Figure 10.
The ciliary remnant in mitotic Nek2 KO cells leads to asynchronous cilium reassembly.(A) Schematic diagram of the experimental procedure and quantification of RPE1 WT and Nek2 KO mitotic cells analyzed using Cep164, Arl13B, and γ-tubulin antibodies. Bar graphs represent mean ± SD. n = 161 (WT) and 232 (Nek2 KO) mitotic cells in three independent experiments. Representative cells are shown to the right. Scale bar, 10 µm. (B) Representative still images of live-cell imaging performed with RPE1 Nek2 KO cells stably expressing Arl13B-GFP and γ-tubulin-mRuby2 (Video 1). Cells were treated as detailed in Fig. 8 A. The insets show magnifications of the basal body area. The numbers inside the panels indicate the position of the two centrosomes. Scale bars, 10 µm (panel) and 2 µm (inset). (C and D) Quantification of Arl13B-GFP remnant inheritance of B. Only ciliated cells that entered mitosis during the time of inspection were analyzed as depicted in C. N indicates the number of inspected cells in three independent experiments. The time of cilia reformation after cell division is plotted for individual cells in D. (E) Nek2 KO cells were treated as shown in Fig. 10 A and serum restimulated in the presence or absence of SAG. Representative image shows Nek2 KO cells stained with Arl13B and Smo as cilia marker and γ-tubulin as basal body marker. DNA was stained with DAPI. The cilium from a neighboring cell (cell boundaries indicated by dashed lines) is marked by an arrow. The graph shows the percentage of mitotic cells with Arl13B- and Smo-positive remnants in n = 43 cells. Scale bars, 10 µm (panel) and 2 µm (inset). (F) Schematic model for the function of Nek2 in cilia and appendage regulation. In cycling cells, Nek2 inhibits cilia assembly in a Kif24-dependent manner and removes DAs from the M-centriole before mitosis independently of Kif24. In ciliated RPE1 cells, cilia disassemble before mitosis. In WT cells, the displacement of “mobile” DAs prevents ciliary vesicle attachment in mitosis. In the absence of Nek2, maintenance of DAs results in a mitotic cilia remnant with the consequence of premature cilia reformation in the next cell cycle. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

The ciliary remnant in mitotic Nek2 KO cells leads to asynchronous cilium reassembly. (A) Schematic diagram of the experimental procedure and quantification of RPE1 WT and Nek2 KO mitotic cells analyzed using Cep164, Arl13B, and γ-tubulin antibodies. Bar graphs represent mean ± SD. n = 161 (WT) and 232 (Nek2 KO) mitotic cells in three independent experiments. Representative cells are shown to the right. Scale bar, 10 µm. (B) Representative still images of live-cell imaging performed with RPE1 Nek2 KO cells stably expressing Arl13B-GFP and γ-tubulin-mRuby2 (Video 1). Cells were treated as detailed in Fig. 8 A. The insets show magnifications of the basal body area. The numbers inside the panels indicate the position of the two centrosomes. Scale bars, 10 µm (panel) and 2 µm (inset). (C and D) Quantification of Arl13B-GFP remnant inheritance of B. Only ciliated cells that entered mitosis during the time of inspection were analyzed as depicted in C. N indicates the number of inspected cells in three independent experiments. The time of cilia reformation after cell division is plotted for individual cells in D. (E) Nek2 KO cells were treated as shown in Fig. 10 A and serum restimulated in the presence or absence of SAG. Representative image shows Nek2 KO cells stained with Arl13B and Smo as cilia marker and γ-tubulin as basal body marker. DNA was stained with DAPI. The cilium from a neighboring cell (cell boundaries indicated by dashed lines) is marked by an arrow. The graph shows the percentage of mitotic cells with Arl13B- and Smo-positive remnants in n = 43 cells. Scale bars, 10 µm (panel) and 2 µm (inset). (F) Schematic model for the function of Nek2 in cilia and appendage regulation. In cycling cells, Nek2 inhibits cilia assembly in a Kif24-dependent manner and removes DAs from the M-centriole before mitosis independently of Kif24. In ciliated RPE1 cells, cilia disassemble before mitosis. In WT cells, the displacement of “mobile” DAs prevents ciliary vesicle attachment in mitosis. In the absence of Nek2, maintenance of DAs results in a mitotic cilia remnant with the consequence of premature cilia reformation in the next cell cycle. A.U., arbitrary units; n.s., not significant. Significance probability values are: n.s., P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

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