Figure 3.
Endocytosis of VSG by clathrin-coated vesicles. (A) Section of a high-pressure frozen, freeze-substituted and Epon-embedded cell showing the FP and flagellum (FL). A clathrin-coated pit (coat marked by arrow heads) pinches off the flagellar pocket membrane and two endocytic vesicles (arrows), which have already lost their clathrin coat, can be seen in the cytoplasm. The VSG surface coat (SC) lines the flagellar pocket membrane and the inner face of coated pits and endocytic vesicles. (B and C) Cryosections labeled with anti-clathrin antibodies and PAG-6. Clathrin covers coated pits (CCP in B) and vesicles (CCV in C); a new coated pit forms at the flagellar pocket membrane in C (arrows). (D) Epon-section prepared as for A was labeled with anti-VSG antibodies and PAG-15. VSG enters a coated pit (arrow heads). (E) Cryosection labeled with rabbit anti-biotin antibodies and PAG-6. Cells were biotinylated at 0°C and then warmed to 26°C for 5 s to initiate endocytosis. Biotinylated VSG (VSGbiot.) enters a clathrin-coated pit and is present in a coated vesicle but the EXC is not labeled. The inset shows a coated pit double labeled for VSG with biotinylated anti-VSG antibodies (PAG-18) and clathrin (PAG-6; for further details, see MATERIALS AND METHODS). Bars (A–D), 250 nm; E, 125 nm.