Supplementary MaterialsFigure 3source data 1: Percentage of cells with the following number of dots/cell respectively for WDR90 and Centrin. 2: Percentage of cells displaying 0, 1, 2 or 4 dots of POC5 based on the number of HsSas-6 dots in U2OS cells treated with control or siRNA. elife-57205-fig3-figsupp2-data2.docx (46K) GUID:?0EB00488-B6D4-4E0A-9476-606543F8C4BF Figure 4source data 1: Diameter at proximal, core and distal region of the centriole. elife-57205-fig4-data1.docx (37K) GUID:?356B847C-B71E-4BB0-975F-9B8BFCC21811 Figure 4source data 2: Inner scaffold proteins coverage. elife-57205-fig4-data2.docx (39K) GUID:?E32A6E9F-9D7E-4386-8898-E5D1B496E40C Figure 6source data 1: Percentage of cells with the following number POC5 dots/cell in siControl and siPOC5 conditions. elife-57205-fig6-data1.docx (13K) GUID:?5066C28D-58C3-4FDA-AEE9-AE80417B6285 Figure 6source data 2: Percentage of cells with the following number WDR90 dots/cell in siControl and siPOC5 conditions. elife-57205-fig6-data2.docx (13K) GUID:?878BB380-8BC5-4314-AEF2-C858F843E00B Figure 6figure supplement 1source data 1: Length of centriole in metaphase and at the end of mitosis in siControl and siPOC5 conditions. elife-57205-fig6-figsupp1-data1.docx (13K) GUID:?A138D11B-DE09-4633-BBDF-6732EEC1B31A Figure 6figure supplement 1source data 2: Percentage of cells with the following number POC5 dots/cell in siControl and siWDR90/POC5 conditions. elife-57205-fig6-figsupp1-data2.docx (13K) GUID:?681F3CD3-0F65-4B21-A6AA-7A3823B9F1FD Figure 6figure supplement 1source data 3: Percentage of cells with the following number WDR90 dots/cell in siControl and siWDR90/POC5 conditions. elife-57205-fig6-figsupp1-data3.docx (13K) GUID:?4AD8652A-73F3-4D5F-A8E5-5842A1E80475 Transparent reporting form. elife-57205-transrepform.docx (246K) GUID:?537FF4C5-623A-466E-9EED-54F56DA2899B Data Availability StatementAll data generated or analysed during this study are included in the manuscript and supporting files. Abstract Centrioles are characterized by a nine-fold arrangement of microtubule triplets held together by an inner protein scaffold. These structurally robust organelles experience strenuous cellular processes such as cell division or ciliary beating while performing their function. However, the molecular mechanisms underlying the stability of microtubule triplets, as well as centriole architectural integrity remain poorly understood. Here, using ultrastructure expansion microscopy for nanoscale protein mapping, we reveal that POC16 and its human homolog WDR90 are components of the microtubule wall along the central core region of the centriole. We further found that WDR90 is an evolutionary microtubule associated protein. Finally, we demonstrate that WDR90 depletion impairs the localization of inner scaffold components, leading to centriole structural abnormalities in human cells. Altogether, this work highlights that WDR90 is an evolutionary conserved molecular player participating in centriole architecture integrity. and human centrioles, suggesting that Onjisaponin B it represents an evolutionary conserved structural feature. Open in a separate window Figure 1. POC16/WDR90 is a conserved central core microtubule wall component.(A) 3D representation of a centriole highlighting the centriolar microtubule wall in light grey and the inner scaffold in yellow. (B) Cryo-EM image of the central core of centrioles from which a microtubule triplet map has been generated (Le Guennec et al., 2020). Schematic representation of the inner junction (IJ) between A- and B-microtubules connecting the inner scaffold. (C) Schematic localization of POC16/WDR90 proteins within the IJ based on its similarity to FAP20. Purple: A-microtubule, green: B microtubule, yellow/gold: inner scaffold Onjisaponin B and stem, orange: DUF667 domain positioned at the IJ. (D) Isolated U-ExM expanded centriole stained for POC16 (yellow) and tubulin (magenta), lateral view. Scale bar: 100 nm. (E) Respective lengths of tubulin and POC16 based on D. Average +/-?SD: Tubulin: 495 nm +/-?33, POC16: 204 nm +/-?53, n?=?46 centrioles from three independent experiments. (F) POC16 length coverage and positioning: 41% +/-?11, n?=?46 centrioles from three independent experiments. (G) Expanded isolated centriole stained for POB15 (green) and tubulin (magenta), lateral view. Scale bar: 100 nm. (H) Respective length of tubulin and POB15 based on G. Average +/-?SD: tubulin?=?497 nm Onjisaponin B +/-?33, POB15?=?200 nm +/-?30, n?=?39 centrioles from three independent DUSP8 experiments. (I) POB15 length coverage and positioning: 40% +/-?6, n?=?39 centrioles from three independent experiments. (J) Expanded human U2OS centriole stained for WDR90 (yellow) and tubulin (magenta), lateral views. (K) Respective lengths of tubulin and Onjisaponin B WDR90 based on J. Average +/-?SD: Tubulin: 432 nm +/-?62, WDR90: 200 nm +/-?80, n?=?35 from three independent experiments. (L) WDR90 length coverage and positioning: 46% +/-?17, n?=?35 from three independent experiments. (M) Isolated U-ExM expanded centriole stained Onjisaponin B for tubulin (magenta) and POC16 (yellow) or POB15 (green), top views. Scale bar: 100 nm. (N) Distance between.