ZIGIR, a Granule-Specific Zn2+ Indicator, Reveals Human Islet α Cell Heterogeneity

Abstract

Numerous mammalian cells contain abundant Zn²⁺ in their secretory granules, yet available Zn²⁺ sensors lack the desired specificity and sensitivity for imaging granular Zn²⁺. We developed a fluorescent zinc granule indicator, ZIGIR, that possesses numerous desired properties for live cell imaging, including >100-fold fluorescence enhancement, membrane permeability, and selective enrichment to acidic granules. The combined advantages endow ZIGIR with superior sensitivity and specificity for imaging granular Zn²⁺. ZIGIR enables separation of heterogenous β cells based on their insulin content and sorting of mouse islets into pure α cells and β cells. In human islets, ZIGIR facilitates sorting of endocrine cells into highly enriched α cells and β cells, reveals unexpectedly high Zn²⁺ activity in the somatostatin granule of some δ cells, and uncovers variation in the glucagon content among human α cells. We expect broad applications of ZIGIR for studying Zn²⁺ biology and Zn²⁺-rich secretory granules and for engineering β cells with high insulin content for treating diabetes.

Keywords: FACS islet cell; TIRF imaging; ZIGIR; Zinc sensor; ZnT8; alpha cell heterogeneity; beta cell; delta cell; dense core granule; zinc imaging.

Figure 1.. Design and Characterization of ZIGIR

(A) Chemical structure of ZIGIR and its mode of action for sensing Zn²⁺. (B) Absorption spectra of ZIGIR in the presence or absence of Zn²⁺. (C) Fluorescence emission spectra of ZIGIR (Exitation = 545 nm) at increasing Zn²⁺ concentrations: 0.63, 40, 160, 400, 630, and 6,300 nM (from bottom to top). (D) Zn²⁺ titration of ZIGIR as measured from its emission at 572 nm. The solid line represents the least-squares exponential fit. (E) ZIGIR is refractory to physiological pH fluctuation and maintains its Zn²⁺ responsivity between pH 5 and pH 9. ZIGIR emission was measured in either nominally Zn²⁺-free solutions or in 10 μM Zn²⁺ solutions. The insert shows the effect of pH on ZIGIR fluorescence from pH 3.5 to pH 9.3 in either the presence (filled square) or the absence of Zn²⁺ (open triangle). (F) Summary of photophysical properties and Zn²⁺ responses. ϕ_fl is the fluorescence quantum yield, ε is the extinction coefficient, N_h is the Hill coefficient, and FC is the fold change in fluorescence brightness (ϕ_fl × ε) from the Zn²⁺-free to the Zn²⁺-bound state.

Figure 2.. ZIGIR Selectively Labels the Insulin Granule and Maintains Zn²⁺ Responsivity in Living β Cells

(A-D) Confocal imaging of MIN6 cells labeled with ZIGIR (live cells) and subsequently fixed and permeabilized for immunofluorescence using antibodies against marker proteins of cellular organelles. The scatterplot (B) of the cellular ZIGIR intensity and the corresponding insulin immunofluorescence showed a Pearson’s R value of 0.80 ± 0.06 (mean ± SD, n = 116 cells). Nuclei are shown in blue with Hoechst 33342. (E and F) Measurement of granule size by ZIGIR imaging. Confocal images of ZIGIR-stained MIN6 cells (E) were analyzed by ImageJ (analyze particles plugin). ZIGIR-positive puncta from 0.01 to 0.2 μm² (particle diameter of 100–500 nm) were analyzed to yield the granule diameter distribution (F) with a mean diameter of 270 ± 100 nm (SD, 252 granules). (G and H) ZIGIR was selectively enriched in the granule and responded to Zn²⁺ elevation. Confocal images of MIN6 cells labeled with ZIGIR in the basal secretion assay buffer (SAB) or after adding Zn/pyrithione (20/10 μM). Quantification of the granular and bulk cytoplasm ZIGIR signal is shown in (H) (mean ± SEM, >600 granules analyzed for each condition; the ZIGIR intensity ratio between granule and cytosol is shown above the bar). (I and J) ZIGIR signal decreased with reducing cellular Zn²⁺ concentration. Confocal images of MIN6 cells labeled with ZIGIR in SAB or after adding TPEN (100 μM). Quantification of the ZIGIR signal at different TPEN concentrations (J; mean ± SEM, >200 granules analyzed for each condition). TPEN, at 25, 50, or 100 μM, dampened the ZIGIR signal by 11-, 37-, or 76-fold, respectively. Scale bar, 5 μm. ZIGIR was loaded to cells at 1 μM for 15 min at 37°C for all experiments.

Figure 3.. A Unique Combination of Properties Endows ZIGIR with Unprecedented Specificity for Labeling Zn²⁺-Rich Secretory Granules

(A-F) Only ZIGIR, not other Zn²⁺ sensors, selectively labels insulin granules in β cells. MIN6 cells were co-loaded with ZIGIR (1 μM) and FluoZin-3/AM (5 μM, A-C) or with ZIGIR and ZnAF3-Ac (2 μM, D-F) and imaged by confocal microscopy first in the basal SAB solution and subsequently in a high Zn²⁺ solution (Zn/pyrithione, 20/10 μM). Cell nuclei are shown in blue with Hoechst 33342. Scale bar, 5 μm. (G-L) ZIGIR is acidotropic and accumulates in acidic granules. Confocal images of H1299 cells (G-I) or U2OS cells (J-L) labeled with ZIGIR in the basal SAB (H and K) or after adding Zn/pyrithione (I and L). LTG (0.4 μM) was added to H1299 cells in the high Zn²⁺ buffer (I, middle image). After ZIGIR imaging, U2OS cells were fixed and stained with antibodies against LAMP2 and GM130 (L, middle image). Scale bar, 10 μm.

Figure 4.. ZIGIR Is a Versatile Probe for Imaging Granule Movement and Exocytosis and for Sorting β Cells Based on Their Insulin Content

(A) Tracking insulin granule exocytosis by TIRF imaging and ZIGIR labeling. Consecutive TIRF images of INS-1 β cells post-KCl stimulation. Images were taken at 1 frame/s. A granule (highlighted by arrows) undergoing exocytosis released ZIGIR into the medium as a cloud (frame 4) and disappeared afterward (cf. Video S5). Scale bar, 1.5 μm. (B-F) ZIGIR as a surrogate marker of insulin granule content for sorting β cells. ZIGIR-high and ZIGIR-low MIN6 β cells were isolated by FACS from the ZIGIR histogram (B) and assayed for their insulin content (C, mean ± SEM of 3 replicates, *p < 0.05). (D-F) Quantification of immunofluorescence of NPC-1 (lysosome), GM130 (Golgi), or insulin of sorted ZIGIR-high or ZIGIR-low MIN6 cells (D, mean ± SEM of 50 cells, ****p < 0.001). Representative confocal immunofluorescence images of sorted MIN6 cells stained with GM130 and insulin (E) or with NPC-1 and insulin (F). Nuclei are marked by DAPI in blue. Scale bar, 10 μm.

Figure 5.. Flow Cytometry Analysis of Granular Zn²⁺ Activity and Sorting of Mouse Islet Cells with ZIGIR

(A) Workflow of cell labeling, FACS, and post-sorting analysis. (B) Flow cytometry histogram of ZIGIR (top) and the corresponding 2D scatterplot (bottom) of mouse islet cells labeled with ZIGIR and Ex4-Cy5. (C) Confocal immunofluorescence images (left column) of sorted islet cells using antibodies against three islet hormones. Cell-type distributions in each subset of sorted cells are shown to the right (mean ± SEM for 3 replicates; >200 cells were analyzed for P1 or P2 and >60 cells were analyzed for P3 or P4 in each replicate). Us, cells unstained by any of the three hormone antibodies. (D) Confocal immunofluorescence images of a mouse pancreas section stained with antibodies against islet hormones and ZnT8. The enlarged images of the area highlighted by the dashed box are shown in the bottom row, with ZnT8 pseudo-colored in red and individual hormones in green to aid visualization of expression overlap. (E) Only ZIGIR, not other Zn²⁺ sensors, could resolve distinct islet endocrine cells according to their granular Zn²⁺ levels. Flow cytometry histograms of mouse islet cells labeled with ZIGIR and three other fluorescent Zn²⁺ sensors. Also see Figures S5B–S5E. Scale bar, 20 μm.

Figure 6.. Flow Cytometry Analysis and Sorting of Human Islet Cells with ZIGIR

(A) Workflow of human islet labeling and analysis. (B) 2D scatterplot of human islet endocrine cells (donor SAMN10737781) by ZIGIR and TM4SF4 labeling. Islet endocrine cells were sorted into three subsets. (C) Cell composition of the sorted P1, P2a, and P2b subsets analyzed by immunofluorescence. Cells that were negatively stained for all three hormones (Ins, Gcg, and Sst) were designated as unstained (us). N is the total number of cells that were imaged and analyzed. (D) Confocal images of Gcg immunofluorescence of sorted P2a and P2b subsets. Scale bar, 50 μm. (E) Quantification of Gcg immunofluorescence of sorted cells. (mean ± SEM, >50 cells for each subset, ****p < 0.0001).

Figure 7.. ZnT8 Is Expressed in Three Major Endocrine Cells of Human Pancreatic Islets

Confocal immunofluorescence images of a human pancreas section stained with antibodies against three islet hormones and ZnT8. The enlarged images of the area highlighted by the dashed box are shown in the panels below, with ZnT8 pseudo-colored in red and individual hormones in green to aid visualization of the expression overlap. Scale bar, 20 μm.