Figure 9.
Characterization of morphological defects of centrioles in cells with perturbed CPAP–CP110 interaction using U-ExM. (A and D–F) U-ExM images of centrioles from host, CPAP-FLWT#3, and CPAP-FLMUT#1 blocked for 24 h in G1/S and stained for acetylated tubulin (blue) combined with CP110 (green) in (A), GFP (magenta) in (D) top image, or CPAP (magenta) in (D) bottom image and (E and F). (A) Normal centrioles from host and CPAP-FLWT#3 and incomplete centriole from CPAP-FLMUT#1 (white arrow). (B) Mean + SD of centriole morphology at G1/S categorized in normal, short, incomplete, and elongated. For host, n = 159, CPAP-FLWT#3, n = 171, and CPAP-FLMUT#1, n = 251 centrioles; nonsignificant (ns); and *P = 0.019 and ****P < 0.0001 calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.77. (C) Mean + SD of CP110 cap on mother and daughter centrioles at G1/S categorized visually as present (clear, bright signal), low levels of (barely visible, low signal), and absent (no signal) CP110. For host, n = 101, CPAP-FLWT#3, n = 114, and CPAP-FLMUT#1, n = 160 centrioles; nonsignificant (ns); and calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.83. (D) Comparison between antibody against GFP (top) or CPAP (bottom) to localize GFP-CPAP (magenta). CPAP is localized to daughter centriole (white arrowhead), inner, proximal (white arrow), and distal cap of mother centriole (black arrowhead). (E) Normal centrioles from host and CPAP-FLWT#3 and incomplete centriole from CPAP-FLMUT#1 (white arrow). (F) Gallery of short or incomplete centrioles from CPAP-FLMUT#1. CPAP population on daughter centriole was unchanged (white arrowhead), and example nine shows minor defect in centriole shaft (white arrow). (G) Mean + SD of CPAP localization on the distal and proximal ends of the mother and daughter centrioles at G1/S, categorized visually as present (clear, bright signal), low levels of (barely visible, low signal), and absent (no signal). For host, n = 140, CPAP-FLWT#3, n = 132, and CPAP-FLMUT#1, n = 179; nonsignificant (ns); and *P = 0.031 or 0.012 and**P = 0.008 calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.13. Scale bar is corrected for ∼4.5 expansion factor.

Characterization of morphological defects of centrioles in cells with perturbed CPAP–CP110 interaction using U-ExM. (A and D–F) U-ExM images of centrioles from host, CPAP-FLWT#3, and CPAP-FLMUT#1 blocked for 24 h in G1/S and stained for acetylated tubulin (blue) combined with CP110 (green) in (A), GFP (magenta) in (D) top image, or CPAP (magenta) in (D) bottom image and (E and F). (A) Normal centrioles from host and CPAP-FLWT#3 and incomplete centriole from CPAP-FLMUT#1 (white arrow). (B) Mean + SD of centriole morphology at G1/S categorized in normal, short, incomplete, and elongated. For host, n = 159, CPAP-FLWT#3, n = 171, and CPAP-FLMUT#1, n = 251 centrioles; nonsignificant (ns); and *P = 0.019 and ****P < 0.0001 calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.77. (C) Mean + SD of CP110 cap on mother and daughter centrioles at G1/S categorized visually as present (clear, bright signal), low levels of (barely visible, low signal), and absent (no signal) CP110. For host, n = 101, CPAP-FLWT#3, n = 114, and CPAP-FLMUT#1, n = 160 centrioles; nonsignificant (ns); and calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.83. (D) Comparison between antibody against GFP (top) or CPAP (bottom) to localize GFP-CPAP (magenta). CPAP is localized to daughter centriole (white arrowhead), inner, proximal (white arrow), and distal cap of mother centriole (black arrowhead). (E) Normal centrioles from host and CPAP-FLWT#3 and incomplete centriole from CPAP-FLMUT#1 (white arrow). (F) Gallery of short or incomplete centrioles from CPAP-FLMUT#1. CPAP population on daughter centriole was unchanged (white arrowhead), and example nine shows minor defect in centriole shaft (white arrow). (G) Mean + SD of CPAP localization on the distal and proximal ends of the mother and daughter centrioles at G1/S, categorized visually as present (clear, bright signal), low levels of (barely visible, low signal), and absent (no signal). For host, n = 140, CPAP-FLWT#3, n = 132, and CPAP-FLMUT#1, n = 179; nonsignificant (ns); and *P = 0.031 or 0.012 and**P = 0.008 calculated using ordinary one-way ANOVA test. Normality tested using Shapiro–Wilk test; P = 0.13. Scale bar is corrected for ∼4.5 expansion factor.

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