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This review will cover recent advances in the knowledge of the composition, structure and toxic mechanisms of the important RTX family of bacterial toxins. I will not discuss their regulation and unusual mechanism of extracellular secretion. There are outstanding recent reviews written by investigators whose research is focused on those topics that can be consulted (Bailey et al. 1997; Young and Holland 1999). After a very general review, the discussion is organized around 13 critical questions and relevant hypotheses involving RTX toxin biology. I will review the results that support or refute these assumptions while proposing several new alternative interpretations of the available data. As foreshadowed in this review’s title, the biology and biochemistry of RTX toxins present unique and difficult experimental problems, which I hope will be effectively elucidated for the reader.

Since the discovery of cellular membrane rafts, the defining of these domains has remained ambiguous due to a great number of isolation procedures proposed for the extraction of the rafts from cells. Characterization of membrane rafts using Triton X-100 insolubility is limited by the fact that weak interactions between proteins and lipids within the membrane rafts cannot be detected. In order to study the role of membrane rafts in cell signal transduction, it is crucial that weak membrane raft–associated proteins are detected. In this report, we demonstrate that by incorporating 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) crosslinking and freezing at −80°C into the membrane raft isolation procedure of HaCaT cells, both membrane raft–associated proteins caveolin-1 and Fas receptor are able to be reproducibly isolated into a single fraction containing the membrane rafts of the cells.

CD47 is a ubiquitously expressed membrane protein with an extracellular Ig domain and a multiple membrane-spanning domain that can synergize with antigen to induce interleukin (IL)-2 secretion by T lymphocytes. Ligation of CD47 induced actin polymerization and increased protein kinase Ctheta (PKCtheta) association with the cytoskeleton independent of antigen receptor ligation, but ligation of mutant forms of the molecule missing either the Ig domain or the multiple membrane-spanning domain did not. Simultaneous ligation of CD47 and CD3 led to additive effects on F-actin and synergistic effects on PKCtheta cytoskeletal association. Disruption of membrane rafts by removal of cholesterol with cyclodextrin blocked CD47-induced actin polymerization, and mutant forms of CD47 that localized poorly to rafts failed to effect cytoskeletal rearrangement. However, raft association alone was not sufficient, because a raft-localized CD47 Ig domain bound to the membrane by a glycan phosphoinositol anchor was unable to induce actin polymerization. A mutant form of CD47 without its Ig domain that did not induce actin polymerization or localize to rafts still enhanced T cell receptor (TCR)-dependent tyrosine phosphorylation of PLCgamma and associated Ca(2+) signaling but did not augment IL-2 secretion. Thus, CD47 synergy with TCR to increase [Ca(2+)](i) is independent of actin and rafts but is insufficient to explain CD47 cooperation with TCR in IL-2 synthesis. Full synergy with TCR requires CD47 localization to membrane rafts where ligation leads to TCR-independent signals causing actin polymerization and PKCtheta translocation.

Annexin II is a member of the annexin family of Ca2+- and phospholipid-binding proteins which is particularly enriched on early endosomal membranes and has been implicated in participating in endocytic events. In contrast to other endosomal annexins the association of annexin II with its target membrane can occur in the absence of Ca2+ in a manner depending on the unique N-terminal domain of the protein. However, endosome binding of annexin II does not require formation of a protein complex with the intracellular ligand S100A10 (p11) as an annexin II mutant protein (PM AnxII) incapable of interacting with p11 is still present on endosomal membranes. Fusion of the N-terminal sequence of this PM AnxII (residues 1–27) to the conserved protein core of annexin I transfers the capability of Ca2+-independent membrane binding to the otherwise Ca2+-sensitive annexin I. These results underscore the importance of the N-terminal sequence of annexin II for the Ca2+-independent endosome association and argue for a direct interaction of this sequence with an endosomal membrane receptor.

Efflux of excess cellular cholesterol mediated by lipid-poor apolipoproteins occurs by an active mechanism distinct from passive diffusion and is controlled by the ATP-binding cassette transporter ABCA1. Here we examined whether ABCA1-mediated lipid efflux involves the selective removal of lipids associated with membrane rafts, plasma membrane domains enriched in cholesterol and sphingomyelin. ABCA1 was not associated with cholesterol and sphingolipid-rich membrane raft domains based on detergent solubility and lack of colocalization with marker proteins associated with raft domains. Lipid efflux to apoA-I was accounted for by decreases in cellular lipids not associated with cholesterol/sphingomyelin-rich membranes. Treating cells with filipin, to disrupt raft structure, or with sphingomyelinase, to digest plasma membrane sphingomyelin, did not impair apoA-I-mediated cholesterol or phosphatidylcholine efflux. In contrast, efflux of cholesterol to high density lipoproteins (HDL) or plasma was partially accounted for by depletion of cholesterol from membrane rafts. Additionally, HDL-mediated cholesterol efflux was partially inhibited by filipin and sphingomyelinase treatment. Apo-A-I-mediated cholesterol efflux was absent from fibroblasts with nonfunctional ABCA1 (Tangier disease cells), despite near normal amounts of cholesterol associated with raft domains and normal abilities of plasma and HDL to deplete cholesterol from these domains. Thus, the involvement of membrane rafts in cholesterol efflux applies to lipidated HDL particles but not to lipid-free apoA-I. We conclude that cholesterol and sphingomyelin-rich membrane rafts do not provide lipid for efflux promoted by apolipoproteins through the ABCA1-mediated lipid secretory pathway and that ABCA1 is not associated with these domains.

NAP-22, a neuronal tissue-enriched acidic membrane protein, is a Ca 2+ -dependent calmodulin binding protein and has similar biochemical characteristics to GAP-43 (neuromodulin). Recent biochemical studies have demonstrated that NAP-22 localizes in the membrane raft domain with a cholesterol-dependent manner. Since the raft domain is assumed to be important to establish and/or to maintain the cell polarity, we have investigated the changes in the localization of NAP-22 during the development of the neuronal polarity in vitro and in vivo, using cultured hippocampal neurons and developing cerebellum neurons, respectively. Cultured hippocampal neurons initially extended several short processes, and at this stage NAP-22 was distributed more or less evenly among them. During the maturation of neuronal cells, NAP-22 was sorted preferentially into the axon. Throughout the developmental stages of hippocampal neurons, the localization change of NAP-22 was quite similar to that of tau, an axonal marker protein, but not to that of microtubule-associated protein-2 (MAP-2), a dendritic marker protein. Further confocal microscopic observation demonstrated the colocalization of NAP-22 and either tau or vesicle-associated protein-2 (VAMP-2). A comparison of the time course of the axonal localization of NAP-22 and GAP-43 showed that NAP-22 localization was much later than that of GAP-43. The correlation between the expression of NAP-22 and synaptogenesis in the cerebellar granular layer, particularly in the synaptic glomeruli, was also investigated. There existed many VAMP-2 positive synapses but no NAP-22 positive ones in 1-week-old cerebellum. On sections of 2-week-old cerebellum, accumulation of NAP-22 to the synaptic glomeruli was clearly observed and this accumulation became clearer during the maturation of the synaptic structure. The present results suggest the possibility that NAP-22 plays an important role in the maturation and/or the maintenance of synapses rather than in the process of the axonal outgrowth, by controlling cholesterol-dependent membrane dynamics.

HIV-1 infection triggers lateral membrane diffusion following interaction of the viral envelope with cell surface receptors. We show that these membrane changes are necessary for infection, as initial gp120–CD4 engagement leads to redistribution and clustering of membrane microdomains, enabling subsequent interaction of this complex with HIV-1 co-receptors. Disruption of cell membrane rafts by cholesterol depletion before viral exposure inhibits entry by both X4 and R5 strains of HIV-1, although viral replication in infected cells is unaffected by this treatment. This inhibitory effect is fully reversed by cholesterol replenishment of the cell membrane. These results indicate a general mechanism for HIV-1 envelope glycoprotein-mediated fusion by reorganization of membrane microdomains in the target cell, and offer new strategies for preventing HIV-1 infection.

Despite CD40's role in stimulating dendritic cells (DCs) for efficient specific T-cell stimulation, its signal transduction components in DCs are still poorly documented. We show that CD40 receptors on human monocyte-derived DCs associate with sphingolipid- and cholesterol-rich plasma membrane microdomains, termed membrane rafts. Following engagement, CD40 utilizes membrane raft-associated Lyn Src family kinase, and possibly other raft-associated Src family kinases, to initiate tyrosine phosphorylation of intracellular substrates. CD40 engagement also leads to a membrane raft-restricted recruitment of tumor necrosis factor (TNF) receptor-associated factor (TRAF) 3 and, to a lesser extent, TRAF2, to CD40's cytoplasmic tail. Thus, the membrane raft structure plays an integral role in proximal events of CD40 signaling in DCs. We demonstrate that stimulation of Src family kinase within membrane rafts initiates a pathway implicating ERK activation, which leads to interleukin (IL)-1alpha/beta and IL-1Ra mRNA production and contributes to p38-dependent IL-12 mRNA production. These results provide the first evidence that membrane rafts play a critical role in initiation of CD40 signaling in DCs, and delineate the outcome of CD40-mediated pathways on cytokine production.

A membrane microdomain enriched in cholesterol and glycosphingolipids, or so called ‘raft’ region, was found to contain many signal transducing proteins such as GPI-anchored cell adhesion molecules, trimeric G proteins, and protein tyrosine kinases. In previous studies, we showed that the raft region obtained from rat brain contains two cytoskeletal proteins, tubulin and actin, as the major components in addition to these signal transducing proteins. In this study, to know the biochemical mechanisms regulating the cytoskeletal organization in this region, actin regulatory activities in raft were surveyed. We found the presence of a Ca 2+ -dependent actin nucleation promoting activity in raft. The solubilization and column fractionation of this activity combined with western blotting and immunoprecipitation showed that gelsolin is one of the actin regulatory proteins in raft.

Signal transduction through the B cell antigen receptor (BCR) is altered in B cells that express a receptor that recognizes self-antigen. To understand the molecular basis for the change in signaling in autoreactive B cells, a transgenic model was used to isolate a homogeneous population of tolerant B lymphocytes. These cells were compared with a similar population of naive B lymphocytes. We show that the BCR from naive B cells enters a detergent-insoluble domain of the cell within 6 s after antigen binding, before a detectable increase in BCR phosphorylation. This fraction appears to be important for signaling because it is enriched for lyn kinase but lacks CD45 tyrosine phosphatase and because the BCR that moves into this domain becomes more highly phosphorylated. Partitioning of the BCR into this fraction is unaffected by src family kinase inhibition. Tolerant B cells do not efficiently partition the BCR into the detergent-insoluble domain, providing an explanation for their reduced tyrosine kinase activation and calcium flux in response to antigen. These results identify an early, regulated step in antigen receptor signaling and self-tolerance.

The acquisition of spatial and functional asymmetry between the rear and the front of the cell is a necessary step for cell chemotaxis. Insulin-like growth factor-I (IGF-I) stimulation of the human adenocarcinoma MCF-7 induces a polarized phenotype characterized by asymmetrical CCR5 chemokine receptor redistribution to the leading cell edge. CCR5 associates with membrane raft microdomains, and its polarization parallels redistribution of raft molecules, including the raft-associated ganglioside GM1, glycosylphosphatidylinositol-anchored green fluorescent protein and ephrinB1, to the leading edge. The non-raft proteins transferrin receptor and a mutant ephrinB1 are distributed homogeneously in migrating MCF-7 cells, supporting the raft localization requirement for polarization. IGF-I stimulation of cholesterol-depleted cells induces projection of multiple pseudopodia over the entire cell periphery, indicating that raft disruption specifically affects the acquisition of cell polarity, but not IGF-I-induced protrusion activity. Cholesterol depletion inhibits MCF-7 chemotaxis, which is restored by replenishing cholesterol. Our results indicate that initial segregation between raft and non-raft membrane proteins mediates the necessary redistribution of specialized molecules for cell migration.

Expansion of commercial micropropagation will require the implementation of scale up and mechanization technology (Vasil, 1991; Vasil, 1994). In developed countries labor cost have been reported to be 65–70 per cent of the cost of production (Chu, 1995; George, 1996). In these countries, mechanization would permit cost-effective micropropagation of transplant vegetable and forestry crops and production of ornamental crops at costs that are competitive with exports of low labor cost countries. In countries where labor costs are low, mechanization would allow standardized production and reduce operator-associated contamination.

The serine-threonine kinase, Akt1/protein kinase Ba is an important mediator of growth, survival and metabolic signaling. Recent studies have implicated cholesterol-rich, lipid raft microdomains in survival signals mediated by Akt1. Here we address the role of lipid raft membranes as a potential site of intersection of androgenic and Akt1 signaling. A subpopulation of androgen receptor (AR) was found to localize to a lipid raft subcellular compartment in LNCaP prostate cancer cells. Endogenous AR interacted with endogenous Akt1 preferentially in lipid raft fractions and androgen substantially enhanced the interaction between the two proteins. The association of AR with Akt1 was inhibited by the anti-androgen, bicalutamide, but was not affected by inhibition of phosphoinositide-3-kinase (PI3K). Androgen promoted endogenous Akt1 activity in lipid raft fractions, in a PI3K-independent manner, within 10 min of treatment. Fusion of a lipid raft targeting sequence to AR enhanced localization of the receptor to rafts, and stimulated Akt1 activity in response to androgen, while reducing the cells’ dependence on constitutive signaling through PI3K for cell survival. These findings suggest that signals channeled through AR and Akt1 intersect by a mechanism involving formation within lipid raft membranes of an androgen-responsive, extranuclear AR/Akt1 complex. Our results indicate that cholesterol-rich membrane microdomains play a role in transmitting nongenomic signals involving androgen and the Akt pathway in prostate cancer cells.

Yews are usually dioecious, evergreen (gymnosperm) trees or shrubs (Fig. 1a). The leaves are simple, flat, linear, often falcate, with distinct but short petioles, arranged spirally though often appearing two-ranked (Fig. 1b). They have a single unbranched midrib and two broad stomate bands on the underside of the leaves. The bark is reddish or purplish to dark chestnut brown, scaly or exfoliating from the trunk and larger branches in thin flakes or long strips. The wood is hard, dense, flexible, elastic, and fine-grained without resin ducts. The winter buds are ovate, axillary, or terminal, with imbricate scales. The flowers are small, solitary, or occasionally twinned, axillary. The female flower resembles an axillary vegetative bud, but is usually decurved or pendent and is easily recognized on close inspection by the micropyle opening in the exposed ovule. The male flower or pollen cone has several sterile scales at the base, with a stalked globose head of 6 to 14 scales, each with 5 to 9 microsporangia or pollen sacs. The solitary seed (which matures in late summer to fall), sometimes called a single-seeded berry or berry-like fruit (Fig. 1b), is surrounded by a fleshy mucilaginous, scarlet outer seed coat or aril (Chadwick and Keen 1976; Silba 1986).

The recent Keystone Symposium on Lipid Rafts and Cell Function (March 23-28, 2006 in Steamboat Springs, CO) brought together biophysicists, biochemists, and cell biologists to discuss the structure and function of lipid rafts. What emerged from the meeting was a consensus definition of a membrane raft: " "Membrane rafts are small (10-200 nm), heterogeneous, highly dynamic, sterol- and sphingolipid-enriched domains that compartmentalize cel- lular processes. Small rafts can sometimes be stabilized to form larger platforms through protein-protein and protein- lipid interactions." " This definition helps to clarify current thinking in a field that has been plagued by the heteroge- neous and sometimes ephemeral nature of its subject.— Pike, L. J. Rafts defined: a report on the Keystone sympo- sium on lipid rafts and cell function. J. Lipid Res. 2006. 47: 1597-1598.

The CD38-ADP-ribosylcyclase-mediated Ca2+ signaling pathway importantly contributes to the vasomotor response in different arteries. Although there is evidence indicating that the activation of CD38-ADP-ribosylcyclase is associated with CD38 internalization, the molecular mechanism mediating CD38 internalization and consequent activation in response to a variety of physiological and pathological stimuli remains poorly understood. Recent studies have shown that CD38 may sense redox signals and is thereby activated to produce cellular response and that the NADPH oxidase isoform, NOX1, is a major resource to produce superoxide (O2 ·−) in coronary arterial myocytes (CAMs) in response to muscarinic receptor agonist, which uses CD38-ADP-ribosylcyclase signaling pathway to exert its action in these CAMs. These findings led us hypothesize that NOX1-derived O2 ·− serves in an autocrine fashion to enhance CD38 internalization, leading to redox activation of CD38-ADP-ribosylcyclase activity in mouse CAMs. To test this hypothesis, confocal microscopy, flow cytometry and a membrane protein biotinylation assay were used in the present study. We first demonstrated that CD38 internalization induced by endothelin-1 (ET-1) was inhibited by silencing of NOX1 gene, but not NOX4 gene. Correspondingly, NOX1 gene silencing abolished ET-1-induced O2 ·− production and increased CD38-ADP-ribosylcyclase activity in CAMs, while activation of NOX1 by overexpression of Rac1 or Vav2 or administration of exogenous O2 ·− significantly increased CD38 internalization in CAMs. Lastly, ET-1 was found to markedly increase membrane raft clustering as shown by increased colocalization of cholera toxin-B with CD38 and NOX1. Taken together, these results provide direct evidence that Rac1-NOX1-dependent O2 ·− production mediates CD38 internalization in CAMs, which may represent an important mechanism linking receptor activation with CD38 activity in these cells.

The steroid hormone progesterone induces the resumption of meiosis of full-grown ovarian oocytes arrested at the first meiotic division. Apart from protein-mediated signaling, progesterone-induced maturation involves enzyme activations that modify membrane fluidity and release a cascade of lipid messengers. Once the oocyte is mature, it enters meiosis II and spontaneously arrests in metaphase until fertilization, in which drastic membrane reorganization occurs when amphibian sperm fuses with the oocyte membrane and exocytosis of cortical granules takes place. The predominant lipids in most membranes are phospholipids and cholesterol. Cholesterol stabilizes membranes at low temperatures and it is an important constituent of membrane rafts. In this study, the cholesterol-binding drug methyl- β -cyclodextrin (M β CD) was used for cholesterol modulation in order to assess membrane raft involvement in amphibian fertilization. The generalized polarization (GP) of the fluorescent probe Laurdan was used to study the effect of cholesterol depletion and fertilization on the biophysical properties of membranes from Rhinella arenarum oocytes evaluated in temperatures ranging from 5 °C to 40 °C. For comparative reasons, cholesterol removal of ovarian oocytes and progesterone-induced maturation were also analyzed. As it was expected for native membranes of complex lipid composition, GP values gradually decreased when temperature increased. Light (L) and Heavy (H) membranes, enriched in rafts and non-raft plasma membrane, respectively, showed two different thermotropic profiles of GP. GP values were higher in L fraction with respect to H indicating a higher membrane lipid order in agreement with the enrichment in cholesterol of these low-density membranes. Cholesterol depletion of ovarian oocytes, mediated by M β CD, caused a decrease in the GP value of L membranes which corresponds to a greater penetration of water molecules and indicates a decrease in membrane lipid order. In contrast, GP values of H membranes increased after M β CD treatment. As a result, both plasma membrane fractions become more similar in their biophysical states. On the other hand, progesterone modified the biophysical properties of the plasma membrane increasing the lipid order of both fractions. Interestingly, different to ovarian oocytes, membranes isolated from ovulated oocytes showed an important lipid order resistance to variations in temperature. In these oocytes, M β CD treatment also decreased lipid order of L membranes but at a lesser extent than ovarian oocytes. In addition, fertilization increased lipid order of non-raft membranes. As we have previously shown for progesterone-induce maturation, functional studies revealed that M β CD treatment inhibited fertilization in a dose-dependent manner. Cholesterol repletion performed using M β CD pre-loaded with cholesterol induced a recovery of the fertilization rate. These results indicate that fertilization competence of amphibian oocytes is sensitive to the cholesterol content of both plasma membrane fractions. Taking into account that the plasma membrane is a major site of cryoinjury during cryopreservation of mammalian oocytes, these results open a discussion on sterol modulation to be able to obtain a stable source of oocytes available for in vitro fertilization and other related biotechnologies.

Abstract 2382 The erythropoietin receptor (EpoR) is a type 1 cytokine receptor and key effector of Epo signaling. Maturation and shuttling of receptor to the plasma membrane is dependent on its association with Janus Kinase 2 (JAK2). Upon ligand binding, EpoR homo-dimerizes to activate JAK2, which in turn phosphorylates tyrosine residues on the receptor cytoplasmic tail permitting recruitment and phosphorylation of the signal transduction and activator of transcription (STAT)-5. Lenalidomide (LEN), a second generation immunomodulatory drug (IMiD), promotes the expansion of primitive erythroid progenitors in vitro and induces the expression of genes involved in erythroid lineage commitment (JCI 2008;118(1):248-58; PLoS medicine 2008;5(2):e35). In del(5q) clones, LEN is cytotoxic by inhibiting the haplodeficient serine/threonine phosphatase PP2A (Wei S, et. al PNAS 2009). To investigate the mechanism by which LEN promotes erythropoiesis, we investigated its action on EpoR cellular dynamics. Using the human erythroleukemia cell line, UT-7, treatment with LEN elicited concentration dependent upregulation of EpoR in whole cell lysates demonstrable by immunoblot (IB) analysis at concentrations ranging from 0.1–10μM, reaching a 2-fold increase vs. controls. Treatment with pomalidomide, an IMiD with superior erythropoietic activity, displayed greater potency, increasing cellular EpoR at concentrations as low as 1nM; whereas thalidomide was ineffective. Upregulation of EpoR was apparent as early as 1 hour (h) after LEN exposure, reaching maximal receptor induction at 8h. Isolation of membrane and cystosolic fractions followed by IB showed that EpoR was upregulated predominantly in the membrane fraction, increasing 4.3-fold. IB of detergent insoluble membrane raft (MR) fractions showed that LEN promoted lipid raft assembly and F-actin polymerization accompanied by translocation of EpoR into MR microdomains with JAK2, STAT-5 and Lyn signal intermediates. Pretreatment with the Rho-GTPase inhibitor, Y27632, suppressed LEN effects on MR and F-actin. Immunoprecipitation (IP) of EpoR showed that JAK2 co-precipitated with the receptor in LEN treated cells, suggesting drug induced release of pre-assembled EpoR/JAK2 complexes from golgi and endosomal compartments. Q-PCR showed no changes in EpoR gene mRNA expression up to 24 hours after drug exposure, supporting post translational drug effects. To confirm that the observed changes in EpoR dynamics in UT-7 cells extends to normal erythroid progenitors, we assessed EpoR expression and MR assembly (cholera toxin B labeled GM1) by fluorescence microscopy in CD71+ erythroid precursors from three normal bone marrow donors. Expression of EpoR increased a mean of 17% (range:0-68%) after 1h LEN [1μM] exposure (p=0.003), accompanied by an increase in MR aggregates. Because the LEN inhibitable target PP2A is a key regulator of Rho-GTPase guided receptor trafficking and AP-1 directed formation of MR targeted clathrin-coated vesicles, we examined its effects on EpoR induction. Pretreatment of UT-7 cells with the PP2A activator FTY720 antagonized the upregulation of EpoR by LEN, whereas treatment with the PP2A inhibitor cantharadin [0.5μM] induced EpoR protein expression. Inhibition of dynamin activity, a PP2A sensitive GTPase involved in type 1 receptor recycling from membrane to endosomes, recapitulated the effects of cantharadin, inducing EpoR expression. In summary, these findings indicate that LEN modulates EpoR equilibrium, mediated in part through its inhibitory effect on PP2A. LEN induces rapid translocation of mature EpoR/JAK2 complexes to the plasma membrane while promoting the assembly of MRs with signaling competent receptor platforms to optimize cytokine signal integrity. Disclosures: List: Celgene: Consultancy.

Background Membrane rafts are small highly dynamic sterol- and sphingolipid-enriched membrane domains that have received considerable attention due to their role in diverse cellular functions. More recently the involvement of membrane rafts in neuronal processes has been highlighted since these specialized membrane domains have been shown to be involved in synapse formation, neuronal polarity and neurodegeneration. Detergent resistance followed by gradient centrifugation is often used as first step in screening putative membrane raft components. Traditional methods of raft isolation employed the nonionic detergent Triton X100. However successful separation of raft from non-raft domains in cells is dependent on matching the detergent used for raft isolation to the specific tissue under investigation. Results We report here the isolation of membrane rafts from primary neuronal culture using a panel of different detergents that gave rise to membrane fractions that differed in respect to cholesterol and protein content. In addition, proteomic profiling of neuronal membrane rafts isolated with different detergents, Triton X100 and CHAPSO, revealed heterogeneity in their protein content. Conclusions These data demonstrate that appropriate selection of detergent for raft isolation is an important consideration for investigating raft protein composition of cultured neurons.

Sphingolipids are a family of membrane lipids that play important roles in the regulation of the fluidity and subdomain structure of the lipid bilayer—especially, lipid rafts.1–4 They play crucial functional roles in membrane raft formation, receptor function, membrane conductance, cell–cell interactions, and internalization of pathogens.2,5,6 Many of them, such as ceramide (CER), ceramide-1-phosphate (C1P), sphingosine (SP), and sphingosine-1-phosphate (S1P), are bioactive molecules that have been implicated in the regulation of cell growth, apoptosis, angiogenesis, vesicular trafficking, and a multitude of specific cell actions and responses.6,7 Aberrant sphingolipid metabolism is associated with inflammation, tumorigenesis, diabetes, and neurodegenerative disorders.8–12 Sphingolipid metabolic defects that cause severe congenital or childhood-onset neurodegenerative diseases, such as Tay-Sachs disease, Fabry's disease, and Niemann-Pick disease, are often associated with blindness, indicating the importance of sphingolipid metabolism in retinal cells.13–15 Patients with Farber's (acid ceramidase), Gaucher's (glucosylceramidase), Krabbe's (galactosylceramidase), and Niemann-Pick (sphingomyelinase) diseases lose vision due to retinal neuronal cell death.16 The processes of apoptosis, neuronal dedifferentiation, and neovascularization are integral to major retinal diseases including retinitis pigmentosa (RP), Stargardt's disease, Leber's congenital amaurosis (LCA), diabetic retinopathy, and age-related macular degeneration (AMD).17–20 Recent evidence suggests a strong correlation between sphingolipid signaling and survival and homeostasis of photoreceptor and retinal pigment epithelial (RPE) cells: (1) CERs are mediators of retinal photoreceptor apoptosis, especially in oxidative stress–induced apoptosis21; (2) reducing the level of free CER through genetic manipulation rescues Drosophila photoreceptor cells from lethal mutations in the phototransduction genes22; (3) aberrant sphingolipid metabolism is reported in diabetic retinopathy16; (4) S1P signaling is involved in pathologic angiogenesis and choroidal neovascularization in the mouse retina23,24; and (5) mutations in the ceramide kinase–like (CERKL) gene, a gene predicted to be involved in CER metabolism, are involved in nonsyndromic retinal degeneration in human (RP26).25–27 These results clearly support the functional role of sphingolipid signaling in retinal physiology and pathophysiology. However, very little is known about their metabolism, abundance, and composition in the retina. As the first step toward understanding their role(s) in the retina, we determined the abundance and molecular composition of retinal sphingolipids. We have recently shown that the human Stargardt 3 (STGD3) retinal dystrophy protein ELOVL4 (elongation of very-long-chain fatty acid 4) is involved in biosynthesis of fatty acids with chain lengths >26 carbons.28 These very-long-chain saturated, mono- and polyunsaturated fatty acids are present in tissues that express ELOVL4, such as retina, skin, and testis, but the specific role that they play is not well understood. Homozygous Elovl4 knockout and knockin mice die after birth as a result of a peculiar skin barrier defect29–31 resulting from a deficiency in a particular sphingolipid (ω-O-acylceramide) that contains very-long-chain fatty acids. As in the skin of these mice, there is a possibility that sphingolipids containing very-long-chain fatty acids are involved in ELOVL4-mediated retinal disease in humans. We, therefore, extended our studies to analyze, for the first time, retinal sphingolipids containing very-long-chain fatty acids.

Lipid rafts, specialized membrane microdomains in the plasma membrane that are rich in cholesterol and sphingolipids, are hot-spots for a number of important cellular processes. The novel acetylcholine receptor (AChR) mutation αC418W was shown to be cholesterol-sensitive (Santiago et al., 2001) and to accumulate in microdomains rich in the membrane raft marker protein caveolin-1 (Baez-Pagan et al., 2008). The objective of this study is to gain insight into the mechanism by which lateral segregation into specialized raft membrane microdomains regulates the activatable pool of AChRs. We performed Fluorescent Recovery After Photobleaching (FRAP) experiments and whole-cell patch clamp recordings of GFP-encoding mus musculus AChRs transfected into HEK 293 cells to assess the role of cholesterol levels in the diffusion and functionality of the AChR (WT and αC418W). Our findings support the hypothesis that a cholesterol-sensitive AChR might reside in a specialized membrane microdomain; however, when cholesterol is depleted in vitro or in vivo, the caveolae disrupt and the cholesterol-sensitive AChRs are released to the pool of activatable receptors. Furthermore, our results suggest that phosphatidylinositol 4,5-bisphosphate (PIP2), which is concentrated in lipid rafts, may be responsible for the increase in whole-cell currents observed upon cholesterol depletion for the αC418W AChR mutant.This work was supported by NIH Grants 2RO1GM56371-12 and 2U54NS43011.

Abstract 2506 Poster Board II-483 Erythropoietin (EPO) is the principal regulator of erythroid progenitor proliferation, differentiation, and survival. Upon ligand engagement, the EPO-receptor (R) homodimerizes to activate the tyrosine kinases, Janus kinase-2 (JAK-2) and Lyn, which in turn phosphorylate the signal transducer and activator of transcription (STAT)-5. Although recent investigations have identified key negative regulators of the EPO-R signal, little is known about the membrane localization and dynamic control of receptor signal fidelity. Here we show a critical role for membrane raft microdomains in the creation of signaling platforms that are essential for signal integrity. Using UT-7 cells, we showed that recombinant human EPO (rhuEPO) stimulation rapidly induced raft formation and aggregation. Confocal microscopy quantitation of the raft ganglioside GM-1 fluorescence showed that raft aggregates increased from a mean of 4.3 ± 1.4 (SE) per cell to 25.6 ± 3.2 aggregates per cell after cytokine stimulation (p≤0.001), accompanied by a greater than 3-fold increase in cluster size (mean stimulated to untreated aggregate size ratio: 3.33 ± 0.11, p≤0.001). Isolation and immunoblot analysis of detergent insoluble raft fractions showed that the EPO-R translocated to lipid rafts after EPO stimulation of UT-7 cells. Confocal microscopy confirmed translocation of the EPO-R into membrane rafts in EPO-stimulated UT-7 cells and normal erythroid bursts. Receptor recruitment into rafts was accompanied by Jak2, Lyn and STAT5 loading into membrane raft fractions upon EPO stimulation. Treatment with methyl-β-cyclodextrin (MBCD) to deplete raft cholesterol and disrupt raft integrity extinguished EPO-induced STAT5 phosphorylation in UT-7 cells and human bone marrow progenitors. Similarly, membrane cholesterol-sequestration by nystatin markedly reduced EPO induced STAT5 phosphorylation in UT-7 cells. MBCD pretreatment of these cells prior to stimulation with phorbol 12-myristate 13-acetate (PMA) did not alter mitogen-activated protein kinase (MAPK) phosphorylation, indicating preservation of non-receptor, non-raft signal integrity despite depletion of cholesterol rich microdomains. These data establish a critical role for membrane raft microdomains in the recruitment and physical assembly of the EPO-R and its signaling intermediates into discrete platforms necessary to optimize signal integrity Disclosures: No relevant conflicts of interest to declare.

We have characterized the membrane properties of inositol phosphorylceramide (IPC) in model membranes with emphasis on calcium induced effects. IPCs are a class of anionic sphingolipids with a single phosphoinositol head group coupled to ceramide. IPCs have been identified in eukaryotic species and also observed in detergent resistant membranes in Leishmania major. Here we report on the properties of N-palmitoyl-IPC (PIPC) in one component bilayers as well as in complex bilayers together with POPC and sterols. According to anisotropy changes reported by DPH and initial DSC experiments, the gel-to-liquid main transition temperature (Tm) of PIPC is about 50 °C. Addition of 5 mM Ca2+ during vesicle preparation markedly increased the Tm to about 65 °C. DPH anisotropy also showed that cholesterol (at 20 mol %) was able to decrease the order in the gel-phase and increase the order in the liquid disordered-phase. Fluorescence quenching studies using the fluorescent sterol analogues cholestatrienol or dehydroergosterol and the doxyl labeled lipid 7SLPC as quencher, showed that PIPC was able to form sterol-enriched ordered domains in the otherwise fluid bilayer environment. Dynamic light scattering studies indicate a clustering of PIPC containing SUVs (PSM:PIPC, 8:2) due to addition of Ca2+ to a final concentration of 5 mM. The increase in Tm and apparent clustering of PIPC containing vesicles shows that PIPC/Ca2+ interactions have substantial effects on the behavior of PIPC in model membranes. IPCs and the calcium induced effects could be important in membrane associated cellular processes such as membrane fusion and membrane raft linked processes.

Hyperactivation of N-methyl-D-aspartate receptors (NRs) is associated with neuronal cell death induced by traumatic brain injury (TBI) and many neurodegenerative conditions. NR signaling efficiency is dependent on receptor localization in membrane raft microdomains. Recently, excitotoxicity has been linked to autophagy, but mechanisms governing signal transduction remain unclear. Here we have identified protein interactions between NR2B signaling intermediates and the autophagic protein Beclin-1 in membrane rafts of the normal rat cerebral cortex. Moderate TBI induced rapid recruitment and association of NR2B and pCaMKII to membrane rafts, and translocation of Beclin-1 out of membrane microdomains. Furthermore, TBI caused significant increases in expression of key autophagic proteins and morphological hallmarks of autophagy that were significantly attenuated by treatment with the NR2B antagonist Ro 25-6981. Thus, stimulation of autophagy by NR2B signaling may be regulated by redistribution of Beclin-1 in membrane rafts after TBI.

By exploiting the biosynthetic pathways of raft lipid constituents, in this study we demonstrate that fluctuations in either sphingolipid or ergosterol levels result in increased drug sensitivity and morphological defects in Candida albicans cells. We show that any change in either ergosterol composition by conditionally disrupting ERG1 or in sphingolipid composition by homozygously disrupting its biosynthetic gene IPT1 leads to improper surface localization of a major ABC (ATP-binding cassette) drug efflux protein, Cdr1p. Results suggest that sterol/sphingolipid-rich membrane microdomains play an important role in positioning and functional maintenance of the integral efflux protein. The impaired ability of erg1/ipt1 mutant cells to efflux drugs mediated through Cdr1p appears to be the main cause of increased drug sensitivity of Candida cells.

The impact of estrogens on the viability of cardiovascular system and their ability to regulate platelet function is still an open and debated question. We have previously shown that estrogen is able to significantly potentiate the aggregation induced by low doses of thrombin and to initiate a rapid and reversible signaling pathway mediated by ERbeta-directed activation of the tyrosine kinases Src and Pyk2 at the level of the plasma membrane. Lipid rafts are critical, cholesterol-enriched membrane domains, which play a major role in blood platelet activation processes. In this work, we investigated the role of lipid rafts in 17beta-estradiol signaling in human platelets. We observed that membrane rafts were essential for both 17beta-estradiol-dependent potentiation of platelet aggregation induced by subthreshold concentrations of thrombin and 17beta-estradiol-induced phosphorylation of Src. 17beta-estradiol caused the reversible translocation of ERbeta to the raft fractions and promoted the rapid and transient recruitment to, and activation within the membrane raft domains of the tyrosine kinases Src and Pyk2. The raft integrity was essential with this respect, as these effects of 17beta-estradiol were completely inhibited by cholesterol depletion. This paper provides evidence for the first time that membrane lipid rafts coordinate estrogen signaling in human platelets.

SummaryNeutrophils rapidly undergo polarization and directional movement to infiltrate the sites of infection and inflammation. Here, we show that an inhibitory MHC I receptor, Ly49Q, was crucial for the swift polarization of and tissue infiltration by neutrophils. During the steady state, Ly49Q inhibited neutrophil adhesion by preventing focal-complex formation, likely by inhibiting Src and PI3 kinases. However, in the presence of inflammatory stimuli, Ly49Q mediated rapid neutrophil polarization and tissue infiltration in an ITIM-domain-dependent manner. These opposite functions appeared to be mediated by distinct use of effector phosphatase SHP-1 and SHP-2. Ly49Q-dependent polarization and migration were affected by Ly49Q regulation of membrane raft functions. We propose that Ly49Q is pivotal in switching neutrophils to their polarized morphology and rapid migration upon inflammation, through its spatiotemporal regulation of membrane rafts and raft-associated signaling molecules.

The Vpu protein of human immunodeficiency virus type 1 (HIV-1) is known to enhance virion release from certain cell types. To accomplish this function, Vpu interacts with the restriction factor known as bone marrow stromal cell antigen 2 (BST-2)/tetherin. In this study, we analyzed whether the Vpu protein is associated with microdomains known as lipid or membrane rafts. Our results indicate that Vpu partially partitions into detergent-resistant membrane (DRM) fractions when expressed alone or in the context of simian–human immunodeficiency virus (SHIV) infection. The ability to be partitioned into rafts was observed with both subtype B and C Vpu proteins. The use of cholesterol lowering lovastatin/M-β-cyclodextrin and co-patching experiments confirmed that Vpu can be detected in cholesterol rich regions of membranes. Finally, we present data showing that raft association-defective transmembrane mutants of Vpu have impaired enhanced virus release function, but still maintain the ability to down-regulate CD4.

The structure and composition of coexisting bilayer phases separated in binary mixtures of dipalmitoylphosphatidylcholine and cholesterol and ternary mixtures of equimolar proportions of dipalmitoyl- and dioleoylphosphatidycholines containing different proportions of cholesterol have been characterized by synchrotron X-ray diffraction methods. The liquid-ordered phase is distinguished from gel and fluid phases by a disordering of the hydrocarbon chains intermediate between the two phases as judged from the wide-angle X-ray scattering profiles. Electron density distribution calculated in coexisting bilayer phases shows that liquid-ordered phase is enriched in dipalmitoylphosphatidylcholine and cholesterol and a higher electron density in the methylene chain region of the bilayer ascribed to the location of the sterol ring of cholesterol. The ratio of the two constituents in the liquid-ordered phase is not constant because the stoichiometry is temperature-dependent as seen by respective changes in bilayer thickness over the range 20 degrees to 36 degrees C where coexisting phases are observed. Three coexisting phases were deconvolved in the ternary mixture at 20 degrees C. From an analysis of the ternary mixtures containing mole fractions of cholesterol from 0.09 to 0.15 it was found that the liquid-crystal and gel phases each contained about 10% of the cholesterol molecules and the liquid-ordered phase was comprised of 30% cholesterol molecules.

Flotillin-1 and flotillin-2 are two homologous, membrane raft associated proteins. Although it has been reported that flotillins are involved in cell adhesion processes and play a role during breast cancer progression, thus making them interesting future therapeutic targets, their precise function has not been well elucidated. The present study investigates the function of these proteins in cell-cell adhesion in non-malignant cells. We have used the non-malignant epithelial MCF10A cells to study the interaction network of flotillins within cell-cell adhesion complexes. RNA interference was used to examine the effect of flotillins on the structure of adherens junctions and on the association of core proteins, such as E-cadherin, with membrane rafts. We here show that the cadherin proteins of the adherens junction associate with flotillin-2 in MCF10A cells and in various human cell lines. In vitro, flotillin-1 and flotillin-2 directly interact with γ-catenin which is so far the only protein known to be present both in the adherens junction and the desmosome. Mapping of the interaction domain within the γ-catenin sequence identified the Armadillo domains 6–8, especially ARM domain 7, to be important for the association with flotillins. Furthermore, depletion of flotillins significantly influenced the morphology of the adherens junction in human epithelial MCF10A cells and altered the association of E-cadherin and γ-catenin with membrane rafts. Taken together, these observations suggest a functional role for flotillins, especially flotillin-2, in cell-cell adhesion in non-malignant epithelial cells.

Membrane rafts are sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Membrane rafts isolated from post-mortem AD brain are enriched in both β-amyloid and phosphorylated tau. Proteolytic processing of APP to generate β-amyloid, the principle component of amyloid plaques, can occur in membrane rafts, implicating them in the pathogenesis of Alzheimer's disease (AD). Secondary to their role in β-amyloid generation, membrane rafts have more recently been implicated in the accumulation, aggregation and degradation of β-amyloid, with evidence supporting a specific role for membrane raft gangliosides in the binding and aggregation of β-amyloid. In addition, membrane domain composition has a direct impact on both the generation of β-amyloid and its subsequent toxic actions and as such is a key target for the development of therapeutic strategies. This mini-review will focus on recent advances in our understanding of the relevance of membrane composition, of both raft and non-raft domains, to AD progression in models and in human disease. We will discuss how manipulation of membrane composition can alter both the proteolytic processing of APP and the subsequent binding and aggregation of β-amyloid peptide.