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 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, was used as a physicochemical model for understanding the formation and behaviors of membraneless organelles in eukaryotic cells. Complex coacervates were formed from oppositely charged biological and synthetic polymers and introduced to aqueous solutions containing neutral crowding agents, including poly(ethylene glycol) (PEG) 8 kDa, ethylene glycol (EG), Ficoll 70 kDa, and sucrose. We found that the presence of these crowders and cosolutes impacted the phase behavior, indicating that coacervation was either enhanced or hindered by the presence of different crowders. This research also highlights the role that crowding agents had on the stability of complex coacervates at increasing concentrations of NaCl, compared to samples lacking crowders. Additionally, we observed evidence that crowding molecules affect the physical morphology of the coacervate phase. These preliminary results demonstrate that crowding-induced effects can influence the phase behavior and physical properties of complex coacervates. These findings also suggest that potential consequences of the high concentrations of intracellular macromolecules and cosolutes should be considered in the formation of membraneless organelles in cells.

Effects of macromolecular crowding on the formation and properties of complex coacervates

ACS Spring 2022

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