Browsing by Author "Thilo, Lutz"
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- ItemOpen AccessComparison between endocytosis and intracanalicular sequestration of cell-surface antigens in human platelets(1992) Jennings, Brent; Holland, Errol; Thilo, LutzHuman 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.
- ItemOpen AccessThe effect of hyperosmolarity on fluid-phase and receptor-mediated endocytosis in P388D1 macrophages(1992) Begg, Michael John; Thilo, LutzExtracellular 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.
- ItemOpen AccessIdentification of putative targeting factors required for specific fusion amongst organelles of the endocytic pathway(2001) Baker, Bienyameen; Thilo, LutzIncludes bibliographical references.
- ItemOpen AccessKinetic analysis of endosome processing : maturation of early endosomes and vesicular traffic to lysosomes(1995) Stroud, Evelyn Joy; Thilo, LutzThe present study was undertaken to establish the mechanism(s) involved in the endocytic pathway, in particular, early endosome processing and delivery to the lysosomes. Two models for endosome processing have previously been proposed in the literature, namely the maturation and vesicular traffic models. The general consensus has been an early phase of intermingling of the endocytic contents markers within early endosomes that mature to form non-fusogenic late endosomes (maturation model). This maturation phase is followed by a segregation phase where intermingling of contents between vesicles no longer takes place. To establish the mechanism(s) involved in early endosome processing and delivery to lysosomes, a kinetic analysis was made using results from cellular fluid-phase uptake assays. This unique approach offers an alternative view to previous studies on the mechanisms in operation during endocytic processing. The results and conclusions made could thus confirm or disprove previously proposed mechanisms.
- ItemOpen AccessThe kinetics of endosome processing(1995) Legalatladi, Seetsela; Thilo, LutzThe 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.
- ItemMetadata onlyMembrane shedding in kidney (MDCK) cells as revealed by covalent markers during quantification of endocytosis and transcytosis(1991) Godenir, Nicole; Thilo, LutzMembrane 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.
- ItemOpen AccessProteomic analysis of the Mycobacterium avium-containing phagosome membrane(2010) Pietersen, Ray-Dean Donovan; Thilo, LutzPathogenic mycobacteria, like [Mycobacterium tuberculosis] M.tb and Mycobacterium avium (M.av), reside intracellularly in phagosomes where they are able to survive, because they block the process of phagosome maturation. ... An approach of characterizing M.av-containing phagosomes in terms of cell surface-derived glycoconjugates, revealed that these phagosomes show a 3- to 4-fold depletion of these glycoconjugates as compared to early endosomes with which the phagosomes continuously fuse and exchange membrane molecules... The aim of this study is to identify the depleted / possibly enriched cell surface derived glycoproteins on the phagosome membrane of M.av-containing phagosomes, because they might play a role in mycobacteria's intracellular survival.
- ItemOpen AccessRegulation of integrin-αM and β2 expression on the surface of macrophages in response to infection with Mycobacterium avium(2007) Itoe, Maurice Ayamba; Thilo, LutzIncludes bibliographical references (leaves 56-69).