Cell signalling
Can fixation fake phase separation?
Open access · cc by · source: Europe PMC
Comparing the same U2OS cells live vs after PFA showed FET-family IDR puncta increase—and can appear where none existed—warning that fixed-cell LLPS can be artefactual.
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Study at a glance
- Design
- Animal / in-vitro — Live vs 4% PFA-fixed imaging of FET-family IDR condensates in U2OS cells
- N
- N=3 · Three FET-family IDRs (FUS, EWS, TAF15) compared live vs fixed
- Population
- U2OS cells transiently expressing EGFP-IDR-NLS constructs
- Outcome
- Fixation-induced changes in apparent LLPS puncta metrics
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Can fixation fake phase separation?
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Three FET IDRs compared live vs fixed.
Results Fixation enhances the LLPS appearance of FET family proteins To investigate the effect of PFA fixation on the appearance of LLPS, we first compared confocal fluorescence images of live and fixed U2OS cells that transiently express an IDR tagged with EGFP and a nuclear localization sequence (NLS).
All three puncta metrics increased after fixation.
We quantified the fixation-induced changes of LLPS appearance by calculating three parameters from the fluorescence images of cells, including the number of puncta, surface roughness, and punctate percentage, and found a significant increase in all three parameters after fixation ( Figure 1D–F , Figure 1—source data 1 ).
Key findings
After fixation, puncta increased in number, size, and contrast; homogeneous EWS-IDR nuclei could gain many discrete puncta; all three quantified metrics rose significantly.
Methodology
Imaged EGFP-tagged FUS/EWS/TAF15 IDRs before and after typical 4% PFA fixation and quantified puncta number, roughness, and punctate percentage.
Limitations
Does not mean all fixed condensates are false, but shows fixation alone can change LLPS appearance.
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