Direct Visualization of Protein Association in Living Cells with Complex‐Edited Electron Microscopy

Abstract

Interactions between and among proteins regulate most cell functions, yet detecting these interactions in living cells, especially at high resolution, remains a challenge. Protein complementation[1] proximity-induced biotinylation[2], FRET[3] and bipartite tetracysteine display[4] can all detect interactions between proteins, but only at the moderate resolution provided by epifluorescent microscopy-approximately 200 nm. Super-resolution imaging has begun to overcome this diffraction limit[5], but it cannot detect protein complexes at the near-atomic-level resolution achievable using electron microscopy (EM).[6] Individual tetracysteine-containing proteins can be visualized using EM by use of the biarsenical dye 4,5-bis(1,3,2-dithiarsolan-2-yl)resorufin (ReAsH).[7-9] Irradiation of a protein•ReAsH complex at 585 nm in the presence of oxygen and 3,3′-diaminobenzidine (DAB) catalyzes the formation of an osmophilic DAB polymer that is opaque to electron beams and appears in the electron microscope as a fine granular precipitate.[7-9] An analogous method able to detect a discrete protein complex within a living cell, followed by fixation and sectioning as required by EM, would provide a powerful tool for visualizing at high resolution the interactions between proteins in their native environments.