Figure 8.
Characterization of the effects of disrupting CPAP–CP110 interaction on centriole length regulation at interphase. (A) Scheme showing the generation of the inducible transgenic cell lines expressing either GFP-tagged WT full-length CPAP (CPAP-FLWT) or full-length CPAP with L149A/K150A mutation (CPAP-FLMUT). U2OS cells (Control) were used to integrate with the Tet repressor, a single FRT site, and the lacZ-Zeocin fusion gene by lentivirus to generate the Flp-In T-REx U2OS host cell line (Host). pcDNA5/FRT/TO vectors for doxycycline-inducible expression of GFP-CPAP-FLWT or GFP-CPAP-FLMUT were co-transfected together with Flp recombinase-encoding pOG44 vector into the Flp-In T-REx U2OS host cell line to induce their integration into the FRT site of the host cell genome in a Flp recombinase-dependent manner. The expression of GFP-CPAP-FLWT or GFP-CPAP-FLMUT was controlled by the inducible hybrid human cytomegalovirus (CMV)/Tet operator 2 (TetO2) promoter. The endogenous CPAP gene was knocked out using a CRISPR/Cas9–based approach. (B) Mean ± SD of the normalized CPAP levels based on western blots shown in Fig. S5 C (n = 3 trials). Cell lines used for quantification are shown in magenta, where cell line pairs 1 and 2 (p1 and p2, respectively) are highlighted. Nonsignificant (ns), P > 0.05 calculated using an unpaired two-tailed Mann–Whitney U test. (C and E) Immunofluorescence images acquired using Airyscan 2 confocal microscope of centrioles at G1/S (C) and G2/M (E) and stained for acetylated tubulin (blue), CP110 (green), and GFP-CPAP (magenta). (D) Median ± IQR of mother centriole length at G1/S measured from proximal end of centriole (determined by acetylated tubulin) to distal end (determined by the geometric center of CP110 signal) (scheme in panel F). n, number of analyzed centrioles: control cell line, n = 113; host, n = 105; CPAP-FLWT#3, n = 132; CPAP-FLWT#4, n = 131; CPAP-FLMUT#1,n = 84; CPAP-FLMUT#5,n = 170; CPAP-FLMUT#4,n = 81; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test. (F) Median ± IQR of centriole length at G2/M measured as in D. n, number of analyzed mother centrioles (MC) and daughter centrioles (DC): control cells, n = 80 MC, 75 DC; host, n = 72 MC, 59 DC; CPAP-FLWT#3,n = 67 MC, 69 DC; CPAP-FLWT#4, n = 64 MC, 57 DC; CPAP-FLMUT#1, n = 71 MC, 80 DC; CPAP-FLMUT#5, n = 78 MC, 79 DC; CPAP-FLMUT#4, n = 79 MC, 77 DC; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test.

Characterization of the effects of disrupting CPAP–CP110 interaction on centriole length regulation at interphase. (A) Scheme showing the generation of the inducible transgenic cell lines expressing either GFP-tagged WT full-length CPAP (CPAP-FLWT) or full-length CPAP with L149A/K150A mutation (CPAP-FLMUT). U2OS cells (Control) were used to integrate with the Tet repressor, a single FRT site, and the lacZ-Zeocin fusion gene by lentivirus to generate the Flp-In T-REx U2OS host cell line (Host). pcDNA5/FRT/TO vectors for doxycycline-inducible expression of GFP-CPAP-FLWT or GFP-CPAP-FLMUT were co-transfected together with Flp recombinase-encoding pOG44 vector into the Flp-In T-REx U2OS host cell line to induce their integration into the FRT site of the host cell genome in a Flp recombinase-dependent manner. The expression of GFP-CPAP-FLWT or GFP-CPAP-FLMUT was controlled by the inducible hybrid human cytomegalovirus (CMV)/Tet operator 2 (TetO2) promoter. The endogenous CPAP gene was knocked out using a CRISPR/Cas9–based approach. (B) Mean ± SD of the normalized CPAP levels based on western blots shown in Fig. S5 C (n = 3 trials). Cell lines used for quantification are shown in magenta, where cell line pairs 1 and 2 (p1 and p2, respectively) are highlighted. Nonsignificant (ns), P > 0.05 calculated using an unpaired two-tailed Mann–Whitney U test. (C and E) Immunofluorescence images acquired using Airyscan 2 confocal microscope of centrioles at G1/S (C) and G2/M (E) and stained for acetylated tubulin (blue), CP110 (green), and GFP-CPAP (magenta). (D) Median ± IQR of mother centriole length at G1/S measured from proximal end of centriole (determined by acetylated tubulin) to distal end (determined by the geometric center of CP110 signal) (scheme in panel F). n, number of analyzed centrioles: control cell line, n = 113; host, n = 105; CPAP-FLWT#3, n = 132; CPAP-FLWT#4, n = 131; CPAP-FLMUT#1,n = 84; CPAP-FLMUT#5,n = 170; CPAP-FLMUT#4,n = 81; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test. (F) Median ± IQR of centriole length at G2/M measured as in D. n, number of analyzed mother centrioles (MC) and daughter centrioles (DC): control cells, n = 80 MC, 75 DC; host, n = 72 MC, 59 DC; CPAP-FLWT#3,n = 67 MC, 69 DC; CPAP-FLWT#4, n = 64 MC, 57 DC; CPAP-FLMUT#1, n = 71 MC, 80 DC; CPAP-FLMUT#5, n = 78 MC, 79 DC; CPAP-FLMUT#4, n = 79 MC, 77 DC; nonsignificant (ns); and ****P < 0.001 calculated using Kruskal–Wallis ANOVA test.

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