Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death
- Hospital for Sick Children, Canada
- Norwegian University of Science and Technology, Norway
- Ragon Institute of MGH, MIT and Harvard, United States
- Boston Children's Hospital, United States
Abstract
First recognized more than 30 years ago, glycine protects cells against rupture from diverse types of injury. This robust and widely observed effect has been speculated to target a late downstream process common to multiple modes of tissue injury. The molecular target of glycine that mediates cytoprotection, however, remains elusive. Here, we show that glycine works at the level of NINJ1, a newly identified executioner of plasma membrane rupture in pyroptosis, necrosis, and post-apoptosis lysis. NINJ1 is thought to cluster within the plasma membrane to cause cell rupture. We demonstrate that the execution of pyroptotic cell rupture is similar for human and mouse NINJ1, and that NINJ1 knockout functionally and morphologically phenocopies glycine cytoprotection in macrophages undergoing lytic cell death. Next, we show that glycine prevents NINJ1 clustering by either direct or indirect mechanisms. In pyroptosis, glycine preserves cellular integrity but does not affect upstream inflammasome activities or accompanying energetic cell death. By positioning NINJ1 clustering as a glycine target, our data resolve a long-standing mechanism for glycine-mediated cytoprotection. This new understanding will inform the development of cell preservation strategies to counter pathologic lytic cell death.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files, which includes the source data for the manuscript figures.
Article and author information
Author details
Benjamin Ethan Steinberg
Funding
International Anesthesia Research Society (Mentored Research Award)
- Benjamin Ethan Steinberg
Department of Anesthesiology and Pain Medicine, University of Toronto (Early Investigator Award)
- Benjamin Ethan Steinberg
Research Council of Norway (287696,223255)
- Trude Helen Flo
Ragon Institute of MGH, MIT and Harvard (Ragon Early Independence Fellowship)
- Charles L Evavold
National Institutes of Health (AI133524,AI093589,AI116550,and P30DK3485)
- Jonathan C Kagan
Boehringer Ingelheim Fonds (PhD Fellowship)
- Pascal Devant
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Ethics
Animal experimentation: All animal studies were approved by the Hospital for Sick Children Animal Care Committee (AUP #47781).
Human subjects: All human studies were conducted according to the principles expressed in the Helsinki Declaration and approved by the Regional Committee for Medical and Health Research Ethics (No. 2009/2245). Informed consent was obtained from all subjects prior to sample collection.
Reviewing Editor
- Carla V Rothlin, Yale University, United States
Version history
- Preprint posted: December 12, 2021 (view preprint)
- Received: March 14, 2022
- Accepted: December 5, 2022
- Accepted Manuscript published: December 5, 2022 (version 1)
- Accepted Manuscript updated: December 8, 2022 (version 2)
- Version of Record published: December 15, 2022 (version 3)
Copyright
© 2022, Borges 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
-
- 3,450
- Page views
-
- 608
- Downloads
-
- 13
- 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)
Glycine inhibits NINJ1 membrane clustering to suppress plasma membrane rupture in cell death
eLife 11:e78609.
https://doi.org/10.7554/eLife.78609
Further reading
-
- 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.
-
- 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.
