Diversifying Interaction Types in Multi-Compartment Models for Intracellular Organization
Open Access
- Author:
- Mountain, Gregory
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- February 27, 2020
- 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
Philip C Bevilacqua, Program Head/Chair - Keywords:
- Liquid-Liquid phase separation
phase separation
coacervate
complex coacervate
membraneless organelle
cellular condensate
phase
granule
liquid phase
polyelectrolyte
ITC
isothermal titration calorimetry
fluorescent labeling - Abstract:
- Biological systems are complex and well organized. The cell utilizes the chemical phenomenon of liquid-liquid phase separation of macromolecular components in order to create membraneless compartments that are held together by associative interaction and present interesting properties such as the ability to concentrate other macromolecules from their environment. These membraneless organelles are highly dynamic and the cell is able to control the assembly and disassembly of these compartments in response to certain stimuli. The cell is also able to create ordered sub-compartments from these liquid assemblies which is thought to facilitate reaction cascades. Macromolecules composing membraneless organelles consist of proteins and nucleic acids that possess a variety of different chemical moieties that allow for many different interaction types such as charge-charge, cation-pi, dipole-dipole, and pi-pi stacking interactions which make fundamentally understanding the properties that define membraneless organelles difficult. The aqueous model systems described here aim to establish a framework for fundamentally understanding the complex properties of macromolecular phase separation. Chapter 1 focuses on background information such as examples of ordered structure within membraneless organelles, and methods of chemical modification used by the cell in order to drive the dynamic behavior of these structures. Complex coacervate systems, which present promising model systems for membraneless organelles, and are the basis for this work are discussed in detail. Chapter 2 describes the generation of multi-compartment complex coacervate systems based on model synthetic, polypeptide, and nucleic acid polyelectrolyte polymers that establish a framework for model systems of coexisting liquid compartments. The factors that drive accumulation of charged macromolecular probes into coacervate systems are found to be dependent on the relative magnitude of associative interactions between the probe and the major components of the coacervate phase with observed accumulation as high as 1000-fold over bulk concentration for small nucleotide probes. In chapter 3 I highlight practical considerations and advice for generating coexisting complex coacervate systems, performing characterization of these systems, and fluorescently labeling polyelectrolytes. I also discuss scenarios resulting in non-liquid complexes and present several methods to overcome this to achieve liquid complex coacervate systems. Chapter 4 discusses the use of isothermal titration calorimetry in combination with UV-melt and turbidity data to probe the different characteristics of polypeptide-RNA complex coacervate systems using arginine, lysine, poly(uridylic acid), and poly(adenylic acid) polyelectrolytes. Availability of additional associative interaction types such as cation-pi, and pi-pi stacking are found to correlate with increased thermodynamic favorability of coacervate formation. Finally, chapter 5 offers general conclusions as well as future directions for coacervate-based model systems and understanding the effects of chemical modification of polyelectrolytes on phase separation.