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. 2011 Dec 27;108(52):21063-8.
doi: 10.1073/pnas.1109773109. Epub 2011 Dec 12.

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

Daliang Li  1 , Shiuhwei Chen, Elisa A Bellomo, Andrei I Tarasov, Callan Kaut, Guy A Rutter, Wen-hong Li
Affiliations

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

Daliang Li et al. Proc Natl Acad Sci U S A. .

Abstract

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Design of ZIMIR. (A) Chemical structure of ZIMIR in the Zn2+-free (nonfluorescent) and Zn2+-bound (strongly fluorescent) states. (B) Mode of action of ZIMIR for reporting local Zn2+ elevation at the membrane surface during exocytotic insulin granule fusion. The two lipophilic alkyl chains (wavy lines) anchor ZIMIR to the outer leaflet of the membrane lipid bilayer.
Fig. 2.
Fig. 2.
Characterization of ZIMIR in vitro and in cells. (A) Zn2+-dependent fluorescence enhancement of ZIMIR-C2. Zn2+ concentrations were 0 nM, 43 nM, 140 nM, 440 nM, 840 nM, 1,640 nM, and 6,440 nM (from bottom to top). (Inset) Absorption spectrum of ZIMIR-C2 changed little with respect to [Zn2+]. (B) Zn2+ titration of ZIMIR-C2 as measured from its emission at 515 nm. The solid line represents the exponential fit. (C) ZIMIR-C2 binds Zn2+ selectively against Ca2+ (1 mM) and Mg2+ (1 mM). All measurements were performed in buffers containing 100 mM Hepes, pH 7.5, with 0.4 μM ZIMIR-C2. Membrane-anchored ZIMIR reports changes of [Zn2+]e. Example ZIMIR fluorescence images of labeled INS-1 cells (D) at different [Zn2+]es and quantification of the average ZIMIR fluorescence intensity along the plasma membrane [IZIMIR(PM)] (E).
Fig. 3.
Fig. 3.
ZIMIR imaging of insulin/Zn2+ release. KCl-stimulated insulin/Zn2+ release in MIN6 cells. Example images of MIN6 cells (A, Nomarski) before (B) and after (C) KCl (40 mM) or Zn2+ (D) addition (1 μM). Scale bar, 10 μm. (E) Time courses of ZIMIR signal changes (F/F0) in two example regions of interest (ROIs, indicated in D by dashed lines) along the plasma membrane. F, fluorescence. Compared with WT β cells (F), ZIMIR responses were greatly reduced in Znt8 KO β cells (G). (H) Average ZIMIR responses [quantified as the area under the curve (AUC) from ZIMIR intensity changes over the averaged baseline signal] to different secretagogues are shown (n = 21 cells from 6 WT mice; n = 29 cells from 6 Znt8 KO mice). TBT, tolbutamide.
Fig. 4.
Fig. 4.
DPAS accelerates Zn2+ dissipation from membranes and facilitates revealing oscillatory activity of insulin/Zn2+ release. (A) Time courses of ZIMIR fluorescence intensity along the plasma membrane [IZIMIR(PM)] decay after washing INS-1 cells (initially bathed in HBS containing 1 μM Zn2+) with HBS containing EDTA (10 μM) and DPAS (0–8 μM). (B) ZIMIR imaging of insulin/Zn2+ release of MIN6 cells bathed in SAB containing EDTA (10 μM) and DPAS (4 μM). Time courses of IZIMIR(PM) fluctuation of four separate regions of interest (ROIs) and example ZIMIR images at different time points are shown (a–e; arrowheads highlight local ZIMIR increases in separate ROIs at different times). F, fluorescence. Scale bar, 10 μm.
Fig. 5.
Fig. 5.
ZIMIR labels cells throughout islets. Confocal images of mouse islets labeled with ZIMIR (A) or calcein/AM (B). Scale bar, 20 μm.
Fig. 6.
Fig. 6.
ZIMIR imaging of GSIS in intact islets. (A) Confocal ZIMIR images of an islet before (Left) and at different time points (B, a–d) after (Right) stimulation with 20 mM glucose. (Right) Images are enlarged views of four subareas containing four example regions of interest (ROIs) along cell membranes that showed strong ZIMIR response. (B) Time courses of ZIMIR fluorescence of ROI-1 through ROI-4.
Fig. 7.
Fig. 7.
Islet β cells secreted insulin at both homologous and heterologous cell-cell contacts. (A) Confocal ZIMIR image of an islet before glucose stimulation. Three regions of interest (ROIs) along intercellular contacts are shown. (B) Confocal immunohistochemical image (red, glucagon; green, insulin) of the same focal plane of the same islet as in A. ROI-1 and ROI-3 correspond to α-β contacts, and ROI-2 corresponds to a β-β contact. (C) Time courses of ZIMIR fluorescence of ROI-1 through ROI-3. Scale bar, 20 μm.
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