Detecting and quantifying liquid–liquid phase separation in living cells by model-free calibrated half-bleaching

Fernando Muzzopappa, Johan Hummert, Michela Anfossi, Stanimir Asenov Tashev, Dirk-Peter Herten and Fabian Erdel ()
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Fernando Muzzopappa: CNRS, UPS
Johan Hummert: University of Birmingham
Michela Anfossi: CNRS, UPS
Stanimir Asenov Tashev: University of Birmingham
Dirk-Peter Herten: University of Birmingham
Fabian Erdel: CNRS, UPS

Nature Communications, 2022, vol. 13, issue 1, 1-15

Abstract: Abstract Cells contain numerous substructures that have been proposed to form via liquid–liquid phase separation (LLPS). It is currently debated how to reliably distinguish LLPS from other mechanisms. Here, we benchmark different methods using well-controlled model systems in vitro and in living cells. We find that 1,6-hexanediol treatment and classical FRAP fail to distinguish LLPS from the alternative scenario of molecules binding to spatially clustered binding sites without phase-separating. In contrast, the preferential internal mixing seen in half-bleach experiments robustly distinguishes both mechanisms. We introduce a workflow termed model-free calibrated half-FRAP (MOCHA-FRAP) to probe the barrier at the condensate interface that is responsible for preferential internal mixing. We use it to study components of heterochromatin foci, nucleoli, stress granules and nuage granules, and show that the strength of the interfacial barrier increases in this order. We anticipate that MOCHA-FRAP will help uncover the mechanistic basis of biomolecular condensates in living cells.

Date: 2022
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DOI: 10.1038/s41467-022-35430-y

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