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The endometrium plays a crucial role in reproduction and, in humans, is cyclically remodeled under hormonal control. Estradiol favors tissue proliferation whereas progesterone inhibits tissue growth and induces morphological changes. Endometriosis is often associated with fertility issues and with exacerbated estrogen and reduced progesterone concentration or response in the eutopic endometrium. However, underlying mechanisms remain unclear. Progesterone Receptor Membrane Component (PGRMC) 1 is a protein able to modulate progesterone response and its murine knockout reduced fertility. However, the precise spatiotemporal pattern of PGRMC1 expression in the human endometrium is still poorly characterized. We investigated variations of eutopic endometrial PGRMC1 expression by combining RT-qPCR, immunofluorescence and in situ hybridization. We found that PGRMC1 expression progressively increases during the proliferative phase and decreases during the secretory phase. However, immunolabeling and identification of mRNA-containing cells were regularly heterogeneous in samples, according to tissue depth, with a gradient extending from the surface epithelium towards the basalis. There was no significant difference in PGRMC1 mRNA amounts between patients with or without endometriosis or adenomyosis, for any phase of the menstrual cycle, but cells with strong or moderate PGRMC1 immunolabeling were reduced during the proliferative phase in endometriotic patients. In conclusion, although the cyclical variation of PGRMC1 expression globally follows fluctuation of ovarian steroids, further work is required to precisely characterize hormonal control and identify the additional levels of regulation responsible for local adjustment of PGRMC1 concentration. This is particularly important in the light of recent studies emphasizing the correlation between adequate PGRMC1 amounts and fertility.

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.

The concept of transient nanometric domains known as lipid rafts has brought interest to reassess the validity of the Singer-Nicholson model of a fluid bilayer for cell membranes. However, this new view is still insufficient to explain the cellular control of surface lipid diversity or membrane deformability. During the past decade, the hypothesis that some lipids form large (submicrometric/mesoscale vs nanometric rafts) and stable (> min vs sec) membrane domains has emerged, largely based on indirect methods. Morphological evidence for stable submicrometric lipid domains, well-accepted for artificial and highly specialized biological membranes, was further reported for a variety of living cells from prokaryotes to yeast and mammalian cells. However, results remained questioned based on limitations of available fluorescent tools, use of poor lipid fixatives, and imaging artifacts due to non-resolved membrane projections. In this review, we will discuss recent evidence generated using powerful and innovative approaches such as lipid-specific toxin fragments that support the existence of submicrometric domains. We will integrate documented mechanisms involved in the formation and maintenance of these domains, and provide a perspective on their relevance on membrane deformability and regulation of membrane protein distribution.

Stearoyl-coenzyme A desaturase (SCD) catalyzes the Δ9-cis desaturation of saturated fatty acids (SFA) to generate monounsaturated fatty acids (MUFA). This enzyme is highly up-regulated by platelet-derived growth factor (PDGF) in human fibroblasts. Accordingly, the analysis of cellular fatty acids by gas chromatography showed that PDGF significantly increased the proportion of MUFA, particularly palmitoleate, in cellular lipids. To further analyze the role of SCD in fibroblasts, we used small hairpin RNA targeting SCD (shSCD), which decreased the MUFA content. SCD down-regulation blunted the proliferation of fibroblasts in response to PDGF. This was confirmed using a pharmacological inhibitor of SCD. In addition, proliferation was blocked by palmitate and stearate (two SCD substrates) but not by palmitoleate and oleate (two SCD products). In the presence of an equal amount of oleate, palmitate had no effect on cell proliferation. SCD inhibition or down-regulation did not decrease PDGF receptor activity or signaling. However, by measuring plasma membrane lipid lateral diffusion by fluorescence recovery after photobleaching, we showed that the modulation of the MUFA/SFA ratio by PDGF and SCD inhibitor was related to modifications of membrane fluidity. Altogether, our data suggest that SCD is required for the response of normal fibroblasts to growth factors.

We recently reported that trace insertion of exogenous fluorescent (green BODIPY) analogs of sphingomyelin into living red blood cells partially spread onto coverslips labels submicrometric domains, visible by confocal microscopy. We here extend this feature to endogenous sphingomyelin, upon binding of a sphingomyelin-specific non-toxic fragment of the earthworm toxin, lysenin, fused to the red monomeric fluorescent protein, mCherry (construct named lysenin*). Specificity of lysenin* binding was verified with composition-defined liposomes and by loss of 125I-lysenin* binding to erythrocytes upon sphingomyelin depletion by sphingomyelinase. 125I-lysenin* binding isotherm indicated saturation at 3.5x10^6molecules/red blood cell, i.e. ~3% of sphingomyelin coverage. Non-saturating lysenin* concentration also labeled submicrometric domains on the plasma membrane of partially spread erythrocytes, co-localizing with inserted green BODIPY-sphingomyelin, and abrogated by sphingomyelinase. Lysenin*-labeled domains were stable in time and space and were regulated by temperature and cholesterol. The abundance, size, positioning and segregation of lysenin*-labeled domains from other lipids (BODIPY-phosphatidylcholine or -glycosphingolipids) depended on membrane tension. Similar lysenin*-labeled domains were evidenced in red blood cells gently suspended in three-dimensional gel. Taken together, these data demonstrate submicrometric compartmentation of endogenous sphingomyelin at the membrane of a living cell in vitro, and suggest it may be a genuine feature of erythrocytes in vivo.

Rapid changes in cell volume characterize macrophage activation, but the role of water channels in inflammation remains unclear. We show here that, in vitro, aquaporin (AQP) blockade or deficiency results in reduced IL-1β release by macrophages activated with a variety of NLRP3 activators. Inhibition of AQP specifically during the regulatory volume decrease process is sufficient to limit IL-1β release by macrophages through the NLRP3 inflammasome axis. The immune-related activity of AQP was confirmed in vivo in a model of acute lung inflammation induced by crystals. AQP1 deficiency is associated with a marked reduction of both lung IL-1β release and neutrophilic inflammation. We conclude that AQP-mediated water transport in macrophages constitutes a general danger signal required for NLRP3-related inflammation. Our findings reveal a new function of AQP in the inflammatory process and suggest a novel therapeutic target for anti-inflammatory therapy.

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.

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.

The elastic properties of membrane bilayers are key parameters that control its deformation and can be affected by pharmacological agents. Our previous atomic force microscopy studies revealed that the macrolide antibiotic, azithromycin, leads to erosion of DPPC domains in a fluid DOPC matrix [A. Berquand, M. P. Mingeot-Leclercq, Y. F. Dufrene, Real-time imaging of drug-membrane interactions by atomic force microscopy, Biochim. Biophys. Acta 1664 (2004) 198-205.]. Since this observation could be due to an effect on DOPC cohesion, we investigated the effect of azithromycin on elastic properties of DOPC giant unilamellar vesicles (GUVs). Microcinematographic and morphometric analyses revealed that azithromycin addition enhanced lipid membranes fluctuations, leading to eventual disruption of the largest GUVs. These effects were related to change of elastic moduli of DOPC, quantified by the micropipette aspiration technique. Azithromycin decreased both the bending modulus (k(c), from 23.1+/-3.5 to 10.6+/-4.5 k(B)T) and the apparent area compressibility modulus (K(app), from 176+/-35 to 113+/-25 mN/m). These data suggested that insertion of azithromycin into the DOPC bilayer reduced the requirement level of both the energy for thermal fluctuations and the stress to stretch the bilayer. Computer modeling of azithromycin interaction with DOPC bilayer, based on minimal energy, independently predicted that azithromycin (i) inserts at the interface of phospholipid bilayers, (ii) decreases the energy of interaction between DOPC molecules, and (iii) increases the mean surface occupied by each phospholipid molecule. We conclude that azithromycin inserts into the DOPC lipid bilayer, so as to decrease its cohesion and to facilitate the merging of DPPC into the DOPC fluid matrix, as observed by atomic force microscopy. These investigations, based on three complementary approaches, provide the first biophysical evidence for the ability of an amphiphilic antibiotic to alter lipid elastic moduli. This may be an important determinant for drug: lipid interactions and cellular pharmacology.

Cancer cells depend on chemotaxis for invasion and frequently overexpress and/or activate Src. We previously reported that v-Src accelerates motility by promoting phosphoinositide 3-kinase (PI3-K) signalling but abrogates chemotaxis. We here addressed the mechanism of the loss of chemotactic response to platelet-derived growth factor (PDGF) gradients in fibroblasts harbouring a thermosensitive v-Src kinase. At non-permissive temperature, PDGF receptor (PDGFR) signalling, assessed by phosphoY(751)-specific antibodies (a docking site for PI3-K), was not detected without PDGF and showed a concentration-dependent PDGF response. Both immunolabeling of PI3-K (p110) and live cell imaging of its product (phosphatidylinositol 3,4,5 tris-phosphate) showed PI3-K recruitment and activation at lamellipodia polarized towards a PDGF gradient. Centrosomes and PDGFR- and Src-bearing endosomes were also oriented towards this gradient. Upon v-Src thermoactivation, (i) Y(751) phosphorylation was moderately induced without PDGF and synergistically increased with PDGF; (ii) PI3-K was recruited and activated all along the plasma membrane without PDGF and did not polarize in response to a PDGF gradient; and (iii) polarization of centrosomes and of PDGFR-bearing endosomes were also abrogated. Thus, PDGF can further increase PDGFR auto-phosphorylation despite strong Src kinase activity, but diffuse downstream activation of PI3-K by Src abrogates cell polarization and chemotaxis: "signalling requires silence".

Although direct evidence for lipid domains in living plasma membranes have been recently provided, their existence is still up for debate as their functions remain unclear. Vital imaging of the smooth erythrocyte plasma membrane allowed us to highlight two distinct populations of submicrometric domains with differential cholesterol and sphingomyelin enrichment. We here report direct evidence for the implication of these two domains in key steps of erythrocyte deformation, combining erythrocyte models exhibiting differential membrane curvature (healthy erythrocytes vs erythrocytes from patients with elliptocytosis and spherocytosis, two erythrocyte membrane fragility diseases), (de)stretching biophysical experiments and the decoration of endogenous cholesterol and sphingomyelin by specific fluorescent toxin fragments. Whereas cholesterol-enriched domains were implicated in the generation/maintenance of high membrane curvature areas needed for membrane bending, sphingomyelin-enriched domains were involved in calcium efflux during erythrocyte shape restoration. Our results indicate that cholesterol- and sphingomyelin-enriched domains play distinct key roles in erythrocyte deformation, as confirmed by the specific domain loss upon erythrocyte aging and erythrocyte deformability impairment upon domain abrogation by pharmacological approaches. Thus, whereas cholesterol- and sphingomyelin-enriched domains were so far highly associated, our findings give direct evidence for differential biophysical properties but also distinct physiopathological roles: (i) membrane bending modulators and/or stabilizers (cholesterol-enriched domains), (ii) platforms for protein sorting and/or activation (e.g. calcium efflux pump; sphingomyelin-enriched domains), and (iii) preferential fragility sites for membrane vesiculation (both domains).

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.

La senescence cellulaire est caracterisee par un arret irreversible de la proliferation, malgre le maintien d’apports nutritionnels et de stimulations mitotiques. Les keratinocytes epidermiques humains normaux atteignent la senescence replicative en culture apres une dizaine de passages, ainsi que le montrent l’augmentation de la proportion des cellules positives a l’activite SA-sgal et le ralentissement de leur proliferation. Des etudes de notre laboratoire ont montre que la perturbation des microdomaines lipidiques par une depletion du cholesterol membranaire via la MβCD induit l’activation de plusieurs voies de signalisation (EGFR, p38MAPK, etc.). L’objectif de notre recherche a ete d’identifier les modifications eventuelles de ces domaines lipidiques au cours de la senescence epidermique. Nos resultats montrent une diminution de l’activation de l’EGFR en reponse a la MβCD lors de la senescence des keratinocytes, sans diminution d’expression d’EGFR. L’imagerie microscopique vitale sur des keratinocytes a differents stades (jeunes, pre-senescents et senescents) nous a permis de denombrer et de determiner la composition et la fonctionnalite des microdomaines lipidiques. Ces observations sont effectuees en utilisant deux sondes fluorescentes specifiques de la sphingomyeline et du cholesterol, les constituants majeurs des microdomaines lipidiques, et permettent d’y confirmer la localisation de l’EGFR. Nous avons demontre par cette approche la diminution du contenu en sphingomyeline dans les microdomaines lipidiques lors de la senescence des keratinocytes humains epidermiques.