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.
Highlights
The cytosol is a complex mixture of macromolecules, including proteins, nucleic acids and polysaccharides
An advantage of polymeric crowders is that they are usually available in a the large variety of sizes (e.g., polyethylene glycol (PEG) can range from 2.5 kDa up to 20 kDa), allowing systematically varying the size of the crowders, while proteins have a fixed size (e.g., bovine serum albumin (BSA) is 68 kDa and lysozyme 14 kDa) [5,52]
These polymeric crowders vary in molecular weight: commonly used Ficoll-70 has an average molecular weight (Mw ) of 70 kDa; PEG is used in a range of 3.35 kDa up to 20 kDa
Summary
The cytosol is a complex mixture of macromolecules, including proteins, nucleic acids and polysaccharides. We first introduce the theoretical framework that has been condensates seem to be dynamically formed and dissolved over time [8] Many of these micro-niches in used extensively to explain crowding effects on biochemical processes, and highlight the factors that the cell have liquid-like properties, such as nucleoli, germ granules, stress granules and paraspeckles [9], are relevant to phase separating proteins. We overview of the use of crowders in in vitro MLO studies, and the observed effects of crowding on refer to several excellent reviews by Ellis [3,12,13,14], Minton [4,15,16,17], and Rivas [5,18] on the physical phase separation, partitioning and the biophysical properties of condensates.
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