Investigations of Phopholipid Membrane Properties Using An Artificial Cell Model for Exocytosis and Membrane Disruption

Open Access
Author:
Eves, Daniel Jacob
Graduate Program:
Chemistry
Degree:
Doctor of Philosophy
Document Type:
Dissertation
Date of Defense:
August 16, 2007
Committee Members:
  • Andrew Ewing, Committee Chair
  • Christine Dolan Keating, Committee Member
  • Erin Elizabeth Sheets, Committee Member
  • Ryan S Clement, Committee Member
Keywords:
  • phospholipids
  • electrochemistry
  • fluorescence
  • liposomes
  • exocytosis
Abstract:
Exocytosis is a fundamental cellular process that propagates neurochemical signals as well as delivers new lipid material to cellular membranes. Exocytosis exhibits various stages which include initial membrane fusion, fusion pore formation and membrane distention or incorporation. This thesis seeks to shed light on the later stages of the exocytotic mechanism following membrane fusion. The first chapter of this thesis gives a framework to interpret experimental results in the subsequent chapters. Properties of biomembranes along with exocytosis and electrochemical means of measuring exocytosis are discussed in Chapter 1. Chapter 2 deals with a quantitative explanation of why and how much catechol escapes the membrane-electrode gap undetected, depending on vesicle size and the size of the detection electrode. The membrane-electrode interface in this model system is similar to a synapse, the liposome as the presynaptic terminal and the electrode acts as the postsynaptic side when it detects the released molecules. This allowed for predictions to be made about coulometric efficiency of amperometric detection in exocytosis in vitro from a variety of cell types. The third chapter investigates the role of lipid headgroups in exocytosis. Various headgroups are added to the soy polar extract and the release characteristics for each lipid additive. This study looks to understand the importance of lipid headgroup composition in the cell membrane from an exocytosis point of view. This study looks toward understanding microdomains in a purely lipidic sense, able to disregard the protein interactions that would be present in a cellular system. To further characterize our lipid model system, in chapter 4 the lipid nanotube that connects the two liposomes for our exocytosis model is measured using steady state electrochemistry. The nanotube has been a fundamental part of the liposome model as it approximates the fusion pore. By measuring the diameter, a better understanding of the forces exerted upon the membrane can be determined. Chapter 5 contains the fabrication of a new microelectrode array that can be used to add spatial resolution to the excellent temporal resolution afforded by amperometry. This electrode array allows for the identification of “hot zones” of exocytosis from a single cell. The characterization of the electrode shows the versatility of this method of detection. In order to further understand membrane dynamics, Chapter 6 investigates the membrane disruption by antibacterial polymers. Based on the antibacterial agents used to kill e-coli, multiple polymers were synthesized to access their relative potency. Then the polymers were applied to the exterior of a giant unilamellar liposome to ascertain their effectiveness in rupturing the membrane. This study gives insight into the relationship between membrane rupture and antibacterial activity. Chapter 7 outlines future directions in modeling exocytosis. This chapter includes preliminary data as well as suggested experiments.