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

Figure 1. Loss of AKAP220 enhances ciliogenesis.

(A) Schematic of AKAP220 interaction with selected binding partners. (B) Disruption of this signaling complex upon removal of AKAP220. Protein kinase A (blue), Glycogen synthase kinase-3 (pink), Protein phosphatase 1 (orange) and IQGAP (yellow) are indicated. (C–H) Immunofluorescent staining of kidney collecting ducts with Arl13b (red), Aquaporin-2 (green) and DAPI (blue) from (C) wild-type and (D) AKAP220KO mice. (E and F) Enlarged sections from wild-type and AKAP220KO mice. (G and H) Gray scale images of Arl13b. (I) Quantification (% ciliated collecting ducts) in wild-type (gray bar) and AKAP220KO (green bar). ****p<0.0001. (J) Quantification of cilia length (µm) in wild-type (gray bar) and AKAP220KO (green bar). ****p<0.0001. Crispr-Cas9 gene editing of AKAP220 in mIMCD3 cells. (K) Immunoblot detection of AKAP220 (top) and GAPDH loading control (bottom) from wild-type (lane 1) and AKAP220KO (lane 2) cell lysates. (L–N) Immunofluorescent detection of primary cilia with acetyl tubulin (green), Arl13b (red) and DAPI (blue) in wild-type, and two independent clones of AKAP220KO mIMCD3 cells. (O) Quantification (% ciliated cells) from wild-type (gray column), AKAP220KO#1 (green column) and AKAP220KO#2 (dark green column). ****p<0.0001, N=3. (P) Schematic depicting reformation of the signaling complex upon rescue with AKAP220. Immunofluorescent detection of Arl13b (pink), GFP (green) and DAPI (blue) in (Q) pEGFP-AKAP220 or (S) GFP-control transfected AKAP220KO mIMCD3 cells. Gray scale image of Arl13b in (R) control cells and (T) AKAP220-rescued cells. (U) Quantification (% ciliated cells) in pEGFP-AKAP220 (black bar) or GFP-control cells (teal bar). ****p<0.0001, N=3. (V) Schematic of wild-type and AKAP220KO mIMCD3 spheroids. Immunofluorescent staining with acetyl tubulin (green) and DAPI (blue) in (W) wild-type and (X) AKAP220KO spheroids. (Y) Quantification (% ciliated cells) in wild-type (gray column) and AKAP220KO (green column) spheroids. ****p<0.0001, N=3. All error bars are s.e.m. p Values were calculated by unpaired two-tailed Student’s t-test. Scale bars (10 µm). Number of cells analyzed indicated below each column.

Figure 1—source data 1. Percent ciliated collecting ducts in kidney sections.
Figure 1—source data 2. Length of primary cilia in kidney sections.
Figure 1—source data 3. Rescue of GFP-AKAP220 in AKAP220KO mIMCD3 cells.
Figure 1—source data 4. Percent ciliated cells measured in spheroids.

Figure 1.

Figure 1—figure supplement 1. Quantification of cilia number in gene-edited mIMCD3 cells.

Figure 1—figure supplement 1.

Immunofluorescent staining of Arl13b (red), acetyl tubulin (green), and DAPI (blue) in serum starved (0.5% FBS, 24 hr) (A) wild-type and (D) AKAP220KO mIMCD3 cells. Gray scale images of Arl13b (B and E) and acetyl tubulin (C and F) show multiple cilia in the AKAP220KO cell. (G) Quantification (% ciliated cells) in wild-type (purple column), AKAP220KO (green column). ****p<0.0001. Number of cells analyzed are indicated below each bar.
Figure 1—figure supplement 2. Deletion of AKAP150 has no effect on primary cilia development.

Figure 1—figure supplement 2.

Crispr-Cas nine gene editing was used to delete the murine anchoring protein AKAP150 in mIMCD3 cells. Double knockout cells were also produced lacking AKAP220 and AKAP150. Immunofluorescent staining with ciliary markers Arl13b (green) and acetyl tubulin (red) in (A) wild-type, (B) AKAP150KO and (C) AKAP220-150KO mIMCD3 cells. DAPI serves as a nuclear marker. (D) Quantification (% ciliated cells) in wild-type (mauve column), AKAP150KO (orange column), AKAP220-150KO (pink column), and AKAP220KO (coral column). Number of cells analyzed are indicated below each bar. ****p<0.0001, ns=non-significant, N=3. All error bars are s.e.m. p Values were calculated by unpaired two-tailed Student’s t-test. Scale bars (10 µm).
Figure 1—figure supplement 3. Sequencing data for CRISPR-Cas9 gene edited mIMCD3 cells.

Figure 1—figure supplement 3.

Sequencing analysis data shows (A) intact AKAP150 in wild-type AKAP150, and deletions of AKAP150 in (B) AKAP150KO cells and (C) AKAP220-150 double knockout cells made in AKAP220KO background.