PROBING SECRETORY VESICLES AND LIPOSOME MODEL SYSTEMS USING NANOSCALE ELECTROCHEMISTRY AND MASS SPECTROMETRY

Doctoral thesis, 2016

Cellular communication is based on the process of exocytosis, regulated release of chemical messengers into the extracellular space. These messengers, neurotransmitters, are packed into vesicles that during exocytosis fuse with the plasma membrane and release their content. Much work has been done to understand the mechanism of exocytosis, whether it is full or transitory transmitter release or a combination of these modes of exocytosis. However, its full comprehension is still under debate. One of the important pieces of the exocytosis puzzle and relevant evidence for the fractional exocytosis, is probing the entire neurotransmitter content of secretory vesicles. Despite the challenge to achieve this, owing to small vesicle size and often less than an attomole of detectable material, advances in bioanalytical techniques have allowed measurements at higher spatial resolution and with decreasing amounts of analytes. In this thesis, I have employed electrochemical techniques and imaging mass spectrometry as analytical tools to study the content of large dense core vesicles of neuroendocrine cells and liposome systems as secretory vesicle models. In the first part of the thesis work, Papers I and II, I applied a new amperometric technique called vesicle electrochemical cytometry to measure the catecholamine content in native vesicles and liposomes. In Paper I, this technique was employed to quantify the content of catecholamines present in single mammalian vesicles isolated from cells of the bovine adrenal gland. Paper II represents the continuation of work done in Paper I, where the same experimental setup was employed with focus on investigation of the mechanism of vesicle rupture onto the electrode by applying a bottom up approach and probing the transmitter loaded liposomes. The second part of this thesis, Papers III, IV and V, describe the application of imaging mass spectrometry to reveal the chemical composition of vesicles and liposomes. In Paper III, time-of-flight secondary ion mass spectrometry (ToF-SIMS) was employed to study the chemistry of micrometer size liposome models containing histamine and liposomes containing an aqueous two-phase system, both to mimic secretory vesicles. Paper IV demonstrates the potential of ToF-SIMS to evaluate the preservation capabilities of chemical fixation, a common approach for sample preparation in subcellular imaging as well as a screening tool for optimization of high-resolution NanoSIMS imaging. In Paper V, NanoSIMS, and electrochemical techniques were used in combination, to study the neurochemistry of large dense core vesicles from PC12 cells. The major goals were to investigate the impact of pharmaceuticals like L-3,4-dihydroxyphenylalanine and reserpine on metabolic pathways of neurotransmitter dopamine and to quantify dopamine content in PC12 vesicles, one vesicle at a time and in subvesicular regions.