Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

Current methods of monitoring insulin secretion lack the required spatial and temporal resolution to adequately map the dynamics of exocytosis of native insulin granules in intact cell populations in three dimensions. Exploiting the fact that insulin granules contain a high level of Zn ²⁺, and that Zn ²⁺ is coreleased with insulin during secretion, we have developed a fluorescent, cell surface-targeted zinc indicator for monitoring induced exocytotic release (ZIMIR). ZIMIR displayed a robust fluorescence enhancement on Zn ²⁺ chelation and bound Zn ²⁺ with high selectivity against Ca ²⁺ and Mg ²⁺. When added to cultured β cells or intact pancreatic islets at low micromolar concentrations, ZIMIR labeled cells rapidly, noninvasively, and stably, and it reliably reported changes in Zn ²⁺ concentration near the sites of granule fusion with high sensitivity that correlated well with membrane capacitance measurement. Fluorescence imaging of ZIMIR-labeled β cells followed the dynamics of exocytotic activity at subcellular resolution, even when using simple epifluorescence microscopy, and located the chief sites of insulin release to intercellular junctions. Moreover, ZIMIR imaging of intact rat islets revealed that Zn ²⁺/insulin release occurred largely in small groups of adjacent β cells, with each forming a "secretory unit." Concurrent imaging of ZIMIR and Fura-2 showed that the amplitude of cytosolic Ca ²⁺ elevation did not necessarily correlate with insulin secretion activity, suggesting that events downstream of Ca ²⁺ signaling underlie the cell-cell heterogeneity in insulin release. In addition to studying stimulation-secretion coupling in cells with Zn ²⁺-containing granules, ZIMIR may find applications in β-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.