A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells
A pH-independent DNA nanodevice for quantifying chloride transport in organelles of living cells
复制标题
DOI：
10.1038/nnano.2015.130
复制
发表时间：
2015-07-01
影响因子：
38.3
通讯作者：
Krishnan, Yamuna
中科院分区：
文献类型：
--
作者：
Saha, Sonali;Prakash, Ved;Krishnan, Yamuna
活细胞细胞质和亚细胞器中氯离子的浓度范围很宽（5-130 mM），并受到细胞内氯离子通道或转运蛋白的严格调节。氯离子敏感蛋白报告基因已被用来研究这些氯离子调节剂的作用，但它们仅限于较小的氯离子浓度范围，并且对 pH 值敏感。在这里，我们证明 DNA 纳米装置可以以与 pH 无关的方式精确测量活细胞中亚细胞氯离子通道和转运蛋白的活性和位置。这种 DNA 纳米装置称为 Clensor，由传感、标准化和靶向模块组成，旨在沿着内溶酶体途径定位在细胞器内。它可以在整个生理状态下对氯离子进行荧光比例传感。我们使用 Clensor 定量果蝇酸性细胞器管腔中的静息氯化物浓度。我们发现，与各种溶酶体贮积病有关的腔内溶酶体氯化物受到细胞内氯化物转运蛋白 DmClC-b 的调节。
The concentration of chloride ions in the cytoplasm and subcellular organelles of living cells spans a wide range (5-130 mM), and is tightly regulated by intracellular chloride channels or transporters. Chloride-sensitive protein reporters have been used to study the role of these chloride regulators, but they are limited to a small range of chloride concentrations and are pH-sensitive. Here, we show that a DNA nanodevice can precisely measure the activity and location of subcellular chloride channels and transporters in living cells in a pH-independent manner. The DNA nanodevice, called Clensor, is composed of sensing, normalizing and targeting modules, and is designed to localize within organelles along the endolysosomal pathway. It allows fluorescent, ratiometric sensing of chloride ions across the entire physiological regime. We used Clensor to quantitate the resting chloride concentration in the lumen of acidic organelles in Drosophila melanogaster. We showed that lumenal lysosomal chloride, which is implicated in various lysosomal storage diseases, is regulated by the intracellular chloride transporter DmClC-b.