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. 2015 Apr 17;10(4):1054-63.
doi: 10.1021/cb5007536. Epub 2015 Jan 27.

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

Daliang Li  1 , Lin Liu  1 , Wen-Hong Li  1
Affiliations

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

Daliang Li et al. ACS Chem Biol. .

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.

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Conflict of interest statement

Notes

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
(A) Structure of ZIMIR and the design of ZIMIR-HaloTag. ZIMIR-HaloTag contains a fluorescent zinc sensor and a reactive chloroalkane handle. (B) Structures of HaloTag AF488 and three fluorescein-based model compounds (Fluo HaloTag-1, -2, and -3) designed to optimize the linker length suitable for ZIMIR-HaloTag.
Figure 2
Figure 2
(A) Schematic of a fusion protein (HaloTag/R-GECO1.2) containing HaloTag (extracellular) and R-GECO1.2 (intracellular) linked by a transmembrane peptide. (B–E) Two-color imaging of cells transfected with HaloTag/R-GECO1.2 and labeled with fluorescent HaloTag substrates (1–2 μM) including HaloTag AF488 (B), Fluo HaloTag-1 (C), Fluo HaloTag-2 (D), or Fluo HaloTag-3 (E). The HaloTag substrates were cell impermeable so they only labeled HaloTag displayed on the cell surface. The fusion protein, however, was found to be localized at both the plasma and intracellular membranes after transient transfection. Scale bar = 5 μm.
Figure 3
Figure 3
Synthesis of ZIMIR-HaloTag. (a) KOtBu, ethyl bromoacetate, THF, r.t., overnight, 60%; (b) TFA, DCM, r.t., overnight; (c) K2CO3, tert-butyl bromoacetate, CHCl3, r.t., overnight, 50% (steps b and c); (d) AlCl3, nitrobenzene, r.t., 16 h, 91%; (e) CH3SO3H, resorcinol, 90 °C, 24 h; (f1) Cs2CO3, Piv2O, DMF, 2 h, 48%; (f2) NBS, BPO, CCl4, 4 h, 84% (both isomers); (g) NaI, proton sponge, CH3CN, reflux, overnight, 70%; (h) NaSH, MeOH/THF/H2O (4:1:1), reflux, 1 h, 90%; (i) Na2SO4, NaCNBH3, MeOH, 34%; (j1) PyBOP, DMF, r.t., overnight, 50%; (j2) TFA, DCM, r.t., 4 h.
Figure 4
Figure 4
Characterization of ZIMIR-HaloTag. (A) Zn2+-dependent fluorescence enhancement of ZIMIR-HaloTag. Zn2+ concentrations (from bottom to top) were 4, 25, 50, 150, 250, 450, 950, and 1950 nM. (B) Zn2+ titration curve of ZIMIR-HaloTag as measured from its emission at 515 nm. The solid line represents the fit using the equation mentioned in the Methods.
Figure 5
Figure 5
Live cell imaging of Zn2+/insulin release with ZIMIR-HaloTag. (A) Example images of MIN6 beta cells cotransfected with HaloTag (pDisplay) and Lyn-R-GECO1.2 and sequentially incubated in the basal medium with 10 μM EDTA and 2 μM DPAS, 40 mM KCl, basal medium, and a high Zn2+ buffer (1 μM). Scale bar = 5 μm. (B) Corresponding time courses of Lyn-R-GECO1.2 and ZIMIR-HaloTag fluorescence at the release site (outlined by the yellow line in A). At the end of the experiment, cells were washed with the basal medium containing 2.5 mM EDTA. (C) Average ZIMIR-HaloTag fluorescence enhancement (peak value over baseline, N = 11 cells) in response to repetitive KCl stimulations or addition of 1 μM Zn2+.
Figure 6
Figure 6
Activation of channelrhodopsin led to Zn2+/insulin secretion events in discrete membrane domains in beta cells. (A) Example images of MIN6 beta cells cotransfected with ChR2(T159C)-R-GECO1.2 and HaloTag (pDisplay). Scale bar = 5 μm. (B) Time courses of R-GECO1.2 and ZIMIR-HaloTag in three example regions of interest (ROIs 1, 2, and 3 as outlined in panel A) during photostimulation (1 s × 9 repetitions, represented by blue bars at the top of the plot during the first 100 s) and post-stimulation (150–250 s). (C) Average number of observed Zn2+/insulin release events per cell either with light stimulation (1 s × 9 repetitions) or without light activation (basal). N = 6 cells. (D) Histogram of cellular membrane subdomains exhibiting different activities of Zn2+/insulin secretion during ChR2(T159C) activation. The analysis arbitrarily divided the entire plasma membrane of individual cells into small ROIs and counted Zn2+/insulin secretion events within each ROI. N = 6 cells with ~16 ROIs/cell. (E) Corresponding cellular Ca2+ activities of three classes of ROI showing different secretory activities analyzed in panel D.
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