Human SFI1 and centrin form a complex critical for centriole architecture and ciliogenesis. (2022)
Figure 4

Figure 4. SFI1 is important for centriole architecture but not for duplication.

(A) Representative confocal images of expanded duplicating centrioles from siCT- and siSFI1- treated U2OS cells. Cells were stained for SFI1 (green) and α/β-tubulin (αβTub, magenta). Inset shows a distal position of the mother centriole where SFI1 signal is visible. White arrowhead indicates the position of SFI1 distal dot in the procentriole of control cell, which is lost in SFI1-depleted cells (yellow arrowhead). Scale bars: 200 nm.

(B) Quantification of the percentage of SFI1-negative procentrioles.

(C) Quantification of the percentage of duplicating centrioles.

(D) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for HsSAS-6 (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of HsSAS-6-positive centrioles in siSFI1-depleted cells compared to control cells.

(E) Representative confocal image of expanded duplicating centrioles from siCT and siSFI1 U2OS treated cells. Cells were stained for STIL (yellow) and α/β-tubulin (αβTub, magenta). Quantification shows no difference in the percentage of STIL-positive centrioles in siSFI1 compared to control cells.

(F) Top views of expanded U2OS centrioles treated with siCT or siSFI1 stained for α/β-tubulin (αβTub, magenta). Scale bars: 200 nm.

(G) Roundness index of centrioles from siCT and siSFI1-treated cells.

(H) Length (circle) and diameter (square) of expanded centrioles in siCT or siSFI1-treated cells.

(I) Representative confocal images of expanded U2OS centrioles from siCT and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta). White stars point to broken microtubule wall. Scale bars: 200 nm.

(J) Percentage of abnormal centrioles in the indicated conditions.

(K) Top views of expanded procentrioles from U2OS cells treated with siCT or siSFI1 and stained for α/β-tubulin (αβTub, magenta). Scale bars: 200 nm.

(L) Roundness index of procentrioles from siCT and siSFI1-treated cells.

(M) Representative confocal images of expanded U2OS centrioles from siCT- and siSFI1-treated cells stained for α/β-tubulin (αβTub, magenta) and Centrin (Cetn2/3, grey). White dashed lines delimitate the proximal, central and distal regions. White star points to the broken microtubule wall. Scale bar: 200 nm.

(N) Centrin coverage (% of the total tubulin length) along the centriole in the indicated conditions.

(O) Representative images of expanded U2OS expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (green) or Centrin2/3 (grey). Red arrowhead indicates abnormal centriole. Scale bar: 250 nm.

(P) Percentage of abnormal centrioles in the indicated conditions.

Data information: Average +/- SD, N, statistical analysis: (B) siCT= 0% +/- 0, siSFI1=71% +/- 11. N = 4 independent experiment (50 centrioles per experiment), Mann-Whitney test (*p=0.028). (C) siCT= 43% +/- 6, siSFI1= 40% +/- 4. N= 7 independent experiment (50 centrioles per experiment), Unpaired t-test (p= 0.3492). (D) siCT= 100% +/- 0, siSFI1= 100% +/- 0. N = 3 independent experiments, Mann-Whitney test (p>0.999). (E) siCT= 100% +/- 0, siSFI1= 100% +/- 0. N = 3 independent experiments, Mann-Whitney test (p>0.999). (G) siCT= 0.88+/-0.05, siSFI1= 0.79+/-0.12. N= 37 and 50 for siCT and siSFI1 respectively from 4 independent experiments, Unpaired t-test (***p= 0.0002). (H) siCT= 417 +/- 45 nm (length) and 188 +/- 10 nm (diameter), siSFI1= 423 +/- 71 nm (length) and 193.5 +/- 13 nm (diameter). N= 50-90 for length and 30-40 for diameter from 4 independent experiments, Mann-Whitney test (p=0.8440 (length), p=0.079 (diameter)). (J) siCT= 0% +/- 0, siSFI1= 35.7% +/- 11. N= 4 and 6 independent experiments for siCT and siSFI1 respectively, Mann-Whitney test (**p=0.0095). (L) siCT= 0.89+/-0.05, siSFI1= 0.87+/-0.06. N= 34 and 33 for siCT and siSFI1 respectively from 4 independent experiments, Unpaired t-test (p= 0.376). (N) siCT: 69% +/-13, siSFI1: 47% +/-13, siSFI1 abnormal: 24% +/- 20. N=25, 34, 29 centrioles for siCT, siSFI1, siSFI1 abnormal respectively from 2 independent experiments. One-way ANOVA followed by Tukey's post-hoc test (siCT vs siSFI ***p<0.0001, siCT vs siSFI1 abnormal ***p<0.0001). (P) siCT-GFP: 1.4% +/- 1.2, siCT-RR: 1% +/- 1.7, siSFI1-GFP: 23.1% +/- 5.8 , siSFI1-RR: 7.2% +/- 2. N=3 independent experiments (>50 centrioles/experiments). One-way ANOVA followed by Tukey's multiple comparison (siCT-GFP vs siSFI1-GFP, ***p=0.0003; siSFI1-GFP vs siSFI1-RR, **p=0.0026).