Electrochemical Detection And Total Internal Reflection Fluorescence Microscopy: Illuminating The Exocytotic Mechanism

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Exocytosis is an essential cellular process wherein membrane-bound packets, or vesicles, of neurotransmitter or hormone molecules are released into the extracellular space. Exocytosis is the method by which neurons communicate with one another at synapses, how molecules such as histamines and adrenaline are released into the bloodstream, and even how cells carry cargo from one intracellular compartment to another. Dysfunction of the exocytotic process is implicated in various neurological disorders such as Parkinson's disease and schizophrenia, and exposure to Tetanus and Botulism toxins. While exocytosis continues to be extensively studied, and links to diseases and toxins such as those mentioned above have been made, much of the molecular mechanism that enables exocytosis remains a mystery. In this dissertation, I will attempt to illuminate some small pieces of this mystery using electrochemical detection techniques, often in combination with Total Internal Reflection Fluorescence (TIRF) microscopy. Microelectrode array devices were fabricated to electrochemically detect release of transmitter molecules from vesicles amperometrically. Amperometry is a technique where an electrode held near a cell at a positive potential will oxidize molecules released from vesicles, generating a transient current spike. Fluorescent labels in the cell could be monitored simultaneously via TIRF microscopy, which excites fluorophores in only a thin layer above a coverslip. In the chapters that follow, I describe the construction of an Annular TIRF microscope. I will explore the kinetics of exocytotic events, were it was found that release of vesicular contents is fast, but diffusion of the released molecules near the cell surface was slower then expected, in contrast to what was previously believed. Then, I will detail the development and use of transparent microelectrodes in combination with TIRF microscopy to observe exocytosis through electrodes. Next I will study the effects of the addition of charged residues to the C-terminus of synaptobrevin II, a vesicle membrane protein essential for exocytosis. The addition of these charged residues inhibited exocytosis, leading to new hypotheses about the function of synaptobrevin II in the exocytotic process. Finally, I report the interaction of suspended carbon nanotubes with cell membranes, which has implications for novel cell biosensors.
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