Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid ordered (L(o)) phase appears to be important for their function. To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropy of two lipid probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We show using model membranes with varying amounts of cholesterol that steady-state fluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest that DPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also depends on the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasma membrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparison with anisotropy values for model membranes entirely in a liquid ordered (L(o)) phase and in a liquid disordered (L(alpha)) phase. This result is consistent with the finding that approximately 30% of plasma membrane phospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolated by sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membrane vesicles, suggesting that they represent a more ordered subset of the plasma membrane. Treatment of plasma membrane vesicles with methyl-beta-cyclodextrin resulting in 75% cholesterol depletion leads to commensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These results demonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amount of lipid order in biological membranes.     

Nuclear import of N-terminal FAK by activation of the FcepsilonRI receptor in RBL-2H3 cells

As FAK integrates membrane receptor signalling, yet is also found in the nucleus, we investigated whether nuclear FAK is regulated by membrane receptor activation. Activation of the mast cell FcepsilonRI receptor leads to the release and synthesis of inflammatory mediators as well as increased proliferation and survival. Using RBL-2H3 basophilic leukaemia cells, FAK and the FcepsilonRI receptor were co-localised following cross-linking of IgE with antigen. This also resulted in a significant increase in the nucleus of several N-terminal FAK fragments, the largest of which included the kinase domain but not the focal adhesion targeting domain. This was confirmed using cells that stably expressed recombinant EGFP-FAK. Furthermore, treatment of EGFP-FAK expressing cells with Leptomycin B, an inhibitor of nuclear export, resulted in increased nuclear localisation of EGFP-FAK. Therefore, FAK can shuttle between the nuclear and cytoplasmic compartments and the cellular distribution of N-terminal FAK is regulated by membrane receptor activation.     

Degranulation in RBL-2H3 cells: regulation by calmodulin pathway

Involvement of the calmodulin pathway in Ca2+-induced degranulation was evaluated in RBL-2H3 mast cells. Pretreatment of RBL-2H3 cells with a calmodulin antagonist, W-13, blocked ionomycin-dependent release of beta-hexosaminidase into the supernatant, although W-13 treatment alone slightly but significantly increased the release. Ca2+/calmodulin activates various protein kinases and phosphatases including myosin-light chain kinase (MLCK), calmodulin-dependent protein kinases (CaMKs), and calcineurin. When RBL-2H3 cells were pretreated with a MLCK inhibitor, ML-7, or a CaMKs inhibitor, KN-93, the ionomycin-dependent release of beta-hexosaminidase into the supernatant was inhibited. In addition, pretreatment with calcineurin inhibitors, cyclosporin A and FR901725, resulted in blockage of the ionomycin-dependent release of beta-hexosaminidase into the supernatant. Our results indicate that Ca2+/calmodulin, activated calmodulin, is indispensable for Ca2+-induced degranulation, and that within the calmodulin pathways, at least MLCK, CaMKs and calcineurin positively regulate the release of granules initiated by increasing cytosolic Ca2+ concentrations in RBL-2H3 cells.     

Phospholipase D2 Modulates the Secretory Pathway in RBL-2H3 Mast Cells

Phospholipase D (PLD) hydrolyses phosphatidylcholine to produce phosphatidic acid (PA) and choline. It has two isoforms, PLD1 and PLD2, which are differentially expressed depending on the cell type. In mast cells it plays an important role in signal transduction. The aim of the present study was to clarify the role of PLD2 in the secretory pathway. RBL-2H3 cells, a mast cell line, transfected to overexpress catalytically active (PLD2CA) and inactive (PLD2CI) forms of PLD2 were used. Previous observations showed that the Golgi complex was well organized in CA cells, but was disorganized and dispersed in CI cells. Furthermore, in CI cells, the microtubule organizing center was difficult to identify and the microtubules were disorganized. These previous observations demonstrated that PLD2 is important for maintaining the morphology and organization of the Golgi complex. To further understand the role of PLD2 in secretory and vesicular trafficking, the role of PLD2 in the secretory process was investigated. Incorporation of sialic acid was used to follow the synthesis and transport of glycoconjugates in the cell lines. The modified sialic acid was subsequently detected by labeling with a fluorophore or biotin to visualize the localization of the molecule after a pulse-chase for various times. Glycoconjugate trafficking was slower in the CI cells and labeled glycans took longer to reach the plasma membrane. Furthermore, in CI cells sialic acid glycans remained at the plasma membrane for longer periods of time compared to RBL-2H3 cells. These results suggest that PLD2 activity plays an important role in regulating glycoconjugate trafficking in mast cells.     

The alpha-galactosyl derivatives of ganglioside GD(1b) are essential for the organization of lipid rafts in RBL-2H3 mast cells

Gangliosides are complex glycosphingolipids that are important in many biological processes. The present study investigated the role of gangliosides in the organization of lipid rafts in RBL-2H3 mast cells and in the modulation of mast cell degranulation via FcRI. The role of gangliosides was examined using two ganglioside deficient cell lines (B6A4A2III-E5 and B6A4C1III-D1) as well as the parent cell line (RBL-2H3). All three cell lines examined express FcRI, Lyn, Syk and LAT. However, only in RBL-2H3 cells were FcRI, LAT and α-galactosyl derivatives of ganglioside GD_1b mobilized to lipid raft domains following FcRI stimulation. The inhibition of glycosphingolipid synthesis in RBL-2H3 cells also resulted in a decrease in the release of β-hexosaminidase activity after FcRI activation. The two mutant cell lines have a reduced release of β-hexosaminidase activity after FcRI stimulation, but not after exposure to calcium ionophore. These results indicate that the α-galactosyl derivatives of ganglioside GD_1b are important in the initial events of FcRI signaling upstream of Ca²⁺ influx. Since the initial signaling events occur in lipid rafts and in the mutant cell lines the rafts are disorganized, these results also suggest that these gangliosides contribute to the correct assembly of lipid rafts and are essential for mast cell activation via FcRI.     

Eotaxin induces migration of RBL-2H3 mast cells via a Rac-ERK-dependent pathway

Eotaxin is a potent chemokine that acts via CC chemokine receptor 3 (CCR3) to induce chemotaxis, mainly on eosinophils. Here we show that eotaxin also induces chemotactic migration in rat basophilic leukemia (RBL-2H3) mast cells. This effect was dose-dependently inhibited by compound X, a selective CCR3 antagonist, indicating that, as in eosinophils, the effect was mediated by CCR3. Eotaxin-induced cell migration was completely blocked in RBL-RacN17 cells expressing a dominant negative Rac1 mutant, suggesting a crucial role for Rac1 in eotaxin signaling to chemotactic migration. ERK activation also proved essential for eotaxin signaling and it too was absent in RBL-RacN17 cells. Finally, we found that activation of Rac and ERK was correlated with eotaxin-induced actin reorganization known to be necessary for cell motility. It thus appears that Rac1 acts upstream of ERK to signal chemotaxis in these cells, and that a Rac-ERK-dependent cascade mediates the eotaxin-induced chemotactic motility of RBL-2H3 mast cells.     

Calmodulin spatial dynamics in RBL-2H3 mast cells

A line of rat basophilic leukaemia (RBL) cells, a model of mast cells, stably expressing EGFP-tagged calmodulin secreted normally in response to standard agonists. As reported for other cell types, calmodulin was concentrated in the mitotic spindle poles of dividing cells. In unstimulated interphase cells calmodulin was concentrated in the cell cortex and at a single central location. Disruption of cortical actin eliminated the concentration of calmodulin at the cortex while the central calmodulin concentration was associated with an enrichment of tubulin and is likely to represent the centrosome. Following stimulation with either an agonist that crosslinks Fc receptors or co-application of phorbol ester and a calcium ionophore the interior of the cells lost calmodulin while cortical fluorescence became more pronounced but also less uniform. After stimulation discrete bright puncta of calmodulin-EGFP (CaM-EGFP) appeared in the cell interior. Puncta colocalised with moving lysotracker-labelled granules, suggesting that calmodulin may play a role in organising their transport. Our results show that in interphase RBL cells a large fraction of the calmodulin pool is associated with targets in the actin cytoskeleton and demonstrate the utility of this model system for studying calmodulin biology.     

Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells

We analyzed the role of gangliosides in the association of the ErbB2 receptor tyrosine-kinase (RTK) with lipid rafts in mammary epithelial HC11 cells. Scanning confocal microscopy experiments revealed a strict ErbB2-GM3 colocalization in wild-type cells. In addition, analysis of membrane fractions obtained using a linear sucrose gradient showed that ErbB2, epidermal growth factor receptor (EGFR) and Shc-p66 (proteins correlated with the ErbB2 signal transduction pathway) were preferentially enriched in lipid rafts together with gangliosides. Blocking of endogenous ganglioside synthesis by (+/-)-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride ([D]-PDMP) induced a drastic cell-surface redistribution of ErbB2, EGFR and Shc-p66, within the Triton-soluble fractions, as revealed by linear sucrose-gradient analysis. This redistribution was partially reverted when exogenous GM3 was added to ganglioside-depleted HC11 cells. The results point out the key role of ganglioside GM3 in retaining ErbB2 and signal-transduction-correlated proteins in lipid rafts.     

Inhibitory Effects of Apple Polyphenol on Induced Histamine Release from RBL-2H3 Cells and Rat Mast Cells

The anti-allergic activities of polyphenol fractions extracted from immature fruits of apple (Rosaceae, Malus sp.) were evaluated by in vitro assays. A crude apple polyphenol (CAP) fraction, which had been obtained from the juice of immature apples by reverse-phase column chromatography, was further purified by LH-20 column chromatography to obtain an apple condensed tannin (ACT) fraction consisting of linear oligomeric epicatechins from the dimer to pentadecamer. ACT strongly inhibited the release of histamine from rat basophilic leukemia (RBL-2H3) cells stimulated by the antigen-stimulation and from rat peritoneal mast cells stimulated by compound 48/80. The IC₅₀ values for histamine release were 30 μg/ml and 25 μg/ml, respectively. ACT also inhibited hyaluronidase activity and the increase in intracellular free calcium concentration in RBL-2H3 cells stimulated with the antigen. These results suggest that ACT affected early signal transduction including the calcium influx.     

Gangliosides Are Important for the Preservation of the Structure and Organization of RBL-2H3 Mast Cells

Gangliosides are known to be important in many biological processes. However, details concerning the exact function of these glycosphingolipids in cell physiology are poorly understood. In this study, the role of gangliosides present on the surface of rodent mast cells in maintaining cell structure was examined using RBL-2H3 mast cells and two mutant cell lines (E5 and D1) deficient in the gangliosides, GM₁ and the α-galactosyl derivatives of the ganglioside GD_1b. The two deficient cell lines were morphologically different from each other as well as from the parental RBL-2H3 cells. Actin filaments in RBL-2H3 and E5 cells were under the plasma membrane following the spindle shape of the cells, whereas in D1 cells, they were concentrated in large membrane ruffles. Microtubules in RBL-2H3 and E5 cells radiated from the centrosome and were organized into long, straight bundles. The bundles in D1 cells were thicker and organized circumferentially under the plasma membrane. The endoplasmic reticulum, the Golgi complex, and the secretory granule matrix were also altered in the mutant cell lines. These results suggest that the mast cell–specific α-galactosyl derivatives of ganglioside GD_1b and GM₁ are important in maintaining normal cell morphology. (J Histochem Cytochem 58:83–93, 2010)     

Microtubule-dependent transport of secretory vesicles in RBL-2H3 cells

Antigen-mediated activation of mast cells results in Ca²⁺-dependent exocytosis of preformed mediators of the inflammatory response. To investigate the role of secretory vesicle motility in this response, we have performed time-lapse confocal microscopy on RBL-2H3 cells transfected with a green fluorescent protein-Fas ligand fusion protein (GFP-FasL). Green fluorescent protein-labeled vesicles exhibit rapid, bidirectional movement in both resting and activated cells and can be localized adjacent to microtubules. Colchicine treatment inhibits the motility of secretory vesicles as measured by fluorescence recovery after photobleaching (FRAP). Colchicine also inhibits both the extent and the rate of exocytosis triggered by receptor activation or by Ca²⁺ ionophore, demonstrating that microtubule-dependent movement of secretory vesicles plays an important role in the exocytic response .     

Mast cell degranulating peptide binds to RBL-2H3 mast cell receptors and inhibits IgE binding

Fluorescent and biotinylated analogs of mast cell degranulating (MCD) peptide were synthesized and the labels fluoresceinisothiocyanate and N-hydroxysuccinimidobiotin were conjugated at position 1 in the MCD peptide sequence. The analogs with these moieties retained histamine-releasing activity as high as that of the parent MCD peptide in rat peritoneal mast cell assays. These labeled analogs were used in rat basophilic leukemia cells (RBL-2H3) to demonstrate by confocal microscopy and flow cytometry the specific binding of MCD peptide to mast cell receptors. Consequently MCD peptide was found to compete with and inhibit the binding of fluorescent IgE on RBL cells as monitored by flow cytometry. Thus MCD peptide may prove to be useful in the study of IgE receptor-bearing cells.     

Relationship of IgE receptor topography to secretion in RBL-2H3 mast cells.

In RBL-2H3 rat leukemic mast cells, cross-linking IgE-receptor complexes with anti-IgE antibody leads to degranulation. Receptor cross-linking also stimulates the redistribution of receptors on the cell surface, a process observed here by labeling the anti-IgE with 15 nm protein A-gold particles that are visible by back-scattered electron imaging in the scanning electron microscope. We report that anti-IgE binding stimulates the redistribution of IgE-receptor complexes at 37 degrees C from a dispersed topography to distributions dominated sequentially by short chains, small clusters, and large aggregates of cross-linked receptors. Cells incubated with 1 microgram/ml anti-IgE, a concentration that stimulates maximum net secretion, redistribute receptors into chains and small clusters during a 15 min incubation period. At 3 and 10 micrograms/ml anti-IgE, net secretion is reduced and the majority of receptors redistribute rapidly into clusters and large aggregates. The addition of Fab fragments with the high anti-IgE concentrations, to reduce cross-linking, delays receptor aggregation and enhances secretion. The progression of receptors from small clusters to large aggregates is prevented in cells treated with dihydrocytochalasin B to prevent F-actin assembly. These results establish that characteristic patterns of receptor topography are correlated with receptor activity. In particular, they link the formation of large receptor aggregates to reduced signalling activity. Cytoskeleton-membrane interaction is implicated in the formation or stabilization of the large receptor clusters.     

Differential uPAR recruitment in caveolar‐lipid rafts by GM1 and GM3 gangliosides regulates endothelial progenitor cells angiogenesis

Gangliosides and the urokinase plasminogen activator receptor (uPAR) tipically partition in specialized membrane microdomains called lipid‐rafts. uPAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells (EPCs) upon recruitment in caveolar‐lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro‐angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of uPAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid‐supported mobile bilayer lipid membranes with raft‐like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance (SPR) the extent of uPAR recruitment. We estimated the apparent dissociation constants of uPAR‐GM1/GM3 complexes. These preliminary observations, indicating that uPAR binds preferentially to GM1‐enriched biomimetic membranes, were validated by identifying a pro‐angiogenic activity of GM1‐enriched EPCs, based on GM1‐dependent uPAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti‐angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar‐raft isolation and Western blotting of uPAR showed that GM1 promotes caveolar‐raft partitioning of uPAR, as opposed to control and GM3‐challenged EPCs. By confocal microscopy, we have shown that in EPCs uPAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of uPAR which is recruited within caveolar rafts thereby triggering angiogenesis.     

Electron spin resonance characterization of liquid ordered phase of detergent-resistant membranes from RBL-2H3 cells.

The dynamic structure of detergent-resistant membranes (DRMs) isolated from RBL-2H3 cells was characterized using two different acyl chain spin-labeled phospholipids (5PC and 16PC), a headgroup labeled sphingomyelin (SM) analog (SD-Tempo) and a spin-labeled cholestane (CSL). It was shown, by comparison to dispersions of SM, dipalmitoylphosphatidylcholine (DPPC), and DPPC/cholesterol of molar ratio 1, that DRM contains a substantial amount of liquid ordered phase: 1) The rotational diffusion rates (R( perpendicular)) of 16PC in DRM between -5 degrees C and 45 degrees C are nearly the same as those in molar ratio DPPC/Chol = 1 dispersions, and they are substantially greater than R( perpendicular) in pure DPPC dispersions in the gel phase studied above 20 degrees C; 2) The order parameters (S) of 16PC in DRM at temperatures above 4 degrees C are comparable to those in DPPC/Chol = 1 dispersions, but are greater than those in DPPC dispersions in both the gel and liquid crystalline phases. 3) Similarly, R( perpendicular) for 5PC and CSL in DRM is greater than in pure SM dispersions in the gel phase, and S for these labels in DRM is greater than in the SM dispersions in both the gel and liquid crystalline phases. 4) R( perpendicular) of SD-Tempo in DRM is greater than in dispersions of SM in both gel and liquid phases, consistent with the liquid-like mobility in the acyl chain region in DRM. However, S of SD-Tempo in DRM is substantially less than that of this spin label in SM in gel and liquid crystalline phases (in absolute values), indicating that the headgroup region in DRMs is less ordered than in pure SM. These results support the hypothesis that plasma membranes contain DRM domains with a liquid ordered phase that may coexist with a liquid crystalline phase. There also appears to be a coexisting region in DRMs in which the chain labels 16PC and 5PC are found to cluster. We suggest that other biological membranes containing high concentrations of cholesterol also contain a liquid ordered phase.     

Epigallocatechin gallate inhibits histamine release from rat basophilic leukemia (RBL-2H3) cells: role of tyrosine phosphorylation pathway

Some tea polyphenolic compounds including (-)-epigallocatechin gallate (EGCG) have been shown to inhibit histamine release from mast cells through poorly understood mechanisms. By using a mast cell model rat basophilic leukemia (RBL-2H3) cells we explored the mechanism of the inhibition. EGCG inhibited histamine release from RBL-2H3 cells in response to antigen or the calcium-ionophore A23187, while (-)-epicatechin (EC) had little effect. Increased tyrosine phosphorylation of several proteins including approximately 120 kDa proteins occurred in parallel with the secretion induced by either stimulation. EGCG also inhibited tyrosine phosphorylation of the approximately 120-kDa proteins induced by either stimulation, whereas EC did not. The tyrosine kinase-specific inhibitor piceatannol inhibited the secretion and tyrosine phosphorylation of these proteins induced by either stimulation also. Further analysis showed that the focal adhesion kinase pp125(FAK) was one of the approximately 120-kDa proteins. These findings suggest that EGCG prevents histamine release from mast cells mainly by inhibiting tyrosine phosphorylation of proteins including pp125(FAK).     

Structure and activation dynamics of RBL-2H3 cells observed with scanning force microscopy.

Surface and subsurface dynamics of Rat Basophilic Leukemia cells, a model system of stimulated secretion, were imaged using Scanning Force Microscopy (SFM) at a rate of 50-60 s/image. Cytoskeletal elements and organelles were tracked within quiescent cells and those activated after IgE receptor crosslinking. In addition, surface waves were observed moving within the plasma membrane. The structures seen in quiescent and activated cells can be correlated with those seen in electron micrographs and topographic SFM images of fixed detergent-extracted cells. Furthermore, images of the detergent-extracted nuclei reveal the presence of numerous nuclear pore complexes. High-magnification images of the nuclear pore complexes show evidence of subunit structure and exhibit dimensions consistent with those reported previously using electron microscopy. The behavior and overall change in morphology of cells observed during activation was consistent with that observed under similar conditions with Differential Interference Contrast microscopy. This study demonstrates that SFM, unlike other techniques, can be used to provide high-resolution information in both fixed and living cells.