Eaglesfield Ross, Fernandez-Vizarra Erika, Lacko Erik, Caldwell Stuart T, Sloan Nikki L, Siciarz Daniel, Hartley Richard C, Tokatlidis Kostas
School of Molecular Biosciences, University of Glasgow, G12 8QQ, UK; National Renewable Energy Laboratory, Golden, CO, USA.
School of Molecular Biosciences, University of Glasgow, G12 8QQ, UK; Department of Biochemistry and Molecular and Cellular Biology, Faculty of Health and Sport Sciences, University of Zaragoza, 22002, Spain.
Redox Biol. 2025 Mar;80:103502. doi: 10.1016/j.redox.2025.103502. Epub 2025 Jan 20.
Mitochondria are major sites of reactive oxygen species (ROS) production within cells. ROS are important signalling molecules, but excessive production can cause cellular damage and dysfunction. It is therefore crucial to accurately determine when, how and where ROS are produced within mitochondria. Previously, ROS detection involved various chemical probes and fluorescent proteins. These have limitations due to accumulation of the molecules only in the mitochondrial matrix, or the need for a new protein to be expressed for every different species. We report dynamic HO flux changes within all mitochondrial sub-compartments with striking spatial resolution. We combined specific targeting of self-labeling proteins with novel HO-reactive probes. The approach is broad-ranging and flexible, with the same expressed proteins loadable with different dyes and sensors. It provides a framework for concomitant analysis of other chemical species, beyond ROS, whose dynamics within mitochondria are yet unknown, without needing to engineer new proteins.
线粒体是细胞内活性氧（ROS）产生的主要场所。ROS是重要的信号分子，但过量产生会导致细胞损伤和功能障碍。因此，准确确定线粒体中ROS何时、如何以及在何处产生至关重要。以前，ROS检测涉及各种化学探针和荧光蛋白。由于这些分子仅在线粒体基质中积累，或者每种不同物种都需要表达一种新的蛋白质，因此存在局限性。我们报告了所有线粒体亚区室中动态的HO通量变化，具有惊人的空间分辨率。我们将自标记蛋白的特异性靶向与新型HO反应探针相结合。该方法具有广泛的适用性和灵活性，相同表达的蛋白质可加载不同的染料和传感器。它为除ROS之外的其他化学物质的伴随分析提供了一个框架，这些化学物质在线粒体内的动态变化尚不清楚，而无需设计新的蛋白质。
