Detection of fluorescently labeled proteins by electron microscopy
- Electron Microscope Unit, The University of New South Wales.
- Institute for Molecular Bioscience, The University of Queensland.
Localising the position of proteins in the cell at high-resolution is critical for determining cellular function. As GFP revolutionized the detection of proteins by light microscopy, similarly new genetic tags for electron microscopy (EM) have great potential for EM visualisation of proteins in cells. We have developed a modular system for enzyme-based protein tagging that facilitates the detection of fluorescently-tagged proteins in the electron microscope by employing a modified soybean ascorbate peroxidase system termed APEX. This system allows for efficient analysis of subcellular protein distributions using existing GFP- and mCherry-tagged protein libraries. We demonstrate we can target APEX to any GFP- or mCherry-tagged protein of interest by engineering and genetically encoding a specific nanobody/binding peptide (BP) fused directly to the APEX-tag. We show that this system is robust, rapid and can be used in animal models. Moreover, this method compatible with correlative light and electron microscopy and can be coupled with sensitive methods for detecting protein-protein interactions through the use of split-GFP. The application of this method to a number of cell biological questions will be addressed in the talk.