Tracking and Measuring Local Protein Synthesis In Vivo

Abstract

Detecting when and how much a protein molecule is synthesized is important for understanding cell function, but current methods have poor cellular or temporal resolution or are destructive to cells. 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 split red fluorescent protein 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 and arbitrarily reset the time of detection of synthesis events, continually over time.

Highly Efficient Fluorescent Probe to Visualize Protein Interactions at the Superresolution

“…We focused on the fact that Dendra2 has a substantially higher photoconversion property than other fluorescent proteins used for the existing split-pcFPs (Figure S2). A previous study has shown that Dendra2 can be divided between strands 10 and 11, enabling protein labeling by taking advantage of its spontaneous complementation . In this study, we develop a pair of nonfluorescent fragments of Dendra2 (split-Dendra2) that is reassembled and then recovers its photocontrollable fluorescence property only when the fragments are brought into proximity by PPIs.…”

“…A previous study has shown that Dendra2 can be divided between strands 10 and 11, enabling protein labeling by taking advantage of its spontaneous complementation. 8 In this study, we develop a pair of nonfluorescent fragments of Dendra2 (split-Dendra2) that is reassembled and then recovers its photocontrollable fluorescence property only when the fragments are brought into proximity by PPIs. To develop split-Dendra2 enabling PPI detection, we comprehensively investigated Dendra2 by dividing it into two fragments separately at their 14 different loop regions.…”