Objectives: 

The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space and allows the release of molecules stored in the granule lumen. After fusion, membrane is retrieved by endocytosis. Our aim is to analyze the kinetics and size of single exo- and endocytotic events in mast cells.

Materials: 

We employed capacitance measurements in the configuration of perforated patch-clamp to detect fusion and fission events. In addition, serotonin release was detected in some recordings simultaneously by amperometry.

Results: 

These measurements revealed novel aspects of the mechanisms controlling fusion and fission of single vesicles. In about 25% of the observed events, fusion occurred transiently, which facilitates rapid recycling of vesicles. The analysis of fusion pore conductance revealed a mean pore diameter of about 2 nm, similar to the initial pore diameter in a full fusion suggesting that the pore size in a transient fusion may affect the subsequent release of vesicle contents. The capacitance measurements also allowed us to investigate formation of the fission pore during endocytosis. The dynamics and regulation of fission pores involve control of the length and diameter of the vesicle neck previous to the final membrane scission. The rate of membrane fission measured as the slope of capacitance off-step was slower than the rate of membrane fusion regardless of whether events are considered reversible or irreversible. In consequence, the fission pore lifetime is longer than the fusion pore lifetime. Finally, we also detected reversible events like capacitance 'flickers' that may be interpreted as reversible endocytosis since they occur after a fission event from a vesicle of similar size.

Conclusions: 

We provided with great temporal precision the dynamics of fission and fusion pores and described the modes of exocytotic and endocytotic events in intact mast cells.