Cilia from digitonin-permeabilized cells are functionally intact. (A) The integrity of the membrane diffusion barrier at the base of cilia was assessed by whole-cilium FRAP. Photobleaching of the distal half of the cilium was performed to test Sstr3 mobility with the ciliary membrane. Averaged fluorescence recovery traces for the bleached region are plotted on the left (n ≥ 14; error bars indicate standard deviation). The Sstr3-GFP diffusion coefficient was determined by fitting observed half-cilium recovery traces to an expected curve (see Materials and methods) and was used to calculate the recovery curve shown in purple. Images on the right show representative data for whole-cilium FRAP. Rel., relative; AU, arbitrary unit. Bar, 2 µm. (B) Reconstitution of IFT in digitonin-permeabilized cells. Kymographs of GFP-IFT88 movement reveal that IFT trains do not move in digitonin-permeabilized cells. Addition of an ATP regenerating system and extracts from bovine retina (left) or Xenopus eggs (middle) reactivates IFT movement in anterograde and retrograde directions. IFT movement in a mock-permeabilized (intact) cell is shown on the right. Bar, 1 µm.