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Why has nature acquired such a huge lipid repertoire? Although it would be theoretically possible to make a lipid bilayer fulfilling barrier functions with only one glycerophospholipid, there are diverse and numerous different lipid species. Lipids are heterogeneously distributed across the evolutionary tree with lipidomes evolving in parallel to organismal complexity. Moreover, lipids are different between organs and tissues and even within the same cell, different organelles have characteristic lipid signatures. At the molecular level, membranes are asymmetric and laterally heterogeneous. This lipid asymmetry at different scales indicates that these molecules may play very specific molecular functions in biology. Some of these roles have been recently uncovered: lipids have been shown to be essential in processes such as hypoxia and ferroptosis or in protein sorting and trafficking but many of them remain still unknown. In this review we will discuss the importance of understanding lipid diversity in biology across scales and we will share a toolbox with some of the emerging technologies that are helping us to uncover new lipid molecular functions in cell biology and, step by step, crack the membrane lipid code., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Suppression of a specific gene effect can be achieved by genetic as well as chemical methods. Each approach may hide unexpected drawbacks, usually in the form of side effects. In the present study, the specific inhibitor myriocin was used to block serine palmitoyltransferase (SPT), the first enzyme in the sphingolipid synthetic pathway, in CHO cells. The subsequent biophysical changes in plasma membranes were measured and compared with results obtained with a genetically modified CHO cell line containing a defective SPT (the LY-B cell line). Similar effects were observed with both approaches: sphingomyelin values were markedly decreased in myriocin-treated CHO cells and, in consequence, their membrane molecular order (measured as laurdan general polarization) and mechanical resistance (AFM-measured breakthrough force values) became lower than in the native, non-treated cells. Cells treated with myriocin reacted homeostatically to maintain membrane order, synthesizing more fully saturated and less polyunsaturated GPL than the non-treated ones, although they achieved it only partially, their plasma membranes remaining slightly more fluid and more penetrable than those from the control cells. The good agreement between results obtained with very different tools, such as genetically modified and chemically treated cells, reinforces the use of both methods and demonstrates that both are adequate for their intended use, i.e. the complete and specific inhibition of sphingolipid synthesis in CHO cells, without apparent unexpected effects., (© 2022. The Author(s).)

This article describes four fluorescent membrane tension probes that have been designed, synthesized, evaluated, commercialized and applied to current biology challenges in the context of the NCCR Chemical Biology. Their names are Flipper-TR ® , ER Flipper-TR ® , Lyso Flipper-TR ® , and Mito Flipper-TR ® . They are available from Spirochrome.

Sphingolipids (SL) are ubiquitous in mammalian cell membranes, yet there is little data on the behavior of cells under SL-restriction conditions. LY-B cells derive from a CHO linein whichserine palmitoyl transferase (SPT), thus de novo SL synthesis, is suppressed, while maintaining the capacity of taking up and metabolizing exogenous sphingoid bases from the culture medium. In this study, LY-B cells were adapted to grow in a fetal bovine serum (FBS)-deficient medium to avoid external uptake of lipids. The lowest FBS concentration that allowed LY-B cell growth, though at a slow rate, under our conditions was 0.04%, that is, 250-fold less than the standard (10%) concentration. Cells grown under limiting SL concentrations remained viable for at least 72 hours. Enriching with sphingomyelin the SL-deficient medium allowed the recovery of growth rates analogous to those of control LY-B cells. Studies including whole cells, plasma membrane preparations, and derived lipid vesicles were carried out. Laurdan fluorescence was recorded to measure membrane molecular order, showing a significant decrease in the rigidity of LY-B cells, not only in plasma membrane but also in whole cell lipid extract, as a result of SL limitation in the growth medium. Plasma membrane preparations and whole cell lipid extracts were also studied using atomic force microscopy in the force spectroscopy mode. Force measurements demonstrated that lower breakthrough forces were required to penetrate samples obtained from SL-poor LY-B cells than those obtained from control cells. Mass-spectroscopic analysis was also a helpful tool to understand the rearrangement undergone by the LY-B cell lipid metabolism. The most abundant SL in LY-B cells, sphingomyelin, decreased by about 85% as a result of SL limitation in the medium, the bioactive lipid ceramide and the ganglioside precursor hexosylceramide decreased similarly, together with cholesterol. Quantitative SL analysis showed that a 250-fold reduction in sphingolipid supply to LY-B cells led only to a sixfold decrease in membrane sphingolipids, underlining the resistance to changes in composition of these cells. Plasma membrane compositions exhibited similar changes, at least qualitatively, as the whole cells with SL restriction. A linear correlation was observed between the sphingomyelin concentration in the membranes, the degree of lipid order as measured by laurdan fluorescence, and membrane breakthrough forces assessed by atomic force microscopy. Smaller, though significant, changes were also detected in glycerophospholipids under SL-restriction conditions., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

To image membrane tension in selected membranes of interest (MOI) inside living systems, the field of mechanobiology requires increasingly elaborated small-molecule chemical tools. We have recently introduced HaloFlipper, i.e., a mechanosensitive flipper probe that can localize in the MOI using HaloTag technology to report local membrane tension changes using fluorescence lifetime imaging microscopy. However, the linker tethering the probe to HaloTag hampers the lateral diffusion of the probe in all the lipid domains of the MOI. For a more global membrane tension measurement in any MOI, we present here a supramolecular chemistry strategy for selective localization and controlled release of flipper into the MOI, using a genetically encoded supramolecular tag. SupraFlippers, functionalized with a desthiobiotin ligand, can selectively accumulate in the organelle having expressed streptavidin. The addition of biotin as a biocompatible external stimulus with a higher affinity for Sav triggers the release of the probe, which spontaneously partitions into the MOI. Freed in the lumen of endoplasmic reticulum (ER), SupraFlippers report the membrane orders along the secretory pathway from the ER over the Golgi apparatus to the plasma membrane. Kinetics of the process are governed by both the probe release and the transport through lipid domains. The concentration of biotin can control the former, while the expression level of a transmembrane protein (Sec12) involved in the stimulation of the vesicular transport from ER to Golgi influences the latter. Finally, the generation of a cell-penetrating and fully functional Sav-flipper complex using cyclic oligochalcogenide (COC) transporters allows us to combine the SupraFlipper strategy and HaloTag technology., Competing Interests: The authors declare the following competing financial interest(s): The University of Geneva has licensed four Flipper-TR probes to Spirochrome for commercialization., (© 2021 The Authors. Published by American Chemical Society.)

Sphingolipids play important roles in physiology and cell biology, but a systematic examination of their functions is lacking. We performed a genome-wide CRISPRi screen in sphingolipid-depleted human cells and identified hypersensitive mutants in genes of membrane trafficking and lipid biosynthesis, including ether lipid synthesis. Systematic lipidomic analysis showed a coordinate regulation of ether lipids with sphingolipids, suggesting an adaptation and functional compensation. Biophysical experiments on model membranes show common properties of these structurally diverse lipids that also share a known function as glycosylphosphatidylinositol (GPI) anchors in different kingdoms of life. Molecular dynamics simulations show a selective enrichment of ether phosphatidylcholine around p24 proteins, which are receptors for the export of GPI-anchored proteins and have been shown to bind a specific sphingomyelin species. Our results support a model of convergent evolution of proteins and lipids, based on their physico-chemical properties, to regulate GPI-anchored protein transport and maintain homeostasis in the early secretory pathway., Competing Interests: Declaration of Interests The University of Geneva has licensed Flipper-TR to Spirochrome for commercialization. J.S.W. consults for and holds equity in KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, and Amgen and is a venture partner at 5AM Ventures., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Tools to image membrane tension in response to mechanical stimuli are badly needed in mechanobiology. We have recently introduced mechanosensitive flipper probes to report quantitatively global membrane tension changes in fluorescence lifetime imaging microscopy (FLIM) images of living cells. However, to address specific questions on physical forces in biology, the probes need to be localized precisely in the membrane of interest (MOI). Herein we present a general strategy to image the tension of the MOI by tagging our newly introduced HaloFlippers to self-labeling HaloTags fused to proteins in this membrane. The critical challenge in the construction of operational HaloFlippers is the tether linking the flipper and the HaloTag: It must be neither too taut nor too loose, be hydrophilic but lipophilic enough to passively diffuse across membranes to reach the HaloTags, and allow partitioning of flippers into the MOI after the reaction. HaloFlippers with the best tether show localized and selective fluorescence after reacting with HaloTags that are close enough to the MOI but remain nonemissive if the MOI cannot be reached. Their fluorescence lifetime in FLIM images varies depending on the nature of the MOI and responds to myriocin-mediated sphingomyelin depletion as well as to osmotic stress. The response to changes in such precisely localized membrane tension follows the validated principles, thus confirming intact mechanosensitivity. Examples covered include HaloTags in the Golgi apparatus, peroxisomes, endolysosomes, and the ER, all thus becoming accessible to the selective fluorescence imaging of membrane tension., Competing Interests: The authors declare the following competing financial interest(s): The University of Geneva has licensed Flipper-TR to Spirochrome for commercialization., (Copyright © 2020 American Chemical Society.)

This study was aimed at preparing and characterizing plasma membranes (PM) from Chinese Hamster Ovary (CHO) cells. Two methods of PM preparation were applied, one based on adhering cells to a poly-lysine-coated surface, followed by hypotonic lysis and removal of intracellular components, so that PM patches remain adhered to each other, and a second one consisting of bleb induction in cells, followed by separation of giant plasma membrane vesicles (GPMV). Both methods gave rise to PM in sufficient amounts to allow biophysical and biochemical characterization. Laurdan generalized polarization was used to measure molecular order in membranes, PM preparations were clearly more ordered than the average cell membranes (GP ≈0.450 vs. ≈0.20 respectively). Atomic force microscopy was used in the force spectroscopy mode to measure breakthrough forces of PM, both PM preparations provided values in the 4-6 nN range, while the corresponding value for whole cell lipid extracts was ≈2 nN. Lipidomic analysis of the PM preparations revealed that, as compared to the average cell membranes, PM were enriched in phospholipids containing 30-32 C atoms in their acyl chains but were relatively poor in those containing 34-40 C atoms. PM contained more saturated and less polyunsaturated fatty acids than the average cell membranes. Blebs (GPMV) and patches were very similar in their lipid composition, except that blebs contained four-fold the amount of cholesterol of patches (≈23 vs. ≈6 mol% total membrane lipids) while the average cell lipids contained 3 mol%. The differences in lipid composition are in agreement with the observed variations in physical properties between PM and whole cell membranes.

Ether lipids are glycerolipids further classified into alkyl-ether and alkenyl-ether (also termed plasmalogens) lipids. The two ether lipid subclasses share the first steps of their synthesis. However, alkyl-ether and alkenyl-ether lipids differ in their structure and physico-chemical properties (featuring different head groups) and, thus, probably in their functions. Ether lipids have intermittent distribution across the evolutionary tree and defects in their synthesis have been shown to perturb cellular homeostasis and lead to disease in humans. Here, we review their structure, their interactions with other lipids, and their potential roles in cellular functions, such as membrane homeostasis and membrane trafficking. Moreover, we discuss still unclear aspects of these lipids such as their subcellular distribution, and the need to unravel their molecular functions as well as how novel tools to study lipid biology will help clarify these aspects., (© 2019 Federation of European Biochemical Societies.)

The mechanisms of Pb(II) toxicity have been studied in human red blood cells using confocal microscopy, immunolabeling, fluorescence-activated cell sorting and atomic force microscopy. The process follows a sequence of events, starting with calcium entry, followed by potassium release, morphological change, generation of ceramide, lipid flip-flop and finally cell lysis. Clotrimazole blocks potassium channels and the whole process is inhibited. Immunolabeling reveals the generation of ceramide-enriched domains linked to a cell morphological change, while the use of a neutral sphingomyelinase inhibitor greatly delays the process after the morphological change, and lipid flip-flop is significantly reduced. These facts point to three major checkpoints in the process: first the upstream exchange of calcium and potassium, then ceramide domain formation, and finally the downstream scramblase activation necessary for cell lysis. In addition, partial non-cytotoxic cholesterol depletion of red blood cells accelerates the process as the morphological change occurs faster. Cholesterol could have a role in modulating the properties of the ceramide-enriched domains. This work is relevant in the context of cell death, heavy metal toxicity and sphingolipid signaling.

We have studied the effect of adding lipid nanovesicles (liposomes) on the aggregation of commercial titanium oxide (TiO2), zinc oxide (ZnO), or cerium oxide (CeO2) nanoparticles (NPs) suspensions in Hepes buffer. Liposomes were prepared with pure phospholipids or mixtures of phospholipids and/or cholesterol. Changes in turbidity were recorded as a function of time, either of metal nanoparticles alone, or for a mixture of nanoparticles and lipidic nanovesicles. Lipid nanovesicles markedly decrease the NPs tendency to sediment irrespective of size or lipid compositions, thus keeping the metal oxide NPs in suspension. Cryo-electron microscopy, fluorescence anisotropy of TMA-DPH and general polarization of laurdan failed to reveal any major effect of the NPs on the lipid bilayer structure or phase state of the lipids. The above data may help in developing studies of the interaction of inhaled particles with lung surfactant lipids and alveolar macrophages., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Ceramides and dihydroceramides are N-acyl derivatives of sphingosine and sphinganine, respectively, which are the major sphingoid-base backbones of mammals. Recent studies have found that mammals, like certain other organisms, also produce 1-deoxy-(dihydro)ceramides (1-deoxyDHCers) that contain sphingoid bases lacking the 1-hydroxyl- or 1-hydroxymethyl- groups. The amounts of these compounds can be substantial-indeed, we have found comparable levels of 1-deoxyDHCers and ceramides in RAW 264.7 cells maintained in culture. The biophysical properties of 1-deoxyDHCers have not yet been reported, although these lipids might play important roles in normal cell regulation and in the pathology of diseases in which they are elevated, such as hereditary sensory autonomic neuropathies or diabetes. This study uses several approaches, including surface-pressure measurements, differential scanning calorimetry, and confocal microscopy, to study the behavior of 1-deoxyDHCers of different N-acyl-chain lengths and their interaction with sphingomyelin (SM). The thermotropic behaviors of 1-deoxyDHCers alone and in mixtures with SM are described, together with their interactions in monolayers and giant unilamellar vesicles. The gel-fluid transition temperatures of the pure compounds increase in the order 1-deoxyceramide < ceramide ≈ 1-deoxyDHCer < 1-(deoxymethyl)DHCer. In general, canonical ceramides are more miscible with SM in bilayers than are 1-deoxyceramides, and 1-(deoxymethyl)DHCers are the most hydrophobic among them, not even capable of forming monolayers at the air-water interface. Thus, these properties suggest that 1-deoxyDHCer can influence the properties of cellular membranes in ways that might affect biological function/malfunction., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Lipid peroxidation plays a central role in the pathogenesis of many diseases like atherosclerosis and multiple sclerosis. We have analyzed the interaction of sphingosine with peroxidized bilayers in model membranes. Cu(2+) induced peroxidation was checked following UV absorbance at 245nm, and also using the novel Avanti snoopers®. Mass spectrometry confirms the oxidation of phospholipid unsaturated chains. Our results show that sphingosine causes aggregation of Cu(2+)-peroxidized vesicles. We observed that aggregation is facilitated by the presence of negatively-charged phospholipids in the membrane, and inhibited by anti-oxidants e.g. BHT. Interestingly, long-chain alkylamines (C18, C16) but not their short-chain analogues (C10, C6, C1) can substitute sphingosine as promoters of vesicle aggregation. Furthermore, sphinganine but not sphingosine-1-phosphate can mimic this effect. Formation of imines in the membrane upon peroxidation was detected by (1)H-NMR and it appeared to be necessary for the aggregation effect. (31)P-NMR spectroscopy reveals that sphingosine facilitates formation of non-lamellar phase in parallel with vesicle aggregation. The data might suggest a role for sphingosine in the pathogenesis of atherosclerosis., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Sphingosine [(2S, 3R, 4E)-2-amino-4-octadecen-1, 3-diol] is the most common sphingoid long chain base in sphingolipids. It is the precursor of important cell signaling molecules, such as ceramides. In the last decade it has been shown to act itself as a potent metabolic signaling molecule, by activating a number of protein kinases. Moreover, sphingosine has been found to permeabilize phospholipid bilayers, giving rise to vesicle leakage. The present contribution intends to analyze the mechanism by which this bioactive lipid induces vesicle contents release, and the effect of negatively charged bilayers in the release process. Fluorescence lifetime measurements and confocal fluorescence microscopy have been applied to observe the mechanism of sphingosine efflux from large and giant unilamellar vesicles; a graded-release efflux has been detected. Additionally, stopped-flow measurements have shown that the rate of vesicle permeabilization increases with sphingosine concentration. Because at the physiological pH sphingosine has a net positive charge, its interaction with negatively charged phospholipids (e.g., bilayers containing phosphatidic acid together with sphingomyelins, phosphatidylethanolamine, and cholesterol) gives rise to a release of vesicular contents, faster than with electrically neutral bilayers. Furthermore, phosphorous 31-NMR and x-ray data show the capacity of sphingosine to facilitate the formation of nonbilayer (cubic phase) intermediates in negatively charged membranes. The data might explain the pathogenesis of Niemann-Pick type C1 disease., (Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

The thermotropic properties of aqueous dispersions of sphingomyelins (SM) and ceramides (Cer) with N-acyl chains varying from C6:0 to C24:1, either pure or in binary mixtures, have been examined by differential scanning calorimetry. Even in the pure state, Cer and particularly SM exhibited complex endotherms, and their thermal properties did not vary in a predictable way with changes in structure. In some cases, e.g. C18:0 SM, atomic force microscopy revealed coexisting lamellar domains made of a single lipid. Partial chain interdigitation and metastable crystalline states were deemed responsible for the complex behavior. SM:Cer mixtures (90:10mol ratio) gave rise to bilayers containing separate SM-rich and Cer-rich domains. In vesicles made of more complex mixtures (SM:PE:Chol, 2:1:1), it is known that sphingomyelinase degradation of SM to Cer is accompanied by vesicle aggregation and release of aqueous contents. These vesicles did not reveal observable domain separation by confocal microscopy. Vesicle aggregation occurred at a faster rate for those bilayers that appeared to be more fluid according to differential scanning calorimetry. Content efflux rates measured by fluorescence spectroscopy were highest with C18:0 and C18:1 SM, and in general those rates did not vary regularly with other physical properties of SM or Cer. In general the individual SM and Cer appear to have particular thermotropic properties, often unrelated to the changes in N-acyl chain., (© 2013.)