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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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.
Flashcards
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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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