Plasma Membrane Sphingomyelin Research Articles

Sphingomyelinase as modulator of neuromuscular transmission via presynaptic mechanism

Secretory sphingomyelinases are released in response to various types of stress stimuli, including inflammation [1]. Excessive activity and release of these enzymes can be pathological and accelerate age-related neurological changes, neuroinflammation, neurodegenerative disorders, and muscle dysfunction [1–3]. In contrast, sphingomyelinases were detected in areas close to exocytosis sites and dendritic spines, and their inhibition reduced synaptic transmission at both the presynaptic and postsynaptic levels in hippocampal synapses. Deficiency of acid sphingomyelinase results in neurodegenerative Niemann–Pick disease type A. Knockouts of neutral sphingomyelinases in model mice cause motor defects and disorders that resemble the Alzheimer’s disease phenotype [1, 2]. These findings suggest the existence of a mechanism regulating synaptic transmission, which is dependent on sphingomyelinases. In this study, we examined the impact of neutral sphingomyelinase on neurotransmission in mouse diaphragm muscles using microelectrode recordings of postsynaptic responses, exo-endocytic FM dyes, and fluorescent probes sensitive to membrane properties. The researchers discovered that adding sphingomyelinase at a low concentration (0.01 u/ml) for 15 minutes causes disruption of the lipid packaging of selectively synaptic membranes. This disruption leads to ceramide accumulation, a decrease in staining with Alexa Fluor 488 cholera toxin subunit B conjugate (a marker of GM1 ganglioside cluster), and a shift to the green part of the F2N12S fluorescence spectrum (an indicator of the expansion of the lipid-disordered phase). Increased fluorescence of 22-NBD-cholesterol, indicating an increase in membrane fluidity, and enhanced incorporation of exogenous ceramide into the outer monolayer were observed in the synaptic membranes. Increasing sphingomyelinase concentration to 0.1 u/ml intensifies changes in synaptic membranes and leads to corresponding alterations in membrane properties in muscle fiber plasmalemma. Subsequent experiments used a synapse-specific effect on membrane properties by applying sphingomyelinase at a low concentration for 15 minutes. The application of sphingomyelinase did not alter the amplitude and temporal parameters of miniature postsynaptic responses or the resting membrane potential. Additionally, sphingomyelinase (0.01 u/ml) did not change spontaneous secretion and evoked neurotransmitter release in response to single stimuli. However, the activity of sphingomyelinase led to a notable increase in the release of neurotransmitters and the rate of exocytotic unloading of FM dye from synaptic vesicles during nerve stimulation at 10, 20, and 70 Hz frequencies. The effect of sphingomyelinase on neurotransmission potentiation was irreversible and more significant at 10 Hz activity. Experiments involving intermittent nerve stimulation with short bursts of 60 stimuli each at 10 or 20 Hz frequencies, along with brief 0.5-second rest intervals, indicated that sphingomyelinase elevated the short-term facilitation of neurotransmitter release at the onset of each subsequent episode at 10 Hz (but not at 20 Hz) stimulation. Experiments with a combination of electrophysiological detection of evoked postsynaptic responses, monitoring of exocytotic release of the FM1-43 dye, and the use of FM1-43 fluorescence quencher that penetrates through the fusion pores have demonstrated that a significant portion of synaptic vesicles release the neurotransmitter through the transient fusion pore (kiss-and-run mechanism) during high-frequency (70 Hz) activity [4, 5]. Treatment with sphingomyelinase inhibited the neurotransmitter release pathway. As a consequence, the majority of neurosecretion events went through exocytosis, completely incorporating the vesicular membrane into the presynaptic membrane. Sphingomyelin in the membranes of presynaptic and synaptic vesicles is believed to have varying functions in regulating neurotransmitter release. To investigate this theory, we stimulated the exo-endocytosis processes of synaptic vesicles using sphingomyelinase, which provided access to the synaptic vesicle membranes. Under these circumstances, the enhancing impact of sphingomyelinase on neurotransmitter release and FM1-43 exocytosis rate were notably inhibited. Based on the obtained data, we hypothesize that sphingomyelinase disrupts the integrity of lipid rafts and selectively forms ceramide accumulations in synaptic membranes within the muscles at low concentrations. This ultimately leads to an increase in the mobilization of synaptic vesicles towards exocytosis sites during 10–70 Hz activity. Presumably, the synaptic vesicles from a distinct pool, which predominantly mediates the neurotransmission at a frequency of 10 Hz, are the most actively involved in exocytosis under these circumstances. Sphingomyelinase modifies membrane properties and can inhibit the release of neurotransmitters through the fusion pore during high-frequency activity, redirecting exocytosis towards the complete fusion pathway. Hydrolysis of sphingomyelin in both plasma and synaptic vesicle membranes reduces the stimulatory effects of sphingomyelinase, indicating a contrasting role of sphingomyelin in presynaptic and vesicular membranes for controlling neurotransmitter release [5].

Sphingomyelinase modulates synaptic vesicle mobilization at the mice neuromuscular junctions

AimsSphingomyelin is an abundant component of the presynaptic membrane and an organizer of lipid rafts. In several pathological conditions, sphingomyelin is hydrolyzed due to an upregulation and release of secretory sphingomyelinases (SMases). Herein, the effects of SMase on exocytotic neurotransmitter release were studied in the diaphragm neuromuscular junctions of mice. Main methodsMicroelectrode recordings of postsynaptic potentials and styryl (FM) dyes were used to estimate neuromuscular transmission. Membrane properties were assessed with fluorescent techniques. Key findingsApplication of SMase at a low concentration (0.01 U ml−1) led to a disruption of lipid-packing in the synaptic membranes. Neither spontaneous exocytosis nor evoked neurotransmitter release (in response to single stimuli) were affected by SMase treatment. However, SMase significantly increased neurotransmitter release and the rate of fluorescent FM-dye loss from the synaptic vesicles at 10, 20 and 70 Hz stimulation of the motor nerve. In addition, SMase treatment prevented a shift of the exocytotic mode from “full-collapse” fusion to “kiss-and-run” during high-frequency (70 Hz) activity. The potentiating effects of SMase on neurotransmitter release and FM-dye unloading were suppressed when synaptic vesicle membranes were also exposed to this enzyme (i.e., stimulation occurred during SMase treatment). SignificanceThus, hydrolysis of the plasma membrane sphingomyelin can enhance mobilization of synaptic vesicles and facilitate full fusion mode of exocytosis, but SMase acting on vesicular membrane had a depressant effect on the neurotransmission. Partially, the effects of SMase can be related with the changes in synaptic membrane properties and intracellular signaling.

Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1.

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca2+-dependent Cl− channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (Cs−1) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca2+ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔGmixπ values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔGmixπ are retained and equalized with the values of ΔGmixπ in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.

55 Sow Milk Lipidome Study Reveals Changes in Fatty Acyl Residues in Triglycerides and Phosphatidylglycerol, but Not in Plasma Membrane Phospholipids Across Lactation

Abstract Sow milk fat content is crucial to neonatal survival, as it is utilized for thermogenesis and nutrition. However, fat is the most variable component of milk in concentration and lipid species. Characterizing lipid changes across the course of a sow’s lactation may help identify molecules or systems to target to help enhance milk fat quality and quantity for neonatal survival and growth. Percent fat variation is greatest in colostrum, the first milk. Little is known regarding colostrum synthesis, other than it accumulates in the gland beginning in mid-late pregnancy, which is prior to the initiation of fatty acid synthesis in lactocytes. The objective of this study was to characterize changes in lipid composition of milk across the course of lactation and determine if there was a relationship between fat percent and lipid species in colostrum and mature milk. Milk was collected from 9 multiparous sows on days 0, 3, 7, and 14 relative to birth. Percent fat was determined by creamatocrit, and found to be different (p< 0.05) between day 0 (12.36 ± 5.90%) and day 3 (16.22 ± 3.65%) but not between day 7 (13.13 ± 2.19%) and 14 (12.13 ± 2.45%). Fat was extracted from milk using the Bligh-Dyer method and profiled using multiple reaction monitoring. Amounts of lipid species were calculated relative to standards and data analysis was performed using Metaboanalyst 4.0. Principle component analysis revealed lactation day had a significant effect on distribution of fats. Triacylglycerides (TAG), phosphatidylglycerol (PG), and plasma membrane lipids were modified from colostrum to mature milk, with a significant increase in PGs and TAGs across the course of lactation. Correlation analysis of percent fat with lipid concentration indicated strong relationships (P < 0.05; |r| >0.80) with eight lipids. No differences are found in the abundance of plasma membrane phospholipids, sphingomyelin, or cholesterol esters across lactation days.

S. aureus alpha-toxin monomer binding and heptamer formation in host cell membranes - Do they determine sensitivity of airway epithelial cells toward the toxin?

Alpha-toxin (Hla) is a major virulence factor of Staphylococcus aureus (S. aureus) and plays an important role in S. aureus-induced pneumonia. It binds as a monomer to the cell surface of eukaryotic host cells and forms heptameric transmembrane pores. Sensitivities toward the toxin of various types of potential host cells have been shown to vary substantially, and the reasons for these differences are unclear. We used three human model airway epithelial cell lines (16HBE14o-, S9, A549) to correlate cell sensitivity (measured as rate of paracellular gap formation in the cell layers) with Hla monomer binding, presence of the potential Hla receptors ADAM10 or α5β1 integrin, presence of the toxin-stabilizing factor caveolin-1 as well as plasma membrane lipid composition (phosphatidylserine/choline, sphingomyelin). The abundance of ADAM10 correlated best with gap formation or cell sensitivities, respectively, when the three cell types were compared. Caveolin-1 or α5β1 integrin did not correlate with toxin sensitivity. The relative abundance of sphingomyelin in plasma membranes may also be used as a proxi for cellular sensitivity against alpha-toxin as sphingomyelin abundances correlated well with the intensities of alpha-toxin mediated gap formation in the cell layers.

Depletion of Host and Viral Sphingomyelin Impairs Influenza Virus Infection

Influenza A virus (IAV) is a major human respiratory pathogen causing annual epidemics as well as periodic pandemics. A complete understanding of the virus pathogenesis and host factors involved in the viral lifecycle is crucial for developing novel therapeutic approaches. Sphingomyelin (SM) is the most abundant membrane sphingolipid. It preferentially associates with cholesterol to form distinct domains named lipid rafts. Sphingomyelinases, including acid sphingomyelinase (ASMase), catalyzes the hydrolysis of membrane SM and consequently transform lipid rafts into ceramide-enriched membrane platforms. In this study, we investigated the effect of SM hydrolysis on IAV propagation. Depleting plasma membrane SM by exogenous bacterial SMase (bSMase) impaired virus infection and reduced virus entry, whereas exogenous SM enhanced infection. Moreover, the depletion of virus envelope SM also reduced virus infectivity and impaired its attachment and internalization. Nonetheless, inhibition of ASMase by desipramine did not affect IAV infection. Similarly, virus replication was not impaired in Niemann-Pick disease type A (NPA) cells, which lack functional ASMase. IAV infection in A549 cells was associated with suppression of ASMase activity starting at 6 h post-infection. Our data reveals that intact cellular and viral envelope SM is required for efficient IAV infection. Therefore, SM metabolism can be a potential target for therapeutic intervention against influenza virus infection.

Воздействие наноразмерных анионов кремний-молибденовой кислоты на плазматическую мембрану фибробластов эмбриона человека

Методом времяпролетной масс-спектрометрии вторичных ионов (ToF-SIMS) исследованы изменения состава плазматических мембран (ПМ) фибробластов эмбриона человека под воздействием наноразмерных анионов кремний-молибденовой кислоты (КМК). Измерены и интерпретированы зависимости масс-спектров основных липидов ПМ от концентрации КМК, коррелирующие с их послойными распределениями и со сродством холестерина к фосфолипидам. Обнаружен новый для биохимии клетки эффект – значительное снижение относительного содержания в ПМ холестерина и сфингомиелина под воздействием многозарядных анионов гетерополикислоты (ГПК). Для водных растворов КМК при концентрации С ~ 10мкМ/л и периода воздействия 48 ч обнаружено, что количество холестерина в ПМ снижается в 2–2.5 раза, при этом количество сфингомиелина уменьшается на 20–25%. Предложен новый механизм начального воздействия ГПК на плазматические мембраны, сводящийся к селективному травлению многозарядными анионами. Согласно предлагаемому механизму на первом этапе взаимодействия аниона полиоксометаллата с клеткой осуществляется экстракция из ПМ холестерина и сфингомиелина – основных регуляторов проницаемости и микровязкости ПМ. Как следствие возросшей проницаемости ПМ в клетках возможно ускорение жизненно важных трансмембранных и латеральных процессов.

The Effect of Nanosized Silicon Molybdate Anions on the Plasma Membrane of Human Fetal Fibroblasts

We used time-of-flight secondary ion mass spectrometry (ToF-SIMS) to study changes in the composition of the plasma membranes of human fetal fibroblasts under the action of nanosized anions of silicon molybdic acid. The dependences of the mass spectra of the main lipids of the plasma membranes on the silicon molybdate concentration were measured and interpreted; the dependences correlate with the layer-by-layer distributions and with the affinity of cholesterol for phospholipids. A new effect for cell biochemistry was discovered, that is, a significant decrease in the relative concentrations of cholesterol and sphingomyelin in plasma membranes under the effect of multiply charged heteropoly anions (HPAs). In aqueous silicon molybdate solutions with a concentration of c ≈ 10 µM/L and an exposure time of 48 h, the amount of cholesterol in plasma membranes decreased by 2–2.5 times, while the amount of sphingomyelin decreased by 20–25%. A new mechanism is proposed for the initial effect of HPA on plasma membranes, which consists of selective etching by multiply charged anions. According to the proposed mechanism, cholesterol and sphingomyelin, the main regulators of permeability and microviscosity of plasma membranes, are extracted from the plasma membrane at the first stage of the interaction of the polyoxometallate anion with the cell. As a consequence of the increased permeability of the plasma membranes in cells, acceleration of vital transmembrane and lateral processes may occur.

Homogeneous and Heterogeneous Bilayers of Ternary Lipid Compositions Containing Equimolar Ceramide and Cholesterol.

Cell membranes have been proposed to be laterally inhomogeneous, particularly in the case of mammalian cells, due to the presence of "domains" enriched in sphingolipids and cholesterol (Chol). Among membrane sphingolipids, sphingomyelin (SM) in the cell plasma membrane is known to be degraded to ceramide (Cer) by acid sphingomyelinases under stress conditions. Since cholesterol (Chol) is abundant in the plasma membrane, the study of ternary mixtures SM:Chol:Cer is interesting from the point of view of membrane biophysics, and it might be physiologically relevant. In previous studies, we have described the homogeneous gel phase formed by phospholipid:Chol:Cer at 54:23:23 mol ratios, where phospholipid was either SM or dipalmitoylphosphatidylcholine (DPPC). We now provide new data, based on trans-parinaric acid and diphenylhexatriene fluorescence, supporting that the gel phase includes all three components in a single bilayer. The main question addressed in this paper is the stability of the ternary gel phase when bilayer composition is changed, specifically when the SM proportion is varied. To this aim, we have prepared bilayers of composition phospholipid:Chol:Cer at X:Y:Y ratios, in which phospholipid increased between 54 and 70 mol %. The N-palmitoyl derivatives of SM (pSM) and Cer (pCer) have been used. We observe that for X = 54 or 60 mol %, a gel phase is clearly predominant. However, when the proportion of phospholipid increases beyond 60 mol %, i.e., in 66:17:17 or 70:15:15 mixtures, a lateral phase separation occurs at the micrometer scale. These data can be interpreted in terms of a pCer:Chol interaction, that would predominate at the lower phospholipid concentrations. The putative pCer:Chol complexes (or nanodomains) would mix well with the phospholipid. At the higher SM concentrations pSM:pCer and pSM:Chol interactions would become more important, giving rise to the coexisting gel and liquid-ordered phases respectively. Heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicating a higher affinity of SM over DPPC for Chol or Cer. The observation that heterogeneity, or lateral phase separation, occurs more easily with pSM than with DPPC, indicates a higher affinity of SM over DPPC for Chol or Cer, and can be related to cell regulation through the sphingolipid signaling pathway.

Molecular Discrimination between Two Conformations of Sphingomyelin in Plasma Membranes

Sphingomyelin and cholesterol are essential lipids that are enriched in plasma membranes of animal cells, where they interact to regulate membrane properties and many intracellular signaling processes. Despite intense study, the interaction between these lipids in membranes is not well understood. Here, structural and biochemical analyses of ostreolysin A (OlyA), a protein that binds to membranes only when they contain both sphingomyelin and cholesterol, reveal that sphingomyelin adopts two distinct conformations in membranes when cholesterol is present. One conformation, bound by OlyA, is induced by stoichiometric, exothermic interactions with cholesterol, properties that are consistent with sphingomyelin/cholesterol complexes. In its second conformation, sphingomyelin is free from cholesterol and does not bind OlyA. A point mutation abolishes OlyA's ability to discriminate between these two conformations. In cells, levels of sphingomyelin/cholesterol complexes are held constant over a wide range of plasma membrane cholesterol concentrations, enabling precise regulation of the chemical activity of cholesterol.

Plasma membrane sphingomyelin modulates thymocyte development by inhibiting TCR-induced apoptosis.

Sphingomyelin (SM) in combination with cholesterol forms specialized membrane lipid microdomains in which specific receptors and signaling molecules are localized or recruited to mediate intracellular signaling. SM-microdomain levels in mouse thymus were low in the early CD4+CD8+ double-positive (DP) stage prior to thymic selection and increased >10-fold during late selection. T-cell receptor (TCR) signal strength is a key factor determining whether DP thymocytes undergo positive or negative selection. We examined the role of SM-microdomains in thymocyte development and related TCR signaling, using SM synthase 1 (SMS1)-deficient (SMS1-/-) mice which display low SM expression in all thymocyte populations. SMS1 deficiency caused reduced cell numbers after late DP stages in TCR transgenic models. TCR-dependent apoptosis induced by anti-CD3 treatment was enhanced in SMS1-/- DP thymocytes both in vivo and in vitro. SMS1-/- DP thymocytes, relative to controls, showed increased phosphorylation of TCR-proximal kinase ZAP-70 and increased expression of Bim and Nur77 proteins involved in negative selection following TCR stimulation. Addition of SM to cultured normal DP thymocytes led to greatly increased surface expression of SM-microdomains, with associated reduction of TCR signaling and TCR-induced apoptosis. Our findings indicate that SM-microdomains are increased in late DP stages, function as negative regulators of TCR signaling and modulate the efficiency of TCR-proximal signaling to promote thymic selection events leading to subsequent developmental stages.

Codetermination of Sphingomyelin and Cholesterol in Cellular Plasma Membrane in Sphingomyelin-Depletion-Induced Cholesterol Efflux.

Quantification of multiple lipids with different contents in plasma membrane in single cells is significant, but challenging, for investigating lipid interactions and the role of dominant protein transporters. In this paper, comonitoring the alteration of low-content sphingomyelin (SM) and high-content cholesterol in plasma membrane of one living cell is realized by use of luminol electrochemiluminescence (ECL) for the first time. Concentrations of SM as low as 0.5 μM are detected, which permits the measurement of low-content membrane SM in single cells. More membrane cholesterol is observed in individual cells after depletion of membrane SM, providing direct evidence about SM-depletion-induced cholesterol efflux. The upregulation of ATP-binding cassette transporters A1 (ABCA1) and G1 (ABCG1) in SM-depleted cells induces a further increase in membrane cholesterol. These results imply that higher expression of ABCA1/G1 promotes cholesterol trafficking, which offers additional information to solve the debate about ABC transporters in cholesterol efflux. Moreover, the established approach offers a special strategy to investigate the correlation of membrane lipids and the role of transporters in cholesterol trafficking.

Gemcitabine kills proliferating endothelial cells exclusively via acid sphingomyelinase activation

Gemcitabine is a widely-used anti-cancer drug with a well-defined mechanism of action in normal and transformed epithelial cells. However, its effect on endothelial cells is largely unknown. Acid sphingomyelinase (ASMase) is highly expressed in endothelial cells, converting plasma membrane sphingomyelin to pro-apoptotic ceramide upon activation by diverse stresses. In the current study, we investigated gemcitabine impact in primary cultures of endothelial cells. We find baseline ASMase increases markedly in bovine aortic endothelial cells (BAEC) as they transit from a proliferative to a confluent growth-arrested state. Further, gemcitabine activates ASMase and induces release of a secretory ASMase form into the media only in proliferating endothelial cells. Additionally, proliferative, but not growth-arrested BAEC, are sensitive to gemcitabine-induced apoptotic death, an effect blocked by inhibiting ASMase with imipramine or by binding ceramide on the cell surface with an anti-ceramide Ab. Confluent growth-arrested BAEC can be re-sensitized to gemcitabine-induced apoptosis by provision of exogenous sphingomyelinase. A highly similar phenotype was observed in primary cultures of human coronary artery endothelial cells. These findings reveal a previously-unrecognized mechanism of gemcitabine cytotoxicity in endothelium that may well contribute to its clinical benefit, and suggest the potential for further improvement of its clinical efficacy via pharmacologic modulation of ASMase/ceramide signaling in proliferative tumor endothelium.

Liver serine palmitoyltransferase activity deficiency in early life impairs adherens junctions and promotes tumorigenesis.

The plasma membrane sphingomyelin level is one of the key factors in regulating hepatocyte polarity and tumorigenesis. (Hepatology 2016;64:2089-2102).

Sphingomyelin-induced inhibition of the plasma membrane calcium ATPase causes neurodegeneration in type A Niemann-Pick disease.

Niemann-Pick disease type A (NPA) is a rare lysosomal storage disorder characterized by severe neurological alterations that leads to death in childhood. Loss-of-function mutations in the acid sphingomyelinase (ASM) gene cause NPA, and result in the accumulation of sphingomyelin (SM) in lysosomes and plasma membrane of neurons. Using ASM knockout (ASMko) mice as a NPA disease model, we investigated how high SM levels contribute to neural pathology in NPA. We found high levels of oxidative stress both in neurons from these mice and a NPA patient. Impaired activity of the plasma membrane calcium ATPase (PMCA) increases intracellular calcium. SM induces PMCA decreased activity, which causes oxidative stress. Incubating ASMko-cultured neurons in the histone deacetylase inhibitor, SAHA, restores PMCA activity and calcium homeostasis and, consequently, reduces the increased levels of oxidative stress. No recovery occurs when PMCA activity is pharmacologically impaired or genetically inhibited in vitro. Oral administration of SAHA prevents oxidative stress and neurodegeneration, and improves behavioral performance in ASMko mice. These results demonstrate a critical role for plasma membrane SM in neuronal calcium regulation. Thus, we identify changes in PMCA-triggered calcium homeostasis as an upstream mediator for NPA pathology. These findings can stimulate new approaches for pharmacological remediation in a disease with no current clinical treatments.

431 Tropomyosin-receptor kinase fused gene (TFG) induces collagen synthesis in human dermal fibroblasts

431 Tropomyosin-receptor kinase fused gene (TFG) induces collagen synthesis in human dermal fibroblasts

Analysis of sphingomyelin in plasma membrane at single cells using luminol electrochemiluminescence

Luminol electrochemiluminescence was applied to detect sphingomyelin in plasma membrane at single cells that exhibited the high cellular heterogeneity.

Identification of Sphingomyelinase on the Surface of Chlamydia pneumoniae: Possible Role in the Entry into Its Host Cells.

We have recently suggested a novel mechanism, autoendocytosis, for the entry of certain microbes into their hosts, with a key role played by the sphingomyelinase-catalyzed topical conversion of sphingomyelin to ceramide, the differences in the biophysical properties of these two lipids providing the driving force. The only requirement for such microbes to utilize this mechanism is that they should have a catalytically active SMase on their outer surface while the target cells should expose sphingomyelin in the external leaflet of their plasma membrane. In pursuit of possible microbial candidates, which could utilize this putative mechanism, we conducted a sequence similarity search for SMase. Because of the intriguing cellular and biochemical characteristics of the poorly understood entry of Chlamydia into its host cells these microbes were of particular interest. SMase activity was measured in vitro from isolated C. pneumoniae elementary bodies (EB) and in the lysate from E. coli cells transfected with a plasmid expressing CPn0300 protein having sequence similarity to SMase. Finally, pretreatment of host cells with exogenous SMase resulting in loss plasma membrane sphingomyelin attenuated attachment of EB.

Raft coalescence and FcγRIIA activation upon sphingomyelin clustering induced by lysenin

Activation of immunoreceptor FcγRIIA by cross-linking with antibodies is accompanied by coalescence of sphingolipid/cholesterol-rich membrane rafts leading to the formation of signaling platforms of the receptor. In this report we examined whether clustering of the raft lipid sphingomyelin can reciprocally induce partition of FcγRIIA to rafts. To induce sphingomyelin clustering, cells were exposed to non-lytic concentrations of GST-lysenin which specifically recognizes sphingomyelin. The lysenin/sphingomyelin complexes formed microscale assemblies composed of GST-lysenin oligomers engaging sphingomyelin of rafts. Upon sphingomyelin clustering, non-cross-linked FcγRIIA associated with raft-derived detergent-resistant membrane fractions as revealed by density gradient centrifugation. Pretreatment of cells with GST-lysenin also increased the size of detergent-insoluble molecular complexes of activated FcγRIIA. Sphingomyelin clustering triggered tyrosine phosphorylation of the receptor and its accompanying proteins, Cbl and NTAL, in the absence of receptor ligands and enhanced phosphorylation of these proteins in the ligand presence. These data indicate that clustering of plasma membrane sphingomyelin induces coalescence of rafts and triggers signaling events analogous to those caused by FcγRIIA activation.

Free cholesterol alters macrophage morphology and mobility by an ABCA1 dependent mechanism

ObjectiveTo investigate whether the morphological and functional changes typical of cell immobilization induced by free cholesterol (FC) accumulation in macrophages is related to the activity of the ATP-binding cassette transporter (ABCA1). Methods and resultsFC loading induced actin rearrangement with ruffling and cell spreading in macrophages that normally express ABCA1, but to a significant lesser extent in ABCA1-KO mouse peritoneal macrophages (MPMs) and in normal cells upon pharmacological inhibition of ABCA1 with probucol. In ABCA1-KO MPMs and in probucol-treated J774 cell migration was inhibited to a lower extent by FC as compared to control cells. Similar results were found in stably ABCA1 knocked down J774 (ABCA1-KD-J774) obtained by RNA interference. FC accessible to cholesterol oxidase, a measure of plasma membrane FC content, was significantly higher in FC-loaded WT MPMs and control J774 than in FC-loaded ABCA1-KO MPMs, ABCA1-KD-J774 or probucol-treated J774. In parallel plasma membrane total phospholids and sphingomyelin increased after cholesterol loading in control J774 but not in ABCA1-KD-J774. In addition, apoA-I, that removes FC from ABCA1 specific pool, partially restored chemotactic response in FC-loaded control J774. No effect was observed with HDL2 that does not interact with ABCA1. Finally, FC-induced Rac activation was more efficient in control J774 than in ABCA1-KD-J774. and was prevented by probucol and apoA-I in control J774. ConclusionIn macrophages ABCA1 activity mediates FC ability to alter plasma membrane organization, to inhibit cell migration, and to activate a Rac-mediated signaling pathway.