Compartmentalization allows for the spatial organization of cellular components and is crucial for numerous biological functions. One recently uncovered strategy for intracellular compartmentalization is phase separation via the de-mixing of biomacromolecules. Membraneless organelles, also referred to as biomolecular condensates, are compartments formed by phase separation and create distinct environments that are essential to cellular processes ranging from cell signaling to gene expression. Biomolecular condensates offer several advantages – for example, dynamic restructuring of internal constituents and diffusion of cellular components into/out of compartments – that make them suitable for applications in biocatalysis or pharmaceutical production. However, the ability to independently engineer the formation and disassembly of condensates in vivo remains a challenge. Here, concepts from polymer science have been used to understand parameters that govern intracellular phase separation. Many biomolecular condensates exhibit physical properties that are similar to complex coacervates as both are liquid-like phase separated mixtures formed via associative phase separation, frequently with oppositely charged polyelectrolytes. We utilize the physical phenomenon of complex coacervation and principles underlying the formation of liquid-like biological condensates to identify design parameters for engineering synthetic, phase separated organelles in E. coli. In this dissertation, we employed a library of cationic charge variants derived from superfolder green fluorescent protein (sfGFP) to elucidate the effects of overall cationic charge on intracellular phase separation. We first investigated the complex coacervation of engineered proteins with biological polyelectrolytes to determine predictive design rules for protein phase separation and translated these design rules in vivo to engineer bacterial condensates. Characterization of the coacervate-like properties and macromolecular composition revealed that these condensates can undergo dynamic restructuring and exhibit biomolecular specificity. To facilitate the engineering of active supercharged proteins, we also developed short, cationic peptide tags, ranging from 6-27 amino acids in length, that can be appended onto any protein of interest to promote intracellular phase separation. We find that overall charge generally determines protein phase behavior and observe the formation and disassembly of condensates near the physiological phase boundary. Interestingly, we find that small modifications in charge density can tune the interaction strength between associating biomacromolecules and thus tune condensate stability. We demonstrate the use of these protein design parameters and cationic peptide tags to sequester catalytic enzymes and manipulate the intracellular localization of multiple proteins. These studies pave the way to building synthetic, functional organelles.

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Ion channels are fundamental and essential molecular machineries located in the membranes of diverse organelles, crucial for maintaining normal cellular function in response to various stimuli. The TRP channel family, discovered in the late 1980s, has been extensively studied for its structures and functions. TRP channels are involved in a broad spectrum of sensory processes such as temperature sensation, touch, pain, and osmolarity regulation. Given their role in sensing diverse stimuli, TRP channels play numerous physiological and pathological roles and have emerged as valuable therapeutic targets for various diseases. As a subfamily of the TRP channel superfamily, TRPML channels also fulfill various physiological functions. Among the TRPML channel subfamilies, TRPML1 and TRPML3 have been identified due to their association with human and mouse disease phenotypes, highlighting their crucial roles in maintaining cellular function and contributing to disease progression when dysfunctional. TRPML1 is extensively studied, likely due to its direct link to human diseases. In contrast, TRPML2 has not been extensively studied because it is not implicated in any disease phenotype. While they are expected to share specific biophysical properties and functions, recent research has increasingly focused on uncovering the unique and essential physiological roles of TRPML2. Studies have revealed its involvement as an osmo/mechanosensitive channel in the immune system and its structure in its apo state. However, further research is needed to fully understand the molecular mechanisms and broader physiological functions of TRPML2. In my thesis, I employ single-particle cryogenic electron microscopy (cryo-EM) to elucidate the structures of human and mouse TRPML2 in the presence of natural and synthetic agonists. These structures highlight distinctive structural characteristics of TRPML2 compared to other TRPML channels and suggest a cooperative and non-canonical activation mechanism involving multiple agonists under experimental conditions. Additionally, electrophysiology experiments were conducted to explore the relationship between the structure and function of human TRPML2. Overall, my thesis work contributes to uncovering unique structural elements and presents the first open-state structure of TRPML2. Furthermore, it offers insights into how TRPML2 interacts with ligands and is activated through a novel activation mechanism.

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.

The mechanisms by which altered processing, distribution and secretion of proteolytic enzymes occur, facilitating degradation of the extracellular matrix in invasive and metastatic cells, are not fully understood. Studies on the MCF-10 A breast epithelial cell line and its premalignant, c-Ha-ras-transfected MCF-10AneoT counterpart have shown that the ras-transfected cell line has a more alkaline pH. The objective of this study was to determine which organelles of the endosome-lysosome route were alkalinized and shifted to the cell periphery after ras-transfection. Antibodies to the hapten 2,4-dinitrophenyl (DNP), required for pH studies, were raised in rabbits and chickens using DNP-ovalbumin (DNP-OVA) as immunogen. Cationised DNP-OVA (DNP-catOVA) was also inoculated to increase antibody titres. Anti-hapten and carrier antibody titres were assessed. In rabbits, cationisation seems useful to increase anti-DNP titres if a non-self carrier protein (OVA) is used. In chickens, cationisation of DNP-OVA seems necessary to produce a sustained anti-OVA (anti-self) response (implying a potential strategy for cancer immunotherapy). Oregon Green® 488 dextran pulse-chase uptake and fluorescent microscopy, and (2,4-dinitroanilino)-3'-amino-N-methyldipropylamine (DAMP) uptake, immunolabelling for DNP (a component of DAMP) and unique markers for the early endosome (early endosome antigen-I, EEAI), the late endosome (cation-independent mannose-6-phosphate receptor, CI-MPR) and the lysosome (small electron dense morphology and lysosome-associated membrane protein-2, LAMP-2) and electron mlcroscopy was performed. The pH of late endosomes and lysosomes in the ras-transfected MCF-10AneoT cell line were found to be relatively alkalinised and Iysosomes shifted toward the cell periphery. The acidic pH of late endosomes is required to release precursor cysteine and aspartic proteases from their receptors (e.g. CI-MPR), process the precursors to active proteases and to allow receptor recycling. The more alkaline pH observed potentially explains the altered processing of proteases in rastransfected cells. Alkalinisation ofthe cytosol may affect the cytoskeleton responsible for, among other things, the positioning and trafficking of various organelles, causing relocation of Iysosomes toward the cell periphery and actin depolymerisation. This may enable fusion of Iysosomes with the plasma membrane and the release of proteolytic enzymes, facilitating the observed invasive phenotype. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.