Can fixation fake phase separation?
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.
Source
Fixation can change the appearance of phase separation in living cells
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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Inspect source passages
Can fixation fake phase separation?
These passages occur verbatim in the stored paper text. They support the points listed below; they do not verify every statement in the explanation.
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 ).
What they did
Imaged EGFP-tagged FUS/EWS/TAF15 IDRs before and after typical 4% PFA fixation and quantified puncta number, roughness, and punctate percentage.
What they found
After fixation, puncta increased in number, size, and contrast; homogeneous EWS-IDR nuclei could gain many discrete puncta; all three quantified metrics rose significantly.
The limits
What it doesn't show
Does not mean all fixed condensates are false, but shows fixation alone can change LLPS appearance.
Key terms
- LLPS
- Liquid–liquid phase separation into condensates.
- PFA fixation
- Paraformaldehyde crosslinking used for fixed-cell imaging.
- IDR
- Intrinsically disordered region prone to condensation.
- FET family
- FUS/EWS/TAF15 proteins with phase-separating IDRs.
- Punctate percentage
- Share of nuclear fluorescence in the concentrated phase.
- Surface roughness
- Intensity SD across the nucleus as an unevenness metric.
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Main warning:
Common questions
Cell type?
U2OS.
Fixation condition emphasized?
4% PFA ~10 min.
Key artifact?
Puncta can appear after fixation where live cells lacked them.
Metrics up after fixation?
Puncta number, roughness, punctate percentage.
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