New Approaches for Chemical Analysis of Single Cells and Vesicles

Doctoral thesis, 2018

Exocytosis is the major cell-to-cell communication process in the nervous system, involving the conversion of an electrical signal (e.g. action potential) to a chemical one. Signaling molecules like neurotransmitters, hormones and/or peptides are stored in vesicles inside the cell. During exocytosis, calcium triggers the release of the vesicular cargo through SNARE-complex mediated fusion of the vesicles with the plasma (outer-membrane) of the cell. Consequently, a transient pore is formed through which the vesicular cargo is released into the extracellular space and is there able to interact with receptors of target cells. Most often, the pore closes again only allowing a fraction of the cargo to be released, so called partial release or kiss-and-run exocytosis. The extent of partial release is modulated by the intracellular calcium concentration, which can be regulated by the strength of the stimuli or with pharmaceuticals. Despite the importance of this process and the efforts that have been done to resolve the fundamental regulatory mechanisms of exocytosis, much remains unknown. In order to gain understanding of how the amount of vesicular cargo released is regulated, information about the total vesicular cargo (quantal content) has to be obtained. Until recently, no method aiming for quantification of the full quantal content existed. Our group has successfully developed an electrochemical method called vesicle impact electrochemical cytometry (VIEC) that allows direct quantification of the vesicular content in secretory granules as demonstrated with large dense-core granules from chromaffin and pheochromocytoma (PC12) cells. Chromaffin cells of the adrenal medulla are the body´s stress response output, and the best studied model system for exocytosis. The large-dense core vesicles of chromaffin cells contain a very important group of neurotransmitters and hormones, namely the catecholamines (e.g. dopamine, norepinephrine and epinephrine). The catecholamines are electroactive, and can readily be oxidized at the surface of a polarized electrode to give away two electrons per molecule. By counting the number of electrons passed through the system (charge) and knowing the charge of one mole of electrons (Faraday´s constant) the number of molecules can be quantified. In the intracellular or IVIEC method a conical nanotip carbon electrode is used to pierce into the cytosol of a living cell, allowing the vesicles to adsorb onto its surface. The vesicles burst open due to the electric field at the polarized electrode and the vesicular cargo is released towards the electrode surface and is oxidized, which allows the full content to be detected. VIEC is an electrochemical method that potentially can be applied to study the quantal content of the electroactive vesicular content of other cell types like mast cells and blood platelets, that contain both histamine and serotonin.