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Fixation Can Change the Appearance of Phase Separation in Living Cells

Shawn Irgen-Gioro, Victoria Walling, View ORCID ProfileShasha Chong
doi: https://doi.org/10.1101/2022.05.06.490956
Shawn Irgen-Gioro
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
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Victoria Walling
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
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Shasha Chong
1Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA
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  • ORCID record for Shasha Chong
  • For correspondence: schong@caltech.edu
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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. 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.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 08, 2022.
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Fixation Can Change the Appearance of Phase Separation in Living Cells
Shawn Irgen-Gioro, Victoria Walling, Shasha Chong
bioRxiv 2022.05.06.490956; doi: https://doi.org/10.1101/2022.05.06.490956
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Fixation Can Change the Appearance of Phase Separation in Living Cells
Shawn Irgen-Gioro, Victoria Walling, Shasha Chong
bioRxiv 2022.05.06.490956; doi: https://doi.org/10.1101/2022.05.06.490956

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