Diffusion of LLPS Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA on Chemically Modified Glass Surface

Abstract

The liquid−liquid phase separation (LLPS) of proteins and RNA molecules has emerged in recent years as an important physicochemical process to explain the organization of membrane-less organelles in living cells and cellular functions and even some fatal neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS) due to the spontaneous condensation and growth of LLPS droplets. In general, the characterization of LLPS droplets has been performed by optical microscopy, where we need transparent substrates. By virtue of the liquid and wetting properties of LLPS droplets on a glass surface, there have been some technical protocols recommended to immobilize droplets on the surfaces. However, interactions between LLPS droplets and glass surfaces still remain unclear. Here, we investigated the surface diffusion of LLPS droplets on the glass surface to understand the interactions of droplets in a dynamic manner, and employed chemically modified glass surface with charges to investigate their Coulombic interaction with the surface. Using the single-particle tracking method, we first analyzed the diffusion of droplets on an untreated glass surface. Then, we compared the diffusion modes of LLPS droplets on each substrate and found that there were two major states of droplets on a solid surface: fix and diffusion mode for the LLPS droplet diffusion. While untreated glass showed a diffusion of droplets mainly, chemically modified glass with positive charges exhibited droplets fixed on the surface. It could arise from the Coulombic interaction between droplets and solid surface, where LLPS droplets have a negative ζ-potential. Our findings on the dynamics of LLPS at the solid/liquid interface could provide a novel insight to advance fundamental studies for understanding the LLPS formation.

Effect of Multivalency on Phase-Separated Droplets Consisting of Poly(PR) Dipeptide Repeats and RNA at the Solid/Liquid Interface

“…The diffusion of the LLPS droplets at the solid/liquid interface could be the switching between diffusion and immobilization mode. 23 To this end, we utilized the linear region of MSD with a time range of less than 0.1 s to estimate the diffusion coefficients for LLPS droplets. For a quantitative comparison between these two peptides, we derived the diffusion coefficients of LLPS droplets on the glass surfaces by fitting with eq 1 .…”

“… 11 Recently, we reported that the chemically modified glass surfaces affect the diffusion of LLPS droplets and their immobilization on the glass surface. 23 This study gave us a way to control the interactions of LLPS droplets with the glass surface via the control of the net charge of the surface by the chemical modification.…”

A material‐based panspermia hypothesis: The potential of polymer gels and membraneless droplets

“…For instance, the surface motion behavior of LLPS droplets has been investigated on a transparent glass surface through fluorescence microscopy. [152] Two surface diffusion modes exist (fix mode and dif- Meanwhile, the distribution of charges in LLPS droplet components also affects droplets assembly (in this case, also made up of peptides and RNA) and their diffusion ability; such a process is related to motor neuronal cell death in amyotrophic lateral sclerosis (ALS) disease. [153] According to fluorescence recovery after photobleaching (FRAP) measurements reflecting diffusion efficiency, droplets of peptide variants with small charge periodicity have a faster FRAP recovery rate than that with larger periodicity, suggesting differences in internal exchange.…”

“…For instance, the surface motion behavior of LLPS droplets has been investigated on a transparent glass surface through fluorescence microscopy. [ 152 ] Two surface diffusion modes exist (fix mode and diffusion mode) and the distribution of these two diffusion modes can be tuned by chemical modification of the glass surface with positive charges due to Coulomb interactions between such droplets and the solid surface. The droplets studied contained a negative ζ‐potential and consisted of peptides and RNA, but it is plausible for gel‐material‐based membraneless droplets to exhibit the same properties and, depending on their ζ‐potential (which can be tuned based on their composition) encapsulate different types of molecules.…”

Increasing complexity of primitive compartments

“…One such function of peptides is the assembly of membraneless coacervates through binding with primitive nucleic acids followed by phase-separation. Such membraneless coacervates have been investigated and could contribute functions such as biopolymer segregation and exchange [11], diffusion [12], growth and division [13], enhancement of compartmentalized reactions [14,15], and even scaffolding of other self-assembled structures [16][17][18] to an evolving prebiotic chemical system. Recent studies have found that more higher-order structures can assemble within peptide-DNA coacervates, leading to potential emergent functions with greater complexity depending on the environmental conditions and identity of the coacervate-forming polymers.…”