Live-Cell Monitoring and Omics Analysis of Liquid–Solid Transitions of Biomolecular Condensates

Biomolecular condensates, or so-called membraneless organelles,
transition from liquid into more solid-like states over time, contributing
to the development of pathological conditions. The present study proposes
a simple method using photoactive yellow protein (PYP) and its specific
fluorescent covalent ligands to distinguish between the liquid and
solid states of protein condensates in live cells. The method, compatible
with fluorescence-activated cell sorting (FACS), correlates the stiffness
of specific protein condensates with their accessibility to PYP ligands,
enabling quantitative multicolor monitoring of condensate solidification.
We applied this technique to 12 phase-separating proteins and their
mutants, finding that TDP-43, particularly its A315T mutant linked
to familial amyotrophic lateral sclerosis, most readily forms solid
aggregates. Furthermore, this FACS-compatible strategy enabled the
isolation of distinct cell populations based on condensate states,
allowing for subsequent proteomic and transcriptomic analyses. Our
findings demonstrate that condensate solidification is accompanied
by the upregulated expression of extracellular matrix proteins, suggesting
a previously unrecognized link between solid aggregate formation and
extracellular matrix hardening.