An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation
An enhanced mutant of red fluorescent protein DsRed for double labeling and developmental timer of neural fiber bundle formation
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DOI：
10.1074/jbc.c100200200
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发表时间：
2001-08-10
影响因子：
4.8
通讯作者：
Ito, K
中科院分区：
文献类型：
--
作者：
Verkhusha, VV;Otsuna, H;Ito, K
我们开发了一种新的珊瑚源红色荧光蛋白DsRed S197 Y，它更亮，基本上没有次级荧光峰。这使其成为用绿色荧光蛋白（GFP）进行双标记的理想报告基因。虽然纯化的蛋白质仅显示出20%的强荧光发射，但表达DsRed S197 Y的培养细胞显示出比表达野生型DsRed的细胞高3-3.5倍的荧光水平。DsRed的荧光成熟比GFP慢得多是荧光发育计时器应用的有益特征。当GFP和DsRed S197 Y同时表达时，发射开始于不同的潜伏期。这提供了关于表达开始后的时间的信息。如果表达在某些类型的细胞分化时被激活，则它反映了细胞分化的顺序。我们将该系统应用于果蝇发育中的大脑，并首次可视化了一个大束内神经纤维的形成顺序。我们的结果表明，新延伸的蘑菇体神经元纤维主要进入核心，而不是现有束的外围。因此，基于DsRed的定时器为模式生物的发育生物学和遗传学提供了不可或缺的工具。
We developed a new variant of coral-derived red fluorescent protein, DsRed S197Y, which is brighter and essentially free from secondary fluorescence peak. This makes it an ideal reporter for double labeling with green fluorescent protein (GFP). Though purified protein shows only 20% stronger fluorescence emission, culture cells that express DsRed S197Y exhibit a 3-3.5 times higher level of fluorescence than the cells that express wild-type DsRed. The much slower fluorescence maturation of DsRed than that of GFP is a beneficial feature for a fluorescent developmental timer application. When GFP and DsRed S197Y are expressed simultaneously, emissions start at different latency. This provides information about the time after the onset of expression. It reflects the order of cell differentiation if the expression is activated upon differentiation of certain types of cells. We applied this system to the developing brain of Drosophila and visualized, for the first time, the formation order of neural fibers within a large bundle. Our results showed that newly extending fibers of the mushroom body neurons mainly run into the core rather than to the periphery of the existing bundle. DsRed-based timer thus presents an indispensable tool for developmental biology and genetics of model organisms.