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

Dieterich, Daniela C. and Hodas, Jennifer J. L. and Gouzer, Géraldine and Shadrin, Ilya Y. and Ngo, John T. and Triller, Antoine and Tirrell, David A. and Schuman, Erin M. (2010) In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons. Nature Neuroscience, 13 (7). pp. 897-905. ISSN 1097-6256. PMCID PMC2920597.

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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.

Item Type:Article
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Tirrell, David A.0000-0003-3175-4596
Schuman, Erin M.0000-0002-7053-1005
Additional Information:© 2010 Nature Publishing Group, a division of Macmillan Publishers Limited. Received 22 January; accepted 17 May; published online 13 June 2010. 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 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.
Funding AgencyGrant Number
Deutsche Akademie der Naturforscher LeopoldinaUNSPECIFIED
Howard Hughes Medical Institute (HHMI)UNSPECIFIED
Ministere de l'Enseignement Superieur et de la RechercheUNSPECIFIED
Nationale de la Recherche MorphoSynDiff-INSERMUNSPECIFIED
Issue or Number:7
PubMed Central ID:PMC2920597
Record Number:CaltechAUTHORS:20100713-132314376
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Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:19032
Deposited By: Tony Diaz
Deposited On:05 Aug 2010 21:54
Last Modified:09 Mar 2020 13:19

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