TIRF and DIC microscopy measurements of IFT in wild-type and dhc1b-3 cells at 21 and 34°C. (A) Kymographs generated from TIRF time series of wild-type and dhc1b-3 cells expressing KAP-GFP at 21 and 34°C, as indicated. (B) Kymographs generated from DIC time series of wild-type and dhc1b-3 cells at 21 and 34°C. For A and B, the x-axis is distance along the length of the flagellum (the flagellar base is on the left and the flagellar tip is on the right) and the y-axis is time (earliest times are at the top). Bars (vertical) 5 s; (horizontal) 5 µm. Quantification of IFT train speeds (C) and frequencies (D) from the TIRF and DIC datasets. Error bars indicate standard deviation. In dhc1b-3 flagella, retrograde IFT is moderately reduced at 21°C and strongly inhibited at 34°C. The large drop in dhc1b-3 retrograde IFT at 34°C is accompanied by a decrease in anterograde IFT frequency. The lower frequency of anterograde trains measured by TIRF vs. DIC likely means that the KAP-GFP fluorescence of some trains was not detected. Nflagella = 231 (TIRF), 109 (DIC).
