Intracellular membraneless organelles commonly arise by phase separation of biomolecules and are essential for cellular processes such as signaling, gene regulation, and reproduction. The abnormal behavior of these biomolecular condensates can lead to cancer and neurodegenerative diseases. Equilibrium thermodynamics can explain condensate properties like internal composition, selective segregation of molecules, and conditions for their assembly.
However, cells are inherently out of equilibrium, and hence, many of the functionalities of intracellular condensates arise from coupling phase separation to active processes such as post-translational modifications. The mechanisms that cells employ to regulate the properties and associated functions of these out-of-equilibrium condensates are poorly understood. In vitro condensate models provide a bottom-up approach to gaining insights into these underlying active mechanisms that may be difficult to isolate in vivo. Complex coacervates formed by liquid-liquid phase separation of oppositely charged polyelectrolytes have been effectively used to emulate condensate properties because of their compatibility with biological processes and their responsiveness to environmental cues. In this dissertation, we aim to use in vitro coacervate systems to reproduce two main characteristics of native condensates - size regulation and stimuli-responsive internal structure.
The two in vitro models described in this thesis utilize the effect of pH on coacervation to demonstrate control over coacervate size or morphology under non-equilibrium conditions. The experimental system consists of an anionic enzyme phase separating with a weak polycation, DEAE-dextran, to form micron-sized coacervate drops. The charges of both the polyelectrolytes are pH sensitive, which makes the electrostatic dominated coacervate assembly pH responsive. The anionic enzyme enriched in the coacervate catalyzes a reaction that changes the solution pH.
In Chapter 2, we demonstrate that the anionic catalase enzyme causes a pH increase that dissolves the coacervates. In turn, the diffusion-limited consumption of substrate within large coacervate drops slows the enzymatic reaction, thereby completing the negative feedback loop. Using a combination of modeling and experiments, we show that drops larger than a critical size can potentially support a localized pH increase that leads to a size-dependent dissolution of coacervates. Although the presence of oxygen bubbles prevents the realization of size control in this system, our work provides a framework to achieve size regulation in active coacervates exhibiting negative feedback between coacervate assembly and enzymatic reactions.
In Chapter 3, we harness the pH-sensitivity of coacervates to elucidate the mechanism of vacuole formation within glucose oxidase/DEAE-dextran coacervates in response to an external pH decrease. We show that the formation of hollow structures or vacuoles within the coacervates depends on the rate of pH decrease and droplet size. Our results reveal that vacuole development occurs under non-equilibrium conditions due to the diffusion-limited mobility of the polycation during a rapid pH decrease. This work serves as a platform to regulate condensate structure in response to changes in the local environment, laying the groundwork for designing synthetic cells with new capabilities.
| Identifer | oai:union.ndltd.org:columbia.edu/oai:academiccommons.columbia.edu:10.7916/gjby-dv95 |
| Date | January 2025 |
| Creators | Modi, Nisha |
| Source Sets | Columbia University |
| Language | English |
| Detected Language | English |
| Type | Theses |
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