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

Butko and colleagues report the invention of fluorescent and photo-oxidizing versions of a molecular probe named TimeSTAMP that allows temporal tagging of newly synthesized proteins of interest. The study uses these new tools to track basal and pharmacologically-induced synthesis of the synaptic protein PDS-95 in real time via live fluorescent imaging and/or with ultrastructural resolution using electron microscopy. 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.