Fye Margret A, Sharma Rahul, Myat Phyu Sin M, Regan Pi'ilani, McKinney Hudson, Gu Guoqiang, Kaverina Irina
Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN, USA.
bioRxiv. 2025 May 27:2025.04.22.650066. doi: 10.1101/2025.04.22.650066.
Accurate detection of insulin secretion from pancreatic beta cells is crucial for understanding normal physiological insulin secretion and its pathophysiological counterpart in diabetic states. Traditional methods using fluorescently labeled insulin granules or dye labeling often struggle to distinguish secretion from insulin granule dynamics. We present an optimized protocol using the cell-impermeable Zn-binding dye FluoZin-3, which fluoresces upon Zn co-secretion with insulin outside of the islet, more accurately representing secretion. FluoZin-3 combined with intact islet attachment to vascular extracellular matrix and TIRF microscopy offers high spatial and temporal resolution as well as a high signal-to-noise ratio in a minimally perturbed system. Additionally, by integrating the cell-permeable Zn-binding dye ZIGIR, we can track insulin granule dynamics alongside secretion events. Our approach generates large datasets, which we efficiently analyze using ilastik machine learning software, enabling fast, accurate, and optionally supervised analysis. This technique builds on our group's previous protocols, detailing a streamlined workflow adaptable to high-resolution, live-cell microscopy for not just insulin but other secretory/granule systems as well. With this method, we investigated secretion behavior of different IG pools in real time during the first phase of insulin secretion: predocked, which appear before high glucose stimulation and are docked at the membrane; docked, which appear upon high glucose stimulation and dock at the membrane; and newcomer, which appear upon high glucose stimulation but don't dock at the membrane. The predocked and newcomer insulin granules are equally secreted and newcomer insulin granules dwell less than one second before secretion upon high glucose stimulation. The predocked and docked insulin granules, however, stay longer at the membrane before secretion. This method is useful for the investigation of functional beta cell heterogeneity of insulin granule secretion in space and time.
准确检测胰腺β细胞的胰岛素分泌对于理解正常生理状态下的胰岛素分泌及其在糖尿病状态下的病理生理对应情况至关重要。使用荧光标记胰岛素颗粒或染料标记的传统方法常常难以区分分泌与胰岛素颗粒动态变化。我们提出了一种优化方案，使用细胞不可渗透的锌结合染料FluoZin-3，它在与胰岛素共同分泌锌离子到胰岛外时会发出荧光，能更准确地代表分泌情况。FluoZin-3与完整胰岛附着于血管细胞外基质以及全内反射荧光显微镜（TIRF显微镜）相结合，在最小扰动系统中提供了高空间和时间分辨率以及高信噪比。此外，通过整合细胞可渗透的锌结合染料ZIGIR，我们可以在追踪分泌事件的同时监测胰岛素颗粒动态变化。我们的方法生成了大量数据集，我们使用ilastik机器学习软件对其进行有效分析，实现快速、准确且可选择进行监督的分析。该技术基于我们团队之前的方案，详细介绍了一种简化的工作流程，适用于高分辨率活细胞显微镜观察，不仅适用于胰岛素，也适用于其他分泌/颗粒系统。通过这种方法，我们实时研究了胰岛素分泌第一阶段不同胰岛素颗粒池的分泌行为：预对接颗粒，在高糖刺激前出现并停靠在细胞膜上；对接颗粒，在高糖刺激时出现并停靠在细胞膜上；新出现颗粒，在高糖刺激时出现但未停靠在细胞膜上。预对接颗粒和新出现颗粒的分泌量相同，新出现颗粒在高糖刺激下分泌前停留时间不到一秒。然而，预对接颗粒和对接颗粒在分泌前在细胞膜上停留的时间更长。该方法有助于研究胰岛素颗粒分泌在空间和时间上的功能性β细胞异质性。
