Engineered ascorbate peroxidase as a genetically-encoded reporter for electron microscopy

By Jeffrey D. Martell, Thomas J. Deerinck, Y. Sancak, Thomas L. Poulos, Vamsi K. Mootha, G. Sosinsky, Mark Ellisman, A. Ting. Nature Biotechnology, 2012

688 citations recorded by the source corpus.

Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments or require light and can be difficult to use. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.

Subfields: Biophysics, Molecular Biology, Structural Biology

View Paper: https://www.semanticscholar.org/paper/363ce9f8bce9990e814d2369eefd2cdfa6debba3

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Citations in the corpus

Antibiotics induce redox-related physiological alterations as part of their lethality (2014, methodology): “To determine directly and specifically if bactericidal antibiotics promote intracellular H2O2 generation, we developed a novel intracellular enzymatic assay using a recently engineered variant of ascorbate peroxidase (APX), AP, which is naturally active in the cytoplasm and exhibits HRP-like kinetic”
Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics (2015, background): “Furthermore, with the advent of genetically encoded markers for electron microscopy (EM) (4, 5), the continued preeminence of SR microscopy for protein-specific structural imaging at the nanoscale is no longer assured.”
Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging (2013, methodology): “To determine if our detection of these proteins in the matrix was accurate, we performed EM imaging, taking advantage of APEX's additional functionality as an EM tag (5).”
Clarifying Tissue Clearing (2015, neutral): “Moreover, it is possible that certain labels that reflect light, such as the horseradish peroxidase (HRP), other peroxidase reaction products (e.g., APEX; Martell et al., 2012), or silver stains (e.”
Site-specific biotinylation of purified proteins using BirA (2015, background): “Also the use of enzymes to achieve promiscuous biotinylation (a BirA mutant or a peroxidase) has enabled labeling of untagged proteins in particular cellular regions or compartments (30,31,32).”
The 4D Nucleome Project (2017, neutral): “These technologies include HRP-labeled antibodies in TSA-SEQ (Box 1), APEX (ascorbate peroxidase; 41 ) and Killer Red 42 .”