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Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy

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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Figure 1: EM reporter scheme and characterization of APEX oligomerization state.
Figure 2: Active-site engineering to boost the activity of APEX.
Figure 3: EM imaging of cellular proteins and organelles with APEX.

References

  1. Hopkins, C., Gibson, A., Stinchcombe, J. & Futter, C. Chimeric molecules employing horseradish peroxidase as reporter enzyme for protein localization in the electron microscope. Methods Enzymol. 327, 35–45 (2000).

    Article  CAS  Google Scholar 

  2. Shu, X. et al. A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol. 9, e1001041 (2011).

    Article  CAS  Google Scholar 

  3. Baughman, J.M. et al. Integrative genomics identifies MCU as an essential component of the mitochondrial calcium uniporter. Nature 476, 341–345 (2011).

    Article  CAS  Google Scholar 

  4. De Stefani, D., Raffaello, A., Teardo, E., Szabo, I. & Rizzuto, R. A forty-kilodalton protein of the inner membrane is the mitochondrial calcium uniporter. Nature 476, 336–340 (2011).

    Article  CAS  Google Scholar 

  5. De Mey, J., Moeremans, M., Geuens, G., Nuydens, R. & De Brabander, M. High resolution light and electron microscopic localization of tubulin with the IGS (immuno gold staining) method. Cell Biol. Int. Rep. 5, 889–899 (1981).

    Article  CAS  Google Scholar 

  6. Giepmans, B.N.G., Deerinck, T.J., Smarr, B.L., Jones, Y.Z. & Ellisman, M.H. Correlated light and electron microscopic imaging of multiple endogenous proteins using Quantum dots. Nat. Methods 2, 743–749 (2005).

    Article  CAS  Google Scholar 

  7. Henderson, D. & Weber, K. Three-dimensional organization of microfilaments and microtubules in the cytoskeleton: Immunoperoxidase labelling and stereo-electron microscopy of detergent-extracted cells. Exp. Cell Res. 124, 301–316 (1979).

    Article  CAS  Google Scholar 

  8. Deerinck, T.J. et al. Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy. J. Cell Biol. 126, 901–910 (1994).

    Article  CAS  Google Scholar 

  9. Ellisman, M.H., Deerinck, T.J., Shu, X. & Sosinsky, G.E. Picking faces out of a crowd: genetic labels for identification of proteins in correlated light and electron microscopy imaging. Methods Cell Biol. 111 139–155 (2012).

  10. Schnell, U., Dijk, F., Sjollema, K.A. & Giepmans, B.N.G. Immunolabeling artifacts and the need for live-cell imaging. Nat. Methods 9, 152–158 (2012).

    Article  CAS  Google Scholar 

  11. Tokuyasu, K.T. Application of cryoultramicrotomy to immunocytochemistry. J. Microsc. 143, 139–149 (1986).

    Article  CAS  Google Scholar 

  12. Porstmann, B., Porstmann, T., Nugel, E. & Evers, U. Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: Horseradish peroxidase, alkaline phosphatase or [beta]-galactosidase? J. Immunol. Methods 79, 27–37 (1985).

    Article  CAS  Google Scholar 

  13. Connolly, C.N., Futter, C.E., Gibson, A., Hopkins, C.R. & Cutler, D.F. Transport into and out of the Golgi complex studied by transfecting cells with cDNAs encoding horseradish peroxidase. J. Cell Biol. 127, 641–652 (1994).

    Article  CAS  Google Scholar 

  14. Li, J., Wang, Y., Chiu, S.-L. & Cline, H. Membrane targeted horseradish peroxidase as a marker for correlative fluorescence and electron microscopy studies. Front. Neural Circuits 4, 6 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Gaietta, G. et al. Multicolor and electron microscopic imaging of connexin trafficking. Science 296, 503–507 (2002).

    Article  CAS  Google Scholar 

  16. Uttamapinant, C. et al. A fluorophore ligase for site-specific protein labeling inside living cells. Proc. Natl. Acad. Sci. USA 107, 10914–10919 (2010).

    Article  CAS  Google Scholar 

  17. Tsien, R. Imagining imaging's future. Nat. Rev. Mol. Cell Biol. 4, SS16–SS21 (2003).

    Google Scholar 

  18. Patterson, W.R. & Poulos, T.L. Crystal structure of recombinant pea cytosolic ascorbate peroxidase. Biochemistry 34, 4331–4341 (1995).

    Article  CAS  Google Scholar 

  19. Mandelman, D., Li, H., Poulos, T.L. & Schwarz, F.P. The role of quaternary interactions on the stability and activity of ascorbate peroxidase. Protein Sci. 7, 2089–2098 (1998).

    Article  CAS  Google Scholar 

  20. McKinney, S.A., Murphy, C.S., Hazelwood, K.L., Davidson, M.W. & Looger, L.L. A bright and photostable photoconvertible fluorescent protein. Nat. Methods 6, 131–133 (2009).

    Article  CAS  Google Scholar 

  21. Koshiba, T. Cytosolic ascorbate peroxidase in seedlings and leaves of maize (Zea mays). Plant Cell Physiol. 34, 713–721 (1993).

    Article  CAS  Google Scholar 

  22. Lauf, U., Lopez, P. & Falk, M.M. Expression of fluorescently tagged connexins: a novel approach to rescue function of oligomeric DsRed-tagged proteins. FEBS Lett. 498, 11–15 (2001).

    Article  CAS  Google Scholar 

  23. Henriksen, A., Smith, A.T. & Gajhede, M. The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates. J. Biol. Chem. 274, 35005–35011 (1999).

    Article  CAS  Google Scholar 

  24. Lad, L., Mewies, M. & Raven, E.L. Substrate binding and catalytic mechanism in ascorbate peroxidase: evidence for two ascorbate binding sites. Biochemistry 41, 13774–13781 (2002).

    Article  CAS  Google Scholar 

  25. Sosinsky, G.E. et al. Tetracysteine genetic tags complexed with biarsenical ligands as a tool for investigating gap junction structure and dynamics. Cell Commun. Adhes. 10, 181–186 (2003).

    Article  CAS  Google Scholar 

  26. Qi, Y.B., Garren, E.J., Shu, X., Tsien, R.Y. & Jin, Y. Photo-inducible cell ablation in Caenorhabditis elegans using the genetically encoded singlet oxygen generating protein miniSOG. Proc. Natl. Acad. Sci. USA 109, 7499–7504 (2012).

    Article  CAS  Google Scholar 

  27. Costantini, L.M., Fossati, M., Francolini, M. & Snapp, E.L. Assessing the tendency of fluorescent proteins to oligomerize under physiologic conditions. Traffic 13, 643–649 (2012).

    Article  CAS  Google Scholar 

  28. Ryan, B., Carolan, N. & Ó'Fágáin, C. Horseradish and soybean peroxidases: comparable tools for alternative niches? Trends Biotechnol. 24, 355–363 (2006).

    Article  CAS  Google Scholar 

  29. Berglund, G. et al. The catalytic pathway of horseradish peroxidase at high resolution. Nature 417, 463–468 (2002).

    Article  CAS  Google Scholar 

  30. Sharp, K.H., Moody, P.C.E., Brown, K.A. & Raven, E.L. Crystal structure of the ascorbate peroxidase salicylhydroxamic acid complex. Biochemistry 43, 8644–8651 (2004).

    Article  CAS  Google Scholar 

  31. Kirmse, R., Bouchet-Marquis, C.d., Page, C., Hoenger, A. & Thomas,, M.R. Three-dimensional cryo-electron microscopy on intermediate filaments. in Methods in Cell Biology, Vol. 96 (eds. Müller-Reichert, T. & Verkade, P.) 565–589 (Academic Press, 2010).

  32. Cheek, J., Mandelman, D., Poulos, T. & Dawson, J. A study of the K+-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side. J. Biol. Inorg. Chem. 4, 64–72 (1999).

    Article  CAS  Google Scholar 

  33. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  34. Richards, M.K. & Marletta, M.A. Characterization of neuronal nitric oxide synthase and a C415H mutant, purified from a baculovirus overexpression system. Biochemistry 33, 14723–14732 (1994).

    Article  CAS  Google Scholar 

  35. Barrows, T.P. & Poulos, T.L. Role of electrostatics and salt bridges in stabilizing the compound i radical in ascorbate peroxidase. Biochemistry 44, 14062–14068 (2005).

    Article  CAS  Google Scholar 

  36. Kremer, J.R., Mastronarde, D.N. & McIntosh, J.R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

    Article  CAS  Google Scholar 

  37. Lawrence, A., Bouwer, J.C., Perkins, G. & Ellisman, M.H. Transform-based backprojection for volume reconstruction of large format electron microscope tilt series. J. Struct. Biol. 154, 144–167 (2006).

    Article  CAS  Google Scholar 

  38. Martone, M.E. et al. A cell-centered database for electron tomographic data. J. Struct. Biol. 138, 145–155 (2002).

    Article  CAS  Google Scholar 

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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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Correspondence to Alice Y Ting.

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The Massachusetts Institute of Technology is seeking to file a patent application covering part of the information contained in this article.

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Supplementary Text and Figures

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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