Timothy J. Stasevich, Associate Professor, BIochemistry & Molecular Biology, Colorado State University, will give a talk on "Real-time quantification of gene expression with single-molecule precision in living cells"

ABSTRACT

My lab is creating technology to image gene expression in real time and with single-molecule precision in living cells. Using tandem-repeat mRNA and protein tags, genetically encoded intrabodies, and single-molecule microscopy, we now regularly image the live-cell translation dynamics of single mRNAs. In this talk, I will introduce our technology and describe how it can be used to amplify fluorescent signals within single translation sites. I will show how we quantify these signals to determine the size, shape, subcellular localization, and mobilities of translation sites, as well as their ribosomal densities and translational initiation and elongation kinetics. I will then highlight a few recent applications of our technology, including the imaging of translation shutdown during cellular stress, HIV-1 translational frameshifting, and non-canonical translation initiation at internal ribosomal entry sites. I will conclude by discussing new intrabodies my lab has engineered that bind the classic HA and FLAG epitopes in living cells. As these intrabodies can be encoded on plasmids, they can easily be adapted by other labs to image translation in multiple colors and in diverse living systems.