CKAP2 is found in the motile and primary cilia of the human male reproductive tract.

(A) Confocal imaging microscopy of human efferent ductules stained for CKAP2, acetylated-α-tubulin (axoneme) and γ-tubulin (basal bodies). Insets indicate CKAP2 localization in multiciliated cells (MMCs), which are characterized by the apical alignment of numerous γ-tubulin-positive basal bodies, and in reabsorptive primary ciliated cells (RPCC). Occasional CKAP2 localization at the ciliary tip can be observed in MMCs. CKAP2 is also present in sperm flagella of the duct lumen. (B) Confocal imaging microscopy of human epididymis stained for CKAP2, acetylated-α-tubulin (axoneme) and γ-tubulin (basal bodies). Insets indicate CKAP2 localization in the primary cilia of principal and basal cells. Occasional CKAP2 localization at the transition zone (TZ) of primary cilia can be observed in principal cells.

CKAP2 is found in the primary cilia of cycling RPE-1 cells.

(A) Wild-type cells were serum starved for 48 h (-FBS), fixed and co-stained for endogenous CKAP2 and acetylated-α-tubulin. Insets indicate different CKAP2 localization patterns, including axoneme (orange arrow) and basal body (white arrow). (B) Quantification of the percentage of cells in which CKAP2 localizes to primary cilia as determined by the axonemal marker acetylated α-tubulin (n = 500 cells). Each dot represents an independent experiment (N = 3). (C) Levels of acetylated α-tubulin at primary cilia in CKAP2-positive (n = 138 cells) and CKAP2-negative cells (n = 151 cells). Results are representative of at least two independent experiments. (D) Ciliary length comparison between CKAP2-expressing (n = 118 cells) and non-expressing cells (n = 128 cells). Results are representative of at least two independent experiments. (E) Serum-starved cells were co-stained for CKAP2 and Ki67, then classified according to the different CKAP2 localization patterns observed in panel A and presented as a percentage of the total cell population (n = 271 cells). (F) Pearson correlation between Ki67 nuclear expression and CKAP2 expression at primary cilia (n = 193 cells). (G) Schematic representation of CKAP2’s dynamic localization throughout the cell cycle. P < 0.05 (statistically significant). Nuclei were stained with DAPI. Measurements are reported as average ± SD. Panel created with BioRender.

Dynamics of CKAP2 localization to primary cilia in cycling cells.

(A) Excerpt of time-lapse imaging showing the dynamic localization of CKAP2 within primary cilia following their assembly. GFP-CKAP2 knock-in RPE-1 cells were serum starved for 32 h and imaged every 10 min for 20 h (see Movie S1). The ciliary probe CenSpark-650 was added to media to visualize the basal bodies and axoneme. (B) Excerpt of time-lapse imaging showing the dynamic localization of CKAP2 within primary cilia during their disassembly. GFP-CKAP2 knock-in RPE-1 cells were serum starved for 32 h and imaged every 10 min for 20 h (see Movie S2). Orange and white arrows indicate the ciliary tip and base, respectively. Blue arrows indicate the centrosomes. (C) Schematic representation of CKAP2 constructs expressed in RPE-1 cells (lower) based on AlphaFold3 model of CKAP2 and two tubulin dimers (upper). (D) Cells were transiently transfected with CKAP2 (full length) or CKAP2 fragments for 24 h, serum-starved for 48 h, fixed and stained for acetylated-α-tubulin. Nuclei were stained with DAPI. Data are representative of at least two independent experiments.

Knockout of CKAP2, CKAP2-Like or both promotes changes in ciliary length, ciliogenesis and signaling.

(A) RPE-1 cells were serum-starved for 48 h, fixed and co-stained for endogenous CEP170 and acetylated-α-tubulin. (B) Quantification of the percentage of cells with cilia (WT n = 1509 cells; CKAP2 KO1 n = 1256 cells; CKAP2 KO2 n = 1454 cells; CKAP2-Like KO1 n = 1455 cells; CKAP2-Like KO2 n = 1310 cells; CKAP2/Like dKO1 n = 693 cells; CKAP2/Like dKO2 n = 751 cells). Data are representative of at least 3 independent experiments. (C) Quantification of ciliary length (WT n = 407 cells; CKAP2 KO1 n = 411 cells; CKAP2 KO2 n = 404 cells; CKAP2-Like KO1 n = 434 cells; CKAP2-Like KO2 n = 481 cells; CKAP2/Like dKO1 n = 451 cells; CKAP2/Like dKO2 n = 659 cells). Data are representative of at least 2 independent experiments. n.s. (not significant), P < 0.05 (statistically significant). (D) RPE-1 cells were serum-starved for 48 h and stimulated with DMSO (0.1%) or SAG (250 nM) for 1 h prior to fixing and co-staining for endogenous Gli2 and acetylated-α-tubulin. (E) Quantification of the percentage of cells with Gli2 at the ciliary tip in control (WT n = 458; CKAP2 KO1 n = 296; CKAP2 KO2 n = 406; CKAP2-Like KO1 n = 286; CKAP2-Like KO2 n = 431; CKAP2/Like dKO1 n = 284; CKAP2/Like dKO2 n = 249) versus SAG-treated cells (WT n = 392; CKAP2 KO1 n = 357; CKAP2 KO2 n = 375; CKAP2-Like KO1 n = 295; CKAP2-Like KO2 n = 319; CKAP2/Like dKO1 n = 258; CKAP2/Like dKO2 n = 226). Data are representative of at least 3 independent experiments. n.s. (not significant), P < 0.05 (statistically significant). Measurements are reported as average ± SD.

CKAP2-Like is found in the motile and primary cilia of the human male reproductive tract.

(A) Confocal imaging microscopy of human efferent ductules stained for CKAP2-Like, acetylated-α-tubulin (axoneme) and γ-tubulin (basal bodies). Insets indicate CKAP2-Like localization in multiciliated cells (MMCs) and in reabsorptive primary ciliated cells (RPCC). Multiciliated cells are characterized by the apical alignment of numerous γ-tubulin-positive basal bodies. CKAP2-Like is also present in sperm flagella of the duct lumen. (B) Confocal imaging microscopy of human epididymis stained for CKAP2-Like, acetylated-α-tubulin (axoneme) and γ-tubulin (basal bodies). Insets indicate CKAP2-Like localization in the primary cilia of principal and basal cells. Occasional CKAP2-Like localization at the transition zone (TZ) and ciliary tip can be observed in principal cells.

CKAP2-Like localization to the primary cilia is linked to cell cycle progression.

(A) Wild-type RPE-1 cells were serum starved for 48 h (-FBS), fixed and co-stained for endogenous CKAP2-Like and acetylated-α-tubulin. Insets indicate different CKAP2-Like localization patterns. (B) GFP-CKAP2 knock-in RPE-1 cells were serum starved for 48 h and CenSpark-650 was added to media prior to live-imaging. (C) Normalized fluorescence intensity measured along the axoneme (from the ciliary base to tip) for n = 22 cells. (D) A Cas9 nuclease and sgRNA against the CKAP2 and CKAP2-Like were transfected into RPE-1 cells by nucleofection. Single cell cloning was then performed into 96-well plates and knockout efficiency was confirmed in at least two clones by DNA sequencing immunofluorescence, and western blot. (E) A Cas9 nuclease and sgRNA against the CKAP2-Like were transfected into CKAP2 KO1 cells by nucleofection. Single cell cloning was then performed into 96-well plates and knockout efficiency was confirmed in at least two clones by DNA sequencing and western blot. Panels D and E created with BioRender.

Validation of CKAP2, CKAP2-Like and CKAP2/CKAP2-Like double knockout clones.

(A) Alignment results for the edited region corresponding to exon 5 in CKAP2-Like sequence of wild-type RPE-1 cells. (B) Alignment results for the edited region corresponding to exon 5 in CKAP2-Like sequence of CKAP2 KO1 clone. (C) Western blot analysis for CKAP2 and CKAP2-Like expression in WT, CKAP2 KO1, CKAP2 KO2, CKAP2-Like KO1, CKAP2-Like KO2, CKAP2/L dKO1 and CKAP2/L dKO2 cells. TUBB3 was used as a loading control.

Knockdown of CKAP2 by RNAi leads to unaltered ciliogenesis and increased ciliary length.

(A) Validation of CKAP2 expression levels at primary cilia in CKAP2 KO1 and CKAP2 KO2 clones by immunofluorescence. (B) Validation of CKAP2-Like expression levels at primary cilia in CKAP2-Like KO1 and CKAP2-Like KO2 clones by immunofluorescence. (C) Immunofluorescence analysis for CKAP2 and acetylated-α-tubulin in siNC and siCKAP2-treated cells. (D) Quantification of the percentage of cells with cilia (siNC n = 702 cells; siCKAP2 n = 768 cells). Data are representative of 2 independent experiments. (E) Quantification of ciliary length (siNC n = 379 cells; siCKAP2 n = 383 cells). Data are representative of 2 independent experiments. n.s. (not-significant), P < 0.05 (statistically significant). Nuclei were stained with DAPI

Knockout of either CKAP2 or CKAP2-Like leads to increased recruitment of its counterpart to the ciliary compartment.

(A) WT and CKAP2 KO RPE-1 cells were serum starved for 48 h, fixed and co-stained for endogenous CKAP2-Like and acetylated-α-tubulin. (B) Quantification of CKAP2-Like fluorescence intensity at primary cilia of WT (n = 286 cells) and CKAP2 KO cells (KO1 n = 226 cells; KO2 n = 219 cells). Data are representative of at least 2 independent experiments. (C) WT and CKAP2-Like KO cells were serum starved for 48 h, fixed and co-stained for endogenous CKAP2 and acetylated-α-tubulin. (D) Quantification of CKAP2 fluorescence intensity at primary cilia of WT (n = 310 cells) and CKAP2-Like KO cells (KO1 n = 297 cells; KO2 n = 278 cells). Data are representative of at least 2 independent experiments. n.s. (not significant), P < 0.05 (statistically significant). Measurements are reported as average ± SD.