Tracking and measuring local protein synthesis in vivo.

Abstract

Detecting when and how much of a protein molecule is synthesized is important for understanding cell function, but current methods either cannot be performed in vivo or have poor temporal resolution. Here, we developed a technique to detect and quantify subcellular protein synthesis events in real time in vivo. This Protein Translation Reporting (PTR) technique uses a genetic tag that produces a stoichiometric ratio of a small peptide portion of a split fluorescent protein and the protein of interest during protein synthesis. We show that the split fluorescent protein peptide can generate fluorescence within milliseconds upon binding the larger portion of the fluorescent protein, and that the fluorescence intensity is directly proportional to the number of molecules of the protein of interest synthesized. Using PTR, we tracked and measured protein synthesis events in single cells over time in vivo. We use different color split fluorescent proteins to detect multiple genes or alleles in single cells simultaneously. We also split a photoswitchable fluorescent protein to photoconvert the reconstituted fluorescent protein to a different channel to continually reset the time of detection of synthesis events.