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DOI	10.1073/pnas.2019071118
	POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos
	Sugizaki A.; Sato K.; Chiba K.; Saito K.; Kawagishi M.; Tomabechi Y.; Mehta S.B.; Ishii H.; Sakai N.; Shirouzu M.; Tani T.; Terada S.
发表日期	2021
ISSN	00278424
卷号	118 期号:11
英文摘要	Biomolecular assemblies govern the physiology of cells. Their function often depends on the changes in molecular arrangements of constituents, both in the positions and orientations. While recent advancements of fluorescence microscopy including super-resolution microscopy have enabled us to determine the positions of fluorophores with unprecedented accuracy, monitoring the orientation of fluorescently labeled molecules within living cells in real time is challenging. Fluorescence polarization microscopy (FPM) reports the orientation of emission dipoles and is therefore a promising solution. For imaging with FPM, target proteins need labeling with fluorescent probes in a sterically constrained manner, but because of difficulties in the rational three-dimensional design of protein connection, a universal method for constrained tagging with fluorophore was not available. Here, we report POLArIS, a genetically encoded and versatile probe for molecular orientation imaging. Instead of using a direct tagging approach, we used a recombinant binder connected to a fluorescent protein in a sterically constrained manner that can target specific biomolecules of interest by combining with phage display screening. As an initial test case, we developed POLArISact, which specifically binds to F-actin in living cells. We confirmed that the orientation of F-actin can be monitored by observing cells expressing POLArISactwith FPM. In living starfish early embryos expressing POLArISact, we found actin filaments radially extending from centrosomes in association with microtubule asters during mitosis. By taking advantage of the genetically encoded nature, POLArIS can be used in a variety of living specimens, including whole bodies of developing embryos and animals, and also be expressed in a cell type/tissue specific manner. © 2021 National Academy of Sciences. All rights reserved.
英文关键词	Actin; Fluorescence microscopy; Fluorescence polarization; Mitosis; Starfish
语种	英语
scopus关键词	F actin; fluorescent dye; actin filament; Article; binding kinetics; embryo development; fluorescence microscopy; fluorescence polarization microscopy; microtubule; mitosis; molecular imaging; molecular orientation imaging; molecular probe; phage display; polarization microscopy; priority journal; protein expression; starfish
来源期刊	Proceedings of the National Academy of Sciences of the United States of America
文献类型	期刊论文
条目标识符	http://gcip.llas.ac.cn/handle/2XKMVOVA/180214
作者单位	Department of Neuroanatomy and Cellular Neurobiology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), Bunkyo-ku, Tokyo, 113-8519, Japan; Center for Brain Integration Research, TMDU, Bunkyo-ku, Tokyo, 113-8519, Japan; Department of Anatomy and Physiological Sciences, Graduate School of Medical and Dental Sciences, TMDU, Bunkyo-ku, Tokyo, 113-8519, Japan; Department of Biological Sciences, Ochanomizu University, Bunkyo-ku, Tokyo, 112-8610, Japan; RIKEN Center for Biosystems Dynamics Research, Yokohama, 230-0045, Japan; Eugene Bell Center for Regenerative Biology and Tissue Engineering, Marine Biological Laboratory, Woods Hole, MA 02543, United States; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology, Ikeda, Osaka, 563-8577, Japan
推荐引用方式 GB/T 7714	Sugizaki A.,Sato K.,Chiba K.,et al. POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos[J],2021,118(11).
APA	Sugizaki A..,Sato K..,Chiba K..,Saito K..,Kawagishi M..,...&Terada S..(2021).POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos.Proceedings of the National Academy of Sciences of the United States of America,118(11).
MLA	Sugizaki A.,et al."POLArIS, a versatile probe for molecular orientation, revealed actin filaments associated with microtubule asters in early embryos".Proceedings of the National Academy of Sciences of the United States of America 118.11(2021).