Cryo electron tomography with Volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei

  1. Simon Imhof
  2. Jiyan Zhang
  3. Hui Wang
  4. Khanh Huy Bui
  5. Hoangkim Nguyen
  6. Ivo Atanosov
  7. Wong H Hui
  8. Shun Kai Yang
  9. Z Hong Zhou  Is a corresponding author
  10. Kent L Hill  Is a corresponding author
  1. University of California, Los Angeles, United States
  2. McGill University, Canada

Abstract

The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structure of the 96-nm repeat from Trypanosoma brucei, a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in T. brucei reflects adaptations to support unique motility needs in diverse host environments.

Data availability

All data generated or analyzed during this study are included in the manuscript ans supporting files. Source data files have been provided for Figure 2F and Figure 3-Suppl. 4. The cryoET sub-tomogram average maps have been deposited in the EM Data Bank under the accession codes EMD-20012, EMD-20013 and EMD-20014, for the wild-type bloodstream form, wild-type and DRC11-knock-down procyclic form, respectively.

Article and author information

Author details

  1. Simon Imhof

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  2. Jiyan Zhang

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Hui Wang

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Khanh Huy Bui

    Department of Anatomy and Cell Biology, McGill University, Montreal, Canada
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2814-9889
  5. Hoangkim Nguyen

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  6. Ivo Atanosov

    California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  7. Wong H Hui

    California NanoSystems Institute, University of California, Los Angeles, Los Angeles, United States
    Competing interests
    The authors declare that no competing interests exist.
  8. Shun Kai Yang

    Department of Anatomy and Cell Biology, McGill University, Montreal, Canada
    Competing interests
    The authors declare that no competing interests exist.
  9. Z Hong Zhou

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    Hong.Zhou@UCLA.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8373-4717
  10. Kent L Hill

    Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, United States
    For correspondence
    kenthill@microbio.ucla.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6529-1273

Funding

Swiss National Science Foundation (P300PA_174358)

  • Simon Imhof

National Science Foundation (DBI-1338135)

  • Z Hong Zhou

National Institutes of Health (R01GM071940)

  • Jiyan Zhang
  • Hui Wang
  • Ivo Atanosov
  • Wong H Hui
  • Z Hong Zhou

National Institutes of Health (S10RR23057)

  • Z Hong Zhou

National Science Foundation (DMR-1548924)

  • Z Hong Zhou

National Institutes of Health (GM007185)

  • Khanh Huy Bui

National Institutes of Health (AI052348)

  • Simon Imhof
  • Hoangkim Nguyen
  • Kent L Hill

Swiss National Science Foundation (P2BEP3_162094)

  • Simon Imhof

National Institutes of Health (S10OD018111)

  • Z Hong Zhou

National Institutes of Health (U24GM116792)

  • Z Hong Zhou

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: September 20, 2019
  2. Accepted: November 11, 2019
  3. Accepted Manuscript published: November 11, 2019 (version 1)
  4. Accepted Manuscript updated: November 14, 2019 (version 2)
  5. Version of Record published: January 21, 2020 (version 3)

Copyright

© 2019, Imhof 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.

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  1. Simon Imhof
  2. Jiyan Zhang
  3. Hui Wang
  4. Khanh Huy Bui
  5. Hoangkim Nguyen
  6. Ivo Atanosov
  7. Wong H Hui
  8. Shun Kai Yang
  9. Z Hong Zhou
  10. Kent L Hill
(2019)
Cryo electron tomography with Volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei
eLife 8:e52058.
https://doi.org/10.7554/eLife.52058

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    Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients.

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    To test whether dystrophin mutations lead to defective cardiac NaV1.5–Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays.

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    Funding:

    Supported by National Institutes of Health R01 HL122352 grant; ‘la Caixa’ Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.