Figure 1.

Mutating tyrosine 819/828 of human PROM1 shortens primary cilia. A–D, polarized WT cells (MDCK), those expressing human PROM1.s1 or PROM1.s2 splice variants, or corresponding Y819/828F mutants were grown for 7 dpc (A–C) or 14 dpc (C and D) and processed for SEM (A and B) and CLSM (C and D). SEM micrographs revealed long (green) and short (red) primary cilia in cells expressing WT and mutated PROM1, respectively (A). Note the presence of very long cilia (>10 μm) in WT PROM1-positive cells (B; see also Fig. S1). Arrow, primary cilium; dashed line, origin of cilium; *, cell without primary cilium; #, artifact of SEM preparation. For the immunofluorescence analysis (C and D), cells were double-immunolabeled for PROM1 and AcTub. The length of AcTub-labeled cilia was evaluated by CLSM and classified into three categories: <3, 3–5, and >5 μm (C). The number of analyzed primary cilia is displayed in parentheses. Data (purple) obtained from cells expressing PROM1.s2 were taken from our recent study (33) and used for comparison. The percentage of total (blue) or PROM1⁺ (black) cells with no cilium is indicated (D). The mean and S.D. (error bars) are presented. Scale bars, 500 nm (top panels) and 2 μm (bottom panels) (A) and 1 μm (B).