Figure 6.
CCDC15 is required for recruitment of inner scaffold proteins to the centriole central core and distal end. (A) Representative U-ExM images of centriolar core proteins POC5 (orange), FAM161A (gray), POC1B (blue), and Centrin-2 (green) in control or CCDC15-depleted RPE1 cells. Cells were expanded 96 h after siRNA transfection and immunostained with the indicated antibodies for inner core proteins and tubulin (magenta) for centrioles. Top view of centrioles for Centrin-2 was represented in addition to the longitudinal views represented for all proteins. Scale bar, 200 nm. (B) Quantification of coverages of the centriolar proteins in A. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from two independent experiments per condition. POC5 coverage: siControl = 52% ± 10, siCCDC15 = 58% ± 12, P = 0.0174; FAM161A coverage: siControl = 50% ± 14, siCCDC15 = 53% ± 15, P = 0.2981; POC1B coverage: siControl = 62% ± 12, siCCDC15 = 53% ± 12, P = 0.0006; Centrin-2 coverage: siControl = 60% ± 12, siCCDC15 = 61% ± 9, P = 0.7254, two-sided t test. (C) Quantification of centrioles positive for Centrin-2 at the distal region of centrioles in control or CCDC15-depleted RPE1 cells based on A. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 81.25% ± 3, siCCDC15 = 52.46% ± 0.96, P < 0.0001, two-sided t test. (D) Representative U-ExM images of SFI1 protein in control, CCDC15, and POC5 siRNA-depleted RPE1 cells. Cells were expanded and immunostained with SFI1 (green) and tubulin (magenta) antibodies. Scale bar, 200 nm. (E) Quantification of centrioles positive for SFI1 at the distal region of centrioles in control and CCDC15-depleted RPE1 cells based on D. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 93.25% ± 0.1, siCCDC15 = 56.38% ± 2.2, P < 0.0001; siControl = 96.5% ± 1.5, siPOC5 = 96.4% ± 1.5, P = 0.9625, two-sided t test. (F) Representative U-ExM images of coverages of CCDC15 in control, POC5, POC1B, and FAM161A-depleted RPE1 cells. Cells were expanded and immunostained with CCDC15 (green) and tubulin (magenta) antibodies. Scale bar, 200 nm. (G) Quantification of CCDC15 coverage with respect to tubulin represented in in control, POC5, POC1B, and FAM161A-depleted RPE1 cells represented in F. Error bars, SD. n > 10 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 48.5% ± 11, siPOC1B = 40.6% ± 12.4, P = 0.0062; siPOC5 = 53.1% ± 8.2, P = 0.0538; siFAM61A = 48.8% ± 10.8, non-significant, two-sided t test. (H) Representative U-ExM images of centrioles from RPE1 cells transfected with control siRNA or CCDC15 and POC1B siRNAs together. Cells were stained for CCDC15 and POC1B in green and tubulin in magenta. Scale bar, 500 nm. (I) Representative U-ExM images of defective centrioles in CCDC15 and POC1B co-depleted cells. Cells were costained with CCDC15 and POC1B in green and tubulin in magenta. Scale bar, 500 nm. (J) Centriole length quantification of I. Error bars, SD. n > 15 centrioles per experiment. Data represents mean value from two independent experiment. siControl: 1 ± 0.13, siCCDC15: 0.87 ± 0.11, P < 0.0001, siCCDC15/siPOC1B: 0.84 ± 0.10, P = 0.2588, two-sided t test. Statistical analysis was done by normalizing the values to the mean of siControl. (K) Percentage of cells with defective centrioles for the indicated cells in CCDC15 and POC1B co-depleted cells. n > 15 centrioles per experiment. Error bars, SD. Data represents mean value from two independent experiments. siControl = 3.35% ± 1.1, siCCDC15 = 12% ± 0.28, siCCDC15/siPOC5 = 23.5% ± 1.13. For siControl-siCCDC15 P = 0.0080, for siCCDC15-siCCDC15/POC1B P = 0.0051, for siControl-siCCDC15/siPOC1B P = 0.0029, two-sided t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant.

CCDC15 is required for recruitment of inner scaffold proteins to the centriole central core and distal end. (A) Representative U-ExM images of centriolar core proteins POC5 (orange), FAM161A (gray), POC1B (blue), and Centrin-2 (green) in control or CCDC15-depleted RPE1 cells. Cells were expanded 96 h after siRNA transfection and immunostained with the indicated antibodies for inner core proteins and tubulin (magenta) for centrioles. Top view of centrioles for Centrin-2 was represented in addition to the longitudinal views represented for all proteins. Scale bar, 200 nm. (B) Quantification of coverages of the centriolar proteins in A. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from two independent experiments per condition. POC5 coverage: siControl = 52% ± 10, siCCDC15 = 58% ± 12, P = 0.0174; FAM161A coverage: siControl = 50% ± 14, siCCDC15 = 53% ± 15, P = 0.2981; POC1B coverage: siControl = 62% ± 12, siCCDC15 = 53% ± 12, P = 0.0006; Centrin-2 coverage: siControl = 60% ± 12, siCCDC15 = 61% ± 9, P = 0.7254, two-sided t test. (C) Quantification of centrioles positive for Centrin-2 at the distal region of centrioles in control or CCDC15-depleted RPE1 cells based on A. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 81.25% ± 3, siCCDC15 = 52.46% ± 0.96, P < 0.0001, two-sided t test. (D) Representative U-ExM images of SFI1 protein in control, CCDC15, and POC5 siRNA-depleted RPE1 cells. Cells were expanded and immunostained with SFI1 (green) and tubulin (magenta) antibodies. Scale bar, 200 nm. (E) Quantification of centrioles positive for SFI1 at the distal region of centrioles in control and CCDC15-depleted RPE1 cells based on D. Error bars, SD. n > 20 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 93.25% ± 0.1, siCCDC15 = 56.38% ± 2.2, P < 0.0001; siControl = 96.5% ± 1.5, siPOC5 = 96.4% ± 1.5, P = 0.9625, two-sided t test. (F) Representative U-ExM images of coverages of CCDC15 in control, POC5, POC1B, and FAM161A-depleted RPE1 cells. Cells were expanded and immunostained with CCDC15 (green) and tubulin (magenta) antibodies. Scale bar, 200 nm. (G) Quantification of CCDC15 coverage with respect to tubulin represented in in control, POC5, POC1B, and FAM161A-depleted RPE1 cells represented in F. Error bars, SD. n > 10 centrioles per experiment. Data represent mean value from three independent experiments per condition. siControl = 48.5% ± 11, siPOC1B = 40.6% ± 12.4, P = 0.0062; siPOC5 = 53.1% ± 8.2, P = 0.0538; siFAM61A = 48.8% ± 10.8, non-significant, two-sided t test. (H) Representative U-ExM images of centrioles from RPE1 cells transfected with control siRNA or CCDC15 and POC1B siRNAs together. Cells were stained for CCDC15 and POC1B in green and tubulin in magenta. Scale bar, 500 nm. (I) Representative U-ExM images of defective centrioles in CCDC15 and POC1B co-depleted cells. Cells were costained with CCDC15 and POC1B in green and tubulin in magenta. Scale bar, 500 nm. (J) Centriole length quantification of I. Error bars, SD. n > 15 centrioles per experiment. Data represents mean value from two independent experiment. siControl: 1 ± 0.13, siCCDC15: 0.87 ± 0.11, P < 0.0001, siCCDC15/siPOC1B: 0.84 ± 0.10, P = 0.2588, two-sided t test. Statistical analysis was done by normalizing the values to the mean of siControl. (K) Percentage of cells with defective centrioles for the indicated cells in CCDC15 and POC1B co-depleted cells. n > 15 centrioles per experiment. Error bars, SD. Data represents mean value from two independent experiments. siControl = 3.35% ± 1.1, siCCDC15 = 12% ± 0.28, siCCDC15/siPOC5 = 23.5% ± 1.13. For siControl-siCCDC15 P = 0.0080, for siCCDC15-siCCDC15/POC1B P = 0.0051, for siControl-siCCDC15/siPOC1B P = 0.0029, two-sided t test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns: non-significant.

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