Abstract
Electron microscopy (EM) is the standard method for imaging cellular structures with nanometer resolution, but existing genetic tags are inactive in most cellular compartments1 or require light and can be difficult to use2. Here we report the development of 'APEX', a genetically encodable EM tag that is active in all cellular compartments and does not require light. APEX is a monomeric 28-kDa peroxidase that withstands strong EM fixation to give excellent ultrastructural preservation. We demonstrate the utility of APEX for high-resolution EM imaging of a variety of mammalian organelles and specific proteins using a simple and robust labeling procedure. We also fused APEX to the N or C terminus of the mitochondrial calcium uniporter (MCU), a recently identified channel whose topology is disputed3,4. These fusions give EM contrast exclusively in the mitochondrial matrix, suggesting that both the N and C termini of MCU face the matrix. Because APEX staining is not dependent on light activation, APEX should make EM imaging of any cellular protein straightforward, regardless of the size or thickness of the specimen.
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Acknowledgements
We thank A. Keating for use of her gel filtration chromatography system and A. Thor, A. Cone and M. Terada for assistance with electron tomography. The IMS-APEX EM data were obtained by H.-W. Rhee, P. Zou, J.D.M. and E.Vasile (Microscopy and Imaging Core Facility, Koch Institute at MIT). C. Uttamapinant (MIT) and H. Fraser (Stanford) provided helpful feedback on the manuscript. Funding was provided by US National Institutes of Health grants DP1 OD003961 (A.Y.T.), P41RR004050 (M.H.E.), P41GM103412 (M.H.E.), GM065937 (G.E.S.), GM072881 (G.E.S.), GM077465 (V.K.M.) and GM42614 (T.L.P.). J.D.M. was supported by a National Science Foundation Graduate Research Fellowship and a National Defense Science and Engineering Graduate Fellowship.
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Supplementary Figures 1–9 and Supplementary Tables 1–2 (PDF 18023 kb)
Supplementary Movie 1
EM tomographic volume of a Cx43-GFP-APEX gap junction. The first animation shows orientations along the three axes of the tomogram and an interpretation of the densities based on the atomic model of Cx26 gap junction channels. The second part of the movie shows progression through 251 XY sections of the tomogram along the Z direction and back again. In some slices, parts of the gap junction shows evidence of a repeat characteristic of a fortuitous cross-section view along the long axis of the gap junction channel. A higher magnification animation of the right hand side of the tomogram shows channel definition. In this field of view, the channel is enclosed by tubular membrane structures, most likely smooth endoplasmic reticulum, on both sides. Full-resolution movies, images and volumes are available for downloading from the Cell Centered Database with Microscopy Product ID # 83884 (http://ccdb.ucsd.edu/CCDBWebSite/index.html). Reference: Martone ME, Gupta A, Wong M, Qian X, Sosinsky G, Ludascher B, Ellisman MH. A cell-centered database for electron tomographic data. J Struct Biol 2002;138:145–155. (MOV 29232 kb)
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Martell, J., Deerinck, T., Sancak, Y. et al. Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30, 1143–1148 (2012). https://doi.org/10.1038/nbt.2375
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DOI: https://doi.org/10.1038/nbt.2375
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