title: Genetic targeting of a small fluorescent zinc indicator to cell surface for monitoring zinc secretion.
abstract: 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.
authors.affiliation: Departments of Cell Biology and Biochemistry, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-9039, United States.
authors.forename: Daliang
authors.initials: D
authors.lastname: Li
authors.affiliation: Departments of Cell Biology and Biochemistry, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-9039, United States.
authors.forename: Lin
authors.initials: L
authors.lastname: Liu
authors.affiliation: Departments of Cell Biology and Biochemistry, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, Texas 75390-9039, United States.
authors.forename: Wen-Hong
authors.initials: WH
authors.lastname: Li
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D060326:Chemistry Techniques, Synthetic; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D007328:Insulin / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D062308:Optogenetics / Q000379:methods; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000032:analysis* / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D007328:Insulin; D011994:Recombinant Proteins; D015032:Zinc