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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.
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