Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis
- University of Edinburgh, United Kingdom
- University of California, San Francisco, United States
- Lunenfeld-Tanenbaum Research Institute, Canada
- University of Valencia, Spain
Abstract
Centrosomes are orbited by centriolar satellites, dynamic multiprotein assemblies nucleated by Pericentriolar Material 1 (PCM1). To study the requirement for centriolar satellites, we generated mice lacking PCM1, a crucial component of satellites. Pcm1-/- mice display partially penetrant perinatal lethality with survivors exhibiting hydrocephalus, oligospermia and cerebellar hypoplasia, and variably expressive phenotypes such as hydronephrosis. As many of these phenotypes have been observed in human ciliopathies and satellites are implicated in cilia biology, we investigated whether cilia were affected. PCM1 was dispensable for ciliogenesis in many cell types, whereas Pcm1-/- multiciliated ependymal cells and human PCM1-/- retinal pigmented epithelial 1 (RPE1) cells showed reduced ciliogenesis. PCM1-/- RPE1 cells displayed reduced docking of the mother centriole to the ciliary vesicle and removal of CP110 and CEP97 from the distal mother centriole, indicating compromised early ciliogenesis. Similarly, Pcm1-/- ependymal cells exhibited reduced removal of CP110 from basal bodies in vivo. We propose that PCM1 and centriolar satellites facilitate efficient trafficking of proteins to and from centrioles, including the departure of CP110 and CEP97 to initiate ciliogenesis, and that the threshold to trigger ciliogenesis differs between cell types.
Data availability
Proteomics data files are be uploaded ProteomeXchange (Identifier: PXD031920), with the accession number is available with the paper.All analysis tools have been made available on GitHub (https://github.com/IGC-Advanced-Imaging-Resource/Hall2022_Paper), as described in Materials and Methods.
Article and author information
Author details
Vicente Herranz-Pérez
Laurence Pelletier
Funding
Medical Research Council (MR_UU_1201018/26)
- Emma A Hall
- Dhivya Kumar
- Patricia L Yeyati
- Lorraine Rose
- Lisa McKie
- Daniel O Dodd
- Peter A Tennant
- Roly Megaw
- Laura C Murphy
- Marisa F Ferreira
- Graeme Grimes
- Lucy Williams
- Tooba Quidwai
- Pleasantine Mill
Sandler Foundation
- Dhivya Kumar
Krembil Foundation
- Suzanna L Prosser
- Laurence Pelletier
European Commission (866355)
- Emma A Hall
- Daniel O Dodd
- Pleasantine Mill
Canadian Institutes of Health Research (167279)
- Suzanna L Prosser
- Laurence Pelletier
European Commission (702601)
- Suzanna L Prosser
National Institutes of Health (R01GM095941)
- Dhivya Kumar
- Vicente Herranz-Pérez
- Jose Manuel García-Verdugo
- Jeremy F Reiter
National Institutes of Health (R01AR054396)
- Dhivya Kumar
- Vicente Herranz-Pérez
- Jose Manuel García-Verdugo
- Jeremy F Reiter
National Institutes of Health (RO1HD089918)
- Dhivya Kumar
- Vicente Herranz-Pérez
- Jose Manuel García-Verdugo
- Jeremy F Reiter
National Institutes of Health (5K99GM140175)
- Dhivya Kumar
Jane Coffin Childs Memorial Fund for Medical Research
- Dhivya Kumar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: Animals were maintained in SPF environment and studies carried out in accordance with the guidance issued by the Medical Research Council in "Responsibility in the Use of Animals in Medical Research" (July 1993) and licensed by the Home Office under the Animals (Scientific Procedures) Act 1986 under project license number P18921CDE in facilities at the University of Edinburgh (PEL 60/6025).
Reviewing Editor
- Lotte B Pedersen, University of Copenhagen, Denmark
Version history
- Preprint posted: April 4, 2022 (view preprint)
- Received: April 6, 2022
- Accepted: February 14, 2023
- Accepted Manuscript published: February 15, 2023 (version 1)
- Version of Record published: March 9, 2023 (version 2)
Copyright
© 2023, Hall 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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Centriolar satellites expedite mother centriole remodeling to promote ciliogenesis
eLife 12:e79299.
https://doi.org/10.7554/eLife.79299
Further reading
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- Cell Biology
- Developmental Biology
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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- 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.
