Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.
Visualization of Neuregulin 1 ectodomain shedding reveals its local processing in vitro and in vivo.
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DOI：
10.1038/srep28873
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发表时间：
2016-07-01
影响因子：
4.6
通讯作者：
Sehara-Fujisawa A
中科院分区：
文献类型：
--
作者：
Kamezaki A;Sato F;Aoki K;Asakawa K;Kawakami K;Matsuzaki F;Sehara-Fujisawa A
神经调节蛋白 1 (NRG1) 发挥着多种发育作用，可能与包括精神分裂症在内的多种神经系统疾病有关。跨膜 NRG1 蛋白被蛋白水解裂解并作为 ErbB 受体的可溶性配体释放。这种翻译后处理被称为“胞外域脱落”，被认为对于 NRG1 功能至关重要。然而，对于体内 NRG1 裂解的调节机制知之甚少。在这里，我们通过将 mCherry 和 GFP 分别融合到 NRG1 的胞外和胞内结构域，开发了一种荧光探针 NRG1 Cleavage Indicating SenSOR (N-CISSOR)。 N-CISSOR 模拟了 NRG1 的亚细胞定位和生化特性，包括培养细胞中的裂解动力学和 ErbB 磷酸化。对 phorbol-12-myristate-13-acetate 刺激的 N-CISSOR 表达 HEK293T 细胞进行 mCherry/GFP 比率成像，能够在亚细胞水平监测 NRG1 的快速胞外域脱落。在斑马鱼胚胎中利用 N-CISSOR 揭示了发育中的运动神经元中优先轴突 NRG1 胞外域脱落，证明 NRG1 胞外域脱落在亚细胞水平上受到空间调节。因此，N-CISSOR 将成为阐明体外和体内 NRG1 胞外域脱落的时空调节的有价值的工具。
Neuregulin1 (NRG1) plays diverse developmental roles and is likely involved in several neurological disorders including schizophrenia. The transmembrane NRG1 protein is proteolytically cleaved and released as a soluble ligand for ErbB receptors. Such post-translational processing, referred to as ‘ectodomain shedding’, is thought to be crucial for NRG1 function. However, little is known regarding the regulatory mechanism of NRG1 cleavage in vivo. Here, we developed a fluorescent probe, NRG1 Cleavage Indicating SenSOR (N-CISSOR), by fusing mCherry and GFP to the extracellular and intracellular domains of NRG1, respectively. N-CISSOR mimicked the subcellular localization and biochemical properties of NRG1 including cleavage dynamics and ErbB phosphorylation in cultured cells. mCherry/GFP ratio imaging of phorbol-12-myristate-13-acetate-stimulated N-CISSOR-expressing HEK293T cells enabled to monitor rapid ectodomain shedding of NRG1 at the subcellular level. Utilizing N-CISSOR in zebrafish embryos revealed preferential axonal NRG1 ectodomain shedding in developing motor neurons, demonstrating that NRG1 ectodomain shedding is spatially regulated at the subcellular level. Thus, N-CISSOR will be a valuable tool for elucidating the spatiotemporal regulation of NRG1 ectodomain shedding, both in vitro and in vivo.