title: Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.
abstract: 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 (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) 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.
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Xia
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China Molecular Imaging Center, University of Georgia, Atlanta, Morehouse School of Medicine, Atlanta, GA 30310.
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Bing
authors.initials: B
authors.lastname: Wu
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Shuyuan
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Xiaoyu
authors.initials: X
authors.lastname: Song
authors.affiliation: Molecular Imaging Center, University of Georgia, Atlanta, Morehouse School of Medicine, Atlanta, GA 30310.
authors.forename: Phil Y
authors.initials: PY
authors.lastname: Yao
authors.affiliation: National Institutes of Health, Bethesda, MD 20892.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Lippincott-Schwartz
authors.affiliation: Anhui Key Laboratory for Cellular Dynamics & Chemical Biology and the Center for Integrated Imaging, Hefei National Laboratory for Physical Sciences at the Nanoscale and University of Science and Technology of China, Hefei, Anhui 230026, China yaoxb@ustc.edu.cn.
authors.forename: Xuebiao
authors.initials: X
authors.lastname: Yao
mesh_terms: D000595:Amino Acid Sequence; D002465:Cell Movement; D006367:HeLa Cells; D006801:Humans; D061986:MCF-7 Cells; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008969:Molecular Sequence Data; D021381:Protein Transport
chemical_list: C500985:MAPRE1 protein, human; D008869:Microtubule-Associated Proteins