scholarly journals Pressure-Jump Kinetics of Liquid–Liquid Phase Separation: Comparison of Two Different Condensed Phases of the RNA-Binding Protein, Fused in Sarcoma

2021 ◽  
Author(s):  
Ryo Kitahara ◽  
Ryota Yamazaki ◽  
Fumika Ide ◽  
Shujie Li ◽  
Yutaro Shiramasa ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Rna Binding Protein ◽  
Liquid Phase Separation ◽  
Pressure Jump ◽  
Condensed Phases ◽  
Fused In Sarcoma ◽  
Kinetics Of ◽  
Liquid Liquid Phase Separation

scholarly journals The phase separation-dependent FUS interactome reveals nuclear and cytoplasmic function of liquid-liquid phase separation

2019 ◽  
Author(s):  
Stefan Reber ◽  
Helen Lindsay ◽  
Anny Devoy ◽  
Daniel Jutzi ◽  
Jonas Mechtersheimer ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Mitochondrial Gene ◽  
Liquid Phase Separation ◽  
Fused In Sarcoma ◽  
Damage Repair ◽  
Interaction Partners ◽  
Lateral Sclerosis ◽  
Liquid Liquid Phase Separation

AbstractLiquid-liquid phase separation (LLPS) of proteins and RNAs has emerged as the driving force underlying the formation of membrane-less organelles. Such biomolecular condensates have various biological functions and have been linked to disease. One of the best studied proteins undergoing LLPS is Fused in Sarcoma (FUS), a predominantly nuclear RNA-binding protein. Mutations in FUS have been causally linked to Amyotrophic Lateral Sclerosis (ALS), an adult-onset motor neuron disease, and LLPS followed by aggregation of cytoplasmic FUS has been proposed to be a crucial disease mechanism. In spite of this, it is currently unclear how LLPS impacts the behaviour of FUS in cells, e.g. its interactome. In order to study the consequences of LLPS on FUS and its interaction partners, we developed a method that allows for the purification of phase separated FUS-containing droplets from cell lysates. We observe substantial alterations in the interactome of FUS, depending on its biophysical state. While non-phase separated FUS interacts mainly with its well-known interaction partners involved in pre-mRNA processing, phase-separated FUS predominantly binds to proteins involved in chromatin remodelling and DNA damage repair. Interestingly, factors with function in mitochondria are strongly enriched with phase-separated FUS, providing a potential explanation for early changes in mitochondrial gene expression observed in mouse models of ALS-FUS. In summary, we present a methodology that allows to investigate the interactome of phase-separating proteins and provide evidence that LLPS strongly shapes the FUS interactome with important implications for function and disease.

scholarly journals The effects of cosolutes and crowding on the kinetics of protein condensate formation based on liquid–liquid phase separation: a pressure-jump relaxation study

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Hasan Cinar ◽  
Roland Winter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Liquid Phase Separation ◽  
Pressure Jump ◽  
Transformation Kinetics ◽  
Biologically Relevant ◽  
Pressure Jump Relaxation ◽  
The Stability ◽  
Kinetics Of ◽  
Liquid Liquid Phase Separation

Abstract Biomolecular assembly processes based on liquid–liquid phase separation (LLPS) are ubiquitous in the biological cell. To fully understand the role of LLPS in biological self-assembly, it is necessary to characterize also their kinetics of formation and dissolution. Here, we introduce the pressure-jump relaxation technique in concert with UV/Vis and FTIR spectroscopy as well as light microscopy to characterize the evolution of LLPS formation and dissolution in a time-dependent manner. As a model system undergoing LLPS we used the globular eye-lens protein γD-crystallin. As cosolutes and macromolecular crowding are known to affect the stability and dynamics of biomolecular condensates in cellulo, we extended our kinetic study by addressing also the impact of urea, the deep-sea osmolyte trimethylamine-N-oxide (TMAO) and a crowding agent on the transformation kinetics of the LLPS system. As a prerequisite for the kinetic studies, the phase diagram of γD-crystallin at the different solution conditions also had to be determined. The formation of the droplet phase was found to be a very rapid process and can be switched on and off on the 1–4 s timescale. Theoretical treatment using the Johnson–Mehl–Avrami–Kolmogorov model indicates that the LLPS proceeds via a diffusion-limited nucleation and growth mechanism at subcritical protein concentrations, a scenario which is also expected to prevail within biologically relevant crowded systems. Compared to the marked effect the cosolutes take on the stability of the LLPS region, their effect at biologically relevant concentrations on the phase transformation kinetics is very small, which might be a particular advantage in the cellular context, as a fast switching capability of the transition should not be compromised by the presence of cellular cosolutes.

scholarly journals Polymer Stiffness Regulates Multivalent Binding and Liquid-Liquid Phase Separation

2020 ◽  
Author(s):  
E. Zumbro ◽  
A. Alexander-Katz
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rational Design ◽  
Rna Binding ◽  
Liquid Phase Separation ◽  
Random Coil ◽  
Small Target ◽  
Toxic Protein ◽  
Multivalent Binding ◽  
Liquid Liquid Phase Separation

AbstractMultivalent binding is essential to many biological processes because it builds high affinity bonds by using several weak binding interactions simultaneously. Multivalent polymers have shown promise as inhibitors of toxins and other pathogens, and they are important components in the formation of biocondensates. Explaining how structural features of these polymers change their binding and subsequent control of phase separation is critical to designing better pathogen inhibitors and also to understanding diseases associated with membraneless organelles. In this work, we will examine the binding of a multivalent polymer to a small target. This scenario could represent a polymeric inhibitor binding to a toxic protein or RNA binding to an RNA-binding protein in the case of liquid-liquid phase separation. We use simulation and theory to show that flexible random-coil polymers bind more strongly than stiff rod-like polymers and that flexible polymers nucleate condensed phases at lower energies than their rigid analogues. We hope these results will provide insight into the rational design of polymeric inhibitors and improve understanding of membraneless organelles.Statement of SignificanceMultivalent polymers are essential for many biological systems, including targeting pathogens and controlling the formation of liquid-liquid phase separated biocondensates. Here, we explain how increasing polymer stiffness can reduce multivalent binding affinity to a small target such as a toxic protein and how modulating polymer stiffness can change the phase boundary for liquid-liquid phase separation. These results have implications for designing stronger pathogen inhibitors and provide insights on neurodegenerative diseases associated with abnormal biocondensate formation.

scholarly journals Liquid-Liquid Phase Separation of Patchy Particles Illuminates Diverse Effects of Regulatory Components on Protein Droplet Formation

2018 ◽  
Author(s):  
Valery Nguemaha ◽  
Huan-Xiang Zhou
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Rna Binding ◽  
Liquid Phase Separation ◽  
Steric Repulsion ◽  
Cellular Functions ◽  
Binding Domains ◽  
Patchy Particles ◽  
Droplet Phase ◽  
Liquid Liquid Phase Separation

AbstractRecently many cellular functions have been associated with membraneless organelles, or protein droplets, formed by liquid-liquid phase separation (LLPS). Proteins in these droplets often contain RNA-binding domains, but the effects of RNA on LLPS have been controversial. To gain better understanding on the roles of RNA, here we used Gibbs-ensemble simulations to determine phase diagrams of two-component patchy particles, as models for mixtures of proteins with RNA or other regulatory components. Protein-like particles have four patches, with attraction strength εPP; regulatory particles experience mutual steric repulsion but have two attractive patches toward proteins, with the strength εPR tunable. At low εPR, the regulator, due to steric repulsion, preferentially partitions in the dispersed phase, thereby displacing the protein into the droplet phase and promoting LLPS. At moderate εPR, the regulator starts to partition and displace the protein in the droplet phase, but only to weaken bonding networks and thereby suppress LLPS. At εPR > εPP, the enhanced bonding ability of the regulator initially promotes LLPS, but at higher amounts, the resulting displacement of the protein suppresses LLPS. These results illustrate how RNA can have disparate effects on LLPS, thus able to perform diverse functions in different organelles.

scholarly journals SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation stimulated by RNA and partitions into phases of human ribonucleoproteins

2020 ◽  
Author(s):  
Theodora Myrto Perdikari ◽  
Anastasia C. Murthy ◽  
Veronica H. Ryan ◽  
Scott Watters ◽  
Mandar T. Naik ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Liquid Phase Separation ◽  
Host Protein ◽  
Host Cells ◽  
Liquid Liquid Phase Separation

AbstractTightly packed complexes of nucleocapsid protein and genomic RNA form the core of viruses and may assemble within viral factories, dynamic compartments formed within the host cells. Here, we examine the possibility that the multivalent RNA-binding nucleocapsid protein (N) from the severe acute respiratory syndrome coronavirus (SARS-CoV-2) compacts RNA via protein-RNA liquid-liquid phase separation (LLPS) and that N interactions with host RNA-binding proteins are mediated by phase separation. To this end, we created a construct expressing recombinant N fused to a N-terminal maltose binding protein tag which helps keep the oligomeric N soluble for purification. Using in vitro phase separation assays, we find that N is assembly-prone and phase separates avidly. Phase separation is modulated by addition of RNA and changes in pH and is disfavored at high concentrations of salt. Furthermore, N enters into in vitro phase separated condensates of full-length human hnRNPs (TDP-43, FUS, and hnRNPA2) and their low complexity domains (LCs). However, N partitioning into the LC of FUS, but not TDP-43 or hnRNPA2, requires cleavage of the solubilizing MBP fusion. Hence, LLPS may be an essential mechanism used for SARS-CoV-2 and other RNA viral genome packing and host protein co-opting, functions necessary for viral replication and hence infectivity.

scholarly journals TDP-43 α-helical structure tunes liquid–liquid phase separation and function

2020 ◽  
Vol 117 (11) ◽  
pp. 5883-5894 ◽  
Author(s):  
Alexander E. Conicella ◽  
Gregory L. Dignon ◽  
Gül H. Zerze ◽  
Hermann Broder Schmidt ◽  
Alexandra M. D’Ordine ◽  
...  
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Structural Changes ◽  
Rna Binding ◽  
Helical Structure ◽  
Liquid Phase Separation ◽  
Control Assembly ◽  
And Function ◽  
Liquid Liquid Phase Separation

Liquid–liquid phase separation (LLPS) is involved in the formation of membraneless organelles (MLOs) associated with RNA processing. The RNA-binding protein TDP-43 is present in several MLOs, undergoes LLPS, and has been linked to the pathogenesis of amyotrophic lateral sclerosis (ALS). While some ALS-associated mutations in TDP-43 disrupt self-interaction and function, here we show that designed single mutations can enhance TDP-43 assembly and function via modulating helical structure. Using molecular simulation and NMR spectroscopy, we observe large structural changes upon dimerization of TDP-43. Two conserved glycine residues (G335 and G338) are potent inhibitors of helical extension and helix–helix interaction, which are removed in part by variants at these positions, including the ALS-associated G335D. Substitution to helix-enhancing alanine at either of these positions dramatically enhances phase separation in vitro and decreases fluidity of phase-separated TDP-43 reporter compartments in cells. Furthermore, G335A increases TDP-43 splicing function in a minigene assay. Therefore, the TDP-43 helical region serves as a short but uniquely tunable module where application of biophysical principles can precisely control assembly and function in cellular and synthetic biology applications of LLPS.

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