Concatenated metallothionein as a clonable gold label for electron microscopy

Abstract

Localization of proteins in cells or complexes using electron microscopy has mainly relied upon the use of heavy metal clusters, which can be difficult to direct to sites of interest. For this reason, we would like to develop a clonable tag analogous to the clonable fluorescent tags common to light microscopy. Instead of fluorescing, such a tag would initiate formation of a heavy metal cluster. To test the feasibility of such a tag, we exploited the metal-binding protein, metallothionein (MT). We created a chimeric protein by fusing one or two copies of the MT gene to the gene for maltose binding protein. These chimeric proteins bound many gold atoms, with a conservative value of 16 gold atoms per copy of metallothionein. Visualization of gold-labeled fusion proteins by scanning electron microscopy required one copy of metallothionein while transmission electron microscopy required two copies. Images of frozen-hydrated samples of simple complexes made with anti-MBP antibodies hint at the usefulness of this method.

Lessons from yeast: the spindle pole body and the centrosome

“…Placement of GFP at the C terminus was not always successful, thus Spc29p-GFP was functional but gave no immunoEM signal; however, when GFP was at the N terminus, staining was seen both at the SPB and the satellite [15]. Given the increasing ease with which homologous recombination can be applied to the genomes of vertebrate and mammalian cells [33][34][35][36], it should be possible to tag genes with GFP or other direct labels [51,52]. CryoEM of centrosomes isolated from cell lines with directly labelled centrosomal components should give high-resolution localization of these components.…”

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

“…Labeling techniques that have been combined with cryo-EM imaging include immunogold labeling (15) and, more recently, clonable tags, which have the advantage of higher labeling specificity and resolution (16,17). To avoid disruption of the assembly or architecture of macromolecular complexes, clonable tags are usually designed to be relatively small (e.g.…”

“…GFP is 27 kDa), but many of them are too small for direct in situ visualization using cryo-ET. Therefore, for EM visualization, small clonable tags are often specifically linked to additional electron-dense probes, such as metal ion binding by metallothionein (7 kDa) (16,18), or tags such as MiniSOG that catalyze the photooxidation of diaminobenzidine into a localized precipitate that can be visualized by OsO 4 treatment (19,20). Despite these recent advances, the techniques used are still limited and/or are not compatible with cryo-EM, because of low contrast, toxicity of metal ion solutions, and/or the requirement to chemically fix and post-stain the cells.…”

Visualizing cellular processes at the molecular level by cryo-electron tomography

“…An approach based on electrondense labeling must be developed to design a clonable tag that can be genetically conjugated to proteins, which would facilitate their localization in cryo-tomograms -that is, we are in need of a GFP analog for cryo-electron microscopy. An elegant example of a clonable tag is metallothionein, a cysteine-rich protein that has been shown to bind to multiple heavy atoms and can be detected by an electron beam (Mercogliano and DeRosier, 2007). Such a labeling strategy would provide a general solution for identifying complexes whose structure is not yet determined or that intimately interact to form large assemblies.…”