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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.

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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.

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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.).

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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.

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Correspondence to Erin M Schuman.

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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

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