The (social) lives,deaths, and biophysical phases of lipid droplets

Lipid droplets (LDs) are major lipid storage organelles, sequestering energy-rich triglycerides and serving as nutrient sinks for cellular homeostasis. Observed for over a century but generally ignored, LDs are now appreciated to play key roles in organismal physiology and disease. They also form numerous functional contacts with other organelles. Here, we highlight recent studies examining LDs from distinct perspectives of their life cycle: their biogenesis, “social” life as they interact with other organelles, and deaths via lipolysis or lipophagy. We also discuss recent work showing how changes in LD lipid content alter the biophysical phases of LD lipids, and how this may fine-tune the LD protein landscape and ultimately LD function.     

Membrane aging during cell growth ascertained by laurdan generalized polarization

The sensitivity of the fluorescent probe Laurdan to the phase state of lipids has been utilized to detect modifications in the composition and physical state of cell membranes during cell growth. In phospholipid vesicles, the Laurdan emission spectrum shows a 50-nm red shift by passing from the gel to the liquid-crystalline phase. The Generalized Polarization (GP) value has been used for the data treatment instead of the ratiometric method common in investigations utilizing other fluorescent probes that display spectral sensitivity to medium properties. The GP value can be measured easily and quickly and possesses all the properties of "classical" polarization, including the additivity rule. Once Laurdan limiting GP values have been established for the gel and the liquid-crystalline phase of lipids, the quantitative determination of coexisting phases in natural samples is possible. In the present work the observation of a relevant decrease in the fractional intensity of the liquid-crystalline phase in K562 cell membranes during 5 days of asynchronous growth is reported. A decrease in the "fluidity" of cell membranes in K562 cells kept in culture for several months is also reported. The procedure developed for labeling cell membranes with Laurdan is reported and the influence of cell metabolism on fluorescence parameters is discussed. Also discussed is the influence of cholesterol on Laurdan GP.     

A study of quercetin effects on phospholipid membranes containing cholesterol using Laurdan fluorescence

Quercetin (QCT) is an important bioactive natural compound found in numerous edible plants. Since the lipid bilayer represents an essential compound of the cell membrane, QCT's direct interaction with this structure is of great interest. Therefore, we proposed to study the effects of QCT on DMPC liposomes containing cholesterol (Chol), and for this purpose Laurdan fluorescence was used. As a fluorescent probe, Laurdan is able to detect changes in membrane phase properties. When incorporated in lipid bilayers, Laurdan emits from two different excited states, a non-relaxed one when the bilayer packing is tight and a relaxed state when the bilayer packing is loose. The main tool for quantifying QCT's effects on phospholipid membranes containing Chol has been the analysis, the decomposition of Laurdan emission spectra in sums of two Gaussian functions on energy. This kind of approach has allowed good analysis of the balance between the two emitting states of Laurdan. Our results show that both Laurdan emission states are present to different extents in a wide temperature range for DMPC liposomes with Chol. QCT is decreasing the phase transition temperature in pure DMPC liposomes as proved by generalized polarization (GP) values. QCT also quenches Laurdan fluorescence, depending on the temperature and the presence of Chol in the membrane. Stern-Volmer constants were calculated for different lipid membrane compositions, and the conclusion was that the relaxed state favors the nonradiative transitions of the fluorophore.     

Plant pentacyclic triterpenic acids as modulators of lipid membrane physical properties

Free triterpenic acids (TTPs) present in plants are bioactive compounds exhibiting multiple nutriceutical activities. The underlying molecular mechanisms have only been examined in part and mainly focused on anti-inflammatory properties, cancer and cardiovascular diseases, in all of which TTPs frequently affect membrane-related proteins. Based on the structural characteristics of TTPs, we assume that their effect on biophysical properties of cell membranes could play a role for their biological activity. In this context, our study is focused on the compounds, oleanolic (3β-hydroxy-12-oleanen-28-oic acid, OLA), maslinic (2α,3β-dihydroxy-12-oleanen-28-oic acid, MSL) and ursolic ((3β)-3-hydroxyurs-12-en-28-oic acid, URL) as the most important TTPs present in orujo olive oil. X-ray diffraction, differential scanning calorimetry, (31)P nuclear magnetic resonance and Laurdan fluorescence data provide experimental evidence that OLA, MSL and URL altered the structural properties of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and DPPC-Cholesterol (Cho) rich membranes, being located into the polar-hydrophobic interphase. Specifically, in DPPC membranes, TTPs altered the structural order of the L(β'), phase without destabilizing the lipid bilayer. The existence of a nonbilayer isotropic phase in coexistence with the liquid crystalline L(α) phase, as observed in DPPC:URL samples, indicated the presence of lipid structures with high curvature (probably inverted micelles). In DPPC:Cho membranes, TTPs affected the membrane phase properties increasing the Laurdan GP values above 40°C. MSL and URL induced segregation of Cho within the bilayer, in contrast to OLA, that reduced the structural organization of the membrane. These results strengthen the relevance of TTP interactions with cell membranes as a molecular mechanism underlying their broad spectrum of biological effects.     

HDAC6 regulates lipid droplet turnover in response to nutrient deprivation via p62-mediated selective autophagy

Autophagy has been evolved as one of the adaptive cellular processes in response to stresses such as nutrient deprivation. Various cellular cargos such as damaged organelles and protein aggregates can be selectively degraded through autophagy. Recently, the lipid storage organelle, lipid droplet(LD), has been reported to be the cargo of starvation-induced autophagy. However, it remains largely unknown how the autophagy machinery recognizes the LDs and whether it can selectively degrade LDs. In this study, we show that Drosophila histone deacetylase 6(dHDAC6), a key regulator of selective autophagy, is required for the LD turnover in the hepatocyte-like oenocytes in response to starvation. HDAC6 regulates LD turnover via p62/SQSTM1(sequestosome 1)-mediated aggresome formation, suggesting that the selective autophagy machinery is required for LD recognition and degradation. Furthermore, our results show that the loss of dHDAC6 causes steatosis in response to starvation. Our findings suggest that there is a potential link between selective autophagy and susceptible predisposition to lipid metabolism associated diseases in stress conditions.     

Visualizing membrane microdomains by Laurdan 2-photon microscopy

Lateral segregation of cell membrane components gives rise to microdomains with a different structure within the membrane. Most prominently, lipid rafts are defined as domains in liquid ordered phase whereas surrounding membranes are more fluid. Here we review a 2-photon fluorescence microscopy approach, which allows the visualization of membrane fluidity. The fluorescent probe Laurdan exhibits a blue shift in emission with increasing membrane condensation caused by an alteration in the dipole moment of the probe as a consequence of exclusion of water molecules from the lipid bilayer. The quantification of membrane order is achieved by the Generalized Polarization (GP) values, which are defined as normalized intensity ratios of two emission channels. GP images are therefore not biased by probe concentrations and membrane ruffles. Furthermore, Laurdan reports membrane structure independently from the lipid and protein cargo of the membrane domains. We give examples where Laurdan microscopy was instrumental in quantifying the formation of condensed membrane domains and their cellular requirements. Moreover we discuss how microdomains identified by Laurdan microscopy are consistent with domains identified by other methodologies and put GP images in the context of current raft hypotheses.     

Variations in carotenoid content and acyl chain composition in exponential,stationary and biofilm states of Staphylococcus aureus,and their influence on membrane biophysical properties

Bacteria are often found in close association with surfaces, resulting in the formation of biofilms. In Staphylococcus aureus (S. aureus), biofilms are implicated in the resilience of chronic infections, presenting a serious clinical problem world-wide. Here, S. aureus biofilms are grown under flow within clinical catheters at 37 °C. The lipid composition and biophysical properties of lipid extracts from these biofilms are compared with those from exponential growth and stationary phase cells. Biofilms show a reduction in iso and anteiso branching compensated by an increase in saturated fatty acids compared to stationary phase. A drastic reduction in carotenoid levels is also observed during biofilm formation. Thermotropic measurements of Laurdan GP and DPH polarization, show a reduction of lipid packing at 37 °C for biofilms compared to stationary phase. We studied the effects of carotenoid content on DMPG and DPPG model membranes showing trends in thermotropic behavior consistent with those observed in bacterial isolates, indicating that carotenoids participate in modulating lipid packing. Additionally, bending elastic constant (k_c) measurements using vesicle fluctuation analysis (VFA) show that the presence of carotenoids can increase membrane bending rigidity. The antimicrobial peptide Magainin H2 was less activity on liposomes composed of stationary phase compared to biofilms or exponential growth isolates. This study contributes to an understanding of how Staphylococcus aureus modulates the composition of its membrane lipids, and how those changes affect the biophysical properties of membranes, which in turn may play a role in its virulence and its resistance to different membrane-active antimicrobial agents.     

高尔基体跨膜糖蛋白73高表达诱发脂肪肝的脂类特性分析

脂肪性肝病是最常见的慢性肝脏疾病,脂质代谢异常是脂肪性肝病发生的重要原因。为研究高尔基体糖蛋白(Golgi protein 73, GP73)对肝脏脂质代谢的影响,选用八周龄C57BL/6J小鼠通过尾静脉注射搭载GP73的腺相关病毒(AAV-GP73),构建肝脏特异性高表达GP73的小鼠,通过对肝脏进行脂质代谢组学分析发现小鼠肝脏中的脂质尤其是甘油三酯明显增加。京都基因与基因组百科全书(kyoto encyclopedia of genes and genomes, KEGG)富集分析显示,GP73通过引起脂代谢产物的改变导致诸多与细胞代谢活动相关的信号通路出现紊乱,特别是与人类密切相关的疾病如Ⅱ型糖尿病、非酒精性脂肪性肝病(NAFLD)和癌细胞胆碱代谢更可能发生失调。研究表明,GP73可能通过参与调控脂类代谢并促进肝脏内脂质积累诱发脂肪肝。     

Osmotic response in Lactobacillus casei ATCC 393: biochemical and biophysical characteristics of membrane

The biochemical and biophysical properties of the membrane and some general characteristics of the response of Lactobacillus casei ATCC 393 (reclassified Lactobacillus zeae) to hyperosmotic conditions were studied. Under hypertonic conditions, the hydrophobicity and the bile salt sensitivity of the cultures were increased. The glycolipid AcylH3DG is only present in membranes of NaCl containing medium, whereas, H4DG undergoes a significant increment and H2DG a significant decrease. The fluidity of both the purified membranes and the total lipid vesicles, as determined with the fluorescent probe DPH, did not change in conditions of high salinity. This was coincident with changes in the fatty acid (FA) composition where an increase in the saturated/unsaturated FA ratio was compensated by a rise in the fluidifying 11,12-methyleneoctadecanoic FA (cyc 19:0). Under osmotic stress conditions, Laurdan and acridine orange in total lipid vesicles showed increased lateral lipid packing and proton permeability, respectively.     

Lipid droplets as ubiquitous fat storage organelles in <Emphasis Type="Italic">C. elegans</Emphasis>

Background  

Lipid droplets are a class of eukaryotic cell organelles for storage of neutral fat such as triacylglycerol (TAG) and cholesterol ester (CE). We and others have recently reported that lysosome-related organelles (LROs) are not fat storage structures in the nematode C. elegans. We also reported the formation of enlarged lipid droplets in a class of peroxisomal fatty acid β-oxidation mutants. In the present study, we seek to provide further evidence on the organelle nature and biophysical properties of fat storage structures in wild-type and mutant C. elegans.     

The impact of lipid polyunsaturation on the physical and mechanical properties of lipid membranes

The lipid composition of cellular membranes and the balance between the different lipid components can be impacted by aging, certain pathologies, specific diets and other factors. This is the case in a subgroup of individuals with psychiatric disorders, such as schizophrenia, where cell membranes of patients have been shown to be deprived in polyunsaturated fatty acids (PUFAs), not only in brain areas where the target receptors are expressed but also in peripheral tissues. This PUFA deprivation thus represents a biomarker of such disorders that might impact not only the interaction of antipsychotic medications with these membranes but also the activation and signaling of the targeted receptors embedded in the lipid membrane. Therefore, it is crucial to understand how PUFAs levels alterations modulate the different physical properties of membranes.In this paper, several biophysical approaches were combined (Laurdan fluorescence spectroscopy, atomic force microscopy, differential scanning calorimetry, molecular modeling) to characterize membrane properties such as fluidity, elasticity and thickness in PUFA-enriched cell membranes and lipid model systems reflecting the PUFA imbalance observed in some diseases. The impact of both the number of unsaturations and their position along the chain on the above properties was investigated. Briefly, data revealed that PUFA presence in membranes increases membrane fluidity, elasticity and flexibility and decreases its thickness and order parameter. Both the level of unsaturation and their position affect these membrane properties.     

Monitoring the organization and dynamics of bovine hippocampal membranes utilizing Laurdan generalized polarization

Organization and dynamics of cellular membranes in the nervous system are crucial for the function of neuronal membrane receptors. The lipid composition of neuronal cells is unique and has been correlated with the increased complexity in the organization of the nervous system during evolution. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors such as the G-protein coupled serotonin_1A receptor. In this paper, we have explored the organization and dynamics of bovine hippocampal membranes using the amphiphilic environment-sensitive fluorescent probe Laurdan. Our results show that the emission spectra of Laurdan display an additional red shifted peak as a function of increasing temperature in native as well as cholesterol-depleted membranes and liposomes made from lipid extracts of the native membrane. Interestingly, wavelength dependence of Laurdan generalized polarization (GP) in native membranes indicates the presence of an ordered gel-like phase at low temperatures, whereas characteristics of the liquid-ordered phase are observed at high temperatures. Similar experiments performed using cholesterol-depleted membranes show fluidization of the membrane with increasing cholesterol depletion. In addition, results from fluorescence polarization of DPH indicate that the hippocampal membrane is fairly ordered even at physiological temperature. The temperature dependence of Laurdan excitation GP provides a measure of the apparent thermal transition temperature and extent of cooperativity in these membranes. Analysis of time-resolved fluorescence measurements of Laurdan shows reduction in mean fluorescence lifetime with increasing temperature due to change in environmental polarity. These results constitute novel information on the dynamics of hippocampal membranes and its modulation by cholesterol depletion monitored using Laurdan fluorescence.     

Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius

The effects of temperature and pH on Laurdan (6-lauroyl-2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) ( approximately 20-150 microm in diameter) composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius have been studied using two-photon excitation. PLFE GUVs made by the electroformation method were stable and well suited for microscopy studies. The generalized polarization (GP) of Laurdan fluorescence in the center cross section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. At all of the temperatures and pHs examined, the GP values are low (below or close to 0), and the GP histograms show a broad distribution width (> 0.3). When excited with light polarized in the y direction, Laurdan fluorescence in the center cross section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x direction of the vesicles, a result opposite that previously obtained on monopolar diester phospholipids. This result indicates that the chromophore of Laurdan in PLFE GUVs is aligned parallel to the membrane surface. The x direction photoselection effect and the low GP values lead us to further propose that the Laurdan chromophore resides in the polar headgroup region of the PLFE liposomes, while the lauroyl tail inserts into the hydrocarbon core of the membrane. This unusual L-shaped disposition is presumably caused by the unique lipid structures and by the rigid and tight membrane packing in PLFE liposomes. The GP exhibited, at both pH values, a small but abrupt decrease near 50 degrees C, suggesting a conformational change in the polar headgroups of PLFE. This transition temperature fully agrees with the d-spacing data recently measured by small-angle x-ray diffraction and with the pyrene-labeled phosphatidylcholine and perylene fluorescence data previously obtained from PLFE multilamellar vesicles. Interestingly, the two-photon Laurdan fluorescence images showed snowflake-like lipid domains in PLFE GUVs at pH 7.23 and low temperatures (<20 degrees C in the cooling scan and <24 degrees C in the heating scan). These domains, attributable to lipid lateral separation, were stable and laterally immobile at low temperatures (<23 degrees C), again suggesting tight membrane packing in the PLFE GUVs.     

Laurdan fluorescence senses mechanical strain in the lipid bilayer membrane

The precise molecular mechanisms by which cells transduce a mechanical stimulus into an intracellular biochemical response have not yet been established. Here, we show for the first time that the fluorescence emission of an environment-sensitive membrane probe Laurdan is modulated by mechanical strain of the lipid bilayer membrane. We have measured fluorescence emission of Laurdan in phospholipid vesicles of 30, 50, and 100 nm diameter to show that osmotically induced membrane tension leads to an increase in polarity (hydration depth) of the phospholipid bilayer interior. Our data indicate that the general polarization of Laurdan emission is linearly dependent on membrane tension. We also show that higher membrane curvature leads to higher hydration levels. We anticipate that the proposed method will facilitate future studies of mechanically induced changes in physical properties of lipid bilayer environment both in vitro and in vivo.     

Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators

Viruses are obligate intracellular parasites that make use of the host metabolic machineries to meet their biosynthetic needs. Thus, identifying the host pathways essential for the virus replication may lead to potential targets for therapeutic intervention. The mechanisms and pathways explored by SARS-CoV-2 to support its replication within host cells are not fully known. Lipid droplets (LD) are organelles with major functions in lipid metabolism, energy homeostasis and intracellular transport, and have multiple roles in infections and inflammation. Here we described that monocytes from COVID-19 patients have an increased LD accumulation compared to SARS-CoV-2 negative donors. In vitro, SARS-CoV-2 infection were seen to modulate pathways of lipid synthesis and uptake as monitored by testing for CD36, SREBP-1, PPARγ, and DGAT-1 expression in monocytes and triggered LD formation in different human cell lines. LDs were found in close apposition with SARS-CoV-2 proteins and double-stranded (ds)-RNA in infected Vero cells. Electron microscopy (EM) analysis of SARS-CoV-2 infected Vero cells show viral particles colocalizing with LDs, suggestive that LDs might serve as an assembly platform. Pharmacological modulation of LD formation by inhibition of DGAT-1 with A922500 significantly inhibited SARS-CoV-2 replication as well as reduced production of mediators pro-inflammatory response. Taken together, we demonstrate the essential role of lipid metabolic reprograming and LD formation in SARS-CoV-2 replication and pathogenesis, opening new opportunities for therapeutic strategies to COVID-19.     

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

Ceramide (Cer) is involved in the regulation of several cellular processes by mechanisms that depend on Cer-induced changes on membrane biophysical properties. Accumulating evidence shows that Cers with different N-acyl chain composition differentially impact cell physiology, which may in part be due to specific alterations in membrane biophysical properties. We now address how the sphingolipid (SL) N-acyl chain affects membrane properties in cultured human embryonic kidney cells by overexpressing different Cer synthases (CerSs). Our results show an increase in the order of cellular membranes in CerS2-transfected cells caused by the enrichment in very long acyl chain SLs. Formation of Cer upon treatment of cells with bacterial sphingomyelinase promoted sequential changes in the properties of the membranes: after an initial increase in the order of the fluid plasma membrane, reorganization into domains with gel-like properties whose characteristics are dependent on the acyl chain structure of the Cer was observed. Moreover, the extent of alterations of membrane properties correlates with the amount of Cer formed. These data reinforce the significance of Cer-induced changes on membrane biophysical properties as a likely molecular mechanism by which different acyl chain Cers exert their specific biological actions.     

Fsp27 promotes lipid droplet growth by lipid exchange and transfer at lipid droplet contact sites

Lipid droplets (LDs) are dynamic cellular organelles that control many biological processes. However, molecular components determining LD growth are poorly understood. Genetic analysis has indicated that Fsp27, an LD-associated protein, is important in controlling LD size and lipid storage in adipocytes. In this paper, we demonstrate that Fsp27 is focally enriched at the LD-LD contacting site (LDCS). Photobleaching revealed the occurrence of lipid exchange between contacted LDs in wild-type adipocytes and Fsp27-overexpressing cells but not Fsp27-deficient adipocytes. Furthermore, live-cell imaging revealed a unique Fsp27-mediated LD growth process involving a directional net lipid transfer from the smaller to larger LDs at LDCSs, which is in accordance with the biophysical analysis of the internal pressure difference between the contacting LD pair. Thus, we have uncovered a novel molecular mechanism of LD growth mediated by Fsp27.     

A brain-liver circuit regulates glucose homeostasis

Increased glucose production (GP) is the major determinant of fasting hyperglycemia in diabetes mellitus. Previous studies suggested that lipid metabolism within specific hypothalamic nuclei is a biochemical sensor for nutrient availability that exerts negative feedback on GP. Here we show that central inhibition of fat oxidation leads to selective activation of brainstem neurons within the nucleus of the solitary tract and the dorsal motor nucleus of the vagus and markedly decreases liver gluconeogenesis, expression of gluconeogenic enzymes, and GP. These effects require central activation of ATP-dependent potassium channels (K(ATP)) and descending fibers within the hepatic branch of the vagus nerve. Thus, hypothalamic lipid sensing potently modulates glucose metabolism via neural circuitry that requires the activation of K(ATP) and selective brainstem neurons and intact vagal input to the liver. This crosstalk between brain and liver couples central nutrient sensing to peripheral nutrient production and its disruption may lead to hyperglycemia.     

Unique effects of different fatty acid species on the physical properties of the torpedo acetylcholine receptor membrane.

To study the effects produced by free fatty acids (FFA) on the biophysical properties of Torpedo marmorata nicotinic acetylcholine receptor-rich native membranes and to investigate the topology of their binding site(s), fluorescence measurements were carried out using the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino) naphthalene) and ADIFAB, an Acrylodan-derivatized intestinal fatty acid-binding protein. The generalized polarization (GP) of the former probe was used to learn about the physical state of the membrane upon FFA binding. Saturated FFA induced a slight increase in GP, whereas cis-unsaturated fatty acids decreased GP. Double bond isomerism could also be distinguished; oleic acid (18:1cis) induced a net disordering effect, whereas elaidic acid (18:1trans) produced no changes in GP. The changes in the efficiency of the F?rster energy transfer from the protein to Laurdan brought about by addition of FFA, together with the distances involved in this process, indicate that all FFA studied share a common site at the lipid-protein interface. However, despite being located at the same site, each class of FFA differs in its effect on the physical properties of the membrane. These data lead us to suggest that it is the direct action of FFA at the lipid-protein interface, displacing essential lipids from their sites rather than changes in bulk properties such as membrane fluidity that accounts for the effect of FFA on the acetylcholine receptor membrane.     

Therapeutic lithium alters polar head-group region of lipid bilayer and prevents lipid peroxidation in forebrain cortex of sleep-deprived rats

Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.