Artificial Cells for Dissecting Exocytosis.

Abstract

The fusion of vesicles and exocytosis release of neurotransmitters into the extracellular space for detection and chemical signal decoding by neighboring cells is the key process in neuronal communication. It is important to understand what regulates exocytosis because the amount of neurotransmitters released into the synaptic cleft has a direct impact on brain function such as cognition learning and memory as well as on brain malfunctions. Much success in molecular biology can be credited for the existence of simplified model systems. Therefore, for gaining deeper insights into the details of exocytosis and what controls vesicle-mediated neurotransmission, functional artificial cells for exocytosis have been developed that can be used for studying various biophysical aspects and roles of molecules affecting exocytosis, which is difficult to study in living cells. Here, we describe the design and fabrication of specific artificial cell models and how chemical measurements at these cells can be implemented for probing dynamics of the exocytosis fusion pore and its effect on the regulation of neurochemical release. We introduce bottom-up synthetic methods for constructing model cells using protein-free giant unilamellar vesicles (GUV) as starting material, which allows further tuning of molecular complexity in a manner that is not possible in living cells and therefore can be used for dissecting the role of essential molecular components affecting the exocytosis process. The experimental setup uses microscopy video recording, micromanipulation and microelectroinjection techniques, and amperometry detection to study neurotransmitter release from these cells mimicking exocytosis.