Abstract
Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
| Original language | English |
|---|---|
| Pages (from-to) | 674-689.e11 |
| Journal | Structure |
| Volume | 28 |
| Issue number | 6 |
| DOIs | |
| State | Published - Jun 2 2020 |
Keywords
- CCDC61
- Chlamydomonas
- SAS6
- VFL3
- XRCC4
- basal body
- centriole
- centrosome
- cilia
- microtubule
- structural biology
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In: Structure, Vol. 28, No. 6, 02.06.2020, p. 674-689.e11.
Research output: Contribution to journal › Article › peer-review
TY - JOUR
T1 - CCDC61/VFL3 Is a Paralog of SAS6 and Promotes Ciliary Functions
AU - Ochi, Takashi
AU - Quarantotti, Valentina
AU - Lin, Huawen
AU - Jullien, Jerome
AU - Rosa e Silva, Ivan
AU - Boselli, Francesco
AU - Barnabas, Deepak D.
AU - Johnson, Christopher M.
AU - McLaughlin, Stephen H.
AU - Freund, Stefan M.V.
AU - Blackford, Andrew N.
AU - Kimata, Yuu
AU - Goldstein, Raymond E.
AU - Jackson, Stephen P.
AU - Blundell, Tom L.
AU - Dutcher, Susan K.
AU - Gergely, Fanni
AU - van Breugel, Mark
N1 - Funding Information: T.O. is supported by a University Academic Fellow start-up fund from the University of Leeds . M.V.B. is supported by the Medical Research Council (MRC file reference MC_UP_1201/3 ). I.R.S. is supported by an MRC LMB César Milstein Fellowship. F.G. is supported by Cancer Research UK ( C14303/A17043 ). F.G. acknowledges support from NIHR Cambridge Biomedical Research Centre , the University of Cambridge , and Hutchison Whampoa. S.K.D receives funds from the National Institute of General Medical Sciences ( GM-032843 and GM-131909 ). T.L.B. is supported by Wellcome Trust program grant ( 09316/Z/10/Z ) and Investigator Award (200814/Z/16/Z) for this research. Research in the S.P.J. laboratory is funded by Cancer Research UK (program grant C6/A18796 ) and a Wellcome Trust Investigator Award ( 206388/Z/17/Z ). A.N.B. is supported by a Cancer Research UK Career Development Fellowship ( C29215/A20772 ). J.J. is funded by a grant from the Wellcome Trust ( 101050/Z/13/Z ) and the MRC ( MR/P00479/1 ). Y.K. was supported by Cancer Research UK ( CRUK-A12874 ) and is currently supported by ShanghaiTech University . R.E.G. is supported by Wellcome Trust Investigator Grant ( 207510/Z/17/Z ). We thank Dr. Francesco Meghini for contributing to the initial characterization of CDCC61 in human cells, Dr. Andy Riddell (Wellcome - MRC Cambridge Stem Cell Institute, Cambridge, UK), Maria Daly and Dr. Fan Zhang (MRC LMB, Cambridge, UK) for FACS, Dr. Mark Skehel (MRC LMB) for mass spectrometry, Drs Jonathan Howe (MRC LMB), Mathias Pasche (MRC LMB), and Ruth Hughes (University of Leeds) for confocal microscopes, Dr. Minmin Yu (MRC LMB) for help with X-ray crystallography, Drs Shaoxia Chen, Christos Savva, and Guiseppe Cannone (MRC LMB) for help with electron microscopy, Mrs Jake Grimmett and Toby Darling (MRC LMB) for help with scientific computing, DLS I02 and I03 beamline scientists for help with collecting X-ray diffraction images, Dr. Andrew Carter (MRC LMB) for tubulin, Dr. Manu Hedge (MRC LMB) for the anti-HA antibody, Dr. Keith Boyle (MRC LMB) for a BFP-containing plasmid, Dr. Antonina Andreeva (MRC LMB) for help with the structure-guided sequence alignment, Dr. Jeffrey L. Salisbury (Mayo Clinic College of Medicine, Rochester, USA) for centrin antibodies, Dr. John Kilmartin (MRC LMB) for Rosetta (DE3) cells and Prof. Colin A. Johnson (University of Leeds) for the human RPE-1 cell line. Funding Information: T.O. is supported by a University Academic Fellow start-up fund from the University of Leeds. M.V.B. is supported by the Medical Research Council (MRC file reference MC_UP_1201/3). I.R.S. is supported by an MRC LMB C?sar Milstein Fellowship. F.G. is supported by Cancer Research UK (C14303/A17043). F.G. acknowledges support from NIHR Cambridge Biomedical Research Centre, the University of Cambridge, and Hutchison Whampoa. S.K.D receives funds from the National Institute of General Medical Sciences (GM-032843 and GM-131909). T.L.B. is supported by Wellcome Trust program grant (09316/Z/10/Z) and Investigator Award (200814/Z/16/Z) for this research. Research in the S.P.J. laboratory is funded by Cancer Research UK (program grant C6/A18796) and a Wellcome Trust Investigator Award (206388/Z/17/Z). A.N.B. is supported by a Cancer Research UK Career Development Fellowship (C29215/A20772). J.J. is funded by a grant from the Wellcome Trust (101050/Z/13/Z) and the MRC (MR/P00479/1). Y.K. was supported by Cancer Research UK (CRUK-A12874) and is currently supported by ShanghaiTech University. R.E.G. is supported by Wellcome Trust Investigator Grant (207510/Z/17/Z). We thank Dr. Francesco Meghini for contributing to the initial characterization of CDCC61 in human cells, Dr. Andy Riddell (Wellcome - MRC Cambridge Stem Cell Institute, Cambridge, UK), Maria Daly and Dr. Fan Zhang (MRC LMB, Cambridge, UK) for FACS, Dr. Mark Skehel (MRC LMB) for mass spectrometry, Drs Jonathan Howe (MRC LMB), Mathias Pasche (MRC LMB), and Ruth Hughes (University of Leeds) for confocal microscopes, Dr. Minmin Yu (MRC LMB) for help with X-ray crystallography, Drs Shaoxia Chen, Christos Savva, and Guiseppe Cannone (MRC LMB) for help with electron microscopy, Mrs Jake Grimmett and Toby Darling (MRC LMB) for help with scientific computing, DLS I02 and I03 beamline scientists for help with collecting X-ray diffraction images, Dr. Andrew Carter (MRC LMB) for tubulin, Dr. Manu Hedge (MRC LMB) for the anti-HA antibody, Dr. Keith Boyle (MRC LMB) for a BFP-containing plasmid, Dr. Antonina Andreeva (MRC LMB) for help with the structure-guided sequence alignment, Dr. Jeffrey L. Salisbury (Mayo Clinic College of Medicine, Rochester, USA) for centrin antibodies, Dr. John Kilmartin (MRC LMB) for Rosetta (DE3) cells and Prof. Colin A. Johnson (University of Leeds) for the human RPE-1 cell line. T.O. performed computational analysis with T.L.B. X-ray crystallography with D.D.B. and M.v.B. electron microscopy, biochemistry experiments with M.v.B. gene knockout and transient expression. V.Q. performed characterizations of knockout and knockdown human cells. H.L. performed Chlamydomonas experiments. J.J. performed Xenopus experiments. I.R.S. performed FACS experiments. F.B. performed light microscopy experiments of Xenopus embryos. D.D.B. determined one of the crystal structures together with T.O. C.M.J and S.H.McL. performed biophysics experiments. S.M.V.F. performed NMR experiments. A.N.B. and Y.K. performed light microscopy experiments of human cells. S.P.J. provided resources for the knockout experiments. T.O. and M.v.B. conceptualized the project. T.O. V.Q. H.L. S.K.D. F.G. and M.v.B. conceived experiments and wrote the original draft of the manuscript. Everyone contributed for reviewing and editing the manuscript. Y.K. R.E.G. S.P.J. T.L.B. S.K.D. F.G. and M.v.B. secured funding. The authors declare no competing interests. Publisher Copyright: © 2020 MRC Laboratory of Molecular Biology
PY - 2020/6/2
Y1 - 2020/6/2
N2 - Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
AB - Centrioles are cylindrical assemblies whose peripheral microtubule array displays a 9-fold rotational symmetry that is established by the scaffolding protein SAS6. Centriole symmetry can be broken by centriole-associated structures, such as the striated fibers in Chlamydomonas that are important for ciliary function. The conserved protein CCDC61/VFL3 is involved in this process, but its exact role is unclear. Here, we show that CCDC61 is a paralog of SAS6. Crystal structures of CCDC61 demonstrate that it contains two homodimerization interfaces that are similar to those found in SAS6, but result in the formation of linear filaments rather than rings. Furthermore, we show that CCDC61 binds microtubules and that residues involved in CCDC61 microtubule binding are important for ciliary function in Chlamydomonas. Together, our findings suggest that CCDC61 and SAS6 functionally diverged from a common ancestor while retaining the ability to scaffold the assembly of basal body-associated structures or centrioles, respectively.
KW - CCDC61
KW - Chlamydomonas
KW - SAS6
KW - VFL3
KW - XRCC4
KW - basal body
KW - centriole
KW - centrosome
KW - cilia
KW - microtubule
KW - structural biology
UR - http://www.scopus.com/inward/record.url?scp=85084859973&partnerID=8YFLogxK
U2 - 10.1016/j.str.2020.04.010
DO - 10.1016/j.str.2020.04.010
M3 - Article
C2 - 32375023
AN - SCOPUS:85084859973
SN - 0969-2126
VL - 28
SP - 674-689.e11
JO - Structure
JF - Structure
IS - 6
ER -