We are developing technology to image single RNA translation dynamics in living cells. Using high-affinity antibody-based probes, multimerized epitope tags, and single molecule microscopy, we are able to visualize and quantify the emergence of nascent protein chains from single premarked RNA1. Here, I'll describe this technology as well as a two color extension useful for comparing translation rates between two different parts of a single open reading frame (ORF) or two different ORFs. Using information from the correlations of fluorescence fluctuations, we can accurately quantify single mRNA translation elongation rates in both tagged and untagged portions of ORFs. By transiently loading probes and reporter DNA into cells in a combinatorial fashion, multiplexed imaging of gene expression is possible. Preliminary application of this technology to the study of viral frameshifting will be discussed.

Timothy J. Stasevich is an Assistant Professor in the Department of Biochemistry at Colorado State University (CSU). His lab uses a combination of advanced fluorescence microscopy, genetic engineering, and computational modeling to study the dynamics of gene regulation in living mammalian cells. Most recently, his lab has pioneered the imaging of real-time single mRNA translation dynamics in living cells [1]. Dr. Stasevich received his B.S. in Physics and Mathematics from the University of Michigan, Dearborn, and his Ph. D. in Physics from the University of Maryland, College Park. He transitioned into experimental biophysics as a post-doctoral research fellow in the laboratory of Dr. James G. McNally at the National Cancer Institute. During this time, he developed technology based on fluorescence microscopy to help establish gold-standard measurements of live-cell protein dynamics. Dr. Stasevich next moved to Osaka University, where he worked with Dr. Hiroshi Kimura as a Japan Society for the Promotion of Science Foreign Postdoctoral Research Fellow. While there, he helped create technology to image endogenous proteins and their post-translation modifications in vivo. This allowed him to image the live-cell dynamics of epigenetic histone modifications during gene activation for the first time [2]. Before joining the faculty at CSU, Dr. Stasevich took a one year hiatus at the HHMI Janelia Research Campus, where he further improved the spatio-temporal resolution of endogenous protein imaging in live-cells.

1. Morisaki, T. et al. Real-time quantification of single RNA translation dynamics in living cells. Science 352, 1425–1429 (2016).

2. Stasevich, T. J. et al. Regulation of RNA polymerase II activation by histone acetylation in single living cells. Nature 516, 272–275 (2014).