Covalent Self-labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings

编号 040025804

推送时间 20200928

研究领域 森林培育 

年份 2020 

类型 期刊 

语种 英语

标题 Covalent Self-labeling of Tagged Proteins with Chemical Fluorescent Dyes in BY-2 Cells and Arabidopsis Seedlings

来源期刊 The Plant Cell

期 第258期

发表时间 20200806

关键词 BY-2 cells;  Arabidopsis;  SNAP-tag;  live cell imaging;  microtubules;  endocytosis;  PIN2;  auxin transporter; 

摘要 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 to use super-resolution microscopy. Despite the 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. Out of 31 synthetic dyes tested here, 26 were taken up into BY-2 cells, while eight were not. This creates sets that can serve to measure endocytosis. Three of the dyes that were able to enter the cells, SNAP-tag ligands of diethylaminocoumarin, tetramethylrhodamine (TMR) and silicon-rhodamine (SiR) 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 seedling. Fluorescence activation-coupled protein labeling (FAPL) with DRBG-488 was used to observe 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.

服务人员 孙小满

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