Visualization of Partial Exocytotic Content Release and Chemical Transport into Nanovesicles in Cells

For decades, "all-or-none" and "kiss-and-run" were thought to be the only major exocytotic release modes in cell-to-cell communication, while the significance of partial release has not yet been widely recognized and accepted owing to the lack of direct evidence for exocytotic partial release. Correlative imaging with transmission electron microscopy and NanoSIMS imaging and a dual stable isotope labeling approach was used to study the cargo status of vesicles before and after exocytosis; demonstrating a measurable loss of transmitter in individual vesicles following stimulation due to partial release. Model secretory cells were incubated with ¹³C-labeled l-3,4-dihydroxyphenylalanine, resulting in the loading of ¹³C-labeled dopamine into their vesicles. A second label, di-N-desethylamiodarone, having the stable isotope ¹²⁷I, was introduced during stimulation. A significant drop in the level of ¹³C-labeled dopamine and a reduction in vesicle size, with an increasing level of ¹²⁷I^-, was observed in vesicles of stimulated cells. Colocalization of ¹³C and ¹²⁷I^- in several vesicles was observed after stimulation. Thus, chemical visualization shows transient opening of vesicles to the exterior of the cell without full release the dopamine cargo. We present a direct calculation for the fraction of neurotransmitter release from combined imaging data. The average vesicular release is 60% of the total catecholamine. An important observation is that extracellular molecules can be introduced to cells during the partial exocytotic release process. This nonendocytic transport process appears to be a general route of entry that might be exploited pharmacologically.