Colloid osmotic parameterization and measurement of subcellular crowding

Abstract

Crowding of the subcellular environment by macromolecules is thought to promote protein aggregation and phase separation. A challenge is how to parameterize the degree of crowding of the cell interior or artificial solutions that is relevant to these reactions. Here I review colloid osmotic pressure as a crowding metric. This pressure is generated by solutions of macromolecules in contact with pores that are permeable to water and ions but not macromolecules. It generates depletion forces that push macromolecules together in crowded solutions and thus promotes aggregation and phase separation. I discuss measurements of colloid osmotic pressure inside cells using the nucleus, the cytoplasmic gel, and fluorescence resonant energy transfer (FRET) biosensors as osmometers, which return a range of values from 1 to 20 kPa. I argue for a low value, 1–2 kPa, in frog eggs and perhaps more generally. This value is close to the linear range on concentration–pressure curves and is thus not crowded from an osmotic perspective. I discuss the implications of a low crowding pressure inside cells for phase separation biology, buffer design, and proteome evolution. I also discuss a pressure–tension model for nuclear shape, where colloid osmotic pressure generated by nuclear protein import inflates the nucleus.

Liquid–liquid phase separation of the Golgi matrix protein GM130

“…S3). Importantly, concerning the above experiments we generally employed conditions approximating the cytoplasm (20 m m HEPES/KOH, pH 7.3, 140 m m KCl, 1 m m MgCl 2 , 1 m m DTT) and notably did not add any crowding agent (such as a polyethylene glycol) needed in many other cases to observe condensate formation in vitro . The droplet evaporation assay yielded similar results even with a simplified buffer composition (5 m m HEPES/KOH, pH 7.3, 140 m m KCl), thereby ruling out that phase separation is induced by increased concentrations of buffer components at the rim.…”

Considerations and Challenges in Studying Liquid-Liquid Phase Separation and Biomolecular Condensates

“…A related issue is the use of synthetic macromolecular crowders such as polyethylene glycol, dextran, or ficoll. In many cases, the amount of crowding agent added to an experiment may exceed the crowding predicted to exist in intracellular contexts (Luby-Phelps, 2013;Mitchison, 2019). It is important to realize that the underlying mechanisms of how crowders enhance phase separation are still unclear.…”

Non-associative phase separation in an evaporating droplet as a model for prebiotic compartmentalization

“…In our model system, both dextran-rich and PEG-rich domains nucleate quickly at the rim of the droplet due to phase separation. However, since PEG is more hydrophobic than dextran 48 and can adsorb onto silica, 49 the dextran-rich compartments, though with a higher density than PEG-rich phase, 38 are advected to the inner part by the fluid flow and PEG-rich compartments stay outside the sessile droplet, as suggested by the SEM images of the deposits (SI Appendix, Fig. S1).…”