The mechanisms of exocytosis currently represent one of the most exciting topics in cell biology. The process of regulated exocytosis is responsible for release of neurotransmitters and neuropeptides by nerve terminals and endocrine cells, release of enzymes or cytotoxic proteins by granulocytes, release of histamine and other mediators by mast cells, as well as several other secretory processes. During exocytosis the membrane of secretory vesicles fuses with the plasma membrane of the cell, allowing the secretory vesicles to release their contents through the fusion pore. We combine of functional biophysical measurements with molecular biology techniques to elucidate the mechanism of membrane fusion.
Before fusion, vesicles are first tethered at the target membrane. We performed the first measurements of tethering forces applying optical tweezers to manipulate natural secretory granules.
Fusion of vesicles with the plasma membrane increases the plasma membrane area leading to proportional changes of membrane capacitance. In whole-cell patch clamp experiments using chromaffin cells expressing genetically modified proteins capacitance measurements are performed to investigate the dynamics of exocytosis stimulated by photorelease of caged calcium.
The highest resolution to investigate single vesicle fusion events is obtained in measurements of membrane capacitance using capacitance measurements on small membrane patches of living cells. In addition, release of oxidizable substances from single vesicles can be studied by amperometry using a carbon fiber electrode. With these methods we can record the opening of single fusion pores having molecular dimensions and the dynamics of transmitter release during pore opening.
For high throughput we develop microchip devices that will record single vesicle fusion events from hundreds of cells simultaneously.
To relate membrane fusion to molecular events we develop transparent electrochemical detector devices for simultanaous electrochemical detection of fusion and fluorescence imaging of molecular events.
