Polymer-Based Experimental Model Systems for Intracellular Organization
Open Access
- Author:
- Marianelli, Allyson
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 15, 2018
- Committee Members:
- Christine Dolan Keating, Dissertation Advisor/Co-Advisor
Christine Dolan Keating, Committee Chair/Co-Chair
Philip C Bevilacqua, Committee Member
Paul S Cremer, Committee Member
Scott Eugene Lindner, Outside Member - Keywords:
- liquid liquid phase separation
macromolecular crowding
coacervation
giant vesicle
protocell
microfluidics - Abstract:
- Living cells are incredibly complex and well organized, making it difficult to investigate any one variable in isolation from the myriad physicochemical and biospecific effects of such an environment. The development of bottom-up experimental model systems that mimic the physicochemical environment of living cells while removing some biospecificity and complexity has become an attractive area of research for understanding the physicochemical driving forces for biological activity. In particular, intracellular organization is crucial for cellular life, providing a means to sequester biomolecules and compartmentalize metabolic reactions. Cells utilize a combination of membrane-bound and membraneless compartments to organize their interiors; this thesis focuses on the development of experimental model systems to better understand the physicochemical driving forces behind intracellular organization. Chapter 1 provides an introduction to relevant background information, including cellular bodies that exist as liquid phases, their in vivo behavior, and work already completed in the literature towards modeling that behavior in vitro. Liquid-liquid phase separation (LLPS) is discussed as a means for both formation of membraneless organelles in living cells and for the development of cellular mimics. The concept of macromolecular crowding is introduced, whereby the presence of macromolecules in solution can dramatically alter solution chemistry, and its theoretical and practical implications are discussed. The prevalence of lipid membranes in cellular organization is also reviewed, along with methods for generating model systems that incorporate lipid membranes. Chapter 2 focuses on the impact of macromolecular crowding on a type of associative LLPS, complex coacervation. A model RNA/polyamine system (composed of poly(uridylic acid) and spermine) was used to mimic nucleic acid-rich liquid organelles in the presence of a variety of neutral macromolecular crowding agents, with turbidimetric analysis and confocal microscopy to probe the phase behavior of the system. Crowding was shown to promote coacervation at both lower polyamine concentrations and temperatures, as well as improve partitioning of an oligonucleotide, with poly(ethylene glycol) (PEG) exerting a more significant impact than Ficoll. Chapter 3 continues to investigate the effect of crowding on complex coacervation of two different systems; a bio-inspired polypeptide system composed of poly(lysine) (polyK) and poly(aspartic acid) (polyD) and a synthetic polyelectrolyte system composed of poly(allylamine) (PAH) and poly(acrylic acid) (PAA). Crowding promoted coacervation at lower polyelectrolyte concentrations and improved the salt stability of the polyK/polyD system, while it hindered phase separation in the PAA/PAH system. Chapter 4 discusses incorporation of lipid membranes into cellular model systems, reporting templated lipid vesicle formation by simple coacervate droplets composed of the arginine-rich protein protamine sulfate. Lipid vesicles exhibit a number of behaviors characteristic of lipid bilayer membranes which are ubiquitous in biology, including lipid molecule alignment and diffusion, selective impermeability, and protection of interior vesicle components. Chapter 5 reports the first use of microfluidic techniques in the Keating group to achieve greater control over the size and content of emulsion droplets to generate large monodisperse populations for use in future experimental cell model systems. A range of emulsion systems was successfully demonstrated including water-in-oil (W/O), aqueous two-phase-in-oil (ATPS/O), and water-in-oil-in-water (W/O/W). Chapter 6 highlights the major findings of this thesis and looks forward to consider future work that will benefit from these results and continue to build on the knowledge gained from this project. Overall, the experiments presented in this dissertation have improved our knowledge of the physicochemical forces governing some aspects of intracellular organization and provided a route to develop an array of bottom-up model cells. This knowledge is widely applicable as it can be used to develop even more complex experimental model systems as well as further understand LLPS in general, which has utility and applications beyond biological mimicry.