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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Animals, Newborn
  • Bacterial Proteins / analysis*
  • Bacterial Proteins / ultrastructure
  • Cells, Cultured
  • Disks Large Homolog 4 Protein
  • Epitopes / analysis
  • Epitopes / ultrastructure
  • Fluorescent Dyes*
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Luminescent Proteins / analysis*
  • Luminescent Proteins / ultrastructure
  • Membrane Proteins / metabolism*
  • Membrane Proteins / ultrastructure*
  • Microscopy, Electron / methods
  • Microscopy, Fluorescence / methods
  • Rats
  • Rats, Sprague-Dawley
  • Time Factors

Substances

  • Bacterial Proteins
  • DLG4 protein, human
  • Disks Large Homolog 4 Protein
  • Epitopes
  • Fluorescent Dyes
  • Intracellular Signaling Peptides and Proteins
  • Luminescent Proteins
  • Membrane Proteins
  • yellow fluorescent protein, Bacteria