A. Effect of the Ift27null1 mutation on IFT protein stability. Protein extracts from wild-type and mutant MEFs were immunoblotted with the antibodies indicated on the right side of each western blot panel. Approximate molecular weights are listed on the left side. Quantitation of IFT protein levels relative to γ-tubulin loading control are listed on the right side of each western blot (n=3 embryos/MEF lines per genotype) (*p<0.05, **p<0.01).
B. E18.5 lung and kidney sections from Ift27+/+ and Ift27null1/null1 mice immunostained with IFT88 (green) and the acetylated tubulin cilia marker 6-11B-1 (red). Scale bar is 5μm and applies to all images. Images are maximum projections of 10 layer Z-stacks acquired every 0.5 μm
C. Immunofluorescence of control and mutant MEFs immunostained with 6-11B-1 (cilia, red) and IFT27, IFT88, IFT140 or Dync2h1 (green). Note the lack of IFT27 staining of mutant cells (25/25 wild type cells and 0/25 mutant cells showed IFT27 staining). Staining of cilia for the other antibodies were similar in both mutant and control cells (25/25 cells positive for each condition).
D. Quantitation of ciliation and ciliary length in MEF cells. Percentage of ciliated cells and ciliary lengths based on ciliary IFT88 immunostaining in serum starved MEFs (n=3 Ift27+/+ and 3 Ift27null1/null1 embryos/MEF lines for % ciliation and n=50 cilia per cell line for length). Differences for percent ciliation were not significantly different, but mutant cilia were slightly longer (** p<0.01).
E. IFT25-Flag (green) localizes to cilia (6-11B-1, red) in wild type cells but not Ift27null1/null1 cells. Inset shows green (IFT25-Flag) channel. Quantification showed 23/25 transfected wild type cells had ciliary-localized IFT25-Flag while 0/25 transfected mutant cells had ciliary-localized IFT25-Flag.
F, G. Interaction between IFT25 and IFT27 are required for IFT25 to enter cilia. F. Immunoprecipitation from IMCD3 cells transfected with Flag-GST, Flag-IFT25 or Flag-IFT25(T40R/T42R/S128E) demonstrate that wild type but not the mutant form of IFT25 bind to IFT27 and IFT88. G. In IMCD3 cells, wild type Flag-IFT25 (Flag, red) localizes to cilia (IFT88, green) but Flag-IFT25(T40R/T42R/S128E) (Flag, red) does not. Quantification showed that 0/50 Flag-GST, 47/50 Flag-IFT25, 0/50 Flag-IFT25(T40R/T42R/S128E) transfected cells had Flag-positive cilia. Scale bar is 5 μm and applies to all images in G.
H. Wild type Flag-IFT25 (top row, Flag, red) but not Flag-IFT25(T40R/T42R/S128E) (bottom row, Flag, red) rescues the BBS9 (left panel, green) and Smo (right panel green) accumulation phenotypes in Ift25null1/null1 mutant MEFs. Arrows mark cilia on transfected cells which are shown in the insets with separate red and green channels. Quantification showed 25/25 cells transfected with wild type IFT25 showed rescue of the accumulation of ciliary BBS9 and Smo phenotypes while 0/25 transfected with IFT25(T40R/T42R/S128E) showed rescue. The * marks a cilium on a non-transfected cell. Scale bar is 5 μm and applies to all images in H.
I. Immunoprecipitation from MEF cells transfected with IFT27-Flag, IFT27(K68L)-Flag, IFT27(T19N)-Flag or Flag-GST demonstrate that all forms of IFT27 bind to IFT25 but only wild type and the K68L forms of IFT27 bind to IFT88. No binding between any form of IFT27 and IFT140 was detected. Note that with this exposure time, IFT88 is not detectable until after immunoprecipitation.
J. Wild type IFT27-Flag (left column, Flag, red), IFT27(K68L)-Flag (middle column, Flag, red) and Flag-IFT27(T19N) (right column, Flag, red) expressed in Ift27+/+ (top row) and Ift27null1/null1 (bottom row) MEFs. Quantification showed that, 80/100 IFT27-Flag, 24/100 IFT27(K68L)-Flag and 0/100 IFT27(T19N)-Flag transfected Ift27+/+ cells had Flag-positive cilia while 72/100 IFT27-Flag, 86/100 IFT27(K68L)-Flag and 36/100 IFT27(T19N)-Flag transfected Ift27null1/null1 cells had Flag-positive cilia. Scale bar is 5 μm and applies to all images in J.