Rapid electron microscopic detection of GFP-tagged proteins in cells and whole organisms

The use of green fluorescent protein (GFP) has revolutionised the study of dynamic cellular processes in cells, tissues, and whole organisms. Laboratories throughout the world have exploited the simplicity of GFP as an everyday tool to determine protein localisation in cell lines and whole organisms. Fluorescence microscopy now dominates the imaging field although the attainable resolution of these methods has always been a limiting factor. Alternative techniques such as electron microscopy offer higher resolution but have traditionally been viewed as slow and technically difficult.

An electron microscopic method that provides rapid, simple, high resolution, and quantitative detection of GFP-tagged proteins would represent a significant advance in the fields of cell and developmental biology. Ideally this method would be compatible with the new 3D electron microscopic techniques now available, including electron tomography and serial blockface scanning electron microscopy and not require new or highly specialised equipment. We have now developed a new technique that we believe fulfils all these requirements (Ariotti et al, 2015).

We have shown that we can target a recently developed modified peroxidase, called APEX (Martell et al, 2012), to any GFP-tagged protein of interest by co-expression with a GFP-binding peptide directly attached to the APEX-tag (Ariotti et al, 2015). The APEX peroxidase produces an electron dense reaction product at the site of the GFP-fusion protein that allows its visualisation in the electron microscope. With this method whole libraries of GFP-tagged proteins could be localised at electron microscopic resolution in a few days. Moreover, the method was shown to generate remarkably high-resolution localisation data. We can localise proteins to distinct subdomains of microdomains of subcellular organelles such as individual intraluminal vesicles within endosomes and specific protein subdomains of the neck region of caveolae. This method proves to be a very powerful and versatile technique for EM analysis. It is quantitative by linescan analysis, compatible with tomography and serial blockface electron microscopy for 3D analysis of whole cell protein distribution, and offers a simplistic alternative to involved correlative light and electron microscopy techniques that are currently available in the literature.

Perhaps of most interest to developmental biologists is the application of this technique to localisation of GFP-fusion proteins in whole organisms (Ariotti et al, 2015). The effort and cost required to generate entirely new and separate gene edited lines for fluorescence and electron microscopic analyses (by conventional APEX tagging methods) are prohibitive. We bypassed this bottleneck by generating new zebrafish lines that express APEX-GFP binding peptide in all tissues. We designed two different zebrafish lines under the control of two different promoters. The first line exploits a beta-actin2 promoter for ubiquitous and continual expression of GFP-binding peptide-APEX fusion in all cells. The second zebrafish line allows for temporal control of the expression of the EM-marker, as it was engineered under the control of the hsp70I promoter that, after a brief and mild heat shock, induces rapid expression and accumulation of the APEX-GFP binding peptide in the cytoplasm of all cells within in the animal. With a single cross between our gene edited lines and any zebrafish stably expressing a GFP-tagged protein we could show rapid electron microscopic localisation of proteins including those expressed at endogenous levels in vivo. By the exploitation of the hsp70I promoter it will be possible to track subcellular protein distribution changes during development to a resolution of approximately 10 nm rapidly and easily. To further simplify the system, both lines were engineered with a red lens to denote expression of GFP-binding peptide-APEX fusion protein for easy genotyping of progeny.

We envisage that this method will provide a rapid localisation strategy in cells and whole organisms. It does not require new or expensive EM equipment, but rather is adapted from an established protocol that has been standard in the electron microscopy field for decades. While we have only demonstrated the utility of the method in zebrafish embryos the technique will not be limited to this particular animal model and we predict will be of use in other species.

References

Ariotti N., Hall T.E., Rae J., Ferguson C., McMahon K.A., Martel N., Webb R.E., Webb R.I., Teasdale R.D., and Parton, R.G. (2015). Modular detection of GFP-labeled proteins for rapid screening by electron microscopy in cells and organisms. Dev. Cell. 35, 513-25.

http://www.ncbi.nlm.nih.gov/pubmed/26585296

Martell, J.D., Deerinck, T.J., Sancak, Y., Poulos, T.L., Mootha, V.K., Sosinsky, G.E., Ellisman, M.H., and Ting, A.Y. (2012). Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat. Biotechnol. 30, 1143-1148.

http://www.ncbi.nlm.nih.gov/pubmed/23086203

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