Molecular speciation and dynamics of oxidized triacylglycerols in lipid droplets: Mass spectrometry and coarse-grained simulations

Lipid droplets (LDs) are ubiquitous and physiologically active organelles regulating storage and mobilization of lipids in response to metabolic demands. Among the constituent LD neutral lipids, such as triacylglycerols, cholesterol esters, and free fatty acids, oxidizable polyunsaturated molecular species may be quite abundant, yet the structural and functional roles of their oxidation products have not been studied. Our previous work documented the presence of these peroxidized species in LDs. Assuming that hydrophilic oxygen-containing functionalities may markedly change the hydrophobic/hydrophilic molecular balance, here we utilized computational modeling to test the hypothesis that lipid peroxidation causes redistribution of lipids between the highly hydrophobic core and the polar surface (phospho)lipid monolayer—the area enriched with integrated enzymatic machinery. Using quantitative liquid chromatography/mass spectrometry, we characterized molecular speciation of oxTAGs in LDs of dendritic cells in cancer and hypoxic trophoblasts cells as two cellular models associated with dyslipidemia. Among the many types of oxidized lipids identified, we found that oxidatively truncated forms and hydroxyl derivatives of TAGs were the prevailing oxidized lipid species in LDs in both cell types. Using coarse-grained molecular dynamics (CG-MD) simulations we established that lipid oxidation changed their partitioning whereby oxidized lipids migrated into the outer monolayer of the LD, where they can affect essential metabolic pathways and undergo conversions, possibly leading to the formation of oxygenated lipid mediators.     

Raman spectroscopy evidence of lipid separation in domestic cat oocytes during freezing

Although lipid droplets are believed to play an important role in cryopreservation of mammalian embryos and oocytes, the effect of low temperatures on lipid droplets and related mechanisms of cryodamage are still obscure. Here, we provide Raman spectroscopy evidence of lipid separation inside the lipid droplets in domestic cat oocytes during slow freezing. It was shown that at −25 °C lipids coexist in two separated phase states inside lipid droplets. The scale of detected domains was a few micrometers size. We also found that under certain conditions these areas have a specific spatial distribution. Lipids with high melting temperatures are distributed near the surface of lipid droplets while fusible lipids are located deep inside. Raman spectroscopy was found to be a prospective approach to study inhomogeneity of lipid phase transition in cells and to reveal effects of this inhomogeneity on cryopreservation of biological cells.     

Do Lipids Show State-dependent Affinity to the Voltage-gated Potassium Channel KvAP?

As all integral membrane proteins, voltage-gated ion channels are embedded in a lipid matrix that regulates their channel behavior either by physicochemical properties or by direct binding. Because manipulation of the lipid composition in cells is difficult, we investigated the influence of different lipids on purified KvAP channels reconstituted in planar lipid bilayers of known composition. Lipids developed two distinct and independent effects on the KvAP channels; lipids interacting with the pore lowered the energy barriers for the final transitions, whereas voltage sensor-bound lipids shifted the midpoint of activation dependent on their electrostatic charge. Above all, the midpoint of activation was determined only by those lipids the channels came in contact with first after purification and can seemingly only be exchanged if the channel resides in the open state. The high affinity of the bound lipids to the binding site has implications not only on our understanding of the gating mechanism but also on the general experimental design of any lipid dependence study.     

Imaging organelle membranes in live cells at the nanoscale with lipid-based fluorescent probes

Understanding how organelles interact, exchange materials, assemble, disassemble, and evolve as a function of space, time, and environment is an exciting area at the very forefront of chemical and cell biology. Here, we bring attention to recent progress in the design and application of lipid-based tools to visualize and interrogate organelles in live cells, especially at super resolution. We highlight strategies that rely on modification of natural lipids or lipid-like small molecules ex cellula, where organelle specificity is provided by the structure of the chemically modified lipid, or in cellula using cellular machinery, where an enzyme labels the lipid in situ. We also describe recent improvements to the chemistry upon which lipid probes rely, many of which have already begun to broaden the scope of biological questions that can be addressed by imaging organelle membranes at the nanoscale.     

Actin-induced perturbation of PS lipid–cholesterol interaction: A possible mechanism of cytoskeleton-based regulation of membrane organization

To obtain insight into the potential role of the cytoskeleton on lipid mixing behavior in plasma membranes, the current study explores the influence of physisorbed actin filaments (F-actin) on lipid–lipid phase separations in planar model membrane systems containing raft-mimicking lipid mixtures of well-defined compositions using a complementary experimental approach of epifluorescence microscopy, fluorescence anisotropy, wide-field single molecule fluorescence microscopy, and interfacial rheometry. In particular, we have explored the impact of F-actin on cholesterol (CHOL)–phospholipid interactions, which are considered important for the formation of CHOL-enriched lipid raft domains. By using epifluorescence microscopy, we show that physisorbed filamentous actin (F-actin) alters the domain size of lipid–lipid phase separations in the presence of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol (CHOL). In contrast, no actin-induced modification in lipid–lipid phase separations is observed in the absence of POPS or when POPS is replaced by another anionic lipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG). Wide-field single molecule fluorescence microscopy on binary lipid mixtures indicate that PS and PG lipids show similar electrostatic interactions with physisorbed actin filaments. Complementary fluorescence anisotropy experiments on binary PS lipid-containing lipid mixtures are provided to illustrate the actin-induced segregation of anionic lipids. The similarity of electrostatic interactions between actin and both anionic lipids suggests that the observed differences in actin-mediated perturbations of lipid phase separations are caused by distinct PS lipid–CHOL versus PG lipid–CHOL interactions. We hypothesize that the actin cytoskeleton and some peripheral membrane proteins may alter lipid–lipid phase separations in plasma membranes in a similar way by interacting with PS lipids.     

Avanti lipid tools: Connecting lipids,technology, and cell biology

Lipid research is challenging owing to the complexity and diversity of the lipidome. Here we review a set of experimental tools developed for the seasoned lipid researcher, as well as, those who are new to the field of lipid research. Novel tools for probing protein–lipid interactions, applications for lipid binding antibodies, enhanced systems for the cellular delivery of lipids, improved visualization of lipid membranes using gold-labeled lipids, and advances in mass spectrometric analysis techniques will be discussed. Because lipid mediators are known to participate in a host of signal transduction and trafficking pathways within the cell, a comprehensive lipid toolbox that aids the science of lipidomics research is essential to better understand the molecular mechanisms of interactions between cellular components. This article is part of a Special Issue entitled Tools to study lipid functions.     

Each liver X receptor (LXR) type has a different purpose in different situations

LXRs, which are nuclear receptors, have 2 isoforms—LXRα and LXRβ. Generally, LXRα is expressed in the liver, kidney, and a limited number of other organs, whereas LXRβ is thought to be expressed ubiquitously. Nevertheless, no clear consensus has been reached on the role of each in kidney lipid metabolism.Many researchers have reported that lipids accumulate in renal tubular epithelial cells during nephrosis. The nephrosis model we used showed the presence of urinary protein 4 days after the induction of illness. Additionally, the model maintained high levels of urinary protein from day 7–14. Lipid accumulation was clearly verified at day 4 and extreme accumulation was observed at day 7. We observed increased expression of LXRα from an early stage of nephrosis. To explore the role of increased LXRα in diseased kidney in vitro, NRK52E, normal kidney tubular epithelial cells, were forced to overexpress LXRα. These cells showed significantly lower lipid accumulation than mock cells did. In contrast, LXRβ knockdown lead to increased lipid accumulation in mock cells, and constancy in overexpressing cells.In normal kidneys, LXRβ is expressed stably to control mainly the intracellular lipids. However, with increasing intracellular lipid accumulation, expression of LXRα and its downstream gene, ABCA1, was upregulated, followed by lipid excretion in an LXRα-dependent manner. This phenomenon strongly suggests the importance of LXRα in lipid metabolism in the diseased kidney.     

The Ether Lipid Precursor Hexadecylglycerol Stimulates the Release and Changes the Composition of Exosomes Derived from PC-3 Cells

Exosomes are vesicles released by cells after fusion of multivesicular bodies with the plasma membrane. In this study, we have investigated whether ether lipids affect the release of exosomes in PC-3 cells. To increase the cellular levels of ether lipids, the ether lipid precursor hexadecylglycerol was added to cells. Lipidomic analysis showed that this compound was in fact able to double the cellular levels of ether lipids in these cells. Furthermore, increased levels of ether lipids were also found in exosomes released by cells containing high levels of these lipids. Interestingly, as measured by nanoparticle tracking analysis, cells containing high levels of ether lipids released more exosomes than control cells, and these exosomes were similar in size to control exosomes. Moreover, silver staining and Western blot analyses showed that the protein composition of exosomes released in the presence of hexadecylglycerol was changed; the levels of some proteins were increased, and the levels of others were reduced. In conclusion, this study clearly shows that an increase in cellular ether lipids is associated with changes in the release and composition of exosomes.     

Oxidized LDL lipids increase β-amyloid production by SH-SY5Y cells through glutathione depletion and lipid raft formation

Elevated total cholesterol in midlife has been associated with increased risk of dementia in later life. We have previously shown that low-density lipoprotein (LDL) is more oxidized in the plasma of dementia patients, although total cholesterol levels are not different from those of age-matched controls. β-Amyloid (Aβ) peptide, which accumulates in Alzheimer disease (AD), arises from the initial cleavage of amyloid precursor protein by β-secretase-1 (BACE1). BACE1 activity is regulated by membrane lipids and raft formation. Given the evidence for altered lipid metabolism in AD, we have investigated a mechanism for enhanced Aβ production by SH-SY5Y neuronal-like cells exposed to oxidized LDL (oxLDL). The viability of SH-SY5Y cells exposed to 4 μg oxLDL and 25 µM 27-hydroxycholesterol (27OH-C) was decreased significantly. Lipids, but not proteins, extracted from oxLDL were more cytotoxic than oxLDL. In parallel, the ratio of reduced glutathione (GSH) to oxidized glutathione was decreased at sublethal concentrations of lipids extracted from native and oxLDL. GSH loss was associated with an increase in acid sphingomyelinase (ASMase) activity and lipid raft formation, which could be inhibited by the ASMase inhibitor desipramine. 27OH-C and total lipids from LDL and oxLDL independently increased Aβ production by SH-SY5Y cells, and Aβ accumulation could be inhibited by desipramine and by N-acetylcysteine. These data suggest a mechanism whereby oxLDL lipids and 27OH-C can drive Aβ production by GSH depletion, ASMase-driven membrane remodeling, and BACE1 activation in neuronal cells.     

The role of lipids and salts in two-dimensional crystallization of the glycine-betaine transporter BetP from Corynebacterium glutamicum

The osmoregulated and chill-sensitive glycine–betaine transporter (BetP) from Corynebacterium glutamicum was reconstituted into lipids to form two-dimensional (2D) crystals. The sensitivity of BetP partly bases on its interaction with lipids. Here we demonstrate that lipids and salts influence crystal morphology and crystallinity of a C-terminally truncated BetP. The salt type and concentration during crystallization determined whether crystals grew in the form of planar-tubes, sheets or vesicles, while the lipid type influenced crystal packing and order. Three different lipid preparations for 2D crystallization were compared. Only the use of lipids extracted from C. glutamicum cells led to the formation of large, well-ordered crystalline areas. To understand the lipid-derived influence on crystallinity, lipid extracts from different stages of the crystallization process were analyzed by quantitative multiple-precursor ion scanning mass spectroscopy (MS). Results show that BetP has a preference for fatty acid moieties 16:0–18:1, and that a phosphatidyl glycerol (PG) 16:0–18:1 rich preparation prevents formation of pseudo crystals.     

Domain coupling in asymmetric lipid bilayers

Biological membranes are heterogeneous assemblies of lipids, proteins, and cholesterol that are organized as asymmetric bimolecular leaflets of lipids with embedded proteins. Modulated by the concentration of cholesterol lipids and proteins may segregate into two or more liquid phases with different physical properties that can coexist in the same membrane. In this review, we summarize recent advances on how this situation can be recreated in a supported bilayer format and how this system has been used to demonstrate the induction of ordered lipid domains in lipid compositions that are typical for the inner leaflet by lipid compositions that are typical for the outer leaflet of mammalian plasma membranes. Proteins are shown to differentially target such induced inner leaflet domains.     

Lipid mimetics: A versatile toolbox for lipid biology and beyond

Being the principal component of biological membranes lipids are essential building blocks of life. Given their huge biological importance, the investigation of lipids, their properties, interactions and metabolic pathways is of prime importance for the fundamental understanding of living cells and organisms as well as the emergence of diseases. Different strategies have been applied to investigate lipid-mediated biological processes, one of them being the use of lipid mimetics. They structurally resemble their natural counterparts but are equipped with functionality that can be used to probe or manipulate lipid-mediated biological processes and biomembranes. Lipid mimetics therefore constitute an indispensable toolbox for lipid biology and membrane research but also beyond for potential applications in medicine or synthetic biology. Herein, we highlight recent advances in the development and application of lipid-mimicking compounds.     

The fatty acid profile changes in marine invertebrate larval cells during cryopreservation

The development of cryopreservation methods for embryonic cells and larvae of sea animals offers a great potential for marine biotechnology. Larval cells of bivalves and sea urchins were frozen to −196 °C using traditional cryoprotectants (Me₂SO and trehalose) and the cryoprotective mixture developed by us. In addition to Me₂SO and trehalose, this mixture contained an exogenous lipid extract from mussel tissues and antioxidants. A positive effect of antioxidants (α-tocopherol acetate, ascorbic acid or echinochrome, the quinoid pigment of sea urchins) on cell viability became significant only in the presence of exogenous lipids. Antioxidants added to cryoprotective mixtures did not reveal visible cryoprotective activity when used separately. To better understand the mechanism of the protective effect of exogenous lipids on cell membranes of sea animals, a comparative analysis of the fatty acid (FA) composition of total lipids in larval cells before and after freezing was carried out using a gas–liquid chromatography. The results indicate that freezing–thawing has direct effects on the FA composition of major lipid classes in marine invertebrate cells, and these effects can vary depending on the provenance of the cells. We have found that (I) both cell viability and the FA profile of cell lipids after cryopreservation depend on the cryoprotectants used; (II) an amount of saturated, monoenic and polyenic FAs changes significantly after cryopreservation. We assume that the addition of the exogenous lipid extract in form of liposomes could promote a renewal of disturbance areas and prevent from membrane damages during freezing–thawing.     

Phospholipids and lipid droplets

Lipid droplets are ubiquitous cellular organelles that allow cells to store large amounts of neutral lipids for membrane synthesis and energy supply in times of starvation. Compared to other cellular organelles, lipid droplets are structurally unique as they are made of a hydrophobic core of neutral lipids and are separated to the cytosol only by a surrounding phospholipid monolayer. This phospholipid monolayer consists of over a hundred different phospholipid molecular species of which phosphatidylcholine is the most abundant lipid class. However, lipid droplets lack some indispensable activities of the phosphatidylcholine biogenic pathways suggesting that they partially depend on other organelles for phosphatidylcholine synthesis.     

An update on genetically encoded lipid biosensors

Specific lipid species play central roles in cell biology. Their presence or enrichment in individual membranes can control properties or direct protein localization and/or activity. Therefore, probes to detect and observe these lipids in intact cells are essential tools in the cell biologist’s freezer box. Herein, we discuss genetically encoded lipid biosensors, which can be expressed as fluorescent protein fusions to track lipids in living cells. We provide a state-of-the-art list of the most widely available and reliable biosensors and highlight new probes (circa 2018–2021). Notably, we focus on advances in biosensors for phosphatidylinositol, phosphatidic acid, and PI 3-kinase lipid products.     

An Integrated Model of Eicosanoid Metabolism and Signaling Based on Lipidomics Flux Analysis

There is increasing evidence for a major and critical involvement of lipids in signal transduction and cellular trafficking, and this has motivated large-scale studies on lipid pathways. The Lipid Metabolites and Pathways Strategy consortium is actively investigating lipid metabolism in mammalian cells and has made available time-course data on various lipids in response to treatment with KDO₂-lipid A (a lipopolysaccharide analog) of macrophage RAW 264.7 cells. The lipids known as eicosanoids play an important role in inflammation. We have reconstructed an integrated network of eicosanoid metabolism and signaling based on the KEGG pathway database and the literature and have developed a kinetic model. A matrix-based approach was used to estimate the rate constants from experimental data and these were further refined using generalized constrained nonlinear optimization. The resulting model fits the experimental data well for all species, and simulated enzyme activities were similar to their literature values. The quantitative model for eicosanoid metabolism that we have developed can be used to design experimental studies utilizing genetic and pharmacological perturbations to probe fluxes in lipid pathways.     

小麦受精过程的细胞化学研究

小麦成熟胚囊卵细胞中存在较多围核分布的淀粉粒和少量散布的脂类颗粒;两个助细胞中积累很多脂类,未见有淀粉粒存在;中央细胞中存在中等量均匀分布的淀粉粒和脂类颗粒。受精时期,胚囊内各细胞中淀粉粒变化不大。精卵核融合时,卵细胞和中央细胞中的脂类分别存在一个积累高峰。合子与相应时期游离核胚乳中的脂类颗粒均较少。原胚初期,每个原胚细胞及胚乳原生质中均积累较多脂类。珠孔附近的内珠被细胞中脂类颗粒较多,并存在一个有规律的变化。在观察的所有发育时期的胚珠中,均未发现贮存蛋白质。胚珠中脂类的一系列变化可能与雌性细胞的营养、胚胎发育初期的养料及花粉管的定向生长等有关     

Regulation of channel function due to physical energetic coupling with a lipid bilayer

Regulation of membrane protein functions due to hydrophobic coupling with a lipid bilayer has been investigated. An energy formula describing interactions between lipid bilayer and integral ion channels with different structures, which is based on the screened Coulomb interaction approximation, has been developed. Here the interaction energy is represented as being due to charge-based interactions between channel and lipid bilayer. The hydrophobic bilayer thickness channel length mismatch is found to induce channel destabilization exponentially while negative lipid curvature linearly. Experimental parameters related to channel dynamics are consistent with theoretical predictions. To measure comparable energy parameters directly in the system and to elucidate the mechanism at an atomistic level we performed molecular dynamics (MD) simulations of the ion channel forming peptide–lipid complexes. MD simulations indicate that peptides and lipids experience electrostatic and van der Waals interactions for short period of time when found within each other’s proximity. The energies from these two interactions are found to be similar to the energies derived theoretically using the screened Coulomb and the van der Waals interactions between peptides (in ion channel) and lipids (in lipid bilayer) due to mainly their charge properties. The results of in silico MD studies taken together with experimental observable parameters and theoretical energetic predictions suggest that the peptides induce ion channels inside lipid membranes due to peptide–lipid physical interactions. This study provides a new insight helping better understand of the underlying mechanisms of membrane protein functions in cell membrane leading to important biological implications.     

Do lipids shape the eukaryotic cell cycle?

Successful passage through the cell cycle presents a number of structural challenges to the cell. Inceptive studies carried out in the last five years have produced clear evidence of modulations in the lipid profile (sometimes referred to as the lipidome) of eukaryotes as a function of the cell cycle. This mounting body of evidence indicates that lipids play key roles in the structural transformations seen across the cycle. The accumulation of this evidence coincides with a revolution in our understanding of how lipid composition regulates a plethora of biological processes ranging from protein activity through to cellular signalling and membrane compartmentalisation. In this review, we discuss evidence from biological, chemical and physical studies of the lipid fraction across the cell cycle that demonstrate that lipids are well-developed cellular components at the heart of the biological machinery responsible for managing progress through the cell cycle. Furthermore, we discuss the mechanisms by which this careful control is exercised.