Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange.
Real-time imaging of exocytotic mucin release and swelling in Calu-3 cells using acridine orange.
复制标题
使用吖啶橙对 Calu-3 细胞中的胞吐粘蛋白释放和肿胀进行实时成像。
DOI：
10.1016/j.ymeth.2013.09.004
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发表时间：
2014
期刊：
影响因子：
--
通讯作者：
Grygorczyk,Ryszard
中科院分区：
文献类型：
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作者：
Shumilov,Dmytro;Popov,Alexander;Fudala,Rafal;Akopova,Irina;Gryczynski,Ignacy;Borejdo,Julian;Gryczynski,Zygmunt;Grygorczyk,Ryszard
黏液分泌是抵御吸入人体肺部的大量刺激物的第一道防线，但异常粘稠的黏液会导致许多呼吸道疾病。理解黏液病理过程受到阻碍，部分原因是缺乏适当的实验工具来标记和研究高灵敏度和时间分辨率的活细胞黏液颗粒分泌。在本报告中，我们介绍了吖啶橙（AO）的原始光谱特性，可以利用各种先进的荧光成像方法研究颗粒释放和粘蛋白肿胀。低浓度（<200 μM） AO溶液在494 nm处吸收最大，在525 nm处发射最大，荧光寿命仅为~ 1.76 ns。相比之下，高浓度（4-30 mM）有利于形成AO聚集体，观察到非常不同的吸收，最大吸收在~ 440 nm，显著红移发射，最大吸收在630 nm，荧光寿命增加10倍以上（~ 20 ns）。为了验证AO在实时成像中的潜在效用，我们对AO染色的Calu-3细胞进行了共聚焦、全内反射荧光（TIRF）和荧光寿命成像（FLIM）。我们发现细胞内颗粒与细胞质中的红移荧光光谱相似，荧光寿命长，与颗粒状AO积累一致。Calu-3细胞的机械刺激导致ao染色颗粒的多次胞外分泌事件，随后是其荧光标记内容物的胞外肿胀，在单线TIRF图像中可见快速扩大的明亮荧光斑块。它们的尺寸膨胀速率符合一级动力学，扩散系数为3.98±0.07 × 10−7cm2/s，与粘液凝胶膨胀的预期一致。随后，由于AO的扩散损失导致荧光降低，与散装水溶液相比，分泌粘液中的荧光降低了约10倍。综上所述，我们发现ao染色可以用于粘蛋白颗粒胞吐和粘蛋白肿胀的实时TIRF成像，具有高灵敏度和时间分辨率。考虑到独特的AO荧光特性，允许AO单体与聚集体选择性激发，我们的研究为未来双色激发方案和双色荧光FLIM活细胞成像分析的发展奠定了基础，具有潜在的许多生物学应用。
Mucus secretion is the first-line of defence against the barrage of irritants inhaled into human lungs, but abnormally thick and viscous mucus results in many respiratory diseases. Understanding the processes underlying mucus pathology is hampered, in part, by lack of appropriate experimental tools for labeling and studying mucin granule secretion from live cells with high sensitivity and temporal resolution. In this report we present original spectroscopic properties of acridine orange (AO) which could be utilized to study granule release and mucin swelling with various advanced fluorescence imaging approaches. Low concentration (<200 μM) AO solutions presented absorption maximum at 494 nm, emission maximum at 525 nm and only ∼1.76 ns fluorescence lifetime. By contrast at high concentrations (4–30 mM) favoring formation of AO aggregates, a very different absorption with maximum at ∼440 nm, dramatically red-shifted emission with maximum at 630 nm, and over 10-fold increased fluorescence lifetime (∼20 ns) was observed. To verify potential utility of AO for real-time imaging we have performed confocal, total internal reflection fluorescence (TIRF) and fluorescence lifetime imaging (FLIM) of AO-stained Calu-3 cells. We found similar red-shifted fluorescence spectra and long fluorescence lifetime in intracellular granules as compared to that in the cytoplasm consistent with granular AO accumulation. Mechanical stimulation of Calu-3 cells resulted in multiple exocytotic secretory events of AO-stained granules followed by post-exocytotic swelling of their fluorescently-labeled content that was seen in single-line TIRF images as rapidly-expanding bright-fluorescence patches. The rate of their size expansion followed first-order kinetics with diffusivity of 3.98 ± 0.07 × 10−7cm2/s, as expected for mucus gel swelling. This was followed by fluorescence decrease due to diffusional loss of AO that was ∼10-fold slower in the secreted mucus compared to bulk aqueous solution. In summary, we showed that AO-staining could be utilized for real-time TIRF imaging of mucin granule exocytosis and mucin swelling with high sensitivity and temporal resolution. Considering unique AO fluorescence properties that permit selective excitation of AO monomers versus aggregates, our study lays the groundwork for future development of two-color excitation scheme and two-color fluorescence FLIM live-cell imaging assay with potentially many biological applications.