Fig. 4
Rapid axonemal deglutamylation preferentially hampers anterograde IFT. a Neon-IFT88 accumulates at the base of deglutamylated cilia. Neon-IFT88-stable NIH3T3 cells were transfected with CCP5CD–Cerulean3–FKBP–P2A–mCherry–FRB–MAP4m. Transfected cells at 80–90% confluency were serum-starved for 24 h and then treated with 100 nM rapamycin for 30 min. Scale bar, 4 μm. b Linescan profile of the indicated proteins from base to the tip of the primary cilium in a. c Representative kymographs of Neon-IFT88 generated from time-lapse imaging of the cilium before and after rapamycin treatment for 30 min. Red, green, and blue lines represent the trajectories of Neon-IFT88 particles in anterograde and retrograde directions, and static Neon-IFT88, respectively. Horizontal scale bar, 10 s. Vertical scale bar, 2 μm. Also, see Supplementary Movie 6. d Translocation of CCP5CD–Cerulean3–FKBP, but not Cerulean3–FKBP or CCP5CDDM–Cerulean3–FKBP, onto the axoneme hampers the IFT only in the anterograde direction. Neon-IFT88-stable NIH3T3 cells were transfected with P2A-based constructs for co-expression of mCherry–FRB–MAP4m and Cerulean3–FKBP-tagged proteins. Transfected cells at 80–90% confluency were serum-starved for 24 h and then treated with 100 nM rapamycin for the indicated times. The velocity of Neon-IFT88 was quantified according to the trajectories shown in the kymographs (see the Methods section; n = 175, 150, and 170 Neon-IFT88 particles for the Cerulean3–FKBP, CCP5CD–Cerulean3–FKBP, and CCP5CDDM–Cerulean3–FKBP groups, respectively; three independent experiments). NS and * indicate no significant difference and P < 0.05, respectively, between the conditions in the presence or absence of rapamycin (Student's t-test)
