Simultaneous independent measurement of endocytosis and exocytosis.

Studies of membrane traffic between the cytoplasm and surface of a cell suggest that membrane internalization is tightly coupled to secretion. Studies using the capacitance technique show that endocytosis can follow evoked exocytosis within a second or less. The capacitance technique, however, measures only the net change in cell surface area, and thus separating exocytosis from endocytosis requires that the two events do not overlap in time. This condition is probably met with small, brief stimuli, but during prolonged stimulation it is more likely that exocytosis and endocytosis occur simultaneously, We used FM1-43 fluorescence, which provides a cumulative measure of exocytosis, independent of endocytosis, in combination with capacitance monitoring to track unidirectional movements of membrane simultaneously and in real time in bovine adrenal chromaffin cells. We confirm that, with relatively small stimuli, exocytosis ceases before endocytosis begins (no overlap). In contrast, during prolonged stimulation, the onset of endocytosis is delayed by 2-3 min, but then the rate of endocytosis quickly grows to equal that of exocytosis. The delayed onset of endocytosis may be an emergency defence against catastrophic cell swelling.