Cytosolically-Active Peroxidases as Reporters for Electron Microscopy

Technology

Professor Alice Ting and colleagues have developed a tool that involves genetically fusing an engineered ascorbate peroxidase (APEX2) to a protein of interest to catalyze the production of an electron-dense precipitate that is easily visualized with either light or electron microscopy. This reaction occurs quickly and locally, thereby attaining very high spatial resolution. The tag is non-toxic and functional in all cellular compartments. Furthermore, it can be fused to fluorescent proteins, allowing for images from electron microscopy to be correlated with those from fluorescence microscopy.APEX2 has been demonstrated for EM imaging of a wide variety of cellular proteins in virtually every subcellular compartment. APEX2 is also useful as a marker of subcellular organelles. Although APEX2 has been most extensively used in cultured cells, it has also been successfully employed in a variety of model organisms, including C. elegans, Drosophila, mice, and zebrafish. See the references for details.  

Problem Addressed      

Determining protein localization in intact cells while maintaining sample integrity is difficult. Traditional techniques involve antibody staining of target proteins, but the permeablization treatments required for antibody access compromise cellular ultrastructure. Alternative methods are limited by an inconvenient requirement for light and oxygen gas (which restricts their use to thin samples), or lack of activity in most cellular compartments.  

Advantages 

•  High spatial resolution 
•  Excellent preservation of cellular ultrastructure with strong contrast on the target of interest
•  Can be used to combine multiple imaging modalities
•  Genetic tag is small, sterically compatible with most proteins, and functions in all cellular compartments
•  Can be used on samples of any thickness (reaction is not light-dependent) 
•  Straightforward method  

Publications

Martell, Jonathan D., Tyler J. Deerinck, Y. Soungyun Kim, et al. "Engineered Ascorbate Peroxidase as a Genetically Encoded Reporter for Electron Microscopy." Nature Biotechnology 30, no. 11 (November 2012): 1143-1148.

"Publications From The Ting Lab On APEX And APEX2 For Electron Microscopy." Nature Biotechnology 30, no. 11 (November 2012): 1143-1148.

Lam, Steve S., Jeffrey D. Martell, Kenan J. Kamer, et al. "Directed Evolution Of APEX2 For Electron Microscopy And Proximity Labeling." Nature Methods 12 (2015): 51-54.

Hung, Shu-Han, Jennifer T. Lam, Kenneth H. Sung, et al. "Electron Microscopy Using The Genetically Encoded APEX2 Tag In Cultured Mammalian Cells." Nature Protocols (2017).