Intracellular environments are crowded with various biological macromolecules, which can contribute to aqueous phase separation within cells. This process can lead to the formation of RNA- and protein-rich liquid membraneless organelles <i>in vivo</i>, such as processing bodies and stress granules in the cytoplasm, as well as Cajal bodies and nucleoli in the nucleus. There are still many unknowns surrounding how liquid organelles form and function, so creating cellular mimics to study membraneless organelles <i>in vitro </i>is an attractive option for scientists, as we can remove some of the complexity of living cells without surrendering a comparable cellular environment. In this research, complex coacervation, a type of associative phase separation, is used as a physicochemical model for understanding the behaviors and compartmentalization abilities of liquid membraneless organelles in eukaryotic cells. Complex coacervates were formed from oppositely charged polypeptides and introduced to aqueous solutions containing neutral crowding agents, including poly(ethylene glycol) (PEG) 8 kDa, ethylene glycol (EG), Ficoll 70 kDa, and sucrose. Increasing the amount of PEG, EG, and Ficoll in samples resulted in a lower charge ratio required for coacervation, indicating that coacervates easily formed. Conversely, sucrose increased the charge ratio required for phase separation, suggesting that interactions between the polypeptides and sucrose molecules hindered coacervation. Macromolecular crowding also stabilized coacervates in increasing concentrations of NaCl, as PEG and Ficoll crowders caused the coacervate phase to persist in higher concentrations of NaCl, compared to samples lacking crowders. Additionally, we observed evidence that crowding molecules impacted the physical morphology of the coacervate phase. These preliminary results demonstrate that crowding-induced effects can influence the phase behavior and physical properties of peptide-based coacervates. These findings also suggest that potential consequences of the high concentrations of intracellular macromolecules and cosolutes should be considered in the formation of liquid organelles in cells.

Complex coacervation as a model for membraneless organelles: Effects of macromolecular crowding on the formation and behaviors of polypeptide coacervates

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

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