1. Cell Biology
Download icon

Actin assembly ruptures the nuclear envelope by prying the lamina away from nuclear pores and nuclear membranes in starfish oocytes

  1. Natalia Wesolowska
  2. Ivan Avilov
  3. Pedro Machado
  4. Celina Geiss
  5. Hiroshi Kondo
  6. Masashi Mori
  7. Peter Lenart  Is a corresponding author
  1. European Molecular Biology Laboratory (EMBL), Germany
  2. Max Planck Institute for Biophysical Chemistry, Germany
Research Article
  • Cited 9
  • Views 2,879
  • Annotations
Cite this article as: eLife 2020;9:e49774 doi: 10.7554/eLife.49774

Abstract

The nucleus of oocytes (germinal vesicle) is unusually large and its nuclear envelope (NE) is densely packed with nuclear pore complexes (NPCs) stockpiled for embryonic development. We showed that breakdown of this specialized NE is mediated by an Arp2/3-nucleated F-actin 'shell' in starfish oocytes, in contrast to microtubule-driven tearing in mammalian fibroblasts. Here, we address the mechanism of F-actin-driven NE rupture by correlated live-cell, super-resolution and electron microscopy. We show that actin is nucleated within the lamina sprouting filopodia-like spikes towards the nuclear membranes. These F-actin spikes protrude pore-free nuclear membranes, whereas the adjoining membrane stretches accumulate NPCs associated with the still-intact lamina. Packed NPCs sort into a distinct membrane network, while breaks appear in ER-like, pore-free regions. Thereby, we reveal a new function for actin-mediated membrane shaping in nuclear rupture that is likely to have implications in other contexts such as nuclear rupture observed in cancer cells.

Data availability

Full resolution EM montages are provided as supplemental files.

Article and author information

Author details

  1. Natalia Wesolowska

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Ivan Avilov

    Max Planck Institute for Biophysical Chemistry, Göttingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Pedro Machado

    Electron Microscopy Core Facility, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Celina Geiss

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Hiroshi Kondo

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Masashi Mori

    Cell Biology and Biophysics Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Peter Lenart

    Cell Biology and Biophysics Unit, Max Planck Institute for Biophysical Chemistry, Heidelberg, Germany
    For correspondence
    plenart@mpibpc.mpg.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3927-248X

Funding

Deutsche Forschungsgemeinschaft (SPP 1464)

  • Natalia Wesolowska

European Molecular Biology Laboratory

  • Natalia Wesolowska
  • Pedro Machado
  • Celina Geiss
  • Hiroshi Kondo
  • Masashi Mori
  • Peter Lenart

Max Planck Society

  • Ivan Avilov
  • Peter Lenart

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

Reviewing Editor

  1. R Dyche Mullins, University of California, San Francisco, United States

Publication history

  1. Received: June 28, 2019
  2. Accepted: January 24, 2020
  3. Accepted Manuscript published: January 28, 2020 (version 1)
  4. Version of Record published: February 18, 2020 (version 2)

Copyright

© 2020, Wesolowska 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

  • 2,879
    Page views
  • 516
    Downloads
  • 9
    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)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)

Further reading

    1. Cell Biology
    James Rae et al.
    Tools and Resources Updated

    Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy (EM). Here, we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal-to-noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins, and cytoskeletal proteins. The method can be combined with different EM techniques including fast freezing and freeze substitution, focussed ion beam scanning EM, and electron tomography. Quantitation of expressed APEX-fusion proteins is achievable using membrane vesicles generated by a cell-free expression system. These membrane vesicles possess a defined quantum of signal, which can act as an internal standard for determination of the absolute density of expressed APEX-fusion proteins. Detection of fusion proteins expressed at low levels in cells from CRISPR-edited mice demonstrates the high sensitivity of the APEX-Gold method.

    1. Cell Biology
    David W Sanders et al.
    Research Article Updated

    Many enveloped viruses induce multinucleated cells (syncytia), reflective of membrane fusion events caused by the same machinery that underlies viral entry. These syncytia are thought to facilitate replication and evasion of the host immune response. Here, we report that co-culture of human cells expressing the receptor ACE2 with cells expressing SARS-CoV-2 spike, results in synapse-like intercellular contacts that initiate cell-cell fusion, producing syncytia resembling those we identify in lungs of COVID-19 patients. To assess the mechanism of spike/ACE2-driven membrane fusion, we developed a microscopy-based, cell-cell fusion assay to screen ~6000 drugs and >30 spike variants. Together with quantitative cell biology approaches, the screen reveals an essential role for biophysical aspects of the membrane, particularly cholesterol-rich regions, in spike-mediated fusion, which extends to replication-competent SARS-CoV-2 isolates. Our findings potentially provide a molecular basis for positive outcomes reported in COVID-19 patients taking statins and suggest new strategies for therapeutics targeting the membrane of SARS-CoV-2 and other fusogenic viruses.