Fixation can change the appearance of phase separation in living cells

  1. Shawn Irgen-Gioro
  2. Shawn Ryohei Yoshida
  3. Victoria Walling
  4. Shasha Chong  Is a corresponding author
  1. California Institute of Technology, United States

Abstract

Fixing cells with paraformaldehyde (PFA) is an essential step in numerous biological techniques as it is thought to preserve a snapshot of biomolecular transactions in living cells. Fixed cell imaging techniques such as immunofluorescence have been widely used to detect liquid-liquid phase separation (LLPS) in vivo. Here, we compared images, before and after fixation, of cells expressing intrinsically disordered proteins that are able to undergo LLPS. Surprisingly, we found that PFA fixation can both enhance and diminish putative LLPS behaviors. For specific proteins, fixation can even cause their droplet-like puncta to artificially appear in cells that do not have any detectable puncta in the live condition. Fixing cells in the presence of glycine, a molecule that modulates fixation rates, can reverse the fixation effect from enhancing to diminishing LLPS appearance. We further established a kinetic model of fixation in the context of dynamic protein-protein interactions. Simulations based on the model suggest that protein localization in fixed cells depends on an intricate balance of protein-protein interaction dynamics, the overall rate of fixation, and notably, the difference between fixation rates of different proteins. Consistent with simulations, live-cell single-molecule imaging experiments showed that a fast overall rate of fixation relative to protein-protein interaction dynamics can minimize fixation artifacts. Our work reveals that PFA fixation changes the appearance of LLPS from living cells, presents a caveat in studying LLPS using fixation-based methods, and suggests a mechanism underlying the fixation artifact.

Data availability

Figure 1 - Source Data 1, Figure 2 - Source Data 1, Figure 3 - Source Data 1, and Figure 6 - Source Data 1 contain the numerical data used to generate the figures. Custom scripts have been uploaded as source code files.

Article and author information

Author details

  1. Shawn Irgen-Gioro

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8638-6191
  2. Shawn Ryohei Yoshida

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  3. Victoria Walling

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    Competing interests
    The authors declare that no competing interests exist.
  4. Shasha Chong

    Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, United States
    For correspondence
    schong@caltech.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5372-311X

Funding

Shurl and Key Curci Foundation (Research Grant)

  • Shasha Chong

John D. Baldeschwieler and Marlene R. Konnar Foundation

  • Shasha Chong

Pew-Stewart Scholars Program for Cancer Research

  • Shasha Chong

Searle Scholars Program

  • Shasha Chong

Merkin Institute for Translational Research (Merkin Innovation Seed Grant)

  • Shasha Chong

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

Reviewing Editor

  1. Felix Campelo, Institute of Photonic Sciences, Spain

Version history

  1. Received: May 2, 2022
  2. Preprint posted: May 8, 2022 (view preprint)
  3. Accepted: November 28, 2022
  4. Accepted Manuscript published: November 29, 2022 (version 1)
  5. Accepted Manuscript updated: November 30, 2022 (version 2)
  6. Version of Record published: January 5, 2023 (version 3)

Copyright

© 2022, Irgen-Gioro 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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  1. Shawn Irgen-Gioro
  2. Shawn Ryohei Yoshida
  3. Victoria Walling
  4. Shasha Chong
(2022)
Fixation can change the appearance of phase separation in living cells
eLife 11:e79903.
https://doi.org/10.7554/eLife.79903

Share this article

https://doi.org/10.7554/eLife.79903

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