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

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

SUBMITTER: Muzzopappa F 

PROVIDER: S-EPMC9758202 | biostudies-literature | 2022 Dec

REPOSITORIES: biostudies-literature