Dynein-2 intermediate 1 chains play crucial but distinct roles in primary cilia formation and function

  1. Laura Vuolo
  2. Nicola L Stevenson
  3. Kate J Heesom
  4. David John Stephens  Is a corresponding author
  1. University of Bristol, United Kingdom

Abstract

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 contains a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilium function. Using quantitative proteomics, we show that WDR34 KO cells can assemble a dynein-2 motor complex that binds IFT proteins yet fails to extend an axoneme, indicating complex function is stalled. In contrast, WDR60 KO cells do extend axonemes but show reduced assembly of dynein-2 and binding to IFT proteins. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport. Our results indicate that the subunit asymmetry within the dynein-2 complex is matched with a functional asymmetry between the dynein-2 intermediate chains. Furthermore, this work reveals that loss of function of dynein-2 leads to defects in transition zone architecture, as well as intraflagellar transport.

Data availability

Proteomics data are included as an Excel file which is readily readable and accessible for the majority of researchers. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE [1] partner repository with the dataset identifier PXD010398.

The following data sets were generated

Article and author information

Author details

  1. Laura Vuolo

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9801-9206
  2. Nicola L Stevenson

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Kate J Heesom

    Proteomics Facility, Faculty of Biomedical Sciences, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. David John Stephens

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    For correspondence
    david.stephens@bristol.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5297-3240

Funding

Medical Research Council (MR/P000177/1)

  • Nicola L Stevenson
  • David John Stephens

Biotechnology and Biological Sciences Research Council (BB/N000420/1)

  • Laura Vuolo
  • David John Stephens

Medical Research Council (MR/K018019/1)

  • Nicola L Stevenson
  • David John Stephens

Biotechnology and Biological Sciences Research Council (BB/L014181/1)

  • David John Stephens

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

Reviewing Editor

  1. Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom

Publication history

  1. Received: June 28, 2018
  2. Accepted: October 14, 2018
  3. Accepted Manuscript published: October 15, 2018 (version 1)
  4. Accepted Manuscript updated: October 16, 2018 (version 2)
  5. Version of Record published: November 1, 2018 (version 3)

Copyright

© 2018, Vuolo 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

  • 2,612
    Page views
  • 476
    Downloads
  • 21
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Laura Vuolo
  2. Nicola L Stevenson
  3. Kate J Heesom
  4. David John Stephens
(2018)
Dynein-2 intermediate 1 chains play crucial but distinct roles in primary cilia formation and function
eLife 7:e39655.
https://doi.org/10.7554/eLife.39655
  1. Further reading

Further reading

    1. Cell Biology
    2. Physics of Living Systems
    Nicola Bellotto, Jaime Agudo-Canalejo ... Victor Sourjik
    Research Article

    Inside prokaryotic cells, passive translational diffusion typically limits the rates with which cytoplasmic proteins can reach their locations. Diffusion is thus fundamental to most cellular processes, but the understanding of protein mobility in the highly crowded and non-homogeneous environment of a bacterial cell is still limited. Here we investigated the mobility of a large set of proteins in the cytoplasm of Escherichia coli, by employing fluorescence correlation spectroscopy (FCS) combined with simulations and theoretical modeling. We conclude that cytoplasmic protein mobility could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. We observed similar size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli. For the faster-diffusing proteins, this size dependence is well consistent with the Stokes-Einstein relation once taking into account the specific dumbbell shape of protein fusions. Pronounced subdiffusion and hindered mobility are only observed for proteins with extensive interactions within the cytoplasm. Finally, while protein diffusion becomes markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity, all of these perturbations affect proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity.

    1. Cell Biology
    Dawafuti Sherpa, Judith Mueller ... Arno F Alpi
    Research Article

    The development of haematopoietic stem cells into mature erythrocytes – erythropoiesis – is a controlled process characterized by cellular reorganization and drastic reshaping of the proteome landscape. Failure of ordered erythropoiesis is associated with anaemias and haematological malignancies. Although the ubiquitin system is a known crucial post-translational regulator in erythropoiesis, how the erythrocyte is reshaped by the ubiquitin system is poorly understood. By measuring the proteomic landscape of in vitro human erythropoiesis models, we found dynamic differential expression of subunits of the CTLH E3 ubiquitin ligase complex that formed maturation stage-dependent assemblies of topologically homologous RANBP9- and RANBP10-CTLH complexes. Moreover, protein abundance of CTLH’s cognate E2 ubiquitin conjugating enzyme UBE2H increased during terminal differentiation, and UBE2H expression depended on catalytically active CTLH E3 complexes. CRISPR-Cas9-mediated inactivation of CTLH E3 assemblies or UBE2H in erythroid progenitors revealed defects, including spontaneous and accelerated erythroid maturation as well as inefficient enucleation. Thus, we propose that dynamic maturation stage-specific changes of UBE2H-CTLH E2-E3 modules control the orderly progression of human erythropoiesis.