Formation of biological condensates via phase separation: Characteristics, analytical methods, and physiological implications

Abstract

Edited by Paul E. FraserLiquid-liquid phase separation (LLPS) facilitates the formation of condensed biological assemblies with well-delineated physical boundaries, but without lipid membrane barriers. LLPS is increasingly recognized as a common mechanism for cells to organize and maintain different cellular compartments in addition to classical membrane-delimited organelles. Membraneless condensates have many distinct features that are not present in membrane-delimited organelles and that are likely indispensable for the viability and function of living cells. Malformation of membraneless condensates is increasingly linked to human diseases. In this review, we summarize commonly used methods to investigate various forms of LLPS occurring both in 3D aqueous solution and on 2D membrane bilayers, such as LLPS condensates arising from intrinsically disordered proteins or structured modular protein domains. We then discuss, in the context of comparisons with membrane-delimited organelles, the potential functional implications of membraneless condensate formation in cells. We close by highlighting some challenges in the field devoted to studying LLPS-mediated membraneless condensate formation.In eukaryotic cells, reaction components are spatiotemporally compartmentalized so that materials are concentrated and activities are localized and protected from damaging activities, such as proteolysis, changes in pH, and undesired covalent modifications. Classical organelles are membrane-enclosed where the lipid bilayer provides a physical barrier to separate their interior contents from the exterior environment. Examples include Golgi apparatus, mitochondria, and endoplasmic reticulum (ER). 3 However, many organelles are not membrane-enclosed (often referred to as membraneless compartments in the literature), and such organelles include but are not limited to germ granules, stress granules, nucleoli, centrosomes, and synapses in neurons. In these membraneless compartments, due to the lack of physical separation, molecules can freely exchange with their counterparts in the surrounding bulk solution. Sharp concentration gradients are maintained between the proteinaceous (and sometimes protein and nucleic acid mixtures) interior and the much more diluted exterior. Reaction machineries can reversibly assemble and disassemble within a short time window, as fast as a few seconds. Reaction constituents can be integrated or removed to control specific activities. While recognized for many years, the mechanisms governing the formation of membraneless organelles have remained unclear until about 10 years ago. The first direct experimental evidence came from the study of P granules in germ cells of Caenorhabditis elegans (1). P granule is a collection of RNA and RNA-binding proteins (RBPs) localized at the posterior cortex of a dividing embryo. P granules appear as spherical droplets with liquid-like properties, and they fuse with one another, deform under shear stress, and flow off the surface of the nucleus. Fluorescence recovery afte...

Limited Dishevelled/Axin oligomerization determines efficiency of Wnt/β-catenin signal transduction

“…It is important to note, however, that understanding the precise connection of the properties of the isolated DIX and DAX domains to the behavior of Dvl and Axin will require experiments using purified, fulllength proteins. For example, both proteins have large unstructured regions and are also regulated by post-translational modifications, features that could contribute to phase separation behavior (Aberti et al, 2019;Feng et al, 2019;Owen and Shewmaker, 2019;Snead and Gladfelter, 2019). Moreover, it is possible that other regions of the Dvl protein affect the extent of DIX domain oligomerization, and likewise there may be intramolecular regulation of the Axin DAX domain (Kim et al, 2013).…”

Spatial control of nucleoporin assembly by Fragile X-related proteins

“…Formation of supramolecular assemblies and membrane-less organelles such as the nucleolus, Cajal bodies, nuclear speckles, stress granules (SG), P-bodies, germ granules and PML bodies are important for cellular homeostasis (Boeynaems et al, 2018). Among the factors controlling their formation and turnover is the presence of intrinsically disordered regions (IDRs) in protein components, their ability to form multivalent protein-protein, and protein-RNA interactions (Feng et al, 2019) and proteins' local concentration. Indeed, many RNA-binding proteins (RBPs) have the ability to demix into liquid states (liquid droplets), which can be subsequently transformed into pathological amyloids both in vitro and in cells (Harrison and Shorter, 2017;Lin et al, 2015;Shorter, 2019).…”

Phase separation-mediated formation of condensed GIT/PIX enzyme complex module for compartmentalized signaling

“…Enzymes can be concentrated at specific subcellular regions by binding to their cognate interacting proteins in accordance to traditional thermodynamic binding equilibrium. Emerging evidence suggests that formation of membrane-less compartments, also known as biomolecular condensates, via liquid-liquid phase separation is a new mechanism used by cells to concentrate biomolecules, including enzymes, at specific subcellular regions [1][2][3][4] . Membrane-less condensates seem to be widespread in cells and include cellular machineries such as P granule 5 , nucleoli 6 , centrosomes 7,8 , pre-and postsynaptic signaling apparatuses [9][10][11][12] , and stress granules 13,14 .…”

“…Membrane-less condensates seem to be widespread in cells and include cellular machineries such as P granule 5 , nucleoli 6 , centrosomes 7,8 , pre-and postsynaptic signaling apparatuses [9][10][11][12] , and stress granules 13,14 . Membrane-less biomolecular condensates display many unique features when compared to traditional stoichiometric assemblies of molecular complexes as well as membrane-enclosed cellular compartments [2][3][4] .…”