Genetic targeting of a small fluorescent zinc indicator to cell surface for monitoring zinc secretion

Numerous mammalian cells contain Zn²⁺ in their secretory granules. During secretion, Zn²⁺ is coreleased with granular cargos into extracellular medium so Zn²⁺ serves as a convenient surrogate marker for tracking the dynamics of secretion. Fluorescent Zn²⁺ sensors that can be selectively targeted to cells of interest would be invaluable tools for imaging Zn²⁺ release in multicellular systems including tissues and live animals. Exploiting the HaloTag labeling technology and using an optimized linker, we have engineered a fluorescent Zn²⁺ indicator that displayed a 15-fold fluorescence enhancement upon Zn²⁺ binding while reacting efficiently with a HaloTag enzyme in a cellular environment. Two-color imaging of ZIMIR-HaloTag and a red-emitting calcium indicator in pancreatic islet beta cells demonstrated that photoactivation of a channelrhodopsin was able to induce exocytosis of Zn²⁺/insulin granules and revealed heterogeneity in secretory activity along the cell membrane that was uncoupled from cellular Ca²⁺ activity. This integrated photonic approach for imaging and controlling the release of large dense core granules provides exquisite cellular selectivity and should facilitate future studies of stimulus-secretion coupling and paracrine signaling in secretory cells.