Liquid–Liquid Phase Separation in Crowded Environments

Abstract

Biomolecular condensates play a key role in organizing cellular fluids such as the cytoplasm and nucleoplasm. Most of these non-membranous organelles show liquid-like properties both in cells and when studied in vitro through liquid–liquid phase separation (LLPS) of purified proteins. In general, LLPS of proteins is known to be sensitive to variations in pH, temperature and ionic strength, but the role of crowding remains underappreciated. Several decades of research have shown that macromolecular crowding can have profound effects on protein interactions, folding and aggregation, and it must, by extension, also impact LLPS. However, the precise role of crowding in LLPS is far from trivial, as most condensate components have a disordered nature and exhibit multiple weak attractive interactions. Here, we discuss which factors determine the scope of LLPS in crowded environments, and we review the evidence for the impact of macromolecular crowding on phase boundaries, partitioning behavior and condensate properties. Based on a comparison of both in vivo and in vitro LLPS studies, we propose that phase separation in cells does not solely rely on attractive interactions, but shows important similarities to segregative phase separation.

Hyperosmotic phase separation: Condensates beyond inclusions, granules and organelles

“…What is the mechanistic relationship between osmotic volume change and phase separation? Osmotic cell compression changes cell volume by exosmosis, resulting in an increase in intracellular crowding and effective protein concentration, both of which influence phase separation as discussed above ( 103 , 104 ). Molecular crowding also has significant effects on protein structure and function ( 105 , 106 , 107 , 108 ).…”

“…Finally, crowding previously has been predicted on theoretical grounds to serve as a cell volume sensor, even though a mechanistic basis is only now emerging ( 96 ). For an in-depth review of crowding effects and phase separation, we refer the reader to Andre and Srpuijt 2020 ( 104 ).…”

Intracellular Ionic Strength Sensing Using NanoLuc

“…Ionic strength determined by the concentrations of different ionic species in a medium, both cations and anions, fundamentally influences electrostatic interactions in and among biomolecules and, therefore, regulates a plethora of cellular processes [ 1 , 2 , 3 , 4 ]. For instance, ionic strength affects the conformational stability and folding/unfolding behavior of highly charged proteins through charge screening, as detected by changes in fluorescence resonance energy transfer (FRET) efficiency [ 5 ] or a ligand-induced conformational change in a multidomain protein as determined from intramolecular bioluminescence resonance energy transfer (BRET) assays [ 6 , 7 , 8 , 9 , 10 ].…”

Glycogen-dependent demixing of frog egg cytoplasm at increased crowding

“…At the same time, diffusion-limited reaction rates increase with concentration but decrease with viscosity, and viscosity increases with concentration (Dill et al, 2011;van den Berg et al, 2017). Crowding tends to promote macromolecular assembly reactions, including linear polymerization and phase-separated condensate assembly (Andre and Spruijt, 2020). Increasing crowding may contribute to a liquid-to-glass transition in bacterial and yeast cytoplasm under energy stress (Joyner et al, 2016;Munder et al, 2016;Parry et al, 2014) or mechanical compression (Okumus et al, 2016).…”