Fig. 7. Comparative phenotypic analysis of inducible RNAi against TbMBAP1, clathrin heavy chain and RAB11. (A) Kinetics of endocytosis of the fluid-phase marker Alexa Fluor^TM 488 dextran in the presence (black circles) or absence (white circles) of tetracycline. Internalization is expressed as the change in relative fluorescence intensity/cell. Analyses were done 6 hours (TbMBAP1, TbCLH) and 10 hours (RAB11) post-induction of RNAi. (B) Localization of the endosomal reporter protein EP:GFP in wild-type cells and after induction of RNAi. The cell surface was stained with AMCA-sulfo-NHS (blue) as described in the legend to Fig. 3B. EP:GFP fluorescence is shown in yellow. The arrows indicate the flagellar pocket. (C) Single frames of time-lapse movies of live cells treated with the plasma membrane dye FM^®2-10. Cells were incubated with FM^®2-10 on ice for 5 minutes and subsequently warmed to 37°C for 10 minutes. While the control cells (left) and TbRAB11-depleted trypanosomes (right) internalize the fluorescent plasma membrane by endocytosis, the TbMBAP1^RNAi (mid left) and TbCLH^RNAi cells (mid right) do not show staining of intracellular membranes. The inset in the right image demonstrates that TbRAB11^RNAi cells are unable to exocytose internalized membrane even after incubation in the absence of FM^®2-10 for 1 hours at 37°C (for details see results section). Arrows indicate the flagellar pocket.