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Fixation Can Change the Appearance of Phase Separation in Living Cells

Irgen-Gioro, Shawn and Walling, Victoria and Chong, Shasha (2022) Fixation Can Change the Appearance of Phase Separation in Living Cells. . (Unpublished)

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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.

Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription Paper
Irgen-Gioro, Shawn0000-0001-8638-6191
Chong, Shasha0000-0002-5372-311X
Additional Information: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. This version posted May 8, 2022. This work was supported by the Shurl and Kay Curci Foundation (to S. Chong) and the John D. Baldeschwieler and Marlene R. Konnar Foundation (to S. Chong). We thank the Caltech Biological Imaging Facility and G. Spigolon for providing technical assistance on confocal fluorescence microscopy. We thank Robert Tjian, Thomas Graham, John Ferrie, Jonathan Karr, and Shawn Yoshida for critical comments on the manuscript. No competing interests declared. The materials described in this study are available on request.
Funding AgencyGrant Number
Shurl and Kay Curci FoundationUNSPECIFIED
John D. Baldeschwieler and Marlene R. Konnar FoundationUNSPECIFIED
Record Number:CaltechAUTHORS:20220509-155322997
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Official Citation:Fixation Can Change the Appearance of Phase Separation in Living Cells. Shawn Irgen-Gioro, Victoria Walling, Shasha Chong. bioRxiv 2022.05.06.490956; doi:
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:114649
Deposited By: Tony Diaz
Deposited On:09 May 2022 16:49
Last Modified:09 May 2022 16:49

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