1. Cell Biology
  2. Microbiology and Infectious Disease

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
https://doi.org/10.7554/eLife.52058
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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

Version 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.

Metrics

  • 3,669
    Page views
  • 573
    Downloads
  • 25
    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. 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

Categories and tags

Further reading

    1. Cell Biology
    2. Medicine

    Glucagon-like peptide-1 receptor activation stimulates PKA-mediated phosphorylation of Raptor and this contributes to the weight loss effect of liraglutide

    Thao DV Le, Dianxin Liu ... Julio E Ayala
    Research Article Updated

    The canonical target of the glucagon-like peptide-1 receptor (GLP-1R), Protein Kinase A (PKA), has been shown to stimulate mechanistic Target of Rapamycin Complex 1 (mTORC1) by phosphorylating the mTOR-regulating protein Raptor at Ser791 following β-adrenergic stimulation. The objective of these studies is to test whether GLP-1R agonists similarly stimulate mTORC1 via PKA phosphorylation of Raptor at Ser791 and whether this contributes to the weight loss effect of the therapeutic GLP-1R agonist liraglutide. We measured phosphorylation of the mTORC1 signaling target ribosomal protein S6 in Chinese Hamster Ovary cells expressing GLP-1R (CHO-Glp1r) treated with liraglutide in combination with PKA inhibitors. We also assessed liraglutide-mediated phosphorylation of the PKA substrate RRXS*/T* motif in CHO-Glp1r cells expressing Myc-tagged wild-type (WT) Raptor or a PKA-resistant (Ser791Ala) Raptor mutant. Finally, we measured the body weight response to liraglutide in WT mice and mice with a targeted knock-in of PKA-resistant Ser791Ala Raptor. Liraglutide increased phosphorylation of S6 and the PKA motif in WT Raptor in a PKA-dependent manner but failed to stimulate phosphorylation of the PKA motif in Ser791Ala Raptor in CHO-Glp1r cells. Lean Ser791Ala Raptor knock-in mice were resistant to liraglutide-induced weight loss but not setmelanotide-induced (melanocortin-4 receptor-dependent) weight loss. Diet-induced obese Ser791Ala Raptor knock-in mice were not resistant to liraglutide-induced weight loss; however, there was weight-dependent variation such that there was a tendency for obese Ser791Ala Raptor knock-in mice of lower relative body weight to be resistant to liraglutide-induced weight loss compared to weight-matched controls. Together, these findings suggest that PKA-mediated phosphorylation of Raptor at Ser791 contributes to liraglutide-induced weight loss.

    1. Cell Biology
    2. Developmental Biology

    Cylicins are a structural component of the sperm calyx being indispensable for male fertility in mice and human

    Simon Schneider, Andjela Kovacevic ... Hubert Schorle
    Research Article

    Cylicins are testis-specific proteins, which are exclusively expressed during spermiogenesis. In mice and humans, two Cylicins, the gonosomal X-linked Cylicin 1 (Cylc1/CYLC1) and the autosomal Cylicin 2 (Cylc2/CYLC2) genes, have been identified. Cylicins are cytoskeletal proteins with an overall positive charge due to lysine-rich repeats. While Cylicins have been localized in the acrosomal region of round spermatids, they resemble a major component of the calyx within the perinuclear theca at the posterior part of mature sperm nuclei. However, the role of Cylicins during spermiogenesis has not yet been investigated. Here, we applied CRISPR/Cas9-mediated gene editing in zygotes to establish Cylc1- and Cylc2-deficient mouse lines as a model to study the function of these proteins. Cylc1 deficiency resulted in male subfertility, whereas Cylc2-/-, Cylc1-/yCylc2+/-, and Cylc1-/yCylc2-/- males were infertile. Phenotypical characterization revealed that loss of Cylicins prevents proper calyx assembly during spermiogenesis. This results in decreased epididymal sperm counts, impaired shedding of excess cytoplasm, and severe structural malformations, ultimately resulting in impaired sperm motility. Furthermore, exome sequencing identified an infertile man with a hemizygous variant in CYLC1 and a heterozygous variant in CYLC2, displaying morphological abnormalities of the sperm including the absence of the acrosome. Thus, our study highlights the relevance and importance of Cylicins for spermiogenic remodeling and male fertility in human and mouse, and provides the basis for further studies on unraveling the complex molecular interactions between perinuclear theca proteins required during spermiogenesis.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Baited reconstruction with 2D template matching for high-resolution structure determination in vitro and in vivo without template bias

    Bronwyn A Lucas, Benjamin A Himes, Nikolaus Grigorieff
    Research Advance

    Previously we showed that 2D template matching (2DTM) can be used to localize macromolecular complexes in images recorded by cryogenic electron microscopy (cryo-EM) with high precision, even in the presence of noise and cellular background (Lucas et al., 2021; Lucas et al., 2022). Here, we show that once localized, these particles may be averaged together to generate high-resolution 3D reconstructions. However, regions included in the template may suffer from template bias, leading to inflated resolution estimates and making the interpretation of high-resolution features unreliable. We evaluate conditions that minimize template bias while retaining the benefits of high-precision localization, and we show that molecular features not present in the template can be reconstructed at high resolution from targets found by 2DTM, extending prior work at low-resolution. Moreover, we present a quantitative metric for template bias to aid the interpretation of 3D reconstructions calculated with particles localized using high-resolution templates and fine angular sampling.