Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
复制标题
超分辨率成像揭示了微管加端跟踪中EB1的结构特征。
DOI：
10.1091/mbc.e14-06-1133
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发表时间：
2014-12-15
影响因子：
3.3
通讯作者：
Yao X
中科院分区：
文献类型：
--
作者：
Xia P;Liu X;Wu B;Zhang S;Song X;Yao PY;Lippincott-Schwartz J;Yao X
介绍了一种利用光激活互补荧光蛋白（PACF）在纳米空间分辨率下对活细胞内蛋白质相互作用进行光学成像的方法。在这里建立的PACF方法将能够在单个活细胞水平上同时可视化同源和异源二聚体相互作用。特定分子及其在真实的时间和空间中的相互作用的可视化对于描绘细胞动力学和信号电路是如何编排的至关重要。蛋白质相互作用的构象动力学和结构可塑性的空间调节是需要重新布线信号电路响应细胞外的线索。我们介绍了一种方法，在纳米空间分辨率的活细胞内的蛋白质相互作用的光学成像，使用光激活互补荧光（PACF）蛋白。互补荧光蛋白分子的子集被激活，本地化，然后漂白，这是由组装的超分辨率图像从总互动分子的聚集位置。使用PACF，我们得到了精确的定位动态微管加端枢纽蛋白EB 1二聚体和它们的不同分布在前缘和迁移细胞的细胞体。我们通过产生EB 1-PACF二聚体（EB 1 wt：EB 1 wt、EB 1 wt：EB 1 mt和EB 1 mt：EB 1 mt）并成像它们在培养细胞中的精确定位，进一步描绘了EB 1的结构-功能关系。令人惊讶的是，我们的分析揭示了以前未表征的EB 1接头区域在跟踪活细胞中微管加末端中的关键作用。因此，PACF提供了一种独特的方法来描绘在纳米尺度上的同源或异源二聚体蛋白质的空间动力学，并建立了一个平台，以报告在活细胞中的蛋白质相互作用的空间和时间的精确调节。
A method is introduced for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells using photoactivatable complementary fluorescent (PACF) proteins. The PACF approach established here will enable the simultaneous visualization of homo- and heterodimeric interactions at the single live-cell level. Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure–function relationship of EB1 by generating EB1-PACF dimers (EB1wt:EB1wt, EB1wt:EB1mt, and EB1mt:EB1mt) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.