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 nati...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.展开更多
基金supported by the Deutsche Forschungsgemeinschaft(grant GRK2223/1)to R.H.and R.R.M.
文摘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.
