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Membrane rafts play a crucial role in the regulation of many important biological processes. Our data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft-domain formation and regulation, including in cells with low expression of MPP1. We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and MS technique. Thereby EFR3A was identified as a candidate protein which interacts with flotillin-2. This was further confirmed via immunoblotting using anti-EFR3A antibody and via co-immunoprecipitation (Co-IP). Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergentresistant membrane (DRM) fraction of HeLa cells, and its presence was sensitive to removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles (GPMVs) derived from KnD cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor (EGFR) and phospholipase C gamma (PLCγ) phosphorylation and affect EGF-dependent cytosolic Ca2+ concentration. Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which appears to be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling.

International audience; Extensive studies showed the crucial role of ATP binding cassette (ABC) transporter ABCA1 in organizing the lipid microenvironment at the plasma membrane (PM) of living cells. However, the exact role of this protein in terms of lipid redistribution and lateral reorganization of the PM is still being discussed. Here, we took advantage of the spot variation fluorescence correlation spectroscopy (svFCS) to investigate the molecular dynamics of the ABCA1 expressed at the PM of Chinese hamster ovary cells (CHO-K1). We confirmed that this protein is strongly confined into the raft nanodomains. Next, in agreement with our previous observations, we showed that amphotericin B does not affect the diffusion properties of an active ABCA1 in contrary to inactive mutant ABCA1MM. We also evidenced that ApoA1 influences the molecular diffusion properties of ABCA1. Finally, we showed that the molecular confinement of ABCA1 depends on the cholesterol content in the PM, but presumably, this is not the only factor responsible for that. We concluded that the molecular dynamics of ABCA1 strongly depends on its activity and the PM composition. We hypothesize that other factors than lipids (i.e., proteins) are responsible for the strong confinement of ABCA1 in PM nanodomains which possibility has to be elucidated.

Amphotericin B (AmB) belongs to a group of polyene antibiotics commonly used in the treatment of systemic mycotic infections. A widely accepted mechanism of action of AmB is based on the formation of an oligomeric pore structure within the plasma membrane (PM) by interaction with membrane sterols. Although AmB binds preferentially to ergosterol, it can also bind to cholesterol in the mammalian PM and cause severe cellular toxicity. The lipid content and its lateral organization at the cell PM appear to be significant for AmB binding. Several ATP-binding cassette (ABC) transporters, including ABCA1, play a crucial role in lipid translocation, cholesterol redistribution and efflux. Here, we demonstrate that cells expressing ABCA1 are more resistant to AmB treatment, while cells lacking ABCA1 expression or expressing non-active ABCA1MM mutant display increased sensitivity. Further, a FLIM analysis of AmB-treated cells reveals a fraction of the antibiotic molecules, characterized by relatively high fluorescence lifetimes (> 6 ns), involved in formation of bulk cholesterol–AmB structures at the surface of ABCA1-expressing cells. Finally, lowering the cellular cholesterol content abolishes resistance of ABCA1-expressing cells to AmB. Therefore, we propose that ABCA1-mediated cholesterol efflux from cells induces formation of bulk cholesterol–AmB structures at the cell surface, preventing AmB cytotoxicity. Electronic supplementary material The online version of this article (10.1007/s00018-019-03154-w) contains supplementary material, which is available to authorized users.

The specific structure and composition of the cell plasma membrane (PM) is crucial for many cellular processes and can be targeted by various substances with potential medical applications. In this context, biosurfactants (BS) constitute a promising group of natural compounds that possess several biological functions, including anticancer activity. Despite the efficiency of BS, their mode of action had never been elucidated before. Here, we demonstrate the influence of cyclic lipopeptide surfactin (SU) on the PM of CHO-K1 cells. Both FLIM and svFCS experiments show that even a low concentration of SU causes significant changes in the membrane fluidity and dynamic molecular organization. Further, we demonstrate that SU causes a relevant dose-dependent reduction of cellular cholesterol by extracting it from the PM. Finally, we show that CHO-25RA cells characterized by increased cholesterol levels are more sensitive to SU treatment than CHO-K1 cells. We propose that sterols organizing the PM raft nanodomains, constitute a potential target for SU and other biosurfactants. In our opinion, the anticancer activity of biosurfactants is directly related with the higher cholesterol content found in many cancer cells.

ATP-binding cassette transporters are involved in the active transport of a wide variety of metabolites in prokaryotes and eukaryotes. One subfamily, the Pleiotropic Drug Resistance (PDR) transporters, or full-size ABCG transporters, are found only in fungi and plants. NtPDR1 was originally identified in Nicotiana tabacum suspension cells (BY2), in which its expression was induced by microbial elicitors. To obtain information on its expression in plants, we generated NtPDR1-specific antibodies and, using Western blotting, found that this transporter is localized in roots, leaves, and flowers and this was confirmed in transgenic plants expressing the ß-glucuronidase reporter gene fused to the NtPDR1 promoter region. Expression was seen in the lateral roots and in the long glandular trichomes of the leaves, stem, and flowers. Western blot analysis and in situ immunolocalization showed NtPDR1 to be localized in the plasma membrane. Induction of NtPDR1 expression by various compounds was tested in N. tabacum BY2 cells. Induction of expression was observed with the hormones methyl jasmonate and naphthalene acetic acid and diterpenes. Constitutive ectopic expression of NtPDR1 in N. tabacum BY2 cells resulted in increased resistance to several diterpenes. Transport tests directly demonstrated the ability of NtPDR1 to transport diterpenes. These data suggest that NtPDR1 is involved in plant defense through diterpene transport.

Pleiotropic drug resistance (PDR) transporters are a group of membrane proteins belonging to the ABCG sub-family of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. Here, we describe a PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Staining of transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting of wild-type plants showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under standard conditions. However, a small increase in susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: silenced plants allowed better growth and faster development of M. sexta larvae than wild-type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory.

Steady-state polarization-resolved fluorescence imaging is used to analyze the molecular orientational order behavior of rigidly labeled major histocompatibility complex class I (MHC I) proteins and lipid probes in cell membranes of living cells. These fluorescent probes report the orientational properties of proteins and their surrounding lipid environment. We present a statistical study of the molecular orientational order, modeled as the width of the angular distribution of the molecules, for the proteins in the cell endomembrane and plasma membrane, as well as for the lipid probes in the plasma membrane. We apply this methodology on cells after treatments affecting the actin and microtubule networks. We find in particular opposite orientational order changes of proteins and lipid probes in the plasma membrane as a response to the cytoskeleton disruption. This suggests that MHC I orientational order is governed by its interaction with the cytoskeleton, whereas the plasma membrane lipid order is governed by the local cell membrane morphology.

Nicotiana plumbaginifolia NpPDR1, a plasma membrane pleiotropic drug resistance-type ATP-binding cassette transporter formerly named NpABC1, has been suggested to transport the diterpene sclareol, an antifungal compound. However, direct evidence for a role of pleiotropic drug resistance transporters in the plant defense is still lacking. In situ immunolocalization and histochemical analysis using the gusA reporter gene showed that NpPDR1 was constitutively expressed in the whole root, in the leaf glandular trichomes, and in the flower petals. However, NpPDR1 expression was induced in the whole leaf following infection with the fungus Botrytis cinerea, and the bacteria Pseudomonas syringae pv tabaci, Pseudomonas fluorescens, and Pseudomonas marginalis pv marginalis, which do not induce a hypersensitive response in N. plumbaginifolia, whereas a weaker response was observed using P. syringae pv syringae, which does induce a hypersensitive response. Induced NpPDR1 expression was more associated with the jasmonic acid than the salicylic acid signaling pathway. These data suggest that NpPDR1 is involved in both constitutive and jasmonic acid-dependent induced defense. Transgenic plants in which NpPDR1 expression was prevented by RNA interference showed increased sensitivity to sclareol and reduced resistance to B. cinerea. These data show that NpPDR1 is involved in pathogen resistance and thus demonstrate a new role for the ATP-binding cassette transporter family.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τ(d) and the squared waist of observation w(2). Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

While intrinsic Brownian agitation within a lipid bilayer does homogenize the molecular distribution, the extremely diverse composition of the plasma membrane, in contrast, favors the development of inhomogeneity due to the propensity of such a system to minimize its total free energy. Precisely, deciphering such inhomogeneous organization with appropriate spatiotemporal resolution remains, however, a challenge. In accordance with its ability to accurately measure diffusion parameters, fluorescence correlation spectroscopy (FCS) has been developed in association with innovative experimental strategies to monitor modes of molecular lateral confinement within the plasma membrane of living cells. Here, we describe a method, namely spot variation FCS (svFCS), to decipher the dynamics of the plasma membrane organization. The method is based on questioning the relationship between the diffusion time τd and the squared waist of observation w2. Theoretical models have been developed to predict how geometrical constraints such as the presence of adjacent or isolated domains affect the svFCS observations. These investigations have allowed significant progress in the characterization of cell membrane lateral organization at the suboptical level, and have provided, for instance, compelling evidence for the in vivo existence of raft nanodomains.

Pleiotropic drug resistance (PDR) transporters are a group of membrane proteins belonging to the ABCG sub-family of ATP binding cassette (ABC) transporters. There is clear evidence for the involvement of plant ABC transporters in resistance to fungal and bacterial pathogens, but not in the biotic stress response to insect or herbivore attack. Here, we describe a PDR transporter, ABCG5/PDR5, from Nicotiana tabacum. GFP fusion and subcellular fractionation studies revealed that ABCG5/PDR5 is localized to the plasma membrane. Staining of transgenic plants expressing the GUS reporter gene under the control of the ABCG5/PDR5 transcription promoter and immunoblotting of wild-type plants showed that, under standard growth conditions, ABCG5/PDR5 is highly expressed in roots, stems and flowers, but is only expressed at marginal levels in leaves. Interestingly, ABCG5/PDR5 expression is induced in leaves by methyl jasmonate, wounding, pathogen infiltration, or herbivory by Manduca sexta. To address the physiological role of ABCG5/PDR5, N. tabacum plants silenced for the expression of ABCG5/PDR5 were obtained. No phenotypic modification was observed under standard conditions. However, a small increase in susceptibility to the fungus Fusarium oxysporum was observed. A stronger effect was observed in relation to herbivory: silenced plants allowed better growth and faster development of M. sexta larvae than wild-type plants, indicating an involvement of this PDR transporter in resistance to M. sexta herbivory.

International audience; Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at characterizing the complex organization and dynamics of the plasma membrane at single-molecule level, by developing analytic tools dedicated to Single-Particle Tracking (SPT) at high density: Multiple-Target Tracing (MTT)(1). Single-molecule videomicroscopy, offering millisecond and nanometric resolution(1-11), allows a detailed representation of membrane organization(12-14) by accurately mapping descriptors such as cell receptors localization, mobility, confinement or interactions.We revisited SPT, both experimentally and algorithmically. Experimental aspects included optimizing setup and cell labeling, with a particular emphasis on reaching the highest possible labeling density, in order to provide a dynamic snapshot of molecular dynamics as it occurs within the membrane. Algorithmic issues concerned each step used for rebuilding trajectories: peaks detection, estimation and reconnection, addressed by specific tools from image analysis(15,16). Implementing deflation after detection allows rescuing peaks initially hidden by neighboring, stronger peaks. Of note, improving detection directly impacts reconnection, by reducing gaps within trajectories. Performances have been evaluated using Monte-Carlo simulations for various labeling density and noise values, which typically represent the two major limitations for parallel measurements at high spatiotemporal resolution.The nanometric accuracy(17) obtained for single molecules, using either successive on/off photoswitching or non-linear optics, can deliver exhaustive observations. This is the basis of nanoscopy methods(17) such as STORM18, PALM(19,20), RESOLFT21 or STED22,23, which may often require imaging fixed samples. The central task is the detection and estimation of diffraction-limited peaks emanating from single-molecules. Hence, providing adequate assumptions such as handling a constant positional accuracy instead of Brownian motion, MTT is straightforwardly suited for nanoscopic analyses. Furthermore, MTT can fundamentally be used at any scale: not only for molecules, but also for cells or animals, for instance. Hence, MTT is a powerful tracking algorithm that finds applications at molecular and cellular scales.

The plasma membrane delineating the cell is a complex multicomponent assembly of lipids and proteins. Deciphering the lateral organization of this supramolecular complex on appropriate length and temporal scales is necessary to unravel its implication in biological function. Here, we show how measurements of diffusion may be taken to shed light on membrane function. We first describe the current methods used to report lateral diffusion of membrane components. We then focus on one fluorescent correlation spectroscopy (FCS)-based method, namely, the spot variation FCS (svFCS), which allows the characterization of the modes of molecular confinement within the plasma membrane of living cells. We next illustrate with different biological systems the progress made toward improving our understanding of cell membrane function. We also discuss the findings with regard to the current view of nanoscale domains/assemblies as a general membrane-organizing principle.

International audience; ABCA1 belongs to the A class of ABC transporter, which is absent in yeast. ABCA1 elicits lipid translocation at the plasma membrane through yet elusive processes. We successfully expressed the mouse Abca1 gene in Saccharomyces cerevisiae. The cloned ABCA1 distributed at the yeast plasma membrane in stable discrete domains that we name MCA (membrane cluster containing ABCA1) and that do not overlap with the previously identified punctate structures MCC (membrane cluster containing Can1p) and MCP (membrane cluster containing Pma1p). By comparison with a nonfunctional mutant, we demonstrated that ABCA1 elicits specific phenotypes in response to compounds known to interact with membrane lipids, such as papuamide B, amphotericin B and pimaricin. The sensitivity of these novel phenotypes to the genetic modification of the membrane lipid composition was studied by the introduction of the cho1 and lcb1-100 mutations involved respectively in phosphatidylserine or sphingolipid biosynthesis in yeast cells. The results, corroborated by the analysis of equivalent mammalian mutant cell lines, demonstrate that membrane composition, in particular its phosphatidylserine content, influences the function of the transporter. We thus have reconstituted in yeast the essential functions associated to the expression of ABCA1 in mammals and characterized new physiological phenotypes prone to genetic analysis. This article is a part of a Special Issue entitled Advances in High Density Lipoprotein Formation and Metabolism: A Tribute to John F. Oram (1945-2010).

International audience; Natural killer (NK) cell tolerance to self is partly ensured by major histocompatibility complex (MHC) class I-specific inhibitory receptors on NK cells, which dampen their reactivity when engaged. However, NK cells that do not detect self MHC class I are not autoreactive. We used dynamic fluorescence correlation spectroscopy to show that MHC class I-independent NK cell tolerance in mice was associated with the presence of hyporesponsive NK cells in which both activating and inhibitory receptors were confined in an actin meshwork at the plasma membrane. In contrast, the recognition of self MHC class I by inhibitory receptors "educated" NK cells to become fully reactive, and activating NK cell receptors became dynamically compartmentalized in membrane nanodomains. We propose that the confinement of activating receptors at the plasma membrane is pivotal to ensuring the self-tolerance of NK cells.

SUMMARY The behaviour of Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 was investigated in response to fungal and oomycete infections. The importance of NpPDR1 in plant defence was demonstrated for two organs in which NpPDR1 is constitutively expressed: the roots and the petal epidermis. The roots of the plantlets of two lines silenced for NpPDR1 expression were clearly more sensitive than those of controls to the fungal pathogens Botrytis cinerea, Fusarium oxysporum sp., F. oxysporum f. sp. nicotianae, F. oxysporum f. sp. melonis and Rhizoctonia solani, as well as to the oomycete pathogen Phytophthora nicotianae race 0. The Ph gene-linked resistance of N. plumbaginifolia to P. nicotianae race 0 was totally ineffective in NpPDR1-silenced lines. In addition, the petals of the NpPDR1-silenced lines were spotted 15%-20% more rapidly by B. cinerea than were the controls. The rapid induction (after 2-4 days) of NpPDR1 expression in N. plumbaginifolia and N. tabacum mature leaves in response to pathogen presence was demonstrated for the first time with fungi and one oomycete: R. solani, F. oxysporum and P. nicotianae. With B. cinerea, such rapid expression was not observed in healthy mature leaves. NpPDR1 expression was not observed during latent infections of B. cinerea in N. plumbaginifolia and N. tabacum, but was induced when conditions facilitated B. cinerea development in leaves, such as leaf ageing or an initial root infection. This work demonstrates the increased sensitivity of NpPDR1-silenced N. plumbaginifolia plants to all of the fungal and oomycete pathogens investigated.

International audience; The ABCA1 transporter orchestrates cellular lipid homeostasis by promoting the release of cholesterol to plasmatic acceptors. The molecular mechanism is, however, unknown. We report here on the biophysical analysis in living HeLa cells of the ABCA1 lipid microenvironment at the plasma membrane. The modifications of membrane attributes induced by ABCA1 were assessed at both the outer and inner leaflet by monitoring either the lifetime of membrane inserted fluorescent lipid analogues by fluorescence lifetime imaging microscopy (FLIM) or, respectively, the membrane translocation of cationic sensors. Analysis of the partitioning of dedicated probes in plasma membrane blebs vesiculated from these cells allowed visualization of ABCA1 partitioning into the liquid disordered-like phase and corroborated the idea that ABCA1 destabilizes the lipid arrangement at the membrane. Specificity was demonstrated by comparison with cells expressing an inactive transporter. The physiological relevance of these modifications was finally demonstrated by the reduced membrane mobility and function of transferrin receptors under the influence of an active ABCA1. Collectively, these data assess that the control of both transversal and lateral lipid distribution at the membrane is the primary function of ABCA1 and positions the effluxes of cholesterol from cell membranes downstream to the redistribution of the sterol into readily extractable membrane pools.

ATP-binding cassette transporters of the pleiotropic drug resistance (PDR) subfamily are composed of five clusters. We have cloned a gene, NpPDR2, belonging to the still uncharacterized cluster IV from Nicotiana plumbaginifolia. NpPDR2 transcripts were found in the roots and mature flowers. In the latter, NpPDR2 expression was restricted to the style and only after pollination. A 1.5-kb genomic sequence containing the putative NpPDR2 transcription promoter was fused to the beta-glucuronidase reporter gene. The GUS expression pattern confirmed the RT-PCR results that NpPDR2 was expressed in roots and the flower style and showed that it was localized around the conductive tissues. Unlike other PDR genes, NpPDR2 expression was not induced in leaf tissues by none of the hormones typically involved in biotic and abiotic stress response. Moreover, unlike NpPDR1 known to be involved in biotic stress response, NpPDR2 expression was not induced in the style upon Botrytis cinerea infection. In N. plumbaginifolia plants in which NpPDR2 expression was prevented by RNA interference, no unusual phenotype was observed, including at the flowering stage, which suggests that NpPDR2 is not essential in the reproductive process under the tested conditions.

Among the ABC transporters, the pleiotropic drug resistance (PDR) family is particular in that its members are found only in fungi and plants and have a reverse domain organization, i.e., the nucleotide binding domain precedes the transmembrane domain. In Arabidopsis and rice, for which the full genome has been sequenced, the family of plant ABC transporters contains 15 and 23 PDR genes, respectively, which can be tentatively organized using the sequence data into five subfamilies. Most of the plant PDR genes so far characterized belong to subfamily I and have been shown to be involved in responses to abiotic and biotic stress, in the latter case, probably by transporting antimicrobial secondary metabolites to the cell surface. Only a single subfamily II member has been characterized. Induction of its expression by iron deficiency suggests its involvement in iron deficiency stress, thus, enlightening a new physiological role for a PDR gene.

Biomolecular orientational organization of lipids and proteins in plasma membrane is a crucial factor in biological processes where functions can be closely related to orientation and ordering mechanisms. The concept of transient nanosized phase separations in ordered and disordered domains, called “lipid rafts” is now widely accepted. Furthermore, the ordered domains are enriched in signalling proteins, which highlights the crucial impact of phase separation during the signalling processes. While this field has been so far largely addressed by studying the translational diffusion behaviour of membrane proteins by Single Molecule Tracking or Fluorescence Correlation Spectroscopy, only little is known about the orientational behaviour of signalling proteins in plasma membranes, mainly due to the lack of appropriate rigid fluorescent label which would be able to act as a proper orientation reporter. In this work we develop a fully polarization-resolved fluorescence imaging technique using a tuneable incident polarization state (“fluorescence polarimetry”), in combination with fluorescence anisotropy imaging, in order to provide orientational order information in very general cell membranes shapes.We apply this technique to the measurement of quantitative orientational distribution of MHC Class I proteins in the plasma and nuclear membranes, benefiting from a rigidly attached GFP probe. The surrounding lipid orientational order in the plasma membrane is additionally probed using the fluorescent reporter di-8-ANEPPQ.The MHC Class I protein is found to be more ordered in the plasma membrane as compared to the nuclear membrane. Both MHC I and di-8-ANEPPQ orientational orders in the plasma membrane are furthermore seen to be highly affected by actin depolymerisation upon Latrunculin A treatment, with variations that indicate both a structural change in the membrane morphology and a disruption of MHC I - actin interactions.