Spatiotemporal visualisation of proHB-EGF ectodomain shedding in living cells
Spatiotemporal visualisation of proHB-EGF ectodomain shedding in living cells
复制标题
活细胞中 proHB-EGF 胞外域脱落的时空可视化
DOI：
10.1093/jb/mvt030
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发表时间：
2013
期刊：
影响因子：
--
通讯作者：
T. Ozawa and S. Higashiyama
中科院分区：
文献类型：
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作者：
H. Inoue;T. Sakaue;T. Ozawa and S. Higashiyama
肝素结合表皮生长因子样生长因子(HB-EGF)是EGF家族的一员，每种生长因子都是作为I型跨膜前体产生的。ProHB-EGF是HB-EGF的前体，其膜旁结构域被去整合素和金属蛋白酶切割。HB-EGF释放到细胞外间隙，强烈激活EGF受体。更好地了解proHB-EGF脱落的相关性与该过程在各种类型细胞的增殖、分化和生存中的重要性有关。通常使用碱性磷酸酶标记的proHB-EGF分析或涉及多个细胞的蛋白质印迹分析来评估proHB-EGF的脱落，这使得很难观察单个细胞活动的时空差异。在这项研究中，我们开发了一种基于proHB-EGF的金属蛋白酶荧光生物传感器，名为Fluhemb，使用一种简单的方法来测量荧光比率的变化，以可视化单个细胞中proHB-EGF脱落的时空调节。在体内外不同条件下检测不同类型细胞中proHB-EGF的脱落活性可能是非常有用的。
Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family, each of which is produced as a type I transmembrane precursor. The juxtamembrane domain of proHB-EGF, a precursor of HB-EGF, is cleaved by a disintegrin and metalloproteases. HB-EGF is released into the extracellular space and strongly activates EGF receptor. The relevance of better understanding proHB-EGF shedding relates to the importance of the process in the proliferation, differentiation and survival of various types of cells. Shedding of proHB-EGF is normally evaluated using an alkaline phosphatase-tagged proHB-EGF assay or a western blotting assay that involves multiple cells, which makes it difficult to observe spatiotemporal differences in the activities of the individual cells. In this study, we developed a fluorescent proHB-EGF-based metalloprotease biosensor, named Fluhemb, to visualize spatiotemporal regulation of proHB-EGF shedding in individual cells using a simple method that measures changes in fluorescence ratios. Fluhemb might be very useful for detecting the activity of proHB-EGF shedding in various types of cells under different conditionsin vitroandin vivo.