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

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.

Metrics

  • 3,415
    Page views
  • 551
    Downloads
  • 19
    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

    Autofluorescence imaging permits label free cell type assignment in unmodified airway and reveals the dynamic formation of airway secretory cell associated antigen passages (SAPs)

    Viral S Shah, Jue Hou ... Jayaraj Rajagopal
    Tools and Resources

    The specific functional properties of a tissue are distributed amongst its component cell types. The various cells act coherently, as an ensemble, in order to execute a physiologic response. Modern approaches for identifying and dissecting novel physiologic mechanisms would benefit from an ability to identify specific cell types in live tissues that could then be imaged in real-time. Current techniques require the use of fluorescent genetic reporters that are not only cumbersome, but which only allow the simultaneous study of 3 or 4 cell types at a time. We report a non-invasive imaging modality that capitalizes on the endogenous autofluorescence signatures of the metabolic cofactors NAD(P)H and FAD. By marrying morphological characteristics with autofluorescence signatures, all seven of the airway epithelial cell types can be distinguished simultaneously in mouse tracheal explant in real-time. Furthermore, we find that this methodology for direct cell type specific identification avoids pitfalls associated with the use of ostensibly cell type-specific markers that are, in fact, altered by clinically relevant physiologic stimuli. Finally, we utilize this methodology to interrogate real-time physiology and identify dynamic secretory cell associated antigen passages (SAPs) that form in response to cholinergic stimulus. The identical process has been well documented in the intestine where the dynamic formation of secretory and goblet cell associated antigen passages (SAPs and GAPs) enable luminal antigen sampling. Given that airway secretory cells can be stimulated to make mucous within hours, we suspect that both SAPs and GAPs are also used for luminal antigen sampling in the airway. This hypothesis is supported by our observation that secretory cells with airway SAPs are frequently juxtaposed to antigen presenting cells.

    1. Cell Biology

    Nuclear SUN1 stabilizes endothelial cell junctions via microtubules to regulate blood vessel formation

    Danielle B Buglak, Pauline Bougaran ... Victoria L Bautch
    Research Article

    Endothelial cells line all blood vessels, where they coordinate blood vessel formation and the blood-tissue barrier via regulation of cell-cell junctions. The nucleus also regulates endothelial cell behaviors, but it is unclear how the nucleus contributes to endothelial cell activities at the cell periphery. Here, we show that the nuclear-localized linker of the nucleoskeleton and cytoskeleton (LINC) complex protein SUN1 regulates vascular sprouting and endothelial cell-cell junction morphology and function. Loss of murine endothelial Sun1 impaired blood vessel formation and destabilized junctions, angiogenic sprouts formed but retracted in SUN1-depleted sprouts, and zebrafish vessels lacking Sun1b had aberrant junctions and defective cell-cell connections. At the cellular level, SUN1 stabilized endothelial cell-cell junctions, promoted junction function, and regulated contractility. Mechanistically, SUN1 depletion altered cell behaviors via the cytoskeleton without changing transcriptional profiles. Reduced peripheral microtubule density, fewer junction contacts, and increased catastrophes accompanied SUN1 loss, and microtubule depolymerization phenocopied effects on junctions. Depletion of GEF-H1, a microtubule-regulated Rho activator, or the LINC complex protein nesprin-1 rescued defective junctions of SUN1-depleted endothelial cells. Thus, endothelial SUN1 regulates peripheral cell-cell junctions from the nucleus via LINC complex-based microtubule interactions that affect peripheral microtubule dynamics and Rho-regulated contractility, and this long-range regulation is important for proper blood vessel sprouting and junction integrity.

    1. Cell Biology

    Defects in lipid homeostasis reflect the function of TANGO2 in phospholipid and neutral lipid metabolism

    Agustin Leonardo Lujan, Ombretta Foresti ... Vivek Malhotra
    Research Article Updated

    We show that TANGO2 in mammalian cells localizes predominantly to mitochondria and partially at mitochondria sites juxtaposed to lipid droplets (LDs) and the endoplasmic reticulum. HepG2 cells and fibroblasts of patients lacking TANGO2 exhibit enlarged LDs. Quantitative lipidomics revealed a marked increase in lysophosphatidic acid (LPA) and a concomitant decrease in its biosynthetic precursor phosphatidic acid (PA). These changes were exacerbated in nutrient-starved cells. Based on our data, we suggest that TANGO2 function is linked to acyl-CoA metabolism, which is necessary for the acylation of LPA to generate PA. The defect in acyl-CoA availability impacts the metabolism of many other fatty acids, generates high levels of reactive oxygen species, and promotes lipid peroxidation. We suggest that the increased size of LDs is a combination of enrichment in peroxidized lipids and a defect in their catabolism. Our findings help explain the physiological consequence of mutations in TANGO2 that induce acute metabolic crises, including rhabdomyolysis, cardiomyopathy, and cardiac arrhythmias, often leading to fatality upon starvation and stress.