PT  - JOURNAL ARTICLE
AU  - Shawn Irgen-Gioro
AU  - Shawn Yoshida
AU  - Victoria Walling
AU  - Shasha Chong
TI  - Fixation Can Change the Appearance of Phase Separation in Living Cells
AID  - 10.1101/2022.05.06.490956
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.05.06.490956
4099  - http://biorxiv.org/content/early/2022/11/23/2022.05.06.490956.short
4100  - http://biorxiv.org/content/early/2022/11/23/2022.05.06.490956.full
AB  - 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.Competing Interest StatementThe authors have declared no competing interest.