Human SFI1 and centrin form a complex critical for centriole architecture and ciliogenesis. (2022)
Figure 5

Figure 5. SFI1 controls CP110 removal and ciliogenesis.

(A) Representative confocal images of serum-starved RPE-1 cells stained for SFI1 (green) and acetylated tubulin (AcTub, magenta). Scale bar: 5µm.

(B, C) Representative confocal images of serum-starved expanded RPE-1 stained for SFI1 (B, green) or Centrins (C, Cetn2/3, grey) and α/β-tubulin (αβTub, magenta). Insets show single channel depicting the distal localization of SFI1 (B, arrowhead) and Centrins (C, arrowhead). Scale bars: 500 nm and 200 nm (inset).

(D) Representative confocal images of serum-starved RPE-1 cells transfected with control or SFI1 siRNA stained for γ-tubulin (γTub, green) and acetylated tubulin (AcTub, magenta) and DNA (DAPI, blue). Scale bar: 5µm.

(E) Percentage of ciliated cells in the indicated conditions.

(F) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and SFI1 (green). Yellow asterisks indicate the absence of SFI1 at the distal tip of the centrioles. Red arrowhead indicates abnormal centriole. Scale bar: 250 nm.

(G) Percentage of RPE-1 cells with centrioles SFI1-positive (intact SFI1), partially depleted (partial SFI1) or totally missing SFI1 (no SFI1) at the distal dot in siSFI-treated cells.

(H) Percentage of abnormal centrioles in RPE-1 cells treated with siSFI1.

(I) Representative widefield images of expanded centrioles during ciliogenesis from siCT and siSFI1 RPE-1 treated cells. Cells were stained for α/β-tubulin (magenta) and CP110 (yellow). Scale bar: 250 nm.

(J) Percentage of ciliated cells observed in U-ExM under the indicated conditions.

(K) Percentage of CP110 capped/uncapped centrioles under the indicated conditions.

(L) Representative widefield images of expanded RPE-1 during ciliogenesis, expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and CP110 (yellow). Arrowheads indicate a cilium. Scale bar: 250 nm.

(M) Percentage of ciliated cells observed in U-ExM in the indicated conditions.

(N) Percentage of CP110 capped/uncapped centrioles in the indicated conditions.

Data information: Average +/- SD, N, statistical analysis: (E) siCT= 75% +/- 3, siSFI1= 26% +/- 6. N= 3 independent experiments (100 cells per experiment), Unpaired t-test (***p=0.0002). (G) siCT= Intact SFI1:97.8% +/- 2.3, Partial SFI1: 2.2% +/- 2.3, No SFI1:0 % +/- 0 , siSFI1=Intact SFI1:9.1% +/- 2.3, Partial SFI1: 40% +/- 4.8, No SFI1:51.6% +/- 7.9. N= 3 independent experiments (>50 centrioles per experiment). Two-way ANOVA (***p<0.0001). (H) siCT: 6.6% +/- 3.2, siSFI1: 39.1% +/- 2.7. N=3 independent experiments (>50 centrioles/experiment). Welch's test (***p=0.002). (J) siCT: 77.9% +/- 0.9, siSFI1: 26.3% +/- 4.6. N=3 independent experiments (>50 centrioles/experiment). Unpaired t test (***p<0.0001). (K) Uncapped= siCT: 90.4% +/- 3.1, siSFI1: 52.6%+/- 3.4; Capped= siCT: 9.6% +/- 3.1, siSFI1: 47.4% +/- 3.4. N=3 independent experiments (>50 centrioles/experiment). Two-way ANOVA (***p<0.0001). (M) siCT-GFP: 66.7% +/- 5, siCT-RR: 71.8% +/- 7.9, siSFI-GFP: 19.4% +/- 1.7, siSFI-RR: 57% +/- 4.6. N=3 independent experiments (>50 centrioles/experiments). One-way ANOVA followed by Tukey's multiple comparison (siCT-GFP vs siSFI-GFP ***p<0.0001, siSFI-GFP vs siSFI1-RR ***p=0.0001). (N) Uncapped= siCT-GFP: 90.9% +/- 4.7, siCT-RR: 94.6% +/- 4, siSFI-GFP: 49.1% +/- 10.5, siSFI-RR: 89.8% +/- 2.1. Capped= siCT-GFP: 9.1% +/- 4.7, siCT-RR: 5.4.6% +/- 4, siSFI-GFP: 50.9% +/- 10.5, siSFI-RR: 10.2% +/- 2.1. N=3 independent experiments (>50 centrioles/experiments). Two-way ANOVA (***p<0.0001).