Scientists have developed a new peroxidise-derived tag that can be used to label proteins for visualization by electron microscopy. The tag, called Apex, is a 28 kDa peroxidise that can withstand the processes needed to fix electron microscopy samples for imaging, doesn’t require an external light source to emit its signal, and is active in any cell compartment, unlike horse radish peroxidise, which isn’t active in the cytosol.
Apex has been developed by a team led by Massachusetts Institute of Technology (MIT) researchers, who were looking to generate a genetically encodable EM tag that addresses the limitations of existing genetic tags. Apex is a monomer derived from the dimeric molecule ascorbate peroxidise APX, a class I cytosolic plant peroxidase that lacks disulfide bonds and calcium ions. Although 40% smaller than HRP, Apex, like HRP, also catalyzes the H2O2-dependent polymerization of 3,3′-diaminobenzidine (DAB) into a localized precipitate that gives EM contrast after treatment with OsO4.
When Alice Y. Ting, Ph.D., Jeffrey D. Martell, Ph.D., et al tested Apex in mammalian cells, they found that the molecule could be used to label target proteins in any cell compartment, including membrane proteins. In one experiment they used the tag to label a recently identified calcium channel protein located within mitochondria, and showed that the protein is embedded in the inner mitochondrial membrane and faces inward to the mitochondrial matrix. Apex staining protocol only requires the addition of DAB and hydrogen peroxide, unlike other labels such as miniSOG, which requires light.
“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,” the researchers write in their published paper in Nature Biotechnology. “Beyond electron microscopy, Apex should have utility for a variety of other imaging and biotechnological applications, such as those for which HRP is currently used. For example, the activity of Apex toward numerous aromatic substrates provides the opportunity not only for EM contrast, but also for colorimetric, fluorescent, and chemiluminescent readouts.”
Drs. Ting, Martell et al describe Apex and their initial experiments in a paper titled “Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy.”