WDR90 is a centriolar microtubule wall protein important for centriole architecture integrity

  1. Emmanuelle Steib
  2. Marine H Laporte
  3. Davide Gambarotto
  4. Natacha Olieric
  5. Celine Zheng
  6. Susanne Borgers
  7. Vincent Olieric
  8. Maeva Le Guennec
  9. France Koll
  10. Anne-Marie Tassin
  11. Michel O Steinmetz
  12. Paul Guichard  Is a corresponding author
  13. Virginie Hamel  Is a corresponding author
  1. University of Geneva, Department of Cell Biology, Sciences III, Switzerland
  2. Laboratory of Biomolecular Research, Division of Biology and Chemistry, Paul Scherrer Institut, Switzerland
  3. Swiss Light Source, Paul Scherrer Institut, Switzerland
  4. Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, France
  5. Biozentrum, University of Basel, Switzerland
6 figures, 2 videos, 1 table and 1 additional file

Figures

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 Paramecium tetraurelia

Figure 2 with 3 supplements
WDR90/POC16-DUF667 directly binds both microtubules and tubulin.

(see also Figure 2—figure supplements 13). (A) Schematic of WDR90/POC16 conservation domains with the Chlamydomonas cilia proteins FAP20 and FAP52/WDR16. DUF667 domain is in orange and WD40 repeats …

Figure 2—figure supplement 1
POC16 conservation across species.

(A) POC16 orthologs DUF667 domain amino acids sequence alignment from seven different species: Chlamydomonas reinhardtii POC16(1-200); homo sapiens WDR90(1-225), Danio rerio drPOC16(1-243), Bovine …

Figure 2—figure supplement 2
Model prediction of POC16 Nter.

(A) POC16 3D model and (B) FAP20 reference structure model (Khalifa et al., 2020). (C) Fitting of POC16 against FAP20 yielding a RMSD value of 1.6 Angs. (D) Fitting of the POC16 model excluding the …

Figure 2—figure supplement 3
POC16 and WDR90 bind microtubules.

(A) Human U2OS cells transiently overexpressing GFP-POC16(1-295) stained for POC16 (green) and tubulin (magenta). Scale bars for panels A-E: 5 µm. (B) Human U2OS cells transiently overexpressing at …

Figure 3 with 2 supplements
WDR90 is recruited in G2 and is important for Centrin and POC5 recruitment to centrioles (See also Figure 3—figure supplements 1 and 2).

(A) Human RPE1 p53- cells synchronized by mitotic shake-off, fixed at different time points for different cell-cycle stages (related to Figure 3—figure supplement 1A, B) and stained with WDR90 …

Figure 3—source data 1

Percentage of cells with the following number of dots/cell respectively for WDR90 and Centrin.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig3-data1-v1.docx
Figure 3—source data 2

Percentage of cells with the following number of dots/cell respectively for WDR90 and HsSAS-6.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig3-data2-v1.docx
Figure 3—figure supplement 1
WDR90 is a satellite and centriolar protein.

(A) FACS profiles of RPE1 p53- cells at different time point post mitotic shake-off, plotted based on propidium iodide (PI) and 5-ethynyl-2´-deoxyuridine (EdU) content. Related to Figure 3(A–E). (B) …

Figure 3—figure supplement 1—source data 1

Percentage of cells in each phase of the cell cycle according to post-mitotic time point.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig3-figsupp1-data1-v1.docx
Figure 3—figure supplement 2
Depletion of WDR90 impairs Centrin and POC5 localization at centrioles.

(A) Human U2OS cell treated with either control or wdr90 siRNA and stained for WDR90 (yellow) and Centrin (magenta). DNA is in blue. Dotted white squares indicate insets. Scale bar: 5 µm. (B) …

Figure 3—figure supplement 2—source data 1

Percentage of cells displaying 0, 1, 2 or 4 dots of WDR90 based on the number of Centrin dots in U2OS cells treated with control or wdr90 siRNA.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig3-figsupp2-data1-v1.docx
Figure 3—figure supplement 2—source data 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 wdr90 siRNA.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig3-figsupp2-data2-v1.docx
Figure 4 with 1 supplement
WDR90 is crucial for inner scaffold components localization (see also Figure 4—figure supplement 1).

(A, B) Expanded centrioles from S-phase U2OS cells treated with either control (A) or wdr90 siRNA (B) stained for tubulin (magenta) and WDR90 (yellow). M stands for mature centriole and P for …

Figure 4—source data 1

Diameter at proximal, core and distal region of the centriole.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig4-data1-v1.docx
Figure 4—source data 2

Inner scaffold proteins coverage.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig4-data2-v1.docx
Figure 4—figure supplement 1
WDR90 depletion affects mainly inner scaffold components.

(A, B) Expanded centrioles from U2OS treated with either control (A) or wdr90 siRNA (B) stained for tubulin (magenta) and POC5+CEP164 (both in green. CEP164 is indicated by a green arrowhead). MC …

Figure 5 with 1 supplement
WDR90 is important for centriole architecture integrity (see also Figure 5—figure supplement 1, Videos 1 and 2).

(A, B) Expanded centrioles from S-phase U2OS cells treated with control (A) or wdr90 siRNA (B), stained for tubulin (magenta) and POC1B (green). White arrowhead: broken microtubule wall of the …

Figure 5—figure supplement 1
WDR90 depletion leads to severe centriolar structure defects.

(A) Quantification of the orientation of mother centrioles in control cells following the appendages marker CEP164. N = 215 centrioles from three independent experiments. (B, C) Percentage of normal …

Figure 6 with 2 supplements
POC5 and WDR90 are important for proper centriole architecture.

(See also Figure 6—figure supplements 1 and 2) (A) Human U2OS cell treated with either control or poc5 siRNA and stained for POC5 (green) and HsSAS-6 (magenta). DNA is in blue. Dotted white squares …

Figure 6—source data 1

Percentage of cells with the following number POC5 dots/cell in siControl and siPOC5 conditions.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig6-data1-v1.docx
Figure 6—source data 2

Percentage of cells with the following number WDR90 dots/cell in siControl and siPOC5 conditions.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig6-data2-v1.docx
Figure 6—figure supplement 1
Characterization of POC5 and POC5/WDR90 depletion.

(A) Quantification of the number of POC5-positive mother and daughter centrioles in siControl and siPOC5. n = 50 centrioles counted from two independent experiments. Average +/- SD: siControl …

Figure 6—figure supplement 1—source data 1

Length of centriole in metaphase and at the end of mitosis in siControl and siPOC5 conditions.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig6-figsupp1-data1-v1.docx
Figure 6—figure supplement 1—source data 2

Percentage of cells with the following number POC5 dots/cell in siControl and siWDR90/POC5 conditions.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig6-figsupp1-data2-v1.docx
Figure 6—figure supplement 1—source data 3

Percentage of cells with the following number WDR90 dots/cell in siControl and siWDR90/POC5 conditions.

https://cdn.elifesciences.org/articles/57205/elife-57205-fig6-figsupp1-data3-v1.docx
Figure 6—figure supplement 2
Loss of the inner scaffold components WDR90 and POC5 leads to centriole breakage.

(A) Expanded centrioles from U2OS treated with control siRNA stained for tubulin (magenta) and POC5+CEP164 (both in green). Scale bar: 250 nm. (B) Expanded centrioles from U2OS treated with poc5

Videos

Video 1
U-ExM expanded control centrioles.

Top viewed expanded centriole from U2OS cell treated with control siRNA and stained for tubulin (magenta) and POC5 (green). Z-stack acquired every 0.12 µm from the proximal to distal end of the …

Video 2
U-ExM expanded centrioles depleted of WDR90.

Top viewed expanded centriole from U2OS cell treated with wdr90 siRNA and stained for tubulin (magenta) and POC5 (green). Z-stack acquired every 0.12 µm from the proximal to distal end of the …

Tables

Key resources table
Reagent type
(species) or resource
DesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Chlamydomonas reinhardtii)WTChlamydomonas Resource CentercMJ030Wild-type
Strain, strain background (Paramecium tetraurelia)7SBeisson et al., 2010doi:10.1101/pdb.prot5364
Cell line (Homo sapiens)U2OSHabedanck et al., 2005PMID:16244668
Cell line (Homo sapiens)RPE-1 p53-Wang et al., 2015PMID:26609813
Cell line (Homo sapiens)U2OS:GFP-WDR90RRThis paperp. 19 of the manuscript (Material and methods)Episomal, puromycine selected, doxycycline-inducible
Transfected construct (Homo sapiens)GFP-WDR90RRThis paperpEBTet-GFP-WDR90RR(FL)
p. 20 of the manuscript (Material and methods)
WDR90RR DNA template from Hamel et al., 2017
Transfected construct (Homo sapiens)GFP-WDR90(1-225)RRThis paperpEBTet-GFP-WDR90RR(1-225)
p. 20 of the manuscript (Material and methods)
WDR90RR DNA template from Hamel et al., 2017
Transfected construct (Homo sapiens)GFP-WDR90This paperGenebank sequence NP_660337, pEGFP-WDR90RT-PCR from human RPE-1 cells, cloned into modified pEGFP-C1 vector using AscI andPacI restriction sites
Transfected construct (Chlamydomonas reinhardtii)POC16 (1-295)This paperpXLG-POC16(1-295), described p.20 of the manuscript in the Material andmethods section.POC16 sequence synthetized by GeneArt using the E. coli codon usage (described in Hamel et al., 2017) cloned into pXLG vector using NotI and BamHI restriction sites
Biological sample (Chlamydomonas reinhardtii)Isolated basal bodiesKlena et al., 2018PMID:30295659
Biological sample (Sus scrofa)TubulinCytoskeletonCat. #: T240Isolated from brain, used for electron microscopy
Biological sample (Bovine taurus)TubulinCentro de Investigastiones Biologicas, Madrid, SpainIsolated from brain, used for pelleting assay
AntibodyTubulin AA345 (mouse monoclonal)Le Guennec et al., 2020PMID:32110738U-ExM
Isolated Basal Bodies (1:500)
U-ExM in cells (1:250)
AntibodyAlpha-Tubulin AA344 (mouse monoclonal)Le Guennec et al., 2020PMID:32110738U-ExM in cells (1:250)
AntibodyPOC16 (rabbit polyclonal)Hamel et al., 2017PMID:28781053U-ExM (1:100)
AntibodyPOB15 (rabbit polyclonal)Hamel et al., 2017PMID:28781053U-ExM (1:100)
AntibodyWDR90 (rabbit polyclonal)NovusBioCat. #: NBP2-31888U-ExM (1:100)
IF (1:250)
AntibodyPOC1B (rabbit polyclonal)ThermoFisherCat. #: PA5-24495U-ExM (1:250)
AntibodyPOC5 (rabbit polyclonal)BethylCat. #: A303-341AU-ExM (1:250)
IF (1:500)
AntibodyFAM161A (rabbit polyclonal)Le Guennec et al., 2020PMID:32110738U-ExM (1:250)
AntibodyCentrin (mouse monoclonal, 20H5)Merck MilliporeCat. #: 04–1624U-ExM (1:250)
IF (1:500)
AntibodyDM1A Tubulin (mouse monoclonal)AbcamCat. #: ab7291IF (1:1000)
AntibodyHsSAS-6 (mouse monoclonal)Santa Cruz BiotechnologyCat. #: sc-81431IF (1:100)
AntibodyPCM1
(rabbit polyclonal)
Santa Cruz BiotechnologyCat. #: sc-67204IF (1:500)
AntibodyCP110
(rabbit polyclonal)
ProteintechCat. #: 12780–1IF (1:500)
AntibodyGFP (mouse monoclonal)AbcamCat. #: ab1218IF (1:500)
AntibodymCherry (rabbit polyclonal)AbcamCat. #: ab167453IF (1:500)
AntibodyptPOC16
(rabbit polyclonal)
This studydescribed p.27 of the manuscript in the Supplemental Methods section.IF (1:50)
AntibodyTubulin 1D5
(mouse mono clonal)
Beisson et al., 2010IF (1:10)
AntibodyAlexa 488 anti-rabbit IgG (goat)ThermoFisherCat. #: A11008U-ExM (1:400)
IF
(1:1000)
AntibodyAlexa 568 anti-mouse IgG (goat)ThermoFisherCat. #: A11004U-ExM (1:400)
IF
(1:1000)
Recombinant DNA reagentpEBTet-EGFP-GWGift from the Gönczy labNa.
Recombinant DNA reagentpENTR-Age-AGTGift from the Gönczy labNa.
Recombinant DNA reagentpEGFP-C1Clontech
Sequence-based reagentsiRNA ControlThermoFisherAM4642Silencer select
Sequence-based reagentsiRNA targetingwdr90 geneThermoFisherS47097Silencer select
Sequence-based reagentsiRNA targetingpcm1 geneThermoFisherADCSU9LSilencer select
Peptide, recombinant proteinPOC16(1-295)This paperUniprot A8JAN3Purified from bacteria
Peptide, recombinant proteinWDR90(1-225)This paperUniprot Q96KV7Purified from bacteria
Peptide, recombinant proteindrPOC16(1-243)This paperUniprot F1RA29Purified from bacteria
Peptide, recombinant proteinbtPOC16(1-224)This paperUniref UPI000572B175Purified from bacteria
Peptide, recombinant proteinptPOC16(2-210)This paperUniprot A0DK60Purified from bacteria
Peptide, recombinant proteinxtPOC16(1-245)This paperUniref UPI0008473371Purified from bacteria
Peptide, recombinant proteinrnPOC16(54-282)This paperUniref UPI0008473371Purified from bacteria
Commercial assay or kitLipofectamine 3000 Transfection kitLifeTechnologyCat. #: L3000015
Commercial assay or kitLipofectamine RNAi max kitLifeTechnologyCat. #: 13778150
Commercial assay or kitClick-EdU-Alexa647 FACS kitCarl RothCat. #: 7783.1
Commercial assay or kitDAPCO Mounting mediumAbcamCat. #: ab188804
Commercial assay or kitAffi-Gel 10Bio-RadCat. #:153–6099
Chemical compound, drugFormaldehyde 36.5–38%SigmaCat. #: F8775
Chemical compound, drugAcrylamide 40%SigmaCat. #: A4058
Chemical compound, drugN,N’-methylbisacrylamide 2%SigmaCat. #: M1533
Chemical compound, drugSodium acrylate 97–99%SigmaCat. #: 408220
Chemical compound, drugAmmonium persulfateThermoFisherCat. #: 17874
Chemical compound, drugTetramethylethyldiamineThermoFisherCat. #: 17919
Chemical compound, drugPoly-D-Lysine 1 mg/mLGibcoCat. #: A3890401
Chemical compound, drugTaxol/PaclitaxelSigma-AldrichCat. #: T7191
Chemical compound, drugCoomassie stainingBiotiumCat. #: 21003
Chemical compound, drugPropidium IodideSigmaCat. #: 81845
Chemical compound, drugRnaseRocheCat. #: 11119915001
Software, algorithmImageJ/FiJiSchindelin et al., 2012doi:10.1038/nmeth.2019
Software, algorithmCentrioleJ plugginGuichard et al., 2013DOI:10.1016/j.cub.2013.06.061
Software, algorithmUnwarpJ plugginSorzano et al., 2005DOI:10.1109/TBME.2005.844030
Software, algorithmGraphPadPrism7GraphPad Software7.0
Software, algorithmPhyre2Kelley and Sternberg, 2009DOI:10.1038/nprot.2015.053
Software, algorithmUCSF ChimeraPettersen et al., 2004DOI:10.1002/jcc.20084
OtherZeiss LSM700 microscopeZeiss
OtherLeica TCS SP8 microscopeLeicaExpansion microscopy
OtherLeica Thunder inverted microscopeLeica
OtherTecnai G2 Sphera microscopeThermofisherNegative stain and cryo-EM

Additional files

Download links