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Subcellular localization of sphingomyelin revealed by two toxin-based probes in mammalian cells
Abstract
Sphingomyelin (SM) is an abundant phospholipid in cell membranes. However, owing to the lack of appropriate probes, the subcellular distribution of SM remains unclear. In this study, we examined the localization of SM in COS-1 cells (green monkey kidney cells) by using two SM probes, lysenin and equinatoxin-II (EqtII). Both toxins stained SM in the plasma membrane (PM), and the stains were abolished by sphingomyelin synthase 2 (SMS2) knockdown or sphingomyelinase (SMase) treatment. Simultaneous labeling by the two toxins showed that the PM has heterogeneous SM pools: a SM pool stained by only lysenin, a SM pool stained only by EqtII, and a SM pool stained by both toxins. In permeabilized cells, lysenin exclusively stained late endosomes (LEs) among intracellular organelles, whereas EqtII stained recycling endosomes (REs) in addition to LEs. The intracellular SM stains by EqtII were abolished by sphingomyelin synthase 1 (SMS1) knockdown, but not by SMS2 knockdown. These results indicate that lysenin and EqtII label different SM pools and that SMS2 and SMS1 are responsible for the synthesis of SM in the PM and endomembranes, respectively, in COS-1 cells. The use of the two SM-binding probes may provide more insights into various sphingomyelin-mediated processes in different topological domains.
... Sphingolipids participate in various biological events, including cell growth, apoptosis, differentiation, and adhesion (reviewed in [15,16]). The choline-containing sphingophospholipid sphingomyelin is a ubiquitous and predominant type of sphingolipid found in In mammalian cells, sphingomyelin is mainly located in the plasma membrane (PM), although the Golgi apparatus (where sphingomyelin is synthesized) and endosomes/lysosomes (to which the PM-derived endocytosis is directed) also have sphingomyelin [17,18]. In the PM phospholipid bilayer, sphingomyelin is predominantly distributed in the exoplasmic leaflet, with less found in the cytoplasmic leaflet. ...
... Sphingomyelin may interact with specific membrane proteins as their functional modulator, while sphingomyelin is also an important metabolic reservoir for sphingolipid mediators such as ceramide, sphingosine, and sphingosine-1-phosphate, which are produced as catabolites of sphingomyelin (reviewed in [15,[22][23][24]). In mammalian cells, sphingomyelin is mainly located in the plasma membrane (PM), although the Golgi apparatus (where sphingomyelin is synthesized) and endosomes/lysosomes (to which the PM-derived endocytosis is directed) also have sphingomyelin [17,18]. In the PM phospholipid bilayer, sphingomyelin is predominantly distributed in the exoplasmic leaflet, with less found in the cytoplasmic leaflet. ...
Lipid transfer proteins (LTPs) are recognized as key players in the inter-organelle trafficking of lipids and are rapidly gaining attention as a novel molecular target for medicinal products. In mammalian cells, ceramide is newly synthesized in the endoplasmic reticulum (ER) and converted to sphingomyelin in the trans-Golgi regions. The ceramide transport protein CERT, a typical LTP, mediates the ER-to-Golgi transport of ceramide at an ER-distal Golgi membrane contact zone. About 20 years ago, a potent inhibitor of CERT, named (1R,3S)-HPA-12, was found by coincidence among ceramide analogs. Since then, various ceramide-resembling compounds have been found to act as CERT inhibitors. Nevertheless, the inevitable issue remains that natural ligand-mimetic compounds might directly bind both to the desired target and to various undesired targets that share the same natural ligand. To resolve this issue, a ceramide-unrelated compound named E16A, or (1S,2R)-HPCB-5, that potently inhibits the function of CERT has recently been developed, employing a series of in silico docking simulations, efficient chemical synthesis, quantitative affinity analysis, protein–ligand co-crystallography, and various in vivo assays. (1R,3S)-HPA-12 and E16A together provide a robust tool to discriminate on-target effects on CERT from off-target effects. This short review article will describe the history of the development of (1R,3S)-HPA-12 and E16A, summarize other CERT inhibitors, and discuss their possible applications.
... Fluorescent protein-conjugated lysenin (Canals et al., 2010;Ishitsuka et al., 2004;Kidani et al., 2012;Yachi et al., 2012), lysenin binding followed by anti-lysenin antibody detection (Kavishwar et al., 2011;Makino et al., 2015;Taksir et al., 2012;Yamaji et al., 1998) and recombinant MBP-or GST-lysenin binding followed by anti-MBP or anti-GST detection (Kiyokawa et al., 2004;Kulma et al., 2012;Makino et al., 2015;Nakai et al., 2000;Skocaj et al., 2014;Yoshida et al., 2001) have been used to localize SM in model membranes (Ishitsuka et al., 2004;Makino et al., 2015), in the plasma membrane of fixed cells (Canals et al., 2010;Kavishwar et al., 2011;Kidani et al., 2012;Kulma et al., 2012;Nakai et al., 2000;Skocaj et al., 2014), in the endocytic compartments of fixed and permeabilized cells (Kiyokawa et al., 2004;Yachi et al., 2012;Yamaji et al., 1998) and the sections of cells and organs Taksir et al., 2012;Yoshida et al., 2001). However, the binding of lysenin at physiological temperature was followed by oligomerization of the protein and pore formation in the membrane, which is accompanied by reorganization of the membrane (Alam et al., 2012;Aoki et al., 2010;Bokori-Brown et al., 2016;Podobnik et al., 2016;Yamaji-Hasegawa et al., 2003;Yilmaz et al., 2013Yilmaz et al., , 2018. ...
... Fluorescent protein-conjugated lysenin (Canals et al., 2010;Ishitsuka et al., 2004;Kidani et al., 2012;Yachi et al., 2012), lysenin binding followed by anti-lysenin antibody detection (Kavishwar et al., 2011;Makino et al., 2015;Taksir et al., 2012;Yamaji et al., 1998) and recombinant MBP-or GST-lysenin binding followed by anti-MBP or anti-GST detection (Kiyokawa et al., 2004;Kulma et al., 2012;Makino et al., 2015;Nakai et al., 2000;Skocaj et al., 2014;Yoshida et al., 2001) have been used to localize SM in model membranes (Ishitsuka et al., 2004;Makino et al., 2015), in the plasma membrane of fixed cells (Canals et al., 2010;Kavishwar et al., 2011;Kidani et al., 2012;Kulma et al., 2012;Nakai et al., 2000;Skocaj et al., 2014), in the endocytic compartments of fixed and permeabilized cells (Kiyokawa et al., 2004;Yachi et al., 2012;Yamaji et al., 1998) and the sections of cells and organs Taksir et al., 2012;Yoshida et al., 2001). However, the binding of lysenin at physiological temperature was followed by oligomerization of the protein and pore formation in the membrane, which is accompanied by reorganization of the membrane (Alam et al., 2012;Aoki et al., 2010;Bokori-Brown et al., 2016;Podobnik et al., 2016;Yamaji-Hasegawa et al., 2003;Yilmaz et al., 2013Yilmaz et al., , 2018. ...
Sphingomyelin (SM) is a major sphingolipid in mammalian cells. Although SM is enriched in the outer leaflet of the cell plasma membrane, lipids are also observed in the inner leaflet of the plasma membrane and intracellular organelles such as endolysosomes, the Golgi apparatus and nuclei. SM is postulated to form clusters with glycosphingolipids (GSLs), cholesterol (Chol), and other SM molecules through hydrophobic interactions and hydrogen bonding. Thus, different clusters composed of SM, SM/Chol, SM/GSL and SM/GSL/Chol with different stoichiometries may exist in biomembranes. In addition, SM monomers may be located in the glycerophospholipid-rich areas of membranes. Recently developed SM-binding proteins (SBPs) distinguish these different SM assemblies. Here, we summarize the effects of intrinsic factors regulating the lipid-binding specificity of SBPs and extrinsic factors, such as the lipid phase and lipid density, on SM recognition by SBPs. The combination of different SBPs revealed the heterogeneity of SM domains in biomembranes.
... Simultaneous labeling of the plasma membrane in COS-1 monkey kidney cells with fluorescent tagged NT-lysenin and EqtII revealed that at least three distinct SM pools exist on the plasma membrane: SM pools stained only with lysenin, SM pools stained only with EqtII, and SM pools stained with both probes [206]. These staining patterns were abolished after SMase treatment and were decreased through SMS2 knockdown. ...
... The segregation of SM pools in the plasma membrane is also observed in LLC-PK1 pig kidney epithelial cells [115]. Concerning the intracellular staining of COS-1 cells, lysenin exclusively stained late endosomes, whereas EqtII stained both late endosomes and recycling endosomes [206]. The intracellular staining with EqtII was only abolished by SMS1 and not SMS2 knockdown. ...
Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively.
... EqtII has also been used to localize SM in cell membranes (19). We previously showed that the membrane labeling of fluorescent protein conjugates of Lys and EqtII does not always overlap (20), suggesting that the 2 proteins bind different pools of SM (21). However, the molecular basis underlying the different labeling has not yet been clarified. ...
... The distribution of SM on the plasma membrane has not been examined in detail. Recently, we showed that EGFP-NT-Lys and EqtII-GFP bind different membrane domains in COS-7 cells using confocal microscopy (20). However, the molecular mechanism underlying the different labeling patterns has not been elucidated. ...
Sphingomyelin (SM) is a major sphingolipid in mammalian cells and is reported to form specific lipid domains together with cholesterol. However, methods to examine the membrane distribution of SM are limited. We demonstrated in model membranes that fluorescent protein conjugates of 2 specific SM-binding toxins, lysenin (Lys) and equinatoxin II (EqtII), recognize different membrane distributions of SM; Lys exclusively binds clustered SM, whereas EqtII preferentially binds dispersed SM. Freeze-fracture immunoelectron microscopy showed that clustered but not dispersed SM formed lipid domains on the cell surface. Glycolipids and the membrane concentration of SM affect the SM distribution pattern on the plasma membrane. Using derivatives of Lys and EqtII as SM distribution-sensitive probes, we revealed the exclusive accumulation of SM clusters in the midbody at the time of cytokinesis. Interestingly, apical membranes of differentiated epithelial cells exhibited dispersed SM distribution, whereas SM was clustered in basolateral membranes. Clustered but not dispersed SM was absent from the cell surface of acid sphingomyelinase-deficient Niemann-Pick type A cells. These data suggest that both the SM content and membrane distribution are crucial for pathophysiological events bringing therapeutic perspective in the role of SM membrane distribution.-Makino, A., Abe, M., Murate, M., Inaba, T., Yilmaz, N., Hullin-Matsuda, F., Kishimoto, T., Schieber, N. L., Taguchi, T., Arai, H., Anderluh, G., Parton, R. G., Kobayashi, T. Visualization of the heterogeneous membrane distribution of sphingomyelin associated with cytokinesis, cell polarity, and sphingolipidosis.
... As one of the major lipids of the vertebrate plasma membrane, SM is mainly located in the plasmalemma outer leaflet, and it can be specifically recognised by lysenin [19,20], a protein that is secreted through the dorsal pores of the earthworm Eisenia foetida [21], and by equinatoxin II (EqTII), a cytolysin from the sea anemone Actinia equina [22]. Although both of these toxins have been shown to bind to SM, they interact with distinct membrane pools of this lipid [23]. The fluorescentlylabelled D4 domain of perfringolysin O (PFO), a cytolysin from the Gram-positive bacterium Clostridium perfringens, was designed for selective labelling of cholesterol-enriched membrane domains [24,25]. ...
... OlyA-mCherry showed different plasma membrane distributions compared to the cholesterol-binding probe PFO-D4-EGPF, and the SM-binding probes GST-lysenin and EqTII-Alexa488. Recent studies have indicated that the lysenin and EqTII derivatives do not even bind to the same population of SM in both the plasma membrane and intracellular membranes [23]. Furthermore, as in our earlier study where mouse somatotrophs were double immunolabelled with an OlyA/PlyB mixture and CT-B-Alexa488 [47], in the present study, OlyA-mCherry and CT-B-Alexa488 were seen to bind to different nanodomains of the MDCK cell membranes. ...
Ostreolysin A (OlyA) is an ∼15-kDa protein that has been shown to bind selectively to membranes rich in cholesterol and sphingomyelin. In this study, we investigated whether OlyA fluorescently tagged at the C-terminal with mCherry (OlyA-mCherry) labels cholesterol/sphingomyelin domains in artificial membrane systems and in membranes of Madin-Darby canine kidney (MDCK) epithelial cells. OlyA-mCherry showed similar lipid binding characteristics to non-tagged OlyA. OlyA-mCherry also stained cholesterol/sphingomyelin domains in the plasma membranes of both fixed and living MDCK cells, and in the living cells, this staining was abolished by pretreatment with either methyl-β-cyclodextrin or sphingomyelinase. Double labelling of MDCK cells with OlyA-mCherry and the sphingomyelin-specific markers equinatoxin II-Alexa488 and GST-lysenin, the cholera toxin B subunit as a probe that binds to the ganglioside GM1, or the cholesterol-specific D4 domain of perfringolysin O fused with EGFP, showed different patterns of binding and distribution of OlyA-mCherry in comparison with these other proteins. Furthermore, we show that OlyA-mCherry is internalised in living MDCK cells, and within 90 min it reaches the juxtanuclear region via caveolin-1-positive structures. No binding to membranes could be seen when OlyA-mCherry was expressed in MDCK cells. Altogether, these data clearly indicate that OlyA-mCherry is a promising tool for labelling a distinct pool of cholesterol/sphingomyelin membrane domains in living and fixed cells, and for following these domains when they are apparently internalised by the cell.
... Local curvature, lipid organization, membrane density, and fluidity, which are all dependent on lipid composition, may all stimulate PFP binding and membrane insertion [85]. This is the case for lysenin, which binds only to clustered SM, and EqtII, which was shown to bind preferentially to SM at the liquid-ordered phase border [75,86]. ...
Pore-forming proteins (PFPs) play a central role in many biological processes related to infection, immunity, cancer, and neurodegeneration. A common feature of PFPs is their ability to form pores that disrupt the membrane permeability barrier and ion homeostasis and generally induce cell death. Some PFPs are part of the genetically encoded machinery of eukaryotic cells that are activated against infection by pathogens or in physiological programs to carry out regulated cell death. PFPs organize into supramolecular transmembrane complexes that perforate membranes through a multistep process involving membrane insertion, protein oligomerization, and finally pore formation. However, the exact mechanism of pore formation varies from PFP to PFP, resulting in different pore structures with different functionalities. Here, we review recent insights into the molecular mechanisms by which PFPs permeabilize membranes and recent methodological advances in their characterization in artificial and cellular membranes. In particular, we focus on single-molecule imaging techniques as powerful tools to unravel the molecular mechanistic details of pore assembly that are often obscured by ensemble measurements, and to determine pore structure and functionality. Uncovering the mechanistic elements of pore formation is critical for understanding the physiological role of PFPs and developing therapeutic approaches.
... For example, pore-forming www.nature.com/scientificreports/ proteins equinatoxin II from a sea anemone and lysenin from an earthworm label sphingomyelin monomers and homomeric sphingomyelin clusters, respectively 21,22 . However, equinatoxin II can also bind sphingomyelin associated with glycosphingolipids 23 , and lysenin can interact with sphingomyelin/cholesterol clusters 24 . ...
An aegerolysin protein ostreolysin A6 (OlyA6) binds to cholesterol-complexed sphingomyelin and can be used for specific labelling of lipid rafts. In addition, OlyA6 interacts with even higher affinity with ceramide phosphoethanolamine (CPE), a sphingolipid that dominates in invertebrate cell membranes. In the presence of pleurotolysin B, a protein bearing the membrane-attack complex/perforin domain, OlyA6 forms pores in insect midgut cell membranes and acts as a potent bioinsecticide. It has been shown that a point mutation of glutamate 69 to alanine (E69A) allows OlyA6 to bind to cholesterol-free sphingomyelin. Using artificial lipid membranes and mammalian MDCK cells, we show that this mutation significantly enhances the interaction of OlyA6 with sphingomyelin and CPE, and allows recognition of these sphingolipids even in the absence of cholesterol. Our results suggest that OlyA6 mutant E69A could serve as complementary tool to detect and study cholesterol-associated and free sphingomyelin or CPE in membranes. However, the mutation does not improve the membrane-permeabilizing activity after addition of pleurotolysin B, which was confirmed in toxicity tests on insect and mammalian cell lines, and on Colorado potato beetle larvae.
... In addition to specific receptor-mediated interactions, PFPs can also bind regions of the plasma membrane characterized by specific physicochemical properties. Specifically, negatively charged phospholipids (e.g., phosphatidylserine, cardiolipin, and phosphatidic acid), lipid organization, and membrane fluidity may all stimulate PFP binding [61][62][63][64]. Furthermore, these properties of the plasma membrane influence the subsequent stages of pore formation [65]. ...
Pore-forming proteins (PFPs) are a heterogeneous group of proteins that are expressed and secreted by a wide range of organisms. PFPs are produced as soluble monomers that bind to a receptor molecule in the host cell membrane. They then assemble into oligomers that are incorporated into the lipid membrane to form transmembrane pores. Such pore formation alters the permeability of the plasma membrane and is one of the most common mechanisms used by PFPs to destroy target cells. Interestingly, PFPs can also indirectly manipulate diverse cellular functions. In recent years, increasing evidence indicates that the interaction of PFPs with lipid membranes is not only limited to pore-induced membrane permeabilization but is also strongly associated with extensive plasma membrane reorganization. This includes lateral rearrangement and deformation of the lipid membrane, which can lead to the disruption of target cell function and finally death. Conversely, these modifications also constitute an essential component of the membrane repair system that protects cells from the lethal consequences of pore formation. Here, we provide an overview of the current knowledge on the changes in lipid membrane organization caused by PFPs from different organisms.
... However, cells exposed to high PFTs concentrations not able to repair their plasma membrane and will ultimately be lysed. For cellular meabilization assays, we titrated aerolysin to sublytic concentrations, to avoid immed Lysenin NT-GFP is a non-toxic form of full-length lysenin and is frequently used as a fluorescent probe for ordered sphingomyelin platforms on the plasma membrane [21]. ...
Pore-forming toxins (PFTs) form multimeric trans-membrane pores in cell membranes that differ in pore channel diameter (PCD). Cellular resistance to large PFTs (>20 nm PCD) was shown to rely on Ca2+ influx activated membrane repair mechanisms. Small PFTs (<2 nm PCD) were shown to exhibit a high cytotoxic activity, but host cell response and membrane repair mechanisms are less well studied. We used monocytic immune cell lines to investigate the cellular resistance and host membrane repair mechanisms to small PFTs lysenin (Eisenia fetida) and aerolysin (Aeromonas hydrophila). Lysenin, but not aerolysin, is shown to induce Ca2+ influx from the extracellular space and to activate Ca2+ dependent membrane repair mechanisms. Moreover, lysenin binds to U937 cells with higher efficiency as compared to THP-1 cells, which is in line with a high sensitivity of U937 cells to lysenin. In contrast, aerolysin equally binds to U937 or THP-1 cells, but in different plasma membrane areas. Increased aerolysin induced cell death of U937 cells, as compared to THP-1 cells, is suggested to be a consequence of cap-like aerolysin binding. We conclude that host cell resistance to small PFTs attack comprises binding efficiency, pore localization, and capability to induce Ca2+ dependent membrane repair mechanisms.
... It is synthesized by the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol as a side product 30,31 . The reaction is catalysed by SMS, which is located at the PM and in the Golgi in most mammalian cells [32][33][34] . However, sphingomyelin has not been detected in plants, as confirmed by our liquid chromatography-mass spectrometry analysis in Col-0 Arabidopsis (data not shown). ...
During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle--endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum--plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.
... It is synthesized by the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol as a side product 30,31 . The reaction is catalysed by SMS, which is located at the PM and in the Golgi in most mammalian cells [32][33][34] . However, sphingomyelin has not been detected in plants, as confirmed by our liquid chromatography-mass spectrometry analysis in Col-0 Arabidopsis (data not shown). ...
During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle–endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum–plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.
... Thus, the efficient delivery of E-cadherin to the lateral cell membrane is critical for the preservation and functionality of AJ in polarized cells [1,43,44]. Different lines of evidences have demonstrated that recycling endosomes are rich in SM and cholesterol, and that SMS 1 is the main enzyme responsible for the generation of recycling endosomes [55,56]. Consistently, in the present study, we observed that the expression of AJ proteins was not altered, as assessed by Western blot analysis of total CD cell homogenates. ...
Epithelial tissue requires that cells attach to each other and to the extracellular matrix by the assembly of adherens junctions (AJ) and focal adhesions (FA) respectively. We have previously shown that, in renal papillary collecting duct (CD) cells, both AJ and FA are located in sphingomyelin (SM)-enriched plasma membrane microdomains. In the present work, we investigated the involvement of SM metabolism in the preservation of the epithelial cell phenotype and tissue organization. To this end, primary cultures of renal papillary CD cells were performed. Cultured cells preserved the fully differentiated epithelial phenotype as reflected by the presence of primary cilia. Cells were then incubated for 24 h with increasing concentrations of D609, a SM synthase (SMS) inhibitor. Knock-down experiments silencing SMS 1 and 2 were also performed. By combining biochemical and immunofluorescence studies, we found experimental evidences suggesting that, in CD cells, SMS 1 activity is essential for the preservation of cell-cell adhesion structures and therefore for the maintenance of CD tissue/tubular organization. The inhibition of SMS 1 activity induced CD cells to lose their epithelial phenotype and to undergo an epithelial-mesenchymal transition (EMT) process.
... [34][35][36][37] Such toxinsh ave been fused to fluorescent proteinso rc hemically labelledw ith dyes, therebyf orming ideal tools to investigate the endocytic pathway of toxin-laden endosomes. [38,39] Sterol-enriched membranese xhibit high-acylc hain order,a kin to the raft principle at the PM. Pioneering work by Maxfield [40] and others [41,42] has demonstrated the differential sorting of lipid analogues based on their preferences form embrane fluidity. ...
Early endosomes are dynamic intracellular compartments that fuse with incoming endocytic carrier vesicles and associated cargoes from the plasma membrane. It has been long known that the lipids confer striking properties and rich biochemistry to bilayers owing to their chemical structures. While the organizational principles of the plasma membrane are relatively more understood, understanding endosomal membranes has been challenging. It has become increasingly apparent that endosomal membranes, owing to their lipid compositions and interactions, use distinct lipid chemistries to function. Here, we discuss the biochemical and biophysical phenomena that are at play at the early endosomal membrane, focusing on cholesterol, phosphoinositides and phosphatidylserine that have clear roles in endosome function. We discuss the various principles and mechanisms of how these lipids are implicated at the functional level in the working of endosomes and summarize the early endosomes as a multi-modal organelle employing distinct lipid specific mechanisms.
... Lipid-binding proteins, such as lysenin (Ishitsuka and Kobayashi, 2004), cholera toxin (Heyningen, 1974), S.V. equinatoxin (Barlic et al., 2004;Yachi et al., 2012) have been useful in studying membrane domains in plasma membrane. These motifs are part of different amphitropic proteins where they help proteins to associate with membranes by binding to unique lipid. ...
Biological membranes are non-covalent assembly of lipids and proteins. Lipids play critical role in determining membrane physical properties and regulate the function of membrane associated proteins. Budding yeast Saccharomyces cerevisiae offers an exceptional advantage to understand the lipid-protein interactions since lipid metabolism and homeostasis are relatively simple and well characterized as compared to other eukaryotes. In addition, a vast array of genetic and cell biological tools are available to determine and understand the role of a particular lipid in various lipid metabolic disorders. Budding yeast has been instrumental in delineating mechanisms related to lipid metabolism, trafficking and their localization in different subcellular compartments at various cell cycle stages. Further, availability of tools and enormous potential for the development of useful reagents and novel technologies to localize a particular lipid in different subcellular compartments in yeast makes it a formidable system to carry out lipid biology. Taken together, yeast provides an outstanding backdrop to characterize lipid metabolic changes under various physiological conditions.
... Other examples of bacterial toxins that bind to specific sphingolipids, are lysenin that binds sphingomyelin [74,75]} or the non-toxic Cholera toxin B subunit that targets the glycosphingolipid, GM1 [76] and the Shiga toxin that binds to Gb3 [77]. Since these lipids are found primarily on the external (luminal) leaflet of membranes they are not listed in Table II. ...
One of the largest challenges in cell biology is to map the lipid composition of the membranes of various organelles and define the exact location of processes that control the synthesis and distribution of lipids between cellular compartments. The critical role of phosphoinositides, low-abundant lipids with rapid metabolism and exceptional regulatory importance in the control of almost all aspects of cellular functions created the need for tools to visualize their localizations and dynamics at the single cell level. However, there is also an increasing need for methods to determine the cellular distribution of other lipids regulatory or structural, such as diacylglycerol, phosphatidic acid, or other phospholipids and cholesterol. This review will summarize recent advances in this research field focusing on the means by which changes can be described in more quantitative terms.
... Lysenin has a rare capacity to bind plasma membrane lipid SM with high affinity 16 . Together with other natural toxins, such as equinatoxin II from sea anemone, it represents an excellent tool for visualizing distribution and dynamics of SM in cells [17][18][19][20] . Monomeric lysenin consists of two distinct domains, the elongated N-terminal domain (pore-forming module, PFM) that shares the fold of all ab-PFTs ( Supplementary Fig. 1), and the C-terminal b-trefoil lectin type domain 21 . ...
The invertebrate cytolysin lysenin is a member of the aerolysin family of pore-forming toxins that includes many representatives from pathogenic bacteria. Here we report the crystal structure of the lysenin pore and provide insights into its assembly mechanism. The lysenin pore is assembled from nine monomers via dramatic reorganization of almost half of the monomeric subunit structure leading to a β-barrel pore ∼10 nm long and 1.6-2.5 nm wide. The lysenin pore is devoid of additional luminal compartments as commonly found in other toxin pores. Mutagenic analysis and atomic force microscopy imaging, together with these structural insights, suggest a mechanism for pore assembly for lysenin. These insights are relevant to the understanding of pore formation by other aerolysin-like pore-forming toxins, which often represent crucial virulence factors in bacteria.
... The ability of PFO to recognize cholesterol has motivated researchers to develop PFO-derived cholesterol biosensors similar to the use of other toxins to visualize lipids. For examples, the non-toxic fragments of the lysenin and equinatoxin II are excellent biosensors of sphingomyelin [21][22][23][24]. Another example of toxins used as tools for cell biology include the non-toxic B subunits of Cholera and Shiga toxins (CTxB and STxB, respectively) that bind to the exofacial glycosphingolipids GM1 and Gb3, respectively [25,26]. ...
Cholesterol is an essential structural component of cellular membranes in eukaryotes. Cholesterol in the exofacial leaflet of the plasma membrane is thought to form membrane nanodomains with sphingolipids and specific proteins. Additionally, cholesterol is found in the intracellular membranes of endosomes and has crucial functions in membrane trafficking. Furthermore, cellular cholesterol homeostasis and regulation of de novo synthesis rely on transport via both vesicular and non-vesicular pathways. Thus, the ability to visualize and detect intracellular cholesterol, especially in the plasma membrane, is critical to understanding the complex biology associated with cholesterol and the nanodomains. Perfringolysin O (PFO) theta toxin is one of the toxins secreted by the anaerobic bacteria Clostridium perfringens and this toxin forms pores in the plasma membrane that causes cell lysis. It is well understood that PFO recognizes and binds to cholesterol in the exofacial leaflets of the plasma membrane, and domain 4 of PFO (D4) is sufficient for the binding of cholesterol. Recent studies have taken advantage of this high-affinity cholesterol-binding domain to create a variety of cholesterol biosensors by using a non-toxic PFO or the D4 in isolation. This review highlights the characteristics and usefulness of, and the principal findings related to, these PFO-derived cholesterol biosensors.
... Les membranes de la cellule ne sont pas homogènes en composition lipidique (sans parler de la composition protéique). Il existe un gradient de teneur en sphingolipides et en cholestérol du réticulum, qui n'en comporte quasiment pas, à la membrane plasmique, où leurs concentrations sont maximales [202][203][204]. L'enrichissement relatif des membranes endosomales en sphingolipides et cholestérol est assuré en partie par un tri lipido-protéique réalisé au niveau du Golgi. ...
The eukaryotic cell physically separates its functions within several membrane-bound organelles, which communicate using vesicles. Vesicular trafficking is under the control of small GTPases that exist as an inactive GDP-bound form and an active GTP-bound form. The switch between GDP and GTP is catalyzed by a guanine nucleotide exchange factor (GEF). On cis-Golgi membranes, Arf1, activated by the large GEF GBF1, recruits the COPI coat. COPI coated vesicles ensure the retrograde transport from the Golgi to the ER. Recently, GBF1 has been implicated in other pathways, such as the life cycle of various viruses and lipid droplet metabolism.Lipid droplets (LD), the major lipid storage organelle, play a major role in lipid homeostasis within the cell. LDs are connected to membrane trafficking and are therefore under the control of GTPases. In previous studies, our team showed that GBF1 localizes around LDs and that it is required for protein loading onto the LD surface. Here, data support the idea that GBF1 localizes to the LD surface. Using cell biology tools and microscopy, we identified, within GBF1, a lipid binding domain. In this domain, a single amphipathic helix is necessary and sufficient for LD targeting in cells. The regulation of GBF1 localization relies on interaction with Rab1 (data support a Rab1-Arf1 cascade between the ER and the Golgi) and on intramolecular interactions between GBF1 domains.
... Nor have they been for other pore-forming toxins such as lysenin, produced by the earthworm Eisenia fetida (22), and cholesterol-dependent hemolysins, listeriolysin O, and perfringolysin O from bacteria Listeria monocytogenes and Clostridium perfringens, respectively (23,24), that target eukaryotic membranes. This receptor-like specificity toward lipids has been exploited for probing SM and cholesterol-enriched domains in eukaryotic cell membranes (25)(26)(27)(28)(29). ...
Equinatoxin II (EqtII) is a soluble, 20 kDa pore-forming protein toxin isolated from the sea anemone Actinia equina. Although pore formation has long been known to occur in distinct stages, including monomeric attachment to phospholipid membranes followed by detachment of the N-terminal helical domain and oligomerization into the final pore assembly, atomistic-level detail of the protein-lipid interactions underlying these events remains elusive. Using high-resolution solution state NMR of uniformly-(15)N-labeled EqtII at the critical micelle concentration of dodecylphosphocholine, we have mapped the lipid-binding site through chemical shift perturbations. Subsequent docking of an EqtII monomer onto a dodecylphosphocholine micelle, followed by 400 ns of all-atom molecular dynamics simulation, saw several high-occupancy lipid-binding pockets stabilized by cation-π, hydrogen bonding, and hydrophobic interactions; and stabilization of the loop housing the conserved arginine-glycine-aspartate motif. Additional simulation of EqtII with an N-acetyl sphingomyelin micelle, for which high-resolution NMR data cannot be obtained due to aggregate formation, revealed that sphingomyelin specificity might occur via hydrogen bonding to the 3-OH and 2-NH groups unique to the ceramide backbone by side chains of D109 and Y113; and main chains of P81 and W112. Furthermore, a binding pocket formed by K30, K77, and P81, proximate to the hinge region of the N-terminal helix, was identified and may be implicated in triggering pore formation.
Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
... On the other hand, Gagescu et al. ( 48 ) reported that recycling endosomes are rich in SM and cholesterol and that this lipid-based sorting mechanism contributes to the maintenance of cell polarity. More recently, Yachi et al. ( 49 ) demonstrated that SMS1 is the main enzyme responsible for the generation of recycling endosomes of SM. All these observations and our present results allow us to suggest that active SMS1-dependent synthesis of SM is necessary to establish mature AJs due to the fact that SM could be essential to form the intracellular vesicles involved in the correct delivery of AJ proteins. ...
Sphingolipids are relevant lipid components of eukaryotic cells. Besides regulating various cellular processes, sphingolipids provide the structural framework for plasma membrane organization. Particularly, sphingomyelin (SM) is associated with detergent-resistant microdomains. We have previously shown that the adherens junction (AJ) complex, the relevant cell-cell adhesion structure involved in cell differentiation and tissue organization, is located in a SM-rich membrane lipid domain. We have also demonstrated that under hypertonic conditions, MDCK cells acquire a differentiated phenotype with changes in sphingolipid metabolism. For these reasons, we decided to evaluate whether SM metabolism is involved in the acquisition of the differentiated phenotype of MDCK cells. We found that SM synthesis mediated by SM Synthase 1 (SMS1) is involved in hypertonicity-induced formation of mature AJs, necessary for correct epithelial cell differentiation. Inhibition of SM synthesis impaired the acquisition of mature AJs, evoking a disintegration-like process reflected by the dissipation of E-cadherin, β- and α-catenins from the AJ complex. As a consequence, MDCK cells did not develop the hypertonicity-induced differentiated epithelial cell phenotype.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... Many of these toxins -or for safety and cell viability reasons, non-toxic domains thereof -can be generated in recombinant form, either as fusion proteins with fluorescent proteins, such as GFP, or chemically conjugated to molecules such as Alexa Fluor succinimidyl ester. These probes have been used extensively to study the outer leaflet of the plasma membrane, as well as the lumen of endocytic pathway vesicles (Yachi et al., 2012). Typically, these types of probes are very specific and can be used for live-cell imaging. ...
Cellular lipids play crucial roles in the cell, including in energy storage, the formation of cellular membranes, and in signaling and vesicular trafficking. To understand the functions and characteristics of lipids within cells, various methods to image lipids have been established. In this Commentary, we discuss the four main types of molecular probes that have significantly contributed to our understanding of the cell biology of lipids. In particular, genetically encoded biosensors and antibodies will be discussed, and how they have been used extensively with traditional light and electron microscopy to determine the subcellular localization of lipids and their spatial and temporal regulation. We highlight some of the recent studies that have investigated the distribution of lipids and their ability to cluster using super-resolution and electron microscopy. We also examine methods for analyzing the movement and dynamics of lipids, including single-particle tracking (SPT), fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). Although the combination of these lipid probes and the various microscopic techniques is very powerful, we also point out several potential caveats and limitations. Finally, we discuss the need for new probes for a variety of phospholipids and cholesterol.
... These results might be reconciled if EqtII preferentially binds to the domain boundaries rather than to the L o domains, or if the L o /L d phase coexistence observed in this work is different from that observed in vivo. Clearly, the situation in cells is more complex, as was shown recently in a comparative study that employed EqtII and another SMspecific pore-forming toxin, lysenin, and found three Biophysical Journal 106(8) 1630-1637 different pools of SM that were stained by either EqtII or lysenin, or both (43). ...
Using phase-separated droplet interface bilayers, we observe membrane binding and pore formation of a eukaryotic cytolysin, Equinatoxin II (EqtII). EqtII activity is known to depend on the presence of sphingomyelin in the target membrane and is enhanced by lipid phase separation. By imaging the ionic flux through individual pores in vitro, we observe that EqtII pores form predominantly within the liquid-disordered phase. We observe preferential binding of labeled EqtII at liquid-ordered/liquid-disordered domain boundaries before it accumulates in the liquid-disordered phase.
... Non-toxic truncated lysenin revealed the heterogeneity of lipid rafts (16) and the role of SM-rich domains during cell division (18). In contrast, Eqt2 labeled selectively SM-rich domains in the cytoplasmic leaflet of the Golgi apparatus (17), indicating the presence of different pools of SM in cell membranes (53). Utilizing D4, a cholesterol-binding probe, and photoactivation localization microscopy, we demonstrated that SM-rich and Chol-rich domains do not always give similar cell surface labeling pattern (38). ...
A mixture of sphingomyelin (SM) and cholesterol (Chol) exhibits a characteristic lipid raft domain of the cell membranes that provides a platform to which various signal molecules as well as virus and bacterial proteins are recruited. Several proteins capable of specifically binding either SM or Chol have been reported. However, proteins that selectively bind to SM/Chol mixtures are less well characterized. In our screening for proteins specifically binding to SM/Chol liposomes, we identified a novel ortholog of Pleurotus ostreatus pleurotolysin A from the extract of edible mushroom Pleurotus eryngii, named pleurotolysin A2 (PlyA2). Enhanced green fluorescent protein (EGFP)-conjugated PlyA2 bound to SM/Chol but not to phosphatidylcholine/Chol liposomes. Cell surface labeling of PlyA2-EGFP was abolished after sphingomyelinase as well as methyl-β-cyclodextrin treatment, removing SM and Chol, respectively, indicating that PlyA2-EGFP specifically binds cell surface SM/Chol rafts. Tryptophan to alanine point mutation of PlyA2 revealed the importance of C-terminal tryptophan residues for SM/Chol binding. Our results indicate that PlyA2-EGFP is a novel protein probe to label SM/Chol lipid domains both in cell and model membranes.
... Mondal et al. (60) showed that sterols are enriched in REs using the fluorescent sterols. Yachi et al. (61) showed that SM is enriched in REs using a SM-binding protein, equinatoxin-II. Therefore, as is the case of evectin-2 that selectively binds PS through its PH domain, having interactions with these lipids may be one mechanism for RE targeting of proteins. ...
Cells internalize extracellular solutes, ligands, and proteins and lipids in the plasma membrane (PM) by endocytosis. The removal of membrane from the PM is counteracted by endosomal recycling pathways that return the endocytosed proteins and lipids back to the PM. Recycling to the PM can occur from early endosomes (EEs). However, many cells have a distinct subpopulation of endosomes that have a mildly acidic pH of 6.5 and are involved in the endosomal recycling. These endosomes are dubbed recycling endosomes (REs). In recent years, studies have begun to reveal that function of REs is not limited to the endosomal recycling. In this review, I summarize the nature of membrane trafficking pathways that pass through REs and the cell biological roles of these pathways.
... Observations performed by using the superresolution microscopy and fluorescently labeled lysenin revealed the involvement of the SM-rich domains in the progression of cytokinesis [76]. Very recently, the structural and functional heterogeneity of SM membrane pools was inspected by comparatively staining COS-1 cells with lysenin and EqtII [77]. The use of both SM probes revealed the existence of at least three different SM pools in plasmalemma: the first stained only with lysenin, the second stained only with EqtII, and the third that could be labeled by both probes. ...
Membrane rafts are transient and unstable membrane microdomains that are enriched in sphingolipids, cholesterol, and specific proteins. They are involved in intracellular trafficking, signal transduction, pathogen entry, and attachment of various ligands. Increasing experimental evidence on the crucial biological roles of membrane rafts under normal and pathological conditions require new techniques for their structural and functional characterization. In particular, fluorescence-labeled cytolytic proteins that interact specifically with molecules enriched in rafts are of increasing interest. Cholera toxin subunit B interacts specifically with raft-residing ganglioside GM1, and it has long been the lipid probe of choice for membrane rafts. Recently, four new pore-forming toxins have been proposed as selective raft markers: (i) equinatoxin II, a cytolysin from the sea anemone Actinia equina, which specifically recognizes free and membrane-embedded sphingomyelin; (ii) a truncated non-toxic mutant of a cytolytic protein, lysenin, from the earthworm Eisenia foetida, which specifically recognizes sphingomyelin-enriched membrane domains; (iii) a non-toxic derivative of the cholesterol-dependent cytolysin perfringolysin O, from the bacterium Clostridium perfringens, which selectively binds to membrane domains enriched in cholesterol; and (iv) ostreolysin, from the mushroom Pleurotus ostreatus, which does not bind to a single raft-enriched lipid component, but requires a specific combination of two of the most important raft-residing lipids: sphingomyelin and cholesterol. Non-toxic, raft-binding derivatives of cytolytic proteins have already been successfully used to explore both the structure and function of membrane rafts, and of raft-associated molecules. Here, we review these four new derivatives of pore-forming toxins as new putative markers of these membrane microdomains.
The coordinated actions of the endosomal uptake and recycling pathways are one of the main cellular mechanisms for controlling the composition of the plasma membrane. Once endocytosed from the cell surface, proteins and membrane lipids typically undergo one of two fates. They can either be sent along the degradative pathway to lysosomes where they are broken down, or they are returned to the plasma membrane by the endosomal recycling pathway. Molecules that are recycled to the plasma membrane must undergo a complex series of sorting events, that occur in several organelles, prior to reaching their destination. Members of the Rab family of small GTPases are key regulators of this process.
Macroscopic lipid observation in the organs of living small animals has not been realized. Here, we visualized sphingomyelin (SM) in the intestines of living mice using an SM-binding protein (EqtII-EGFP-His) under two-photon microscopy. The SM was identified as 10 μm spots in glands of the lamina propria of the mucosa in the large and small intestines. The spots vertically penetrated from the serosa toward the mucosal side. At the edge of the mucosal side in the small intestine, these spots connected with each other and formed horizontal lines. For the large intestine, the horizontal lines became a surface, indicating that SM covered the whole crypt membrane. Detailed observation revealed thin SM-positive lines that connected the spots and the blood vessels in the small intestine. Thus, SM exists at crypt surfaces and inside crypts of the intestines and can regulate the functions of the digestion system.
Sphingolipids (SLs) are a family of bioactive lipids implicated in a variety of cellular processes, and whose levels are controlled by an interlinked network of enzymes. While the spatial distribution of SL metabolism throughout the cell has been understood for some time, the implications of this for SL signaling and biological outcomes have only recently begun to be fully explored. In this review, we outline the compartmentalization of SL metabolism and describe advances in tools for investigating and probing compartment-specific SL functions. We also briefly discuss the implications of SL compartmentalization for cell signaling and therapeutic approaches to targeting the SL network.
Very few proteins are reported to bind specific lipids. Because of the high selectivity and strong binding to specific lipids, lipid-targeting pore forming toxins (PFTs) have been employed to study the distribution of lipids in cell- and model-membranes. Non-toxic and monomeric PFT-derivatives are especially useful to study living cells. In this chapter we highlight sphingomyelin (SM)-binding PFT, lysenin (Lys), its derivatives, and newly identified SM/cholesterol binding protein, nakanori. We describe the preparation of non-toxic mutant of Lys (NT-Lys) and its application in optical and super resolution microscopy. We also discuss the observation of nanometer scale lipid domains labeled with nakanori and maltose-binding protein (MBP)-Lys in electron microscopy.
Sphingolipid metabolism plays a critical role in regulating processes that control cellular fate. This dynamic pathway can generate and degrade the central players: ceramide, sphingosine and sphingosine-1-phosphate in almost any membrane in the cell, adding an unexpected level of complexity in deciphering signaling events. While in vitro assays have been developed for most enzymes in SL metabolism, these assays are setup for optimal activity conditions and can fail to take into account regulatory components such as compartmentalization, substrate limitations, and binding partners that can affect cellular enzymatic activity. Therefore, many in-cell assays have been developed to derive results that are authentic to the cellular situation which may give context to alteration in SL mass. This review will discuss approaches for utilizing probes for mammalian in-cell assays to interrogate most enzymatic steps central to SL metabolism. The use of inhibitors in conjunction with these probes can verify the specificity of cellular assays as well as provide valuable insight into flux in the SL network. The use of inhibitors specific to each of the central sphingolipid enzymes are also discussed to assist researchers in further interrogation of these pathways.
Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially colocalize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile alpha motif of SMS1 reduced the stability of the SMS1/GCS complex, resulting in a significant reduction in SM synthesis in vivo. In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo. These results suggest that formation of the SMS1/GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1/GCS complex was confirmed by CRISPR/Cas9–mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.
Sphingomyelin (SM) is a major sphingolipid in mammalian cells whereas its analog, ceramide phosphoethanolamine (CPE) is found in trace amounts in mammalian cells and in larger amounts in invertebrates such as insect cells like Drosophila melanogaster. To visualize endogenous SM or CPE, we need specific probes able to recognize the chemical structure of the lipid, rather than its physical property. A limited number of proteins is known to specifically and strongly bind SM or CPE. These proteins are either toxins produced by non-mammalian organisms, subunits or fragments of toxins or a protein that has similar structure to a toxin. These proteins labeled with small fluorophore (e.g Alexa Fluor) or conjugated to fluorescent proteins (e.g mCherry) or other types of markers (e.g 125I, maltose-binding protein) are used to detect SM or CPE. Here we summarize the characteristics of specific SM-binding proteins, lysenin and equinatoxin; CPE- and SM/cholesterol (Chol) binding aegerolysin proteins, pleurotolysin A2, ostreolysin and erylysin A and SM/Chol-binding protein, nakanori. Then we give examples of their applications including their limitations related not only to their lipid specificity and binding constants, but also to the lipid organization in the membrane.
Sphingomyelin (SM) biosynthesis represents a complex, finely regulated process, mostly occurring in vertebrates. It is intimately linked to lipid transport and it is ultimately carried out by two enzymes, SM synthase 1 and 2, selectively localized in the Golgi and plasma membrane. In the course of the SM biosynthetic reaction, various lipids are metabolized. Because these lipids have both structural and signaling functions, the SM biosynthetic process has the potential to affect diverse important cellular processes (such as cell proliferation, cell survival, and migration). Thus defects in SM biosynthesis might directly or indirectly impact the normal physiology of the cell and eventually of the organism. In this chapter, we will focus on evidence supporting a role for SM biosynthesis in specific cellular functions and how its dysregulation can affect neoplastic transformation.
The ancient phylum of Cnidaria contains many aquatic species with peculiar lifestyle. In order to survive, these organisms have evolved attack and defense mechanisms that are enabled by specialized cells and highly developed venoms. Pore-forming toxins are an important part of their venomous arsenal. Along some other types, the most representative are examples of four protein families that are commonly found in other kingdoms of life: actinoporins, Cry-like proteins, aerolysin-like toxins and MACPF/CDC toxins. Some of the homologues of pore-forming toxins may serve other functions, such as in food digestion, development and response against pathogenic organisms. Due to their interesting physico-chemical properties, the cnidarian pore-forming toxins may also serve as tools in medical research and nanobiotechnological applications.
Although sphingomyelin and cholesterol are major lipids of mammalian cells, the detailed distribution of these lipids in cellular membranes remains still obscure. However, the recent development of protein probes that specifically bind sphingomyelin and/or cholesterol provides new information about the landscape of the lipid domains that are enriched with sphingomyelin or cholesterol or both. Here, we critically summarize the tools to study distribution and dynamics of sphingomyelin and cholesterol.(2).
Pore forming toxins (PFTs) evolved to permeate the plasma membrane of target cells. This is achieved in a multistep mechanism that usually involves binding of soluble protein monomer to the lipid membrane, oligomerization at the plane of the membrane, and insertion of part of the polypeptide chain across the lipid membrane to form a conductive channel. Introduced pores allow uncontrolled transport of solutes across the membrane, inflicting damage to the target cell. PFTs are usually studied from the perspective of structure-function relationships, often neglecting the important role of the bulk membrane properties on the PFT mechanism of action. In this Account, we discuss how membrane lateral heterogeneity, thickness, and fluidity influence the pore forming process of PFTs. In general, lipid molecules are more accessible for binding in fluid membranes due to steric reasons. When PFT specifically binds ordered domains, it usually recognizes a specific lipid distribution pattern, like sphingomyelin (SM) clusters or SM/cholesterol complexes, and not individual lipid species. Lipid domains were also suggested to act as an additional concentration platform facilitating PFT oligomerization, but this is yet to be shown. The last stage in PFT action is the insertion of the transmembrane segment across the membranes to build the transmembrane pore walls. Conformational changes are a spontaneous process, and sufficient free energy has to be available for efficient membrane penetration. Therefore, fluid bilayers are permeabilized more readily in comparison to highly ordered and thicker liquid ordered lipid phase (Lo). Energetically more costly insertion into the Lo phase can be driven by the hydrophobic mismatch between the thinner liquid disordered phase (Ld) and large protein complexes, which are unable to tilt like single transmembrane segments. In the case of proteolipid pores, membrane properties can directly modulate pore size, stability, and even selectivity. Finally, events associated with pore formation can modulate properties of the lipid membrane and affect its organization. Model membranes do not necessarily reproduce the physicochemical properties of the native cellular membrane, and caution is needed when transferring results from model to native lipid membranes. In this context, the utilization of novel approaches that enable studying PFTs on living cells at a single molecule level should reveal complex protein-lipid membrane interactions in greater detail.
Actinoporins (APs) from sea anemones are ~ 20 kDa pore forming toxins with a β-sandwich structure flanked by two α-helices. The molecular mechanism of APs pore formation is composed of several well-defined steps. APs bind to membrane by interfacial binding site composed of several aromatic amino acid residues that allow binding to phosphatidylcholine and specific recognition of sphingomyelin. Subsequently, the N-terminal α-helix from the β-sandwich has to be inserted into the lipid/water interphase in order to form a functional pore. Functional studies and single molecule imaging revealed that only several monomers, 3-4, oligomerise to form a functional pore. In this model the α-helices and surrounding lipid molecules build toroidal pore. In agreement, APs pores are transient and electrically heterogeneous. On the contrary, crystalized oligomers of actinoporin fragaceatoxin C, were found to be composed of eight monomers with no lipids present between the adjacent α-helices. This article is part of a Special Issue entitled: Pore- Forming Toxins.
Whereas asymmetric transbilayer lipid distribution in the plasma membrane is well recognized, methods to examine the precise localization of lipids are limited. In this review, we critically evaluate the methods that are applied to study transbilayer asymmetry of lipids, summarizing the factors that influence the measurement. Although none of the present methods is perfect, the current application of immunoelectron microscopy-based technique provides a new picture of lipid asymmetry. Next, we summarize the transbilayer distribution of individual lipid in both erythrocytes and nucleated cells. Finally we discuss the concept of the interbilayer communication of lipids.
Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Lipids play an essential role in the structure of the endosomal membranes as well as in their dynamic rearrangement during the transport of internalized cargoes along the endocytic pathway. In this review, we discuss the function of endosomal lipids mainly in mammalian cells, focusing on two well-known components of the lipid rafts, sphingomyelin and cholesterol, as well as on three anionic phospholipids, phosphatidylserine, polyphosphoinositides and the atypical phospholipid, bis(monoacylglycero)phosphate/lysobisphosphatidic acid. We detail the structure, metabolism, distribution and role of these lipids in the endosome system as well as their importance in pathological conditions where modification of the endosomal membrane flow can lead to various diseases such as lipid-storage diseases, myopathies and neuropathies.
Membrane lipids not only provide the structural framework of cellular membranes but also influence protein functions in several different ways. In comparison to proteins, however, relatively little is known about distribution of membrane lipids because of the insufficiency of microscopic methods. The difficulty in studying lipid distribution results from several factors, including their unresponsiveness to chemical fixation, fast translational movement, small molecular size, and high packing density. In this Current Topic, we consider the major microscopic methods and discuss whether and to what degree of precision these methods can reveal membrane lipid distribution in situ. We highlight two fixation methods, chemical and physical, and compare the theoretical limitations to their spatial resolution. Recognizing the strengths and weaknesses of each method should help researchers interpret their microscopic results and increase our understanding of the physiological functions of lipids.
Sphingomyelin (SM) is one of the major lipids in the mammalian plasma membrane. Multiple lines of evidence suggest that SM plays at least two functional roles in the cell, as a reservoir of lipid second messengers and a platform for signaling molecules. To understand the molecular organization and dynamics of the SM-rich membrane domains, new approaches have been developed utilizing newly characterized specific SM-binding probes and state-of-the-art microscopy techniques. The toxic protein from the sea anemone, equinatoxin II, has been characterized as a specific probe for SM. The cytolytic protein from the earthworm, lysenin, has also been used as a SM-specific probe for the analysis of the heterogeneity of SM-rich membrane domains. Recently, using a non-toxic form of lysenin, we showed the spatial and temporal localization of SM in the plasma membrane by confocal and super-resolution microscopy. New microscopy techniques have also been introduced by other groups to help visualize membrane lipid domains. Here we review the most recent studies on imaging the SM-rich domains in biological membranes. This article is part of a Special Issue entitled New frontiers in sphingolipid biology.
The COG (conserved oligomeric Golgi complex) is a Golgi-associated tethering complex involved in retrograde trafficking of multiple Golgi enzymes. COG deficiencies lead to misorganization of the Golgi, defective trafficking of glycosylation enzymes, and abnormal N-, O- and ceramide-linked oligosaccharides. Here, we show that in Cog2 null mutant ldlC cells, the content of sphingomyelin (SM) is reduced to ∼25% of WT cells. Sphingomyelin synthase (SMS) activity is essentially normal in ldlC cells, but in contrast with the typical Golgi localization in WT cells, in ldlC cells, transfected SMS1 localizes to vesicular structures scattered throughout the cytoplasm, which show almost no signal of co-transfected ceramide transfer protein (CERT). Cog2 transfection restores SM formation and the typical SMS1 Golgi localization phenotype. Adding exogenous N-6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]hexanoyl-4-d-erythro-sphingosine (C(6)-NBD-ceramide) to ldlC cell cultures results in normal SM formation. Endogenous ceramide levels were 3-fold higher in ldlC cells than in WT cells, indicating that Golgi misorganization caused by Cog2 deficiency affects the delivery of ceramide to sites of SM synthesis by SMS1. Considering the importance of SM as a structural component of membranes, this finding is also worth of consideration in relation to a possible contribution to the clinical phenotype of patients suffering congenital disorders of glycosylation type II.
Although sphingomyelin is an important cellular lipid, its subcellular distribution is not precisely known. Here we use a
sea anemone cytolysin, equinatoxin II (EqtII), which specifically binds sphingomyelin, as a new marker to detect cellular
sphingomyelin. A purified fusion protein composed of EqtII and green fluorescent protein (EqtII-GFP) binds to the SM rich
apical membrane of Madin-Darby canine kidney (MDCK) II cells when added exogenously, but not to the SM-free basolateral membrane.
When expressed intracellularly within MDCK II cells, EqtII-GFP colocalizes with markers for Golgi apparatus and not with those
for nucleus, mitochondria, endoplasmic reticulum or plasma membrane. Colocalization with the Golgi apparatus was confirmed
by also using NIH 3T3 fibroblasts. Moreover, EqtII-GFP was enriched in cis-Golgi compartments isolated by gradient ultracentrifugation. The data reveal that EqtII-GFP is a sensitive probe for membrane
sphingomyelin, which provides new information on cytosolic exposure, essential to understand its diverse physiological roles.
Cell membranes display a tremendous complexity of lipids and proteins designed to perform the functions cells require. To
coordinate these functions, the membrane is able to laterally segregate its constituents. This capability is based on dynamic
liquid-liquid immiscibility and underlies the raft concept of membrane subcompartmentalization. Lipid rafts are fluctuating
nanoscale assemblies of sphingolipid, cholesterol, and proteins that can be stabilized to coalesce, forming platforms that
function in membrane signaling and trafficking. Here we review the evidence for how this principle combines the potential
for sphingolipid-cholesterol self-assembly with protein specificity to selectively focus membrane bioactivity.
The plasma membrane is organized into various subdomains of clustered macromolecules. Such domains include adhesive structures (cellular synapses, substrate adhesions, and cell-cell junctions) and membrane invaginations (clathrin-coated pits and caveolae), as well as less well-defined domains such as lipid rafts and lectin-glycoprotein lattices. Domains are organized by specialized scaffold proteins including the intramembranous caveolins, which stabilize lipid raft domains, and the galectins, a family of animal lectins that cross-link glycoproteins forming molecular lattices. We review evidence that these heterogeneous microdomains interact to regulate substratum adhesion and cytokine receptor dynamics at the cell surface.
Lysenin, a novel 41-kDa protein purified from coelomic fluid of the earthworm Eisenia foetida, induced erythrocyte lysis. Preincubation of lysenin with vesicles containing sphingomyelin inhibited lysenin-induced hemolysis completely, whereas vesicles containing phospholipids other than sphingomyelin showed no inhibitory activity, suggesting that lysenin bound specifically to sphingomyelin on erythrocyte membranes. The specific binding of lysenin to sphingomyelin was confirmed by enzyme-linked immunosorbent assay, TLC immunostaining, and liposome lysis assay. In these assays, lysenin bound specifically to sphingomyelin and did not show any cross-reaction with other phospholipids including sphingomyelin analogs such as sphingosine, ceramide, and sphingosylphosphorylcholine, indicating that it recognized a precise molecular structure of sphingomyelin. Kinetic analysis of the lysenin-sphingomyelin interaction by surface plasmon resonance measurements using BIAcoreTM system showed that lysenin associated with membrane surfaces composed of sphingomyelin (kon = 3.2 x 10(4) M-1 s-1) and dissociated extremely slowly (koff = 1.7 x 10(-4) s-1), giving a low dissociation constant (KD = 5.3 x 10(-9) M). Incorporation of cholesterol into the sphingomyelin membrane significantly increased the total amount of lysenin bound to the membrane, whereas it did not change the kinetic parameters of the lysenin-membrane interaction, suggesting that lysenin specifically recognized sphingomyelin and cholesterol incorporation changed the topological distribution of sphingomyelin in the membranes, thereby increasing the accessibility of sphingomyelin to lysenin. Immunofluorescence staining of fibroblasts derived from a patient with Niemann-Pick disease type A showed that lysenin stained the surfaces of the fibroblasts uniformly, whereas intense lysosomal staining was observed when the cells were permeabilized by digitonin treatment. Preincubation of lysenin with vesicles containing sphingomyelin abolished lysenin immunostaining. This study demonstrated that lysenin bound specifically to sphingomyelin on cellular membranes and should be a useful tool to probe the molecular motion and function of sphingomyelin in biological membranes.
We present a biochemical and morphological characterization of recycling endosomes containing the transferrin receptor in the epithelial Madin-Darby canine kidney cell line. We find that recycling endosomes are enriched in molecules known to regulate transferrin recycling but lack proteins involved in early endosome membrane dynamics, indicating that recycling endosomes are distinct from conventional early endosomes. We also find that recycling endosomes are less acidic than early endosomes because they lack a functional vacuolar ATPase. Furthermore, we show that recycling endosomes can be reached by apically internalized tracers, confirming that the apical endocytic pathway intersects the transferrin pathway. Strikingly, recycling endosomes are enriched in the raft lipids sphingomyelin and cholesterol as well as in the raft-associated proteins caveolin-1 and flotillin-1. These observations may suggest that a lipid-based sorting mechanism operates along the Madin-Darby canine kidney recycling pathway, contributing to the maintenance of cell polarity. Altogether, our data indicate that recycling endosomes and early endosomes differ functionally and biochemically and thus that different molecular mechanisms regulate protein sorting and membrane traffic at each step of the receptor recycling pathway.
Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.
Sphingomyelin (SM) is a major component of animal plasma membranes. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, yielding diacylglycerol as a side product. This reaction is catalysed by SM synthase, an enzyme whose biological potential can be judged from the roles of diacylglycerol and ceramide as anti- and proapoptotic stimuli, respectively. SM synthesis occurs in the lumen of the Golgi as well as on the cell surface. As no gene for SM synthase has been cloned so far, it is unclear whether different enzymes are present at these locations. Using a functional cloning strategy in yeast, we identified a novel family of integral membrane proteins exhibiting all enzymatic features previously attributed to animal SM synthase. Strikingly, human, mouse and Caenorhabditis elegans genomes each contain at least two different SM synthase (SMS) genes. Whereas human SMS1 is localised to the Golgi, SMS2 resides primarily at the plasma membrane. Collectively, these findings open up important new avenues for studying sphingolipid function in animals.
Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-beta-cyclodextrin (MbetaCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MbetaCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 (putative molecular mass, 48.6 kDa), which contains a sterile alpha motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MbetaCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.
Equinatoxin-II is a eukaryotic pore-forming toxin belonging to the family of actinoporins. Its interaction with model membranes
is largely modulated by the presence of sphingomyelin. We have used large unilamellar vesicles and lipid monolayers to gain
further information about this interaction. The coexistence of gel and liquid-crystal lipid phases in sphingomyelin/phosphatidylcholine
mixtures and the coexistence of liquid-ordered and liquid-disordered lipid phases in phosphatidylcholine/cholesterol or sphingomyelin/phosphatidylcholine/cholesterol
mixtures favor membrane insertion of equinatoxin-II. Phosphatidylcholine vesicles are not permeabilized by equinatoxin-II.
However, the localized accumulation of phospholipase C-generated diacylglycerol creates conditions for toxin activity. By
using epifluorescence microscopy of transferred monolayers, it seems that lipid packing defects arising at the interfaces
between coexisting lipid phases may function as preferential binding sites for the toxin. The possible implications of such
a mechanism in the assembly of a toroidal pore are discussed.
Sphingomyelin (SM) is a vital component of cellular membranes in organisms ranging from mammals to protozoa. Its production involves the transfer of phosphocholine from phosphatidylcholine to ceramide, yielding diacylglycerol in the process. The mammalian genome encodes two known SM synthase (SMS) isoforms, SMS1 and SMS2. However, the relative contributions of these enzymes to SM production in mammalian cells remained to be established. Here we show that SMS1 and SMS2 are co-expressed in a variety of cell types and function as the key Golgi- and plasma membrane-associated SM synthases in human cervical carcinoma HeLa cells, respectively. RNA interference-mediated depletion of either SMS1 or SMS2 caused a substantial decrease in SM production levels, an accumulation of ceramides, and a block in cell growth. Although SMS-depleted cells displayed a reduced SM content, external addition of SM did not restore growth. These results indicate that the biological role of SM synthases goes beyond formation of SM.
It has become increasingly difficult to find an area of cell biology in which lipids do not have important, if not key, roles as signalling and regulatory molecules. The rapidly expanding field of bioactive lipids is exemplified by many sphingolipids, such as ceramide, sphingosine, sphingosine-1-phosphate (S1P), ceramide-1-phosphate and lyso-sphingomyelin, which have roles in the regulation of cell growth, death, senescence, adhesion, migration, inflammation, angiogenesis and intracellular trafficking. Deciphering the mechanisms of these varied cell functions necessitates an understanding of the complex pathways of sphingolipid metabolism and the mechanisms that regulate lipid generation and lipid action.
Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.
Cytokinesis is a crucial step in the creation of two daughter cells by the formation and ingression of the cleavage furrow.
Here, we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization
of phosphatidylinositol-4,5-bisphosphate (PIP2) to the cleavage furrow during cytokinesis. Real-time observation with a labeled SM-specific protein, lysenin, revealed that
SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Superresolution fluorescence microscopy
analysis indicates a transbilayer colocalization between the SM-rich domains in the outer leaflet and PIP2-rich domains in the inner leaflet of the plasma membrane. The depletion of SM disperses PIP2 and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results
suggest that the formation of SM-rich domains is required for the accumulation of PIP2 to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.
Sphingomyelin (SM) plays important roles in regulating structure and function of plasma membrane, but how intracellular localization of SM is regulated in neuronal cells is not understood. Here we show that two isoforms of SM synthase (SMS) are differentially expressed in neuronal subtypes and that only SMS2 proteins localize in neurites of hippocampal neurons. Moreover, SMS proteins induce Lysenin-binding SM clusters exclusively in their vicinity although neurons hardly contain such cluster under control condition. These findings indicate three important notions about SM metabolism in neurons. First, the activity of SMS is the rate-limiting step of SM cluster formation. Second, the SM content or clustering can be modulated by SMS activity. Third, SMS1 and SMS2 play distinct roles in regulating local SM clustering. Particularly, SMS2, rather than SMS1, is likely to be the major enzyme that is important for SM synthesis in the long neurites and its tip, the growth cone.
The potassium xanthate D609 is widely accepted as a selective inhibitor of PC-specific phospholipase C (PC-PLC). The tricyclo[5.2.1.02,6]decane skeleton present in D609 can lead to four diastereomeric paris, but the diastereoselectivity of PC-PLC inhibition has never been reported. In this article, the synthesis of racemic D609 diastereomers and that of other xanthates, as well as their inhibitory effect on PC-PLC is reported. All xanthates obtained were competitive inhibitors of PC-PLC from Bacillus cereus (PLCBc). No significant differences were found in the activity of D609 diastereomers (K
i 13–17 μM), suggesting the absence of a diastereochemical control of the enzyme by xanthate inhibitors. This result was confirmed after obtaining other potassium xanthates differing from D609 in the aliphatic chain. Among them, the potassium O-n-decenylxanthate was the most active inhibitor of PLCBc (K
i 10 μM). These data indicate that the essential structural requirements for PLCBc in vitro inhibition by xanthates are the presence of a Zn-chelating dithiocarbonate head and a sufficiently hydrophobic aliphatic moiety.
Synthesis and sorting of lipids are essential for membrane biogenesis; however, the mechanisms underlying the transport of membrane lipids remain little understood. Ceramide is synthesized at the endoplasmic reticulum and translocated to the Golgi compartment for conversion to sphingomyelin. The main pathway of ceramide transport to the Golgi is genetically impaired in a mammalian mutant cell line, LY-A. Here we identify CERT as the factor defective in LY-A cells. CERT, which is identical to a splicing variant of Goodpasture antigen-binding protein, is a cytoplasmic protein with a phosphatidylinositol-4-monophosphate-binding (PtdIns4P) domain and a putative domain for catalysing lipid transfer. In vitro assays show that this lipid-transfer-catalysing domain specifically extracts ceramide from phospholipid bilayers. CERT expressed in LY-A cells has an amino acid substitution that destroys its PtdIns4P-binding activity, thereby impairing its Golgi-targeting function. We conclude that CERT mediates the intracellular trafficking of ceramide in a non-vesicular manner.
Little is known about the heterogenous organization of lipids in biological membranes. Sphingomyelin (SM) is a major plasma membrane lipid that forms lipid domains together with cholesterol and glycolipids. Using SM-specific toxin, lysenin, we showed that in cultured epithelial cells the accessibility of the toxin to SM is different between apical and basolateral membranes. Apical membranes are highly enriched with glycolipids. The inhibitory role of glycolipids in the binding of lysenin to SM was confirmed by comparing the glycolipid-deficient mutant melanoma cell line with its parent cell. Model membrane experiments indicated that glycolipid altered the local density of SM so that the affinity of the lipid for lysenin was decreased. Our results indicate that lysenin recognizes the heterogenous organization of SM in biomembranes and that the organization of SM differs between different cell types and between different membrane domains within the same cell. Isothermal titration calorimetry suggests that lysenin binding to SM is presumably the result of a SM-lysenin complex formation of specific stoichiometry, thus supporting the idea of the existence of small condensed lipid complexes consisting of just a few lipid molecules in living cells.
Tricyclodecan-9-yl-xanthogenate (D609) is a selective tumor cytotoxic agent. However, the mechanisms of action of D609 against tumor cells have not been well established. Using U937 human monocytic leukemia cells, we examined the ability of D609 to inhibit sphingomyelin synthase (SMS), since inhibition of SMS may contribute to D609-induced tumor cell cytotoxicity via modulating the cellular levels of ceramide and diacylglycerol (DAG). The results showed that D609 is capable of inducing U937 cell death by apoptosis in a dose- and time-dependent manner. The induction of U937 cell apoptosis was associated with an inhibition of SMS activity and a significant increase in the intracellular level of ceramide and decrease in that of sphingomyelin (SM) and DAG, which resulted in an elevation of the ratio between ceramide and DAG favoring the induction of apoptosis. In addition, incubation of U937 cells with C(6)-ceramide and/or H7 (a selective PKC inhibitor) reduced U937 cell viability; whereas pretreatment of the cells with a PKC activator, PMA or 1-oleoyl-2-acetylglycerol (OAG), attenuated D609-induced U937 cell apoptosis. These results suggest that SMS is a potential target of D609 and inhibition of SMS may contribute to D609-induced tumor cell death via modulation of the cellular levels of ceramide and DAG.
After endocytosis, most membrane proteins and lipids return to the plasma membrane (recycling pathway), but some membrane components are delivered to lysosomes (degradation pathway). These two pathways diverge in early endosomes. The recycling pathway involves recycling endosomes and the degradation pathway incorporates late endosomes and lysosomes. In many cell lines, these organelles often are located in the perinuclear region where they visually intermix. The present study, by tracking specific ligands (epidermal growth factor and transferrin) and expression of Rab proteins (Rab5, Rab7, and Rab11), demonstrated that, in COS-1 cells, the two pathways were spatially segregated. Recycling endosomes were mostly confined within the ring-shaped structure of the Golgi complex ("the Golgi ring"), whereas late endosomes and lysosomes were excluded from inside the Golgi ring. Thus, the unique organization of endocytic organelles in COS-1 cells can be utilized to visualize endocytic trafficking pathways in detail.
Equinatoxin II is a pore-forming protein of the actinoporin family. After membrane binding, it inserts its N-terminal alpha-helix and forms a protein/lipid pore. Equinatoxin II activity depends on the presence of sphingomyelin in the target membrane; however, the role of this specificity is unknown. On the other hand, sphingomyelin is considered an essential ingredient of lipid rafts and promotes liquid-ordered/liquid-disordered phase separation in model membranes that mimic raft composition. Here, we used giant unilamellar vesicles to simultaneously investigate the effect of sphingomyelin and phase separation on the membrane binding and permeabilizing activity of Equinatoxin II. Our results show that Equinatoxin II binds preferentially to the liquid-ordered phase over the liquid-disordered one and that it tends to concentrate at domain interfaces. In addition, sphingomyelin strongly enhances membrane binding of the toxin but is not sufficient for membrane permeabilization. Under the same experimental conditions, Equinatoxin II formed pores in giant unilamellar vesicles containing sphingomyelin only when liquid-ordered and -disordered phases coexisted. Our observations demonstrate the importance of phase boundaries for Equinatoxin II activity and suggest a double role of sphingomyelin as a specific receptor for the toxin and as a promoter of the membrane organization necessary for Equinatoxin II action.
Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.
The Authors Journal compilation © 2012 by the Molecular Biology Society of Japan
The Authors
Journal compilation © 2012 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2012) 17, 720–727