Browsing by Subject "Endocytosis"

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    Comparison between endocytosis and intracanalicular sequestration of cell-surface antigens in human platelets
    (1992) Jennings, Brent; Holland, Errol; Thilo, Lutz
    Human platelets respond to various macromolecules in the plasma. Uptake of specific ligands, and antibodies to various epitopes on the platelet plasma membrane, has been observed. The platelet canalicular system has been shown to be involved with this uptake. Recently, investigators have speculated on the role of endocytosis in platelets to account for the presence of plasma proteins such as fibrinogen and immunoglobulin within platelet organelles. Antibodies binding to cell-surface antigens on platelets can lead to a redistribution of these antigens. When antibodies, specific for platelet cell-surface receptors, bind to platelets they may either undergo endocytosis into intracellular vacuoles, or may merely become sequestered within the canalicular system of platelets. The present study investigated whether endocytosis occurs in platelets. Such a process would lead to the endocytic uptake of a fluid-phase marker and would involve internalization and recycling of cell surface membrane. A fluid-phase marker (FITC-dextran) was used to measure any constitutive endocytic activity. In addition, a suitable membrane marker was used to determine whether membrane internalization occurred. This involved a technique whereby radioactive galactose was covalently attached to cell-surface glycoconjugates. A monoclonal antibody to the platelet receptor, GPIIbIIIa, was used in conjunction with the membrane marker in order to determine if membrane internalization was involved during the subsequent redistribution of the receptor-antibody complex. Immunocytochemical techniques using electron-dense probes were employed to localise the sites to which this receptor-antibody complex became redistributed. In comparison with reported rates of endocytic uptake of fluid-phase marker in other cell types, no significant endocytic activity could be detected with platelets, after taking their relatively small volume into account. Similarly, membrane internalization was not detected with resting platelets. Following challenge of the platelets with anti-GPIIbIIIa antibody, no membrane internalization could be measured during redistribution of the receptor-antibody complex. The compartment to which the receptor-antibody complex was redistributed could be identified morphologically as the canalicular system. The present data provide evidence for a process of sequestration of receptor-antibody in the canalicular system of resting platelets. It remains possible that other mechanisms exist within the platelet system for uptake of extracellular material as this study dealt exclusively with the platelet response to a specific antibody. These results may have implications with respect to the interaction of platelets with anti-platelet antibodies in the normal state, as well as with clinical disorders involving elevated levels of platelet-associated IgG. As far as can be deduced from the available literature, these data represent the first use of a covalent membrane marker in conjunction with uptake of macromolecules to study endocytic events in human platelets.
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    Differential Internalization of Mammalian and Non-mammalian Gonadotropin-releasing Hormone Receptors: UNCOUPLING OF DYNAMIN-DEPENDENT INTERNALIZATION FROM MITOGEN-ACTIVATED PROTEIN KINASE SIGNALING
    (2001) Hislop, James N; Everest, Helen M; Flynn, Andrea; Harding, Tom; Uney, James B; Troskie, Brigitte E; Millar, Robert P; McArdle, Craig A
    Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.
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    The effect of hyperosmolarity on fluid-phase and receptor-mediated endocytosis in P388D1 macrophages
    (1992) Begg, Michael John; Thilo, Lutz
    Extracellular components can be internalized by either receptor-mediated or fluid-phase endocytosis. Receptor-mediated endocytosis involves the internalization of receptor-ligand complexes into coated vesicles of about 0.1 μm in diameter. The average diameter of primary pinocytic vesicles has been calculated to be 0.24 - 0.28 μm. The discrepancy in size between coated vesicles and the average pinosome diameter can be explained if, in addition to coated vesicles, another endocytic process involving vesicles larger than 0.28 μm in diameter takes place. These two vesicle types could together produce an average diameter of 0.24 μm. This hypothesis suggests that coated vesicles cannot fully account for fluid-phase uptake. Hypertonic conditions can selectively inhibit receptor-mediated endocytosis, leaving fluid-phase uptake unaffected, again suggesting that an alternative to coated pit-mediated uptake exists. In this study we determined the volume-weighted average diameter of primary pinocytic vesicles under hypertonic conditions (0.52 osm) where receptor-mediated uptake of transferrin was selectively inhibited by 42%. Fluid-phase uptake of FITC-dextran was unaffected by 0.52 osm medium. The internalization rate of ³H-galactose-labelled plasma membrane was reduced from 2.6 %/min to 1.5 %/min. The decrease in the rate of membrane internalization, without a reduction in the rate of fluid uptake at hypertonicity, implied a reduced surface to volume ratio of the pinocytic vesicles formed under these conditions. This suggested an increase in the average diameter of primary pinocytic vesicles. Membrane internalization rates were calculated on the assumption that all labelled cell-surface constituents were internalized to the same relative extent, as has been shown previously for isotonic conditions. This assumption was also shown to hold true under isotonic conditions. The reduced rate of membrane internalization under hypertonic conditions was shown not to be due to the exclusion of any labelled protein species from internalized vesicles. The larger average vesicle size determined under conditions of selective reduction of coated vesicle formation (i.e. hypertonicity), demonstrates the existence of a population of larger pinosomes involved in a possible alternative mechanism to coated-pit-mediated endocytosis.
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    The kinetics of endosome processing
    (1995) Legalatladi, Seetsela; Thilo, Lutz
    The present thesis looks at the behaviour of internalised cell surface-derived membrane marker in comparison with the behaviour of endocytosed HRP (horse-radish peroxidase) as a fluid-phase contents marker. The pooling and/or segregation in the endosome was measured by determining co-localization with HRP. Colocalization of the two markers in the endosome is studied by using the ability of HRP to catalyse the crosslinking of membrane marker in endosomes with DAB (3,3'-diaminobenzidine), rendering the membrane marker detergent insoluble. To study the kinetic behaviour of membrane marker, radioactive galactose was covalently bound to cell-surface glycoconjugates on mouse macrophage-cells, P388D₁, as catalysed by galactosyltransferase. This provided a general membrane marker. After endocytosis-derived redistribution of membrane marker between the cell surface and endosomal membrane, a steady state was established with about 16% of the label on internal membranes. The bulk of the label on the cell surface was removable by subsequent treatment with β-galactosidase.
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    Membrane shedding in kidney (MDCK) cells as revealed by covalent markers during quantification of endocytosis and transcytosis
    (1991) Godenir, Nicole; Thilo, Lutz
    Membrane traffic in polarised cells was investigated by growing Madin-Darby canine kidney (MOCK) cells on ·permeable polycarbonate filter supports which allowed access to both sides of the cell monolayer. Membrane glycoconjugates on the apical and basolateral cell surfaces were labelled enzymatically with ³H- and ¹⁴C-galactose, respectively, to provide covalent membrane markers. Experiments were done to quantitate membrane traffic during endocytosis at the respective plasma membrane domains and that due to transcytosis. Internalized label was quantitatively distinguished from label on the respective cell surface by its resistance to removal by glycosidases.
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    Receptor-mediated endocytosis of low density lipoproteins in aortic endothelial cells
    (1986) Sanan, David Austin; Coetzee, G A
    Lipoprotein binding and metabolism in actively-dividing (subconfluent) and quiescent (postconfluent) bovine aortic endothelial cells (ECs) were qualitatively investigated by fluorescence microscopy using dioctadecylindocarbocyanine-labelled lipoproteins and by indirect immunofluorescence microscopy. LDL and acetylated-LDL (AcLDL) were seen bound to the surfaces of subconfluent ECs (at 4°C or at 37°C), as a random distribution of punctate foci. ECs therefore closely resembled fibroblasts in the distribution of LDL receptors on their surfaces. No binding of LDL was seen on postconfluent EC surfaces by either direct or indirect fluorescence microscopy. The patterns of AcLDL binding on postconfluent ECs resembled those on subconfluent ECs. Intracellular LDL and AcLDL occurred as perinuclear accumulations of large fluorescent disc-shaped profiles in subconfluent ECs. These accumulations were shown to arise from surface-bound material by pulse-chase experiments. Intracellular LDL was absent in the majority of postconfluent ECs, while AcLDL accumulation was massive. "Wounding" of cultures allowed simultaneous assessment of lipoprotein metabolism in quiescent and actively-dividing areas of the same culture. Quantitative assessments of the above-mentioned phenomena were made using ¹²⁵I-labelled lipoproteins. Receptor-mediated binding of LDL decreased five to ten-fold as the cultures modulated from subconfluent to postconfluent morphology. No receptor-bound LDL was detected in postconfluent ECs. Conversely, the amount of AcLDL bound increased at least fivefold during EC growth in parallel cultures. The amounts of lipoproteins endocytosed and metabolised were generally related proportionately to the amounts bound in each case. The distribution of LDL receptors on cultured cells was also investigated at the ultrastructural level using colloidal gold-conjugated LDL as a probe, and similarly labelled antibodies as probes. Whole-mounted cells with receptor probes bound to them were examined directly in the transmission electron microscope. The topographical distribution of LDL receptors has not been investigated by these techniques before. A novel method of preparing cytochemically-labelled, whole-mounted cells from styrene culture dishes was developed and used in this study. LDL Receptors expressed on the surfaces of human skin fibroblasts served to standardise these colloidal gold techniques and fortuitously led to new information on receptor distribution. Normal (FGo) and LDL receptor-negative mutant fibroblasts (GM 2000) acted as positive and negative controls respectively. Normal fibroblast LDL receptors were grouped into clusters consistent in size with coated pits (200 - 500 nm in diameter). A novel finding was the presence of a diffuse population of receptors scattered randomly amongst the clustered receptors. Another mutant fibroblast, GM 2408A, known to have an aberrant LDL receptor distribution, was also examined. Its receptors were shown to be dispersed singly, and in occasional groups of two and three, at random over the cell surfaces. No clusters were detected. The receptor-negative GM 2000 bound virtually no probes. While not as sensitive as the colloidal gold-conjugated LDL probe, an antireceptor monoclonal antibody (IgG-C7), localised by indirect immunogold labelling, gave similar results when applied to the above cells. This was taken as strong corroborative evidence that the LDL receptor distributions as determined by colloidal gold-conjugated LDL were correct. It is suggested that the dispersed population of receptors on normal fibroblasts may represent newly-emerged recycling receptors which have yet to cluster in coated pits. A further new finding reported here is the existence of the same two patterns of LD L receptors, dispersed and clustered, on the surface of subconfluent ECs. It was noted, from the study of whole-mounted and thin-sectioned cells, that the receptors were preferentially arranged in rings following the circumference of coated pit areas on the cell surface. Often these rings associated in groups or even coalesced into compound clusters. The significance of these groupings is not yet understood. In sharp contrast to the situation on subconfluent ECs, no LDL receptors (probed with the extremely sensitive colloidal-gold conjugated LDL) could be detected at the EM level on the surface of postconfluent ECs. Active cells in wounded postconfluent monolayers expressed abundant receptors detected at the EM level. It is concluded that postconfluent quiescent bovine aortic ECs in vitro metabolise virtually no LDL via the LDL-receptor pathway due to a vanishingly low number of LDL receptors. This contrasts with the ability of postconfluent cells to metabolise relatively large amounts of AcLDL via a receptor-mediated mechanism. The significance of these conclusions is discussed with respect to the interaction of plasma lipoproteins with the endothelium in vivo.