Effect of GFP tags on the localization of EB1 and EB1 fragments in vivo.
Effect of GFP tags on the localization of EB1 and EB1 fragments in vivo.
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DOI：
10.1002/cm.20409
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发表时间：
2010-01
期刊：
影响因子：
2.9
通讯作者：
Goodson, Holly V.
中科院分区：
文献类型：
--
作者：
Skube, Susan B.;Chaverri, Jose M.;Goodson, Holly V.
EB1是一种微管正端追踪蛋白，在微管（MT）动力学的调节中起核心作用。绿色荧光蛋白（GFP）标记的EB1构建体通常用于研究EB1本身，也用作动态微管正端的标记。为了正确解读这些研究，了解标签在体内对EB1和其他蛋白质行为的影响是很重要的。为了解决这个问题并增进对EB1功能的理解，我们调查了所表达的EB1片段的定位，并研究了GFP标签是否会改变这些定位。我们发现，N端和C端标签都不是无害的：标记的EB1和EB1片段通常与未标记的对应物行为不同。N端标签显著损害所表达的EB1蛋白结合微管和/或追踪微管正端的能力，尽管它们保留了一些微管结合能力。C端标记的EB1构建体具有与未标记构建体相似的定位，这最初表明它们是无害的。然而，大多数在任一端标记的构建体都会导致CLIP - 170从微管正端消失。这种效应与未标记的全长EB1相反，未标记的全长EB1会将CLIP - 170招募到微管上。这些观察结果表明，尽管EB1 - GFP可以是研究微管动力学的有力工具，但应谨慎使用，因为它可能会改变正在用于研究的系统。此外，一些未标记的片段具有意想不到的定位。特别是，一种缺乏卷曲螺旋结构的EB1构建体虽然微弱但也能追踪微管正端，这为EB1 +TIP行为需要二聚化的观点提供了反证。
EB1 is a microtubule plus-end tracking protein that plays a central role in the regulation of microtubule (MT) dynamics. GFP-tagged EB1 constructs are commonly used to study EB1 itself and also as markers of dynamic MT plus ends. To properly interpret these studies, it is important to understand the impact of tags on the behavior of EB1 and other proteins in vivo. To address this problem and improve understanding of EB1 function, we surveyed the localization of expressed EB1 fragments and investigated whether GFP tags alter these localizations. We found that neither N-terminal nor C-terminal tags are benign: tagged EB1 and EB1 fragments generally behave differently from their untagged counterparts. N-terminal tags significantly compromise the ability of expressed EB1 proteins to bind MTs and/or track MT plus ends, although they leave some MT-binding ability intact. C-terminally tagged EB1 constructs have localizations similar to the untagged constructs, initially suggesting that they are benign. However, most constructs tagged at either end cause CLIP-170 to disappear from MT plus ends. This effect is opposite to that of untagged full-length EB1, which recruits CLIP-170 to MTs. These observations demonstrate that although EB1-GFP can be a powerful tool for studying microtubule dynamics, it should be used carefully because it may alter the system that it is being used to study. In addition, some untagged fragments had unexpected localizations. In particular, an EB1 construct lacking the coiled-coil tracks MT plus ends, though weakly, providing evidence against the idea that EB1 +TIP behavior requires dimerization.
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