A drug-controllable tag for visualizing newly synthesized proteins in cells and whole animals.
Research on basic cellular processes involving local production or delivery of proteins, such as activity-dependent synaptic modification in neurons, would benefit greatly from a robust, nontoxic method to visualize selectively newly synthesized copies of proteins of interest within cells, tissues, or animals. We report a technique for covalent labeling of newly synthesized proteins of interest based on drug-dependent preservation of epitope tags. Epitope tags are removed from proteins of interest immediately after translation by the activity of a sequence-specific protease until the time a protease inhibitor is added, after which newly synthesized protein copies retain their tags. This method, which we call TimeSTAMP for time-specific tagging for the age measurement of proteins, allows sensitive and nonperturbative visualization and quantification of newly synthesized proteins of interest with exceptionally tight temporal control. We demonstrate applications of TimeSTAMP in retrospectively identifying growing synapses in cultured neurons and in visualizing the distribution of recently synthesized proteins in intact fly brains.
Lin
Michael Z
MZ
Department of Pharmacology and Howard Hughes Medical Institute, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0647, USA. mlin@ucsd.edu
Glenn
Jeffrey S
JS
Tsien
Roger Y
RY
D000818:Animals; D002219:Carbamates / Q000494:pharmacology; D002460:Cell Line; D004331:Drosophila melanogaster; D000939:Epitopes / Q000737:chemistry / Q000235:genetics; D006801:Humans; D047028:Macrocyclic Compounds / Q000494:pharmacology; D008722:Methods; D009474:Neurons / Q000737:chemistry / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014176:Protein Biosynthesis; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D013569:Synapses / Q000737:chemistry / Q000378:metabolism*; D013844:Thiazoles / Q000494:pharmacology; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
D002219:Carbamates; D000939:Epitopes; D047028:Macrocyclic Compounds; D011480:Protease Inhibitors; D011804:Quinolines; D011993:Recombinant Fusion Proteins; D013844:Thiazoles; D017361:Viral Nonstructural Proteins; C479555:BILN 2061; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases