3D visualization of mitochondrial solid-phase calcium stores in whole cells

Abstract

The entry of calcium into mitochondria is central to metabolism, inter-organelle communication, and cell life/death decisions. Long-sought transporters involved in mitochondrial calcium influx and efflux have recently been identified. To obtain a unified picture of mitochondrial calcium utilization, a parallel advance in understanding the forms and quantities of mitochondrial calcium stores is needed. We present here the direct 3D visualization of mitochondrial calcium in intact mammalian cells using cryo-scanning transmission electron tomography (CSTET). Amorphous solid granules containing calcium and phosphorus were pervasive in the mitochondrial matrices of a variety of mammalian cell types. Analysis based on quantitative electron scattering revealed that these repositories are equivalent to molar concentrations of dissolved ions. These results demonstrate conclusively that calcium buffering in the mitochondrial matrix in live cells occurs by phase separation, and that solid-phase stores provide a major ion reservoir that can be mobilized for bioenergetics and signaling.

Article and author information

Author details

  1. Sharon Grayer Wolf

    Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel
    For correspondence
    sharon.wolf@weizmann.ac.il
    Competing interests
    The authors declare that no competing interests exist.
  2. Yael Mutsafi

    Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  3. Tali Dadosh

    Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  4. Tal Ilani

    Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  5. Zipora Lansky

    Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  6. Ben Horowitz

    Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  7. Sarah Rubin

    Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
  8. Michael Elbaum

    Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot, Israel
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7915-5512
  9. Deborah Fass

    Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel
    For correspondence
    Deborah.Fass@weizmann.ac.il
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9418-6069

Funding

European Research Council under the European Union's Seventh Framework Programme, grand number 310649 (310649)

  • Deborah Fass

I-CORE Program of the Planning and Budgeting Committee and the Israel Science Foundation (1775/12)

  • Deborah Fass

Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science

  • Sharon Grayer Wolf

Israel Science Foundation (1285/14)

  • Michael Elbaum

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Werner Kühlbrandt, Max Planck Institute of Biophysics, Germany

Publication history

  1. Received: June 28, 2017
  2. Accepted: November 3, 2017
  3. Accepted Manuscript published: November 6, 2017 (version 1)
  4. Version of Record published: November 27, 2017 (version 2)

Copyright

© 2017, Wolf 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

  • 4,404
    Page views
  • 695
    Downloads
  • 43
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, Scopus, PubMed Central.

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. Sharon Grayer Wolf
  2. Yael Mutsafi
  3. Tali Dadosh
  4. Tal Ilani
  5. Zipora Lansky
  6. Ben Horowitz
  7. Sarah Rubin
  8. Michael Elbaum
  9. Deborah Fass
(2017)
3D visualization of mitochondrial solid-phase calcium stores in whole cells
eLife 6:e29929.
https://doi.org/10.7554/eLife.29929

Further reading

    1. Cell Biology
    Lei Yuan et al.
    Research Article

    The phagocytic receptor CED-1 mediates apoptotic cell recognition by phagocytic cells, enabling cell corpse clearance in Caenorhabditis elegans. Whether appropriate levels of CED-1 are maintained for executing the engulfment function remains unknown. Here, we identified the C. elegans E3 ubiquitin ligase tripartite motif containing-21 (TRIM-21) as a component of the CED-1 pathway for apoptotic cell clearance. When the NPXY motif of CED-1 was bound to the adaptor protein CED-6 or the YXXL motif of CED-1 was phosphorylated by tyrosine kinase SRC-1 and subsequently bound to the adaptor protein NCK-1 containing the SH2 domain, TRIM-21 functioned in conjunction with UBC-21 to catalyze K48-linked poly-ubiquitination on CED-1, targeting it for proteasomal degradation. In the absence of TRIM-21, CED-1 accumulated post-translationally and drove cell corpse degradation defects, as evidenced by direct binding to VHA-10. These findings reveal a unique mechanism for the maintenance of appropriate levels of CED-1 to regulate apoptotic cell clearance.

    1. Cell Biology
    2. Genetics and Genomics
    Anthony J Veltri et al.
    Research Article Updated

    Key protein adapters couple translation to mRNA decay on specific classes of problematic mRNAs in eukaryotes. Slow decoding on non-optimal codons leads to codon-optimality-mediated decay (COMD) and prolonged arrest at stall sites leads to no-go decay (NGD). The identities of the decay factors underlying these processes and the mechanisms by which they respond to translational distress remain open areas of investigation. We use carefully designed reporter mRNAs to perform genetic screens and functional assays in Saccharomyces cerevisiae. We characterize the roles of Hel2, Syh1, and Smy2 in coordinating translational repression and mRNA decay on NGD reporter mRNAs, finding that Syh1 and, to a lesser extent its paralog Smy2, act in a distinct pathway from Hel2. This Syh1/Smy2-mediated pathway acts as a redundant, compensatory pathway to elicit NGD when Hel2-dependent NGD is impaired. Importantly, we observe that these NGD factors are not involved in the degradation of mRNAs enriched in non-optimal codons. Further, we establish that a key factor previously implicated in COMD, Not5, contributes modestly to the degradation of an NGD-targeted mRNA. Finally, we use ribosome profiling to reveal distinct ribosomal states associated with each reporter mRNA that readily rationalize the contributions of NGD and COMD factors to degradation of these reporters. Taken together, these results provide new insight into the role of Syh1 and Smy2 in NGD and into the ribosomal states that correlate with the activation of distinct pathways targeting mRNAs for degradation in yeast.