Strategies for Elucidating the Biophysical Mechanisms of Exocytosis using Liposomes and PC12 Cells

Open Access

Author:

Wittenberg, Nathan James

Graduate Program:

Chemistry

Degree:

Doctor of Philosophy

Document Type:

Dissertation

Date of Defense:

June 30, 2006

Committee Members:

• Andrew Ewing, Committee Chair
• Mary Beth Williams, Committee Member
• Nicholas Winograd, Committee Member
• Erwin A Vogler, Committee Member

Keywords:

• membrane biophysics
• liposomes
• PC12 cells
• amperometry
• exocytosis

Abstract:

Exocytosis is a process that is essential for cellular chemical communication and is also the process by which new portions of membrane are delivered to the cellular surface. Exocytosis in the classical sense can be broken down into stages that include initial membrane fusion, fusion pore formation and membrane incorporation or distention. This thesis seeks to shed light on the later stages of the exocytotic mechanism following membrane fusion, and in particular the roles of the plasma and vesicular membranes in said mechanism. The first chapter of this thesis provides the introductory framework necessary for the interpretation of experimental results in the subsequent chapters. Biomembranes and their properties, along with cellular secretion and electrochemical methods in biological environments are discussed in Chapter 1. Chapter 2 describes a protein free liposome-based model system used to mimic exocytosis. The model, termed an “artificial cell,” consists of a unilamellar liposome that protrudes from a multilamellar liposome which itself is adherent to a glass coverslip. Then, by careful micromanipulation the unilamellar liposome is transformed into a network of two liposomes, one inside the other, with a lipid nanotube connection. This network has the same membrane geometry as a cell that has opened a fusion pore and is about to undergo the final stage of exocytosis. In this system the interior vesicle is filled with catechol that can be detected electrochemically as it is released in a fashion akin to cellular exocytosis. The experiments presented in the second chapter have led to the conclusion that the final stages of exocytosis can proceed on biological timescales without protein intervention and seem to suggest that if proteins are indeed involved in the final stages, they may be inhibitory. In addition, the second chapter also contains a lipid nanotube based explanation for neurotransmitter release through the fusion pore. In the course of doing the experiments described in the second chapter, it was noticed that not all of the catechol released from the interior vesicles was being detected amperometrically. So, the third chapter 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 much like a cellular synapse, with the liposome as the neurotransmitter-releasing presynaptic cell and the amperometric electrode functioning as the postsynaptic cell by “capturing” the released molecules. This kind of treatment allowed inferences to be made about the nature of neurotransmitter escape from synapses in vivo as well as predictions to be made about coulometric efficiency of amperometric detection of exocytosis in vitro from a variety of cell types. Another goal of the work with the artificial cell system was to determine how the kinetics of release can be altered and controlled by incorporation of new lipids to the liposome preparation or by changing the liposome’s solution environment. In chapter 4 a method of accelerating release from the liposomal system is described. Addition of short chain alcohols, for example, ethanol and propanol, accelerates the release process in a manner dependent on the alcohol type and concentration. Follow-up experiments to characterize this effect included compression isotherms of lipid monolayers, which showed a marked decrease in lipid packing density in alcoholic solutions. The increase in lipid disorder due to alcohol exposure is most likely the root cause of the accelerated membrane distention because previous work has shown that an increase in disorder is correlated with an increase in membrane flexibility. This would result in a membrane that would undergo distention on shorter timescales. Chapters 5 and 6 of this thesis relate to exocytosis from living PC12 cells. PC12 cells synthesize, store and release dopamine and are very useful as neuronal models. In these two chapters, PC12 cells are treated with drugs to manipulate the size of the secretory vesicles as well as incubated in high osmolarity solutions to lower the tension on the plasma membrane. Experimental results obtained with these pharmacological manipulations have led to the development of a model of neurotransmitter release through the fusion pore that takes into account transpore membrane tensions. This model assumes the fusion pore in some cases takes on a transient, nanotubular geometry and behaves like a lipid nanotube in liposome micropipette aspiration experiments. Chapter 7 outlines future directions in modeling exocytosis. This chapter includes preliminary data as well as suggested experiments.