Giant Lipid Vesicles Encapsulating A Synthetic Cytoplasm As A Model System To Investigate Cellular Fission and Differentiation
Open Access
- Author:
- Koback, Meghan Jo
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- December 12, 2011
- Committee Members:
- Christine Dolan Keating, Dissertation Advisor/Co-Advisor
Barbara Jane Garrison, Committee Chair/Co-Chair
Nicholas Winograd, Committee Member
Scott A Showalter, Committee Member
Peter J Butler, Special Member - Keywords:
- ATPS
Aqueous phase separation
Aqueous Two-Phase System
Polyethylene glycol
Dextran
Giant Vesicles (GVs)
lipid bilayer
vesicles
liposomes - Abstract:
- Biological cells exhibit chemical heterogeneity in both their cytoplasmic interiors and their plasma membranes. This heterogeneity has consequences for cell function, such as polarity and asymmetric division, yet studying the underlying mechanisms has proven difficult due to the complexity of even the simplest living cell. Models of biological cells provide a tool for testing hypotheses in cellular biology. For example, giant lipid vesicles (GVs) are cell-sized structures composed of a lipid bilayer membrane surrounding an aqueous core, that serve as model cells or parts of cells. By encapsulating an aqueous two-phase system (ATPS) it has been possible to generate microcompartments within GVs. This work is interesting in that aqueous phase separation is one proposed mechanism for dynamic microcompartmentalization in the cytoplasm of biological cells, and our model provides a means for examining the consequences of this type of heterogeneity in a simple system. Previous work has demonstrated osmotic deflation, temperature, and pH-driven dynamic microcompartmentation within the GV interior, as well as structural and cytoplasmic polarity. This thesis examines the consequences of this initial polarity, focusing on asymmetric fission and differentiation in this model cell. While the overall goal of this thesis is concerned with polar ATPS GVs, Chapter 5 answers fundamental questions about the non-ideal behavior of two-polymer solutions, which has direct consequences for our artificial cell work. Chapter 1 provides a brief introduction to ATPS GVs as a simple model system for phase-separation driven microcompartmentation of the cytoplasm. Background information on microcompartmentation in biological cells and its consequences for cell function is presented. Examples of experimental models of the cytoplasm are reported, and shortcomings of these models are discussed. An introduction to GVs as membrane models, and model cells is provided. The chapter concludes with a summary of previous work incorporating an ATPS cytoplasm mimic inside GV model cells. Chapter 2 describes ATPS GVs presenting micron-scale domains in their bilayer membranes. It describes the effects of PEGylated lipids and temperature on domain localization, as well as the interplay between the membrane and interior chemistry. Additionally, osmotic deflation induced budding of these ATPS GVs with heterogeneous membranes is reported, which resulted in vesicles that were asymmetric, or polar, in their structure as well as the membrane and interior compositions. Chapter 3 builds on this initial polarity and describes the consequences of osmotic deflation of these asymmetric GVs. Further increases in external osmolality drove fission of polar GVs, to produce two non-identical daughter vesicles that were different in their membrane and interior “cytoplasmic” compositions. Additionally, osmotic-stress induced aqueous phase separation and polarity in daughter vesicles is reported. In Chapter 4, the differential segregation of denatured proteins and preferential accumulation in one daughter vesicle, but not the other, upon asymmetric fission of an ATPS GV is presented. The asymmetric segregation of aggregates is believed to play a role in cellular aging, and the work described in Chapter 4 provides insight into possible mechanisms by which this biased aggregate accumulation may occur in living systems. Chapter 5 describes observed non-ideal properties of ATPS, and measured polymer-polymer interactions, in order to gain insight into the driving forces of ATPS phase separation- information that is fundamentally important for our synthetic cell work. Overall, this thesis presents important findings on ATPS phase behavior, and demonstrates a model cell capable of polarity and asymmetric cell division. This model cell could potentially serve as a test bed for examining hypotheses in cell biology (e.g. the role of membrane composition in cellular polarity; malfunctions in asymmetric inheritance; spatial and organization cues in polarity induction, etc.).