The Mechanisms of Exocytosis Studied in Cells and Models with Amperometry
Abstract
Exocytosis is the universal basis for neuronal communication, allowing the controlled release of neurotransmitter molecules from the presynaptic cell. Exocytosis has been widely studied in many systems. However, the details of this vital process are not fully understood. Exocytosis is quantal in its nature in that the neurotransmitter is released in packages. These packages were initially assumed to be comprised of the entire vesicular content. More recently, this simplistic view has been replaced with a more complex one with several different suggested modes of release. Vesicles can, in addition to the irreversible complete collapse of the vesicle into the cell membrane, fuse transiently with the cell membrane in a process that has been termed kiss-and-run. Transient fusion has the advantage of allowing the vesicle not to release its entire content but instead potentially control the fraction of neurotransmitter molecules it lets out. To elucidate the details of the exocytotic process, it is necessary to resolve individual release events. Amperometric detection at micrometer sized electrodes is an excellent tool for this purpose, providing quantitative and detailed kinetic information about single release events. In this thesis I have employed amperometry to study the mechanisms of exocytosis at PC12 cells and in an artificial cell model.
In paper I, detection of exocytosis using amperometry and fast scan cyclic voltammetry at a microelectrode array is compared and evaluated. In paper II, amperometric detection of exocytosis is compared at a disk and a ring-shaped microelectrode, and the results are used to evaluate the diffusion coefficient of dopamine in the extracellular matrix surrounding the cell. In paper III, I define and explore a new feature of amperometric peaks recorded at PC12 cells, the post spike foot. This feature is used to show that changing the lipid composition of the cell membrane can alter the fraction of neurotransmitter released per event. In paper IV the influence of membrane lipid composition on lipid nanotube dimensions is studied using amperometry at a lipid nanotube-liposome network. It has been demonstrated that sorting of membrane lipids based on structure occurs and a dynamic method for controlling lipid nanotube diameter has been established. The same experimental setup is used in paper V, but here it is applied to PC12 cell plasma membrane vesicles and used to study the membrane dynamics of exocytosis. In this system it has been demonstrated that release occurs though two distinct mechanisms; full and partial distension of the initial fusion pore.
Parts of work
I. Zhang et al. Temporal Resolution in Electrochemical Imaging on Single PC12 Cells Using Amperometry and Voltammetry at Microelectrode Arrays. Anal. Chem. 2011 Jan;83(2):571-577 ::DOI::10.1021/ac102502g II. Trouillon et al. Evaluating the diffusion coefficient of dopamine at the cell surface during amperometric detection: disk vs. ring microelectrodes. III. Mellander et al. Amperometric post spike feet reveal most exocytosis is via extended kiss-and-run fusion. Nature Scientific Reports 2012 Nov; 2,907 ::doi::10.1038/srep00907 IV. Kurczy et al. Composition based strategies for controlling radii in lipid nanotubes. V. Mellander et al. Observation of two modes of exocytosis in an artificial cell model.
Degree
Doctor of Philosophy
University
University of Gothenburg. Faculty of Science
Institution
Department of Chemistry and Molecular Biology ; Institutionen för kemi och molekylärbiologi
Disputation
kl. 10:00 i föreläsningssal KB, Institutionen för kemi och molekylärbiologi, Kemigården 4, Göteborg.
Date of defence
2013-06-05
Date
2013-05-15Author
Mellander, Lisa
Keywords
Exocytosis
Electrochemistry
Cells
Cell models
Phospholipids
Publication type
Doctoral thesis
ISBN
978-91-628-8710-0
Language
eng