CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels

  1. André B Goncalves
  2. Sarah K Hasselbalch
  3. Beinta B Joensen
  4. Sebastian Patzke
  5. Pernille Martens
  6. Signe K Ohlsen
  7. Mathieu Quinodoz
  8. Konstantinos Nikopoulos
  9. Reem Suleiman
  10. Magnus P Damso Jeppesen
  11. Catja Weiss
  12. Søren Tvorup Christensen
  13. Carlo Rivolta
  14. Jens S Andersen
  15. Pietro Farinelli  Is a corresponding author
  16. Lotte B Pedersen  Is a corresponding author
  1. University of Copenhagen, Denmark
  2. Oslo University Hospital, Norway
  3. University of Southern Denmark, Denmark
  4. University of Basel, Switzerland
  5. University of Lausanne, Switzerland

Abstract

CEP78 is a centrosomal protein implicated in ciliogenesis and ciliary length control, and mutations in the CEP78 gene cause retinal cone-rod dystrophy associated with hearing loss. However, the mechanism by which CEP78 affects cilia formation is unknown. Based on a recently discovered disease-causing CEP78 p.L150S mutation, we identified the disease-relevant interactome of CEP78. We confirmed that CEP78 interacts with the EDD1-DYRK2-DDB1VPRBP E3 ubiquitin ligase complex, which is involved in CP110 ubiquitination and degradation, and identified a novel interaction between CEP78 and CEP350 that is weakened by the CEP78L150S mutation. We show that CEP350 promotes centrosomal recruitment and stability of CEP78, which in turn leads to centrosomal recruitment of EDD1. Consistently, cells lacking CEP78 display significantly increased cellular and centrosomal levels of CP110, and depletion of CP110 in CEP78-deficient cells restored ciliation frequency to normal. We propose that CEP78 functions downstream of CEP350 to promote ciliogenesis by negatively regulating CP110 levels via an EDD1-dependent mechanism.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 3A and Figure 7-figure supplement 2.

Article and author information

Author details

  1. André B Goncalves

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  2. Sarah K Hasselbalch

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  3. Beinta B Joensen

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  4. Sebastian Patzke

    Oslo University Hospital, Oslo, Norway
    Competing interests
    The authors declare that no competing interests exist.
  5. Pernille Martens

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  6. Signe K Ohlsen

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  7. Mathieu Quinodoz

    Institute of Molecular and Clinical Ophthalmology Basel (IOB); Department of Ophthalmology, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  8. Konstantinos Nikopoulos

    Department of Computational Biology, University of Lausanne, Lausanne, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  9. Reem Suleiman

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  10. Magnus P Damso Jeppesen

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  11. Catja Weiss

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  12. Søren Tvorup Christensen

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5004-304X
  13. Carlo Rivolta

    Institute of Molecular and Clinical Ophthalmology Basel (IOB); Department of Ophthalmology, University of Basel, Basel, Switzerland
    Competing interests
    The authors declare that no competing interests exist.
  14. Jens S Andersen

    Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark
    Competing interests
    The authors declare that no competing interests exist.
  15. Pietro Farinelli

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    Pietro.Farinelli@twelve.bio
    Competing interests
    The authors declare that no competing interests exist.
  16. Lotte B Pedersen

    Department of Biology, University of Copenhagen, Copenhagen, Denmark
    For correspondence
    lbpedersen@bio.ku.dk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9749-3758

Funding

Independent Research Fund Denmark (8020‐00162B)

  • Pietro Farinelli
  • Lotte B Pedersen

Carlsberg Foundation (CF18‐0294)

  • Lotte B Pedersen

Independent Research Fund Denmark (8021-00425A)

  • Jens S Andersen

Swiss National Science Foundation (176097)

  • Carlo Rivolta

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Goncalves et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,028
    views
  • 311
    downloads
  • 33
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. André B Goncalves
  2. Sarah K Hasselbalch
  3. Beinta B Joensen
  4. Sebastian Patzke
  5. Pernille Martens
  6. Signe K Ohlsen
  7. Mathieu Quinodoz
  8. Konstantinos Nikopoulos
  9. Reem Suleiman
  10. Magnus P Damso Jeppesen
  11. Catja Weiss
  12. Søren Tvorup Christensen
  13. Carlo Rivolta
  14. Jens S Andersen
  15. Pietro Farinelli
  16. Lotte B Pedersen
(2021)
CEP78 functions downstream of CEP350 to control biogenesis of primary cilia by negatively regulating CP110 levels
eLife 10:e63731.
https://doi.org/10.7554/eLife.63731

Share this article

https://doi.org/10.7554/eLife.63731

Further reading

    1. Cell Biology
    2. Evolutionary Biology
    Paul Richard J Yulo, Nicolas Desprat ... Heather L Hendrickson
    Research Article

    Maintenance of rod-shape in bacterial cells depends on the actin-like protein MreB. Deletion of mreB from Pseudomonas fluorescens SBW25 results in viable spherical cells of variable volume and reduced fitness. Using a combination of time-resolved microscopy and biochemical assay of peptidoglycan synthesis, we show that reduced fitness is a consequence of perturbed cell size homeostasis that arises primarily from differential growth of daughter cells. A 1000-generation selection experiment resulted in rapid restoration of fitness with derived cells retaining spherical shape. Mutations in the peptidoglycan synthesis protein Pbp1A were identified as the main route for evolutionary rescue with genetic reconstructions demonstrating causality. Compensatory pbp1A mutations that targeted transpeptidase activity enhanced homogeneity of cell wall synthesis on lateral surfaces and restored cell size homeostasis. Mechanistic explanations require enhanced understanding of why deletion of mreB causes heterogeneity in cell wall synthesis. We conclude by presenting two testable hypotheses, one of which posits that heterogeneity stems from non-functional cell wall synthesis machinery, while the second posits that the machinery is functional, albeit stalled. Overall, our data provide support for the second hypothesis and draw attention to the importance of balance between transpeptidase and glycosyltransferase functions of peptidoglycan building enzymes for cell shape determination.

    1. Cell Biology
    Kaima Tsukada, Rikiya Imamura ... Mikio Shimada
    Research Article

    Polynucleotide kinase phosphatase (PNKP) has enzymatic activities as 3′-phosphatase and 5′-kinase of DNA ends to promote DNA ligation and repair. Here, we show that cyclin-dependent kinases (CDKs) regulate the phosphorylation of threonine 118 (T118) in PNKP. This phosphorylation allows recruitment to the gapped DNA structure found in single-strand DNA (ssDNA) nicks and/or gaps between Okazaki fragments (OFs) during DNA replication. T118A (alanine)-substituted PNKP-expressing cells exhibited an accumulation of ssDNA gaps in S phase and accelerated replication fork progression. Furthermore, PNKP is involved in poly (ADP-ribose) polymerase 1 (PARP1)-dependent replication gap filling as part of a backup pathway in the absence of OFs ligation. Altogether, our data suggest that CDK-mediated PNKP phosphorylation at T118 is important for its recruitment to ssDNA gaps to proceed with OFs ligation and its backup repairs via the gap-filling pathway to maintain genome stability.