Bidirectional IFT trafficking takes place on two sides of the axoneme in live trypanosomes expressing mNG::IFT81. (A) Temporal projection of a stack of images corresponding with Video 5 showing the presence of two parallel lines for IFT in the flagellum in addition to the pool of IFT at the base. The left (green) and right (magenta) sides were defined after orientating the cell with the posterior end on top of the image and the flagellum on the left-hand side. (B) Still images from Video 5 of live trypanosomes expressing mNG::IFT81 imaged at high resolution. Green and magenta arrowheads indicate trains on the left and right sides, with light arrowheads indicating anterograde trains and darker arrowheads showing retrograde trains. The time point for each image is indicated. (C) Kymograph analysis of the same cell showing trafficking on the left side (green), on the right side (magenta), and the merged images for the region of interest indicated in A. Bars: 2.5 µm (horizontal bar); 2.5 s (vertical bar). (D) Dot plot of the frequency of anterograde IFT trains visible on the left (green) and the right (magenta) side, the sum of both (cyan), and from videos where only one track was visible (unresolved; gray). Values are coming from 18 different cells containing a total of 529 trains. *, P = 0.02. (E) Same representation but for the speed of anterograde trains. Only statistically significant differences are shown (one-way ANOVA test).