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Fixation can change the appearance of phase separation in living cells

Irgen-Gioro, Shawn and Yoshida, Shawn and Walling, Victoria and Chong, Shasha (2022) Fixation can change the appearance of phase separation in living cells. eLife, 11 . Art. No. e79903. ISSN 2050-084X. PMCID PMC9817179. doi:10.7554/elife.79903.

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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. 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.

Item Type:Article
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URLURL TypeDescription CentralArticle ItemDiscussion Paper
Irgen-Gioro, Shawn0000-0001-8638-6191
Yoshida, Shawn0000-0002-0866-2741
Chong, Shasha0000-0002-5372-311X
Additional Information:This work was supported by the Shurl and Kay Curci Foundation Research Grant (to S Chong), the John D Baldeschwieler and Marlene R Konnar Foundation (to S Chong), Pew-Stewart Scholars Program for Cancer Research (to S Chong), Searle Scholars Program (to S Chong), and Merkin Innovation Seed Grant (to S Chong). We thank Luke Lavis for providing fluorescent HaloTag ligands; the Caltech Biological Imaging Facility and Giada Spigolon for providing technical assistance on confocal fluorescence microscopy; and Robert Tjian, Thomas Graham, John Ferrie, and Jonathan Karr for critical comments on the manuscript. 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. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Group:Richard N. Merkin Institute for Translational Research
Funding AgencyGrant Number
Shurl and Key Curci FoundationUNSPECIFIED
John D. Baldeschwieler and Marlene R. Konnar FoundationUNSPECIFIED
Pew-Stewart Scholars Program for Cancer ResearchUNSPECIFIED
Searle Scholars ProgramUNSPECIFIED
Caltech Merkin Institute for Translational ResearchUNSPECIFIED
PubMed Central ID:PMC9817179
Record Number:CaltechAUTHORS:20230125-514169000.3
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:118927
Deposited By: Research Services Depository
Deposited On:17 Feb 2023 21:38
Last Modified:21 Feb 2023 17:41

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