Fig 4.

TbRME-8 RNAi delays endocytosis in BSF. (A) Control and tetracycline-induced RNAi lines for TbRME-8 were allowed to take up FITC-conjugated ConA (red) at the indicated temperatures, fixed, and counterstained with DAPI for DNA (blue). ConA staining was assessed by immunofluorescence. In control cells, at 4°C ConA is not internalized and remains at the flagellar pocket, at 12°C ConA is preferentially blocked at the early endosome, and at 37°C ConA stains the lysosome (39). Induced RNAi lines for TbRME-8 were analyzed at 24 (Day 1) and 48 h (Day 2) postinduction. Representative images show a delay in delivery of ConA to the lysosome at 37°C in the RNAi lines. The scale bar, bottom right, is 2 μm. (B) Quantification of ConA trafficking at 12°C and 37°C, assessed by counting ConA puncta in 50 cells per sample, 1 day postinduction. Most control cells show two or three ConA puncta at 12°C, representing staining at the flagellar pocket and early endosomal compartments. In TbRME-8 RNAi cells, there is an overrepresentation of cells with only one ConA spot, possibly representing a delay of trafficking from the flagellar pocket. Furthermore, most control cells show a single ConA spot at 37°C, representing staining restricted to the lysosome. In TbRME-8 RNAi cells, there is an overrepresentation of cells with two or three ConA spots, likely representing a delay in trafficking through the endosomes. (C) Quantification of ConA lysosomal uptake dynamics at 37°C, 1 day postinduction. In control cells, ConA staining overlaps with the lysosomal marker p67 in the majority of cells within 30 min of exposure to ConA, while in the RNAi-induced cell line (day 1: 24 h postinduction), there is a marked delay in ConA uptake and overlap of p67. Data and error bars represent results for two different TbRME-8 RNAi clones; at least 30 cells were examined for each sample at each time point. Representative images are shown in Fig. S3 in the supplemental material.