Kinetics of dhc1b-3 flagellar loss in asynchronous vegetative cells, during gametogenesis, and in fla10 dhc1b-3 double mutants. Bar graphs depict the percentage of deflagellated cells, and line graphs show the average flagellar length of flagellated cells. Error bars indicate standard deviation. (A) Vegetative wild-type and dhc1b-3 cells were grown in TAP media for 2 d at 21°C (dense, post-log phase cultures) and then shifted to 34°C. In this experiment, dhc1b-3 flagella were maintained with only mild shortening (10–20%) for 30 h. In less dense, rapidly dividing cultures, some dhc1b-3 cells lost flagella as early as 18 h at 34°C (not depicted). The cause of this variability is unknown, but it is likely related to the growth phase of the culture. It was not determined whether flagellar loss in these asynchronous cultures was due to cell division (as shown in Fig. S4 A) or other factors, such as retrograde IFT dropping below a critical threshold. (B) Vegetative cells were grown in TAP media at 21°C for 1 d, incubated for increasing periods of time at 34°C, and then induced to differentiate synchronously into gametes by diluting cells into M-N media followed by 18 h incubation at 34°C. Pre-incubation for 4–8 h at 34°C inhibited the assembly of new flagella in dhc1b-3 gametes. (C) Vegetative fla10 and fla10 dhc1b-3 double mutant strains were grown in TAP media for 2 d at 21°C and then shifted to 34°C. Both strains had the same kinetics of flagellar shortening and loss, which was far more rapid than flagellar loss in the dhc1b-3 single mutant. Nflagella = 1,849 (A), 833 (B), 940 (C) from single experiments.
