Novel acid-activated fluorophores reveal a dynamic wave of protons in the intestine of Caenorhabditis elegans.
Novel acid-activated fluorophores reveal a dynamic wave of protons in the intestine of Caenorhabditis elegans.
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DOI：
10.1021/cb300396j
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发表时间：
2013-03-15
影响因子：
4
通讯作者：
Peterson, Blake R.
中科院分区：
文献类型：
--
作者：
Bender, Aaron;Woydziak, Zachary R.;Fu, Liqiang;Branden, Michael;Zhou, Zhenguo;Ackley, Brian D.;Peterson, Blake R.
与脊椎动物的消化系统不同，秀丽隐杆线虫的消化道管腔是不分段的，呈弱酸性（pH ~ 4.4），每 45 至 50 秒超电波动至 pH > 6。为了探究这种酸度的动态，我们合成了称为堪萨斯红的新型酸激活荧光团。这些罗丹明 B 的二价衍生物集中在活体秀丽隐杆线虫的肠腔中，并表现出可调节的 pKa 值 (2.3-5.4)，该值由烷基胺取代基的氟化程度控制，从而可以对体内一系列酸性液体进行成像。对自由进食这些荧光团的动物进行的荧光视频显微镜显示，秀丽隐杆线虫肠道中的酸度是不连续的；后肠包含一个大的酸性部分，其两侧是最后端具有较高 pH 值的较小区域。值得注意的是，在排便运动过程中，这种酸性热点迅速从后部肠道移动到最前部肠道，每 45 至 50 秒它就会局部化长达 7 秒。对 pH 不敏感和碱激活的荧光团以及突变和转基因动物的研究表明，这种动态的酸度波需要质子交换器 PBO-4，不涉及液体的大量运动，并且可能涉及肠细胞顶端表面质子转运蛋白的顺序激活。由于缺乏隔离低 pH 值的特定器官，线虫通过产生动态质子热点来区分酸度，这些质子有节奏地从后肠迁移到前肠。
Unlike the digestive systems of vertebrate animals, the lumen of the alimentary canal of C. elegans is unsegmented and weakly acidic (pH ~ 4.4), with ultradian fluctuations to pH > 6 every 45 to 50 seconds. To probe the dynamics of this acidity, we synthesized novel acid-activated fluorophores termed Kansas Reds. These dicationic derivatives of rhodamine B become concentrated in the lumen of the intestine of living C. elegans and exhibit tunable pKa values (2.3–5.4), controlled by the extent of fluorination of an alkylamine substituent, that allow imaging of a range of acidic fluids in vivo. Fluorescence video microscopy of animals freely feeding on these fluorophores revealed that acidity in the C. elegans intestine is discontinuous; the posterior intestine contains a large acidic segment flanked by a smaller region of higher pH at the posterior-most end. Remarkably, during the defecation motor program, this hot spot of acidity rapidly moves from the posterior intestine to the anterior-most intestine where it becomes localized for up to 7 seconds every 45 to 50 seconds. Studies of pH-insensitive and base-activated fluorophores as well as mutant and transgenic animals revealed that this dynamic wave of acidity requires the proton exchanger PBO-4, does not involve substantial movement of fluid, and likely involves the sequential activation of proton transporters on the apical surface of intestinal cells. Lacking a specific organ that sequesters low pH, C. elegans compartmentalizes acidity by producing of a dynamic hot spot of protons that rhythmically migrates from the posterior to anterior intestine.
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