Single molecule microscopy reveals key physical features of repair foci in living cells

  1. Judith Miné-Hattab  Is a corresponding author
  2. Mathias Heltberg
  3. Marie Villemeur
  4. Chloé Guedj
  5. Thierry Mora
  6. Aleksandra M Walczak
  7. Maxime Dahan
  8. Angela Taddei  Is a corresponding author
  1. Institut Curie, France
  2. Ecole Normale Supérieure, France
  3. École Normale Supérieure, France

Abstract

In response to double strand breaks (DSB), repair proteins accumulate at damaged sites, forming membrane-less sub-compartments or foci. Here we explored the physical nature of these foci, using single molecule microscopy in living cells. Rad52, the functional homolog of BRCA2 in yeast, accumulates at DSB sites and diffuses ~6 times faster within repair foci than the focus itself, exhibiting confined motion. The Rad52 confinement radius coincides with the focus size: foci resulting from 2 DSBs are twice larger in volume that the ones induced by a unique DSB and the Rad52 confinement radius scales accordingly. In contrast, molecules of the single strand binding protein Rfa1 follow anomalous diffusion similar to the focus itself or damaged chromatin. We conclude that while most Rfa1 molecules are bound to the ssDNA, Rad52 molecules are free to explore the entire focus reflecting the existence of a liquid droplet around damaged DNA.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files are available on zenodo using the following link: https://zenodo.org/record/4495116.

The following data sets were generated

Article and author information

Author details

  1. Judith Miné-Hattab

    UMR 3664 - Nuclear Dynamics, Institut Curie, Paris, France
    For correspondence
    judith.Mine@curie.fr
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9986-4092
  2. Mathias Heltberg

    UMR 3664 - Nuclear Dynamics, Institut Curie, paris, France
    Competing interests
    No competing interests declared.
  3. Marie Villemeur

    UMR3664 - Nuclear Dynamics, Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  4. Chloé Guedj

    UMR3664 - Nuclear Dynamics, Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  5. Thierry Mora

    Laboratoire de physique, Ecole Normale Supérieure, Paris, France
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5456-9361
  6. Aleksandra M Walczak

    Laboratoire de Physique Theorique, École Normale Supérieure, Paris, France
    Competing interests
    Aleksandra M Walczak, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-2686-5702
  7. Maxime Dahan

    Division of Genetics, Genomics & Development, Department of Molecular and Cell Biology, Institut Curie, Paris, France
    Competing interests
    No competing interests declared.
  8. Angela Taddei

    UMR3664, Institut Curie, Paris, France
    For correspondence
    angela.taddei@curie.fr
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3217-0739

Funding

Agence Nationale de la Recherche (ANR-11-LABEX-0044 DEEP)

  • Angela Taddei

Agence Nationale de la Recherche (ANR-10-IDEX-0001-02 PSL)

  • Angela Taddei

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

Reviewing Editor

  1. Irene E Chiolo, University of Southern California, United States

Version history

  1. Received: June 30, 2020
  2. Accepted: January 26, 2021
  3. Accepted Manuscript published: February 5, 2021 (version 1)
  4. Version of Record published: March 1, 2021 (version 2)
  5. Version of Record updated: March 5, 2021 (version 3)

Copyright

© 2021, Miné-Hattab 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

  • 3,260
    Page views
  • 487
    Downloads
  • 41
    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. Judith Miné-Hattab
  2. Mathias Heltberg
  3. Marie Villemeur
  4. Chloé Guedj
  5. Thierry Mora
  6. Aleksandra M Walczak
  7. Maxime Dahan
  8. Angela Taddei
(2021)
Single molecule microscopy reveals key physical features of repair foci in living cells
eLife 10:e60577.
https://doi.org/10.7554/eLife.60577

Share this article

https://doi.org/10.7554/eLife.60577

Further reading

    1. Physics of Living Systems
    Yangfan Zhang, George V Lauder
    Research Article

    Many animals moving through fluids exhibit highly coordinated group movement that is thought to reduce the cost of locomotion. However, direct energetic measurements demonstrating the energy-saving benefits of fluid-mediated collective movements remain elusive. By characterizing both aerobic and anaerobic metabolic energy contributions in schools of giant danio (Devario aequipinnatus), we discovered that fish schools have a concave upward shaped metabolism–speed curve, with a minimum metabolic cost at ~1 body length s-1. We demonstrate that fish schools reduce total energy expenditure (TEE) per tail beat by up to 56% compared to solitary fish. When reaching their maximum sustained swimming speed, fish swimming in schools had a 44% higher maximum aerobic performance and used 65% less non-aerobic energy compared to solitary individuals, which lowered the TEE and total cost of transport by up to 53%, near the lowest recorded for any aquatic organism. Fish in schools also recovered from exercise 43% faster than solitary fish. The non-aerobic energetic savings that occur when fish in schools actively swim at high speed can considerably improve both peak and repeated performance which is likely to be beneficial for evading predators. These energetic savings may underlie the prevalence of coordinated group locomotion in fishes.

    1. Computational and Systems Biology
    2. Physics of Living Systems
    Nicholas M Boffi, Yipei Guo ... Ariel Amir
    Research Article

    The adaptive dynamics of evolving microbial populations takes place on a complex fitness landscape generated by epistatic interactions. The population generically consists of multiple competing strains, a phenomenon known as clonal interference. Microscopic epistasis and clonal interference are central aspects of evolution in microbes, but their combined effects on the functional form of the population’s mean fitness are poorly understood. Here, we develop a computational method that resolves the full microscopic complexity of a simulated evolving population subject to a standard serial dilution protocol. Through extensive numerical experimentation, we find that stronger microscopic epistasis gives rise to fitness trajectories with slower growth independent of the number of competing strains, which we quantify with power-law fits and understand mechanistically via a random walk model that neglects dynamical correlations between genes. We show that increasing the level of clonal interference leads to fitness trajectories with faster growth (in functional form) without microscopic epistasis, but leaves the rate of growth invariant when epistasis is sufficiently strong, indicating that the role of clonal interference depends intimately on the underlying fitness landscape. The simulation package for this work may be found at https://github.com/nmboffi/spin_glass_evodyn.