Human SFI1 and centrin form a complex critical for centriole architecture and ciliogenesis. (2022)
Figure 3

Figure 3. SFI1 depletion prevents distal Centrin recruitment at centrioles.

(A-F) Representative confocal images of expanded U2OS centrioles treated with siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) stained for α/β-tubulin (αβTub, magenta) and SFI1 (A-C, green) or Centrin2/3 (D-F, grey). Insets show top views of expanded centrioles at different positions along the centriole (P= proximal, C= central and D= distal). Note that in the absence of SFI1 and Centrin staining at the distal tip, orientation of the centriole was decided based on the larger diameter of the proximal region compared to the distal one, as previously observed in cryo-tomography (Greenan et al, 2020). White arrowheads points to the distal dot of SFI1 and Centrin that disappear in SFI1-depleted (yellow arrowheads) but not in POC5-depleted centrioles. Scale bars: 200 nm and 100 nm (inset). Longitudinal and radial localization of SFI1 and Centrin2/3 in siCT (A, D), siSFI1 (B, E) and siPOC5 (C, F) are presented below the corresponding image. N=25, 40 and 60 centrioles from 3 independent experiments. N=29, 34, 34 centrioles from 3 independent experiments.

(G, H) Percentage of cells with centrioles SFI1-positive (intact SFI1), partially depleted (partial SFI1) or totally missing SFI1 (no SFI1) at the distal dot in siSFI1 (G) and siPOC5 (H) compared to control cells.

(I, J) Percentage of centriole with a distal Centrin 2/3 signal in siSFI1 (I) and siPOC5 (J) compared to control cells.

(K-N) Representative images of expanded U2OS cells expressing GFP alone or GFP+SFI1-RR and treated with siCT or siSFI1. Cells were stained for α/β-tubulin (αβTub, magenta) and SFI1 (K, green) or Centrin2/3 (M, grey). Yellow asterisk indicates the centriole lacking SFI1 and Centrin distal dots. Scale bar: 250 nm. Expression of GFP+SFI1-RR in siSFI1 treated cell rescues the presence of SFI1 and Centrin at the distal tip of the centriole.

(O, P) Representative confocal images of expanded Cetn2 WT, Cetn KO or Cetn2 rescue RPE-1. Cells were stained for α/β-tubulin (αβTub, magenta) and Centrin2 (O, grey) or SFI1 (P, green). Arrowheads indicate the presence (white) or the absence (yellow) of Centrin2 and SFI1. Scale bar: 200nm. Quantification shows the total absence of Centrin2 in the KO cells correlating with the absence of SFI1. Both Centrin2 and SFI1 localization are rescued when Centrin2 is re-expressed in KO cells.

Data information: Average +/- SD, N, statistical analysis: (G) siCT= Intact SFI1:99.3% +/- 0.9, Partial SFI1: 0.7% +/- 1, No SFI1:0% +/- 0 , siSFI1=Intact SFI1:13.1% +/- 14.5, Partial SFI1: 35.1% +/- 7.9, No SFI1:51.8% +/- 21.5. N= 3 independent experiments (>80 centrioles per experiment). Two-way ANOVA (***p<0.0001). (H) siCT= Intact SFI1:97% +/- 2.8, Partial SFI1: 3% +/- 2.8, No SFI1:0% +/- 0 , siSFI1=Intact SFI1:100% +/- 0, Partial SFI1: 0% +/- 0, No SFI1:0% +/- 0. N= 2 independent experiments (>80 centrioles per experiment). Two-way ANOVA (p=0.104) (I) siCT= Intact SFI1:99.2% +/- 1, Partial SFI1: 0.8% +/- 1, No SFI1:0% +/- 0 , siSFI1=Intact SFI1:15.6% +/- 12.4, Partial SFI1: 39% +/- 10.5, No SFI1:45.4% +/- 5.7. N= 3 independent experiments (>80 centrioles per experiment). Two-way ANOVA (***p<0.0001) (J) siCT= Intact SFI1:99% +/- 1.7, Partial SFI1: 1% +/- 1.7, No SFI1:0% +/- 0 , siSFI1=Intact SFI1:99.3% +/- 1.15, Partial SFI1: 0.7% +/- 1.15, No SFI1:0% +/- 0. N= 2 independent experiments (>80 centrioles per experiment). Two-way ANOVA (p=0.892). (L) siCT-GFP= Intact SFI1: 100% +/- 0, Partial SFI1: 0% +/- 0, No SFI1: 0% +/- 0. siCT-RR= Intact SFI1: 94.4% +/- 7.8, Partial SFI1: 5.6% +/- 7.8, No SFI1: 0% +/- 0. siSFI1-GFP= Intact SFI1: 24% +/- 14.4, Partial SFI1: 41.6% +/- 11.8, No SFI1: 34.6% +/- 26.5. siSFI1-RR= Intact SFI1: 70.9% +/- 7.4, Partial SFI1: 26.5% +/- 6.7, No SFI1: 2.6% +/- 0.6. N=2 independent experiments (>50 centrioles/experiments). Two way ANOVA followed by Tukey's multiple comparison (siCT-GFP vs siSFI1-GFP, ***p<0.0001; siSFI1-GFP vs siSFI1-RR, **p=0.0032). (N) siCT-GFP= Intact Cetn: 95.8% +/- 5.9, Partial Cetn: 4.2% +/- 5.9, No Cetn: 0% +/- 0. siCT-RR= Intact Cetn: 95.8% +/- 5.9, Partial Cetn: 4.2% +/- 5.9, No Cetn: 0% +/- 0. siSFI1-GFP= Intact Cetn: 21.7% +/- 7.1, Partial Cetn: 36.7% +/- 9.4, No Cetn: 41.7% +/- 16.5. siSFI1-RR= Intact Cetn: 77% +/- 1.1, Partial Cetn: 20.6% +/- 2.2, No Cetn: 2.4% +/- 3.4. N=2 independent experiments (>50 centrioles/experiments). Two way ANOVA followed by Tukey's multiple comparison (siCT-GFP vs siSFI1-GFP, ***p<0.0001; siSFI1-GFP vs siSFI1-RR, ***p<0.0001).(O) Cetn2 WT= Intact Cetn2: 89.2% +/- 5.7 , No Cetn2: 10.8% +/- 5.7; Cetn2 KO= Intact Cetn2: 0% +/- 0 , No Cetn2: 100% +/- 0; Cetn2 KO + Cetn2 = Intact Cetn2: 90.9% +/- 8.6 , No Cetn2: 9.1% +/- 8.6. Two way ANOVA followed by Tukey's multiple comparison (Cetn2 WT vs Cetn2 KO, p<0.0001; Cetn2 KO vs Cetn2 KO + Cetn2, p<0.0001). (P) Cetn2 WT= Intact SFI1: 100% +/- 0 , No SFI1: 0% +/- 0; Cetn2 KO= Intact SFI1: 3.8% +/- 4.3 , No SFI1: 96.2% +/- 4.3; Cetn2 KO + Cetn2 = Intact SFI1: 100% +/- 0, No SFI1: 0% +/- 0. N=3 independent experiment (>20 centrioles/experiment). Two way ANOVA followed by Tukey's multiple comparison (Cetn2 WT vs Cetn2 KO, p<0.0001; Cetn2 KO vs Cetn2 KO + Cetn2, p<0.0001).