Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings.
Covalent Self-Labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings.
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DOI：
10.1105/tpc.20.00439
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发表时间：
2020-10
期刊：
影响因子：
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通讯作者：
Nakamura M
中科院分区：
文献类型：
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作者：
Iwatate RJ;Yoshinari A;Yagi N;Grzybowski M;Ogasawara H;Kamiya M;Komatsu T;Taki M;Yamaguchi S;Frommer WB;Nakamura M
snap标记的微管蛋白的多色成像和化学染料对PIN2内吞作用的监测表明，特定的自标记蛋白可以有效地用于植物。合成化学荧光染料在生物学上有许多应用前景。共价的靶向标记，例如使用snap标签，使用合成染料标记体内的特定蛋白质，用于研究内吞作用等过程或通过超分辨率显微镜成像。尽管具有潜力，但这种化学标记尚未在植物中得到有效应用。一个主要的缺点是有限的知识关于细胞壁和膜的渗透性可用的合成染料。在这里测试的31种合成染料中，23种被BY-2细胞吸收，而8种没有。这就产生了一组可以用来测量内吞作用的染料。三种能够进入细胞的染料，二乙基氨基香豆素，四甲基罗丹明和硅罗丹明647的snap -标签配体，被用于snap -标签α-微管蛋白。通过对拟南芥（Arabidopsis thaliana）幼苗间期和有丝分裂期间微管阵列的活细胞成像和可视化验证了标记的成功。用DRBG-488荧光激活偶联蛋白标记法观察有丝分裂过程中PIN-FORMED2 （PIN2）的内吞作用和向液泡的传递，以及新合成的PIN2优先传递到积极形成的细胞板。总之，这些数据表明，蛋白质的特异性自我标记可以有效地用于植物中各种细胞和生物过程的研究。
Multicolor imaging with SNAP-tagged tubulin and monitoring of PIN2 endocytosis with a chemical dye demonstrate that specific self-labeling of proteins can be used effectively in plants. Synthetic chemical fluorescent dyes promise to be useful for many applications in biology. Covalent, targeted labeling, such as with a SNAP-tag, uses synthetic dyes to label specific proteins in vivo for studying processes such as endocytosis or for imaging via super-resolution microscopy. Despite its potential, such chemical tagging has not been used effectively in plants. A major drawback has been the limited knowledge regarding cell wall and membrane permeability of the available synthetic dyes. Of 31 synthetic dyes tested here, 23 were taken up into BY-2 cells, while eight were not. This creates sets of dyes that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine, and silicon-rhodamine 647, were used to SNAP-tag α-tubulin. Successful tagging was verified by live cell imaging and visualization of microtubule arrays in interphase and during mitosis in Arabidopsis (Arabidopsis thaliana) seedlings. Fluorescence activation-coupled protein labeling with DRBG-488 was used to observe PIN-FORMED2 (PIN2) endocytosis and delivery to the vacuole as well as preferential delivery of newly synthesized PIN2 to the actively forming cell plate during mitosis. Together, the data demonstrate that specific self-labeling of proteins can be used effectively in plants to study a wide variety of cellular and biological processes.