Dynamic intracellular distribution of Notch during activation and asymmetric cell division revealed by functional fluorescent fusion proteins
Dynamic intracellular distribution of Notch during activation and asymmetric cell division revealed by functional fluorescent fusion proteins
复制标题
DOI：
10.1111/j.1365-2443.2010.01412.x
复制
发表时间：
2010-07-01
期刊：
影响因子：
2.1
通讯作者：
Hayashi, Shigeo
中科院分区：
文献类型：
--
作者：
Kawahashi, Koji;Hayashi, Shigeo
Notch信号传导介导细胞命运特化所需的细胞间通信。由于大多数Notch蛋白以细胞内库的形式存在，因此关于Notch的细胞内运输的信息对于理解其调控是必不可少的。为了观察Notch蛋白在体内的运输，我们构建了一个Notch蛋白，其中绿色荧光蛋白（GFP）插入到细胞内结构域。该构建体拯救了Notch突变体胚胎的神经原性表型，并且当在翅成虫盘中过表达时引起典型的Notch功能获得性表型。使用这种和类似构建的Notch-mCherry融合，我们显示了Notch在内体中的快速时空动力学。动力学分析表明，Notch-GFP的激活胞外脱落导致快速和不受限制的Notch细胞内结构域的核转位。在感觉器官前体细胞的不对称细胞分裂期间，Notch-GFP的分布在子细胞的界面处显示出短暂的不对称性，证实了使子细胞之间的Notch信号传导水平偏置的可能的调节位点。
Notch signaling mediates intercellular communication required for cell fate specification. Because the majority of Notch proteins exist as an intracellular pool, information on intracellular trafficking of Notch is essential for understanding its regulation. To observe trafficking of Notch protein in vivo, we constructed a Notch protein with green fluorescent protein (GFP) inserted into the intracellular domain. This construct rescued the neurogenic phenotype of Notch mutant embryos and caused the typical Notch gain-of-function phenotype when over-expressed in the wing imaginal disc. Using this and similarly constructed Notch-mCherry fusion, we showed rapid spatio-temporal dynamics of Notch in endosomes. Kinetic analysis revealed that activation of Notch-GFP by ectodomain shedding causes fast and unrestricted nuclear translocation of Notch intracellular domain. During asymmetric cell division of sensory organ precursor cells, distribution of Notch-GFP showed transient asymmetry at the interface of daughter cells, confirming a possible site of regulation that biases Notch signaling level among daughter cells.