Direct demonstration of the endocytic function of caveolae by a cell-free assay.
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The endocytic function of caveolae was challenged by taking advantage of a cell-free assay directly measuring the detachment of receptor-containing vesicles from isolated plasma membranes. Plasma membranes from cultured cells surface-labeled with 125I-cholera toxin (segregating in caveolae) were isolated as described previously. Following incubation of these labeled membranes in the presence of nucleotide(s) and cytosol, a significant proportion of the initially membrane-associated radioactivity was released into the incubation medium in sedimentable form (14*10(6 )g). Results of biochemical, morphological, and fractionation analysis of the material containing the released radioactivity directly demonstrated that caveolae are plasma membrane domains involved in an endocytic process and resulting in the formation of caveolae-derived vesicles. In addition, these studies allowed a direct comparison of caveolae- and clathrin-coated pit-mediated endocytosis and reveal that these two processes diverge in terms of kinetics, cytosol and nucleotide requirements as well as in terms of the density and size of the endocytic vesicles formed. (+info)
The pentaene macrolide antibiotic filipin prefers more rigid DPPC bilayers: a fluorescence pressure dependence study.
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Filipin is a pentaene macrolide antibiotic which was previously shown to incorporate more extensively into DPPC bilayers below the main phase transition temperature than above this temperature. This result was extremely unusual because drugs tend to be expelled from ordered gel phases. However, such results could not be safely attributed to the phase change of the bilayer itself because the temperature was changing concomitantly. In this work we changed the bilayer phase isothermally (53 degrees C) by hydrostatic pressure variation and discovered that filipin has a slightly more extensive incorporation in the pure DPPC gel phase (P>ca. 54.4 MPa): Kp,lc approximately 3x10(3) vs. Kp,gel approximately 6x10(3). The presence of sterols (45% molar ergosterol or cholesterol) caused an increase in the partition coefficients, regardless of pressure, ergosterol having a more pronounced effect (Kp approximately 2x10(4)-6x10(4)). Kp was pressure dependent in both cases, but mainly with cholesterol (Kp approximately 2x10(3)-2x10(4)). At variance with cholesterol, when ergosterol was used, no phase transition was detected. This difference cannot be due to a more extended uptake of filipin by cholesterol-containing membranes, and so must be due to specific interactions with cholesterol. In agreement with this finding, we discovered that filipin is more tightly packed (lower partial molar volume) in the cholesterol-rich phase than in the ergosterol-rich phase. Our results also point to a 2:1 DPPC:cholesterol stoichiometry in the cholesterol-rich phase (17% molar cholesterol). All partition coefficients were calculated from steady-state fluorescence anisotropy measurements. (+info)
Involvement of caveolae in the uptake of respiratory syncytial virus antigen by dendritic cells.
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The uptake of respiratory syncytial virus (RSV) antigen by cattle dendritic cells was investigated. Pathways of antigen uptake were monitored by flow cytometry using specific tracers and by proliferation assays, which were used to measure the presentation of RSV antigen and ovalbumin. Inhibitors that differentially affected pathways were used to distinguish them. Presentation of RSV antigen, but not ovalbumin, was inhibited by phorbol myristate acetate and filipin, which have been reported to inhibit caveolae, but not by cytochalasin D, amiloride, or mannose. These inhibitors have been reported to block macropinocytosis and other actin-dependent uptake mechanisms, endocytic pathways involving clathrin-coated pits, and the mannose receptor. Furthermore, co-localization of RSV antigen and caveolae was observed by confocal microscopy. Thus, the major route for uptake of RSV antigen by cattle dendritic cells is one mediated by caveolae, adding a pathway of antigen uptake by dendritic cells to those established. (+info)
Histamine induces tyrosine phosphorylation of endothelial cell-to-cell adherens junctions.
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Endothelial adherens junctions (AJ) promote intercellular adhesion and may contribute to the control of vascular permeability. These structures are formed by a transmembrane and cell-specific adhesive protein, vascular endothelial (VE)-cadherin, which is linked by its cytoplasmic tail to intracellular proteins called catenins (alpha-catenin, beta-catenin, and plakoglobin) and to the actin cytoskeleton. Little is known about the functional regulation of AJ in endothelial cells. In this study, we analyzed the effect of histamine on AJ organization in cultured endothelial cells. We first observed that histamine induced detectable intercellular gaps only in loosely-confluent cells, whereas this effect was strongly reduced or absent in long-confluent cultures. Despite this difference, in vitro permeability was augmented by histamine in both conditions. In resting conditions, tyrosine phosphorylation of AJ components and permeability values were higher in recently-confluent cells as compared with long-confluent cells. Histamine did not affect the phosphorylation state of AJ in recently-confluent cells but strongly increased this parameter in long-confluent cultures. In addition, in long-confluent cells, histamine caused dissociation of VE-cadherin from the actin cytoskeleton measured by a decrease of the amount of the molecule in the detergent-insoluble fraction of the cell extracts. Dibutyryl cAMP was able to prevent the effect of histamine on both tyrosine phosphorylation of AJ components and on endothelial permeability. The effect of histamine was specific for VE-cadherin because the phosphorylation state of neural (N)-cadherin, the other major endothelial cadherin, was unchanged by this agent. Hence AJ components are a target of histamine activation cascade; we suggest that induction of tyrosine phosphorylation of VE-cadherin and catenins contributes to the histamine effect on permeability, even in absence of frank intercellular gaps and cell retraction. (+info)
Cholesterol efflux-mediated signal transduction in mammalian sperm: cholesterol release signals an increase in protein tyrosine phosphorylation during mouse sperm capacitation.
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We previously demonstrated that mouse sperm capacitation is accompanied by a time-dependent increase in protein tyrosine phosphorylation that is dependent on the presence of BSA, Ca2+, and NaHCO(3), all three of which are also required for this maturational event. We also demonstrated that activation of protein kinase A (PK-A) is upstream of this capacitation-associated increase in protein tyrosine phosphorylation. BSA is hypothesized to modulate capacitation through the removal of cholesterol from the sperm plasma membrane. In this report, we demonstrate that incubation of mouse sperm medium containing BSA results in a release of cholesterol from the sperm plasma membrane to the medium; release of this sterol does not occur in medium devoid of BSA. We next determined whether cholesterol release leads to changes in protein tyrosine phosphorylation. Blocking the action of BSA by adding exogenous cholesterol-SO-(4) to the BSA-containing medium inhibits the increase in protein tyrosine phosphorylation as well as capacitation. This inhibitory effect is overcome by (1) the addition of increasing concentrations of BSA at a given concentration of cholesterol-SO-(4) and (2) the addition of dibutyryl cAMP plus IBMX. High-density lipoprotein (HDL), another cholesterol binding protein, also supports the capacitation-associated increase in protein tyrosine phosphorylation through a cAMP-dependent pathway, whereas proteins that do not interact with cholesterol have no effect. HDL also supports sperm capacitation, as assessed by fertilization in vitro. Finally, we previously demonstrated that HCO-(3) is necessary for the capacitation-associated increase in protein tyrosine phosphorylation and demonstrate here, by examining the effectiveness of HCO-(3) or BSA addition to sperm on protein tyrosine phosphorylation, that the HCO-(3) effect is downstream of the site of BSA action. Taken together, these data demonstrate that cholesterol release is associated with the activation of a transmembrane signal transduction pathway involving PK-A and protein tyrosine phosphorylation, leading to functional maturation of the sperm. (+info)
Activation of Src family kinase yes induced by Shiga toxin binding to globotriaosyl ceramide (Gb3/CD77) in low density, detergent-insoluble microdomains.
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Shiga toxin (Stx) is an enterotoxin produced by Shigella dysenteriae serotype 1 and enterohemorrhagic Escherichia coli, which binds specifically to globotriaosylceramide, Gb3, on the cell surface and causes cell death. We previously demonstrated that Stx induced apoptosis in human renal tubular cell line ACHN cells (Taguchi, T., Uchida, H., Kiyokawa, N., Mori, T., Sato, N., Horie, H., Takeda, T and Fujimoto, J. (1998) Kidney Int. 53, 1681-1688). To study the early signal transduction after Stx addition, Gb3-enriched microdomains were prepared from ACHN cells by sucrose density gradient centrifugation of Triton X-100 lysate as buoyant, detergent-insoluble microdomains (DIM). Gb3 was only recovered in DIM and was associated with Src family kinase Yes. Phosphorylation of tyrosine residues of proteins in the DIM fraction increased by 10 min and returned to the resting level by 30 min after the addition of Stx. Since the kinase activity of Yes changed with the same kinetics, Yes was thought to be responsible for the hyperphosphorylation observed in DIM proteins. Unexpectedly, however, all of the Yes kinase activity was obtained in the high density, detergent-soluble fraction. Yes was assumed to be activated and show increased Triton X-100 solubility in the early phase of retrograde endocytosis of Stx-Gb3 complex. Since Yes activation by the Stx addition was suppressed by filipin pretreatment, Gb3-enriched microdomains containing cholesterol were deeply involved in Stx signal transduction. (+info)
cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera.
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To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling. (+info)
Role of Niemann-Pick type C1 protein in intracellular trafficking of low density lipoprotein-derived cholesterol.
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Niemann-Pick type C (NPC) is a disease that affects intracellular cholesterol-trafficking pathways. By cloning the hamster ortholog of NPC1, we identified the molecular lesions in two independently isolated Chinese hamster ovary cell mutants, CT60 and CT43. Both mutants lead to premature translational terminations of the NPC1 protein. Transfecting hamster NPC1 cDNA complemented the defects of the mutants. Investigation of the CT mutants, their parental cells, and an NPC1-stable transfectant allow us to present evidence that NPC1 is involved in a post-plasma membrane cholesterol-trafficking pathway. We found that the initial movement of low density lipoprotein (LDL)-derived cholesterol to the plasma membrane (PM) did not require NPC1. After reaching the PM and subsequent internalization, however, cholesterol trafficking back to the PM did involve NPC1. Both LDL-derived cholesterol and cholesterol originating from the PM accumulated in a dense, intracellular compartment in the CT mutants. Cholesterol movement from this compartment to the PM or endoplasmic reticulum was defective in the CT mutants. Our results functionally distinguish the dense, intracellular compartment from the early endocytic hydrolytic organelle and imply that NPC1 is involved in sorting cholesterol from the intracellular compartment back to the PM or to the endoplasmic reticulum. (+info)