Glycogen-dependent demixing of frog egg cytoplasm at increased crowding

Crowding increases the tendency of macromolecules to aggregate and phase separate, and regulated crowding contributes to subcellular organization and stress response. To explore the effect of crowding in a well-characterized model cytoplasm, we developed methods to concentrate the macromolecule components of Xenopus egg extracts without changing small molecules. Egg cytoplasm contains a high concentration of glycogen that serves as an energy store for early development. When crowding was increased 1.4×, the egg cytoplasm demixed into two liquid phases of approximately equal volume, one of which was highly enriched in glycogen. Glycogen hydrolysis prevented and reversed demixing. Quantitative proteomics showed that the glycogen-rich phase was enriched in proteins that bind glycogen, participate in carbohydrate metabolism, or are part of very high-molecular-weight complexes. The glycogen-depleted phase was enriched in ribosomes, endoplasmic reticulum (ER), and mitochondria. Smaller soluble proteins were approximately equipartitioned. Glycogen is usually observed in aggregates in intact cells, and recent work suggested a role for phase separation in its localization. Our results show that glycogen particles can spontaneously demix and suggest that demixing may be regulated by crowding.