Skip to main content
SpringerLink
Log in

Advances in molecular probe-based labeling tools and their application to multiscale multimodal correlated microscopies

  • Review
  • Published:
Journal of Chemical Biology

Abstract

The need to determine the precise subcellular distribution of specific proteins and macromolecular complexes in cells and tissues has been the major driving force behind the development of new molecular-genetic and chemical-labeling approaches applicable to high-resolution, correlated, multidimensional microscopy. This short review is intended to provide an overview of recently developed and widely used electron microscopy (EM)-compatible probes, including tetracysteine tags, mini singlet oxygen generator (MiniSOG), time-specific tag for the age measurement of proteins (TimeSTAMP) with MiniSOG, and enhanced ascorbate peroxidase (APEX). We describe how these highly specific and genetically introduced EM probes are now used, in conjunction with lower resolution light microscopic methods, to obtain wide field or dynamic records of live preparation or of large maps in 3D using recently developed laboratory-scale X-ray microscopes. The article is intended to enable researchers through a high-level view of the toolbox of labels available today for studies aiming to analyze dynamic subcellular and molecular processes in cell culture systems as well as in animal tissues—and ultimately allow investigators to determine the precise location of macromolecular complexes by EM.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9

Similar content being viewed by others

References

  1. Boassa D, Berlanga ML, Yang MA, Terada M, Hu J, Bushong EA, Hwang M, Masliah E, George JM, Ellisman MH (2013) Mapping the subcellular distribution of alpha-synuclein in neurons using genetically encoded probes for correlated light and electron microscopy: implications for Parkinson’s disease pathogenesis. J Neurosci 33(6):2605–2615

    Article  CAS  Google Scholar 

  2. Boassa D, Nguyen P, Hu J, Ellisman MH, Sosinsky GE (2015) Pannexin2 oligomers localize in the membranes of endosomal vesicles in mammalian cells while Pannexin1 channels traffic to the plasma membrane. Front Cell Neurosci 8:468

    Article  Google Scholar 

  3. Burgers PP, Ma Y, Margarucci L, Mackey M, van der Heyden MA, Ellisman M, Scholten A, Taylor SS, Heck AJ (2012) A small novel A-kinase anchoring protein (AKAP) that localizes specifically protein kinase A-regulatory subunit I (PKA-RI) to the plasma membrane. J Biol Chem 287(52):43789–43797

    Article  CAS  Google Scholar 

  4. Bushong EA, Johnson DD, Kim KY, Terada M, Hatori M, Peltier ST, Panda S, Merkle A, Ellisman MH (2014) X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens. Microsc Microanal 13:1–8

    Google Scholar 

  5. Butko MT, Yang J, Geng Y, Kim HJ, Jeon NL, Shu X, Mackey MR, Ellisman MH, Tsien RY, Lin MZ (2012) Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy. Nat Neurosci 15(12):1742–51

    Article  CAS  Google Scholar 

  6. Cleyrat C, Darehshouri A, Steinkamp MP, Vilaine M, Boassa D, Ellisman MH, Hermouet S, Wilson BS (2014) Mpl traffics to the cell surface through conventional and unconventional routes. Traffic 15(9):961–982

    Article  CAS  Google Scholar 

  7. Denk W, Horstmann H (2004) Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure. PLoS Biol 2(11), e329

    Article  Google Scholar 

  8. Ellisman MH, Deerinck TJ, Shu X, Sosinsky GE (2012) 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

    Article  CAS  Google Scholar 

  9. Gaietta G, Deerinck TJ, Adams SR, Bouwer J, Tour O, Laird DW, Sosinsky GE, Tsien RY, Ellisman MH (2002) Multicolor and electron microscopic imaging of connexin trafficking. Science 296:503–507

    Article  CAS  Google Scholar 

  10. Gaietta GM, Giepmans BN, Deerinck TJ, Smith WB, Ngan L, Llopis J, Adams SR, Tsien RY, Ellisman MH (2006) Golgi twins in late mitosis revealed by genetically encoded tags for live cell imaging and correlated electron microscopy. Proc Natl Acad Sci U S A 103:17777–17782

    Article  CAS  Google Scholar 

  11. Heim R, Tsien RY (1996) Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer. Curr Biol 6(2):178–182

    Article  CAS  Google Scholar 

  12. Hoffmann C, Gaietta G, Zürn A, Adams SR, Terrillon S, Ellisman MH, Tsien RY, Lohse MJ (2010) Fluorescent labeling of tetracysteine-tagged proteins in intact cells. Nat Protoc 5(10):1666–1677

    Article  CAS  Google Scholar 

  13. Kuipers J, van Ham TJ, Kalicharan RD, Veenstra-Algra A, Sjollema KA, Dijk F, Schnell U, Giepmans BN (2015) FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles. Cell Tissue Res. 360(1):61–70

  14. Lam SS, Martell JD, Kamer KJ, Deerinck TJ, Ellisman MH, Mootha VK, Ting AY (2015) Directed evolution of APEX2 for electron microscopy and proximity labeling. Nat Methods 12(1):51–54

    Article  CAS  Google Scholar 

  15. Lelek M, Di Nunzio F, Henriques R, Charneau P, Arhel N, Zimmer C (2012) Superresolution imaging of HIV in infected cells with FlAsH-PALM. Proc Natl Acad Sci U S A 109(22):8564–8569

    Article  CAS  Google Scholar 

  16. Lin MZ, Tsien RY (2010) TimeSTAMP tagging of newly synthesized proteins. Curr Protoc Protein Sci Chapter 26:Unit 26.5

  17. Ludwig A, Howard G, Mendoza-Topaz C, Deerinck T, Mackey M, Sandin S, Ellisman MH, Nichols BJ (2013) Molecular composition and ultrastructure of the caveolar coat complex. PLoS Biol 11(8), e1001640

    Article  CAS  Google Scholar 

  18. Martell JD, Deerinck TJ, Sancak Y, Poulos TL, Mootha VK, Sosinsky GE, Ellisman MH, Ting AY (2012) Engineered ascorbate peroxidase as a genetically encoded reporter for electron microscopy. Nat Biotechnol 30:1143–1150

    Article  CAS  Google Scholar 

  19. Ou HD, Kwiatkowski W, Deerinck TJ, Noske A, Blain KY, Land HS, Soria C, Powers CJ, May AP, Shu X, Tsien RY, Fitzpatrick JA, Long JA, Ellisman MH, Choe S, O’Shea CC (2012) A structural basis for the assembly and functions of a viral polymer that inactivates multiple tumor suppressors. Cell 151(2):304–319

    Article  CAS  Google Scholar 

  20. Shu X, Lev-Ram V, Deerinck TJ, Qi Y, Ramko EB, Davidson MW, Jin Y, Ellisman MH, Tsien RY (2011) A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms. PLoS Biol 9(4):e1001041

    Article  CAS  Google Scholar 

  21. Tsien RY (1998) The green fluorescent protein. Annu Rev Biochem 67:509–544, Review

    Article  CAS  Google Scholar 

  22. Whitt MA, Mire CE (2011) Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy. Methods 55(2):127–136

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work presented here was conducted at the National Center for Microscopy and Imaging Research at San Diego, which is supported by NIH Grant GM103412 awarded to Dr. Mark Ellisman. National Institutes of Health grants P41RR004050 (Mark Ellisman) and GM086197 (Roger Y. Tsien and Mark Ellisman) and AHA Grant 10SDG2610281 (Daniela Boassa) provided funding for this research.

Compliance with ethical standards

ᅟ

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. National Center for Microscopy and Imaging Research and Center for Research on Biological Systems, University of California, San Diego, La Jolla, San Diego, CA, 92093, USA

    M. H. Ellisman, T. J. Deerinck, K. Y. Kim, E. A. Bushong, S. Phan & D. Boassa

  2. Department of Neurosciences, University of California, San Diego, La Jolla, San Diego, CA, 92093, USA

    M. H. Ellisman

  3. Salk Institute for Biological Studies, San Diego, CA, USA

    M. H. Ellisman

  4. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    A. Y. Ting

Authors
  1. M. H. Ellisman
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. T. J. Deerinck
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. K. Y. Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. A. Bushong
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. S. Phan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. Y. Ting
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. D. Boassa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to D. Boassa.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ellisman, M.H., Deerinck, T.J., Kim, K.Y. et al. Advances in molecular probe-based labeling tools and their application to multiscale multimodal correlated microscopies. J Chem Biol 8, 143–151 (2015). https://doi.org/10.1007/s12154-015-0132-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12154-015-0132-6

Keywords

Search

Navigation