Cryo electron tomography with Volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen Trypanosoma brucei
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
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
- Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom
Version history
- Received: September 20, 2019
- Accepted: November 11, 2019
- Accepted Manuscript published: November 11, 2019 (version 1)
- Accepted Manuscript updated: November 14, 2019 (version 2)
- 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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Further reading
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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.
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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.