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Nat Neurosci. 2012 Dec;15(12):1742-51. doi: 10.1038/nn.3246. Epub 2012 Oct 28.

Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy.

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1
Department of Pharmacology and Howard Hughes Medical Institute, University of California San Diego, La Jolla, California, USA.

Abstract

Protein synthesis is highly regulated throughout nervous system development, plasticity and regeneration. However, tracking the distributions of specific new protein species has not been possible in living neurons or at the ultrastructural level. Previously we created TimeSTAMP epitope tags, drug-controlled tags for immunohistochemical detection of specific new proteins synthesized at defined times. Here we extend TimeSTAMP to label new protein copies by fluorescence or photo-oxidation. Live microscopy of a fluorescent TimeSTAMP tag reveals that copies of the synaptic protein PSD95 are synthesized in response to local activation of growth factor and neurotransmitter receptors, and preferentially localize to stimulated synapses in rat neurons. Electron microscopy of a photo-oxidizing TimeSTAMP tag reveals new PSD95 at developing dendritic structures of immature neurons and at synapses in differentiated neurons. These results demonstrate the versatility of the TimeSTAMP approach for visualizing newly synthesized proteins in neurons.

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PMID:
23103964
PMCID:
PMC3509268
DOI:
10.1038/nn.3246
[Indexed for MEDLINE]
Free PMC Article

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