In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport

Abstract

Export of mRNA occurs via nuclear pores, large nano-machines with diameters of roughly 120 nm that are the only link between nucleus and cytoplasm1. 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 NPC2–7, but transport selectivity and dynamics of mRNA export at nuclear pores remain unknown8. We developed a super-registration approach using fluorescence microscopy that can overcome the current limitations of colocalization by means of measuring intermolecular distances of chromatically different fluorescent molecules with nm precision. With this method we achieve 20 ms time- and at least26 nm spatial precision, rendering the capture of highly transient interactions in living cells possible. With this method we were able to spatially resolve the kinetics of mRNA transport and present a three step model consisting of docking (80ms), transport (5–20ms) and release (80ms), totalling 180 ± 10 ms. Importantly, 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.