The interior of a cell is highly crowded with different macromolecules. In order for these macromolecules to interact in meaningful ways they need to be organized into compartments. Some are confined inside membrane-bound organelles, while others form membraneless organelles (MLOs) through liquid-liquid phase separation (LLPS). Compartments formed through LLPS arise either as densely packed coacervates or segregated aqueous two-phase systems (ATPSs). While LLPS and ATPSs have been studied for decades, coacervates and MLOs in cells are a relatively new field of study and few have studied how coacervates act within an ATPS. This thesis aims to combine these two fundamentally antagonistic forms of LLPS, with the hypothesis that it will elucidate methods for molecular distribution within cells. This study combined an ATPS of PEG and dextran with a coacervating system of PLL and ATP. Their interplay was studied using cell-free models, primarily water-in-oil microfluidic droplets. Observations were collected using optical and fluorescence microscopy. The location of coacervates and their individual components were strongly influenced by the ATPS, with both PLL and ATP/PLL coacervates partitioning into the dextran-rich phase. It was further found that dextran was sequestered inside of the coacervates, potentially leading to the ATPS outside the coacervates to mix. Further testing in artificial vesicles may lead to pathways for cycles of confinement and dissolution as well a method for artificial cell transport.

Like impressionist art, a multitude of subtle and complex interactions determine the behavior of biological systems but a generalized perception loses significant resolution. This is true within the discipline of soft matter, where the chemical multiplicity of the involved components and their resulting physical consequences within lamellar mesophase assemblies are commonly ignored. Therefore, it is imperative to investigate the minute thermodynamic considerations between varied lipidic lamellar mesophases and biologically relevant dopants (like surface-active agents and proteinaceous content) and their resulting physical behaviors. By using various microscopy techniques and x-ray diffraction measurements, lamellar mesophase behavior can be monitored upon the addition (in real-time or post-doping event) of these surface-active and biological substances, and the resulting analyses elucidate the microscopic information others have commonly missed. Such studies can not only enlighten scientists with a higher-level understanding of amphiphilic systems but also lead to the development of unique structural assemblies for various applications.In a larger perspective, this dissertation aims to assemble structurally-diverse lamellar mesophases and expose them to surface-active molecules (also written as surfactant or detergent) and proteinaceous content in varying methods to connect macroscopic and microscopic information. Employing various methodologies like directed aqueous hydration, water vapor hydration, and electroformation, both multilamellar and unilamellar mesophases populated by common lipids and fluorescently-tagged phospholipids were assembled. Such assemblies were then perturbed by symmetric (or internalized) or asymmetric (or external) doping of the focal substances. The consequential physical and structural properties of the mixed-component systems were then investigated to understand the impacts of chemical multiplicity. This dissertation only begins to expound the subtleties of chemistry within lamellar mesophases and question the value of generalized models of membrane behavior. Through these efforts, a new, adaptable, and inclusive intellectual framework of membranes can be developed and considered.

Cells, the smallest structural and functional units of life, are constantly on the move. They engage in multitudes of activities ranging from nutrient procurement, communication, growth to replication and even migration. Cellular membrane, an encasement made of phospholipid bilayer not only compartmentalizes the cellular interior from the extracellular environment, playing a critical role in cell’s viability, but it is also the hub for all essential cellular communications with the surroundings. These often involve molecular level reorganization and morphological remodeling of the membrane interface - an energetically intensive cellular activity most commonly mediated by a host of protein machinery. This was not the case for early cells- a coagulated mass of informational macromolecules bound by lipid membrane- that lacked this advanced protein toolkit, however still survived to evolve into the modern cells that we know. How could the primitive cell-like systems make their ends meet?This dissertation investigates how protocells might have performed essential life functions through alternative mechanisms. Using giant unilamellar vesicles (GUVs) as models, my research explores the impact of osmotic stresses – those that arise merely by local gradients of concentration between inside and the outside of vesicular compartments – on membrane organization, dynamics, and function. My findings showcase how osmotic cycling of the GUVs can lead to the formation of multiple invaginations in vesicle, successfully engulfing and transferring solutes from the vesicle exteriors to the interiors. This simple physical process thus mimics the versatile biological mechanism of endocytosis for movement of macromolecules and materials across the living cell. Introducing compositional degrees of freedom in the make-up of these GUVs reveals how membrane molecules become selectively involved during these processes. During the formation of osmotically induced invaginations, different lipids sort differently into invaginations producing compositionally differentiated liquid-ordered and liquid-disordered domains. This compositional sorting eventually leads to materially asymmetric division of invaginations producing daughter compartments of distinctly different molecular signatures. In this same vein, creating phase separating vesicles with distinct lipid domains shows that disparity in domain water permeabilities can induce directional fluxes and eventual vectorial GUV propulsion under applied osmotic stress, mirroring motility mechanisms cells often adopt to migrate. These results provide insights into how primitive cell-like structures may have achieved critical functions in absence of proteins. Not only does this study help in understanding plausible protocellular activity but it also paves way for creating synthetic cellular systems with life-like adaptive responses in fluctuating environments.