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As sessile organisms, plants must directly deal with an often complex and adverse environment in which hyperosmotic stress is one of the most serious abiotic factors, challenging cellular physiology and integrity. The plasma membrane (PM) is the hydrophobic barrier between the inside and outside environments of cells and is considered a central compartment in cellular adaptation to diverse stress conditions through dynamic PM remodeling. Endocytosis is a powerful method for rapid remodeling of the PM. In animal cells, different endocytic pathways are activated in response to osmotic stress, while only a few reports are related to the endocytosis response pathway and involve a mechanism in plant cells upon hyperosmotic stress. In this study, using different endocytosis inhibitors, the microdomain-specific dye di-4-ANEPPDHQ, variable-angle total internal reflection fluorescence microscopy (VA-TIRFM), and confocal microscopy, we discovered that internalized Clathrin Light Chain-Green Fluorescent Protein (CLC-GFP) increased under hyperosmotic conditions, accompanied by decreased fluorescence intensity of CLC-GFP at the PM. CLC-GFP tended to have higher diffusion coefficients and a fraction of CLC-GFP molecules underwent slower diffusion upon hyperosmotic stress. Meanwhile, an increased motion range of CLC-GFP was found under hyperosmotic treatment compared with the control. In addition, the order of the PM decreased, but the order of the endosome increased when cells were in hyperosmotic conditions. Hence, our results demonstrated that clathrin-mediated endocytosis and membrane microdomain-associated endocytosis both participate in the adaptation to hyperosmotic stress. These findings will help to further understand the role and the regulatory mechanism involved in plant endocytosis in helping plants adapt to osmotic stress.

International audience; The plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants.

Membrane microdomains or rafts, sterol- and sphingolipid-rich microdomains in the plasma membrane have been studied extensively in mammalian cells. Recently, rafts were found to mediate virulence in a variety of parasites, including Toxoplasma gondii. However, it has been difficult to examine a two-dimensional distribution of lipid molecules at a nanometer scale. We tried to determine the distribution of glycosphingolipids GM1 and GM3, putative raft components in the T. gondii cell membrane in this study, using a rapid-frozen and freeze-fractured immuno-electron microscopy method. This method physically stabilized molecules in situ, to minimize the probability of artefactual disruption. Labeling of GM3, but not GM1, was observed in the exoplasmic (or luminal), but not the cytoplasmic, leaflet of the inner membrane complex (IMC) in T. gondii infected in human foreskin fibroblast-1 (HFF-1). No labeling was detected in any leaflet of the T. gondii plasma membrane. In contrast to HFF-1, T. gondii infected in mouse fibroblast (MF), labelings of both GM1 and GM3 were detected in the IMC luminal leaflet, although GM1's gold labeling density was very low. The same freeze-fracture EM method showed that both GM1 and GM3 were expressed in the exoplasmic leaflet of the MF plasma membrane. However, labeling of only GM3, but not GM1, was detected in the exoplasmic leaflet of the HFF-1 plasma membrane. These results suggest that GM1 or GM3, localized in the IMC, is obtained from the plasma membranes of infected host mammalian cells. Furthermore, the localization of microdomains or rafts in the luminal leaflets of the intracellular confined space IMC organelle of T. gondii suggests a novel characteristic of rafts.

Acidic glycosphingolipids, i.e., gangliosides, are predominantly and consistently expressed in nervous tissues of vertebrates at high levels. Therefore, they are considered to be involved in the development and function of nervous systems. Recent studies involving genetic engineering of glycosyltransferase genes have revealed novel aspects of the roles of gangliosides in the regulation of nervous tissues. In this review, novel findings regarding ganglioside functions and their modes of action elucidated mainly by studies of gene knockout mice are summarized. In particular, the roles of gangliosides in the regulation of lipid rafts to maintain the integrity of nervous systems are reported with a focus on the roles in the regulation of neuro-inflammation and neurodegeneration via complement systems. In addition, recent advances in studies of congenital neurological disorders due to genetic mutations of ganglioside synthase genes and also in the techniques for the analysis of ganglioside functions are introduced.

3′,5′-Cyclic adenosine monophosphate (cAMP) is a ubiquitous second messenger which plays critical roles in cardiac function and disease. In adult mouse ventricular myocytes (AMVMs), several distinct functionally relevant microdomains with tightly compartmentalized cAMP signaling have been described. At least two types of microdomains reside in AMVM plasma membrane which are associated with caveolin-rich raft and non-raft sarcolemma, each with distinct cAMP dynamics and their differential regulation by receptors and cAMP degrading enzymes phosphodiesterases (PDEs). However, it is still unclear how cardiac disease such as hypertrophy leading to heart failure affects cAMP signals specifically in the non-raft membrane microdomains. To answer this question, we generated a novel transgenic mouse line expressing a highly sensitive Förster resonance energy transfer (FRET)-based biosensor E1-CAAX targeted to non-lipid raft membrane microdomains of AMVMs and subjected these mice to pressure overload induced cardiac hypertrophy. We could detect specific changes in PDE3-dependent compartmentation of β-adrenergic receptor induced cAMP in non-raft membrane microdomains which were clearly different from those occurring in caveolin-rich sarcolemma. This indicates differential regulation and distinct responses of these membrane microdomains to cardiac remodeling.

Mammalian sphingolipids, primarily with C24 or C16 acyl chains, reside in the outer leaflet of the plasma membrane. Curiously, little is known how C24 sphingolipids impact cholesterol and membrane microdomains. Here, we present evidence that C24 sphingomyelin, when placed in the outer leaflet, suppresses microdomains in giant unilamellar vesicles and also suppresses submicron domains in the plasma membrane of HeLa cells. Free energy calculations suggested that cholesterol has a preference for the inner leaflet if C24 sphingomyelin is in the outer leaflet. We indeed observe that cholesterol enriches in the inner leaflet (80%) if C24 sphingomyelin is in the outer leaflet. Similarly, cholesterol primarily resides in the cytoplasmic leaflet (80%) in the plasma membrane of human erythrocytes where C24 sphingolipids are naturally abundant in the outer leaflet. We conclude that C24 sphingomyelin uniquely interacts with cholesterol and regulates the lateral organization in asymmetric membranes, potentially by generating cholesterol asymmetry.

Four-transmembrane-domain proteins of the tetraspanin superfamily are the organizers of specific microdomains at the membrane [TERMs (tetraspanin-enriched microdomains)] that incorporate various transmembrane receptors and modulate their activities. The structural aspects of the organization of TERM are poorly understood. In the present study, we investigated the role of gangliosides in the assembly and stability of TERM. We demonstrated that inhibition of the glycosphingolipid biosynthetic pathway with specific inhibitors of glucosylceramide synthase [NB-DGJ (N-butyldeoxygalactonojirimycin) and PPMP (D-threo-1-phenyl-2-hexadecanoylamino-3-morpholino-1-propanol·HCl)] resulted in specific weakening of the interactions involving tetraspanin CD82. Furthermore, ectopic expression of the plasma-membrane-bound sialidase Neu3 in mammary epithelial cells also affected stability of the complexes containing CD82: its association with tetraspanin CD151 was decreased, but the association with EGFR [EGF (epidermal growth factor) receptor] was enhanced. The destabilization of the CD82-containing complexes upon ganglioside depletion correlated with the re-distribution of the proteins within plasma membrane. Importantly, depletion of gangliosides affected EGF-induced signalling only in the presence of CD82. Taken together, our results provide strong evidence that gangliosides play an important role in supporting the integrity of CD82-enriched microdomains. Furthermore, these results demonstrate that the association between different tetraspanins in TERM is controlled by distinct mechanisms and identify Neu3 as a first physiological regulator of the integrity of these microdomains.

The two proteins reggie-1 and reggie-2 (flotillins) were identified in axon-regenerating neurons in the central nervous system and shown to be essential for neurite growth and regeneration in fish and mammals. Reggies/flotillins are microdomain scaffolding proteins sharing biochemical properties with lipid raft molecules, form clusters at the cytoplasmic face of the plasma membrane and interact with signaling molecules in a cell type specific manner. In this review, reggie microdomains, lipid rafts, related scaffolding proteins and caveolin—which, however, are responsible for their own microdomains and functions—are introduced. Moreover, the function of the reggies in axon growth is demonstrated: neurons fail to extend axons after reggie knockdown. Furthermore, our current concept of the molecular mechanism underlying reggie function is presented: the association of glycosyl-phophatidyl inositol (GPJ)-anchored surface proteins with reggie microdomains elicits signals which activate src tyrosine and mitogen-activated protein kinases, as well as small guanosine 5′-triphosphate-hydrolyzing enzymes. This leads to the mobilization of intracellular vesicles and to the recruitment of bulk membrane and specific cargo proteins, such as cadherin, to specific sites of the plasma membrane such as the growth cone of elongating axons. Thus, reggies regulate the targeted delivery of cargo—a process which is required for process extension and growth. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases.

Endomembranes modify the progression of the cytosolic Ca2+ wave and contribute to generate Ca2+ microdomains, both in the cytosol and inside the own organella. The concentration of Ca2+ in the cytosol ([Ca2+]C), the mitochondria ([Ca2+]M) and the nucleus ([Ca2+]N) are similar at rest, but may become very different during cell activation. Mitochondria avidly take up Ca2+ from the high [Ca2+]C microdomains generated during cell activation near Ca2+ channels of the plasma membrane and/or the endomembranes and prevent propagation of the high Ca2+ signal to the bulk cytosol. This shaping of [Ca2+]C signaling is essential for independent regulation of compartmentalized cell functions. On the other hand, a high [Ca2+]M signal is generated selectively in the mitochondria close to the active areas, which tunes up respiration to the increased local needs. The progression of the [Ca2+]C signal to the nucleus may be dampened by mitochondria, the nuclear envelope or higher buffering power inside the nucleoplasm. On the other hand, selective [Ca2+]N signals could be generated by direct release of stored Ca2+ into the nucleoplasm. Ca2+ release could even be restricted to subnuclear domains. Putative Ca2+ stores include the nuclear envelope, their invaginations inside the nucleoplasm (nucleoplasmic reticulum) and nuclear microvesicles. Inositol trisphosphate, cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate have all been reported to produce release of Ca2+ into the nucleoplasm, but contribution of these mechanisms under physiological conditions is still uncertain. © 2006 Elsevier Ltd. All rights reserved., This work was funded by grants from the Ministerio de Educación y Ciencia (BFU2004-02765/BFI and BFU2005-05464) and from Junta de Castilla y León (VA016A05). Pablo Chamero holded a predoctoral fellowship from the Basque government.

The EGF receptor is a transmembrane receptor tyrosine kinase that is enriched in lipid rafts. Subdomains I, II and III of the extracellular domain of the EGF receptor participate in ligand binding and dimer formation. However, the function of the cysteine-rich subdomain IV has not been elucidated. In this study, we analyzed the role of the membrane-proximal portion of subdomain IV in EGF binding and signal transduction. A double Cys→Ala mutation that breaks the most membrane-proximal disulfide bond (Cys600 to Cys612), ablated high affinity ligand binding and substantially reduced signal transduction. A similar mutation that breaks the overlapping Cys596 to Cys604 disulfide had little effect on receptor function. Mutation of residues within the Cys600 to Cys612 disulfide loop did not alter the ligand binding or signal transducing activities of the receptor. Despite the fact that the C600,612A EGF receptor was significantly impaired functionally, this receptor as well as all of the other receptors with mutations in the region of residues 596 to 612 localized normally to lipid rafts. These data suggest that the disulfide-bonded structure of the membrane-proximal portion of the EGF receptor, rather than its primary sequence, is important for EGF binding and signaling but is not involved in localizing the receptor to lipid rafts.

Ras proteins are compartmentalized by dynamic interactions with both plasma membrane microdomains and intracellular membranes. The mechanisms underlying Ras compartmentalization involve a series of protein/lipid, lipid/lipid and cytoskeleton interactions, resulting in the generation of discrete microdomains from which Ras operates. Segregation of Ras proteins to these different platforms regulates the formation of Ras signaling complexes and the generation of discrete signal outputs. This temporal and spatial modulation of Ras signal transduction provides a mechanism for the generation of different biological outcomes from different Ras isoforms, as well as flexibility in the signal output from a single activated isoform.

When human platelets are chilled below 20 degrees C, they undergo cold-induced activation. We have previously shown that cold activation correlates with the main phospholipid phase transition (10-20 degrees C) and induces the formation of large raft aggregates. In addition, we found that the glycoprotein CD36 is selectively enriched within detergent-resistant membranes (DRMs) of cold-activated platelets and is extremely sensitive to treatment with methyl-beta-cyclodextrin (MbetaCD). Here, we further studied the partitioning of downstream signaling molecules within the DRMs. We found that the phospholipase Cgamma2 (PLCgamma2) and the protein tyrosine kinase Syk do not partition exclusively within the DRMs, but their distribution is perturbed by cholesterol extraction. In addition, PLCgamma2 activity increases in cold-activated cells compared to resting platelets and is entirely inhibited after treatment with MbetaCD. The Src-family protein tyrosine kinases Src and Lyn preferentially partition within the DRMs and are profoundly affected by removal of cholesterol. These kinases are non-redundant in cold-activation. CD36, active Lyn, along with inactive Src and PLCgamma2 co-localize in small raft complexes in resting platelets. Cold-activation induces raft aggregation, resulting in changes in the activity of these proteins. These data suggest a crucial role of raft aggregation in the early events of cold-induced platelet activation.

Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing Lo nanodomains.

International audience; Interleukin(IL)-7 is the main homeostatic regulator of CD4 T-lymphocytes (helper) at both central and peripheral levels. Upon activation by IL-7, several signalling pathways, mainly Jak/STAT, PI3K/Akt and MAPK, induce the expression of genes involved in T-cell differentiation, activation and proliferation. We have analyzed the early events of CD4 T-cell activation by IL-7. We have shown that IL-7 in the first few minutes induces the formation of cholesterol-enriched membrane microdomains that compartmentalize its activated receptor and initiate its anchoring to the cytoskeleton supporting the formation of the signalling complex-the signalosome-on the IL-7-receptor cytoplasmic-domains. Here we describe by stimulated emission depletion (STED) microscopy, the key roles played by membrane microdomains and cytoskeleton transient organization in the IL-7-regulated Jak/STAT signalling pathway. We image phospho-STAT5 and cytoskeleton components along IL-7-activation kinetics using appropriate inhibitors. We show that lipid raft inhibitors delay and reduce IL-7-induced Jak1 and Jak3 phosphorylation. Drug-induced disassembly of cytoskeleton inhibits phospho-STAT5 formation, transport and translocation into the nucleus that controls the transcription of genes involved in T-cell activation and proliferation. We fit together the results of these quantitative analyses and propose the following mechanism: activated IL-7-receptors embedded in membrane microdomains induce actin-microfilament meshwork formation, anchoring microtubules that grow radially from rafted receptors to the nuclear membrane. STAT5 phosphorylated by signalosomes are loaded on kinesins and glide along the microtubules across the cytoplasm to reach the nucleus two minutes after IL-7-stimulation. Radial microtubules disappear 15 minutes later while transversal microtubules, independent of phospho-STAT5 transport, begin to bud from the microtubule organization center.

International audience; Plants encounter throughout their life all kinds of microorganisms, such as bacteria, fungi, or oomycetes, with either friendly or unfriendly intentions. During evolution, plants have developed a wide range of defense mechanisms against attackers. In return, adapted microbes have developed strategies to overcome the plant lines of defense, some of these microbes engaging in mutualistic or parasitic endosymbioses. By sensing microbe presence and activating signaling cascades, the plasma membrane through its dynamics plays a crucial role in the ongoing molecular dialogue between plants and microbes. This review describes the contribution of endocytosis to different aspects of plant–microbe interactions, microbe recognition and development of a basal immune response, and colonization of plant cells by endosymbionts. The putative endocytic routes for the entry of microbe molecules or microbes themselves are explored with a special emphasis on clathrin-mediated endocytosis. Finally, we evaluate recent findings that suggest a link between the compartmentalization of plant plasma membrane into microdomains and endocytosis.

Lactosylceramide (LacCer), a neutral glycosphingolipid, is abundantly expressed on human neutrophils, and specifically recognizes several pathogenic microorganisms. LacCer forms membrane microdomains coupled with the Src family kinase Lyn on the plasma membrane, and ligand binding to LacCer activates Lyn, resulting in neutrophil functions. In contrast, neutrophilic differentiated HL-60 cells do not have Lyn-associated LacCer-enriched microdomains and lack LacCer-mediated functions. In neutrophil plasma membranes, the very long fatty acid C24:0 and C24:1 chains are the main components of LacCer, whereas plasma membrane of D-HL-60 cells mainly includes C16-LacCer species. Here, we suggest that LacCer species containing very long fatty acid chains are indispensable for the association of Lyn with LacCer-enriched microdomains and LacCer-mediated functions.

DJ-1 is secreted into the serum and plasma of patients with various diseases. In this study, DJ-1 was found to be secreted into culture media of various cells and the amount of wild-type DJ-1 secreted was two-fold greater than that of mutant DJ-1 of cysteine at 106 (C106). Furthermore, the oxidative status of more than 90% of the DJ-1 secreted from HeLa cells was SOH and SO2H forms of C106. A portion of DJ-1 in cells was localized in microdomains of the membrane. These findings suggest that DJ-1 is secreted through microdomains and that oxidation of DJ-1 at C106 facilitates the secretion.

Item does not contain fulltext Clustering of cell surface receptors into micro-domains in the plasma membrane is an important mechanism for regulating cellular functions. Unfortunately, these domains are often too small to be resolved with conventional optical microscopy. Near-field scanning optical microscopy (NSOM) is a relatively new technique that combines ultra high optical resolution, down to 70 nm, with single molecule detection sensitivity. As such, the technique holds great potential for direct visualisation of domains at the cell surface. Yet, NSOM operation under liquid conditions is far from trivial. In this contribution, we show that the performance of NSOM can be extended to measurements in liquid environments using a diving bell concept. For the first time, individual fluorescent molecules on the membrane of cells in solution are imaged with a spatial resolution of 90 nm. Furthermore, using this technique we have been able to directly visualise nanometric sized domains of the C-type lectin DC-SIGN on the membrane of dendritic cells, both in air and in liquid.

Phosphoprotein associated with GEMs (PAG), also known as Csk-binding protein (Cbp), is a broadly expressed palmitoylated transmembrane adapter protein found in membrane rafts, also called GEMs (glycosphingolipid-enriched membrane microdomains). PAG is known to bind and activate the essential regulator of Src-family kinases, cytoplasmic protein tyrosine kinase Csk. In the present study we used the yeast 2-hybrid system to search for additional proteins which might bind to PAG. We have identified the abundant cytoplasmic adapter protein EBP50 (ezrin/radixin/moesin (ERM)-binding phosphoprotein of 50 kDa), also known as NHERF (Na+/H+ exchanger regulatory factor), as a specific PAG-binding partner. The interaction involves the C-terminal sequence (TRL) of PAG and N-terminal PDZ domain(s) of EBP50. As EBP50 is known to interact via its C-terminal domain with the ERM-family proteins, which in turn bind to actin cytoskeleton, the PAG–EBP50 interaction may be important for connecting membrane rafts to the actin cytoskeleton.

We produced mice lacking the sperm surface protein cyritestin (ADAM 3) and found mutant males are infertile. Similar to fertilin beta (ADAM 2) null sperm (C. Cho et al., 1998, Science 281, 1857–1859), cyritestin null sperm are drastically deficient in adhesion to the egg zona pellucida (0.3% of wild type) and to the egg plasma membrane (9% of wild type). Thus sperm from male mice with a gene deletion of either ADAM have a loss of adhesive function in at least two steps of fertilization. We found deletion of either ADAM gene resulted in the loss of multiple gene products. This loss of multiple gene products (sperm membrane proteins) appears to result from a novel, developmental mechanism during sperm differentiation. Because the altered sperm protein expression must be responsible for the fertilization defects, our data suggest new models for the molecular basis of the affected steps in fertilization.

Plasma membranes regulate the influx and efflux of molecules across themselves and are also responsible for primary signal transduction between cells or within the same cell. Presence of lateral heterogeneity and the ability of reorganization are essential requirements for effective functioning of biomembranes. Lipid rafts are small, heterogeneous, dynamic domains enriched in glycosphingolipids, sphingomyelin and cholesterol, and profoundly influence membrane organization. Glycosphingolipids are inclined towards formation of liquid-ordered phases in membranes, both with and without cholesterol; they are therefore prime players in domain formation. Here, we discuss the role of glycosphingolipids in microdomain formation and their spatial organization within these rafts.

© 2015 Elsevier B.V. Abstract Photosynthesis is the key bioenergetic process taking place in the chloroplast. The components of the photosynthetic machinery are embedded in a highly dynamic matrix the thylakoid membrane. This membrane has the capacity to adapt during developmental transitions and under stress conditions. The galactolipids are the major polar lipid components of the thylakoid membrane conferring bilayer properties while neutral thylakoid lipids such as the prenyllipids and carotenoids contribute to essential functions such as electron transport and photoprotection. Despite a large number of studies the intriguing processes of thylakoid membrane biogenesis and dynamics remain unsolved. Plastoglobules thylakoid associated lipid droplets appear to actively participate in thylakoid function from biogenesis to senescence. Recruitment of specific proteins enables the plastoglobules to act in metabolite synthesis repair and disposal under changing environmental conditions and developmental stages. In this review we describe plastoglobules as thylakoid membrane microdomains and discuss their involvement in lipid remodeling during stress and in the conversion from one plastid type to another. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

Tetraspanins (Tspans) are a family of four-span transmembrane proteins, known as plasma membrane “master organizers.” They form Tspan-enriched microdomains (TEMs or TERMs) through lateral association with one another and other membrane proteins. If multiple microdomains associate with each other, larger platforms can form. For infection, viruses interact with multiple cell surface components, including receptors, activating proteases, and signaling molecules. It appears that Tspans, such as CD151, CD82, CD81, CD63, CD9, Tspan9, and Tspan7, coordinate these associations by concentrating the interacting partners into Tspan platforms. In addition to mediating viral attachment and entry, these platforms may also be involved in intracellular trafficking of internalized viruses and assist in defining virus assembly and exit sites. In conclusion, Tspans play a role in viral infection at different stages of the virus replication cycle. The present review highlights recently published data on this topic, with a focus on events at the plasma membrane. In light of these findings, we propose a model for how Tspan interactions may organize cofactors for viral infection into distinct molecular platforms.