Involvement Of Lipid Rafts Research Articles

Involvement of Lipid Rafts and Caveolae in gas6-Axl Signaling in Endothelial Cells

Abstract 2211Lipid rafts are localized regions of plasma membranes that contain primarily cholesterol and glycosphingolipids. Many receptor tyrosine kinases including the epidermal growth factor receptor, the platelet derived growth factor receptor, and the insulin receptor, localize specifically to these structures. Lipid rafts have been shown to play a role in intracellular signaling events. More specifically, diverse processes such as dimer formation, autophosphorylation, and intracellular adaptor protein recruitment/activation have been specifically involved in receptor tyrosine kinase/lipid raft interactions. Caveolae are defined as a subclass of lipid rafts and form well-shape invaginations on the cell surface. Caveolin-1 is the major constitutive protein of caveolae. Axl is a receptor tyrosine kinase whose association with lipid rafts and more specifically with caveolae has not been elucidated. Gas6, the ligand for Axl, is a vitamin K dependent protein that is post-translationally modified by an enzymatic process called g-carboxylation. We hypothesize that gas6/Axl mediated intracellular signaling is dependent on its localization to lipid rafts/caveolae in endothelial cells. First, we show that gas6-induced c-Src, Akt and ERK1/2 phosphorylation is mediated via Axl by using Axl siRNA in human umbilical vein endothelial cells (HUVECs). Then, lipid rafts were isolated from HUVECs by a detergent-free lysis method followed by a sucrose gradient centrifugation and western blot analysis. We found that Axl moved into the lipid raft-enriched fractions after 5 and 10 min of gas6 treatment as shown by its colocalization with caveolin-1. Interestingly, after gas6 treatment, c-Src, which is known to act as a transient docking platform for signaling molecules in lipid rafts, follow the same localization pattern to lipid raft-enriched fractions. The requirement of caveolae was then evaluated by transfecting HUVECs with caveolin-1 siRNA. Caveolin-1 knock down abolished completely gas6-induced phosphorylation of c-Src, Akt and ERK1/2 thus highlighting the role of caveolae in gas6-Axl signaling. Taken together these results demonstrate that activation of gas6-dependent signaling pathways involves Axl trafficking to lipid rafts/caveolae. These novel findings identify lipid rafts/caveolae as dynamic scaffolding systems for gas6/Axl interactions in endothelial cells. Disclosures:No relevant conflicts of interest to declare.

Lipid-Mediated Endocytosis

Receptor-mediated endocytosis is used by a number of viruses and toxins to gain entry into cells. Some have evolved to use specific lipids in the plasma membrane as their receptors. They include bacterial toxins such as Shiga and Cholera toxin and viruses such as mouse polyoma virus and simian virus 40. Through multivalent binding to glycosphingolipids, they induce lipid clustering and changes in membrane properties. Internalization occurs by unusual endocytic mechanisms involving lipid rafts, induction of membrane curvature, trans-bilayer coupling, and activation of signaling pathways. Once delivered to early endosomes, they follow diverse intracellular routes to the lumen of the ER, from which they penetrate into the cytosol. The role of the lipid receptors is central in these well-studied processes.

Involvement of lipid rafts in the localization and dysfunction effect of the antitumor ether phospholipid edelfosine in mitochondria

Lipid rafts and mitochondria are promising targets in cancer therapy. The synthetic antitumor alkyl-lysophospholipid analog edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) has been reported to target lipid rafts. Here, we have found that edelfosine induced loss of mitochondrial membrane potential and apoptosis in human cervical carcinoma HeLa cells, both responses being abrogated by Bcl-xL overexpression. We synthesized a number of new fluorescent edelfosine analogs, which preserved the proapoptotic activity of the parent drug, and colocalized with mitochondria in HeLa cells. Edelfosine induced swelling in isolated mitochondria, indicating an increase in mitochondrial membrane permeability. This mitochondrial swelling was independent of reactive oxygen species generation. A structurally related inactive analog was unable to promote mitochondrial swelling, highlighting the importance of edelfosine molecular structure in its effect on mitochondria. Raft disruption inhibited mitochondrial localization of the drug in cells and edelfosine-induced swelling in isolated mitochondria. Edelfosine promoted a redistribution of lipid rafts from the plasma membrane to mitochondria, suggesting a raft-mediated link between plasma membrane and mitochondria. Our data suggest that direct interaction of edelfosine with mitochondria eventually leads to mitochondrial dysfunction and apoptosis. These observations unveil a new framework in cancer chemotherapy that involves a link between lipid rafts and mitochondria in the mechanism of action of an antitumor drug, thus opening new avenues for cancer treatment.

Fish Oils Inhibit EGFR Signals in Normal Mouse Colon and Suppress up-Regulated EGFR Signals in AOM Rat Colon Cancers. In Vitro Dissection of Mechanism Involving Lipid Rafts

Fish Oils Inhibit EGFR Signals in Normal Mouse Colon and Suppress up-Regulated EGFR Signals in AOM Rat Colon Cancers. In Vitro Dissection of Mechanism Involving Lipid Rafts

Involvement of lipid rafts in macrophage apoptosis induced by cationic liposomes

We have demonstrated that protein kinase Cδ (PKCδ) could be involved in macrophage apoptosis induced by cationic liposomes composed of stearylamine (SA-liposomes), but the detailed mechanism of how SA-liposomes activate PKCδ has remained unclear. In this paper, we clarified whether lipid rafts are involved in the PKCδ activation induced by SA-liposomes. Co-localization of SA-liposomes and Cholera toxin B subunit (CBT), which specifically binds to ganglioside GM1 on lipid rafts, was found by microscopic observation. The incorporation of SA-liposomes into lipid rafts was clearly inhibited by the pretreatment of cells with an agent, 2,6-di-O-methyl-α-cyclodextrin (DM-α-CD) which disrupts lipid rafts. Activation of PKCδ and externalization of phosphatidylserine induced by SA-liposomes were also suppressed by DM-α-CD, which extracts sphingolipids and proteins from lipid rafts. Reactive oxygen species (ROS) generation, which could be involved in the macrophage apoptosis, was also inhibited by DM-α-CD. Furthermore, apoptosis induced by SA-liposomes was clearly inhibited when the cells were pre-treated with DM-α-CD, but not nystatin, a cholesterol-sequestering agent that disrupt lipid rafts. These findings suggest that sphingolipids in lipid rafts are involved in the activation of PKCδ which leads to apoptosis induced by cationic liposomes, SA-liposomes.

Involvement of lipid rafts in adhesion-induced activation of Met and EGFR

BackgroundCell adhesion has been shown to induce activation of certain growth factor receptors in a ligand-independent manner. However, the mechanism for such activation remains obscure.MethodsHuman epidermal carcinoma A431 cells were used as a model to examine the mechanism for adhesion-induced activation of hepatocyte growth factor receptor Met and epidermal growth factor receptor (EGFR). The cells were suspended and replated on culture dishes under various conditions. The phosphorylation of Met at Y1234/1235 and EGFR at Y1173 were used as indicators for their activation. The distribution of the receptors and lipid rafts on the plasma membrane were visualized by confocal fluorescent microscopy and total internal reflection microscopy.ResultsWe demonstrate that Met and EGFR are constitutively activated in A431 cells, which confers proliferative and invasive potentials to the cells. The ligand-independent activation of Met and EGFR in A431 cells relies on cell adhesion to a substratum, but is independent of cell spreading, extracellular matrix proteins, and substratum stiffness. This adhesion-induced activation of Met and EGFR cannot be attributed to Src activation, production of reactive oxygen species, and the integrity of the cytoskeleton. In addition, we demonstrate that Met and EGFR are independently activated upon cell adhesion. However, partial depletion of Met and EGFR prevents their activation upon cell adhesion, suggesting that overexpression of the receptors is a prerequisite for their self-activation upon cell adhesion. Although Met and EGFR are largely distributed in 0.04% Triton-insoluble fractions (i.e. raft fraction), their activated forms are detected mainly in 0.04% Triton-soluble fractions (i.e. non-raft fraction). Upon cell adhesion, lipid rafts are accumulated at the cell surface close to the cell-substratum interface, while Met and EGFR are mostly excluded from the membrane enriched by lipid rafts.ConclusionsOur results suggest for the first time that cell adhesion to a substratum may induce a polarized distribution of lipid rafts to the cell-substratum interface, which may allow Met and EGFR to be released from lipid rafts, thus leading to their activation in a ligand-independent manner.

Aβ Behavior on Neuronal Membranes: Aggregation and Toxicities

A growing body of evidence suggests that the aggregation and toxic potentials of amyloidogenic proteins, including amyloid β-protein (Aβ), α-synuclein, and prion protein, emerge through the interaction of these proteins with neuronal and/or glial membranes. The aggregation and deposition of Aβ are the initial events of Alzheimer's disease (AD), and the toxicity of aggregated Aβ is the basis for the neuronal loss in AD brains. Thus, the Aβ behavior on neuronal membranes should be one of the critical issues to be clarified for our further understanding of the pathogenesis of AD and to develop therapeutic strategies. To accelerate studies in this field, we have invited original research articles as well as review articles that will provide novel information for our special issue. The first three papers of this special issue describe the crucial involvement of lipid rafts, which are specific membrane microdomains on the cell surface that are rich in sphingolipids and cholesterol, in the production, aggregation, and toxicities of Aβ. The subsequent three papers focus on the gangliosides, which are the major constituent of lipid rafts, particularly in terms of their role in the induction of conformational changes of Aβ, leading to their aggregation and emerging toxicities. The next two articles address how Aβ causes neuronal injury by showing the possibility of formation of amyloid channels in the neuronal membranes, resulting in the disruption of calcium homeostasis that is critical for the function and survival of neurons, and the possibility of generation of radicals. In regard to the Aβ toxicities, much attention has been paid to the argument that the accumulation of Aβ inside neurons may be the critical step. In this context, the next two papers propose a mechanism by which Aβ enters the neurons, which are followed by another two papers showing how the internalized Aβ acts pathologically inside neurons, emphasizing the possibility that the mitochondria may be a target of intraneuronal Aβ. A further argument for the possible interaction between Aβ and neuronal membranes is presented in the next four papers. In these papers, it is presented how Aβ affects the properties of neuronal membranes or, conversely, how the alteration of membrane properties affects the processing of amyloid precursor protein (APP) leading to Aβ generation. Note that the metabolism of neuronal lipids, particularly sphingolipids and ceramide, can be regulated in association with APP processing. The final paper of this special issue describes a foresighted aspect of science and technology of nanochemistry with respect to the pathological protein aggregation, which is likely based on the catalysts of membrane lipids, suggesting an opportunity for developing novel nanomedicines and nanodiagnostics for various amyloidoses. We all look forward to seeing further expansion of studies in this field in the near future. Katsuhiko Yanagisawa Jacques Fantini Avijit Chakrabartty Anne Eckert

The role of lipid rafts in prion protein biology.

The conformational conversion of the cellular prion protein, PrP(C), to the misfolded isoform PrP(Sc )is the central pathogenic event in the uniquely transmissible neurodegenerative prion diseases. As both PrP(C) and PrP(Sc) are associated with membranes, the nature of the membrane microenvironment may well play a significant role in both the conformational conversion process as well as the normal functions of PrP(C). Within the membrane are various microdomains, areas of distinct lipid and protein composition, the best studied of which are the cholesterol- and sphingolipid-rich lipid rafts. These domains are characterized biochemically by their relative resistance to solubilization in certain detergents at low temperature. In this article we review the evidence for the involvement of lipid rafts in the localization and trafficking of PrP(C), in the cellular signaling, neuroprotective and metal binding functions of PrP(C), and as sites for the conversion of PrP(C) to PrP(Sc) and in cell-to-cell prion transmission.

Mechanisms of Amyloid‐Beta Peptide Uptake by Neurons: The Role of Lipid Rafts and Lipid Raft‐Associated Proteins

A hallmark pathological feature of Alzheimer's disease (AD) is the accumulation of extracellular plaques composed of the amyloid-beta (Aβ) peptide. Thus, classically experiments were designed to examine Aβ toxicities within the central nervous system (CNS) from the extracellular space. However, a significant amount of evidence now suggests that intraneuronal accumulation of Aβ is neurotoxic and may play an important role in the disease progression of AD. One of the means by which neurons accumulate intracellular Aβ is through uptake of extracellular Aβ peptides, and this process may be a potential link between Aβ generation, synaptic dysfunction, and AD pathology. Recent studies have found that neuronal internalization of Aβ involves lipid rafts and various lipid raft-associated receptor proteins. Uptake mechanisms independent of lipid rafts have also been implicated. The aim of this paper is to summarize these findings and discuss their significance in the pathogenesis of AD.

Unsaturated fatty acids prevent desensitization of the human growth hormone secretagogue receptor by blocking its internalization

The composition of the plasma membrane affects the responsiveness of cells to metabolically important hormones such as insulin and vasoactive intestinal peptide. Ghrelin is a metabolically regulated hormone that activates the G protein-coupled receptor GH secretagogue receptor type 1a (GHSR) not only in the pituitary gland but also in peripheral tissues such as the pancreas, stomach, and T cells in the circulation. We have investigated the effects of lipids and altered plasma membrane composition on GHSR activation. Oligounsaturated fatty acids (OFAs) disrupt the structure of membranes and make them more fluid. Prolonged (96 h), but not acute, treatment of the GHSR cells with the 18C OFAs oleic and linoleic acid caused a significant increase in sensitivity of the receptor to ghrelin (EC(50) reduced by a factor of 2.4 and 2.9 at 60 and 120 microM OFAs, respectively). OFAs were found to block the inhibitory effects of ghrelin pretreatment on subsequent ghrelin responsiveness, suggesting that OFAs suppress desensitization of GHSR. Radioligand displacement studies did not show a significant shift in receptor binding after incubation with OFAs. However, it was found that OFA treatment suppressed GHSR internalization, likely explaining OFA-induced refractoriness to ligand-induced desensitization. The involvement of lipid rafts in this process was indicated by the altered responsiveness of GHSR under conditions that alter membrane cholesterol. In conclusion, our findings demonstrate the importance of membrane composition for GHSR activation and desensitization and indicate at least part of the mechanism through which OFAs and cholesterol could affect ghrelin's activity in vivo.

P2Y2 Receptor-Gq/11 Signaling at Lipid Rafts Is Required for UTP-Induced Cell Migration in NG 108-15 Cells

Lipid rafts, formed by sphingolipids and cholesterol within the membrane bilayer, are believed to have a critical role in signal transduction. P2Y(2) receptors are known to couple with G(q) family G proteins, causing the activation of phospholipase C (PLC) and an increase in intracellular Ca(2+) ([Ca(2+)](i)) levels. In the present study, we investigated the involvement of lipid rafts in P2Y(2) receptor-mediated signaling and cell migration in NG 108-15 cells. When NG 108-15 cell lysates were fractionated by sucrose density gradient centrifugation, Galpha(q/11) and a part of P2Y(2) receptors were distributed in a fraction where the lipid raft markers, cholesterol, flotillin-1, and ganglioside GM1 were abundant. Methyl-beta-cyclodextrin (CD) disrupted not only lipid raft markers but also Galpha(q/11) and P2Y(2) receptors in this fraction. In the presence of CD, P2Y(2) receptor-mediated phosphoinositide hydrolysis and [Ca(2+)](i) elevation were inhibited. It is noteworthy that UTP-induced cell migration was inhibited by CD or the G(q/11)-selective inhibitor YM254890 [(1R)-1-{(3S,6S,9S,12S,18R,21S,22R)-21-acetamido-18-benzyl-3-[(1R)-1-methoxyethyl]-4,9,10,12,16, 22-hexamethyl-15-methylene-2,5,8,11,14,17,-20-heptaoxo-1,19-dioxa-4,7,10,13,16-pentaazacyclodocosan-6-yl}-2-methylpropyl rel-(2S,3R)-2-acetamido-3-hydroxy-4-methylpentanoate]. Moreover CD and YM254890 completely inhibited Rho-A activation. Downstream of Rho-A signaling, stress fiber formation and phosphorylation of cofilin were also inhibited by CD or YM254890. However, UTP-induced phosphorylation of cofilin was not affected by the expression of p115-regulator of G protein signaling, which inhibits the G(12/13) signaling pathway. This implies that UTP-induced Rho-A activation was relatively regulated by the G(q/11) signaling pathway. These results suggest that lipid rafts are critical for P2Y(2) receptor-mediated G(q/11)-PLC-Ca(2+) signaling and this cascade is important for cell migration in NG 108-15 cells.

The Rafts of the Medusa: cholesterol targeting in cancer therapy

In this issue of Oncogene, Mollinedo and co-workers present promising evidence that cholesterol-sensitive signaling pathways involving lipid rafts can be therapeutically targeted in multiple myeloma. Because the pathways considered in their study are used by other types of tumor cells, one implication of this report is that cholesterol-targeting approaches may be applicable to other malignancies.

Selective export of human GPI-anchored proteins from the endoplasmic reticulum

Selective export of transmembrane proteins from the endoplasmic reticulum (ER) relies on recognition of cytosolic-domain-localized transport signals by the Sec24 subunit of the COPII vesicle coat. Human cells express four Sec24 isoforms, termed Sec24A, Sec24B, Sec24C and Sec24D that are differentially required for selective, signal-mediated ER export of transmembrane proteins. By contrast, luminally exposed glycosylphosphatidylinositol (GPI)-anchored membrane proteins cannot bind directly to Sec24 and must either use membrane-spanning cargo receptors or alternative mechanisms for ER export. Little is known about the mechanism underlying export of GPI-anchored proteins from the ER in higher eukaryotes. Using siRNA-based silencing, we identified that ER-to-Golgi transport of the human GPI-anchored protein CD59 requires Sec24, with preference for the Sec24C and Sec24D isoforms, and the recycling transmembrane protein complex p24-p23 that exhibited the same Sec24C-Sec24D isoform preference for ER export. Co-immunoprecipitation indicated unprecedented physical interaction of CD59 as well as a GFP-folate-receptor-GPI-anchor hybrid with a p24-p23 complex. Density gradient centrifugation revealed co-partitioning of CD59 and p24-p23 into biosynthetically early lipid raft fractions, and CD59 transport to the Golgi was cholesterol dependent. The results suggest that the 24p-23p complex acts as a cargo receptor for GPI-anchored proteins by facilitating their export from the ER in a Sec24-isoform-selective manner involving lipid rafts as early sorting platforms.

In vitro and In vivo Selective Antitumor Activity of Edelfosine against Mantle Cell Lymphoma and Chronic Lymphocytic Leukemia Involving Lipid Rafts

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) remain B-cell malignancies with limited therapeutic options. The present study investigates the in vitro and in vivo effect of the phospholipid ether edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) in MCL and CLL. Several cell lines, patient-derived tumor cells, and xenografts in severe combined immunodeficient mice were used to examine the anti-MCL and anti-CLL activity of edelfosine. Furthermore, we analyzed the mechanism of action and drug biodistribution of edelfosine in MCL and CLL tumor-bearing severe combined immunodeficient mice. Here, we have found that the phospholipid ether edelfosine was the most potent alkyl-lysophospholipid analogue in killing MCL and CLL cells, including patient-derived primary cells, while sparing normal resting lymphocytes. Alkyl-lysophospholipid analogues ranked edelfosine > perifosine >> erucylphosphocholine > or = miltefosine in their capacity to elicit apoptosis in MCL and CLL cells. Edelfosine induced coclustering of Fas/CD95 death receptor and rafts in MCL and CLL cells. Edelfosine was taken up by malignant cells, whereas normal resting lymphocytes hardly incorporated the drug. Raft disruption by cholesterol depletion inhibited drug uptake, Fas/CD95 clustering, and edelfosine-induced apoptosis. Edelfosine oral administration showed a potent in vivo anticancer activity in MCL and CLL xenograft mouse models, and the drug accumulated dramatically and preferentially in the tumor. Our data indicate that edelfosine accumulates and kills MCL and CLL cells in a rather selective way, and set coclustering of Fas/CD95 and lipid rafts as a new framework in MCL and CLL therapy. Our data support a selective antitumor action of edelfosine.

β‐Amyloid activates presynaptic α7 nicotinic acetylcholine receptors reconstituted into a model nerve cell system: involvement of lipid rafts

Beta amyloid (Abeta) plays a central role in the pathogenesis of Alzheimer's disease. Abeta is the major constituent of senile plaques, but there is a significant presence of Abeta in the brain in soluble forms. The results of functional studies indicate that soluble Abeta interacts with the alpha7 nicotinic acetylcholine receptor (nAChR) complex with apparent high affinity. However, conflicting data exist as to the nature of the Abeta-alpha7 nAChR interaction, and whether it is the result of specific binding. Moreover, both agonist-like and antagonist-like effects have been reported. In particular, agonist-like effects have been observed for presynaptic nAChRs. Here, we demonstrate Abeta(1-42)-evoked stimulatory changes in presynaptic Ca(2+) level via exogenous alpha7 nAChRs expressed in the axonal varicosities of differentiated hybrid neuroblastoma NG108-15 cells as a model, presynaptic system. The Abeta(1-42)-evoked responses were concentration-dependent and were sensitive to the highly selective alpha7 nAChR antagonist alpha-bungarotoxin. Voltage-gated Ca(2+) channels and internal Ca(2+) stores were both involved in Abeta(1-42)-evoked increases in presynaptic Ca(2+) following activation of alpha7 nAChRs. In addition, disruption of lipid rafts by cholesterol depletion led to substantially attenuated responses to Abeta(1-42), whereas responses to nicotine were largely intact. These results directly implicate the nicotinic receptor complex as a target for the agonist-like action of pico- to nanomolar concentrations of soluble Abeta(1-42) on the presynaptic nerve terminal, including the possible involvement of receptor-associated lipid rafts. This interaction probably plays an important neuromodulatory role in synaptic dynamics.

High Molecular Weight Polyethylene Glycol Preserves Intestinal Integrity during Tissue Harvest for Transplantation

Introduction: During organ harvest for transplantation (TX) tissues undergo cold ischemia (C) during preservation and reperfusion during implantation. Cooling to 4°C with specialized solutions is routinely employed to decrease tissue edema, oxidative stress and preserve membrane integrity. We have previously reported that high molecular weight polyethylene glycol (15-20,000 MW, PEG15-20) protects the intestinal epithelium against a variety of cellular stresses including radiation injury and microbial invasion by mechanisms that appear to involve lipid rafts.

Lipid Rafts and Caveolin-1 Coordinate Interleukin-1β (IL-1β)-dependent Activation of NFκB by Controlling Endocytosis of Nox2 and IL-1β Receptor 1 from the Plasma Membrane

Recent evidence suggests that signaling by the proinflammatory cytokine interleukin-1beta (IL-1beta) is dependent on reactive oxygen species derived from NADPH oxidase. Redox signaling in response to IL-1beta is known to require endocytosis of its cognate receptor (IL-1R1) following ligand binding and the formation of redox-active signaling endosomes that contain Nox2 (also called redoxosomes). The consequent generation of reactive oxygen species by redoxosomes is responsible for the downstream recruitment of IL-1R1 effectors (IRAK, TRAF6, and IkappaB kinase kinases) and ultimately for activation of the transcription factor NFkappaB. Despite this knowledge of the signaling events that occur downstream of redoxosome formation, an understanding of the mechanisms that coordinate the genesis of redoxosomes following IL-1beta stimulation has been lacking. Here, we demonstrate that lipid rafts play an important role in this process. We show that Nox2 and IL-1R1 localize to plasma membrane lipid rafts in the unstimulated state and that IL-1beta signals caveolin-1-dependent endocytosis of both proteins into the redoxosome. We also show that inhibiting lipid raft-mediated endocytosis prevents NFkappaB activation. Finally, we demonstrate that Vav1, a Rac1 guanine exchange factor and activator of Nox2, is recruited to lipid rafts following IL-1beta stimulation and that it is required for NFkappaB activation. Our results fill in an important mechanistic gap in the understanding of early IL-1R1 and Nox2 signaling events that control NFkappaB activation, a redox-dependent process important in inflammation.

Involvement of lipid rafts and cellular actin in AcMNPV GP64 distribution and virus budding

GP64 is the major envelope glycoprotein associated with the budded virus (BV) of Autographa californica nucleopolyhedrovirus (AcMNPV) and is essential for attachment and budding of BV particles. Confocal microscopy and flotation assays established the presence of lipid raft domains within the plasma membranes of AcMNPV-infected Sf9 cells and suggested the association of GP64 with lipid rafts during infection. GP64 and filamentous actin (F-actin) were found to co-localise at the cell cortex at 24 and 48 hpi and an additional restructuring of F-actin during infection was visualised, resulting in a strongly polarised distribution of both F-actin and GP64 at the cell cortex. Depletion of F-actin, achieved by treatment of Sf9 cells with latrunculin B (LB), resulted in the redistribution of GP64 with significant cytoplasmic aggregation and reduced presence at the plasma membrane. Treatment with LB also resulted in reduced production of BV in Sf9 cells. Analysis of virus gene transcription confirmed this reduction was not due to decreased trafficking of nucleocapsids to the nucleus or to decreased production of infectious progeny nucleocapsids. Reduced BV production due to a lack of GP64 at the plasma membrane of AcMNPV-infected Sf9 cells treated with LB, suggests a key role for F-actin in the egress of BV.

S1584 Carbon Monoxide Reduces Cholestatic Liver Injury Induced By Bile Duct Ligation

stained with a commercially available kit and co-localization with RhoA was determined by confocal microscopy. RESULTS: Simvastatin significantly decreased cell number in all of the six CCA lines (40 to 80%; 5 μM for 48 hrs) and HiBEC as compared to untreated cells. To distinguish whether the decrease was due to apoptosis or cell cycle arrest, cell proliferation was assessed by BrdU uptake. Simvastatin (5 μM for 4-6 hrs) induced a significant increase in apoptosis in Mz-ChA-1, but not BrdU uptake. However, simvastatin while not triggering apoptosis in HiBEC did reduce BrdU uptake or proliferation in HiBEC. Pretreatment of MzChA-1 with Mev, FPP and GPP did not reverse the effects of simvastatin on Mz-ChA-1 cell viability suggesting that simvastatin-induced apoptosis is not heavily dependent upon HMGCoA reductase inhibition. In contrast, cholesterol reversed simvastatin-induced apoptosis indicating involvement of lipid rafts. Simvastatin disrupts lipid rafts in Mz-ChA-1 (present in higher levels than in HiBEC) resulting in a loss of RhoA colocalization and the potential disruption of a RhoA dependent cell survival pathway. In fact, disruption of lipid rafts with MBCD or inhibition of the upstream activator of RhoA, GGTase, with GGTI-298 also reduced Mz-ChA-1 viability to that achieved by simvastatin alone. CONCLUSION: Targeting of lipid rafts and RhoA may represent a new target for the treatment of CCA.

Transmembrane Form Agrin-induced Process Formation Requires Lipid Rafts and the Activation of Fyn and MAPK

Overexpression or clustering of the transmembrane form of the extracellular matrix heparan sulfate proteoglycan agrin (TM-agrin) induces the formation of highly dynamic filopodia-like processes on axons and dendrites from central and peripheral nervous system-derived neurons. Here we show that the formation of these processes is paralleled by a partitioning of TM-agrin into lipid rafts, that lipid rafts and transmembrane-agrin colocalize on the processes, that extraction of lipid rafts with methyl-beta-cyclodextrin leads to a dose-dependent reduction of process formation, that inhibition of lipid raft synthesis prevents process formation, and that the continuous presence of lipid rafts is required for the maintenance of the processes. Association of TM-agrin with lipid rafts results in the phosphorylation and activation of the Src family kinase Fyn and subsequently in the phosphorylation and activation of MAPK. Inhibition of Fyn or MAPK activation inhibits process formation. These results demonstrate that the formation of filopodia-like processes by TM-agrin is the result of the activation of a complex intracellular signaling cascade, supporting the hypothesis that TM-agrin is a receptor or coreceptor on neurons.