My lab is creating technology to image and quantify gene activity in real time and with single-molecule precision in living cells. Using new mRNA and protein tags, fluorescent probes, and single-molecule microscopy, we image DNA transcription and mRNA translation live. In this talk, I will briefly introduce our technology and describe how it can be used to amplify fluorescence from newly synthesized proteins as they are being translated from single mRNAs. I will show how we quantify these signals to determine the size, shape, subcellular localization, and mobilities of mRNA translation sites, as well as their protein synthesis dynamics. I will then highlight some of our most recent advances, including (1) single-molecule co-imaging of transcription and translation of CRISPR-edited endogenous genes, (2) optogenetic tools to tether mRNA during translation, (3) open-source software to quantify single-mRNA translation, and (4) a palette of genetically-encoded fluorescent probes to light up translation in multiple colors with high fidelity.
Support or Funding Information
NIH: R35GM119728 and R56AI155897; NSF: MCB-1845761
CRISPR’d cell lines. To co-image single-gene transcription and translation kinetics, CRISPR was used in HeLa cells to tag both MYH9 alleles (diploid in HeLa) encoding myosin-2A (Myo2A) with an N-terminal 6xFLAG tag (green) and a 24xMS2 tag in the 3’UTR (purple). Image Credit: O'Neil Wiggan, CSU Stasevich Lab
