Mapping translation 'hot-spots' in live cells by tracking single molecules of mRNA and ribosomes
Zachary B Katz,
Brian P English,
Timothée Lionnet,
Young J Yoon,
Nilah Monnier,
Ben Ovryn,
Mark Bathe,
Robert H Singer
Affiliations
Zachary B Katz
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States; Salk Institute for Biological Studies, La Jolla, United States
Brian P English
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
Timothée Lionnet
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
Young J Yoon
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States
Nilah Monnier
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
Ben Ovryn
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States
Mark Bathe
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, United States
Robert H Singer
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, New York, United States; Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, United States
Messenger RNA localization is important for cell motility by local protein translation. However, while single mRNAs can be imaged and their movements tracked in single cells, it has not yet been possible to determine whether these mRNAs are actively translating. Therefore, we imaged single β-actin mRNAs tagged with MS2 stem loops colocalizing with labeled ribosomes to determine when polysomes formed. A dataset of tracking information consisting of thousands of trajectories per cell demonstrated that mRNAs co-moving with ribosomes have significantly different diffusion properties from non-translating mRNAs that were exposed to translation inhibitors. These data indicate that ribosome load changes mRNA movement and therefore highly translating mRNAs move slower. Importantly, β-actin mRNA near focal adhesions exhibited sub-diffusive corralled movement characteristic of increased translation. This method can identify where ribosomes become engaged for local protein production and how spatial regulation of mRNA-protein interactions mediates cell directionality.
