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Sitosterolemia is a metabolic disorder leading to excessive accumulation of phytosterols. Hemolytic stomatocytosis and macrothrombocytopenia are part of the clinical picture. However, the impact of phytosterols on red blood cell (RBC) deformability, membrane lipid composition and distribution and the efficiency of the reference treatment, Ezetimibe, are largely unknown. This study addresses these issues using RBCs from three patients with sitosterolemia and healthy RBCs exposed to β-sitosterol. Patients presented an increased proportion of stomatocytes, decreased RBC deformability and increased RBC hydration and osmotic fragility compared to healthy donors. At the membrane level, patient RBCs showed (i) very high content in β-sitosterols, (ii) increased proportions of saturated fatty acids and polyunsaturated fatty acid species with long and unsaturated carbon chains, and (iii) decreased content in phosphatidylethanolamine species. These lipid changes were accompanied by an almost complete abrogation of cholesterol-enriched domains, which could result from: (i) the reduced phosphatidylethanolamine content which positively correlated with domain abundance; and (ii) the fatty acid modifications and increased phytosterol content, both compatible with higher membrane stiffness. The role of β-sitosterol was supported by comparable changes in RBC morphology and cholesterol-enriched domains upon β-sitosterol integration at the healthy RBC membrane. Finally, Ezetimibe treatment combined with a sterol restricted diet lowered phytosterols and improved anemia and RBC deformability and hydration. However, this treatment had no or limited effect on RBC morphology and cholesterol-enriched domain abundance. This study reveals for the first time that phytosterols affect RBC membrane lipid composition and distribution but also RBC morphology, hydration, deformability and fragility.

The membrane activity of some saponins, such as digitonin or alpha-hederin, is usually attributed to their interaction with membrane cholesterol (Chol). This contrasts with our recent publication showing that Chol, contrary to sphingomyelin (SM), can delay the cytotoxicity of the saponin ginsenoside Rh2, challenging the usual view that most saponins mediate their membrane effects through interaction with Chol. The aim of the present study was to elucidate the respective importance of Chol and SM as compared to phosphatidylcholine (PC) species in the membrane-related effects of Rh2. On simple lipid monolayers, Rh2 interacted more favorably with eggSM and DOPC than with Chol and eggPC. Using Large Unilamellar Vesicles (LUVs) of binary or ternary lipid compositions, we showed that Rh2 increased vesicle size, decreased membrane fluidity and induced membrane permeability with the following preference: eggSM:eggPC > eggSM:eggPC:Chol > eggPC:Chol. On Giant Unilamellar Vesicles (GUVs), we evidenced that Rh2 generated positive curvatures in eggSM-containing GUVs and small buds followed by intra-luminal vesicles in eggSM-free GUVs. Altogether, our data indicate that eggSM promotes and accelerates membrane-related effects induced by Rh2 whereas Chol slows down and depresses these effects. This study reconsiders the theory that Chol is the only responsible for the activity of saponins.

Background/Aims: Red blood cells (RBC) have been shown to exhibit stable submicrometric lipid domains enriched in cholesterol (chol), sphingomyelin (SM), phosphatidylcholine (PC) or ganglioside GM1, which represent the four main lipid classes of their outer plasma membrane leaflet. However, whether those lipid domains co-exist at the RBC surface or are spatially related and whether and how they are subjected to reorganization upon RBC deformation are not known. Methods: Using fluorescence and/or confocal microscopy and well-validated probes, we compared these four lipid-enriched domains for their abundance, curvature association, lipid order, temperature dependence, spatial dissociation and sensitivity to RBC mechanical stimulation. Results: Our data suggest that three populations of lipid domains with decreasing abundance coexist at the RBC surface: (i) chol-enriched ones, associated with RBC high curvature areas; (ii) GM1/PC/chol-enriched ones, present in low curvature areas; and (iii) SM/PC/chol-enriched ones, also found in low curvature areas. Whereas chol-enriched domains gather in increased curvature areas upon RBC deformation, low curvature-associated lipid domains increase in abundance either upon calcium influx during RBC deformation (GM1/PC/chol-enriched domains) or upon secondary calcium efflux during RBC shape restoration (SM/PC/chol-enriched domains). Hence, abrogation of these two domain populations is accompanied by a strong impairment of the intracellular calcium balance. Conclusion: Lipid domains could contribute to calcium influx and efflux by controlling the membrane distribution and/or the activity of the mechano-activated ion channel Piezo1 and the calcium pump PMCA. Whether this results from lipid domain biophysical properties, the strength of their anchorage to the underlying cytoskeleton and/or their correspondence with inner plasma membrane leaflet lipids remains to be demonstrated.

[Authors equally contributed to the work : Hélène Pollet, Anne-Sophie Cloos] Red blood cell (RBC) deformability is altered in inherited RBC disorders but the mechanism behind this is poorly understood. Here, we explored the molecular, biophysical, morphological, and functional consequences of α-spectrin mutations in a patient with hereditary elliptocytosis (pEl) almost exclusively expressing the Pro260 variant of SPTA1 and her mother (pElm), heterozygous for this mutation. At the molecular level, the pEI RBC proteome was globally preserved but spectrin density at cell edges was increased. Decreased phosphatidylserine vs. increased lysophosphatidylserine species, and enhanced lipid peroxidation, methemoglobin, and plasma acid sphingomyelinase (aSMase) activity were observed. At the biophysical level, although membrane transversal asymmetry was preserved, curvature at RBC edges and rigidity were increased. Lipid domains were altered for membrane:cytoskeleton anchorage, cholesterol content and response to Ca2+ exchange stimulation. At the morphological and functional levels, pEl RBCs exhibited reduced size and circularity, increased fragility and impaired membrane Ca2+ exchanges. The contribution of increased membrane curvature to the pEl phenotype was shown by mechanistic experiments in healthy RBCs upon lysophosphatidylserine membrane insertion. The role of lipid domain defects was proved by cholesterol depletion and aSMase inhibition in pEl. The data indicate that aberrant membrane content and biophysical properties alter pEl RBC morphology and functionality.

The shedding of extracellular vesicles (EVs) from the red blood cell (RBC) surface is observed during senescence in vivo and RBC storage in vitro. Two main models for EV shedding, respectively based on calcium rise and oxidative stress, have been proposed in the literature but the role of the plasma membrane lipid composition and properties is not understood. Using blood in K+/EDTA tubes stored for up to 4 weeks at 4°C as a relevant RBC vesiculation model, we showed here that the RBC plasma membrane lipid composition, organization in domains and biophysical properties were progressively modified during storage and contributed to the RBC vesiculation. First, the membrane content in cholesterol and linoleic acid decreased whereas lipid peroxidation and spectrin:membrane occupancy increased, all compatible with higher membrane rigidity. Second, phosphatidylserine surface exposure showed a first rapid rise due to membrane cholesterol decrease, followed by a second calcium-dependent increase. Third, lipid domains mainly enriched in GM1 or sphingomyelin strongly increased from the 1st week while those mainly enriched in cholesterol or ceramide decreased during the 1st and 4th week, respectively. Fourth, the plasmatic acid sphingomyelinase activity considerably increased upon storage following the sphingomyelin-enriched domain rise and potentially inducing the loss of ceramide-enriched domains. Fifth, in support of the shedding of cholesterol- and ceramide-enriched domains from the RBC surface, the number of cholesterol-enriched domains lost and the abundance of EVs released during the 1st week perfectly matched. Moreover, RBC-derived EVs were enriched in ceramide at the 4th week but depleted in sphingomyelin. Then, using K+/EDTA tubes supplemented with glucose to longer preserve the ATP content, we better defined the sequence of events. Altogether, we showed that EV shedding from lipid domains only represents part of the global vesiculation mechanistics, for which we propose four successive events (cholesterol domain decrease, oxidative stress, sphingomyelin/sphingomyelinase/ceramide/calcium alteration and phosphatidylserine exposure).

Background/Aims: Transient nanometric cholesterol- and sphingolipid-enriched domains, called rafts, are characterized by higher lipid order as compared to surrounding lipids. Here, we asked whether the seminal concept of highly ordered rafts could be refined with the presence of lipid domains exhibiting different enrichment in cholesterol and sphingomyelin and association with erythrocyte curvature areas. We also investigated how differences in lipid order between domains and surrounding membrane (bulk) are regulated and whether changes in order differences could participate to erythrocyte deformation and vesiculation. Methods: We used the fluorescent hydration- and membrane packing-sensitive probe Laurdan to determine by imaging mode the Generalized Polarization (GP) values of lipid domains vs the surrounding membrane. Results: Laurdan revealed the majority of sphingomyelin-enriched domains associated to low erythrocyte curvature areas and part of the cholesterol-enriched domains associated with high curvature. Both lipid domains were less ordered than the surrounding lipids in erythrocytes at resting state. Upon erythrocyte deformation (elliptocytes and stimulation of calcium exchanges) or membrane vesiculation (storage at 4°C), lipid domains became more ordered than the bulk. Upon aging and in membrane fragility diseases (spherocytosis), an increase in the difference of lipid order between domains and the surrounding lipids contributed to the initiation of domain vesiculation. Conclusion: The critical role of domain-bulk differential lipid order modulation for erythrocyte reshaping is discussed in relation with the pressure exerted by the cytoskeleton on the membrane.

Saponins exhibit several biological and pharmacological activities, such as antibacterial, anti-inflammatory and anticancer effects. Many studies attribute their activities to their interactions with cholesterol. In this study, we focus on the steroid saponin ginsenoside Rh2, one of the active principles of Panax ginseng root. Some evidence suggests that lipid rafts, defined as nanodomains enriched in cholesterol and sphingolipids, could be involved in the Rh2-induced apoptosis. However, the role of membrane lipids, especially cholesterol, in this process is still poorly understood. Here, we demonstrate that (i) A549, THP-1 and U937 cells are all susceptible to the Rh2-induced apoptosis but to a differential extent and (ii) the cytotoxic effect inversely correlates with the cell membrane cholesterol content. Upon cholesterol depletion via methyl-β-cyclodextrin, those three cells lines become more sensitive to Rh2-induced apoptosis. Then, focusing on the cholesterol-auxotroph U937 cell line, we showed that Rh2 alters plasma membrane fluidity by compacting the hydrophobic core of lipid bilayer (DPH anisotropy) and relaxing the interfacial packaging of the polar head of phospholipids (TMA-DPH anisotropy). The treatment with Rh2 conducts to the dephosphorylation of Akt and the activation of the intrinsic pathway of apoptosis (loss of mitochondrial membrane potential, caspase-9 and -3 activation). All these features are induced faster in cholesterol-depleted cells, which could be explained by faster cell accumulation of Rh2 in these conditions. This work is the first reporting that membrane cholesterol could delay the activity of ginsenoside Rh2, renewing the idea that saponin cytotoxicity is ascribed to an interaction with membrane cholesterol.

Cellular membrane lateral organization, in particular the assembly of lipids in domains, is difficult to evaluate at high resolution. Here, we used atomic force microscopy (AFM) to investigate at high-resolution lipid membranes containing variable amounts of sphingomyelin (SM) and cholesterol (Chol), two abundant membrane lipids. To this end, we developed new AFM tip functionalization strategies to specifically probe SM and Chol. Multiparametric AFM imaging allowed us to highlight the lateral submicrometric organization of these two lipids within lipid bilayers through the simultaneous topographic evidence of different phase regimes together with the extraction of their nanomechanical properties and the specific detection of lipid moieties by functionalized AFM probes. The combination of AFM topography and nanomechanical mapping with specific probes for molecular recognition of lipids represents a novel approach to identify lipid-enriched domains in supported bilayers and offers a unique perspective to directly observe lipid assemblies in living cells.

Calcium ions (Ca2+) are major messengers in cell signaling, impacting nearly every aspect of cellular life. Those signals are generated within a wide spatial and temporal range through a large variety of Ca2+ channels, pumps, and exchangers. More and more evidences suggest that Ca2+ exchanges are regulated by their surrounding lipid environment. In this review, we point out the technical challenges that are currently being overcome and those that still need to be defeated to analyze the Ca2+ transport protein−lipid interactions. We then provide evidences for the modulation of Ca2+ transport proteins by lipids, including cholesterol, acidic phospholipids, sphingolipids, and their metabolites. We also integrate documented mechanisms involved in the regulation of Ca2+ transport proteins by the lipid environment. Those include: (i) Direct interaction inside the protein with non-annular lipids; (ii) close interaction with the first shell of annular lipids; (iii) regulation of membrane biophysical properties (e.g., membrane lipid packing, thickness, and curvature) directly around the protein through annular lipids; and (iv) gathering and downstream signaling of several proteins inside lipid domains. We finally discuss recent reports supporting the related alteration of Ca2+ and lipids in different pathophysiological events and the possibility to target lipids in Ca2+-related diseases.

The purpose of this study was to assess whether cationic nanoliposomes could address tumor vaccines to dendritic cells in the lungs in vivo. Nanoliposomes were prepared using a cationic lipid, dimethylaminoethanecarbamoyl-cholesterol (DC-cholesterol) or dioleoyltrimethylammoniumpropane (DOTAP), and dipalmitoylphosphatidylcholine (DPPC), the most abundant phospholipid in lung surfactant. The liposomes presented a size below 175 nm and they effectively entrapped tumor antigens, an oligodeoxynucletotide containing CpG motifs (CpG) and the fluorescent dye calcein used as a tracer. Although the liposomes could permanently entrap a large fraction of the actives, they could not sustain their release in vitro. Liposomes made of DOTAP were safe to respiratory cells in vitro, while liposomes composed of DC-cholesterol were cytotoxic. DOTAP nanoliposomes were mainly taken up by alveolar macrophages following delivery to the lungs in mice. Few dendritic cells took up the liposomes, and interstitial macrophages did not take up liposomal calcein more than they took up soluble calcein. Stimulation of the innate immune system using liposomal CpG strongly enhanced uptake of calcein liposomes by all phagocytes in the lungs. Although a small percentage of dendritic cells took up the nanoliposomes, alveolar macrophages represented a major barrier to dendritic cell access in the lungs.

Tumor cells present profound alterations in their composition, structural organization, and functional properties. A landmark of cancer cells is an overall altered mechanical phenotype, which so far are linked to changes in their cytoskeletal regulation and organization. Evidence exists that the plasma membrane (PM) of cancer cells also shows drastic changes in its composition and organization. However, biomechanical characterization of PM remains limited mainly due to the difficulties encountered to investigate it in a quantitative and label‐free manner. Here, the biomechanical properties of PM of a series of MCF10 cell lines, used as a model of breast cancer progression, are investigated. Notably, a strong correlation between the cell PM elasticity and oncogenesis is observed. The altered membrane composition under cancer progression, as emphasized by the PM‐associated cholesterol levels, leads to a stiffening of the PM that is uncoupled from the elastic cytoskeletal properties. Conversely, cholesterol depletion of metastatic cells leads to a softening of their PM, restoring biomechanical properties similar to benign cells. As novel therapies based on targeting membrane lipids in cancer cells represent a promising approach in the field of anticancer drug development, this method contributes to deciphering the functional link between PM lipid content and disease., Here, invasive malignant breast cancer cells are demonstrated to be softer than their healthy and premalignant counterparts and have an increased plasma membrane cholesterol content, increasing its tension and stiffness, which are critical parameters in the mediation of cellular responses.

Membrane lipid raft model has long been debated, but recently the concept of lipid submicrometric domains has emerged to characterize larger (micrometric) and more stable lipid membrane domains. Such domains organize signaling platforms involved in normal or pathological conditions. In this study, adhering human keratinocytes were investigated for their ability to organize such specialized lipid domains. Successful fluorescent probing of lipid domains, by either inserting exogenous sphingomyelin (BODIPY-SM) or using detoxified fragments of lysenin and theta toxins fused to mCherry, allowed specific, sensitive and quantitative detection of sphingomyelin and cholesterol and demonstrated for the first time submicrometric organization of lipid domains in living keratinocytes. Potential functionality of such domains was additionally assessed during replicative senescence, notably through gradual disappearance of SM-rich domains in senescent keratinocytes. Indeed, SM-rich domains were found critical to preserve keratinocyte migration before senescence, because sphingomyelin or cholesterol depletion in keratinocytes significantly alters lipid domains and reduce migration ability.

Although lipid domains have been evidenced in several living cell plasma membranes, their roles remain largely unclear. We here investigated whether they could contribute to function-associated cell (re)shaping. To address this question, we used erythrocytes as cellular model since they (i) exhibit a specific biconcave shape, allowing for reversible deformation in blood circulation, which is lost by membrane vesiculation upon aging; and (ii) display at their outer plasma membrane leaflet two types of submicrometric domains differently enriched in cholesterol and sphingomyelin. We here reveal the specific association of cholesterol- and sphingomyelin-enriched domains with distinct curvature areas of the erythrocyte biconcave membrane. Upon erythrocyte deformation, cholesterol-enriched domains gathered in high curvature areas. In contrast, sphingomyelin-enriched domains increased in abundance upon calcium efflux during shape restoration. Upon erythrocyte storage at 4 °C (to mimick aging), lipid domains appeared as specific vesiculation sites. Altogether, our data indicate that lipid domains could contribute to erythrocyte function-associated (re)shaping.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY (*) analogs of glucosylceramide (GlcCer*), sphingomyelin (SM*) and phosphatidylcholine (PC*). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-β-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC* and SM*, but not GlcCer*, domains, suggesting preferential interaction with lipid* assemblies with the highest vulnerability to methyl-β-cyclodextrin. Surfactin not only reversed disappearance of SM* domains upon cholesterol depletion but further increased PC* domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC* and SM* domains, suggesting increased lipid* recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid* domains is a visual powerful tool, revealing that intrinsic lipid* domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

Micrometric membrane lipid segregation is controversial. We addressed this issue in attached erythrocytes and found that fluorescent boron dipyrromethene (BODIPY) analogs of glycosphingolipids (GSLs) [glucosylceramide (BODIPY-GlcCer) and monosialotetrahexosylganglioside (GM1BODIPY)], sphingomyelin (BODIPY-SM), and phosphatidylcholine (BODIPY-PC inserted into the plasma membrane spontaneously gathered into distinct submicrometric domains. GM1BODIPY domains colocalized with endogenous GM1 labeled by cholera toxin. All BODIPY-lipid domains disappeared upon erythrocyte stretching, indicating control by membrane tension. Minor cholesterol depletion suppressed BODIPY-SM and BODIPY-PC but preserved BODIPY-GlcCer domains. Each type of domain exchanged constituents but assumed fixed positions, suggesting self-clustering and anchorage to spectrin. Domains showed differential association with 4.1R versus ankyrin complexes upon antibody patching. BODIPY-lipid domains also responded differentially to uncoupling at 4.1R complexes [protein kinase C (PKC) activation] and ankyrin complexes (in spherocytosis, a membrane fragility disease). These data point to micrometric compartmentation of polar BODIPY-lipids modulated by membrane tension, cholesterol, and differential association to the two nonredundant membrane:spectrin anchorage complexes. Micrometric compartmentation might play a role in erythrocyte membrane deformability and fragility.

Extracellular vesicles (EVs) contribute to several pathophysiological processes and appear as emerging targets for disease diagnosis and therapy. However, successful translation from bench to bedside requires deeper understanding of EVs, in particular their diversity, composition, biogenesis and shedding mechanisms. In this review, we focus on plasma membrane-derived microvesicles (MVs), far less appreciated than exosomes. We integrate documented mechanisms involved in MV biogenesis and shedding, focusing on the red blood cell as a model. We then provide a perspective for the relevance of plasma membrane lipid composition and biophysical properties in microvesiculation on red blood cells but also platelets, immune and nervous cells as well as tumor cells. Although only a few data are available in this respect, most of them appear to converge to the idea that modulation of plasma membrane lipid content, transversal asymmetry and lateral heterogeneity in lipid domains may play a significant role in the vesiculation process. We suggest that lipid domains may represent platforms for inclusion/exclusion of membrane lipids and proteins into MVs and that MVs could originate from distinct domains during physiological processes and disease evolution.

Micrometric lipid compartmentation at the plasma membrane is disputed. Using live confocal imaging, we found that three unrelated fluorescent sphingomyelin (SM) analogs spontaneously clustered at the outer leaflet into micrometric domains, contrasting with homogeneous labelling by DiIC18 and TMA-DPH. In erythrocytes, these domains were round, randomly distributed, and reversibly coalesced under hypotonicity. BODIPY-SM and -glucosylceramide showed distinct temperature-dependence, in the same ranking as Tm for corresponding natural lipids, indicating phase behaviour. Scanning electron microscopy excluded micrometric surface structural features. In CHO cells, similar surface micrometric patches were produced by either direct BODIPY-SM insertion or intracellular processing from BODIPY-ceramide, ruling out aggregation artefacts. BODIPY-SM surface micrometric patches were refractory to endocytosis block or actin depolymerization and clustered upon cholesterol deprivation, indicating self-clustering at the plasma membrane. BODIPY-SM excimers further suggested clustering in ordered domains. Segregation of BODIPY-SM and -lactosylceramide micrometric domains showed coexistence of distinct phases. Consistent with micrometric domain boundaries, fluorescence recovery after photobleaching (FRAP) revealed restriction of BODIPY-SM lateral diffusion over long-range, but not short-range, contrasting with comparable high mobile fraction of BODIPY-lactosylceramide in both ranges. Controlled perturbations of endogenous SM pool similarly affected BODIPY-SM domain size by confocal imaging and its mobile fraction by FRAP. The latter evidence supports the hypothesis that, as shown for BODIPY-SM, endogenous SM spontaneously clusters at the plasmalemma outer leaflet of living cells into ordered micrometric domains, defined in shape by liquid-phase coexistence and in size by membrane tension and cholesterol. This proposal remains speculative and calls for further investigations.

Cholesterol is a major regulator of plasma membrane fluidity and deformability but its lateral distribution is poorly understood due to lack of suitable vital probes. The objective of our study was...

Although cholesterol is essential for membrane fluidity and deformability, the level of its lateral heterogeneity at the plasma membrane of living cells is poorly understood due to lack of appropriate probe. We here report on the usefulness of the D4 fragment of Clostridium perfringens toxin fused to mCherry (theta*), as specific, non-toxic, sensitive and quantitative cholesterol-labeling tool, using erythrocyte flat membrane. By confocal microscopy, theta* labels cholesterol-enriched submicrometric domains in coverslip-spread but also gel-suspended (non-stretched) fresh erythrocytes, suggesting in vivo relevance. Cholesterol domains on spread erythrocytes are stable in time and space, restricted by membrane:spectrin anchorage via 4.1R complexes, and depend on temperature and sphingomyelin, indicating combined regulation by extrinsic membrane:cytoskeleton interaction and by intrinsic lipid packing. Cholesterol domains partially co-localize with BODIPY-sphingomyelin-enriched domains. In conclusion, we show that theta* is a useful vital probe to study cholesterol organization and demonstrate that cholesterol forms submicrometric domains in living cells.

Perturbation of lipid metabolism favours progression of Alzheimer disease, in which processing of Amyloid Precursor Protein (APP) has important implications. APP cleavage is tightly regulated by cholesterol and APP fragments regulate lipid homeostasis. Here, we investigated whether up or down regulation of full-length APP expression affected neuronal lipid metabolism. Expression of APP decreased HMG-CoA reductase (HMGCR)-mediated cholesterol biosynthesis and SREBP mRNA levels, while its down regulation had opposite effects. APP and SREBP1 co-immunoprecipitated and co-localized in the Golgi. This interaction prevented Site-2 protease-mediated processing of SREBP1, leading to inhibition of transcription of its target genes. A GXXXG motif in APP sequence was critical for regulation of HMGCR expression. In astrocytes, APP and SREBP1 did not interact nor did APP affect cholesterol biosynthesis. Neuronal expression of APP decreased both HMGCR and cholesterol 24-hydroxylase mRNA levels and consequently cholesterol turnover, leading to inhibition of neuronal activity, which was rescued by geranylgeraniol, generated in the mevalonate pathway, in both APP expressing and mevastatin treated neurons. We conclude that APP controls cholesterol turnover needed for neuronal activity., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Low-density lipoprotein receptor-related protein-1 (LRP-1) is a plasma membrane scavenger and signaling receptor, composed of a large ligand-binding subunit (515-kDa α-chain) linked to a shorter transmembrane subunit (85-kDa β-chain). LRP-1 cell-surface level and function are controlled by proteolytic shedding of its ectodomain. Here, we identified ectodomain sheddases in human HT1080 cells and demonstrated regulation of the cleavage by cholesterol by comparing the classical fibroblastoid type with a spontaneous epithelioid variant, enriched ∼2-fold in cholesterol. Two membrane-associated metalloproteinases were involved in LRP-1 shedding: a disintegrin and metalloproteinase-12 (ADAM-12) and membrane-type 1 matrix metalloproteinase (MT1-MMP). Although both variants expressed similar levels of LRP-1, ADAM-12, MT1-MMP, and specific tissue inhibitor of metalloproteinases-2 (TIMP-2), LRP-1 shedding from epithelioid cells was ∼4-fold lower than from fibroblastoid cells. Release of the ectodomain was triggered by cholesterol depletion in epithelioid cells and impaired by cholesterol overload in fibroblastoid cells. Modulation of LRP-1 shedding on clearance was reflected by accumulation of gelatinases (MMP-2 and MMP-9) in the medium. We conclude that cholesterol exerts an important control on LRP-1 levels and function at the plasma membrane by modulating shedding of its ectodomain, and therefore represents a novel regulator of extracellular proteolytic activities.—Selvais, C., D'Auria, L., Tyteca, D., Perrot, G, Lemoine, P., Troeberg, L., Dedieu, S., Noël, A., Nagase, H., Henriet, P., Courtoy, P. J., Marbaix, E., Emonard, H. Cell cholesterol modulates metalloproteinase-dependent shedding of low-density lipoprotein receptor-related protein-1 (LRP-1) and clearance function.

Most Src family members are diacylated and constitutively associate with membrane "lipid rafts" that coordinate signalling. Whether the monoacylated Src, frequently hyperactive in carcinomas, also localizes at "rafts" remains controversial. Using polarized MDCK cells expressing the thermosensitive v-Src/tsLA31 variant, we here addressed how Src tyrosine-kinase activation may impact on its (i) membrane recruitment, in particular to "lipid rafts"; (ii) subcellular localization; and (iii) signalling. The kinetics of Src-kinase thermoactivation correlated with its recruitment from the cytosol to sedimentable membranes where Src largely resisted solubilisation by non-ionic detergents at 4 degrees C and floated into sucrose density gradients like caveolin-1 and flotillin-2, i.e. "lipid rafts". By immunofluorescence, activated Src showed a dual localization, at apical endosomes/macropinosomes and at the apical plasma membrane. The plasma membrane Src pool did not colocalize with caveolin-1 and flotillin-2, but extensively overlapped GM1 labelling by cholera toxin. Severe ( approximately 70%) cholesterol extraction with methyl-beta-cyclodextrin (MbetaCD) did not abolish "rafts" floatation, but strongly decreased Src association with floating "rafts" and abolished its localization at the apical plasma membrane. Src activation independently activated first the MAP-kinase - ERK1/2 pathway, then the PI3-kinase - Akt pathway. MAP-kinase - ERK1/2 activation was insensitive to MbetaCD, which suppressed Akt phosphorylation and apical endocytosis induced by Src, both depending on the PI3-kinase pathway. We therefore suggest that activated Src is recruited at two membrane compartments, allowing differential signalling, first via ERK1/2 at "non-raft" domains on endosomes, then via PI3-kinase-Akt on a distinct set of "rafts" at the apical plasma membrane. Whether this model is applicable to c-Src remains to be examined.