Fig. 2. TbDLP is required for endocytosis. (A) Ablation of TbDLP by RNAi results in enlarged FPs. FP in living cells were visualized by Fluorescein-conjugated tomato lectin (TLect). Nomarski (Nom) images and the merged pictures of the tomato lectin and the DAPIstaining of uninduced (0 h) and induced cells (24 h) are shown. Bars, 5 µm. (B), Analysis of the TbDLP-RNAi cell line by transmission electron microscopy. Bars, 1 µm. (C) Kinetics of appearance of enlarged FPs and loss of endocytic activity during induction of TbDLP-RNAi. FPs were visualized by AMCA sulfo-NHS labeling of surface proteins as described (Engstler et al., 2004). Enlarged FPs in uninduced (–Tet, ) and induced (+Tet, ) TbDLP-RNAi cells were automatically detected using a series of scripted digital image segmentation steps. Total endocytic activity was measured in the same culture by quantifying the internalized AMCA-labeled surface proteins (+Tet, grey symbols). All values were normalized to the corresponding total cell numbers (n>300 cells) and expressed relative to that of the corresponding uninduced culture.