In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transportBiochemistry and Molecular Pharmacology Publications and Presentations
UMMS AffiliationDepartment of Biochemistry and Molecular Pharmacology; RNA Therapeutics Institute
Medical Subject HeadingsActins; Active Transport, Cell Nucleus; Animals; Binding Sites; Cell Line; Cell Nucleus; Cytoplasm; Kinetics; Mice; Mice, Transgenic; Microscopy, Fluorescence; Molecular Imaging; Nuclear Pore; *RNA Transport; RNA, Messenger; Staining and Labeling; Substrate Specificity
AbstractExport of messenger RNA occurs via nuclear pores, which are large nanomachines with diameters of roughly 120 nm that are the only link between the nucleus and cytoplasm. Hence, mRNA export occurs over distances smaller than the optical resolution of conventional light microscopes. There is extensive knowledge on the physical structure and composition of the nuclear pore complex, but transport selectivity and the dynamics of mRNA export at nuclear pores remain unknown. Here we developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of co-localization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nanometre precision. With this method we achieve 20-ms time-precision and at least 26-nm spatial precision, enabling the capture of highly transient interactions in living cells. Using this approach we were able to spatially resolve the kinetics of mRNA transport in mammalian cells and present a three-step model consisting of docking (80 ms), transport (5-20 ms) and release (80 ms), totalling 180 +/- 10 ms. Notably, the translocation through the channel was not the rate-limiting step, mRNAs can move bi-directionally in the pore complex and not all pores are equally active.
Related ResourcesLink to Article in PubMed
Citation InformationDavid Grunwald and Robert H. Singer. "In vivo imaging of labelled endogenous beta-actin mRNA during nucleocytoplasmic transport" Vol. 467 Iss. 7315 (2010) ISSN: 0028-0836 (Linking)
Available at: http://works.bepress.com/david_grunwald/5/