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First published online March 2, 2004
doi: 10.1242/10.1242/jcs.00938

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Kinetics of endocytosis and recycling of the GPI-anchored variant surface glycoprotein in Trypanosoma brucei

¹ Ludwigs-Maximilians-Universität, Department Biologie I, Bereich Genetik, Maria-Ward-Strasse 1a, D-80638 München, Germany
² Max-Planck-Institut für Biologie, Abteilung Membranbiochemie, Corrensstrasse 38, D-72076 Tübingen, Germany
³ Division of Medical Biochemistry, University Cape Town Medical School, Anzio Road, ZA-7925 Observatory, South Africa
⁴ Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35, D-72076 Tübingen, Germany

View larger version (19K): [in a new window]   Fig. 1. Kinetics of internalisation and recycling of VSG and endocytosis of fluid-phase marker. (A) Rate of internalisation. VSGbiotin uptake is expressed as the fraction of surface-associated VSGbiotin prior to endocytosis (t=0). The data represent the average (±s.e.) of five independent experiments. (B) Rate of recycling. Exocytosis of VSG is expressed as the non-acetylated VSG transported from the cell interior to the acetylated surface normalised to the total surface VSG without treatment. The data represent the average (±s.e.) of five independent measurements. (C) Rate of fluid-phase uptake of Alexa Fluor 488 dextran. Internalisation is expressed as the change in relative fluorescence intensity/cell. Data from three different experiments performed at a range of concentrations (1.25 to 5 mg/ml) were normalised to 1 mg/ml and averaged.

View larger version (48K): [in a new window]   Fig. 2. Endocytosis and recycling of VSG and separation of VSG and fluid-phase cargo. Digitally deconvolved 3D-fluorescence images of representative cells at different time points are shown. (A) Kinetics of endocytosis of VSGbiotin at 37°C detected with Alexa Fluor 488 Streptavidin (yellow). Control refers to a surface-biotinylated cell before endocytosis. In the other samples, surface-associated biotin label has been removed prior to immuno-labelling. The lysosome (L) is marked by an anti-p67 monoclonal antibody and Alexa Fluor 594-labelled goat anti-mouse IgG (red); the kinetoplast (K) and nucleus (N) are visualised with DAPI (blue). (B) Kinetics of recycling of VSGbiotin (yellow). Biotinylated cells were allowed to endocytose VSGbiotin for 3 minutes at 37°C, then stripped of surface label at 0°C and finally incubated at 37°C for the indicated times. The flagellar pocket and the cell surface are marked by open and closed arrows, respectively. The kinetoplast (K) and nucleus (N) are visualised with DAPI (blue). (C) Kinetics of segregation of VSGbiotin and a fluid-phase marker. Cells were labelled at the surface with sulfo-NHS-SS-biotin and AMCA-sulfo-NHS (blue). Endocytosis was allowed in the presence of Alexa Fluor 594 dextran (red) at 37°C, the biotin label at the surface was stripped at 0°C and the cells were then processed for staining with Alexa Fluor 488 Streptavidin (green). The flagellar pocket is marked by open arrows. Blue size bars: 3 µm.

View larger version (23K): [in a new window]   Fig. 3. Time-resolved, quantitative co-localisation analysis of VSG during endocytosis. Increase of co-localisation or of segregation in intracellular space was described empirically by first-order processes of the form Ax(1-e-kxt), and where this was followed by a second slower phase that derived from the first (D,E), by Ax(l-e-kxt)x{1+Bx[1-e-hxt]}. A represents the amplitude of the first phase, B is a measure for the efficiency at which A contributes to the extent of the second phase, and k and h are the rate constants. Values in brackets in E were not obtained independently from the data, but were fixed as taken from the corresponding values in D. VSGbiotin gains access to intracellular compartments as defined by clathrin (A), RAB5 (B), RAB7 (C) or RAB11 (D). The kinetics of the percentage of co-localisation between endocytosed VSGbiotin and the four markers are shown for cells taken from the same experiment. (E) Segregation of endocytosed VSGbiotin from Alexa Fluor 488-conjugated dextran.

View larger version (81K): [in a new window]   Fig. 4. Endosomal compartments of T. brucei. A-C show deconvolved 3D immunofluorescence images of cells labelled with primary and fluorophore-tagged secondary antibodies. (A) The RAB5A-positive compartment stained by rabbit antibodies (red) is located close to the lysosome (labelled by a mouse antibody against p67; green). The kinetoplast (K) and nucleus (N) are visualised by DAPI. (B) The RAB7-positive compartment stained by rabbit antibodies (red) is located close to the lysosome (labelled by p67; green). 3D inspection indicates that the two compartments do not overlap. (C) Visualisation of the RAB7-compartment with rat antibodies (green) and the RAB5A-compartment with rabbit antibodies (red) shows that the two compartments are closely associated but distinct. The cell surface and flagellar pocket are labelled by AMCA (blue). (D) Schematic diagram of endocytic structures. (E) Electron micrograph of cryosection labelled with rabbit-anti-RAB5A antibodies and 6 nm Protein A-gold complexes. cEC is positive, whereas linear EC and EXCs (asterisks) are not labelled. (F) Cryosection labelled with rat anti-RAB7 antibodies and goat anti-rat IgG/IgM-12 nm gold conjugate. The antibodies bind to a complex membrane structure. (G) Cells were incubated for 3 minutes at 37°C in the presence of 10 mg/ml horseradish peroxidase (HRP) and processed for diaminobenzidine-staining and Epon embedding (Webster, 1989). The enzyme is detected in cisternae (EC and cEC), class I (filled arrow head) and class II CCVs (open arrow heads) and in EXCs (asterisks). (H) Same as G. A class II CCV (arrow head) budding from the rim of an EC is strongly labelled. (I) Cells were incubated for 5 minutes at 37°C with ferritin (50 mg/ml) and then processed for Epon embedding (Langreth and Balber, 1975). Ferritin is excluded from the narrow luminal parts of the cisternae but present in abundance in clathrin-coated buds (arrow head) and in class II CCVs (inset). CCV I, class I clathrin-coated vesicle; CCV II, class II clathrin-coated vesicle; cEC, circular endosomal profiles; EC, endosomal cisterna; ER, endoplasmic reticulum; EXC, exocytic carrier; F, flagellum; FP, flagellar pocket; G, Golgi complex; K, kinetoplast; L, lysosome; N, nucleus; PM, plasma membrane. Bars: A-C, 2 µm; E-I, 0.5 µm; I (inset), 0.2 µm.

View larger version (11K): [in a new window]   Fig. 5. Flow diagram of VSG or fluid-phase cargo through endosomal structures. CCV I and II, clathrin-coated vesicles of classes I and II; EE, early endosome; EXC, exocytic carrier; FP, flagellar pocket; L, lysosome; LE, late endosome; RE, recycling endosome. The markers defining these compartments are indicated.

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