van Manen Henk-Jan, van Bruggen Robin, Roos Dirk, Otto Cees
Biophysical Engineering Group, Faculty of Science & Technology, Institute for Biomedical Technology, BMTI, and MESA+ Institute for Nanotechnology, University of Twente, Enschede, The Netherlands.
Antioxid Redox Signal. 2006 Sep-Oct;8(9-10):1509-22. doi: 10.1089/ars.2006.8.1509.
The phagocyte NADPH oxidase is a key component of the innate immune response against invading microorganisms, because the generation of superoxide (O(2)(-)) inside the phagocytic vacuole by this enzyme is responsible for microbial killing by mechanisms that are directly or indirectly dependent on reactive oxygen species (ROS) formation. Most of what is known about the membrane-embedded and cytosolic NADPH oxidase subunits and their intricate network of interactions on assembly and activation has been derived from biochemical and biophysical studies involving subcellular fractionation or reconstituted cell-free systems. Such investigations can be complemented by single-cell microscopy on phagocytes, which may reveal spatial and/or temporal details about NADPH oxidase assembly that cannot be obtained from fractionated-cell assays. In recent years, we have investigated the NADPH oxidase in neutrophils using two complementary optical imaging techniques: Raman microscopy, a vibrational spectroscopic technique that does not require protein labeling, and live-cell fluorescence microscopy, which sheds light on the dynamics of NADPH oxidase assembly in individual cells. Here, we briefly introduce these techniques, compare their characteristics, and show their potential for studying NADPH oxidase at the single-cell level. New microscopy data are presented to illustrate the versatility of Raman and fluorescence microscopy on intact neutrophils.
吞噬细胞NADPH氧化酶是针对入侵微生物的固有免疫反应的关键组成部分，因为该酶在吞噬泡内产生超氧化物（O₂⁻），通过直接或间接依赖活性氧（ROS）形成的机制负责杀灭微生物。关于膜嵌入和胞质NADPH氧化酶亚基及其在组装和激活时复杂的相互作用网络的大部分知识，都来自涉及亚细胞分级分离或重组无细胞系统的生化和生物物理研究。吞噬细胞的单细胞显微镜检查可以补充此类研究，它可能揭示NADPH氧化酶组装的空间和/或时间细节，而这些细节无法从分级细胞测定中获得。近年来，我们使用两种互补的光学成像技术研究了中性粒细胞中的NADPH氧化酶：拉曼显微镜，一种不需要蛋白质标记的振动光谱技术，以及活细胞荧光显微镜，它揭示了单个细胞中NADPH氧化酶组装的动力学。在这里，我们简要介绍这些技术，比较它们的特点，并展示它们在单细胞水平研究NADPH氧化酶的潜力。还展示了新的显微镜数据，以说明拉曼和荧光显微镜在完整中性粒细胞上的多功能性。
