Cilium length regulation by ICK and MOK interacts with cAMP and mTORC1 signaling.

<p>(A) Average lengths of primary cilia of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or treated with 100 µM forskolin (Fsk) for 24 hours. (B) Immunofluorescence images of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or forskolin-treated, stained with anti-acetylated tubulin. (C) Average anterograde velocity of IFT20-GFP in IMCD-3 control cells, and cells depleted of ICK, untreated and forskolin-treated. (D) Average lengths of primary cilia of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or treated with 0.5 µM rapamycin (rapa) for 24 hours. (E) Immunofluorescence images of IMCD-3 cells expressing a control shRNA, ICKsh #01, or MOKsh #01, untreated or rapamycin-treated, stained with anti-acetylated tubulin. (F) Average anterograde velocity of IFT20-GFP in IMCD-3 control cells, and cells depleted of ICK, untreated and rapamycin-treated. Statistically significant differences (p<0.001) compared to untreated cells are indicated with a black asterisk, and compared to the control shRNA are indicated with a red asterisk. Error bars indicate SD. Numbers in the bars indicate number of cilia (A and D) or particles (C and F) analyzed. Scale bar, 10 µm.</p

ICK and MOK control cilium length.

<p>(A) Immunoblot of cell lysates of IMCD-3 cells transduced with shRNA constructs targeting ICK and MOK. Actin was used as loading control. (B) Average length of primary cilia of IMCD-3 cells depleted of ICK. (C) Average length of primary cilia of IMCD-3 cells depleted of MOK. (D) Average length of primary cilia of IMCD-3 cells depleted of ICK and MOK, combining ICKsh #01 and MOKsh #01 or ICKsh #02 and MOKsh #02. (E) Immunofluorescence images of IMCD-3 cells expressing control shRNA, ICKsh #01, or MOKsh #01 stained with anti-acetylated tubulin. Scale bar 10 µm. (F) Average length of primary cilia of IMCD-3 cells overexpressing GFP-C1, wild type (wt) or kinase-dead (kd) GFP-ICK, or GFP-MOK. (G) Immunofluorescence images of IMCD-3 cells expressing GFP-C1, wt or kd GFP-ICK, or GFP-MOK stained with anti-acetylated tubulin. Scale bar 1 µm. Numbers in the red bars indicate number of cilia measured. Data were obtained in at least 2 independent experiments. Statistically significant differences (p<0.001) compared to control cells are indicated with a black asterisk. Error bars indicate SD.</p

Depletion of ICK results in increased anterograde IFT velocity.

<p>(A) Average anterograde velocities of IFT components in control cells, and cells depleted of ICK or MOK using two independent shRNA constructs for each. Statistically significant differences (p<0.001) compared to the velocity of the same IFT component in control cells are indicated with a black asterisk. (B) Average retrograde velocities of IFT components in control cells, and cells depleted of ICK and MOK. (C) Representative kymograph of IFT43-YFP in cells depleted of ICK. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding, drawing anterograde trajectories are shown in red and retrograde trajectories are shown in blue. Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p

ICK and MOK localize to the primary cilium.

<p>IMCD-3 cells expressing (A) GFP-ICK and CFP-centrin-2, or (B) GFP-MOK and CFP-centrin-2, serum-starved for 48 hours and immunostained for acetylated tubulin (acTub). Insets show enlargements of the region containing the cilium. Scale bars 10 µm.</p

Live imaging of fluorescently tagged components of the IFT machinery.

<p>(A) Fluorescence images of the cilia of IMCD-3 cells expressing mCit-KIF3B, IFT43-YFP, GFP-BBS8, IFT20-GFP, and KIF17-mCit. The basal body is indicated with an arrowhead. Scale bar 1 µm. (B) Representative kymograph of IFT43-YFP in cilia of control cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing, anterograde trajectories are shown in red and retrograde trajectories are shown in blue. (C) Average anterograde and retrograde velocities of IFT components in control cells. Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p

Overexpression of ICK results in decreased retrograde IFT velocity.

<p>(A) Representative example of kymograph of GFP-ICK in IMCD-3 cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing anterograde trajectories are shown in red, and retrograde trajectories are shown in blue. (B) Representative example of kymograph of GFP-MOK in IMCD-3 cells. The basal body (b.b.) and the distal tip (d.t.) of the cilium are indicated. In the corresponding drawing anterograde trajectories are shown in red, and retrograde trajectories are shown in blue. (C) Average anterograde velocities of wt or kd GFP-ICK or GFP-MOK. (D) Average retrograde velocities of wt or kd GFP-ICK or GFP-MOK. The velocity of GFP-ICK is statistically significantly different (p<0.001) from those of GFP-ICKkd and wt or kd GFP-MOK (indicated with a black asterisk). Error bars indicate SD. Numbers in the bars indicate number of particles analyzed.</p