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.

How to operate a nuclear pore complex by Kap-centric control

“…68 Finally, the increased confinement within the pore may promote relatively fast transport via quasi one-dimensional diffusion. 67 All in all these scenarios are consistent with the notion that passage through the NPC is not a rate-limiting step 57 except when long-lived Kaps are depleted from the pore.…”

“…40 Similar bimodal kinetics has also been reported for GFP-Kapb1 42 in permeabilized cells and mRNA export in living cells. 57 Interestingly, it was found that additions of exogenous Kapb1, 40 as well as other receptors 58 further reduced NPC permeability against passive cargoes, thereby recapitulating the role of Kaps as bona fide constituents of the NPC. Moreover, Yang and Musser, 36 as well as Timney et al 45 showed in vitro and in vivo respectively, that increasing amounts of Kaps expedited cargo import rates, rather than slowing it down.…”

“…66 Nevertheless, from the perspective of Kap-centric control a displacement of preloaded Kaps from the FG Nups would result in the effective retraction/reduction of the NPC barrier. We speculate that the presence of multiple Kaps on large cargoes 67,68 (or receptors that shuttle messenger ribonucleic proteins 57,69 ) may be able to displace preloaded Kaps because of increased binding avidity with the FG Nups. Indeed, the presence of multiple Kaps has also shown to improve the transport efficiency of large cargoes.…”

Imaging Translation in Single Cells Using Fluorescent Microscopy

“…1C) (Shav-Tal et al 2004). Transcripts diffuse within the nucleoplasm for times ranging from 5 to 40 minutes; however, once a transcript engages a nuclear pore, export into the cytoplasm is fast (less than 0.2 sec) (Grunwald and Singer 2010;Mor et al 2010). Interestingly, transport through the nuclear pore takes only 5-20 min and docking on the nuclear side and release into the cytoplasm both take approximately 80 min, indicating that translocation through the nuclear pore is not the rate-limiting step (Grunwald and Singer 2010).…”

Nuclear export, enlightened

“…Before the work of Siebrasse et al (1), two studies of mRNA export in mammalian cells used mRNAs genetically engineered to contain a tandem array of fluorescent tags (10,11), together with a superregistration microscopy innovation in the latter (11). In contrast, Siebrasse et al…”