Song Xinbo, Li Ning, Wang Chao, Xiao Yi
State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian 116024, China.
J Mater Chem B. 2017 Jan 14;5(2):360-368. doi: 10.1039/c6tb02524b. Epub 2016 Dec 12.
It is meaningful to accurately quantify the changes in local viscosity within the mitochondria of living cells, because viscosity influences mitochondrial network organization and metabolite diffusion. Although many molecular probes targeting mitochondria have been reported, almost all of them are not fixed to the mitochondria. Thus, they may not be suitable for sensing in abnormal mitochondria with lowered potential. In order to monitor viscosity in all mitochondria, no matter their working or health status, we develop the first fixable BODIPY (boron-dipyrromethene) rotor, named Vis-A. Vis-A contains an aldehyde group as an anchor to react with amino groups of proteins, which make it immobilizable in mitochondria. Vis-B, the reference compound without such anchor unit, is also synthesized. Both Vis-A and Vis-Bshow excellent mitochondrial targetability, as good as the commercially available mitochondrial dye (Mito Tracker Deep Red). However, when cells are chemically treated to decrease the mitochondrial potential, only Vis-A continues emitting strong fluorescence in mitochondria, but the signals of Vis-B and Mito Tracker Deep Red completely disappear. This comparison confirms that Vis-A not only specifically localizes in mitochondria, but also can be stably retained there. Then, Vis-A is applied to detect the mitochondrial viscosity of living cells by Fluorescence Lifetime Imaging (FLIM). Especially, with the aid of Vis-A, the changes in viscosity under typical pathological conditions (i.e., treatment with rotenone and carbonylcyanide-m-chlorophenylhydrazone (CCCP)) for mitochondria are monitored by FLIM.
准确量化活细胞线粒体内局部粘度的变化具有重要意义，因为粘度会影响线粒体网络组织和代谢物扩散。尽管已经报道了许多靶向线粒体的分子探针，但几乎所有探针都不能固定在线粒体上。因此，它们可能不适用于检测电位降低的异常线粒体中的粘度。为了监测所有线粒体的粘度，无论其工作状态或健康状况如何，我们开发了第一种可固定的硼二吡咯亚甲基（BODIPY）转子，命名为Vis-A。Vis-A含有一个醛基作为锚定基团，可与蛋白质的氨基反应，使其能够固定在线粒体中。还合成了没有这种锚定单元的参考化合物Vis-B。Vis-A和Vis-B都表现出优异的线粒体靶向性，与市售线粒体染料（Mito Tracker Deep Red）一样好。然而，当对细胞进行化学处理以降低线粒体电位时，只有Vis-A继续在线粒体中发出强烈荧光，而Vis-B和Mito Tracker Deep Red的信号完全消失。这种比较证实，Vis-A不仅能特异性地定位于线粒体，还能稳定地保留在那里。然后，将Vis-A应用于通过荧光寿命成像（FLIM）检测活细胞的线粒体粘度。特别是，借助Vis-A，通过FLIM监测了线粒体在典型病理条件下（即用鱼藤酮和羰基氰化物间氯苯腙（CCCP）处理）粘度的变化。
