Split-HaloTag Imaging Assay for in vivo 3D-Microscopy and Subdiffractional Analyses of Protein-Protein Interactions

Abstract

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 visualisation of protein complex localisation. Due to its simple protocols, it has become one of the most frequently used methods. However, standard fluorescent proteins entail several drawbacks for sophisticated microscopy. With the HaloTag(R) 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 established the Split-HaloTag(R) imaging assay in plants which is based on the reconstitution of a functional HaloTag(R) protein upon protein-protein interaction and subsequent covalent binding of an added fluorescent ligand. Its suitability and robustness were demonstrated using well-characterised interactions as an example for protein-protein interaction at cellular structures: the molybdenum cofactor biosynthesis complex anchoring to filamentous actin. Additionally, a specific interaction was visualised with subdiffractional polarisation microscopy in a more distinctive manner as example for sophisticated imaging. Split-GFP and Split-HaloTag(R) can complement one another as Split-HaloTag(R) represents an alternative option and an addition to the large toolbox of in vivo methods. Therefore, this promising new Split-HaloTag(R) imaging assay provides a unique and sensitive approach for more detailed characterization of protein-protein interaction with specific microscopic techniques such as 3D-imaging, single molecule tracking and super-resolution microscopy.