SIMULTANEOUS INDEPENDENT MEASUREMENT OF ENDOCYTOSIS AND EXOCYTOSIS

STUDIES of membrane traffic between the cytoplasm and surface of a cel l suggest that membrane internalization is tightly coupled to secretio n. Studies using the capacitance technique show that endocytosis can f ollow evoked exocytosis within a second or less(1-3). 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 sm all, brief stimuli(1-5), but during prolonged stimulation it is more l ikely that exocytosis and endocytosis occur simultaneously(6). We used FM1-43 fluorescence, which provides a cumulative measure of exocytosi s, independent of endocytosis, in combination with capacitance monitor ing to track unidirectional movements of membrane simultaneously and i n 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 qu ickly grows to equal that of exocytosis. The delayed onset of endocyto sis may be an emergency defence against catastrophic cell swelling.