Figure 7

Ciliogenesis Is Markedly Reduced by the JS (V194A) and JATD (A199P) Mutations In Vivo

(A) Cilia numbers are much reduced in V194A and A199P mutant RPE-1 cell lines. Serum-starved (48 hr) RPE-1 cells were stained by antibodies against the ciliary membrane protein ARL13b and acetylated α-tubulin. Images correspond to maximum intensity projections of confocal micrographs. Scale bars, 5 μm.

(B) Quantification of the experiment shown in (A). Number of cilia was counted in serum-starved (48 hr or 72 hr) A199P#1, A199P#2, V194A, and CEP120 null (ΔCEP120) RPE-1 cell clones.

(C) TALPID3 is detectable in the basal bodies of ciliated V194A, A199P, and control RPE-1 cell lines. Serum-starved (24 hr) RPE-1 cells were stained by antibodies against TALPID3 and acetylated-α-tubulin. Images to the right show basal bodies and cilia at high (2×) magnification. Images correspond to maximum intensity projections of confocal micrographs. Scale bars, 5 μm.

(D) TALPID3 localizes to the distal ends of mother and daughter centrioles in control and A199P mutant RPE-1 cell lines, but not in V194A cells. Serum-starved (24 hr) RPE-1 cells were stained by antibodies against TALPID3 and γ-tubulin. Insets show centrosomes at high (4×) magnification. Numbering is included to aid identification of centrosomes. Images correspond to maximum intensity projections of confocal micrographs. Scale bars, 5 μm.

(E) Centrosomal TALPID3 levels are reduced in V194A mutant RPE-1 cell lines. Quantification of total centrosomal TALPID3 levels from maximum intensity projections obtained in experiment shown in (D). Quantification was performed as in Figure 4D. Swarm plots represent values from single cells, with horizontal lines marking the median, and error bars represent SD. p values were obtained from an unpaired, two-tailed Student’s t test (^∗p < 0.05, ^∗∗∗∗p < 0.0001). Similar results were obtained from two biological replicates.

See also Figures S4, S6, and S7 and Table S4.