Marschall Robert, Tudzynski Paul
Institut fuer Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität Muenster, Hindenburgplatz 55, D-48143 Muenster, Germany.
Institut fuer Biologie und Biotechnologie der Pflanzen, Westf. Wilhelms-Universität Muenster, Hindenburgplatz 55, D-48143 Muenster, Germany.
Fungal Genet Biol. 2014 Oct;71:68-75. doi: 10.1016/j.fgb.2014.08.009. Epub 2014 Sep 16.
Reactive oxygen species (ROS) are produced in conserved cellular processes either as by-products of the cellular respiration in mitochondria, or purposefully for defense mechanisms, signaling cascades or cell homeostasis. ROS have two diametrically opposed attributes due to their highly damaging potential for DNA, lipids and other molecules and due to their indispensability for signaling and developmental processes. In filamentous fungi, the role of ROS in growth and development has been studied in detail, but these analyses were often hampered by the lack of reliable and specific techniques to monitor different activities of ROS in living cells. Here, we present a new method for live cell imaging of ROS in filamentous fungi. We demonstrate that by use of a mixture of two fluorescent dyes it is possible to monitor H2O2 and superoxide specifically and simultaneously in distinct cellular structures during various hyphal differentiation processes. In addition, the method allows for reliable fluorometric quantification of ROS. We demonstrate that this can be used to characterize different mutants with respect to their ROS production/scavenging potential.
活性氧（ROS）在保守的细胞过程中产生，要么作为线粒体细胞呼吸的副产物，要么是为了防御机制、信号级联反应或细胞内稳态而特意产生。由于ROS对DNA、脂质和其他分子具有高度破坏性潜力，同时又在信号传导和发育过程中不可或缺，因此具有两个截然相反的特性。在丝状真菌中，ROS在生长和发育中的作用已得到详细研究，但这些分析常常因缺乏可靠且特异的技术来监测活细胞中ROS的不同活性而受阻。在此，我们提出一种用于丝状真菌中ROS活细胞成像的新方法。我们证明，通过使用两种荧光染料的混合物，可以在各种菌丝分化过程中，在不同的细胞结构中特异性地同时监测过氧化氢（H2O2）和超氧阴离子。此外，该方法还能对ROS进行可靠的荧光定量。我们证明，这可用于根据不同突变体的ROS产生/清除潜力对其进行表征。
