Disruption Of Membrane Rafts Research Articles

Insights into the Surface Binding and Structural Interference of Polyphenols with the Membrane Raft Domains in Relation to Their Distinctive Ability to Inhibit Preadipocyte Differentiation in 3T3-L1 Cells.

Polyphenols with different structures have shown distinct variations in their ability to inhibit the differentiation of 3T3-L1 preadipocytes. However, the underlying mechanisms for these differences remain unclear. In the present study, the surface binding of polyphenols to different membrane domains was explored using coarse-grained molecular dynamics simulation (CG-MDs). Subsequently, this surface binding was confirmed in the liposome system by microscale thermophoresis. Additionally, the interference of polyphenols on the membrane raft's structure was studied through atomic force microscopy and high-content screening fluorescence microscopy. The results indicated that polyphenols with a differentiation-inhibitory ability, such as epicatechin-3-gallate (ECG) and epicatechin-3-gallate-(4β → 8, 2β → O → 7)-epicatechin-3-gallate (A-type ECG dimer), exhibited strong binding to ordered domains enriched in sphingolipids and cholesterol. This binding led to the structural disruption of membrane rafts by altering their size and shape, with the binding constant of 3.8 μM for ECG and 0.3 μM for A-type ECG dimer, respectively. In contrast, epicatechin (EC) with little differentiation-inhibitory ability had no effects on membrane rafts, and its binding constant with the ordered domain was 380.6 μM. Overall, the surface binding of polyphenols to ordered domains and the resulting disruption of membrane rafts structure might be a fundamental mechanism by which polyphenols inhibited the differentiation of 3T3-L1 preadipocytes.

Involvement of Lipid Rafts in the Invasion of Opportunistic Bacteria Serratia into Eukaryotic Cells.

Cell membrane rafts form signaling platforms on the cell surface, controlling numerous protein-protein and lipid-protein interactions. Bacteria invading eukaryotic cells trigger cell signaling to induce their own uptake by non-phagocytic cells. The aim of this work was to reveal the involvement of membrane rafts in the penetration of the bacteria Serratia grimesii and Serratia proteamaculans into eukaryotic cells. Our results show that the disruption of membrane rafts by MβCD in the three cell lines tested, M-HeLa, MCF-7 and Caco-2, resulted in a time-dependent decrease in the intensity of Serratia invasion. MβCD treatment produced a more rapid effect on the bacterial susceptibility of M-HeLa cells compared to other cell lines. This effect correlated with a faster assembly of the actin cytoskeleton upon treatment with MβCD in M-HeLa cells in contrast to that in Caco-2 cells. Moreover, the 30 min treatment of Caco-2 cells with MβCD produced an increase in the intensity of S. proteamaculans invasion. This effect correlated with an increase in EGFR expression. Together with the evidence that EGFR is involved in S. proteamaculans invasion but not in S. grimesii invasion, these results led to the conclusion that an increase in EGFR amount on the plasma membrane with the undisassembled rafts of Caco-2 cells after 30 min of treatment with MβCD may increase the intensity of S. proteamaculans but not of S. grimesii invasion. Thus, the MβCD-dependent degradation of lipid rafts, which enhances actin polymerization and disrupts signaling pathways from receptors on the host cell's surface, reduces Serratia invasion.

Integrated metabolomics and lipidomics reveals high accumulation of polyunsaturated lysoglycerophospholipids in human lung fibroblasts exposed to fine particulate matter

Exposure to fine particulate matter (PM) comprising toxic compounds arising from air pollution is a major human health concern. It is linked to increased mortality and incidence of various lung diseases. However, the mechanisms underlying the toxic effects of PM on lung fibroblasts have not been fully explored. We used targeted quantitative metabolomics and lipidomics analysis along with cytotoxicity studies to comprehensively characterize the alterations in the metabolite profiles of human lung fibroblasts (HEL 299) upon exposure to PM2.5 and PM10. This exposure at 50 μg/mL for 72 h induced an abnormally high apoptotic response via triggering intracellular reactive oxygen species (ROS) production and mitochondrial dysfunction through an imbalance between pro- and anti-apoptotic signaling pathways. The cytotoxic effects of PM2.5 were more severe than those of PM10. Metabolomics and lipidomics analyses revealed that PM exposure triggered substantial changes in the cellular metabolite profile, which involved reduced mitochondria-related metabolites such as tricarboxylic acid (TCA) cycle intermediates, amino acids, and free fatty acids as well as increased lysoglycerophospholipids (LPLs) containing polyunsaturated fatty acids. The decrease in mitochondria-related metabolites suggested that PM exposure led to reduced TCA cycle capacity and energy production. Apoptotic and inflammatory responses as well as mitochondrial dysfunction were likely to be accelerated because of excessive accumulation of LPLs, contributing to the disruption of membrane rafts and Ca2+ homeostasis and causing increased mitochondrial ROS formation. These results provide valuable insights regarding the toxic effects of PM exposure. Our study also provides a new direction for research on PM exposure-related health disorders using different cell lines.

Membrane rafts regulate sperm acrosome reaction via cAMP-dependent pathway in chickens (Gallus gallus domesticus).

Both transcriptionally and translationally inactive sperm need preassembled pathways into specific cellular compartments to function. Although initiation of the acrosome reaction (AR) involves several signaling pathways including protein kinase A (PKA) activation, how these are regulated remains poorly understood in avian sperm. Membrane rafts are specific membrane regions enriched in sterols and functional proteins and play important roles in diverse cellular processes, including signal transduction. Our recent studies on chicken sperm demonstrated that membrane rafts exist and play a role in multistage fertilization. These, combined with the functional importance of membrane rafts in mammalian sperm AR, prompted us to investigate the roles of membrane rafts in signaling pathways leading to AR in chicken sperm. Using 2-hydroxypropyl-β-cyclodextrin (2-OHCD), we found that the disruption of membrane rafts inhibits PKA activity and AR without affecting protein tyrosine phosphorylation; however, these inhibitions were abolished in the presence of a cyclic 3,5-adenosine monophosphate (cAMP) analog. In addition, biochemical experiments showed a decrease in cAMP content in 2-OHCD-treated sperm, suggesting the involvement of soluble adenylyl cyclase (sAC) and transmembrane adenylyl cyclase (tmAC). Pharmacological experiments, combined with transcriptome analysis, showed that sAC and tmAC are present and involved in AR induction in chicken sperm. Furthermore, stimulation of both isoforms reversed the inhibition of PKA activity and AR in 2-OHCD-treated sperm. In conclusion, our results demonstrated that membrane rafts play an important role in AR induction by regulating the cAMP-dependent pathway and that they provide a mechanistic insight into membrane regulation of AR and sperm function in birds.

Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease.

In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe’s disease. A model of lipid raft disruption helped explaining psychosine’s effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe’s disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine’s toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders.

Histoplasma capsulatum-Induced Cytokine Secretion in Lung Epithelial Cells Is Dependent on Host Integrins, Src-Family Kinase Activation, and Membrane Raft Recruitment.

Histoplasma capsulatum var. capsulatum is a dimorphic fungus that causes histoplasmosis, a human systemic mycosis with worldwide distribution. In the present work, we demonstrate that H. capsulatum yeasts are able to induce cytokine secretion by the human lung epithelial cell line A549 in integrin- and Src-family kinase (SFK)-dependent manners. This conclusion is supported by small interfering RNA (siRNA) directed to α3 and α5 integrins, and PP2, an inhibitor of SFK activation. siRNA and PP2 reduced IL-6 and IL-8 secretion in H. capsulatum-infected A549 cell cultures. In addition, α3 and α5 integrins from A549 cells were capable of associating with H. capsulatum yeasts, and this fungus promotes recruitment of these integrins and SFKs to A549 cell membrane rafts. Corroborating this finding, membrane raft disruption with the cholesterol-chelator methyl-β-cyclodextrin reduced the levels of integrins and SFKs in these cell membrane domains. Finally, pretreatment of A549 cells with the cholesterol-binding compound, and also a membrane raft disruptor, filipin, significantly reduced IL-6 and IL-8 levels in A549-H.capsulatum cultures. Taken together, these results indicate that H. capsulatum yeasts induce secretion of IL-6 and IL-8 in human lung epithelial cells by interacting with α3 and α5 integrins, recruiting these integrins to membrane rafts, and promoting SFK activation.

Paracoccidioides brasiliensis induces recruitment of α3 and α5 integrins into epithelial cell membrane rafts, leading to cytokine secretion

Paracoccidioides brasiliensis is one of the etiological agents of paracoccidioidomycosis, a human systemic mycosis, highly prevalent in Latin America. In the present work, we demonstrate that P. brasiliensis yeasts promote IL-6 and IL-8 secretion by the human lung epithelial cell line A549 in an integrin-dependent manner. In fact, small interfering RNA directed to α3 and α5 integrins decreased IL-6 and IL-8 levels in P. brasiliensis-infected A549 cell cultures. This fungus also led to an increase in the expression of α3 and α5 integrins in this epithelial cell line. In addition, P. brasiliensis yeasts promoted α3 and α5 integrins clustering into A549 cell membrane rafts. Furthermore, epithelial cell membrane raft disruption with nystatin decreased IL-6 and IL-8 levels in P. brasiliensis-A549 cell cultures. Therefore, by increasing host α3 and α5 integrins levels and clustering these receptors into membrane rafts, P. brasiliensis yeasts may modulate host inflammation.

Membrane raft disruption results in neuritic retraction prior to neuronal death in cortical neurons

Membrane rafts, rich in sphingolipids and cholesterol, play an important role in neuronal membrane domain-specific signaling events, maintaining synapses and dendritic spines. The purpose of this study is to examine the neuronal response to membrane raft disruption. Membrane rafts of 8 DIV primary neuronal cultures were isolated based on the resistance to Triton X-100 and ability to float in sucrose gradients. Membrane rafts from primary cortical neurons were also imaged using the membrane raft marker, cholera toxin subunit-B (CTxB), and were co-immunolabelled with the dendritic microtubule associated protein marker, MAP-2, the dendritic and axonal microtubule protein, β-III-Tubulin, and the axonal microtubule protein, Tau. Exposure of cortical neurons to either the cholesterol depleting compound, methyl-beta-cyclodextrin (MBC), or to the glycosphingolipid metabolism inhibiting agent D-threo-1-phenyl-2-decanoylamino-3- morpholino-1-propanol (D-PDMP), resulted in neuritic retraction prior to the appearance of neuronal death. Further investigation into the effects of MBC revealed a pronounced perturbation of microtubule protein association with membrane rafts during neuritic retraction. Interestingly, stabilizing microtubules with Paclitaxel did not prevent MBC induced neuritic retraction, suggesting that neuritic retraction occurred independently of microtubule disassembly and that microtubule association with membrane rafts is critical for maintaining neuritic stability. Overall, the data indicated that membrane rafts play an important role in neurite stability and neuronal viability.

Cholesterol-Lowering Drugs Inhibit Lectin-Like Oxidized Low-Density Lipoprotein-1 Receptor Function by Membrane Raft Disruption

Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for oxidized low-density lipoprotein (ox-LDL) in endothelial cells, is up-regulated in atherosclerotic lesions. Statins are the principal therapeutic agents for cardiovascular diseases and are known to down-regulate LOX-1 expression. Whether the effect on the LOX-1 receptor is related to statin-mediated cholesterol-lowering activity is unknown. We investigate the requirement of cholesterol for LOX-1-mediated lipid particle internalization, trafficking, and processing and the role of statins as inhibitors of LOX-1 function. Disruption of cholesterol-rich membrane microdomains by acute exposure of cells to methyl-β-cyclodextrin or chronic exposure to different statins (lovastatin and atorvastatin) led to a spatial disorganization of LOX-1 in plasma membranes and a marked loss of specific LOX-1 function in terms of ox-LDL binding and internalization. Subcellular fractionation and immunochemical studies indicate that LOX-1 is naturally present in caveolae-enriched lipid rafts and, by cholesterol reduction, the amount of LOX-1 in this fraction is highly decreased (≥60%). In contrast, isoprenylation inhibition had no effect on the distribution and function of LOX-1 receptors. Furthermore, in primary cultures from atherosclerotic human aorta lesions, we confirm the presence of LOX-1 in caveolae-enriched lipid rafts and demonstrate that lovastatin treatment led to down-regulation of LOX-1 in lipid rafts and rescue of the ox-LDL-induced apoptotic phenotype. Taken together, our data reveal a previously unrecognized essential role of membrane cholesterol for LOX-1 receptor activity and suggest that statins protect vascular endothelium against the adverse effect of ox-LDL by disruption of membrane rafts and impairment of LOX-1 receptor function.

Visualization of PKA activity in plasma membrane microdomains

Membrane rafts are sphingolipid- and cholesterol-rich microdomains that contain dynamic arrangements of signaling proteins. Notably, various components of the ubiquitous cAMP/Protein Kinase A (PKA) pathway, including β-adrenergic receptors (β-ARs), G proteins, and adenylyl cyclases (ACs), have been shown to localize differentially between membrane rafts and non-raft regions of the plasma membrane. As PKA participates in regulating diverse fundamental cellular functions, a number of which require membrane rafts, it is important to understand how PKA activity is specifically regulated in these membrane microdomains. To this end, we developed an improved FRET-based PKA activity biosensor, and targeted it to both membrane raft and non-raft regions of the plasma membrane to examine PKA activity dynamics in different plasma membrane microdomains. Disruption of membrane rafts via cholesterol depletion was shown to enhance β-AR stimulated PKA activity at the plasma membrane, suggesting that membrane rafts play a negative role in β-AR stimulated PKA activation. Furthermore, we found that membrane rafts possess higher basal PKA activity in the resting state compared to non-raft regions, which depends on the integrity of membrane rafts and proper localization of PKA. This study shows that membrane rafts play an important role in regulating the activity of PKA at the plasma membrane, and demonstrates the ability of live-cell FRET-based assays to reveal dynamic differences amongst plasma membrane microdomains, laying a foundation for further dissection of membrane regulated signal transduction.

Membrane Rafts Are Involved in Intracellular Miconazole Accumulation in Yeast Cells

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

Lipido‐sterolic extract of Serenoa repens (LSESr, Permixon®) treatment affects human prostate cancer cell membrane organization

The molecular mechanism by which the lipido-sterolic extract of Serenoa repens (LSESr, Permixon) affects prostate cells remains to be fully elucidated. In androgen-independent PC3 prostate cancer cells, the LSESr-induced effects on proliferation and apoptosis were evaluated by counting cells and using a FACScan cytofluorimeter. PC3 cells were stained with JC-1 dye to detect mitochondrial membrane potential. Cell membrane lipid composition was evaluated by thin layer chromatography and gas chromatographic analysis. Akt phosphorylation was analyzed by Western blotting and cellular ultrastructure through electron microscopy. LSESr (12.5 and 25 microg/ml) administration exerted a biphasic action by both inhibiting proliferation and stimulating apoptosis. After 1 h, it caused a marked reduction in the mitochondrial potential, decreased cholesterol content and modified phospholipid composition. A decrease in phosphatidylinositol-4,5-bisphosphate (PIP2) level was coupled with reduced Akt phosphorylation. After 24 h, all of these effects were restored to pre-treatment conditions; however, the saturated (SFA)/unsaturated fatty acid (UFA) ratio increased, mainly due to a significant decrease in omega 6 content. The reduction in cholesterol content could be responsible for both membrane raft disruption and redistribution of signaling complexes, allowing for a decrease of PIP2 levels, reduction of Akt phosphorylation and apoptosis induction. The decrease in omega 6 content appears to be responsible for the prolonged and more consistent increase in the apoptosis rate and inhibition of proliferation observed after 2-3 days of LSESr treatment. In conclusion, LSESr administration results in complex changes in cell membrane organization and fluidity of prostate cancer cells that have progressed to hormone-independent status.

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galβ1,4Manα-PO 4 attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.

Lipid Rafts Determine Clustering of STIM1 in Endoplasmic Reticulum-Plasma Membrane Junctions and Regulation of Store-operated Ca2+ Entry (SOCE)

Store depletion induces STIM1 to aggregate and relocate into clusters at ER-plasma membrane junctions where it functionally interacts with and activates plasma membrane channels that mediate store-operated Ca(2+) entry (SOCE). Thus, the site of peripheral STIM1 clusters is critical for the regulation of SOCE. However, what determines the location of the STIM1 clusters in the ER-PM junctional regions, and whether these represent specific sites in the cell is not yet known. Here we report that clustering of STIM1 in the subplasma membrane region of the cell and activation of TRPC1-dependent SOCE are determined by lipid raft domains (LRD). We show that store depletion increased partitioning of TRPC1 and STIM1 into plasma membrane LRD. TRPC1 and STIM1 associated with each other within the LRD, and this association was dynamically regulated by the status of the ER Ca(2+) store. Peripheral STIM1 clustering was independent of TRPC1. However, sequestration of membrane cholesterol attenuated thapsigargin-induced clustering of STIM1 as well as SOCE in HSG and HEK293 cells. Recruitment and association of STIM1 and TRPC1 in LRD was also decreased. Additionally STIM1(D76A), which is peripherally localized and constitutively activates SOCE in unstimulated cells, displayed a relatively higher partitioning into LRD and interaction with TRPC1, as compared with STIM1. Disruption of membrane rafts decreased peripheral STIM1(D76A) puncta, its association with TRPC1 and the constitutive SOCE. Together, these data demonstrate that intact LRD determine targeting of STIM1 clusters to ER-plasma membrane junctions following store depletion. This facilitates the functional interaction of STIM1 with TRPC1 and activation of SOCE.

Membrane Raft Disruption Promotes Axonogenesis in N2a Neuroblastoma Cells

Membrane rafts are discrete microdomains found in cell membranes that contain cholesterol and glycosphingolipids such as gangliosides. As cholesterol is a major component of membrane rafts, its sequestration by the polyene filipin can be used to disrupt them. In previous work we observed that membrane raft disruption by filipin treatment of murine neuroblastoma N2a cells led to changes in expression of cell processes. In this study, we determined the type of process formation induced by filipin treatment as well as whether their expression was accompanied by changes in ganglioside content or subcellular distribution. The results indicate that the processes formed were axonal in nature and their expression was accompanied by changes in both ganglioside content as well as the subcellular localization of GM1.

Interaction of epithelial cell membrane rafts with Paracoccidioides brasiliensis leads to fungal adhesion and Src-family kinase activation

Membrane rafts are cholesterol- and sphingolipid-enriched cell membrane domains, which are ubiquitous in mammals and play an essential role in different cellular functions, including host cell-pathogen interaction. In this work, by using several approaches, we demonstrated the involvement of epithelial cell membrane rafts in adhesion process of the pathogenic fungus Paracoccidioides brasiliensis. This conclusion was supported by the localization of ganglioside GM1, a membrane raft marker, at P. brasiliensis-epithelial cell contact sites, and by the inhibition of this fungus adhesion to host cells pre-treated with cholesterol-extractor (methyl-β-cyclodextrin, MβCD) or -binding (nystatin) agents. In addition, at a very early stage of P. brasiliensis-A549 cell interaction, this fungus promoted activation of Src-family kinases (SFKs) and extracellular signal-regulated kinase 1/2 (ERK1/2) of these epithelial cells. Whereas SFKs were partially responsible for activation of ERK1/2, membrane raft disruption with MβCD in A549 cells led to total inhibition of SFK activation. Taking together, these data indicate for the first time that epithelial cell membrane rafts are essential for P. brasiliensis adhesion and activation of cell signaling molecules.

The α1a-Adrenergic Receptor Occupies Membrane Rafts with Its G Protein Effectors but Internalizes via Clathrin-coated Pits

The alpha(1a)-adrenergic receptor (alpha(1a)AR) occupies intracellular and plasma membranes in both native and heterologous expression systems. Based on multiple independent lines of evidence, we demonstrate the alpha(1a)AR at the cell surface occupies membrane rafts but exits from rafts following stimulation. In non-detergent raft preparations, basal alpha(1a)AR is present in low density membrane rafts and colocalizes with its G protein effectors on density gradients. Raft disruption by cholesterol depletion with methyl-beta-cyclodextrin eliminates these light rafts. To confirm the presence of the alpha(1a)AR in plasma membrane rafts, fluorescence resonance energy transfer measurements were used to demonstrate colocalization of surface receptor and the raft marker, cholera toxin B. This colocalization was largely lost following alpha(1a)AR stimulation with phenylephrine. Similarly, receptor stimulation causes exit of the alpha(1a)AR from light rafts within 3-10 min in contrast to the G proteins, which largely remain in light rafts. Importantly, this delayed exit of the alpha(1a)AR suggests acute receptor signaling and desensitization occur entirely within rafts. Interestingly, both confocal analysis and measurement of surface alpha(1a)AR levels indicate modest receptor internalization during the 10 min following stimulation, suggesting most of the receptor has entered non-raft plasma membrane. Nevertheless, activation does increase the rate of receptor internalization as does disruption of rafts with methyl-beta-cyclodextrin, suggesting raft exit enables internalization. Confocal analysis of surface-labeled hemagglutinin-alpha(1a)AR reveals that basal and stimulated receptor occupies clathrin pits in fixed cells consistent with previous indirect evidence. The evidence presented here strongly suggests the alpha(1a)AR is a lipid raft protein under basal conditions and implies agonist-mediated signaling occurs from rafts.

The Roles of Membrane Rafts in CD32A-Mediated Phagocytosis

AbstractMembrane rafts are highly dynamic heterogeneous sterol- and sphingolipid-rich micro-domains on cell surfaces. They are generally believed to provide residency for cell surface molecules (e.g., adhesion and signaling molecules) and scaffolding to facilitate the functions of these molecules such as membrane trafficking, receptor transport, cell signaling, and endocytosis.The governing, or overall hypothesis, for this project is that membrane rafts provide residency for Fc[gamma]RIIA (CD32A) on K562 cells, and that by doing so they provide a platform from which Fc[gamma]RIIA initiate or carry out their functions, which include migration, signaling, phagocytic synapse formation, and internalization of IgG opsonized targets.Using immuno-fluorescent laser scanning confocal microscopy and reflection interference microscopy (RIM), we studied the spatial and temporal distributions of membrane rafts and surface receptors, signaling molecules, and cell organelles during the formation of phagocytic contact areas. K562 cells, which naturally express CD32A, a cell surface receptor for the Fc portion of Immuno-globulin G(IgG), was chosen as a model for neutrophils. An opsonized target was modeled using a glass supported lipid bilayer reconstituted with IgG. CD32A was found to cluster and co-localize with membrane rafts. Placing the K562 cells on the lipid bilayer triggered a process of contact area formation that includes binding between receptors and ligands, their recruitment to the contact area, a concurrent membrane raft movement to and concentration in the contact area, and transport of CD32A, IgG, and membrane rafts to the Golgi complex. Characterization of these processes was performed using agents known to disrupt detergent resistant membranes (DRMs), dissolve actin microfilaments, and inhibit myosin motor activity, which abolished the CD32A clusters and prevented the contact area formation. The relevance to phagocytosis of contact area formation between K562 cells and lipid bilayers was demonstrated using micro-beads coated with a lipid bilayer reconstituted with IgG as the opsonized target instead of the glass supported planar lipid bilayer. Disruption of membrane rafts, salvation of the actin cytoskeleton, and inhibition of myosin II activity were found to inhibit phagocytosis.These data suggest membrane rafts play several important roles in CD32A mediated phagocytosis including pre-clustering CD32A, transport of CD32A to the phagocytic cup, and transport of the opsonized target towards the Golgi complex. Here we have provided evidence that membrane rafts serve as platforms which are used to cluster CD32A and transport CD32A along the actin cytoskeleton to the site of phagocytic synapse formation thus allowing for the quick assembly of a phagocytic synapse.

Membrane Mobility and Microdomain Association of the Dopamine Transporter Studied with Fluorescence Correlation Spectroscopy and Fluorescence Recovery after Photobleaching

To investigate microdomain association of the dopamine transporter (DAT), we employed FCS (fluorescence correlation spectroscopy) and FRAP (fluorescence recovery after photobleaching). In non-neuronal cells (HEK293), FCS measurements revealed for the YFP-DAT (DAT tagged with yellow fluorescent protein) a diffusion coefficient (D) of approximately 3.6 x 10(-9) cm2/s, consistent with a relatively freely diffusible protein. In neuronally derived cells (N2a), we were unable to perform FCS measurements on plasma membrane-associated protein due to photobleaching, suggesting partial immobilization. This was supported by FRAP measurements that revealed a lower D and a mobile fraction of the YFP-DAT in N2a cells compared to HEK293 cells. Comparison with the EGFP-EGFR (epidermal growth factor receptor) and the EGFP-beta2AR (beta2 adrenergic receptor) demonstrated that this observation was DAT specific. Both the cytoskeleton-disrupting agent cytochalasin D and the cholesterol-depleting agent methyl-beta-cyclodextrin (mbetaCD) increased the lateral mobility of the YFP-DAT but not that of the EGFP-EGFR. The DAT associated in part with membrane raft markers both in the N2a cells and in rat striatal synaptosomes as assessed by sucrose density gradient centrifugation. Raft association was further confirmed in the N2a cells by cholera toxin B patching. It was, moreover, observed that cholesterol depletion, and thereby membrane raft disruption, decreased both the Vmax and KM values for [3H]dopamine uptake without altering DAT surface expression. In summary, we propose that association of the DAT with lipid microdomains in the plasma membrane and/or the cytoskeleton serves to regulate both the lateral mobility of the transporter and its transport capacity.

Kininostatin Associates With Membrane Rafts and Inhibits αvβ3 Integrin Activation in Human Umbilical Vein Endothelial Cells

The cleaved form of high molecular weight kininogen (HKa) is a potent inhibitor of angiogenesis and tumor growth in vivo; the functional domain has been identified as domain 5 (D5, named as kininostatin). We now identify the subcellular targeting site for D5 on endothelial cells (ECs), and investigate D5 inhibition of integrin functions. Endothelial membrane rafts were isolated using sucrose density gradient centrifugation. D5, bound to ECs, was predominantly associated with membrane rafts, in which uPAR, a HKa receptor, was also localized. In contrast, other HKa receptors, cytokeratin-1 and gC1q receptor, were not detected in membrane rafts. Colocalization of D5 with caveolin-1 was demonstrated on ECs by confocal microscopy. Disruption of membrane rafts by cholesterol removal decreased D5 binding to ECs. On stimulation with vascular endothelial growth factor, alpha(v)beta3 integrin formed a complex with uPAR and caveolin-1, which was accompanied by an increase in ligand binding affinity of alpha(v)beta3 integrin. These events were inhibited by D5. Consistently, D5 suppressed specific alpha(v)beta3 integrin-mediated EC adhesion and spreading as well as small guanosine triphosphatase Rac1 activation. D5 binds to ECs via membrane rafts and downregulates alpha(v)beta3 integrin bidirectional signaling and the downstream Rac1 activation pathway.