Morita Maya, Sawaki Kazumasa, Kinoshita Daiki, Sakurai Chiye, Hori Naohiro, Hatsuzawa Kiyotaka
Division of Molecular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Tottori 683-8503, Japan.
J Biochem. 2017 Nov 1;162(5):309-316. doi: 10.1093/jb/mvx034.
Phagosome formation and maturation are essential innate immune mechanisms to engulf and digest foreign particles. To analyze these processes quantitatively, we established a specific Escherichia coli probe expressing a tandem fluorescent protein, comprising glutathione S-transferase fused with monomeric Cherry (mCherry) and monomeric Venus (mVenus). We demonstrated that mVenus was more susceptible to bleaching in an acidic environment than mCherry, and that the mVenus:mCherry fluorescence intensity ratio can be used to monitor phagosomal pH changes during maturation. Using this probe, we revealed that synaptosomal-associated protein of 23 kDa, a plasma membrane soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein, actively regulated phagocytosis of E. coli and subsequent phagosome maturation in macrophages. Our results indicated that this probe has the potential to be a powerful tool in understanding the molecular mechanisms of phagosome formation and maturation.
吞噬体的形成和成熟是吞噬和消化外来颗粒的重要先天性免疫机制。为了定量分析这些过程，我们构建了一种表达串联荧光蛋白的特异性大肠杆菌探针，该探针由与单体樱桃红蛋白（mCherry）和单体金星蛋白（mVenus）融合的谷胱甘肽S-转移酶组成。我们证明，在酸性环境中，mVenus比mCherry更容易发生光漂白，并且mVenus:mCherry荧光强度比可用于监测吞噬体成熟过程中的pH变化。利用该探针，我们发现23 kDa的突触体相关蛋白（一种质膜可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体蛋白）可积极调节巨噬细胞中大肠杆菌的吞噬作用及随后的吞噬体成熟。我们的结果表明，该探针有可能成为理解吞噬体形成和成熟分子机制的有力工具。
