skeletal muscle cells interaction increases lipid droplet biogenesis and positively modulates the production of IL-12, IFN-g and PGE2

    PubMed Central

    2014-01-01

    Background The interest in the mechanisms involved in Toxoplasma gondii lipid acquisition has steadily increased during the past few decades, but it remains not completely understood. Here, we investigated the biogenesis and the fate of lipid droplets (LD) of skeletal muscle cells (SkMC) during their interaction with T. gondii by confocal and electron microscopy. We also evaluated whether infected SkMC modulates the production of prostaglandin E2 (PGE2), cytokines interleukin-12 (IL-12) and interferon-gamma (INF-g), and also the cyclooxygenase-2 (COX-2) gene induction. Methods Primary culture of skeletal muscle cells were infected with tachyzoites of T. gondii and analysed by confocal microscopy for observation of LD. Ultrastructural cytochemistry was also used for lipid and sarcoplasmatic reticulum (SR) detection. Dosage of cytokines (IL-12 and INF-g) by ELISA technique and enzyme-linked immunoassay (EIA) for PGE2 measurement were employed. The COX-2 gene expression analysis was performed by real time reverse transcriptase polymerase chain reaction (qRT-PCR). Results We demonstrated that T. gondii infection of SkMC leads to increase in LD number and area in a time course dependent manner. Moreover, the ultrastructural analysis demonstrated that SR and LD are in direct contact with parasitophorous vacuole membrane (PVM), within the vacuolar matrix, around it and interacting directly with the membrane of parasite, indicating that LD are recruited and deliver their content inside the parasitophorous vacuole (PV) in T. gondii-infected SkMC. We also observed a positive modulation of the production of IL-12 and IFN-g, increase of COX-2 mRNA levels in the first hour of T. gondii-SkMC interaction and an increase of prostaglandin E2 (PGE2) synthesis from 6 h up to 48 h of infection. Conclusions Taken together, the close association between SR and LD with PV could represent a source of lipids as well as other nutrients for the parasite survival, and together with the

  20. RAB GTPASES ASSOCIATE WITH ISOLATED LIPID DROPLETS (LDS) AND SHOW ALTERED CONTENT AFTER ETHANOL ADMINISTRATION: POTENTIAL ROLE IN ALCOHOL-IMPAIRED LD METABOLISM

    PubMed Central

    Rasineni, Karuna; McVicker, Benita L.; Tuma, Dean J.; McNiven, Mark A.; Casey, Carol A.

    2013-01-01

    Background Alcoholic liver disease is manifested by the presence of fatty liver, primarily due to accumulation of hepatocellular lipid droplets (LDs). The presence of membrane-trafficking proteins (e.g. Rab GTPases) with LDs indicates that LDs may be involved in trafficking pathways known to be altered in ethanol damaged hepatocytes. Since these Rab GTPases are crucial regulators of protein trafficking, we examined the effect ethanol administration has on hepatic Rab protein content and association with LDs. Methods Male Wistar rats were pair-fed Lieber-DeCarli diets for 5 to 8 weeks. Whole liver and isolated LD fractions were analyzed. Identification of LDs and associated Rab proteins was performed in frozen liver or paraffin-embedded sections followed by immunohistochemical analysis. Results Lipid accumulation was characterized by larger LD vacuoles and increased total triglyceride content in ethanol-fed rats. Rabs 1, 2, 3d, 5, 7 and 18 were analyzed in post-nuclear supernatant (PNS) as well as LDs. All of the Rabs were found in the PNS, and Rabs 1, 2, 5 and 7 did not show alcohol-altered content, while Rab 3d content was reduced by over 80%, and Rab 18 also showed ethanol-induced reduction in content. Rab 3d was not found to associate with LDs, while all other Rabs were found in the LD fractions, and several showed an ethanol-related decrease (Rabs 2, 5, 7, 18). Immunohistochemical analysis revealed the enhanced content of a LD-associated protein, perilipin 2 (PLIN2) that was paralleled with an associated decrease of Rab 18 in ethanol-fed rat sections. Conclusion Chronic ethanol feeding was associated with increased PLIN2 and altered Rab GTPase content in enriched LD fractions. Although mechanisms driving these changes are not established, further studies on intracellular protein trafficking and LD biology after alcohol administration will likely contribute to our understanding of fatty liver disease. PMID:24117505

  1. Identification of Plants That Inhibit Lipid Droplet Formation in Liver Cells: Rubus suavissimus Leaf Extract Protects Mice from High-Fat Diet-Induced Fatty Liver by Directly Affecting Liver Cells

    PubMed Central

    Takahashi, Tomohiro; Sugawara, Wataru; Takiguchi, Yuya; Takizawa, Kento; Nakabayashi, Ami; Nakamura, Mitsuo; Nagano-Ito, Michiyo; Ichikawa, Shinichi

    2016-01-01

    Fatty liver disease is a condition in which abnormally large numbers of lipid droplets accumulate in liver cells. Fatty liver disease induces inflammation under conditions of oxidative stress and may result in cancer. To identify plants that protect against fatty liver disease, we examined the inhibitory effects of plant extracts on lipid droplet formation in mouse hepatoma cells. A screen of 98 water extracts of plants revealed 4 extracts with inhibitory effects. One of these extracts, Rubus suavissimus S. Lee (Tien-cha or Chinese sweet tea) leaf extract, which showed strong inhibitory effects, was tested in a mouse fatty liver model. In these mouse experiments, intake of the plant extract significantly protected mice against fatty liver disease without affecting body weight gain. Our results suggest that RSE directly affects liver cells and protects them from fatty liver disease. PMID:27429636

  2. Identification of Plants That Inhibit Lipid Droplet Formation in Liver Cells: Rubus suavissimus Leaf Extract Protects Mice from High-Fat Diet-Induced Fatty Liver by Directly Affecting Liver Cells.

    PubMed

    Takahashi, Tomohiro; Sugawara, Wataru; Takiguchi, Yuya; Takizawa, Kento; Nakabayashi, Ami; Nakamura, Mitsuo; Nagano-Ito, Michiyo; Ichikawa, Shinichi

    2016-01-01

    Fatty liver disease is a condition in which abnormally large numbers of lipid droplets accumulate in liver cells. Fatty liver disease induces inflammation under conditions of oxidative stress and may result in cancer. To identify plants that protect against fatty liver disease, we examined the inhibitory effects of plant extracts on lipid droplet formation in mouse hepatoma cells. A screen of 98 water extracts of plants revealed 4 extracts with inhibitory effects. One of these extracts, Rubus suavissimus S. Lee (Tien-cha or Chinese sweet tea) leaf extract, which showed strong inhibitory effects, was tested in a mouse fatty liver model. In these mouse experiments, intake of the plant extract significantly protected mice against fatty liver disease without affecting body weight gain. Our results suggest that RSE directly affects liver cells and protects them from fatty liver disease. PMID:27429636

  3. Spg20−/− mice reveal multimodal functions for Troyer syndrome protein spartin in lipid droplet maintenance, cytokinesis and BMP signaling

    PubMed Central

    Renvoisé, Benoît; Stadler, Julia; Singh, Rajat; Bakowska, Joanna C.; Blackstone, Craig

    2012-01-01

    Hereditary spastic paraplegias (HSPs; SPG1-48) are inherited neurological disorders characterized by lower extremity spasticity and weakness. Loss-of-function mutations in the SPG20 gene encoding spartin cause autosomal recessive Troyer syndrome (SPG20), which has additional features of short stature, cognitive deficits and distal amyotrophy. To identify cellular impairments underlying Troyer syndrome, we generated Spg20−/− mice, which exhibit progressive gait defects. Although gross central nervous system pathology appeared largely normal, cerebral cortical neurons cultured from neonatal Spg20−/− mice exhibited increased axon branching, a phenotype suppressed by reintroducing spartin and which required its interaction with the endosomal sorting complex required for transport (ESCRT)-III protein IST1. Analysis of the bone morphogenetic protein (BMP) signaling pathway in Spg20−/− embryonic fibroblasts indicated that Smad1/5 phosphorylation is modestly elevated, possibly due to alterations in BMP receptor trafficking. Cytokinesis was impaired in embryonic fibroblasts cultured from Spg20−/− mice, and binucleated chondrocytes were prominent in epiphyseal growth plates of bones in Spg20−/− mice, perhaps explaining the short stature of patients. Finally, adipose tissue from Spg20−/− female mice exhibited increased lipid droplet (LD) numbers and alterations in perilipin levels, supporting a role for spartin in LD maintenance. Taken together, our results support multimodal functions for spartin that provide important insights into HSP pathogenesis. PMID:22619377

  4. Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase.

    PubMed

    Bouchez, Isabelle; Pouteaux, Marie; Canonge, Michel; Genet, Mélanie; Chardot, Thierry; Guillot, Alain; Froissard, Marine

    2015-01-01

    It has now been clearly shown that lipid droplets (LDs) play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+) ATPase (V-ATPase). Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS) complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

  5. Evidence Suggesting That Francisella tularensis O-Antigen Capsule Contains a Lipid A-Like Molecule That Is Structurally Distinct from the More Abundant Free Lipid A

    PubMed Central

    Barker, Jason H.; Kaufman, Justin W.; Apicella, Michael A.; Weiss, Jerrold P.

    2016-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, produces a high molecular weight capsule that is immunologically distinct from Francisella lipopolysaccharide but contains the same O-antigen tetrasaccharide. To pursue the possibility that the capsule of Francisella live vaccine strain (LVS) has a structurally unique lipid anchor, we have metabolically labeled Francisella with [14C]acetate to facilitate highly sensitive compositional analysis of capsule-associated lipids. Capsule was purified by two independent methods and yielded similar results. Autoradiographic and immunologic analysis confirmed that this purified material was largely devoid of low molecular weight LPS and of the copious amounts of free lipid A that the Francisellae accumulate. Chemical hydrolysis yielded [14C]-labeled free fatty acids characteristic of Francisella lipid A but with a different molar ratio of 3-OH C18:0 to 3-OH C16:0 and different composition of non-hydroxylated fatty acids (mainly C14:0 rather than C16:0) than that of free Francisella lipid A. Mild acid hydrolysis to induce selective cleavage of KDO-lipid A linkage yielded a [14C]-labeled product that partitioned during Bligh/Dyer extraction and migrated during thin-layer chromatography like lipid A. These findings suggest that the O-antigen capsule of Francisella contains a covalently linked and structurally distinct lipid A species. The presence of a discrete lipid A-like molecule associated with capsule raises the possibility that Francisella selectively exploits lipid A structural heterogeneity to regulate synthesis, transport, and stable bacterial surface association of the O-antigen capsular layer. PMID:27326857

  6. Dynamic morphologies of microscale droplet interface bilayers.

    PubMed

    Mruetusatorn, Prachya; Boreyko, Jonathan B; Venkatesan, Guru A; Sarles, Stephen A; Hayes, Douglas G; Collier, C Patrick

    2014-04-21

    Droplet interface bilayers (DIBs) are a powerful platform for studying the dynamics of synthetic cellular membranes; however, very little has been done to exploit the unique dynamical features of DIBs. Here, we generate microscale droplet interface bilayers (μDIBs) by bringing together femtoliter-volume water droplets in a microfluidic oil channel, and characterize morphological changes of the μDIBs as the droplets shrink due to evaporation. By varying the initial conditions of the system, we identify three distinct classes of dynamic morphology. (1) Buckling and fission: when forming μDIBs using the lipid-out method (lipids in oil phase), lipids in the shrinking monolayers continually pair together and slide into the bilayer to conserve their mass. As the bilayer continues to grow, it becomes confined, buckles, and eventually fissions one or more vesicles. (2) Uniform shrinking: when using the lipid-in method (lipids in water phase) to form μDIBs, lipids uniformly transfer from the monolayers and bilayer into vesicles contained inside the water droplets. (3) Stretching and unzipping: finally, when the droplets are pinned to the wall(s) of the microfluidic channel, the droplets become stretched during evaporation, culminating in the unzipping of the bilayer and droplet separation. These findings offer a better understanding of the dynamics of coupled lipid interfaces. PMID:24647872

  7. Dynamic Morphologies of Microscale Droplet Interface Bilayers

    SciTech Connect

    Mruetusatorn, Prachya; Boreyko, Jonathan B; Sarles, Stephen A; Venkatesan, Guru; Hayes, Douglas G; Collier, Pat

    2014-01-01

    Droplet interface bilayers (DIBs) are a powerful platform for studying the dynamics of synthetic cellular membranes; however, very little has been done to exploit the unique dynamical features of DIBs. Here, we generate microscale droplet interface bilayers ( DIBs) by bringing together femtoliter-volume water droplets in a microfluidic oil channel, and characterize morphological changes of the DIBs as the droplets shrink due to evaporation. By varying the initial conditions of the system, we identify three distinct classes of dynamic morphology. (1) Buckling and Fission: When forming DIBs using the lipid-out method (lipids in oil phase), lipids in the shrinking monolayers continually pair together and slide into the bilayer to conserve their mass. As the bilayer continues to grow, it becomes confined, buckles, and eventually fissions one or more vesicles. (2) Uniform Shrinking: When using the lipid-in method (lipids in water phase) to form DIBs, lipids uniformly transfer from the monolayers and bilayer into vesicles contained inside the water droplets. (3) Stretching and Unzipping: Finally, when the droplets are pinned to the wall(s) of the microfluidic channel, the droplets become stretched during evaporation, culminating in the unzipping of the bilayer and droplet separation. These findings offer a better understanding of the dynamics of coupled lipid interfaces.

  8. Dengue virus capsid protein binding to hepatic lipid droplets (LD) is potassium ion dependent and is mediated by LD surface proteins.

    PubMed

    Carvalho, Filomena A; Carneiro, Fabiana A; Martins, Ivo C; Assunção-Miranda, Iranaia; Faustino, André F; Pereira, Renata M; Bozza, Patricia T; Castanho, Miguel A R B; Mohana-Borges, Ronaldo; Da Poian, Andrea T; Santos, Nuno C

    2012-02-01

    Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of -19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na(+)/K(+)-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs.

  9. Dual N- and C-Terminal Helices Are Required for Endoplasmic Reticulum and Lipid Droplet Association of Alcohol Acetyltransferases in Saccharomyces cerevisiae

    PubMed Central

    Lin, Jyun-Liang; Wheeldon, Ian

    2014-01-01

    In the yeast Saccharomyces cerevisiae two alcohol acetyltransferases (AATases), Atf1 and Atf2, condense short chain alcohols with acetyl-CoA to produce volatile acetate esters. Such esters are, in large part, responsible for the distinctive flavors and aromas of fermented beverages including beer, wine, and sake. Atf1 and Atf2 localize to the endoplasmic reticulum (ER) and Atf1 is known to localize to lipid droplets (LDs). The mechanism and function of these localizations are unknown. Here, we investigate potential mechanisms of Atf1 and Atf2 membrane association. Segments of the N- and C-terminal domains of Atf1 (residues 24–41 and 508–525, respectively) are predicted to be amphipathic helices. Truncations of these helices revealed that the terminal domains are essential for ER and LD association. Moreover, mutations of the basic or hydrophobic residues in the N-terminal helix and hydrophobic residues in the C-terminal helix disrupted ER association and subsequent sorting from the ER to LDs. Similar amphipathic helices are found at both ends of Atf2, enabling ER and LD association. As was the case with Atf1, mutations to the N- and C-terminal helices of Atf2 prevented membrane association. Sequence comparison of the AATases from Saccharomyces, non-Saccharomyces yeast (K. lactis and P. anomala) and fruits species (C. melo and S. lycopersicum) showed that only AATases from Saccharomyces evolved terminal amphipathic helices. Heterologous expression of these orthologs in S. cerevisiae revealed that the absence of terminal amphipathic helices eliminates LD association. Combined, the results of this study suggest a common mechanism of membrane association for AATases via dual N- and C-terminal amphipathic helices. PMID:25093817

  10. Dengue Virus Capsid Protein Binding to Hepatic Lipid Droplets (LD) Is Potassium Ion Dependent and Is Mediated by LD Surface Proteins

    PubMed Central

    Carvalho, Filomena A.; Carneiro, Fabiana A.; Martins, Ivo C.; Assunção-Miranda, Iranaia; Faustino, André F.; Pereira, Renata M.; Bozza, Patricia T.; Castanho, Miguel A. R. B.; Mohana-Borges, Ronaldo; Da Poian, Andrea T.

    2012-01-01

    Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of −19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at +13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C protein-functionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na+/K+-ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. PMID:22130547

  11. Fat area and lipid droplet morphology of porcine oocytes during in vitro maturation with trans-10, cis-12 conjugated linoleic acid and forskolin.

    PubMed

    Prates, E G; Marques, C C; Baptista, M C; Vasques, M I; Carolino, N; Horta, A E M; Charneca, R; Nunes, J T; Pereira, R M

    2013-04-01

    Lipid droplets (LD) in porcine oocytes form a dark mass reaching almost all cytoplasm. Herein we investigated changes in fat areas, cytoplasmic tone and LD morphology during in vitro maturation (IVM) of porcine oocytes cultured with 100 μM trans-10, cis-12 conjugated linoleic acid (t10,c12 CLA) or 10 μM forskolin at different time periods. Four groups were constituted: control, excipient, t10,c12 CLA and forskolin, with drugs being supplemented during 44 to 48 h and the initial 22 to 24 h in Experiments 1 and 2, respectively. In Experiment 3, forskolin was supplemented for the first 2 h. Matured oocytes were inseminated with frozen-thawed boar semen and cleavage rate recorded. Before and during IVM, samples of oocytes were evaluated for LD, total and fat areas and fat gray value or for meiotic progression. Results showed that forskolin supplementation during 44 to 48 h or 22 to 24 h inhibits oocyte maturation (exp. 1: forskolin = 5.1 ± 8.0%, control = 72.6 ± 5.0%; exp. 2: forskolin = 24.3 ± 7.4%, control = 71.6 ± 5.6%) and cleavage (exp. 1: forskolin = 0.0 ± 0.0%, control = 55.4 ± 4.1%; exp. 2: forskolin = 8.3 ± 3.3%, control = 54.5 ± 3.0%). Forskolin also reduced oocyte and fat areas. In Experiment 3, forskolin negative effect on oocyte maturation and cleavage disappeared, although minor (P ⩽ 0.03) LD and oocyte fat areas were identified at 22 to 24 h of IVM. Oocytes supplemented with t10,c12 CLA during 44 to 48 h presented a lighter (P ⩽ 0.04) colour tone cytoplasm than those of control and forskolin. In conclusion, t10,c12 CLA and forskolin were capable of modifying the distribution and morphology of cytoplasmic LD during porcine oocyte maturation, thus reducing its lipid content in a time-dependent manner.

  12. Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in Sphagnum-Dominated Northern Wetlands.

    PubMed

    Moore, Eli K; Villanueva, Laura; Hopmans, Ellen C; Rijpstra, W Irene C; Mets, Anchelique; Dedysh, Svetlana N; Sinninghe Damsté, Jaap S

    2015-09-01

    Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface.

  13. Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in Sphagnum-Dominated Northern Wetlands

    PubMed Central

    Villanueva, Laura; Hopmans, Ellen C.; Rijpstra, W. Irene C.; Mets, Anchelique; Dedysh, Svetlana N.; Sinninghe Damsté, Jaap S.

    2015-01-01

    Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface. PMID:26150465

  14. Influence of Growth Phase, pH, and Temperature on the Abundance and Composition of Tetraether Lipids in the Thermoacidophile Picrophilus torridus.

    PubMed

    Feyhl-Buska, Jayme; Chen, Yufei; Jia, Chengling; Wang, Jin-Xiang; Zhang, Chuanlun L; Boyd, Eric S

    2016-01-01

    The abundance and composition of glycerol dibiphytanyl glycerol tetraether (GDGT) and glycerol tribiphytanyl glycerol tetraether (GTGT) lipids were determined as a function of growth phase as a proxy for nutrient availability, the pH of growth medium, and incubation temperature in cultures of the thermoacidophile Picrophilus torridus. Regardless of the cultivation condition, the abundance of GDGTs and GTGTs was greater in the polar than core fraction, with a marked decrease in core GDGTs in cultures harvested during log phase growth. These data are consistent with previous suggestions indicating that core GDGTs are re-functionalized during polar lipid synthesis. Under all conditions examined, polar lipids were enriched in a GDGT with 2 cyclopentyl rings (GDGT-2), indicating GDGT-2 is the preferred lipid in this taxon. However, lag or stationary phase grown cells or cells subjected to pH or thermal stress were enriched in GDGTs with 4, 5, or 6 rings and depleted in GDGTs with 1, 2, 3, rings relative to log phase cells grown under optimal conditions. Variation in the composition of polar GDGT lipids in cells harvested during various growth phases tended to be greater than in cells cultivated over a pH range of 0.3-1.1 and a temperature range of 53-63°C. These results suggest that the growth phase, the pH of growth medium, and incubation temperature are all important factors that shape the composition of tetraether lipids in Picrophilus. The similarity in enrichment of GDGTs with more rings in cultures undergoing nutrient, pH, and thermal stress points to GDGT cyclization as a generalized physiological response to stress in this taxon. PMID:27625636

  15. Influence of Growth Phase, pH, and Temperature on the Abundance and Composition of Tetraether Lipids in the Thermoacidophile Picrophilus torridus

    PubMed Central

    Feyhl-Buska, Jayme; Chen, Yufei; Jia, Chengling; Wang, Jin-Xiang; Zhang, Chuanlun L.; Boyd, Eric S.

    2016-01-01

    The abundance and composition of glycerol dibiphytanyl glycerol tetraether (GDGT) and glycerol tribiphytanyl glycerol tetraether (GTGT) lipids were determined as a function of growth phase as a proxy for nutrient availability, the pH of growth medium, and incubation temperature in cultures of the thermoacidophile Picrophilus torridus. Regardless of the cultivation condition, the abundance of GDGTs and GTGTs was greater in the polar than core fraction, with a marked decrease in core GDGTs in cultures harvested during log phase growth. These data are consistent with previous suggestions indicating that core GDGTs are re-functionalized during polar lipid synthesis. Under all conditions examined, polar lipids were enriched in a GDGT with 2 cyclopentyl rings (GDGT-2), indicating GDGT-2 is the preferred lipid in this taxon. However, lag or stationary phase grown cells or cells subjected to pH or thermal stress were enriched in GDGTs with 4, 5, or 6 rings and depleted in GDGTs with 1, 2, 3, rings relative to log phase cells grown under optimal conditions. Variation in the composition of polar GDGT lipids in cells harvested during various growth phases tended to be greater than in cells cultivated over a pH range of 0.3–1.1 and a temperature range of 53–63°C. These results suggest that the growth phase, the pH of growth medium, and incubation temperature are all important factors that shape the composition of tetraether lipids in Picrophilus. The similarity in enrichment of GDGTs with more rings in cultures undergoing nutrient, pH, and thermal stress points to GDGT cyclization as a generalized physiological response to stress in this taxon.

  16. Influence of Growth Phase, pH, and Temperature on the Abundance and Composition of Tetraether Lipids in the Thermoacidophile Picrophilus torridus

    PubMed Central

    Feyhl-Buska, Jayme; Chen, Yufei; Jia, Chengling; Wang, Jin-Xiang; Zhang, Chuanlun L.; Boyd, Eric S.

    2016-01-01

    The abundance and composition of glycerol dibiphytanyl glycerol tetraether (GDGT) and glycerol tribiphytanyl glycerol tetraether (GTGT) lipids were determined as a function of growth phase as a proxy for nutrient availability, the pH of growth medium, and incubation temperature in cultures of the thermoacidophile Picrophilus torridus. Regardless of the cultivation condition, the abundance of GDGTs and GTGTs was greater in the polar than core fraction, with a marked decrease in core GDGTs in cultures harvested during log phase growth. These data are consistent with previous suggestions indicating that core GDGTs are re-functionalized during polar lipid synthesis. Under all conditions examined, polar lipids were enriched in a GDGT with 2 cyclopentyl rings (GDGT-2), indicating GDGT-2 is the preferred lipid in this taxon. However, lag or stationary phase grown cells or cells subjected to pH or thermal stress were enriched in GDGTs with 4, 5, or 6 rings and depleted in GDGTs with 1, 2, 3, rings relative to log phase cells grown under optimal conditions. Variation in the composition of polar GDGT lipids in cells harvested during various growth phases tended to be greater than in cells cultivated over a pH range of 0.3–1.1 and a temperature range of 53–63°C. These results suggest that the growth phase, the pH of growth medium, and incubation temperature are all important factors that shape the composition of tetraether lipids in Picrophilus. The similarity in enrichment of GDGTs with more rings in cultures undergoing nutrient, pH, and thermal stress points to GDGT cyclization as a generalized physiological response to stress in this taxon. PMID:27625636

  17. Dancing Droplets

    NASA Astrophysics Data System (ADS)

    Cira, Nate; Prakash, Manu

    2013-11-01

    Inspired by the observation of intricate and beautifully dynamic patterns generated by food coloring on corona treated glass slides, we have investigated the behavior of propylene glycol and water droplets on clean glass surfaces. These droplets exhibit a range of interesting behaviors including long distance attraction or repulsion, and chasing/fleeing upon contact. We present explanations for each of these behaviors, and propose a detailed model for the long distance interactions based on vapor facilitated coupling. Finally we use our understanding to create several novel devices which: passively sort droplets by surface tension, spontaneously align droplets, drive droplets in circles, and cause droplets to bounce on a vertical surface. The simplicity of this system lends it particularly well to application as a toy model for physical systems with force fields and biological systems such as chemotaxis and motility.

  18. Swimming Droplets

    NASA Astrophysics Data System (ADS)

    Maass, Corinna C.; Krüger, Carsten; Herminghaus, Stephan; Bahr, Christian

    2016-03-01

    Swimming droplets are artificial microswimmers based on liquid droplets that show self-propelled motion when immersed in a second liquid. These systems are of tremendous interest as experimental models for the study of collective dynamics far from thermal equilibrium. For biological systems, such as bacterial colonies, plankton, or fish swarms, swimming droplets can provide a vital link between simulations and real life. We review the experimental systems and discuss the mechanisms of self-propulsion. Most systems are based on surfactant-stabilized droplets, the surfactant layer of which is modified in a way that leads to a steady Marangoni stress resulting in an autonomous motion of the droplet. The modification of the surfactant layer is caused either by the advection of a chemical reactant or by a solubilization process. Some types of swimming droplets possess a very simple design and long active periods, rendering them promising model systems for future studies of collective behavior.

  19. Droplet microfluidics.

    PubMed

    Teh, Shia-Yen; Lin, Robert; Hung, Lung-Hsin; Lee, Abraham P

    2008-02-01

    Droplet-based microfluidic systems have been shown to be compatible with many chemical and biological reagents and capable of performing a variety of "digital fluidic" operations that can be rendered programmable and reconfigurable. This platform has dimensional scaling benefits that have enabled controlled and rapid mixing of fluids in the droplet reactors, resulting in decreased reaction times. This, coupled with the precise generation and repeatability of droplet operations, has made the droplet-based microfluidic system a potent high throughput platform for biomedical research and applications. In addition to being used as microreactors ranging from the nano- to femtoliter range; droplet-based systems have also been used to directly synthesize particles and encapsulate many biological entities for biomedicine and biotechnology applications. This review will focus on the various droplet operations, as well as the numerous applications of the system. Due to advantages unique to droplet-based systems, this technology has the potential to provide novel solutions to today's biomedical engineering challenges for advanced diagnostics and therapeutics.

  20. Light Driven Formation and Rupture of Droplet Bilayers

    PubMed Central

    Dixit, Sanhita S.; Kim, Hanyoup; Vasilyev, Arseny; Eid, Aya; Faris, Gregory W.

    2010-01-01

    We demonstrate optical manipulation of nanoliter aqueous droplets containing surfactant or lipid molecules and immersed in an organic liquid using near infrared light. The resulting emulsion droplets are manipulated using both the thermocapillary effect and convective fluid motion. Droplet pair-interactions induced in the emulsion upon optical initiation and control provide direct observations of the coalescence steps in intricate detail. Droplet-droplet adhesion (bilayer formation) is observed under several conditions. Selective bilayer rupture is also realized using the same infrared laser. The technique provides a novel approach to study thin film drainage and interface stability in emulsion dynamics. The formation of stable lipid bilayers at the adhesion interface between interacting water droplets can provide an optical platform to build droplet-based lipid bilayer assays. The technique also has relevance for understanding and improving microfluidics applications by devising Petri dish based droplet assays requiring no substrate fabrication. PMID:20361732

  1. Droplet Growth

    NASA Astrophysics Data System (ADS)

    Marder, Michael Paolo

    When a mixture of two materials, such as aluminum and tin, or alcohol and water, is cooled below a certain temperature, the two components begin to separate. If one component is dilute in the other, it may separate out in the form of small spheres, and these will begin to enlarge, depleting the supersaturated material around them. If the dynamics is sufficiently slow, thermodynamics gives one considerable information about how the droplets grow. Two types of experiment have explored this behavior and given puzzling results. Nucleation experiments measure the rate at which droplets initially appear from a seemingly homogeneous mixture. Near the critical point in binary liquids, experiments conducted in the 1960's and early 1970's showed that nucleation was vastly slower than theory seemed to predict. The resolution of this problem arises by considering in detail the dynamics of growing droplets and comparing it with what experiments actually measure. Here will be presented a more detailed comparison of theory and experiment than has before been completed, obtaining satisfactory agreement with no free parameters needed. A second type of experiment measures droplet size distributions after long times. In the late stage, droplets compete with each other for material, a few growing at the expense of others. A theory first proposed by Lifshitz and Slyozov claims that this distribution, properly scaled, should be universal, and independent of properties of materials. Yet experimental measurements consistently find distributions that are more broad and squat than the theory would predict. Satisfactory agreement with experiment can be achieved by considering two points. First, one must study the complete time development of droplet size distributions, to understand when the asymptotic regime obtains. Second, droplet size distributions are spread by correlations between droplets. If one finds a small droplet, it is small because large droplets nearby are competing with it

  2. Droplet microactuator system

    NASA Technical Reports Server (NTRS)

    Pamula, Vamsee K. (Inventor); Srinivasan, Vijay (Inventor); Pollack, Michael G. (Inventor); Eckhardt, Allen E. (Inventor); Paik, Philip Y. (Inventor)

    2010-01-01

    The present invention relates to a droplet microactuator system. According to one embodiment, the droplet microactuator system includes: (a) a droplet microactuator configured to conduct droplet operations; (b) a magnetic field source arranged to immobilize magnetically responsive beads in a droplet during droplet operations; (c) a sensor configured in a sensing relationship with the droplet microactuator, such that the sensor is capable of sensing a signal from and/or a property of one or more droplets on the droplet microactuator; and (d) one or more processors electronically coupled to the droplet microactuator and programmed to control electrowetting-mediated droplet operations on the droplet actuator and process electronic signals from the sensor.

  3. Sessile nanofluid droplet drying.

    PubMed

    Zhong, Xin; Crivoi, Alexandru; Duan, Fei

    2015-03-01

    Nanofluid droplet evaporation has gained much audience nowadays due to its wide applications in painting, coating, surface patterning, particle deposition, etc. This paper reviews the drying progress and deposition formation from the evaporative sessile droplets with the suspended insoluble solutes, especially nanoparticles. The main content covers the evaporation fundamental, the particle self-assembly, and deposition patterns in sessile nanofluid droplet. Both experimental and theoretical studies are presented. The effects of the type, concentration and size of nanoparticles on the spreading and evaporative dynamics are elucidated at first, serving the basis for the understanding of particle motion and deposition process which are introduced afterward. Stressing on particle assembly and production of desirable residue patterns, we express abundant experimental interventions, various types of deposits, and the effects on nanoparticle deposition. The review ends with the introduction of theoretical investigations, including the Navier-Stokes equations in terms of solutions, the Diffusion Limited Aggregation approach, the Kinetic Monte Carlo method, and the Dynamical Density Functional Theory. Nanoparticles have shown great influences in spreading, evaporation rate, evaporation regime, fluid flow and pattern formation of sessile droplets. Under different experimental conditions, various deposition patterns can be formed. The existing theoretical approaches are able to predict fluid dynamics, particle motion and deposition patterns in the particular cases. On the basis of further understanding of the effects of fluid dynamics and particle motion, the desirable patterns can be obtained with appropriate experimental regulations. PMID:25578408

  4. Yeast Droplets

    NASA Astrophysics Data System (ADS)

    Nguyen, Baochi; Upadhyaya, Arpita; van Oudenaarden, Alexander; Brenner, Michael

    2002-11-01

    It is well known that the Young's law and surface tension govern the shape of liquid droplets on solid surfaces. Here we address through experiments and theory the shape of growing aggregates of yeast on agar substrates, and assess whether these ideas still hold. Experiments are carried out on Baker's yeast, with different levels of expressions of an adhesive protein governing cell-cell and cell-substrate adhesion. Changing either the agar concentration or the expression of this protein modifies the local contact angle of a yeast droplet. When the colony is small, the shape is a spherical cap with the contact angle obeying Young's law. However, above a critical volume this structure is unstable, and the droplet becomes nonspherical. We present a theoretical model where this instability is caused by bulk elastic effects. The model predicts that the transition depends on both volume and contact angle, in a manner quantitatively consistent with our experiments.

  5. Modular droplet actuator drive

    NASA Technical Reports Server (NTRS)

    Pollack, Michael G. (Inventor); Paik, Philip (Inventor)

    2011-01-01

    A droplet actuator drive including a detection apparatus for sensing a property of a droplet on a droplet actuator; circuitry for controlling the detection apparatus electronically coupled to the detection apparatus; a droplet actuator cartridge connector arranged so that when a droplet actuator cartridge electronically is coupled thereto: the droplet actuator cartridge is aligned with the detection apparatus; and the detection apparatus can sense the property of the droplet on a droplet actuator; circuitry for controlling a droplet actuator coupled to the droplet actuator connector; and the droplet actuator circuitry may be coupled to a processor.

  6. Construction and manipulation of functional three-dimensional droplet networks.

    PubMed

    Wauer, Tobias; Gerlach, Holger; Mantri, Shiksha; Hill, Jamie; Bayley, Hagan; Sapra, K Tanuj

    2014-01-28

    Previously, we reported the manual assembly of lipid-coated aqueous droplets in oil to form two-dimensional (2D) networks in which the droplets are connected through single lipid bilayers. Here we assemble lipid-coated droplets in robust, freestanding 3D geometries: for example, a 14-droplet pyramidal assembly. The networks are designed, and each droplet is placed in a designated position. When protein pores are inserted in the bilayers between specific constituent droplets, electrical and chemical communication pathways are generated. We further describe an improved means to construct 3D droplet networks with defined organizations by the manipulation of aqueous droplets containing encapsulated magnetic beads. The droplets are maneuvered in a magnetic field to form simple construction modules, which are then used to form larger 2D and 3D structures including a 10-droplet pyramid. A methodology to construct freestanding, functional 3D droplet networks is an important step toward the programmed and automated manufacture of synthetic minimal tissues. PMID:24341760

  7. Acidophilic green alga Pseudochlorella sp. YKT1 accumulates high amount of lipid droplets under a nitrogen-depleted condition at a low-pH.

    PubMed

    Hirooka, Shunsuke; Higuchi, Sumio; Uzuka, Akihiro; Nozaki, Hisayoshi; Miyagishima, Shin-ya

    2014-01-01

    Microalgal storage lipids are considered to be a promising source for next-generation biofuel feedstock. However, microalgal biodiesel is not yet economically feasible due to the high cost of production. One of the reasons for this is that the use of a low-cost open pond system is currently limited because of the unavoidable contamination with undesirable organisms. Extremophiles have an advantage in culturing in an open pond system because they grow in extreme environments toxic to other organisms. In this study, we isolated the acidophilic green alga Pseudochlorella sp. YKT1 from sulfuric acid mine drainage in Nagano Prefecture, Japan. The vegetative cells of YKT1 display the morphological characteristics of Trebouxiophyceae and molecular phylogenetic analyses indicated it to be most closely related to Pseudochlorella pringsheimii. The optimal pH and temperature for the growth of YKT1 are pH 3.0-5.0 and a temperature 20-25°C, respectively. Further, YKT1 is able to grow at pH 2.0 and at 32°C, which corresponds to the usual water temperature in the outdoors in summer in many countries. YKT1 accumulates a large amount of storage lipids (∼30% of dry weigh) under a nitrogen-depleted condition at low-pH (pH 3.0). These results show that acidophilic green algae will be useful for industrial applications by acidic open culture systems.

  8. Acidophilic green alga Pseudochlorella sp. YKT1 accumulates high amount of lipid droplets under a nitrogen-depleted condition at a low-pH.

    PubMed

    Hirooka, Shunsuke; Higuchi, Sumio; Uzuka, Akihiro; Nozaki, Hisayoshi; Miyagishima, Shin-ya

    2014-01-01

    Microalgal storage lipids are considered to be a promising source for next-generation biofuel feedstock. However, microalgal biodiesel is not yet economically feasible due to the high cost of production. One of the reasons for this is that the use of a low-cost open pond system is currently limited because of the unavoidable contamination with undesirable organisms. Extremophiles have an advantage in culturing in an open pond system because they grow in extreme environments toxic to other organisms. In this study, we isolated the acidophilic green alga Pseudochlorella sp. YKT1 from sulfuric acid mine drainage in Nagano Prefecture, Japan. The vegetative cells of YKT1 display the morphological characteristics of Trebouxiophyceae and molecular phylogenetic analyses indicated it to be most closely related to Pseudochlorella pringsheimii. The optimal pH and temperature for the growth of YKT1 are pH 3.0-5.0 and a temperature 20-25°C, respectively. Further, YKT1 is able to grow at pH 2.0 and at 32°C, which corresponds to the usual water temperature in the outdoors in summer in many countries. YKT1 accumulates a large amount of storage lipids (∼30% of dry weigh) under a nitrogen-depleted condition at low-pH (pH 3.0). These results show that acidophilic green algae will be useful for industrial applications by acidic open culture systems. PMID:25221913

  9. A Lys49 Phospholipase A2, Isolated from Bothrops asper Snake Venom, Induces Lipid Droplet Formation in Macrophages Which Depends on Distinct Signaling Pathways and the C-Terminal Region

    PubMed Central

    Cristina Giannotti, Karina; Leiguez, Elbio; Moreira, Vanessa; Nascimento, Neide Galvão; Lomonte, Bruno; Gutiérrez, José Maria; Lopes de Melo, Robson; Teixeira, Catarina

    2013-01-01

    MT-II, a Lys49PLA2 homologue devoid of catalytic activity from B. asper venom, stimulates inflammatory events in macrophages. We investigated the ability of MT-II to induce formation of lipid droplets (LDs), key elements of inflammatory responses, in isolated macrophages and participation of protein kinases and intracellular PLA2s in this effect. Influence of MT-II on PLIN2 recruitment and expression was assessed, and the effects of some synthetic peptides on LD formation were further evaluated. At noncytotoxic concentrations, MT-II directly activated macrophages to form LDs. This effect was reproduced by a synthetic peptide corresponding to the C-terminal sequence 115–129 of MT-II, evidencing the critical role of C-terminus for MT-II-induced effect. Moreover, MT-II induced expression and recruitment of PLIN2. Pharmacological interventions with specific inhibitors showed that PKC, PI3K, ERK1/2, and iPLA2, but not P38MAPK or cPLA2, signaling pathways are involved in LD formation induced by MT-II. This sPLA2 homologue also induced synthesis of PGE2 that colocalized to LDs. In conclusion, MT-II is able to induce formation of LDs committed to PGE2 formation in a process dependent on C-terminal loop engagement and regulated by distinct protein kinases and iPLA2. LDs may constitute an important inflammatory mechanism triggered by MT-II in macrophages. PMID:23509782

  10. Multifactorial resistance to aminopeptidase inhibitor prodrug CHR2863 in myeloid leukemia cells: down-regulation of carboxylesterase 1, drug sequestration in lipid droplets and pro-survival activation ERK/Akt/mTOR

    PubMed Central

    Verbrugge, Sue Ellen; Al, Marjon; Assaraf, Yehuda G.; Kammerer, Sarah; Chandrupatla, Durga M.S.H.; Honeywell, Richard; Musters, Rene P.J.; Giovannetti, Elisa; O'Toole, Tom; Scheffer, George L.; Krige, David; de Gruijl, Tanja D.; Niessen, Hans W.M.; Lems, Willem F.; Kramer, Pieternella A.; Scheper, Rik J.; Cloos, Jacqueline; Ossenkoppele, Gert J.; Peters, Godefridus J.; Jansen, Gerrit

    2016-01-01

    Aminopeptidase inhibitors are receiving attention as combination chemotherapeutic agents for the treatment of refractory acute myeloid leukemia. However, the factors determining therapeutic efficacy remain elusive. Here we identified the molecular basis of acquired resistance to CHR2863, an orally available hydrophobic aminopeptidase inhibitor prodrug with an esterase-sensitive motif, in myeloid leukemia cells. CHR2863 enters cells by diffusion and is retained therein upon esterase activity-mediated conversion to its hydrophilic active metabolite drug CHR6768, thereby exerting amino acid depletion. Carboxylesterases (CES) serve as candidate prodrug activating enzymes given CES1 expression in acute myeloid leukemia specimens. We established two novel myeloid leukemia sublines U937/CHR2863(200) and U937/CHR2863(5uM), with low (14-fold) and high level (270-fold) CHR2863 resistance. The latter drug resistant cells displayed: (i) complete loss of CES1-mediated drug activation associated with down-regulation of CES1 mRNA and protein, (ii) marked retention/sequestration of the prodrug, (iii) a substantial increase in intracellular lipid droplets, and (iv) a dominant activation of the pro-survival Akt/mTOR pathway. Remarkably, the latter feature coincided with a gain of sensitivity to the mTOR inhibitor rapamycin. These finding delineate the molecular basis of CHR2863 resistance and offer a novel modality to overcome this drug resistance in myeloid leukemia cells. PMID:26496029

  11. On microtransport phenomena in minute droplets: A critical review

    SciTech Connect

    Aydin, O.; Yang, W.J.

    2000-05-01

    Liquid droplets are abundant in nature and industry. Their industrial applications are very broad. They appear in the forms of sessile, impinging, and hanging/suspending droplets, undergoing evaporation or solidification depending upon ambient conditions. In the present article, a critical review is presented for the important literature pertinent to microtransport phenomena in minute droplets. Thermocapillarity is the principal motivating force in convective heat and mass transfer, phase change, and instability inside the droplets, supplemented in part by the buoyancy force. The dimensionless governing parameters are identified and their roles in droplet transport phenomena are determined. This article includes 135 references.

  12. Dynamics of skirting droplets

    NASA Astrophysics Data System (ADS)

    Akers, Caleb; Hale, Jacob

    2014-11-01

    It has been observed that non-coalescence between a droplet and pool of like fluid can be prolonged or inhibited by sustained relative motion between the two fluids. In this study, we quantitatively describe the motion of freely moving droplets that skirt across the surface of a still pool of like fluid. Droplets of different sizes and small Weber number were directed horizontally onto the pool surface. After stabilization of the droplet shape after impact, the droplets smoothly moved across the surface, slowing until coalescence. Using high-speed imaging, we recorded the droplet's trajectory from a top-down view as well as side views both slightly above and below the fluid surface. The droplets' speed is observed to decrease exponentially, with the smaller droplets slowing down at a greater rate. Droplets infused with neutral density micro beads showed that the droplet rolls along the surface of the pool. A qualitative model of this motion is presented.

  13. Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism

    PubMed Central

    Falcinelli, Silvia; Picchietti, Simona; Rodiles, Ana; Cossignani, Lina; Merrifield, Daniel L.; Taddei, Anna Rita; Maradonna, Francesca; Olivotto, Ike; Gioacchini, Giorgia; Carnevali, Oliana

    2015-01-01

    The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders. PMID:25822072

  14. Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism.

    PubMed

    Falcinelli, Silvia; Picchietti, Simona; Rodiles, Ana; Cossignani, Lina; Merrifield, Daniel L; Taddei, Anna Rita; Maradonna, Francesca; Olivotto, Ike; Gioacchini, Giorgia; Carnevali, Oliana

    2015-01-01

    The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders. PMID:25822072

  15. Lactobacillus rhamnosus lowers zebrafish lipid content by changing gut microbiota and host transcription of genes involved in lipid metabolism.

    PubMed

    Falcinelli, Silvia; Picchietti, Simona; Rodiles, Ana; Cossignani, Lina; Merrifield, Daniel L; Taddei, Anna Rita; Maradonna, Francesca; Olivotto, Ike; Gioacchini, Giorgia; Carnevali, Oliana

    2015-03-30

    The microbiome plays an important role in lipid metabolism but how the introduction of probiotic communities affects host lipid metabolism is poorly understood. Using a multidisciplinary approach we addressed this knowledge gap using the zebrafish model by coupling high-throughput sequencing with biochemical, molecular and morphological analysis to evaluate the changes in the intestine. Analysis of bacterial 16S libraries revealed that Lactobacillus rhamnosus was able to modulate the gut microbiome of zebrafish larvae, elevating the abundance of Firmicutes sequences and reducing the abundance of Actinobacteria. The gut microbiome changes modulated host lipid processing by inducing transcriptional down-regulation of genes involved in cholesterol and triglycerides metabolism (fit2, agpat4, dgat2, mgll, hnf4α, scap, and cck) concomitantly decreasing total body cholesterol and triglyceride content and increasing fatty acid levels. L. rhamnosus treatment also increased microvilli and enterocyte lengths and decreased lipid droplet size in the intestinal epithelium. These changes resulted in elevated zebrafish larval growth. This integrated system investigation demonstrates probiotic modulation of the gut microbiome, highlights a novel gene network involved in lipid metabolism, provides an insight into how the microbiome regulates molecules involved in lipid metabolism, and reveals a new potential role for L. rhamnosus in the treatment of lipid disorders.

  16. Shape-Shifting Droplet Networks.

    PubMed

    Zhang, T; Wan, Duanduan; Schwarz, J M; Bowick, M J

    2016-03-11

    We consider a three-dimensional network of aqueous droplets joined by single lipid bilayers to form a cohesive, tissuelike material. The droplets in these networks can be programed to have distinct osmolarities so that osmotic gradients generate internal stresses via local fluid flows to cause the network to change shape. We discover, using molecular dynamics simulations, a reversible folding-unfolding process by adding an osmotic interaction with the surrounding environment which necessarily evolves dynamically as the shape of the network changes. This discovery is the next important step towards osmotic robotics in this system. We also explore analytically and numerically how the networks become faceted via buckling and how quasi-one-dimensional networks become three dimensional. PMID:27015513

  17. Shape-Shifting Droplet Networks

    NASA Astrophysics Data System (ADS)

    Zhang, T.; Wan, Duanduan; Schwarz, J. M.; Bowick, M. J.

    2016-03-01

    We consider a three-dimensional network of aqueous droplets joined by single lipid bilayers to form a cohesive, tissuelike material. The droplets in these networks can be programed to have distinct osmolarities so that osmotic gradients generate internal stresses via local fluid flows to cause the network to change shape. We discover, using molecular dynamics simulations, a reversible folding-unfolding process by adding an osmotic interaction with the surrounding environment which necessarily evolves dynamically as the shape of the network changes. This discovery is the next important step towards osmotic robotics in this system. We also explore analytically and numerically how the networks become faceted via buckling and how quasi-one-dimensional networks become three dimensional.

  18. A new droplet generator

    NASA Technical Reports Server (NTRS)

    Slack, W. E.

    1982-01-01

    A new droplet generator is described. A loud speaker driven extractor needle was immersed in a pendant drop. Pulsing the speaker extracted the needle forming a fluid ligament which will decay into a droplet. The droplets were sized by stroboscopic photographs. The droplet's size was changed by varying the amplitude of the speaker pulses and the extractor needle diameter. The mechanism of droplet formation is discussed and photographs of ligament decay are presented. The droplet generator worked well on both oil and water based pesticide formulations. Current applications and results are discussed.

  19. A new droplet generator

    NASA Astrophysics Data System (ADS)

    Slack, W. E.

    1982-03-01

    A new droplet generator is described. A loud speaker driven extractor needle was immersed in a pendant drop. Pulsing the speaker extracted the needle forming a fluid ligament which will decay into a droplet. The droplets were sized by stroboscopic photographs. The droplet's size was changed by varying the amplitude of the speaker pulses and the extractor needle diameter. The mechanism of droplet formation is discussed and photographs of ligament decay are presented. The droplet generator worked well on both oil and water based pesticide formulations. Current applications and results are discussed.

  20. Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin.

    PubMed

    Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Rahman, Farzana; Blee, Elizabeth; Murphy, Denis J

    2016-01-01

    Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL(-1). Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32-37, 20-24, and 16-18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log K LB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins, which

  1. Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin.

    PubMed

    Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Rahman, Farzana; Blee, Elizabeth; Murphy, Denis J

    2016-01-01

    Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL(-1). Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32-37, 20-24, and 16-18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log K LB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins, which

  2. Biochemical, Transcriptional, and Bioinformatic Analysis of Lipid Droplets from Seeds of Date Palm (Phoenix dactylifera L.) and Their Use as Potent Sequestration Agents against the Toxic Pollutant, 2,3,7,8-Tetrachlorinated Dibenzo-p-Dioxin

    PubMed Central

    Hanano, Abdulsamie; Almousally, Ibrahem; Shaban, Mouhnad; Rahman, Farzana; Blee, Elizabeth; Murphy, Denis J.

    2016-01-01

    Contamination of aquatic environments with dioxins, the most toxic group of persistent organic pollutants (POPs), is a major ecological issue. Dioxins are highly lipophilic and bioaccumulate in fatty tissues of marine organisms used for seafood where they constitute a potential risk for human health. Lipid droplets (LDs) purified from date palm, Phoenix dactylifera, seeds were characterized and their capacity to extract dioxins from aquatic systems was assessed. The bioaffinity of date palm LDs toward 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), the most toxic congener of dioxins was determined. Fractioned LDs were spheroidal with mean diameters of 2.5 µm, enclosing an oil-rich core of 392.5 mg mL-1. Isolated LDs did not aggregate and/or coalesce unless placed in acidic media and were strongly associated with three major groups of polypeptides of relative mass 32–37, 20–24, and 16–18 kDa. These masses correspond to the LD-associated proteins, oleosins, caleosins, and steroleosins, respectively. Efficient partitioning of TCDD into LDs occurred with a coefficient of log KLB/w,TCDD = 7.528 ± 0.024; it was optimal at neutral pH and was dependent on the presence of the oil-rich core, but was independent of the presence of LD-associated proteins. Bioinformatic analysis of the date palm genome revealed nine oleosin-like, five caleosin-like, and five steroleosin-like sequences, with predicted structures having putative lipid-binding domains that match their LD stabilizing roles and use as bio-based encapsulation systems. Transcriptomic analysis of date palm seedlings exposed to TCDD showed strong up-regulation of several caleosin and steroleosin genes, consistent with increased LD formation. The results suggest that the plant LDs could be used in ecological remediation strategies to remove POPs from aquatic environments. Recent reports suggest that several fungal and algal species also use LDs to sequester both external and internally derived hydrophobic toxins

  3. Droplet-Based Production of Liposomes

    NASA Technical Reports Server (NTRS)

    Ackley, Donald E.; Forster, Anita

    2009-01-01

    A process for making monodisperse liposomes having lipid bilayer membranes involves fewer, simpler process steps than do related prior methods. First, a microfluidic, cross junction droplet generator is used to produce vesicles comprising aqueous solution droplets contained in single layer lipid membranes. The vesicles are collected in a lipid-solvent mix that is at most partially soluble in water and is less dense than is water. A layer of water is dispensed on top of the solvent. By virtue of the difference in densities, the water sinks to the bottom and the solvent floats to the top. The vesicles, which have almost the same density as that of water, become exchanged into the water instead of floating to the top. As there are excess lipids in the solvent solution, in order for the vesicles to remain in the water, the addition of a second lipid layer to each vesicle is energetically favored. The resulting lipid bilayers present the hydrophilic ends of the lipid molecules to both the inner and outer membrane surfaces. If lipids of a second kind are dissolved in the solvent in sufficient excess before use, then asymmetric liposomes may be formed.

  4. Dynamic interactions of Leidenfrost droplets on liquid metal surface

    NASA Astrophysics Data System (ADS)

    Ding, Yujie; Liu, Jing

    2016-09-01

    Leidenfrost dynamic interaction effects of the isopentane droplets on the surface of heated liquid metal were disclosed. Unlike conventional rigid metal, such conductive and deformable liquid metal surface enables the levitating droplets to demonstrate rather abundant and complex dynamics. The Leidenfrost droplets at different diameters present diverse morphologies and behaviors like rotation and oscillation. Depending on the distance between the evaporating droplets, they attract and repulse each other through the curved surfaces beneath them and their vapor flows. With high boiling point up to 2000 °C, liquid metal offers a unique platform for testing the evaporating properties of a wide variety of liquid even solid.

  5. In vitro characterization of perfluorocarbon droplets for focused ultrasound therapy

    NASA Astrophysics Data System (ADS)

    Schad, Kelly C.; Hynynen, Kullervo

    2010-09-01

    Focused ultrasound therapy can be enhanced with microbubbles by thermal and cavitation effects. However, localization of treatment is difficult as bioeffects can occur outside of the target region. Spatial control of bubbles can be achieved by ultrasound-induced conversion of liquid perfluorocarbon droplets to gas bubbles. This study was undertaken to determine the acoustic parameters for bubble production by droplet conversion and how it depends on the acoustic conditions and droplet physical parameters. Lipid-encapsulated droplets containing dodecafluoropentane were manufactured with sizes ranging from 1.9 to 7.2 µm in diameter and diluted to a concentration of 8 × 106 droplets mL-1. The droplets were sonicated in vitro with a focused ultrasound transducer and varying frequency and exposure under flow conditions through an acoustically transparent vessel. The sonications were 10 ms in duration at frequencies of 0.578, 1.736 and 2.855 MHz. The pressure threshold for droplet conversion was measured with an active transducer operating in pulse-echo mode and simultaneous measurements of broadband acoustic emissions were performed with passive acoustic detection. The results show that droplets cannot be converted at low frequency without broadband emissions occurring. However, the pressure threshold for droplet conversion decreased with increasing frequency, exposure and droplet size. The pressure threshold for broadband emissions was independent of the droplet size and was 2.9, 4.4 and 5.3 MPa for 0.578, 1736 and 2.855 MHz, respectively. In summary, we have demonstrated that droplet conversion is feasible for clinically relevant sized droplets and acoustic exposures.

  6. How coalescing droplets jump.

    PubMed

    Enright, Ryan; Miljkovic, Nenad; Sprittles, James; Nolan, Kevin; Mitchell, Robert; Wang, Evelyn N

    2014-10-28

    Surface engineering at the nanoscale is a rapidly developing field that promises to impact a range of applications including energy production, water desalination, self-cleaning and anti-icing surfaces, thermal management of electronics, microfluidic platforms, and environmental pollution control. As the area advances, more detailed insights of dynamic wetting interactions on these surfaces are needed. In particular, the coalescence of two or more droplets on ultra-low adhesion surfaces leads to droplet jumping. Here we show, through detailed measurements of jumping droplets during water condensation coupled with numerical simulations of binary droplet coalescence, that this process is fundamentally inefficient with only a small fraction of the available excess surface energy (≲ 6%) convertible into translational kinetic energy. These findings clarify the role of internal fluid dynamics during the jumping droplet coalescence process and underpin the development of systems that can harness jumping droplets for a wide range of applications.

  7. Droplet transport system and methods

    NASA Technical Reports Server (NTRS)

    Neitzel, G. Paul (Inventor)

    2010-01-01

    Embodiments of droplet transport systems and methods are disclosed for levitating and transporting single or encapsulated droplets using thermocapillary convection. One method embodiment, among others comprises providing a droplet of a first liquid; and applying thermocapillary convection to the droplet to levitate and move the droplet.

  8. Wettability effects on droplet coalescence

    NASA Astrophysics Data System (ADS)

    Graham, Percival; de Pauw, Dennis; Dolatabadi, Ali

    2012-11-01

    Droplet impingement has been studied since 1895, with the works of A.M. Worthington. Throughout the past century, a variety of interesting phenomena have been uncovered. These include the bouncing of droplets off of each other or liquid pools, intricate droplet splashing mechanics, and droplets bouncing off of superhydrophobic surfaces; to name a few. In addition to intricate phenomena, droplet dynamics are relevant to many engineering applications, such as painting, spray coating ink-jet printing, and ice accumulation. These fields all involve interactions between droplets; therefore, studying droplet coalescence would benefit them greatly. The works presented include the coalescence of droplets with different impact conditions, various offsets, and at different wettabilities. Surface wettabilities studied are hydrophilic, hydrophobic and superhydrophobic. Fascinating phenomena observed include, bouncing of the impinging droplet off of the sessile droplet, sliding of the impinging droplet along the sessile droplet, and induced detachment on the sessile droplet on superhydrophobic surfaces. In order to capture the maximum spreading of the merged droplets, models related to coalescence of droplets in air and maximum spreading of a single droplet are combined to yield a new model to predict the maximum spreading of head-on droplet impact. Based on the free surface, and accuracy of the analytical model, droplet impact could be viewed as a mix of droplet coalescence in a gaseous media and droplet impact on a dry surface. Funding from NSERC.

  9. Water droplets also swim!

    NASA Astrophysics Data System (ADS)

    van der Linden, Marjolein; Izri, Ziane; Michelin, Sébastien; Dauchot, Olivier

    2015-03-01

    Recently there has been a surge of interest in producing artificial swimmers. One possible path is to produce self-propelling droplets in a liquid phase. The self-propulsion often relies on complex mechanisms at the droplet interface, involving chemical reactions and the adsorption-desorption kinetics of the surfactant. Here, we report the spontaneous swimming of droplets in a very simple system: water droplets immersed in an oil-surfactant medium. The swimmers consist of pure water, with no additional chemical species inside: water droplets also swim! The swimming is very robust: the droplets are able to transport cargo such as large colloids, salt crystals, and even cells. In this talk we discuss the origin of the spontaneous motion. Water from the droplet is solubilized by the reverse micellar solution, creating a concentration gradient of swollen reverse micelles around each droplet. By generalizing a recently proposed instability mechanism, we explain how spontaneous motion emerges in this system at sufficiently large Péclet number. Our water droplets in an oil-surfactant medium constitute the first experimental realization of spontaneous motion of isotropic particles driven by this instability mechanism.

  10. Droplet Combustion Experiment (DCE)

    NASA Technical Reports Server (NTRS)

    Haggard, John B., Jr.; Nayagan, Vedha; Dryer, Frederick L.; Williams, Forman A.

    1998-01-01

    The first space-based experiments were performed on the combustion of free, individual liquid fuel droplets in oxidizing atmospheres. The fuel was heptane, with initial droplet diameters ranging about from 1 mm to 4 mm. The atmospheres were mixtures of helium and oxygen, at pressures of 1.00, 0.50 and 0.25 bar, with oxygen mole fractions between 20% and 40%, as well as normal Spacelab cabin air. The temperatures of the atmospheres and of the initial liquid fuel were nominally 300 K. A total of 44 droplets were burned successfully on the two flights, 8 on the shortened STS-83 mission and 36 on STS-94. The results spanned the full range of heptane droplet combustion behavior, from radiative flame extinction at larger droplet diameters in the more dilute atmospheres to diffusive extinction in the less dilute atmospheres, with the droplet disappearing prior to flame extinction at the highest oxygen concentrations. Quasisteady histories of droplet diameters were observed along with unsteady histories of flame diameters. New and detailed information was obtained on burning rates, flame characteristics and soot behavior. The results have motivated new computational and theoretical investigations of droplet combustion, improving knowledge of the chemical kinetics, fluid mechanics and heat and mass transfer processes involved in burning liquid fuels.

  11. Relative abundance of Delta(5)-sterols in plasma membrane lipids of root-tip cells correlates with aluminum tolerance of rice.

    PubMed

    Khan, M Shahadat Hossain; Tawaraya, Keitarou; Sekimoto, Hiroshi; Koyama, Hiroyuki; Kobayashi, Yuriko; Murayama, Tetsuya; Chuba, Masaru; Kambayashi, Mihoko; Shiono, Yoshihito; Uemura, Matsuo; Ishikawa, Satoru; Wagatsuma, Tadao

    2009-01-01

    We investigated variations in aluminum (Al) tolerance among rice plants, using ancestor cultivars from the family line of the Al-tolerant and widely cultivated Japonica cultivar, Sasanishiki. The cultivar Rikuu-20 was Al sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. These two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the plasma membrane (PM) and greater Al uptake within 1 h of Al treatment. This could not be explained by organic acid release. Lipid composition of the PM differed between these cultivars, and may account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 had a lower ratio of phospholipids to Delta(5)-sterols than the sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitors of Delta(5)-sterol synthesis to alter the ratio of phospholipids to Delta(5)-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 and its Al-tolerant ancestor cultivars Kamenoo and Kyoku. In addition, Rikuu-132 showed a similar level of Al sensitivity when the ratio of phospholipids to Delta(5)-sterols was increased to match that of Rikuu-20 after treatment with uniconazole-P, an inhibitor of obtusifoliol-14alpha-demethylase. These results indicate that PM lipid composition is a factor underlying variations in Al tolerance among rice cultivars.

  12. Liquid droplet generation

    NASA Technical Reports Server (NTRS)

    Muntz, E. P.; Orme, Melissa; Farnham, Tony; Vandiep, G. Pham; Huerre, P.

    1989-01-01

    A pre-prototype segment of a droplet sheet generator for a liquid droplet radiator was designed, constructed and tested. The ability to achieve a uniform, non-diverging droplet sheet is limited by manufacturing tolerances on nozzle parallelism. For an array of 100, 100 micrometer diameters nozzles spaced 5 stream diameters apart, typical standard deviations in stream alignment were plus or minus 10 mrad. The drop to drop fractional speed variations of the drops in typical streams were similar and independent of position in the array. The absolute value of the speed dispersion depended on the amplitude of the disturbance applied to the stream. A second generation preliminary design of a 5200 stream segment of a droplet sheet generator was completed. The design is based on information developed during testing of the pre-prototype segment, along with the results of an acoustical analysis for the stagnation cavity pressure fluctuations used to break-up the streams into droplets.

  13. Universal fluid droplet ejector

    DOEpatents

    Lee, E.R.; Perl, M.L.

    1999-08-24

    A droplet generator comprises a fluid reservoir having a side wall made of glass or quartz, and an end cap made from a silicon plate. The end cap contains a micromachined aperture through which the fluid is ejected. The side wall is thermally fused to the end cap, and no adhesive is necessary. This means that the fluid only comes into contact with the side wall and the end cap, both of which are chemically inert. Amplitudes of drive pulses received by reservoir determine the horizontal displacements of droplets relative to the ejection aperture. The drive pulses are varied such that the dropper generates a two-dimensional array of vertically-falling droplets. Vertical and horizontal inter-droplet spacings may be varied in real time. Applications include droplet analysis experiments such as Millikan fractional charge searches and aerosol characterization, as well as material deposition applications. 8 figs.

  14. Supercritical microgravity droplet vaporization

    NASA Technical Reports Server (NTRS)

    Hartfield, J.; Curtis, E.; Farrell, P.

    1990-01-01

    Supercritical droplet vaporization is an important issue in many combustion systems, such as liquid fueled rockets and compression-ignition (diesel) engines. In order to study the details of droplet behavior at these conditions, an experiment was designed to provide a gas phase environment which is above the critical pressure and critical temperature of a single liquid droplet. In general, the droplet begins as a cold droplet in the hot, high pressure environment. In order to eliminate disruptions to the droplet by convective motion in the gas, forced and natural convection gas motion are required to be small. Implementation of this requirement for forced convection is straightforward, while reduction of natural convection is achieved by reduction in the g-level for the experiment. The resulting experiment consists of a rig which can stably position a droplet without restraint in a high-pressure, high temperature gas field in microgravity. The microgravity field is currently achieved by dropping the device in the NASA Lewis 2.2 second drop tower. The performance of the experimental device and results to date are presented.

  15. Reactive Leidenfrost droplets

    NASA Astrophysics Data System (ADS)

    Raufaste, C.; Bouret, Y.; Celestini, F.

    2016-05-01

    We experimentally investigate the reactivity of Leidenfrost droplets with their supporting substrates. Several organic liquids are put into contact with a copper substrate heated above their Leidenfrost temperature. As the liquid evaporates, the gaseous flow cleans the superficial copper oxide formed at the substrate surface and the reaction maintains a native copper spot below the evaporating droplet. The copper spot can reach several times the droplet size for the most reactive organic compounds. This study shows an interesting coupling between the physics of the Leidenfrost effect and the mechanics of reactive flows. Different applications are proposed such as drop motion tracking and vapor flow monitoring.

  16. Studying interfacial reactions of cholesterol sulfate in an unsaturated phosphatidylglycerol layer with ozone using field induced droplet ionization mass spectrometry.

    PubMed

    Ko, Jae Yoon; Choi, Sun Mi; Rhee, Young Min; Beauchamp, J L; Kim, Hugh I

    2012-01-01

    Field-induced droplet ionization (FIDI) is a recently developed ionization technique that can transfer ions from the surface of microliter droplets to the gas phase intact. The air-liquid interfacial reactions of cholesterol sulfate (CholSO(4)) in a 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) surfactant layer with ozone (O(3)) are investigated using field-induced droplet ionization mass spectrometry (FIDI-MS). Time-resolved studies of interfacial ozonolysis of CholSO(4) reveal that water plays an important role in forming oxygenated products. An epoxide derivative is observed as a major product of CholSO(4) oxidation in the FIDI-MS spectrum after exposure of the droplet to O(3) for 5 s. The abundance of the epoxide product then decreases with continued O(3) exposure as the finite number of water molecules at the air-liquid interface becomes exhausted. Competitive oxidation of CholSO(4) and POPG is observed when they are present together in a lipid surfactant layer at the air-liquid interface. Competitive reactions of CholSO(4) and POPG with O(3) suggest that CholSO(4) is present with POPG as a well-mixed interfacial layer. Compared with CholSO(4) and POPG alone, the overall ozonolysis rates of both CholSO(4) and POPG are reduced in a mixed layer, suggesting the double bonds of both molecules are shielded by additional hydrocarbons from one another. Molecular dynamics simulations of a monolayer comprising POPG and CholSO(4) correlate well with experimental observations and provide a detailed picture of the interactions between CholSO(4), lipids, and water molecules in the interfacial region.

  17. Active droplet generation in microfluidics.

    PubMed

    Chong, Zhuang Zhi; Tan, Say Hwa; Gañán-Calvo, Alfonso M; Tor, Shu Beng; Loh, Ngiap Hiang; Nguyen, Nam-Trung

    2016-01-01

    The reliable generation of micron-sized droplets is an important process for various applications in droplet-based microfluidics. The generated droplets work as a self-contained reaction platform in droplet-based lab-on-a-chip systems. With the maturity of this platform technology, sophisticated and delicate control of the droplet generation process is needed to address increasingly complex applications. This review presents the state of the art of active droplet generation concepts, which are categorized according to the nature of the induced energy. At the liquid/liquid interface, an energy imbalance leads to instability and droplet breakup.

  18. Active droplet generation in microfluidics.

    PubMed

    Chong, Zhuang Zhi; Tan, Say Hwa; Gañán-Calvo, Alfonso M; Tor, Shu Beng; Loh, Ngiap Hiang; Nguyen, Nam-Trung

    2016-01-01

    The reliable generation of micron-sized droplets is an important process for various applications in droplet-based microfluidics. The generated droplets work as a self-contained reaction platform in droplet-based lab-on-a-chip systems. With the maturity of this platform technology, sophisticated and delicate control of the droplet generation process is needed to address increasingly complex applications. This review presents the state of the art of active droplet generation concepts, which are categorized according to the nature of the induced energy. At the liquid/liquid interface, an energy imbalance leads to instability and droplet breakup. PMID:26555381

  19. Universal fluid droplet ejector

    DOEpatents

    Lee, Eric R.; Perl, Martin L.

    1999-08-24

    A droplet generator comprises a fluid reservoir having a side wall made of glass or quartz, and an end cap made from a silicon plate. The end cap contains a micromachined aperture through which the fluid is ejected. The side wall is thermally fused to the end cap, and no adhesive is necessary. This means that the fluid only comes into contact with the side wall and the end cap, both of which are chemically inert. Amplitudes of drive pulses received by reservoir determine the horizontal displacements of droplets relative to the ejection aperture. The drive pulses are varied such that the dropper generates a two-dimensional array of vertically-falling droplets. Vertical and horizontal interdroplet spacings may be varied in real time. Applications include droplet analysis experiments such as Millikan fractional charge searches and aerosol characterization, as well as material deposition applications.

  20. Functions of the Coacervate Droplets

    NASA Astrophysics Data System (ADS)

    Okihana, Hiroyuki; Ponnamperuma, Cyril

    1982-12-01

    Functions of coacervate droplets as protocells are studied by using synthetic polymers. The coacervate droplets were made from PVA-A and PVA-S. When glycine or diglycine were in the surrounding medium, the coacervate droplets concentrated them. The concentration of glycine in the coacervate droplets was higher than that of diglycine. When this mixture was irradiated by UV light, the coacervate droplets protected them from the photochemical decomposition.

  1. Microscopic Rayleigh Droplet Beams

    NASA Astrophysics Data System (ADS)

    Doak, R. B.

    2005-11-01

    A periodically triggered Rayleigh Droplet Beam (RDB) delivers a perfectly linear and periodic stream of identical, monoenergetic droplets that are phase-locked to the trigger signal. The droplet diameter and spacing are easily adjusted of choice of nozzle diameter and trigger frequency. Any liquid of low viscosity may be emloyed as the beam fluid. Although the field of nanofluidics is expanding rapidly, little effort has yet been devoted to ``external flows'' such as RDB's. At ASU we have generated RDB's of water and methanol down to 2 microns in droplet diameter. Nozzle clogging is the sole impediment to smaller droplets. Microscopic Rayleigh droplet beams offer tremendous potential for fundamental physical measurements, fluid dynamics research, and nanofabrication. This talk will describe the apparatus and techniques used at ASU to generate RDB's (surprisingly simple and inexpensive), discuss the triboelectric phenomena that play a role (surprisingly significant), present some initial experimental fluid dynamics measurements, and briefly survey RDB applications. Our particular interest in RDB's is as microscopic transport systems to deliver hydrated, undenatured proteins into vacuum for structure determination via serial diffraction of x-rays or electrons. This may offer the first general method for structure determination of non-crystallizable proteins.

  2. Lipid synthesis in chick epidermis.

    PubMed

    Lavker, R M

    1975-07-01

    Lipid synthesis in newborn chick epidermis was studied by electron microscopic autoradiography after injection of tritiated palmitate. The labeled lipid product in the tissue was identified as mostly triglyceride. At the earliest time after injection (6 hr), the radioactive precursor was taken up by all viable cells of the epidermis. Grain density was heaviest over basal cells, moderate over spinous cells, and slight over granular cells; thus lipid incorporation is highest in the basal and spinous regions of the chick epidermis. As time after injection progressed, the increasing amounts of grains over the granular and horny cells and decreasing amounts over the basal and spinous cells reflected the continuous upward displacement of cells from one layer into the next. From the distribution of silver grains within the epidermal cells, it has been concluded that, with the passage of time, triglycerides synthesized by the epidermal cells were mainly located in lipid droplets. The numerous grains associated with the elements of the endoplasmic reticulum indicated that this organelle is involved in aggregating triglyceride molecules into lipid droplets. The fact that grains were seen within the horny cells indicated that part of the horny cell consists of lipid probably derived from the lipid droplets retained by the cells during keratinization. PMID:1151110

  3. Fuel Droplet Burning During Droplet Combustion Experiment

    NASA Technical Reports Server (NTRS)

    2003-01-01

    Fuel ignites and burns in the Droplet Combustion Experiment (DCE) on STS-94 on July 4 1997, MET:2/05:40 (approximate). The DCE was designed to investigate the fundamental combustion aspects of single, isolated droplets under different pressures and ambient oxygen concentrations for a range of droplet sizes varying between 2 and 5 mm. DCE used various fuels -- in drops ranging from 1 mm (0.04 inches) to 5 mm (0.2 inches) -- and mixtures of oxidizers and inert gases to learn more about the physics of combustion in the simplest burning configuration, a sphere. The experiment elapsed time is shown at the bottom of the composite image. The DCE principal investigator was Forman Williams, University of California, San Diego. The experiment was part of the space research investigations conducted during the Microgravity Science Laboratory-1R mission (STS-94, July 1-17 1997). Advanced combustion experiments will be a part of investigations plarned for the International Space Station. (1.4MB, 13-second MPEG, screen 320 x 240 pixels; downlinked video, higher quality not available)A still JPG composite of this movie is available at http://mix.msfc.nasa.gov/ABSTRACTS/MSFC-0300168.html.

  4. Transcript profiling reveals mechanisms for lipid conservation during diapause in the mosquito, Aedes albopictus

    PubMed Central

    Reynolds, Julie A.; Poelchau, Monica F.; Rahman, Zahra; Armbruster, Peter A.; Denlinger, David L.

    2012-01-01

    The Asian tiger mosquito, Aedes albopictus, is a medically important invasive species whose geographic distribution has expanded dramatically during the past 20 years, and one of the key elements of its success is its capacity to survive long distance transport as a diapausing pharate first instar larva, encased within the chorion of the egg. We report that pharate larvae entering diapause are larger and contain 30% more lipid than their nondiapausing counterparts. To improve our understanding of the molecular regulation of lipid metabolism during diapause, we assessed the relative mRNA abundance of 21 genes using qRT-PCR. Elevated expression of lipid storage droplet protein 2 during embryonic development likely contributes to the higher amounts of lipid we noted in diapausing individuals. The conservation of lipids during diapause is reflected in downregulation of genes involved in lipid catabolism, including lipase 2, lipase 3, lipase 4, acyl-CoA dehydrogenase 4, and isovaleryl-CoA dehydrogenase. Two genes involved in fatty acid synthesis and modification, Δ(9)-desaturase, and fatty acyl-CoA elongase, were both upregulated in diapausing pharate larvae, suggesting roles for their gene products in generating unsaturated fatty acids to enhance membrane fluidity at low temperatures and generating precursors to the surface hydrocarbons needed to resist desiccation, respectively. Together, the results point to substantial distinctions in lipid metabolism within the embryo as a consequence of the diapause program, and these differences occur both before the actual onset of diapause as well as during the diapause state. PMID:22579567

  5. Hybrid lipid-silica microcapsules engineered by phase coacervation of Pickering emulsions to enhance lipid hydrolysis.

    PubMed

    Simovic, Spomenka; Heard, Peter; Prestidge, Clive A

    2010-07-14

    We report on the fabrication of dry hybrid lipid-silica microcapsules for enhanced lipid hydrolysis using Pickering emulsion templates formed by interfacial nanoparticle-emulsifier electrostatic interaction. The microcapsules are produced by controlled precipitation of emulsion droplets by oppositely charged silica nanoparticles at room temperature. Microcapsule formation is driven by the interfacial structure of the initial Pickering emulsion, which is in turn controlled by the nanoparticle to lipid ratio. In the region of charge reversed, precipitated and aggregated droplets, droplet-nanoparticle networks have been identified by freeze-fracture SEM imaging. The microcapsules have diameters in the range 20-50 mum and contain approximately 65% oil distributed within an internal matrix structure composed of a labyrinth of interconnected pores approximately 20-100 nm. Pore distribution and diameters depend on the silica to nanoparticle ratio that in turn determines droplet coating and stability. The microcapsules facilitate enhanced lipid hydrolysis kinetics, i.e. their pseudo first-order rate constant for lipid hydrolysis is approximately 3 times greater than for equivalent submicron lipid droplets. This behaviour is attributed to the increased oil surface area within the microcapsule due to the specific porous structure that causes rapid release of submicron and micron size oil droplets. The simple route for fabrication of porous microcapsule morphologies may present new opportunities for applications in encapsulation, delivery, coatings, and catalysis.

  6. Nematic droplets on fibers

    NASA Astrophysics Data System (ADS)

    Batista, V. M. O.; Silvestre, N. M.; Telo da Gama, M. M.

    2015-12-01

    The emergence of new techniques for the fabrication of nematic droplets with nontrivial topology provides new routes for the assembly of responsive devices. Here we explore some of the properties of nematic droplets on fibers, which constitute the basic units of a type of device that is able to respond to external stimuli, including the detection of gases. We perform a numerical study of spherical nematic droplets on fibers. We analyze the equilibrium textures for homogeneous and hybrid boundary conditions and find that in some cases the nematic avoids the nucleation of topological defects, which would provide a different optical response. We consider in detail a homeotropic nematic droplet wrapped around a fiber with planar anchoring. We investigate the effect of an electric field on the texture of this droplet. In the presence of a dc field, the system undergoes an orientational transition above a given threshold Ec, for which a ring defect is transformed into a figure-eight defect. We also consider ac fields, at high and low frequencies, and find that the textures are similar to those observed for static fields, in contrast with recently reported experiments.

  7. Microfluidic devices for droplet injection

    NASA Astrophysics Data System (ADS)

    Aubrecht, Donald; Akartuna, Ilke; Weitz, David

    2012-02-01

    As picoliter-scale reaction vessels, microfluidic water-in-oil emulsions have found application for high-throughput, large-sample number analyses. Often, the biological or chemical system under investigation needs to be encapsulated into droplets to prevent cross contamination prior to the introduction of reaction reagents. Previous techniques of picoinjection or droplet synchronization and merging enable the addition of reagents to individual droplets, but present limitations on what can be added to each droplet. We present microfluidic devices that couple the strengths of picoinjection and droplet merging, allowing us to selectively add precise volume to our droplet reactions.

  8. Droplet lasing spectroscopy applied to droplet stream flames

    SciTech Connect

    Santangelo, P.J.; Kennedy, I.M.

    1999-04-01

    Droplet lasing spectroscopy (DLS) has been applied to the measurement of droplet size and vaporization rates in both reacting and non-reacting rectilinear droplet streams. A Berglund-Liu droplet generator was used to generate a stream of droplets, approximately 63 microns in diameter and 6.5 droplet diameters apart. Ethanol, methanol, and a pentane/ethanol mixture were doped with Rhodamine 6G. Lasing spectra were examined in the steady-state combustion regime. In the pentane/ethanol case the measurements were carried out in a sooting region of the flame. In some cases, vaporization rates were high enough to measure the rate from consecutive droplets, yielding a quasi-instantaneous measurement. In all cases, the D{sup 2} law of droplet vaporization was evident. In addition, photographs of the flames yielded measurements of flame height and thickness.

  9. Droplet Combustion Experiment movie

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Droplet Combustion Experiment (DCE) was designed to investigate the fundamental combustion aspects of single, isolated droplets under different pressures and ambient oxygen concentrations for a range of droplet sizes varying between 2 and 5 mm. The DCE principal investigator was Forman Williams, University of California, San Diego. The experiment was part of the space research investigations conducted during the Microgravity Science Laboratory-1 mission (STS-83, April 4-8 1997; the shortened mission was reflown as MSL-1R on STS-94). Advanced combustion experiments will be a part of investigations plarned for the International Space Station. (1.1 MB, 12-second MPEG, screen 320 x 240 pixels; downlinked video, higher quality not available)A still JPG composite of this movie is available at http://mix.msfc.nasa.gov/ABSTRACTS/MSFC-0300164.html.

  10. The Walking Droplet Instability

    NASA Astrophysics Data System (ADS)

    Bostwick, Joshua; Steen, Paul

    2013-11-01

    A droplet of liquid that partially wets a solid substrate assumes a spherical-cap equilibrium shape. We show that the spherical-cap with a mobile contact-line is unstable to a non-axisymmetric disturbance and we characterize the instability mechanism, as it depends upon the wetting properties of the substrate. We then solve the hydrodynamic problem for inviscid motions showing that the flow associated with the instability correlates with horizontal motion of the droplet's center-of-mass. We calculate the resulting ``walking speed.'' A novel feature is that the energy conversion mechanism is not unique, so long as the contact-line is mobilized. Hence, the walking droplet instability is potentially significant to a number of industrial applications, such as self-cleansing surfaces or energy harvesting devices.

  11. Chip-based droplet sorting

    DOEpatents

    Beer, Neil Reginald; Lee, Abraham; Hatch, Andrew

    2014-07-01

    A non-contact system for sorting monodisperse water-in-oil emulsion droplets in a microfluidic device based on the droplet's contents and their interaction with an applied electromagnetic field or by identification and sorting.

  12. Effect of Mannosylerythritol lipid-A on light scattering of AOT/D2O/Octane

    NASA Astrophysics Data System (ADS)

    Sharifi, Soheil

    2016-09-01

    The light scattering technique is used for the study of interaction of Mannosylerythritol lipid-A on AOT/D2O/Octane. The collective diffusion of AOT/D2O droplets soluble in Octane mixed with lipid is founded from a correlation function of light scattering. We focus on the variation of the dynamic behavior of droplets as a function of the lipid concentrations and the size of droplets. The increase of concentration of Mannosylerythritol lipid-A on microemulsion decreases the dynamic of droplets. The SAXS experiment shows the size and the interaction of the droplets change by increase of Mannosylerythritol lipid-A concentration. A hard sphere model can describe the interaction of lipid with AOT/D2O droplets.

  13. Microfluidic fabrication of asymmetric giant lipid vesicles

    PubMed Central

    Hu, Peichi C.; Li, Su; Malmstadt, Noah

    2011-01-01

    We have developed a microfluidic technology for the fabrication of compositionally asymmetric giant unilamellar vesicles (GUVs). The vesicles are assembled in two independent steps. In each step, a lipid monolayer is formed at a water-oil interface. The first monolayer is formed inside of a microfluidic device with a multiphase droplet flow configuration consisting of a continuous oil stream in which water droplets are formed. These droplets are dispensed into a vessel containing a layer of oil over a layer of water. The second lipid monolayer is formed by transferring the droplets through this second oil-water interface by centrifugation. By dissolving different lipid compositions in the different oil phases, the composition of each leaflet of the resulting lipid bilayer can be controlled. We have demonstrated membrane asymmetry by showing differential fluorescence quenching of labeled lipids in each leaflet and by demonstrating that asymmetric GUVs will bind an avidin-coated surface only when biotinylated lipids are targeted to the outer leaflet. In addition, we have demonstrated the successful asymmetric targeting of phosphatidylserine lipids to each leaflet, producing membranes with a biomimetic and physiologically relevant compositional asymmetry. PMID:21449588

  14. Evaporation-Induced Buckling and Fission of Microscale Droplet Interface Bilayers

    SciTech Connect

    Boreyko, Jonathan B; Mruetusatorn, Prachya; Sarles, Stephen A; Retterer, Scott T; Collier, Pat

    2013-01-01

    Droplet interface bilayers (DIBs) are a robust platform for studying synthetic cellular membranes; however, to date no DIBs have been produced at cellular length scales. Here, we create microscale droplet interface bilayers ( DIBs) at the interface between aqueous femtoliter-volume droplets within an oil-filled microfluidic channel. The uniquely large area-to-volume ratio of the droplets results in strong evaporation effects, causing the system to transition through three distinct regimes. First, the two adjacent droplets shrink into the shape of a single spherical droplet, where an augmented lipid bilayer partitions two hemi-spherical volumes. In the second regime, the combined effects of the shrinking monolayers and growing bilayer force the confined bilayer to buckle to conserve its mass. Finally, at a bending moment corresponding to a critical shear stress, the buckling bilayer fissions a vesicle to regulate its shape and stress. The DIBs produced here enable evaporation-induced bilayer dynamics reminiscent of endo- and exocytosis in cells.

  15. Regulation of renal lipid metabolism, lipid accumulation, and glomerulosclerosis in FVBdb/db mice with type 2 diabetes.

    PubMed

    Wang, Zhuowei; Jiang, Tao; Li, Jinping; Proctor, Gregory; McManaman, James L; Lucia, Scott; Chua, Streamson; Levi, Moshe

    2005-08-01

    Diabetic kidney disease has been associated with the presence of lipid deposits, but the mechanisms for the lipid accumulation have not been fully determined. In the present study, we found that db/db mice on the FVB genetic background with loss-of-function mutation of the leptin receptor (FVB-Lepr(db) mice or FVBdb/db) develop severe diabetic nephropathy, including glomerulosclerosis, tubulointerstitial fibrosis, increased expression of type IV collagen and fibronectin, and proteinuria, which is associated with increased renal mRNA abundance of transforming growth factor-beta, plasminogen activator inhibitor-1, and vascular endothelial growth factor. Electron microscopy demonstrates increases in glomerular basement membrane thickness and foot process (podocyte) length. We found that there is a marked increase in neutral lipid deposits in glomeruli and tubules by oil red O staining and biochemical analysis for cholesterol and triglycerides. We also detected a significant increase in the renal expression of adipocyte differentiation-related protein (adipophilin), a marker of cytoplasmic lipid droplets. We examined the expression of sterol regulatory element-binding protein (SREBP)-1 and -2, transcriptional factors that play an important role in the regulation of fatty acid, triglyceride, and cholesterol synthesis. We found significant increases in SREBP-1 and -2 protein levels in nuclear extracts from the kidneys of FVBdb/db mice, with increases in the mRNA abundance of acetyl-CoA carboxylase, fatty acid synthase, and 3-hydroxy-3-methylglutaryl-CoA reductase, which mediates the increase in renal triglyceride and cholesterol content. Our results indicate that in FVBdb/db mice, renal triglyceride and cholesterol accumulation is mediated by increased activity of SREBP-1 and -2. Based on our previous results with transgenic mice overexpressing SREBP-1 in the kidney, we propose that increased expression of SREBPs plays an important role in causing renal lipid

  16. HIF-2α dependent lipid storage promotes endoplasmic reticulum homeostasis in clear cell renal cell carcinoma

    PubMed Central

    Qiu, Bo; Ackerman, Daniel; Sanchez, Danielle J.; Li, Bo; Ochocki, Joshua D.; Grazioli, Alison; Bobrovnikova-Marjon, Ekaterina; Diehl, J. Alan; Keith, Brian; Simon, M. Celeste

    2015-01-01

    Two hallmarks of clear cell renal cell carcinoma (ccRCC) are constitutive hypoxia inducible factor (HIF) signaling and abundant intracellular lipid droplets (LDs). However, regulation of lipid storage and its role in ccRCC are incompletely understood. Transcriptional profiling of primary ccRCC samples revealed that expression of the LD coat protein gene PLIN2 was elevated in tumors and correlated with HIF-2α, but not HIF-1α, activation. HIF-2α dependent PLIN2 expression promoted lipid storage, proliferation, and viability in xenograft tumors. Mechanistically, lipid storage maintained integrity of the endoplasmic reticulum (ER), which is functionally and physically associated with LDs. Specifically, PLIN2 dependent lipid storage suppressed cytotoxic ER stress responses that otherwise result from elevated protein synthetic activity characteristic of ccRCC cells. Thus, in addition to promoting ccRCC proliferation and anabolic metabolism, HIF-2α modulates lipid storage to sustain ER homeostasis, particularly under conditions of nutrient and oxygen limitation, thereby promoting tumor cell survival. PMID:25829424

  17. Diffraction of walking droplets

    NASA Astrophysics Data System (ADS)

    Harris, Daniel M.; Pucci, Giuseppe; Bush, John W. M.

    2014-11-01

    We present results from our revisitation of the experiment of a walking droplet passing through a single slit, originally investigated by Couder & Fort (PRL, 2006). On each passage, the walker's trajectory is deviated as a result of the spatial confinement of its guiding wave. We explore the role of the droplet size and the bath's vibration amplitude on both the dynamics and statistics. We find the behavior to be remarkably sensitive to these control parameters. A complex physical picture emerges. The authors gratefully acknowledge the financial support of the NSF through Grant CMMI-1333242, DMH through the NSF Graduate Research Fellowship Program, and GP through the Programma Operativo Regionale (POR) Calabria - FSE 2007/2013.

  18. Trehalose 6,6′-Dimycolate and Lipid in the Pathogenesis of Caseating Granulomas of Tuberculosis in Mice

    PubMed Central

    Hunter, Robert L.; Olsen, Margaret; Jagannath, Chinnaswamy; Actor, Jeffrey K.

    2006-01-01

    Trehalose 6,6′-dimycolate (TDM) is the most abundant, most granulomagenic, and most toxic lipid extractable from the surface of virulent Mycobacterium tuberculosis (MTB). We further examined its toxicity, which requires activation by oily surfaces. Injections of MTB and/or TDM into sensitized mice induced caseating granulomas that centered on oil droplets. If large doses of MTB were injected in saline, caseating granulomas developed in adipose tissue, but MTB with surface TDM removed induced only acute inflammation that did not persist. Variations in protocols produced several variants of caseating granulomas, each with characteristics of human tuberculosis. In each instance, MTB were localized in fat cells or oil drops during initiation of caseating granulomas suggesting that necrosis was caused by activation of the toxicity of TDM toxicity. Evidence extending these findings to the lung was derived from the observation that in sensitized mice, as in humans, tuberculosis development stimulates accumulation of lipid selectively in alveoli. MTB preferentially associated with lipid droplets in developing necrotic foci in late-stage murine tuberculosis. This supports the hypothesis that pulmonary tuberculosis sequesters MTB in a protected environment that accumulates lipid until it is able to activate the toxicity of TDM and initiate necrosis that results in caseating granulomas. PMID:16565499

  19. High-Voltage Droplet Dispenser

    NASA Technical Reports Server (NTRS)

    Eichenberg, Dennis J.

    2003-01-01

    An apparatus that is extremely effective in dispensing a wide range of droplets has been developed. This droplet dispenser is unique in that it utilizes a droplet bias voltage, as well as an ionization pulse, to release a droplet. Apparatuses that deploy individual droplets have been used in many applications, including, notably, study of combustion of liquid fuels. Experiments on isolated droplets are useful in that they enable the study of droplet phenomena under well-controlled and simplified conditions. In this apparatus, a syringe dispenses a known value of liquid, which emerges from, and hangs onto, the outer end of a flat-tipped, stainless steel needle. Somewhat below the needle tip and droplet is a ring electrode. A bias high voltage, followed by a high-voltage pulse, is applied so as to attract the droplet sufficiently to pull it off the needle. The voltages are such that the droplet and needle are negatively charged and the ring electrode is positively charged.

  20. Monodisperse Micro-Oil Droplets Stabilized by Polymerizable Phospholipid Coatings as Potential Drug Carriers.

    PubMed

    Park, Yoonjee; Pham, Tuan A; Beigie, Carl; Cabodi, Mario; Cleveland, Robin O; Nagy, Jon O; Wong, Joyce Y

    2015-09-15

    There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug

  1. Perilipin-related protein regulates lipid metabolism in C. elegans

    PubMed Central

    Chughtai, Ahmed Ali; Kaššák, Filip; Kostrouchová, Markéta; Novotný, Jan Philipp; Krause, Michael W.; Kostrouch, Zdenek

    2015-01-01

    Perilipins are lipid droplet surface proteins that contribute to fat metabolism by controlling the access of lipids to lipolytic enzymes. Perilipins have been identified in organisms as diverse as metazoa, fungi, and amoebas but strikingly not in nematodes. Here we identify the protein encoded by the W01A8.1 gene in Caenorhabditis elegans as the closest homologue and likely orthologue of metazoan perilipin. We demonstrate that nematode W01A8.1 is a cytoplasmic protein residing on lipid droplets similarly as human perilipins 1 and 2. Downregulation or elimination of W01A8.1 affects the appearance of lipid droplets resulting in the formation of large lipid droplets localized around the dividing nucleus during the early zygotic divisions. Visualization of lipid containing structures by CARS microscopy in vivo showed that lipid-containing structures become gradually enlarged during oogenesis and relocate during the first zygotic division around the dividing nucleus. In mutant embryos, the lipid containing structures show defective intracellular distribution in subsequent embryonic divisions and become gradually smaller during further development. In contrast to embryos, lipid-containing structures in enterocytes and in epidermal cells of adult animals are smaller in mutants than in wild type animals. Our results demonstrate the existence of a perilipin-related regulation of fat metabolism in nematodes and provide new possibilities for functional studies of lipid metabolism. PMID:26357594

  2. Droplet microfluidics based microseparation systems.

    PubMed

    Xiao, Zhiliang; Niu, Menglei; Zhang, Bo

    2012-06-01

    Lab on a chip (LOC) technology is a promising miniaturization approach. The feature that it significantly reduced sample consumption makes great sense in analytical and bioanalytical chemistry. Since the start of LOC technology, much attention has been focused on continuous flow microfluidic systems. At the turn of the century, droplet microfluidics, which was also termed segmented flow microfluidics, was introduced. Droplet microfluidics employs two immiscible phases to form discrete droplets, which are ideal vessels with confined volume, restricted dispersion, limited cross-contamination, and high surface area. Due to these unique features, droplet microfluidics proves to be a versatile tool in microscale sample handling. This article reviews the utility of droplet microfluidics in microanalytical systems with an emphasize on separation science, including sample encapsulation at ultra-small volume, compartmentalization of separation bands, isolation of droplet contents, and related detection techniques.

  3. Rapidly pulsed helium droplet source

    SciTech Connect

    Pentlehner, Dominik; Riechers, Ricarda; Dick, Bernhard; Slenczka, Alkwin; Even, Uzi; Lavie, Nachum; Brown, Raviv; Luria, Kfir

    2009-04-15

    A pulsed valve connected to a closed-cycle cryostat was optimized for producing helium droplets. The pulsed droplet beam appeared with a bimodal size distribution. The leading part of the pulse consists of droplets suitable for doping with molecules. The average size of this part can be varied between 10{sup 4} and 10{sup 6} helium atoms, and the width of the distribution is smaller as compared to a continuous-flow droplet source. The system has been tested in a single pulse mode and at repetition rates of up to 500 Hz with almost constant intensity. The droplet density was found to be increased by more than an order of magnitude as compared to a continuous-flow droplet source.

  4. Maze Solving by Chemotactic Droplets

    SciTech Connect

    Lagzi, Istvan; Soh, Siowling; Wesson, Paul J.; Browne, Kevin P.; Grzybowski, Bartosz A.

    2010-01-11

    Droplets emitting surface-active chemicals exhibit chemotaxis toward low-pH regions. Such droplets are self-propelled and navigate through a complex maze to seek a source of acid placed at one of the maze’s exits. In doing so, the droplets find the shortest path through the maze. Chemotaxis and maze solving are due to an interplay between acid/base chemistry and surface tension effects.

  5. Lipoprotein-like particles in a prokaryote: quinone droplets of Thermoplasma acidophilum.

    PubMed

    Nagy, István; Knispel, Roland Wilhelm; Kofler, Christine; Orsini, Massimiliano; Boicu, Marius; Varga, Sándor; Weyher-Stingl, Elisabeth; Sun, Na; Fernandez-Busnadiego, Ruben; Kukolya, József; Nickell, Stephan; Baumeister, Wolfgang

    2016-09-01

    Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet.

  6. Lipoprotein-like particles in a prokaryote: quinone droplets of Thermoplasma acidophilum.

    PubMed

    Nagy, István; Knispel, Roland Wilhelm; Kofler, Christine; Orsini, Massimiliano; Boicu, Marius; Varga, Sándor; Weyher-Stingl, Elisabeth; Sun, Na; Fernandez-Busnadiego, Ruben; Kukolya, József; Nickell, Stephan; Baumeister, Wolfgang

    2016-09-01

    Cytosolic, globular droplets with an average diameter of 50 nm were observed in vitrified Thermoplasma acidophilum cells by means of cryo-electron tomography. These droplets were isolated by column chromatography and immunoprecipitation protein purification methods. Subsequent chemical and biochemical analyses identified lipid and protein components, respectively. Two major lipid components, comigrating menaquinones at the solvent front and the slower migrating Thermoplasma polar lipid U4, were detected by TLC experiments. The major protein component was identified as the 153 amino acid long Ta0547 vitellogenin-N domain protein. This domain has been found so far exclusively in large lipid transport proteins of vertebrates and non-vertebrates. Blast protein database homology searches with Ta0547 did not return any eukaryal hits; homologous sequences were found only in thermo-acidophilic archaeons. However, a profile-sequence domain search performed with the vitellogenin-N domain (PF01347) hmm-profile against the T. acidophilum proteome returned Ta0547 as hit. Electron microscopy appearance of isolated droplets resembled to lipoprotein particles. However, no (tetraether) lipid layer could be detected on the droplets surface, rather hydrophobic compounds of the electron dense lumen were surrounded by a denser discontinuous protein boundary. Based on described features, these particles qualify for a novel lipoprotein particle category, what we nominated Thermoplasma Quinone Droplet. PMID:27405311

  7. Enhanced Jumping-Droplet Departure.

    PubMed

    Kim, Moon-Kyung; Cha, Hyeongyun; Birbarah, Patrick; Chavan, Shreyas; Zhong, Chen; Xu, Yuehan; Miljkovic, Nenad

    2015-12-15

    Water vapor condensation on superhydrophobic surfaces has received much attention in recent years because of its ability to shed water droplets at length scales 3 decades smaller than the capillary length (∼1 mm) via coalescence-induced droplet jumping. Jumping-droplet condensation has been demonstrated to enhance heat transfer, anti-icing, and self-cleaning efficiency and is governed by the theoretical inertial-capillary scaled jumping speed (U). When two droplets coalesce, the experimentally measured jumping speed (Uexp) is fundamentally limited by the internal fluid dynamics during the coalescence process (Uexp < 0.23U). Here, we theoretically and experimentally demonstrate multidroplet (>2) coalescence as an avenue to break the two-droplet speed limit. Using side-view and top-view high-speed imaging to study more than 1000 jumping events on a copper oxide nanostructured superhydrophobic surface, we verify that droplet jumping occurs as a result of three fundamentally different mechanisms: (1) coalescence between two droplets, (2) coalescence among more than two droplets (multidroplet), and (3) coalescence between one or more droplets on the surface and a returning droplet that has already departed (multihop). We measured droplet-jumping speeds for a wide range of droplet radii (5-50 μm) and demonstrated that while the two-droplet capillary-to-inertial energy conversion mechanism is not identical to that of multidroplet jumping, speeds above the theoretical two-droplet limit (>0.23U) can be achieved. However, we discovered that multihop coalescence resulted in drastically reduced jumping speeds (≪0.23U) due to adverse momentum contributions from returning droplets. To quantify the impact of enhanced jumping speed on heat-transfer performance, we developed a condensation critical heat flux model to show that modest jumping speed enhancements of 50% using multidroplet jumping can enhance performance by up to 40%. Our results provide a starting point for the

  8. Uranium droplet core nuclear rocket

    NASA Technical Reports Server (NTRS)

    Anghaie, Samim

    1991-01-01

    Uranium droplet nuclear rocket is conceptually designed to utilize the broad temperature range ofthe liquid phase of metallic uranium in droplet configuration which maximizes the energy transfer area per unit fuel volume. In a baseline system dissociated hydrogen at 100 bar is heated to 6000 K, providing 2000 second of Isp. Fission fragments and intense radian field enhance the dissociation of molecular hydrogen beyond the equilibrium thermodynamic level. Uranium droplets in the core are confined and separated by an axisymmetric vortex flow generated by high velocity tangential injection of hydrogen in the mid-core regions. Droplet uranium flow to the core is controlled and adjusted by a twin flow nozzle injection system.

  9. Significance of droplet-droplet interactions in droplet streams: Atmospheric to supercritical conditions

    NASA Astrophysics Data System (ADS)

    Connon, Corinne Shirley

    In an effort to optimize liquid fuel combustion a considerable amount of research has been directed towards the atomization of large liquid masses into small droplets to increase the surface area available for vaporization. The current work uses a single linear array of moving droplets of uniform size and spacing to investigate the behavior of interacting droplets. A series of experiments, over a range of ambient conditions, demonstrate how a lead droplet alters the environment experienced by its trailing neighbor. This behavior is of particular interest for droplet groups under high pressure and temperature, where experimental data has been limited. Gas phase velocity and vapor concentration measurements show that as the space between adjacent droplets decreases entrainment of fluid towards the axis of motion is reduced. Trapped gases create a gaseous cylinder, composed of ambient gas and fuel vapor, which surrounds and moves with the droplet stream. As ambient pressure increase, the oscillatory behavior of the lead droplet wake begins to interfere with its trailing neighbor. Loss of stream stability and enhanced droplet stripping in part result from these oscillating wakes. However, acceleration of droplet stripping is mainly produced by liquid and gas density similarity, which increases the centrifugal stress and the growth rate of capillary waves. Further, injection of subcritical droplets into an ambient environment at temperatures and pressures above the liquid droplet critical point shows behavior not greatly different from the results obtained at high ambient pressures. The similarity results from thermal heatup times exceeding the breakup times generated from the severe aerodynamics encountered at high ambient density and high liquid-gas relative velocities.

  10. A microfluidic platform for probing single cell plasma membranes using optically trapped Smart Droplet Microtools (SDMs).

    PubMed

    Lanigan, Peter M P; Ninkovic, Tanja; Chan, Karen; de Mello, Andrew J; Willison, Keith R; Klug, David R; Templer, Richard H; Neil, Mark A A; Ces, Oscar

    2009-04-21

    We recently introduced a novel platform based upon optically trapped lipid coated oil droplets (Smart Droplet Microtools-SDMs) that were able to form membrane tethers upon fusion with the plasma membrane of single cells. Material transfer from the plasma membrane to the droplet via the tether was seen to occur. Here we present a customised version of the SDM approach based upon detergent coated droplets deployed within a microfluidic format. These droplets are able to differentially solubilise the plasma membrane of single cells with spatial selectivity and without forming membrane tethers. The microfluidic format facilitates separation of the target cells from the bulk SDM population and from downstream analysis modules. Material transfer from the cell to the SDM was monitored by tracking membrane localized EGFP.

  11. Chemistry and biology in femtoliter and picoliter volume droplets.

    PubMed

    Chiu, Daniel T; Lorenz, Robert M

    2009-05-19

    The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases; in cancer metastasis, for example, a single cell seeds the formation of a distant tumor. Although tiny, a cell is a highly heterogeneous and compartmentalized structure: proteins, lipids, RNA, and small-molecule metabolites constantly traffic among intracellular organelles. Gaining detailed information about the spatiotemporal distribution of these biomolecules is crucial to our understanding of cellular function and dysfunction. To access this information, we need sensitive tools that are capable of extracting comprehensive biochemical information from single cells and subcellular organelles. In this Account, we outline our approach and highlight our progress toward mapping the spatiotemporal organization of information flow in single cells. Our technique is centered on the use of femtoliter- and picoliter-sized droplets as nanolabs for manipulating single cells and subcellular compartments. We have developed a single-cell nanosurgical technique for isolating select subcellular structures from live cells, a capability that is needed for the high-resolution manipulation and chemical analysis of single cells. Our microfluidic approaches for generating single femtoliter-sized droplets on demand include both pressure and electric field methods; we have also explored a design for the on-demand generation of multiple aqueous droplets to increase throughput. Droplet formation is only the first step in a sequence that requires manipulation, fusion, transport, and analysis. Optical approaches provide the most convenient and precise control over the formed droplets with our technology platform; we describe aqueous droplet manipulation with optical vortex traps, which enable the remarkable ability to dynamically "tune" the concentration of the contents. Integration of thermoelectric manipulations with these techniques affords further control. The

  12. Chemistry and Biology in Femtoliter and Picoliter Volume Droplets

    PubMed Central

    Chiu, Daniel T.; Lorenz, Robert M.

    2009-01-01

    Conspectus The basic unit of any biological system is the cell, and malfunctions at the single-cell level can result in devastating diseases; in cancer metastasis, for example, a single cell seeds the formation of a distant tumor. Although tiny, a cell is a highly heterogeneous and compartmentalized structure: proteins, lipids, RNA, and small-molecule metabolites constantly traffic among intracellular organelles. Gaining detailed information about the spatiotemporal distribution of these biomolecules is crucial to our understanding of cellular function and dysfunction. To access this information, we need sensitive tools that are capable of extracting comprehensive biochemical information from single cells and subcellular organelles. In this Account, we outline our approach and highlight our progress towards mapping the spatiotemporal organization of information flow in single cells. Our technique is centered on the use of femtoliter- and picoliter-sized droplets as nanolabs for manipulating single cells and subcellular compartments. We have developed a single-cell nanosurgical technique for isolating select subcellular structures from live cells, a capability that is needed for the high-resolution manipulation and chemical analysis of single cells. Our microfluidic approaches for generating single femtoliter-sized droplets on demand include both pressure and electric field methods; we have also explored a design for the on-demand generation of multiple aqueous droplets to increase throughput. Droplet formation is only the first step in a sequence that requires manipulation, fusion, transport, and analysis. Optical approaches provide the most convenient and precise control over the formed droplets with our technology platform; we describe aqueous droplet manipulation with optical vortex traps, which enable the remarkable ability to dynamically “tune” the concentration of the contents. Integration of thermoelectric manipulations with these techniques affords

  13. A comprehensive flexoelectric model for droplet interface bilayers acting as sensors and energy harvesters

    NASA Astrophysics Data System (ADS)

    Kancharala, Ashok; Freeman, Eric; Philen, Michael

    2016-10-01

    Droplet interface bilayers have found applications in the development of biologically-inspired mechanosensors. In this research, a comprehensive flexoelectric framework has been developed to predict the mechanoelectric capabilities of the biological membrane under mechanical excitation for sensing and energy harvesting applications. The dynamic behavior of the droplets has been modeled using nonlinear finite element analysis, coupled with a flexoelectric model for predicting the resulting material polarization. This coupled model allows for the prediction of the mechanoelectrical response of the droplets under excitation. Using the developed framework, the potential for sensing and energy harvesting through lipid membranes is investigated.

  14. Experiments examining drag in linear droplet packets

    NASA Astrophysics Data System (ADS)

    Nguyen, Q. V.; Dunn-Rankin, D.

    1992-01-01

    This paper presents an experimental study of vertically traveling droplet packets, where the droplets in each packet are aligned linearly, one behind another. The paper describes in detail, an experimental apparatus that produces repeatable, linearly aligned, and isolated droplet packets containing 1 6 droplets per packet. The apparatus is suitable for examining aerodynamic interactions between droplets within each packet. This paper demonstrates the performance of the apparatus by examining the drag reduction and collision of droplets traveling in the wake of a lead droplet. Comparison of a calculated single droplet trajectory with the detailed droplet position versus time data for a droplet packet provides the average drag reduction experienced by the trailing droplets due to the aerodynamic wake of the lead droplet. For the conditions of our experiment (4 droplet packet, 145 μm methanol droplets, 10 m/s initial velocity, initial droplet spacing of 5.2 droplet diameters, Reynolds number approx. 80) the average drag on the first trailing droplet was found to be 75% of the drag on the lead droplet.

  15. A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails

    PubMed Central

    Carreras, P.; Elani, Y.; Law, R. V.; Brooks, N. J.; Seddon, J. M.; Ces, O.

    2015-01-01

    Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together—oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300–1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or “rails,” and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication. PMID:26759638

  16. A microfluidic platform for size-dependent generation of droplet interface bilayer networks on rails.

    PubMed

    Carreras, P; Elani, Y; Law, R V; Brooks, N J; Seddon, J M; Ces, O

    2015-11-01

    Droplet interface bilayer (DIB) networks are emerging as a cornerstone technology for the bottom up construction of cell-like and tissue-like structures and bio-devices. They are an exciting and versatile model-membrane platform, seeing increasing use in the disciplines of synthetic biology, chemical biology, and membrane biophysics. DIBs are formed when lipid-coated water-in-oil droplets are brought together-oil is excluded from the interface, resulting in a bilayer. Perhaps the greatest feature of the DIB platform is the ability to generate bilayer networks by connecting multiple droplets together, which can in turn be used in applications ranging from tissue mimics, multicellular models, and bio-devices. For such applications, the construction and release of DIB networks of defined size and composition on-demand is crucial. We have developed a droplet-based microfluidic method for the generation of different sized DIB networks (300-1500 pl droplets) on-chip. We do this by employing a droplet-on-rails strategy where droplets are guided down designated paths of a chip with the aid of microfabricated grooves or "rails," and droplets of set sizes are selectively directed to specific rails using auxiliary flows. In this way we can uniquely produce parallel bilayer networks of defined sizes. By trapping several droplets in a rail, extended DIB networks containing up to 20 sequential bilayers could be constructed. The trapped DIB arrays can be composed of different lipid types and can be released on-demand and regenerated within seconds. We show that chemical signals can be propagated across the bio-network by transplanting enzymatic reaction cascades for inter-droplet communication. PMID:26759638

  17. Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-dimensional Gel Electrophoresis and HPLC-ESI-MS/MS

    PubMed Central

    Xie, Xitao; Yi, Zhengping; Bowen, Benjamin; Wolf, Cassandra; Flynn, Charles R.; Sinha, Sandeep; Mandarino, Lawrence J.; Meyer, Christian

    2010-01-01

    Abnormalities in adipocytes play an important role in various conditions, including the metabolic syndrome, type 2 diabetes mellitus and cardiovascular disease, but little is known about alterations at the protein level. We therefore sought to 1) comprehensively characterize the human adipocyte proteome for the first time, and 2) demonstrate feasibility of measuring adipocyte protein abundances by one-dimensional SDS-PAGE and High Performance Liquid Chromatography -Electron Spray Ionization - tandem Mass Spectrometry (HPLC-ESI-MS/MS). In adipocytes isolated from ~0.5 g subcutaneous abdominal adipose tissue of three healthy, lean subjects we identified a total of 1493 proteins. Triplicate analysis indicated a 22.5% coefficient of variation of protein abundances. Proteins ranged from 5.8 to 629 kDa and included a large number of proteins involved in lipid metabolism, such as fatty acid transport, fatty acid oxidation, lipid storage, lipolysis and lipid droplet maintenance. Furthermore, we found most glycolysis enzymes and numerous proteins associated with oxidative stress, protein synthesis and degradation as well as some adipokines. 22% of all proteins were of mitochondrial origin. These results provide the first detailed characterization of the human adipocyte proteome, suggest an important role of adipocyte mitochondria, and demonstrate feasibility of this approach to examine alterations of adipocyte protein abundances in human diseases. PMID:20812759

  18. Droplet Burns in the Fiber-Supported Droplet Combustion Experiment

    NASA Technical Reports Server (NTRS)

    2003-01-01

    A fuel droplet burns in the Fiber-Supported Droplet Combustion (FSDC) Experiment on STS-94, July 4 1997, MET:02/19:20 (approximate). This experiment, performed in the Middeck Glovebox, allows us to study the burning of fuels such as n-heptane, n-decane, methanol, ethanol, methanol/water mixtures, and heptane/hexadecane mixtures in droplets as large as 6 mm (nearly 1/4 inch). In this sequence, you see the burn of a 5mm droplet of n-heptane, in a 30% O2/He environment at 1 atmosphere pressure. The droplet (looking bright pink because of reflected light) hangs suspended from the supporting fiber. FSDC-2 studied fundamental phenomena related to liquid fuel droplet combustion in air. Pure fuels and mixtures of fuels were burned as isolated single and dual droplets with and without forced air convection. The FSDC guest investigator was Forman Williams, University of California, San Diego. The experiment was part of the space research investigations conducted during the Microgravity Science Laboratory-1R mission (STS-94, July 1-17 1997). Advanced combustion experiments will be a part of investigations plarned for the International Space Station. (279KB JPEG, 1350 x 2026 pixels; downlinked video, higher quality not available) The MPG from which this composite was made is available at http://mix.msfc.nasa.gov/ABSTRACTS/MSFC-0300175.html.

  19. Leidenfrost levitation: beyond droplets

    PubMed Central

    Hashmi, Ali; Xu, Yuhao; Coder, Benjamin; Osborne, Paul A.; Spafford, Jonathon; Michael, Grant E.; Yu, Gan; Xu, Jie

    2012-01-01

    Friction is a major inhibitor in almost every mechanical system. Enlightened by the Leidenfrost effect – a droplet can be levitated by its own vapor layer on a sufficiently hot surface – we demonstrate for the first time that a small cart can also be levitated by Leidenfrost vapor. The levitated cart can carry certain amount of load and move frictionlessly over the hot surface. The maximum load that the cart can carry is experimentally tested over a range of surface temperatures. We show that the levitated cart can be propelled not only by gravitational force over a slanted flat surface, but also self-propelled over a ratchet shaped horizontal surface. In the end, we experimentally tested water consumption rate for sustaining the levitated cart, and compared the results to theoretical calculations. If perfected, this frictionless Leidenfrost cart could be used in numerous engineering applications where relative motion exists between surfaces. PMID:23150770

  20. Leidenfrost levitation: beyond droplets.

    PubMed

    Hashmi, Ali; Xu, Yuhao; Coder, Benjamin; Osborne, Paul A; Spafford, Jonathon; Michael, Grant E; Yu, Gan; Xu, Jie

    2012-01-01

    Friction is a major inhibitor in almost every mechanical system. Enlightened by the Leidenfrost effect - a droplet can be levitated by its own vapor layer on a sufficiently hot surface - we demonstrate for the first time that a small cart can also be levitated by Leidenfrost vapor. The levitated cart can carry certain amount of load and move frictionlessly over the hot surface. The maximum load that the cart can carry is experimentally tested over a range of surface temperatures. We show that the levitated cart can be propelled not only by gravitational force over a slanted flat surface, but also self-propelled over a ratchet shaped horizontal surface. In the end, we experimentally tested water consumption rate for sustaining the levitated cart, and compared the results to theoretical calculations. If perfected, this frictionless Leidenfrost cart could be used in numerous engineering applications where relative motion exists between surfaces.

  1. Lepidopteran defence droplets - a composite physical and chemical weapon against potential predators

    PubMed Central

    Pentzold, Stefan; Zagrobelny, Mika; Khakimov, Bekzod; Engelsen, Søren Balling; Clausen, Henrik; Petersen, Bent Larsen; Borch, Jonas; Møller, Birger Lindberg; Bak, Søren

    2016-01-01

    Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG11-NH2) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin β-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific β-glucosidase, the only β-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific β-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel β-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and β-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide. PMID:26940001

  2. The storage lipids in Tangier disease. A physical chemical study.

    PubMed

    Katz, S S; Small, D M; Brook, J G; Lees, R S

    1977-06-01

    The physical states and phase behavior of the lipids of the spleen, liver, and splenic artery from a 38-yr-old man with Tangier disease were studied. Many intracellular lipid droplets in the smectic liquid crystalline state were identified by polarizing microscopy in macrophages in both the spleen and liver, but not in the splenic artery. The droplets within individual cells melted sharply over a narrow temperature range, indicating a uniform lipid composition of the droplets of each cell. However different cells melted over a wide range, 20-53 degrees C indicating heterogeneity of lipid droplet composition between cells. Furthermore, most of the cells (81%) had droplets in the liquid crystalline state at 37 degrees C. X-ray diffraction studies of splenic tissue at 37 degrees C revealed a diffraction pattern typical of cholesterol esters in the smectic liquid crystalline state. Differential scanning calorimetry of spleen showed a broad reversible transition from 29-52 degrees C, with a maximum mean transition temperature at 42 degrees C, correlating closely with the polarizing microscopy observations. The enthalpy of the transition, 0.86+/-0.07 cal/g of cholesterol ester, was quantitatively similar to that of the liquid crystalline to liquid transition of pure cholesterol esters indicating that nearly all of the cholesterol esters in the tissue were free to undergo the smectic-isotropic phase transition. Lipid compositions of spleen and liver were determined, and when plotted on the cholesterol-phospholipid-cholesterol ester phase diagram, fell within the two phase zone. The two phases, cholesterol ester droplets and phospholipid bilayers were isolated by ultracentrifugation of tissue homogenates. Lipid compositions of the separated phases approximated those predicted by the phase diagram. Extracted lipids from the spleen, when dispersed in water and ultracentrifuged, underwent phase separation in a similar way. Thus (a) most of the storage lipids in the liver and

  3. The storage lipids in Tangier disease. A physical chemical study.

    PubMed

    Katz, S S; Small, D M; Brook, J G; Lees, R S

    1977-06-01

    The physical states and phase behavior of the lipids of the spleen, liver, and splenic artery from a 38-yr-old man with Tangier disease were studied. Many intracellular lipid droplets in the smectic liquid crystalline state were identified by polarizing microscopy in macrophages in both the spleen and liver, but not in the splenic artery. The droplets within individual cells melted sharply over a narrow temperature range, indicating a uniform lipid composition of the droplets of each cell. However different cells melted over a wide range, 20-53 degrees C indicating heterogeneity of lipid droplet composition between cells. Furthermore, most of the cells (81%) had droplets in the liquid crystalline state at 37 degrees C. X-ray diffraction studies of splenic tissue at 37 degrees C revealed a diffraction pattern typical of cholesterol esters in the smectic liquid crystalline state. Differential scanning calorimetry of spleen showed a broad reversible transition from 29-52 degrees C, with a maximum mean transition temperature at 42 degrees C, correlating closely with the polarizing microscopy observations. The enthalpy of the transition, 0.86+/-0.07 cal/g of cholesterol ester, was quantitatively similar to that of the liquid crystalline to liquid transition of pure cholesterol esters indicating that nearly all of the cholesterol esters in the tissue were free to undergo the smectic-isotropic phase transition. Lipid compositions of spleen and liver were determined, and when plotted on the cholesterol-phospholipid-cholesterol ester phase diagram, fell within the two phase zone. The two phases, cholesterol ester droplets and phospholipid bilayers were isolated by ultracentrifugation of tissue homogenates. Lipid compositions of the separated phases approximated those predicted by the phase diagram. Extracted lipids from the spleen, when dispersed in water and ultracentrifuged, underwent phase separation in a similar way. Thus (a) most of the storage lipids in the liver and

  4. Lossless droplet transfer of droplet-based microfluidic analysis

    DOEpatents

    Kelly, Ryan T; Tang, Keqi; Page, Jason S; Smith, Richard D

    2011-11-22

    A transfer structure for droplet-based microfluidic analysis is characterized by a first conduit containing a first stream having at least one immiscible droplet of aqueous material and a second conduit containing a second stream comprising an aqueous fluid. The interface between the first conduit and the second conduit can define a plurality of apertures, wherein the apertures are sized to prevent exchange of the first and second streams between conduits while allowing lossless transfer of droplets from the first conduit to the second conduit through contact between the first and second streams.

  5. Droplets, Bubbles and Ultrasound Interactions.

    PubMed

    Shpak, Oleksandr; Verweij, Martin; de Jong, Nico; Versluis, Michel

    2016-01-01

    The interaction of droplets and bubbles with ultrasound has been studied extensively in the last 25 years. Microbubbles are broadly used in diagnostic and therapeutic medical applications, for instance, as ultrasound contrast agents. They have a similar size as red blood cells, and thus are able to circulate within blood vessels. Perfluorocarbon liquid droplets can be a potential new generation of microbubble agents as ultrasound can trigger their conversion into gas bubbles. Prior to activation, they are at least five times smaller in diameter than the resulting bubbles. Together with the violent nature of the phase-transition, the droplets can be used for local drug delivery, embolotherapy, HIFU enhancement and tumor imaging. Here we explain the basics of bubble dynamics, described by the Rayleigh-Plesset equation, bubble resonance frequency, damping and quality factor. We show the elegant calculation of the above characteristics for the case of small amplitude oscillations by linearizing the equations. The effect and importance of a bubble coating and effective surface tension are also discussed. We give the main characteristics of the power spectrum of bubble oscillations. Preceding bubble dynamics, ultrasound propagation is introduced. We explain the speed of sound, nonlinearity and attenuation terms. We examine bubble ultrasound scattering and how it depends on the wave-shape of the incident wave. Finally, we introduce droplet interaction with ultrasound. We elucidate the ultrasound-focusing concept within a droplets sphere, droplet shaking due to media compressibility and droplet phase-conversion dynamics.

  6. Droplet resonator based optofluidic microlasers

    NASA Astrophysics Data System (ADS)

    Kiraz, Alper; Jonáš, Alexandr; Aas, Mehdi; Karadag, Yasin; Brzobohatý, Oto; Ježek, Jan; Pilát, Zdeněk.; Zemánek, Pavel; Anand, Suman; McGloin, David

    2014-03-01

    We introduce tunable optofluidic microlasers based on active optical resonant cavities formed by optically stretched, dye-doped emulsion droplets confined in a dual-beam optical trap. To achieve tunable dye lasing, optically pumped droplets of oil dispersed in water are stretched by light in the dual-beam trap. Subsequently, resonant path lengths of whispering gallery modes (WGMs) propagating in the droplet are modified, leading to shifts in the microlaser emission wavelengths. We also report lasing in airborne, Rhodamine B-doped glycerolwater droplets which were localized using optical tweezers. While being trapped near the focal point of an infrared laser, the droplets were pumped with a Q-switched green laser. Furthermore, biological lasing in droplets supported by a superhydrophobic surface is demonstrated using a solution of Venus variant of the yellow fluorescent protein or E. Coli bacterial cells expressing stably the Venus protein. Our results may lead to new ways of probing airborne particles, exploiting the high sensitivity of stimulated emission to small perturbations in the droplet laser cavity and the gain medium.

  7. Levitation of liquid sodium droplets

    SciTech Connect

    Roy, S.S.; Cramb, A.W.; Hoburg, J.F.; Lally, B.

    1995-12-01

    Droplets of liquid sodium ranging from 1.2 to 2.1 g, immersed in mineral oil, were levitated in an electromagnetic field. The experimental setup was designed and constructed to levitate small metal droplets at audio frequencies. The levitated droplet was found to be very stable inside the inductor, and the equilibrium shape attained by the droplet in the electromagnetic field was measured during the experiment. A surface coupled mathematical model was used to calculate the self-consistent equilibrium droplet shape of liquid sodium under the influence of an electromagnetic field. The predicted shapes of the metal droplet and the position of the droplet inside the inductor compare well with the experimental data. The idea of casting metals and alloys without any physical contact has generated a lot of interest in the metals industry, especially in the production of metals/alloys that are highly reactive and have a very high melting point. Containerless casting can be achieved by levitating or pushing the liquid metal from the surface of the container.

  8. Bacterial encountering with oil droplet

    NASA Astrophysics Data System (ADS)

    Sheng, Jian; Molaei, Mehdi

    2014-11-01

    Encountering of microorganisms with rising oil droplets in aqueous environments is the first and one of the critical steps in the biodegradation of crude oil. Several factors such as droplet sizes, rising velocity, surfactant, and motility of bacteria are expected to affect the encounter rate. We establish well controlled microfluidic devices by applying layer-by-layer technique that allows us to produce horizontal micro droplets with different sizes. The encounter rates of passive particles, motile and non-motile bacteria with these droplets are measured by high speed microscopy. The effects of mobility and motility of these particles on encounter rates are assessed quantitatively. Meanwhile, we visualize reorientation of the particle due to flow filed around the oil droplet. Results show that the motile bacteria have higher probabilities to interact with an oil droplet compare to the passive particles. Ongoing analyses focus on the effect of shear rates, angular dispersion, curvatures of streamlines, and the swimming velocity of bacteria. The ratios of the encounter area to the entire droplet surface at various flow regimes will also been measured. GoMRI.

  9. Involvement of the Saccharomyces cerevisiae hydrolase Ldh1p in lipid homeostasis.

    PubMed

    Debelyy, Mykhaylo O; Thoms, Sven; Connerth, Melanie; Daum, Günther; Erdmann, Ralf

    2011-06-01

    Here, we report the functional characterization of the newly identified lipid droplet hydrolase Ldh1p. Recombinant Ldh1p exhibits esterase and triacylglycerol lipase activities. Mutation of the serine in the hydrolase/lipase motif GXSXG completely abolished esterase activity. Ldh1p is required for the maintenance of a steady-state level of the nonpolar and polar lipids of lipid droplets. A characteristic feature of the Saccharomyces cerevisiae Δldh1 strain is the appearance of giant lipid droplets and an excessive accumulation of nonpolar lipids and phospholipids upon growth on medium containing oleic acid as a sole carbon source. Ldh1p is thought to play a role in maintaining the lipid homeostasis in yeast by regulating both phospholipid and nonpolar lipid levels. PMID:21478434

  10. Blood droplet dynamics--I.

    PubMed

    Pizzola, P A; Roth, S; De Forest, P R

    1986-01-01

    The interpretation of bloodstain patterns at crime scenes has received increased attention in recent years. Important to an understanding of this is knowledge of the fundamentals of blood droplet formation and impact dynamics. A review of the literature reveals that a considerable amount of work has been done with aqueous drop dynamics. Workers in the forensic science area seem to have been unaware of this. In addition, some of the most important and comprehensive early work with blood droplet dynamics seems to have been forgotten. It is not cited in more recent publications dealing with bloodstain pattern interpretation. This literature is reviewed and discussed as well. The present study presents results of experiments with blood droplet dynamics and high-speed photographs of blood droplet impacts on stationary target surfaces. Some longstanding misconceptions of importance to forensic scientists engaged in crime scene reconstruction are discussed. PMID:3944577

  11. Droplet combustion at reduced gravity

    NASA Technical Reports Server (NTRS)

    Dryer, F. L.; Williams, F. A.

    1988-01-01

    The current work involves theoretical analyses of the effects identified, experiments in the NASA Lewis drop towers performed in the middeck areas of the Space Shuttle. In addition, there is laboratory work associated with the design of the flight apparatus. Calculations have shown that some of the test-matrix data can be obtained in drop towers, and some are achievable only in the space experiments. The apparatus consists of a droplet dispensing device (syringes), a droplet positioning device (opposing, retractable, hollow needles), a droplet ignition device (two matched pairs of retractable spark electrodes), gas and liquid handling systems, a data acquisition system (mainly giving motion-picture records of the combustion in two orthogonal views, one with backlighting for droplet resolution), and associated electronics.

  12. Hdac3 Deficiency Increases Marrow Adiposity and Induces Lipid Storage and Glucocorticoid Metabolism in Osteochondroprogenitor Cells

    PubMed Central

    McGee-Lawrence, Meghan E; Carpio, Lomeli R; Schulze, Ryan J; Pierce, Jessica L; McNiven, Mark A; Farr, Joshua N; Khosla, Sundeep; Oursler, Merry Jo; Westendorf, Jennifer J

    2016-01-01

    Bone loss and increased marrow adiposity are hallmarks of aging skeletons. Conditional deletion of histone deacetylase 3 (Hdac3) in murine osteochondroprogenitor cells causes osteopenia and increases marrow adiposity, even in young animals, but the origins of the increased adiposity are unclear. To explore this, bone marrow stromal cells (BMSCs) from Hdac3-depleted and control mice were cultured in osteogenic medium. Hdac3-deficient cultures accumulated lipid droplets in greater abundance than control cultures and expressed high levels of genes related to lipid storage (Fsp27/Cidec, Plin1) and glucocorticoid metabolism (Hsd11b1) despite normal levels of Pparγ2. Approximately 5% of the lipid containing cells in the wild-type cultures expressed the master osteoblast transcription factor Runx2, but this population was threefold greater in the Hdac3-depleted cultures. Adenoviral expression of Hdac3 restored normal gene expression, indicating that Hdac3 controls glucocorticoid activation and lipid storage within osteoblast lineage cells. HDAC3 expression was reduced in bone cells from postmenopausal as compared to young women, and in osteoblasts from aged as compared to younger mice. Moreover, phosphorylation of S424 in Hdac3, a posttranslational mark necessary for deacetylase activity, was suppressed in osseous cells from old mice. Thus, concurrent declines in transcription and phosphorylation combine to suppress Hdac3 activity in aging bone, and reduced Hdac3 activity in osteochondroprogenitor cells contributes to increased marrow adiposity associated with aging. PMID:26211746

  13. Droplets engulfing on a filament

    NASA Astrophysics Data System (ADS)

    Wu, Xiang-Fa; Yu, Meng; Zhou, Zhengping; Bedarkar, Amol; Zhao, Youhao

    2014-03-01

    Two immiscible droplets wetting on a filament may assume engulfing, partial-engulfing, or non-engulfing morphology that depends on the wetting behavior and geometries of the resulting droplet-on-filament system. This paper studies the wetting behavior of two immiscible droplets contacting and sitting symmetrically on a straight filament. A set of ordinary differential equations (ODEs) is formulated for determining the wetting morphology of the droplet-on-filament system. In the limiting case of engulfing or non-engulfing, the morphology of the droplet-on-filament system is determined in explicit form. In the case of partial-engulfing, surface finite element method is further employed for determining the wetting morphology, surface energy, and internal pressures of droplets of the system. Numerical scaling study is performed to explore their dependencies upon the wetting properties and geometries of the system. The study can be applicable for analysis and design of textiles with tailorable wetting properties and development of novel multifunctional fibrous materials for environmental protection such as oil-spill sorption, etc.

  14. Analysis of lipid profile in lipid storage myopathy.

    PubMed

    Aguennouz, M'hammed; Beccaria, Marco; Purcaro, Giorgia; Oteri, Marianna; Micalizzi, Giuseppe; Musumesci, Olimpia; Ciranni, Annmaria; Di Giorgio, Rosa Maria; Toscano, Antonio; Dugo, Paola; Mondello, Luigi

    2016-09-01

    Lipid dysmetabolism disease is a condition in which lipids are stored abnormally in organs and tissues throughout the body, causing muscle weakness (myopathy). Usually, the diagnosis of this disease and its characterization goes through dosage of Acyl CoA in plasma accompanied with evidence of droplets of intra-fibrils lipids in the patient muscle biopsy. However, to understand the pathophysiological mechanisms of lipid storage diseases, it is useful to identify the nature of lipids deposited in muscle fiber. In this work fatty acids and triglycerides profile of lipid accumulated in the muscle of people suffering from myopathies syndromes was characterized. In particular, the analyses were carried out on the muscle biopsy of people afflicted by lipid storage myopathy, such as multiple acyl-coenzyme A dehydrogenase deficiency, and neutral lipid storage disease with myopathy, and by the intramitochondrial lipid storage dysfunctions, such as deficiencies of carnitine palmitoyltransferase II enzyme. A single step extraction and derivatization procedure was applied to analyze fatty acids from muscle tissues by gas chromatography with a flame ionization detector and with an electronic impact mass spectrometer. Triglycerides, extracted by using n-hexane, were analyzed by high performance liquid chromatography coupled to mass spectrometer equipped with an atmospheric pressure chemical ionization interface. The most representative fatty acids in all samples were: C16:0 in the 13-24% range, C18:1n9 in the 20-52% range, and C18:2n6 in the 10-25% range. These fatty acids were part of the most representative triglycerides in all samples. The data obtained was statistically elaborated performing a principal component analysis. A satisfactory discrimination was obtained among the different diseases. Using component 1 vs component 3 a 43.3% of total variance was explained. Such results suggest the important role that lipid profile characterization can have in supporting a correct

  15. Reversible, voltage-activated formation of biomimetic membranes between triblock copolymer-coated aqueous droplets in good solvents.

    PubMed

    Tamaddoni, Nima; Taylor, Graham; Hepburn, Trevor; Michael Kilbey, S; Sarles, Stephen A

    2016-06-21

    Biomimetic membranes assembled from block copolymers attract considerable interest because they exhibit greater stability and longetivity compared to lipid bilayers, and some enable the reconstitution of functional transmembrane biomolecules. Yet to-date, block copolymer membranes have not been achieved using the droplet interface bilayer (DIB) method, which uniquely allows assembling single- and multi-membrane networks between water droplets in oil. Herein, we investigate the formation of poly(ethylene oxide)-b-poly(dimethyl siloxane)-b-poly(ethylene oxide) triblock copolymer-stabilized interfaces (CSIs) between polymer-coated aqueous droplets in solutions comprising combinations of decane, hexadecane and AR20 silicone oil. We demonstrate that triblock-coated droplets do not spontaneously adhere in these oils because all are thermodynamically good solvents for the hydrophobic PDMS middle block. However, thinned planar membranes are reversibly formed at the interface between droplets upon the application of a sufficient transmembrane voltage, which removes excess solvent from between droplets through electrocompression. At applied voltages above the threshold required to initiate membrane thinning, electrowetting causes the area of the CSI between droplets to increase while thickness remains constant; the CSI electrowetting response is similar to that encountered with lipid-based DIBs. In combination, these results reveal that stable membranes can be assembled in a manner that is completely reversible when an external pressure is used to overcome a barrier to adhesion caused by solvent-chain interactions, and they demonstrate new capability for connecting and disconnecting aqueous droplets via polymer-stabilized membranes.

  16. Genetics of Lipid-Storage Management in Caenorhabditis elegans Embryos.

    PubMed

    Schmökel, Verena; Memar, Nadin; Wiekenberg, Anne; Trotzmüller, Martin; Schnabel, Ralf; Döring, Frank

    2016-03-01

    Lipids play a pivotal role in embryogenesis as structural components of cellular membranes, as a source of energy, and as signaling molecules. On the basis of a collection of temperature-sensitive embryonic lethal mutants, a systematic database search, and a subsequent microscopic analysis of >300 interference RNA (RNAi)-treated/mutant worms, we identified a couple of evolutionary conserved genes associated with lipid storage in Caenorhabditis elegans embryos. The genes include cpl-1 (cathepsin L-like cysteine protease), ccz-1 (guanine nucleotide exchange factor subunit), and asm-3 (acid sphingomyelinase), which is closely related to the human Niemann-Pick disease-causing gene SMPD1. The respective mutant embryos accumulate enlarged droplets of neutral lipids (cpl-1) and yolk-containing lipid droplets (ccz-1) or have larger genuine lipid droplets (asm-3). The asm-3 mutant embryos additionally showed an enhanced resistance against C band ultraviolet (UV-C) light. Herein we propose that cpl-1, ccz-1, and asm-3 are genes required for the processing of lipid-containing droplets in C. elegans embryos. Owing to the high levels of conservation, the identified genes are also useful in studies of embryonic lipid storage in other organisms. PMID:26773047

  17. Uniform-droplet spray forming

    SciTech Connect

    Blue, C.A.; Sikka, V.K.; Chun, Jung-Hoon; Ando, T.

    1997-04-01

    The uniform-droplet process is a new method of liquid-metal atomization that results in single droplets that can be used to produce mono-size powders or sprayed-on to substrates to produce near-net shapes with tailored microstructure. The mono-sized powder-production capability of the uniform-droplet process also has the potential of permitting engineered powder blends to produce components of controlled porosity. Metal and alloy powders are commercially produced by at least three different methods: gas atomization, water atomization, and rotating disk. All three methods produce powders of a broad range in size with a very small yield of fine powders with single-sized droplets that can be used to produce mono-size powders or sprayed-on substrates to produce near-net shapes with tailored microstructures. The economical analysis has shown the process to have the potential of reducing capital cost by 50% and operating cost by 37.5% when applied to powder making. For the spray-forming process, a 25% savings is expected in both the capital and operating costs. The project is jointly carried out at Massachusetts Institute of Technology (MIT), Tuffs University, and Oak Ridge National Laboratory (ORNL). Preliminary interactions with both finished parts and powder producers have shown a strong interest in the uniform-droplet process. Systematic studies are being conducted to optimize the process parameters, understand the solidification of droplets and spray deposits, and develop a uniform-droplet-system (UDS) apparatus appropriate for processing engineering alloys.

  18. Development of an Acoustic Droplet Vaporization, Ultrasound Drug Delivery Emulsion

    NASA Astrophysics Data System (ADS)

    Fabiilli, Mario L.; Sebastian, Ian E.; Fowlkes, J. Brian

    2010-03-01

    Many therapeutic applications of ultrasound (US) include the use of pefluorocarbon (PFC) microbubbles or emulsions. These colloidal systems can be activated in the presence of US, which in the case of emulsions, results in the production of bubbles—a process known as acoustic droplet vaporization (ADV). ADV can be used as a drug delivery mechanism, thereby yielding the localized release of toxic agents such a chemotherapeutics. In this work, emulsions that contain PFC and chlorambucil, a chemotherapy drug, are formulated using albumin or lipid shells. For albumin droplets, the oil phase—which contained CHL—clearly enveloped the PFC phase. The albumin emulsion also displayed better retention of CHL in the absence of US, which was evaluated by incubating Chinese hamster ovary cells with the various formulations. Thus, the developed emulsions are suitable for further testing in ADV-induced release of CHL.

  19. Control of Nanomaterial Self-Assembly in Ultrasonically Levitated Droplets.

    PubMed

    Seddon, Annela M; Richardson, Sam J; Rastogi, Kunal; Plivelic, Tomás S; Squires, Adam M; Pfrang, Christian

    2016-04-01

    We demonstrate that acoustic trapping can be used to levitate and manipulate droplets of soft matter, in particular, lyotropic mesophases formed from self-assembly of different surfactants and lipids, which can be analyzed in a contact-less manner by X-ray scattering in a controlled gas-phase environment. On the macroscopic length scale, the dimensions and the orientation of the particle are shaped by the ultrasonic field, while on the microscopic length scale the nanostructure can be controlled by varying the humidity of the atmosphere around the droplet. We demonstrate levitation and in situ phase transitions of micellar, hexagonal, bicontinuous cubic, and lamellar phases. The technique opens up a wide range of new experimental approaches of fundamental importance for environmental, biological, and chemical research.

  20. Control of Nanomaterial Self-Assembly in Ultrasonically Levitated Droplets.

    PubMed

    Seddon, Annela M; Richardson, Sam J; Rastogi, Kunal; Plivelic, Tomás S; Squires, Adam M; Pfrang, Christian

    2016-04-01

    We demonstrate that acoustic trapping can be used to levitate and manipulate droplets of soft matter, in particular, lyotropic mesophases formed from self-assembly of different surfactants and lipids, which can be analyzed in a contact-less manner by X-ray scattering in a controlled gas-phase environment. On the macroscopic length scale, the dimensions and the orientation of the particle are shaped by the ultrasonic field, while on the microscopic length scale the nanostructure can be controlled by varying the humidity of the atmosphere around the droplet. We demonstrate levitation and in situ phase transitions of micellar, hexagonal, bicontinuous cubic, and lamellar phases. The technique opens up a wide range of new experimental approaches of fundamental importance for environmental, biological, and chemical research. PMID:26979408

  1. Incorporation and characterization of biological molecules in droplet-interface bilayer networks for novel active systems

    NASA Astrophysics Data System (ADS)

    Sarles, Stephen A.; Ghanbari Bavarsad, Pegah; Leo, Donald J.

    2009-03-01

    Biological molecules including phospholipids and proteins offer scientists and engineers a diverse selection of materials to develop new types of active materials and smart systems based on ion conduction. The inherent energy-coupling abilities of these components create novel kinds of transduction elements. Networks formed from droplet-interface bilayers (DIB) are a promising construct for creating cell mimics that allow for the assembly and study of these active biological molecules. The current-voltage relationship of symmetric, "lipid-in" dropletinterface bilayers are characterized using electrical impedance spectroscopy (EIS) and cyclic voltammetry (CV). "Lipid-in" diphytanoyl phosphatidylcholine (DPhPC) droplet-interface bilayers have specific resistances of nearly 10MΩ•cm2 and rupture at applied potentials greater than 300mV, indicating the "lipid-in" approach produces higher quality interfacial membranes than created using the original "lipid-out" method. The incorporation of phospholipids into the droplet interior allows for faster monolayer formation but does not inhibit the selfinsertion of transmembrane proteins into bilayer interfaces that separate adjacent droplets. Alamethicin proteins inserted into single and multi-DIB networks produce a voltage-dependent membrane conductance and current measurements on bilayers containing this type of protein exhibit a reversible, 3-4 order-of-magnitude conductance increase upon application of voltage.

  2. Chronic ethanol consumption in mice alters hepatocyte lipid droplet properties

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Hepatosteatosis is a common pathological feature of impaired hepatic metabolism following chronic alcohol consumption. Although often benign and reversible, it is widely believed that steatosis is a risk factor for development of advanced liver pathologies, including steatohepatitis and ...

  3. Droplets merging through wireless ultrasonic actuation.

    PubMed

    Nayak, Praveen Priyaranjan; Kar, Durga Prasanna; Bhuyan, Satyanarayan

    2016-01-01

    A new technique of droplets merging through wireless ultrasonic actuation has been proposed and experimentally investigated in this work. The proposed method is based on the principle of resonant inductive coupling and piezoelectric resonance. When a mechanical vibration is excited in a piezoelectric plate, the ultrasonic vibration transmitted to the droplets placed on its surface and induces merging. It has been observed that the merging rate of water droplets depends on the operating frequency, mechanical vibration of piezoelectric plate, separation distance between the droplets, and volume of droplets. The investigated technique of droplets merging through piezoelectric actuation is quite useful for microfluidics, chemical and biomedical engineering applications.

  4. Obesogens beyond Vertebrates: Lipid Perturbation by Tributyltin in the Crustacean Daphnia magna

    PubMed Central

    Jordão, Rita; Casas, Josefina; Fabrias, Gemma; Campos, Bruno; Piña, Benjamín; Lemos, Marco F.L.; Soares, Amadeu M.V.M.; Tauler, Romà

    2015-01-01

    Background The analysis of obesogenic effects in invertebrates is limited by our poor knowledge of the regulatory pathways of lipid metabolism. Recent data from the crustacean Daphnia magna points to three signaling hormonal pathways related to the molting and reproductive cycles [retinoic X receptor (RXR), juvenile hormone (JH), and ecdysone] as putative targets for exogenous obesogens. Objective The present study addresses the disruptive effects of the model obesogen tributyltin (TBT) on the lipid homeostasis in Daphnia during the molting and reproductive cycle, its genetic control, and health consequences of its disruption. Methods D. magna individuals were exposed to low and high levels of TBT. Reproductive effects were assessed by Life History analysis methods. Quantitative and qualitative changes in lipid droplets during molting and the reproductive cycle were studied using Nile red staining. Lipid composition and dynamics were analyzed by ultra-performance liquid chromatography coupled to a time-of-flight mass spectrometer. Relative abundances of mRNA from different genes related to RXR, ecdysone, and JH signaling pathways were studied by qRT-PCR. Results and Conclusions TBT disrupted the dynamics of neutral lipids, impairing the transfer of triacylglycerols to eggs and hence promoting their accumulation in adult individuals. TBT’s disruptive effects translated into a lower fitness for offspring and adults. Co-regulation of gene transcripts suggests that TBT activates the ecdysone, JH, and RXR receptor signaling pathways, presumably through the already proposed interaction with RXR. These findings indicate the presence of obesogenic effects in a nonvertebrate species. Citation Jordão R, Casas J, Fabrias G, Campos B, Piña B, Lemos MF, Soares AM, Tauler R, Barata C. 2015. Obesogens beyond vertebrates: lipid perturbation by tributyltin in the crustacean Daphnia magna. Environ Health Perspect 123:813–819; http://dx.doi.org/10.1289/ehp.1409163 PMID

  5. Vibration-Induced Droplet Atomization

    NASA Technical Reports Server (NTRS)

    Smith, M. K.; James, A.; Vukasinovic, B.; Glezer, A.

    1999-01-01

    Thermal management is critical to a number of technologies used in a microgravity environment and in Earth-based systems. Examples include electronic cooling, power generation systems, metal forming and extrusion, and HVAC (heating, venting, and air conditioning) systems. One technique that can deliver the large heat fluxes required for many of these technologies is two-phase heat transfer. This type of heat transfer is seen in the boiling or evaporation of a liquid and in the condensation of a vapor. Such processes provide very large heat fluxes with small temperature differences. Our research program is directed toward the development of a new, two-phase heat transfer cell for use in a microgravity environment. In this paper, we consider the main technology used in this cell, a novel technique for the atomization of a liquid called vibration-induced droplet atomization. In this process, a small liquid droplet is placed on a thin metal diaphragm that is made to vibrate by an attached piezoelectric transducer. The vibration induces capillary waves on the free surface of the droplet that grow in amplitude and then begin to eject small secondary droplets from the wave crests. In some situations, this ejection process develops so rapidly that the entire droplet seems to burst into a small cloud of atomized droplets that move away from the diaphragm at speeds of up to 50 cm/s. By incorporating this process into a heat transfer cell, the active atomization and transport of the small liquid droplets could provide a large heat flux capability for the device. Experimental results are presented that document the behavior of the diaphragm and the droplet during the course of a typical bursting event. In addition, a simple mathematical model is presented that qualitatively reproduces all of the essential features we have seen in a burst event. From these two investigations, we have shown that delayed droplet bursting results when the system passes through a resonance

  6. Droplet burning at zero G

    NASA Technical Reports Server (NTRS)

    Williams, F. A.

    1978-01-01

    Questions of the importance and feasibility of performing experiments on droplet burning at zero gravity in Spacelab were studied. Information on the physics and chemistry of droplet combustion, with attention directed specifically to the chemical kinetics, heat and mass transfer, and fluid mechanics of the phenomena involved, are presented. The work was divided into three phases, the justification, the feasibility, and the conceptual development of a preliminary design. Results from the experiments performed revealed a few new facts concerning droplet burning, notably burning rates in excess of theoretical prediction and a phenomenon of flash extinction, both likely traceable to accumulation of carbon produced by gas-phase pyrolysis in the fuel-rich zone enclosed by the reaction surface. These experiments also showed that they were primarily due to timing difficulties.

  7. The Droplet Combustion Experiment (DCE)

    NASA Technical Reports Server (NTRS)

    2003-01-01

    The Droplet Combustion Experiment (DCE) was designed to investigate the fundamental combustion aspects of single, isolated droplets under different pressures and ambient oxygen concentrations for a range of droplet sizes varying between 2 and 5 mm. The DCE principal investigator was Forman Williams, University of California, San Diego. The experiment was part of the space research investigations conducted during the Microgravity Science Laboratory-1R mission (STS-94, July 1-17 1997). Advanced combustion experiments will be a part of investigations plarned for the International Space Station. (167KB, 5-second MPEG, screen 160 x 120 pixels; downlinked video, higher quality not available)A still JPG composite of this movie is available at http://mix.msfc.nasa.gov/ABSTRACTS/MSFC-0300166.html.

  8. The emerging role of autophagy in peroxisome dynamics and lipid metabolism of phyllosphere microorganisms.

    PubMed

    Oku, Masahide; Takano, Yoshitaka; Sakai, Yasuyoshi

    2014-01-01

    Eukaryotic microorganisms resident in the phyllosphere (above-ground, plant-surface environments) undergo dynamic changes in nutrient conditions and adapt their metabolic pathways during proliferation or in the course of infection of host plants. Some of these metabolic switches are accomplished by regulation of organelle abundance. Recent studies have shown that autophagy plays a major role in reducing the organelle quantity, thereby contributing to the metabolic switch required for survival or virulence of the microorganisms in the phyllosphere. In this mini review the metabolic pathways in several phytopathogenic fungi and the non-infectious asporogenous yeast Candida boidinii, which involve lipid droplets and peroxisomes, are summarized. The physiological functions of Atg (Autophagy-related) proteins in these organisms are discussed in relation to the dynamics of these two important organelles. PMID:24653730

  9. Film boiling of mercury droplets

    NASA Technical Reports Server (NTRS)

    Baumeister, K. J.; Schoessow, G. J.; Chmielewski, C. E.

    1975-01-01

    Vaporization times of mercury droplets in Leidenfrost film boiling on a flat horizontal plate are measured in an air atmosphere. Extreme care was used to prevent large amplitude droplet vibrations and surface wetting; therefore, these data can be compared to film boiling theory. Diffusion from the upper surface of the drop appears as a dominant mode of mass transfer from the drop. A closed-form analytical film boiling theory is developed to account for the diffusive evaporation. Reasonable agreement between data and theory is seen.

  10. Film boiling of mercury droplets

    NASA Technical Reports Server (NTRS)

    Baumeister, K. J.; Schoessow, G. J.; Chmielewski, C. E.

    1975-01-01

    Vaporization times of mercury droplets in Leidenfrost film boiling on a flat horizontal plate are measured in an air atmosphere. Extreme care was used to prevent large amplitude droplet vibrations and surface wetting; therefore, these data can be compared to film boiling theory. For these data, diffusion from the upper surface of the drop is a dominant mode of mass transfer from the drop. A closed-form analytical film boiling theory is developed to account for the diffusive evaporation. Reasonable agreement between data and theory is seen.

  11. Lipid metabolism and signaling in cardiac lipotoxicity.

    PubMed

    D'Souza, Kenneth; Nzirorera, Carine; Kienesberger, Petra C

    2016-10-01

    The heart balances uptake, metabolism and oxidation of fatty acids (FAs) to maintain ATP production, membrane biosynthesis and lipid signaling. Under conditions where FA uptake outpaces FA oxidation and FA sequestration as triacylglycerols in lipid droplets, toxic FA metabolites such as ceramides, diacylglycerols, long-chain acyl-CoAs, and acylcarnitines can accumulate in cardiomyocytes and cause cardiomyopathy. Moreover, studies using mutant mice have shown that dysregulation of enzymes involved in triacylglycerol, phospholipid, and sphingolipid metabolism in the heart can lead to the excess deposition of toxic lipid species that adversely affect cardiomyocyte function. This review summarizes our current understanding of lipid uptake, metabolism and signaling pathways that have been implicated in the development of lipotoxic cardiomyopathy under conditions including obesity, diabetes, aging, and myocardial ischemia-reperfusion. This article is part of a Special Issue entitled: Heart Lipid Metabolism edited by G.D. Lopaschuk.

  12. From Leaf Synthesis to Senescence: n-Alkyl Lipid Abundance and D/H Composition Among Plant Species in a Temperate Deciduous Forest at Brown's Lake Bog, Ohio, USA

    NASA Astrophysics Data System (ADS)

    Freimuth, E. J.; Diefendorf, A. F.; Lowell, T. V.

    2014-12-01

    The hydrogen isotope composition (D/H, δD) of terrestrial plant leaf waxes is a promising paleohydrology proxy because meteoric water (e.g., precipitation) is the primary hydrogen source for wax synthesis. However, secondary environmental and biological factors modify the net apparent fractionation between precipitation δD and leaf wax δD, limiting quantitative reconstruction of paleohydrology. These secondary factors include soil evaporation, leaf transpiration, biosynthetic fractionation, and the seasonal timing of lipid synthesis. Here, we investigate the influence of each of these factors on n-alkyl lipid δD in five dominant deciduous angiosperm tree species as well as shrubs, ferns and grasses in the watershed surrounding Brown's Lake Bog, Ohio, USA. We quantified n-alkane and n-alkanoic acid concentrations and δD in replicate individuals of each species at weekly to monthly intervals from March to October 2014 to assess inter- and intraspecific isotope variability throughout the growing season. We present soil, xylem and leaf water δD from each individual, and precipitation and atmospheric water vapor δD throughout the season to directly examine the relationship between source water and lipid isotope composition. These data allow us to assess the relative influence of soil evaporation and leaf transpiration among plant types, within species, and along a soil moisture gradient throughout the catchment. We use leaf water δD to approximate biosynthetic fractionation for each individual and test whether this is a species-specific and seasonal constant, and to evaluate variation among plant types with identical growth conditions. Our high frequency sampling approach provides new insights into the seasonal timing of n-alkane and n-alkanoic acid synthesis and subsequent fluctuations in concentration and δD in a temperate deciduous forest. These results will advance understanding of the magnitude and timing of secondary influences on the modern leaf wax

  13. Persisting water droplets on water surfaces.

    PubMed

    Klyuzhin, Ivan S; Ienna, Federico; Roeder, Brandon; Wexler, Adam; Pollack, Gerald H

    2010-11-11

    Droplets of various liquids may float on the respective surfaces for extended periods of time prior to coalescence. We explored the features of delayed coalescence in highly purified water. Droplets several millimeters in diameter were released from a nozzle onto a water surface. Results showed that droplets had float times up to hundreds of milliseconds. When the droplets did coalesce, they did so in stepwise fashion, with periods of quiescence interspersed between periods of coalescence. Up to six steps were noted before the droplet finally vanished. Droplets were released in a series, which allowed the detection of unexpected abrupt float-time changes throughout the duration of the series. Factors such as electrostatic charge, droplet size, and sideways motion had considerable effect on droplet lifetime, as did reduction of pressure, which also diminished the number of steps needed for coalescence. On the basis of present observations and recent reports, a possible mechanism for noncoalescence is considered. PMID:20961076

  14. Fungal Morphology, Iron Homeostasis, and Lipid Metabolism Regulated by a GATA Transcription Factor in Blastomyces dermatitidis

    PubMed Central

    Marty, Amber J.; Broman, Aimee T.; Zarnowski, Robert; Dwyer, Teigan G.; Bond, Laura M.; Lounes-Hadj Sahraoui, Anissa; Fontaine, Joël; Ntambi, James M.; Keleş, Sündüz; Kendziorski, Christina; Gauthier, Gregory M.

    2015-01-01

    In response to temperature, Blastomyces dermatitidis converts between yeast and mold forms. Knowledge of the mechanism(s) underlying this response to temperature remains limited. In B. dermatitidis, we identified a GATA transcription factor, SREB, important for the transition to mold. Null mutants (SREBΔ) fail to fully complete the conversion to mold and cannot properly regulate siderophore biosynthesis. To capture the transcriptional response regulated by SREB early in the phase transition (0–48 hours), gene expression microarrays were used to compare SREB∆ to an isogenic wild type isolate. Analysis of the time course microarray data demonstrated SREB functioned as a transcriptional regulator at 37°C and 22°C. Bioinformatic and biochemical analyses indicated SREB was involved in diverse biological processes including iron homeostasis, biosynthesis of triacylglycerol and ergosterol, and lipid droplet formation. Integration of microarray data, bioinformatics, and chromatin immunoprecipitation identified a subset of genes directly bound and regulated by SREB in vivo in yeast (37°C) and during the phase transition to mold (22°C). This included genes involved with siderophore biosynthesis and uptake, iron homeostasis, and genes unrelated to iron assimilation. Functional analysis suggested that lipid droplets were actively metabolized during the phase transition and lipid metabolism may contribute to filamentous growth at 22°C. Chromatin immunoprecipitation, RNA interference, and overexpression analyses suggested that SREB was in a negative regulatory circuit with the bZIP transcription factor encoded by HAPX. Both SREB and HAPX affected morphogenesis at 22°C; however, large changes in transcript abundance by gene deletion for SREB or strong overexpression for HAPX were required to alter the phase transition. PMID:26114571

  15. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations.

    PubMed

    You, David J; Yoon, Jeong-Yeol

    2012-01-01

    A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using "wire-guided" method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of

  16. Droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling for simpler and faster PCR assay using wire-guided manipulations

    PubMed Central

    2012-01-01

    A computer numerical control (CNC) apparatus was used to perform droplet centrifugation, droplet DNA extraction, and rapid droplet thermocycling on a single superhydrophobic surface and a multi-chambered PCB heater. Droplets were manipulated using “wire-guided” method (a pipette tip was used in this study). This methodology can be easily adapted to existing commercial robotic pipetting system, while demonstrated added capabilities such as vibrational mixing, high-speed centrifuging of droplets, simple DNA extraction utilizing the hydrophobicity difference between the tip and the superhydrophobic surface, and rapid thermocycling with a moving droplet, all with wire-guided droplet manipulations on a superhydrophobic surface and a multi-chambered PCB heater (i.e., not on a 96-well plate). Serial dilutions were demonstrated for diluting sample matrix. Centrifuging was demonstrated by rotating a 10 μL droplet at 2300 round per minute, concentrating E. coli by more than 3-fold within 3 min. DNA extraction was demonstrated from E. coli sample utilizing the disposable pipette tip to cleverly attract the extracted DNA from the droplet residing on a superhydrophobic surface, which took less than 10 min. Following extraction, the 1500 bp sequence of Peptidase D from E. coli was amplified using rapid droplet thermocycling, which took 10 min for 30 cycles. The total assay time was 23 min, including droplet centrifugation, droplet DNA extraction and rapid droplet thermocycling. Evaporation from of 10 μL droplets was not significant during these procedures, since the longest time exposure to air and the vibrations was less than 5 min (during DNA extraction). The results of these sequentially executed processes were analyzed using gel electrophoresis. Thus, this work demonstrates the adaptability of the system to replace many common laboratory tasks on a single platform (through re-programmability), in rapid succession (using droplets), and with a high level of

  17. Water droplet impact on elastic superhydrophobic surfaces.

    PubMed

    Weisensee, Patricia B; Tian, Junjiao; Miljkovic, Nenad; King, William P

    2016-01-01

    Water droplet impact on surfaces is a ubiquitous phenomenon in nature and industry, where the time of contact between droplet and surface influences the transfer of mass, momentum and energy. To manipulate and reduce the contact time of impacting droplets, previous publications report tailoring of surface microstructures that influence the droplet - surface interface. Here we show that surface elasticity also affects droplet impact, where a droplet impacting an elastic superhydrophobic surface can lead to a two-fold reduction in contact time compared to equivalent rigid surfaces. Using high speed imaging, we investigated the impact dynamics on elastic nanostructured superhydrophobic substrates having membrane and cantilever designs with stiffness 0.5-7630 N/m. Upon impact, the droplet excites the substrate to oscillate, while during liquid retraction, the substrate imparts vertical momentum back to the droplet with a springboard effect, causing early droplet lift-off with reduced contact time. Through detailed experimental and theoretical analysis, we show that this novel springboarding phenomenon is achieved for a specific range of Weber numbers (We >40) and droplet Froude numbers during spreading (Fr >1). The observation of the substrate elasticity-mediated droplet springboard effect provides new insight into droplet impact physics.

  18. Droplet Combustion and Soot Formation in Microgravity

    NASA Technical Reports Server (NTRS)

    Avedisian, C. Thomas

    1994-01-01

    One of the most complex processes involved in the combustion ot liquid fuels is the formation of soot. A well characterized flow field and simplified flame structure can improve considerably the understanding of soot formation processes. The simplest flame shape to analyze for a droplet is spherical with its associated one-dimensional flow field. It is a fundamental limit and the oldest and most often analyzed configuration of droplet combustion. Spherical symmetry in the droplet burning process will arise when there is no relative motion between the droplet and ambience or uneven heating around the droplet periphery, and buoyancy effects are negligible. The flame and droplet are then concentric with each other and there is no liquid circulation within the droplet. An understanding of the effect of soot on droplet combustion should therefore benefit from this simplified configuration. Soot formed during spherically symmetric droplet combustion, however, has only recently drawn attention and it appears to be one of the few aspects associated with droplet combustion which have not yet been thoroughly investigated. For this review, the broad subject of droplet combustion is narrowed considerably by restricting attention specifically to soot combined with spherically symmetric droplet burning processes that are promoted.

  19. Water droplet impact on elastic superhydrophobic surfaces.

    PubMed

    Weisensee, Patricia B; Tian, Junjiao; Miljkovic, Nenad; King, William P

    2016-01-01

    Water droplet impact on surfaces is a ubiquitous phenomenon in nature and industry, where the time of contact between droplet and surface influences the transfer of mass, momentum and energy. To manipulate and reduce the contact time of impacting droplets, previous publications report tailoring of surface microstructures that influence the droplet - surface interface. Here we show that surface elasticity also affects droplet impact, where a droplet impacting an elastic superhydrophobic surface can lead to a two-fold reduction in contact time compared to equivalent rigid surfaces. Using high speed imaging, we investigated the impact dynamics on elastic nanostructured superhydrophobic substrates having membrane and cantilever designs with stiffness 0.5-7630 N/m. Upon impact, the droplet excites the substrate to oscillate, while during liquid retraction, the substrate imparts vertical momentum back to the droplet with a springboard effect, causing early droplet lift-off with reduced contact time. Through detailed experimental and theoretical analysis, we show that this novel springboarding phenomenon is achieved for a specific range of Weber numbers (We >40) and droplet Froude numbers during spreading (Fr >1). The observation of the substrate elasticity-mediated droplet springboard effect provides new insight into droplet impact physics. PMID:27461899

  20. Sophisticated compound droplets on fiber networks

    NASA Astrophysics Data System (ADS)

    Weyer, Floriane; Lismont, Marjorie; Dreesen, Laurent; Vandewalle, Nicolas

    2015-11-01

    Droplets on fibers are part of our everyday lives. Indeed, many phenomena involve drops and fibers such as the formation of dew droplets on a spiderweb, the trapping of water droplets on cactus spines or the dyeing of cotton or wool fibers. Therefore, this topic has been widely studied in the recent years and it appears that droplets on fibers can be the starting point for an open digital microfluidics. We study the behavior of soapy water droplets on a fiber array. When a droplet slides along a vertical fiber and encounters a horizontal fiber, it can either stick there or continue its way. In the latter case, the droplet releases a tiny residue. We study the volume of these residues depending on the geometry of the node. By using this technique, a large number of small droplets can be trapped at the nodes of a fiber array. These residues can be encapsulated and collected by an oil droplet in order to create a multicompound droplet. Moreover, by using optical fibers, we can provoke and detect the fluorescen