Electron cryo-tomography provides insight into procentriole architecture and assembly mechanism
Abstract
Centriole is an essential structure with multiple functions in cellular processes. Centriole biogenesis and homeostasis is tightly regulated. Using electron cryo-tomography (cryoET) we present the structure of procentrioles from Chlamydomonas reinhardtii. We identified a set of non-tubulin components attached to the triplet microtubule (MT), many are at the junctions of tubules likely to reinforce the triplet. We describe structure of the A-C linker that bridges neighboring triplets. The structure infers that POC1 is likely an integral component of A-C linker. Its conserved WD40 β-propeller domain provides attachment sites for other A-C linker components. The twist of A-C linker results in an iris diaphragm-like motion of the triplets in the longitudinal direction of procentriole. Finally, we identified two assembly intermediates at the growing ends of procentriole allowing us to propose a model for the procentriole assembly. Our results provide a comprehensive structural framework for understanding the molecular mechanisms underpinning procentriole biogenesis and assembly.
Data availability
8 structures based on the subtomogram averaging have been deposited in the EMDB under the accession codes: EMD-9167, EMD-9168, EMD-9169, EMD-9170, EMD-9171, EMD-9172, EMD-9173, EMD-9174
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9167.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9168.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9169.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9170.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9171.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9172.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9173.
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Electron cryo-tomography and subtomogram averaging of microtubule triplet from procentrioleElectron Microscopy Data Bank, EMD-9174.
Article and author information
Author details
Funding
Howard Hughes Medical Institute
- David A Agard
National Institutes of Health (GM031627)
- David A Agard
National Institutes of Health (GM118099)
- David A Agard
National Institutes of Health (PO1 GM105537)
- David A Agard
National Institutes of Health (GM113602)
- Wallace F Marshall
Fundacion Ramon Areces
- Jose-Jesus Fernandez
Spanish AEI/FEDER (SAF2017-84565-R)
- Jose-Jesus Fernandez
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Sriram Subramaniam, University of British Columbia, Canada
Version history
- Received: November 6, 2018
- Accepted: February 10, 2019
- Accepted Manuscript published: February 11, 2019 (version 1)
- Version of Record published: February 21, 2019 (version 2)
Copyright
© 2019, Li 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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- Cell Biology
- Structural Biology and Molecular Biophysics
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.
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Dendritic cells (DCs), the key antigen-presenting cells, are primary regulators of immune responses. Transcriptional regulation of DC development had been one of the major research interests in DC biology, however, the epigenetic regulatory mechanisms during DC development remains unclear. Here, we report that Histone deacetylase 3 (Hdac3), an important epigenetic regulator, is highly expressed in pDCs, and its deficiency profoundly impaired the development of pDCs. Significant disturbance of homeostasis of hematopoietic progenitors was also observed in HDAC3-deficient mice, manifested by altered cell numbers of these progenitors and defective differentiation potentials for pDCs. Using the in vitro Flt3L supplemented DC culture system, we further demonstrated that HDAC3 was required for the differentiation of pDCs from progenitors at all developmental stages. Mechanistically, HDAC3 deficiency resulted in enhanced expression of cDC1-associated genes, owing to markedly elevated H3K27 acetylation (H3K27ac) at these gene sites in BM pDCs. In contrast, the expression of pDC-associated genes was significantly downregulated, leading to defective pDC differentiation.