Glycogen-dependent demixing of frog egg cytoplasm at increased crowding

Crowding increases the tendency of macromolecules to aggregate and phase separate, and high crowding can induce glass-like states of cytoplasm. To explore the effect of crowding in a well-characterized model cytoplasm we developed methods to selectively concentrate components larger than 25 kDa from Xenopus egg extracts. When crowding was increased 1.4x, the egg cytoplasm demixed into two liquid phases of approximately equal volume. One of the phases was highly enriched in glycogen while the other had a higher protein concentration. Glycogen hydrolysis blocked or reversed demixing. Quantitative proteomics showed that the glycogen phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are in complexes with especially high native molecular weight. The glycogen phase was depleted of ribosomes, ER and mitochondria. These results inform on the physical nature of a glycogen-rich cytoplasm and suggest a role of demixing in the localization of glycogen particles in tissue cells.