Real-Time Study of Protein Phase Separation with Spatiotemporal Analysis of Single-Nanoparticle Trajectories.

Liquid-liquid phase separation (LLPS) underlies the formation mechanism of membraneless biomolecular condensates locally to perform important physiological functions such as selective autophagy, but little is known about the relationship between their dynamic structural organization and biophysical properties. Here, a dark-field microscopy based single plasmonic nanoparticle tracking (DFSPT) technique was introduced to simultaneously monitor the diffusion dynamics of multiple gold nanorod (AuNR) probes in a protein LLPS system and to quantitatively characterize the spatiotemporal heterogeneity of the LLPS condensates during their phase transformation. Based on spatially and temporally resolved analysis of the diffusional behavior of the AuNRs, structure and material properties of p62 condensates, such as the viscoelasticity, the compartmentalization, and the recruitment of protein-covered nanoparticles into the large droplet, have been observed. Moreover, the nonsmooth droplet interface, its solidification after further phase transition or maturation, and the size effect of the inner vacuoles have also been revealed. Our method can be potentially applied to investigation of different reconstituted membrane-free biomolecular condensates and study of their dynamic evolution.