ReAsH is the variant that is useful for EM, because it can generate singlet oxygen upon illumination. Singlet oxygen drives localized polymerization of the substrate diaminobenzidene (DAB) into an insoluble form that can be viewed by EM. “Because the fluorescent label binds directly to the protein you are trying to localize, and the DAB polymer deposits directly nearby the fluorophore, the resolution is better [than immunogold labeling],” says Ellisman. Additionally, this technique does not require the diffusion of large antibodies into the fixed specimens.
The group put their new label to work by examining the protein connexin43, which multimerizes to form gap junctions. Pulse-chase with first FlAsH and then ReAsH revealed newer (i.e., red) molecules of connexin at the outer edge of large clusters of gap junctions known as plaques. Green molecules were concentrated in the center, indicating that older connexins are endocytosed from the middle of the plaques. EM demonstrated that newly synthesized connexins were first sent to nonplaque sites on the cell surface before flowing to the edge of a plaque.
The team expects there will be many uses for the FlAsH-ReAsH system, especially in revealing how large complexes, such as receptor patches at synapses, are formed. “Anyone who has a question about how a cell manages the assembly of large macromolecular structures of a protein should benefit from the use of this system,” says Ellisman. ▪