title: Fluorescent and photo-oxidizing TimeSTAMP tags track protein fates in light and electron microscopy.
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.
authors.affiliation: Department of Pharmacology and Howard Hughes Medical Institute, University of California San Diego, La Jolla, California, USA.
authors.forename: Margaret T
authors.initials: MT
authors.lastname: Butko
authors.forename: Jin
authors.initials: J
authors.lastname: Yang
authors.forename: Yang
authors.initials: Y
authors.lastname: Geng
authors.forename: Hyung Joon
authors.initials: HJ
authors.lastname: Kim
authors.forename: Noo Li
authors.initials: NL
authors.lastname: Jeon
authors.forename: Xiaokun
authors.initials: X
authors.lastname: Shu
authors.forename: Mason R
authors.initials: MR
authors.lastname: Mackey
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.forename: Roger Y
authors.initials: RY
authors.lastname: Tsien
authors.forename: Michael Z
authors.initials: MZ
authors.lastname: Lin
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001426:Bacterial Proteins / Q000032:analysis* / Q000648:ultrastructure; D002478:Cells, Cultured; D000076122:Disks Large Homolog 4 Protein; D000939:Epitopes / Q000032:analysis / Q000648:ultrastructure; D005456:Fluorescent Dyes; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000032:analysis* / Q000648:ultrastructure; D008565:Membrane Proteins / Q000378:metabolism* / Q000648:ultrastructure*; D008854:Microscopy, Electron / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
chemical_list: D001426:Bacterial Proteins; C501444:DLG4 protein, human; D000076122:Disks Large Homolog 4 Protein; D000939:Epitopes; D005456:Fluorescent Dyes; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; C054562:yellow fluorescent protein, Bacteria