Split-HaloTag imaging assay for sophisticated microscopy of protein–protein interactions in planta  

作　　者：Rieke Minner-Meinen Jan-Niklas Weber Andreas Albrecht Rainer Matis Maria Behnecke Cindy Tietge Stefan Frank Jutta Schulze Henrik Buschmann Peter Jomo Walla Ralf-R.Mendel Robert Hänsch David Kaufholdt 

机构地区：[1]Institut für Pflanzenbiologie,Technische Universität Braunschweig,Humboldtstrasse 1,38106 Braunschweig,Germany [2]Institut für Physikalische und Theoretische Chemie,Technische Universität Braunschweig,Hagenring 30.023c,38106 Braunschweig,Germany [3]Botany Department,Universität Osnabrück,Barbara Strasse 11,49076 Osnabrück,Germany [4]Center of Molecular Ecophysiology(CMEP),College of Resources and Environment,Southwest University,Tiansheng Road No.2,Beibei District,400715 Chongqing,P.R.China

出　　处：《Plant Communications》2021年第5期148-159,共12页植物通讯（英文）

基　　金：supported by the Deutsche Forschungsgemeinschaft(grant GRK2223/1)to R.H.and R.R.M.

摘　　要：An ever-increasing number of intracellular multi-protein networks have been identified in plant cells.Split-GFP-based protein–protein interaction assays combine the advantages of in vivo interaction studies in a native environment with additional visualization of protein complex localization.Because of their simple protocols,they have become some of the most frequently used methods.However,standard fluorescent proteins present several drawbacks for sophisticated microscopy.With the HaloTag system,these drawbacks can be overcome,as this reporter forms covalent irreversible bonds with synthetic photostable fluorescent ligands.Dyes can be used in adjustable concentrations and are suitable for advanced microscopy methods.Therefore,we have established the Split-HaloTag imaging assay in plants,which is based on the reconstitution of a functional HaloTag protein upon protein–protein interaction and the subsequent covalent binding of an added fluorescent ligand.Its suitability and robustness were demonstrated using a well-characterized interaction as an example of protein–protein interaction at cellular structures:the anchoring of the molybdenumcofactor biosynthesis complex to filamentous actin.In addition,a specific interactionwas visualized in a more distinctivemannerwith subdiffractional polarizationmicroscopy,Airyscan,and structured illumination microscopy to provide examples of sophisticated imaging.Split-GFPand Split-HaloTag can complement one another,as Split-HaloTag represents an alternative option and an addition to the large toolbox of in vivo methods.Therefore,this promising new Split-HaloTag imaging assay provides a unique and sensitive approach formore detailed characterization of protein–protein interactions using specific microscopy techniques,such as 3D imaging,single-molecule tracking,and super-resolution microscopy.

关 键 词：advanced microscopy cytoskeleton photostable fluorescent dyes gateway cloning molybdenum cofactor biosynthesis complex protein-protein interaction Split-HaloTag imaging assay 

分 类 号：Q94[生物学—植物学]