Live-Cell Imaging to Resolve Salt-Induced Liquid–Liquid Phase Separation of FUS Protein by Dye Self-Labeling

Abstract

The aggregation of fusion in sarcoma (FUS) in the cytoplasm and nucleus is a pathological feature of Amyotrophic lateral sclerosis (ALS) and Frontotemporal Dementia (FTD). Genetic mutations, abnormal protein synthesis, environmental stress, and aging have all been implicated as causative factors in this process. Salt ions are essential to many physiological processes in the body, and the imbalance of them is an important environmental stress factor in cells. However, their effect on liquid−liquid phase separation (LLPS) of FUS proteins in living cells is not well understood. Here, we map the various salt-induced LLPS of FUS in living cells by genetically coding and self-labeling FUS with organic dyes. The brightness and photostability of the dyes enable long-term imaging to track the mechanism of the assembly and disappearance of FUS phase separation. The FUS protein showed a better phase separation tendency under 0.3 M salt stimulation, and there was a large amount of FUS shuttling from the nucleus to the cytoplasm. At this concentration, various salt solutions displayed different effects on the phase separation of FUS protein, following the Hofmeister effects. We further observed that the assembly of FUS droplets underwent a process of rapid formation of small droplets, plateaus, and mutual fusion. Strikingly, The CsCl-stimulated FUS droplets were not completely reversible after washing, and some solid-like granules remained in the nucleus. Taken together, these results help broaden our understanding of the LLPS of FUS proteins in cellular stress responses.

Ionic Effect on the Microenvironment of Biomolecular Condensates

“…The addition of ions into aqueous solutions induces the rearrangement of protein–ion, ion–water, and protein–water interaction, ,− thus significantly affecting the phase separation behavior of LLPS-prone proteins. − Ions could increase the surface tension of the solution, , effectively triggering phase separation which releases water from the protein hydration layer into the surrounding environment. , On the contrary, the translational entropy of ions counteracts the tendency of phase separation . In addition, the specific binding of the ions contributes greatly to the phase separation process.…”

Modulation of Biomolecular Liquid–Liquid Phase Separation by Preferential Hydration and Interaction of Small Osmolytes with Proteins

“…FUS is an intrinsically disordered RNA-binding protein that forms liquid droplets through LLPS and has been used as a model system in many biophysical and biochemical studies. − Irreversible aggregation of FUS in the cytosol causes familial amyotrophic lateral sclerosis (ALS); therefore, elucidating the aggregation mechanism and development of aggregation inhibitors are strongly desired . We identified two LLPS states for FUS: normal LLPS (i.e., LP-LLPS), which is stable below 2 kbar, and aberrant LLPS (i.e., HP-LLPS), which is stable above 2 kbar and accelerates irreversible aggregation. − Furthermore, FUS-LLPS is destabilized with increasing salt concentration and stabilizes again at higher salt concentrations; reentrant LLPS was observed at concentrations above 2 M of NaCl in a buffer solution, known as high-salt LLPS .…”