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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)
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Imaging dynamic insulin release using a fluorescent zinc indicator for monitoring induced exocytotic release (ZIMIR)

机译：使用荧光锌指示剂对动态胰岛素释放进行成像，以监测诱导的胞吐释放（ZIMIR）

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摘要

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+) che-lation 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, noninva-sively, 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 p-cell engineering and screening for molecules regulating insulin secretion on high-throughput platforms.
机译：当前监测胰岛素分泌的方法缺乏所需的空间和时间分辨率，无法在三个维度上完整地描绘完整细胞群体中天然胰岛素颗粒的胞吐动力学。利用胰岛素颗粒含有高水平的Zn〜（2+）以及在分泌过程中Zn〜（2+）与胰岛素共释放这一事实，我们开发了一种荧光的，面向细胞表面的锌指示剂，用于监测诱导的胞吐释放（ZIMIR）。 ZIMIR对Zn〜（2+）的螯合和结合的Zn〜（2+）具有较强的荧光增强作用，对Ca〜（2+）和Mg〜（2+）具有很高的选择性。当以低微摩尔浓度添加到培养的β细胞或完整的胰岛中时，ZIMIR标记的细胞迅速，无创且稳定，并以高灵敏度可靠地报告了颗粒融合部位附近Zn〜（2+）浓度的变化，与膜电容测量密切相关。 ZIMIR标记的β细胞的荧光成像即使在使用简单的落射荧光显微镜检查时，也能以亚细胞分辨率跟踪胞吐活性的动态变化，并将胰岛素释放的主要部位定位在细胞间连接处。此外，对完整大鼠胰岛的ZIMIR成像显示，Zn〜（2 +）/胰岛素的释放主要发生在一小群相邻的β细胞中，每一个都形成一个“分泌单位”。 ZIMIR和Fura-2的并发成像显示，胞浆Ca〜（2+）升高的幅度不一定与胰岛素分泌活性相关，这表明Ca〜（2+）信号下游的事件是细胞内异质性的基础。胰岛素释放。除了研究含Zn〜（2+）的颗粒在细胞中的刺激-分泌偶联外，ZIMIR还可用于p细胞工程和筛选高通量平台上调节胰岛素分泌的分子。

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  • 作者

    Daliang Li; Shiuhwei Chen; Elisa A. Bellomo; Andrei I. Tarasov; Callan Kaut; Guy A. Rutter; Wen-hong Li;

  • 作者单位

    Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039;

    Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039;

    Section of Cell Biology, Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Imperial College London, London SW7 2AZ, United Kingdom;

    Section of Cell Biology, Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Imperial College London, London SW7 2AZ, United Kingdom;

    Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039;

    Section of Cell Biology, Division of Diabetes, Endocrinology, and Metabolism, Department of Medicine, Imperial College London, London SW7 2AZ, United Kingdom;

    Departments of Cell Biology and of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390-9039;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    probe development; zinc imaging; hormone secretion assay;

    机译：探针开发;锌成像激素分泌测定;

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