The human phosphatase CDC14A modulates primary cilium length by regulating centrosomal actin nucleation (2018)
Figure 1

Figure 1. Ablation of hCDC14A phosphatase activity in human RPE1 cells leads to the elongation of the primary cilium

WT and hCDC14APD RPE1 cells were serum starved for 48 h for inducing ciliogenesis prior to fixation. Cilia were stained with Arl13B (green) while the basal bodies were marked with C-Nap1 (red). DNA was stained with DAPI. Cilia in the magenta box are shown enlarged on the right picture.

and C. Percent of ciliated cells, as well as the length of cilia, was quantified from A. Cilia length was measured by a semi-automated ImageJ macro as described in methods. Data points are the average of three independent experiments with N = 150 cilia for each experiment and condition. Error bars represent the mean ± SD. **** P≤ 0.0001. High means 80-90% and low 40-50% confluency of RPE1 cells.

Elongated cilia phenotype was confirmed by siRNA-mediated knockdown of hCDC14A. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SD. **** P≤ 0.0001. For definition of confluency see (C).

hCDC14A-GFP and hCDC14AC278S-GFP under control of the TetON promoter were stably integrated into RPE1 cells. hCDC14A-GFP and hCDC14AC278S-GFP localized to the C-Nap1 marked basal body upon Dox induction. In addition, expression of hCDC14A-GFP but not the phosphatase dead hCDC14AC278S-GFP mutant inhibited cilia formation. Ciliated cells were quantified 48 h after serum starvation with and without Dox addition. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SD. *** P≤ 0.001; ns: not significant.

Expression of hCDC14A-GFP and hCDC14AC278S-GFP was validated by immunoblot analysis with anti-GFP antibodies. GAPDH was the loading control.

RPE1 cells over-expressing hCDC14AC278S have longer cilia than those over-expressing hCDC14A. Three independent experiments, N = 100 cilia for hCDC14A expressing cells and 200 for hCDC14AC278S expressing cells. Mean ± SD. *** P≤ 0.001.

The dynamics of ciliogenesis was determined by serum starving WT and hCDC14APD cells for different time-points. The cilia from hCDC14APD cells continue to elongate even after 48 h of starvation whereas those from WT cells reached constant length within 12 to 24 h. Three independent experiments; N = 150 cilia for each experiment and condition. Mean ± SEM. * P≤ 0.05, **; P≤ 0.01; **** P≤ 0.0001.

In a time course experiment, the cilia disassembly kinetics was measured by first inducing ciliation through 48 h of serum starvation prior to incubation with serum containing media for different time points. Measurement of cilia length indicated the comparable rates of cilia disassembly in RPE1 WT and hCDC14APD cells. Cilia disassembly rates are shown in Fig. EV1D. Three independent experiments, N = 150 cilia for each experiment and condition. Mean ± SEM. **** P<0.0001.

Images of cilia of WT RPE1 and hCDC14APD RPE1 cells from I, 0, 6 and 24 h after serum addition. Fixed cells were analyzed for cilia length.

Data information: (A, E, J) Sizes of the scale bars are indicated next to the designated images. (C, D) One-way ANOVA. (E, G, H, I) Unpaired Student's t-test.