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. 2021 Jul 12;10:e67828. doi: 10.7554/eLife.67828

Figure 6. Loss of phosphatase anchoring promotes cilium elongation.

(A) Schematic of cylindrical and retracted cilia morphologies (purple). Super-resolution fixed-cell imaging of Arl13b (gray scale) in (B) wild-type, (C) AKAP220KO, and (D) AKAP220-ΔPP1 cilia. (E) Quantification (% bulbed cilia) in wild-type (gray), AKAP220KO (green), and AKAP220-ΔPP1 (blue) cells. ****p<0.0001, N=3. Scale bars (1 µm). Super-resolution live-cell images of Arl13b-GFP in (F) wild-type and (G) AKAP220-ΔPP1 cilia. (H) Quantification of cilia length in wild-type (gray), AKAP220KO (green), and AKAP220-ΔPP1 (blue) cells. ****p<0.0001, N=3. Scale bars (5 µm). (I) Kidney-on-a-chip device. Location of kidney tubule (Tubule) and extracellular matrix (ECM) are indicated. Confocal imaging of (J) Wild-type and (L) AKAP220-ΔPP1cells cultured in chip device with Arl13b (red), acetyl tubulin (green), and DAPI (blue). Cilia (white circles) are marked. (Insets) Magnified images of representative cilia (cyan circles) from (K) wild-type and (M) AKAP220-ΔPP1 pseudotubules. (N) Quantification of cilia length in wild-type (gray) and AKAP220-ΔPP1 (blue). ****p<0.0001, N=3. Scale bars (10 µm). Inset scale bars (5 µm). (O–U) Fluorescence recovery after photobleaching (FRAP) of Arl13b-GFP in primary cilia. (O) Schematic of how FRAP was measured. (P–R) Super-resolution live-cell images of Arl13b-GFP in (P) wild-type, (Q) AKAP220KO and (R) AKAP220-ΔPP1 cells. Circles mark bleached portion of cilia. (S) FRAP recovery curves of Arl13b over time (3500 ms) in wild-type (gray), AKAP220KO (green), and AKAP220-ΔPP1 (blue) cells. Scale bars (5 µm). (T) Quantification of mobile fraction in wild-type (gray), AKAP220KO (green), and AKAP220-ΔPP1 (blue) cilia. ****p<0.0001, N=3. (U) Half-life of Arl13b in wild-type (gray), AKAP220KO (green), and AKAP220-ΔPP1 (blue) cilia. ****p<0.0001, N=3. (V) Schematic depicting how AKAP220 modulation of the actin barrier influences movement of proteins in and out of primary cilia. All error bars are s.e.m. p Values were calculated by unpaired two-tailed Student’s t-test. Number of cilia analyzed indicated below each column.

Figure 6—source data 1. Primary cilia length from live cell experiments in mIMCD3 cells.
Figure 6—source data 2. Length of primary cilia in kidney on a chip device.
Figure 6—source data 3. FRAP curved for Arl13b-GFP in mIMCD3 cell lines.
Figure 6—source data 4. Mobile fraction of Arl13b-GFP 3 s after photobleaching.
Figure 6—source data 5. Half-life of Arl13b-GFP in mIMCD3 cell lines.

Figure 6.

Figure 6—figure supplement 1. Super resolution videos depicting flexibility of AKAP220KO cilia.

Figure 6—figure supplement 1.

(A) Wild-type and (B) AKAP220KO primary cilia from mIMCD3 cells transduced with Arl13b-GFP were monitored across a time course of 4 s. The AKAP220 cilium is longer and more flexible compared to the wild-type.