TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells.
TagBiFC technique allows long-term single-molecule tracking of protein-protein interactions in living cells.
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TagBiFC 技术允许对活细胞中蛋白质-蛋白质相互作用进行长期单分子追踪
DOI：
10.1038/s42003-021-01896-7
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发表时间：
2021-03-19
影响因子：
5.9
通讯作者：
Sun Y
中科院分区：
文献类型：
--
作者：
Shao S;Zhang H;Zeng Y;Li Y;Sun C;Sun Y
蛋白质-蛋白质相互作用(PPI)是细胞活动调节的关键。利用双分子荧光互补(BIFC)技术可视化PPI有助于理解PPI如何实现其功能。然而，目前的BIFC是基于荧光蛋白质的，其亮度和光稳定性对于单分子示踪实验来说是次优的，导致要么时空分辨率低，要么无法对延长的时间过程进行跟踪。在这里，我们开发了基于Split HaloTag的TagBiFC技术，Split HaloTag是一种自我标记标签，可以连接有机染料分子，因此在活细胞内的PPI可视化中提供比荧光蛋白更好的亮度和光稳定性。通过筛选和优化，证明重组的HaloTag比以前的方法具有更高的定位精度和更长的跟踪长度。利用TagBiFC，我们揭示了转录因子二聚体与染色质DNA的动态相互作用是明显的，并与它们的二聚体状态密切相关，表明了这类转录因子的一般调控机制。此外，我们还展示了TagBiFC在单核小体成像、单个mRNA的轻负荷成像、细胞结构的低本底成像等方面的优势应用。我们相信，我们的TagBiFC系统的这些优越性能将在活细胞内的单分子成像研究中有广泛的应用。
Protein-protein interactions (PPIs) are critical for cellular activity regulation. Visualization of PPIs using bimolecular fluorescence complementation (BiFC) techniques helps to understand how PPIs implement their functions. However, current BiFC is based on fluorescent proteins and the brightness and photostability are suboptimal for single molecule tracking experiments, resulting in either low spatiotemporal resolution or incapability of tracking for extended time course. Here, we developed the TagBiFC technique based on split HaloTag, a self-labeling tag that could conjugate an organic dye molecule and thus offered better brightness and photostability than fluorescent proteins for PPI visualization inside living cells. Through screening and optimization, we demonstrated that the reconstituted HaloTag exhibited higher localization precision and longer tracking length than previous methods. Using TagBiFC, we reveal that the dynamic interactions of transcription factor dimers with chromatin DNA are distinct and closely related to their dimeric states, indicating a general regulatory mechanism for these kinds of transcription factors. In addition, we also demonstrated the advantageous applications of TagBiFC in single nucleosome imaging, light-burden imaging of single mRNA, low background imaging of cellular structures. We believe these superior properties of our TagBiFC system will have broad applications in the studies of single molecule imaging inside living cells.
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改进的荧光蛋白用于内源性蛋白质标记。
影响因子：
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作者：
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Tjian, Robert
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Waldo, GS
影响因子：
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作者：
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通讯作者：
Lavis LD