APEX-Gold: A genetically-encoded particulate marker for robust 3D electron microscopy

Abstract

Genetic tags allow rapid localization of tagged proteins in cells and tissues. APEX, an ascorbate peroxidase, has proven to be one of the most versatile and robust genetic tags for ultrastructural localization by electron microscopy. Here we describe a simple method, APEX-Gold, which converts the diffuse oxidized diaminobenzidine reaction product of APEX into a silver/gold particle akin to that used for immunogold labelling. The method increases the signal to noise ratio for EM detection, providing unambiguous detection of the tagged protein, and creates a readily quantifiable particulate signal. We demonstrate the wide applicability of this method for detection of membrane proteins, cytoplasmic proteins and cytoskeletal proteins. The method can be combined with different electron microscopic techniques including fast freezing and freeze substitution, focussed ion beam scanning electron microscopy, and electron tomography. The method allows detection of endogenously expressed proteins in genome-edited cells. We make use of a cell-free expression system to generate membrane particles with a defined quantum of an APEX-fusion protein. These particles can be added to cells to provide an internal standard for estimating absolute density of expressed APEX-fusion proteins.

Advances in enzyme-mediated proximity labeling and its potential for plant research

“…Many sample preparation methods have been developed for PL-assisted light and electron microscopy ( Martell et al, 2017 ; Tsang et al, 2018 ; Sengupta et al, 2019 ; Rae et al, 2020 ). Notably, since labeling happens in fixed tissue, plants are amenable to DAB staining ( Graham and Karnovsky, 1966 ; Ekes, 1971 ) and methods for (cryo)EM, cryo-electron tomography, and correlated light and electron microscopy (CLEM) are generally accessible to plant tissues ( Otegui and Pennington, 2019 ; Wang et al, 2019a ), some APEX-cryoEM methods ( Tsang et al, 2018 ; Sengupta et al, 2019 ) hold promise for plant use.…”