Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Technical Report
  • Published:

In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons

Abstract

Protein translation has been implicated in different forms of synaptic plasticity, but direct in situ visualization of new proteins is limited to one or two proteins at a time. Here we describe a metabolic labeling approach based on incorporation of noncanonical amino acids into proteins followed by chemoselective fluorescence tagging by means of 'click chemistry'. After a brief incubation with azidohomoalanine or homopropargylglycine, a robust fluorescent signal was detected in somata and dendrites. Pulse-chase application of azidohomoalanine and homopropargylglycine allowed visualization of proteins synthesized in two sequential time periods. This technique can be used to detect changes in protein synthesis and to evaluate the fate of proteins synthesized in different cellular compartments. Moreover, using strain-promoted cycloaddition, we explored the dynamics of newly synthesized membrane proteins using single-particle tracking and quantum dots. The newly synthesized proteins showed a broad range of diffusive behaviors, as would be expected for a pool of labeled proteins that is heterogeneous.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Chemical components and FUNCAT procedure.
Figure 2: Visualization of newly synthesized proteins in dissociated primary hippocampal neurons.
Figure 3: Sequential labeling of two newly synthesized protein pools with two metabolic markers.
Figure 4: Time course for the detection of newly synthesized proteins in somata and dendrites.
Figure 5: BDNF-induced increases in protein synthesis.
Figure 6: BDNF-induced increase in dendritic protein synthesis.
Figure 7: Local BDNF-induced increase in dendritic protein synthesis.
Figure 8: Diffusion properties of newly synthesized proteins at the surface of dissociated primary hippocampal neurons.

Similar content being viewed by others

References

  1. Sutton, M.A. & Schuman, E.M. Dendritic protein synthesis, synaptic plasticity, and memory. Cell 127, 49–58 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Nguyen, P.V., Abel, T. & Kandel, E.R. Requirement of a critical period of transcription for induction of a late phase of LTP. Science 265, 1104–1107 (1994).

    Article  CAS  PubMed  Google Scholar 

  3. Bito, H., Deisseroth, K. & Tsien, R.W. CREB phosphorylation and dephosphorylation: a Ca2+- and stimulus duration-dependent switch for hippocampal gene expression. Cell 87, 1203–1214 (1996).

    Article  CAS  PubMed  Google Scholar 

  4. Casadio, A. et al. A transient, neuron-wide form of CREB-mediated long-term facilitation can be stabilized at specific synapses by local protein synthesis. Cell 99, 221–237 (1999).

    Article  CAS  PubMed  Google Scholar 

  5. Kang, H. & Schuman, E.M. A requirement for local protein synthesis in neurotrophin-induced hippocampal synaptic plasticity. Science 273, 1402–1406 (1996).

    Article  CAS  PubMed  Google Scholar 

  6. Martin, K.C. et al. Synapse-specific, long-term facilitation of Aplysia sensory to motor synapses: a function for local protein synthesis in memory storage. Cell 91, 927–938 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Weiler, I.J., Wang, X. & Greenough, W.T. Synapse-activated protein synthesis as a possible mechanism of plastic neural change. Prog. Brain Res. 100, 189–194 (1994).

    Article  CAS  PubMed  Google Scholar 

  8. Steward, O. mRNA localization in neurons: a multipurpose mechanism? Neuron 18, 9–12 (1997).

    Article  CAS  PubMed  Google Scholar 

  9. Liao, L. et al. BDNF induces widespread changes in synaptic protein content and up-regulates components of the translation machinery: an analysis using high-throughput proteomics. J. Proteome Res. 6, 1059–1071 (2007).

    Article  CAS  PubMed  Google Scholar 

  10. Manadas, B. et al. BDNF-induced changes in the expression of the translation machinery in hippocampal neurons: protein levels and dendritic mRNA. J. Proteome Res. 8, 4536–4552 (2009).

    Article  CAS  PubMed  Google Scholar 

  11. Takei, N. et al. Brain-derived neurotrophic factor induces mammalian target of rapamycin-dependent local activation of translation machinery and protein synthesis in neuronal dendrites. J. Neurosci. 24, 9760–9769 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Aakalu, G., Smith, W.B., Nguyen, N., Jiang, C. & Schuman, E.M. Dynamic visualization of local protein synthesis in hippocampal neurons. Neuron 30, 489–502 (2001).

    Article  CAS  PubMed  Google Scholar 

  13. Macchi, P. et al. A GFP-based system to uncouple mRNA transport from translation in a single living neuron. Mol. Biol. Cell 14, 1570–1582 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wang, D.O. et al. Synapse- and stimulus-specific local translation during long-term neuronal plasticity. Science 324, 1536–1540 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lin, M.Z., Glenn, J.S. & Tsien, R.Y. A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals. Proc. Natl. Acad. Sci. USA 105, 7744–7749 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Kiick, K.L., Saxon, E., Tirrell, D.A. & Bertozzi, C.R. Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation. Proc. Natl. Acad. Sci. USA 99, 19–24 (2002).

    Article  CAS  PubMed  Google Scholar 

  17. Link, A.J., Mock, M.L. & Tirrell, D.A. Non-canonical amino acids in protein engineering. Curr. Opin. Biotechnol. 14, 603–609 (2003).

    Article  CAS  PubMed  Google Scholar 

  18. Link, A.J. & Tirrell, D.A. Cell surface labeling of Escherichia coli via copper(I)-catalyzed [3+2] cycloaddition. J. Am. Chem. Soc. 125, 11164–11165 (2003).

    Article  CAS  PubMed  Google Scholar 

  19. Zhang, Z. et al. A new strategy for the site-specific modification of proteins in vivo. Biochemistry 42, 6735–6746 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Beatty, K.E. & Tirrell, D.A. Two-color labeling of temporally defined protein populations in mammalian cells. Bioorg. Med. Chem. Lett. 18, 5995–5999 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Dieterich, D.C. et al. Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging. Nat. Protoc. 2, 532–540 (2007).

    Article  PubMed  Google Scholar 

  22. Dieterich, D.C., Link, A.J., Graumann, J., Tirrell, D.A. & Schuman, E.M. Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal noncanonical amino acid tagging (BONCAT). Proc. Natl. Acad. Sci. USA 103, 9482–9487 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Rostovtsev, V.V., Green, L.G., Fokin, V.V. & Sharpless, K.B. A stepwise Huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Edn. Engl. 41, 2596–2599 (2002).

    Article  CAS  Google Scholar 

  24. Whitaker, J.E., Haugland, R.P., Ryan, D., Hewitt, P.C. & Prendergast, F.G. Fluorescent rhodol derivatives: versatile, photostable labels and tracers. Anal. Biochem. 207, 267–279 (1992).

    Article  CAS  PubMed  Google Scholar 

  25. Lewin, G.R. & Barde, Y.A. Physiology of the neurotrophins. Annu. Rev. Neurosci. 19, 289–317 (1996).

    Article  CAS  PubMed  Google Scholar 

  26. Alcor, D., Gouzer, G. & Triller, A. Single-particle tracking methods for the study of membrane receptors dynamics. Eur. J. Neurosci. 30, 987–997 (2009).

    Article  PubMed  Google Scholar 

  27. Baskin, J.M. et al. Copper-free click chemistry for dynamic in vivo imaging. Proc. Natl. Acad. Sci. USA 104, 16793–16797 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Dahan, M. et al. Diffusion dynamics of glycine receptors revealed by single-quantum dot tracking. Science 302, 442–445 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Renner, M., Choquet, D. & Triller, A. Control of the postsynaptic membrane viscosity. J. Neurosci. 29, 2926–2937 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Sergé, A., Fourgeaud, L., Hemar, A. & Choquet, D. Receptor activation and Homer differentially control the lateral mobility of metabotropic glutamate receptor 5 in the neuronal membrane. J. Neurosci. 22, 3910–3920 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

  31. Renner, M. et al. Deleterious effects of amyloid beta oligomers acting as an extracellular scaffold for mGluR5. Neuron (in the press).

  32. Renner, M.L., Cognet, L., Lounis, B., Triller, A. & Choquet, D. The excitatory postsynaptic density is a size exclusion diffusion environment. Neuropharmacology 56, 30–36 (2009).

    Article  CAS  PubMed  Google Scholar 

  33. Ouyang, Y., Kantor, D., Harris, K.M., Schuman, E.M. & Kennedy, M.B. Visualization of the distribution of autophosphorylated calcium/calmodulin-dependent protein kinase II after tetanic stimulation in the CA1 area of the hippocampus. J. Neurosci. 17, 5416–5427 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Waung, M.W., Pfeiffer, B.E., Nosyreva, E.D., Ronesi, J.A. & Huber, K.M. Rapid translation of Arc/Arg3.1 selectively mediates mGluR-dependent LTD through persistent increases in AMPAR endocytosis rate. Neuron 59, 84–97 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Ouyang, Y., Rosenstein, A., Kreiman, G., Schuman, E.M. & Kennedy, M.B. Tetanic stimulation leads to increased accumulation of Ca2+/calmodulin-dependent protein kinase II via dendritic protein synthesis in hippocampal neurons. J. Neurosci. 19, 7823–7833 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Villareal, G., Li, Q., Cai, D. & Glanzman, D.L. The role of rapid, local, postsynaptic protein synthesis in learning-related synaptic facilitation in Aplysia. Curr. Biol. 17, 2073–2080 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Beatty, K.E. et al. Fluorescence visualization of newly synthesized proteins in mammalian cells. Angew. Chem. Int. Edn. Engl. 45, 7364–7367 (2006).

    Article  CAS  Google Scholar 

  38. Agard, N.J., Prescher, J.A. & Bertozzi, C.R. A strain-promoted [3 + 2] azidealkyne cycloaddition for covalent modification of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046–15047 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Taylor, A.M., Dieterich, D.C., Ito, H.T., Kim, S.A. & Schuman, E.M. Microfluidic local perfusion chambers for the visualization and manipulation of synapses. Neuron 66, 57–68 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Frey, U. & Morris, R.G. Synaptic tagging and long-term potentiation. Nature 385, 533–536 (1997).

    Article  CAS  PubMed  Google Scholar 

  41. Huang, T., McDonough, C.B. & Abel, T. Compartmentalized PKA signaling events are required for synaptic tagging and capture during hippocampal late-phase long-term potentiation. Eur. J. Cell Biol. 85, 635–642 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Sajikumar, S., Navakkode, S. & Frey, J.U. Identification of compartment- and process-specific molecules required for “synaptic tagging” during long-term potentiation and long-term depression in hippocampal CA1. J. Neurosci. 27, 5068–5080 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Ngo, J.T. et al. Cell-selective metabolic labeling of proteins. Nat. Chem. Biol. 5, 715–717 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Flexner, J.B., Flexner, L.B. & Stellar, E. Memory in mice as affected by intracerebral puromycin. Science 141, 57–59 (1963).

    Article  CAS  PubMed  Google Scholar 

  45. Agranoff, B.W. & Klinger, P.D. Puromycin effect on memory fixation in the goldfish. Science 146, 952–953 (1964).

    Article  CAS  PubMed  Google Scholar 

  46. Renner, M., Specht, C.G. & Triller, A. Molecular dynamics of postsynaptic receptors and scaffold proteins. Curr. Opin. Neurobiol. 18, 532–540 (2008).

    Article  CAS  PubMed  Google Scholar 

  47. Wang, Q. et al. Bioconjugation by copper(I)-catalyzed azide–alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125, 3192–3193 (2003).

    Article  CAS  PubMed  Google Scholar 

  48. Gogolla, N., Galimberti, I., DePaola, V. & Caroni, P. Long-term live imaging of neuronal circuits in organotypic hippocampal slice cultures. Nat. Protoc. 1, 1223–1226 (2006).

    Article  CAS  PubMed  Google Scholar 

  49. Bannai, H., Levi, S., Schweizer, C., Dahan, M. & Triller, A. Imaging the lateral diffusion of membrane molecules with quantum dots. Nat. Protoc. 1, 2628–2634 (2007).

    Article  Google Scholar 

Download references

Acknowledgements

We thank L. Chen for making beautiful cultured hippocampal neurons. We thank A.J. Link for discussions and help with the tag syntheses. We are grateful to O. Kobler for help with Imaris software. We are extremely grateful to both C. Bertozzi and J. Baskin for providing the difluorinated cyclooctyne-biotin and advising on its use. This work was supported by the German Academy for Natural Scientists Leopoldina (D.C.D.), the US National Institutes of Health (E.M.S. and D.A.T.), the Howard Hughes Medical Institute (E.M.S.), the Ministère de l'Enseignement Supérieur et de la Recherche (G.G.) and the Nationale de la Recherche MorphoSynDiff–INSERM (A.T.).

Author information

Authors and Affiliations

Authors

Contributions

D.C.D., J.J.L.H., G.G. and E.M.S. performed experiments; D.C.D., G.G., A.T. and E.M.S. designed experiments; D.C.D., J.J.L.H., I.Y.S., G.G. and E.M.S. analyzed data; D.C.D., G.G., A.T. and E.M.S. wrote the paper; J.T.N. and D.A.T. provided reagents.

Corresponding author

Correspondence to Erin M Schuman.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 11714 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Dieterich, D., Hodas, J., Gouzer, G. et al. In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nat Neurosci 13, 897–905 (2010). https://doi.org/10.1038/nn.2580

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.2580

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing