In vivo imaging of labelled endogenous β-actin mRNA during nucleocytoplasmic transport - PubMed (original) (raw)

. 2010 Sep 30;467(7315):604-7.

doi: 10.1038/nature09438. Epub 2010 Sep 15.

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

David Grünwald et al. Nature. 2010.

Abstract

Export 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.

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Figures

Fig. 1

Fig. 1

Super-Registration Precision and Detection of Nuclear mRNA. a–g) the registration precision achieved in this experiment was based on imaging nuclear pores on two cameras immediately before data acquisition (SI). Data from both cameras (a red, b green), merged image (c) after registration. A filtered merged image (d) with 21 nuclear pores, white circles outlined in red (e). f) Co-registration precision between the best aligned 6 (black bars and black line in inset), 10 (light grey) and 15 (dark grey) nuclear pores. Fit (inset), Gaussian fit to the ‘15 pore’ data set: registration= 10±1 nm, 13±1 nm FWHM. g) Distances between pores in e). Peak= 7.5 nm. h) mRNAs interacted with nuclear pores infrequently and not all interactions resulted in export of mRNAs from the nucleus (frame numbers in SI Movie 1, mRNA scanning pores, t = 40 ms. i) full length traces h) and SI Movie 1; first (blue), last (cyan). j) Intensity trace (green),tracked mRNA, background (black). k) slow export images from SI Movie 2 (frame indicated). l) fast export (SI Movie 3). m) Distances between mRNA and pore from l) colocalization precision, 26 nm total (SI). Nucleoplasmic mRNA (+) cytoplasmic mRNA (−) (SI & Fig. S5). n) Intensity mRNA signal (green) vs. background. o) mRNA positions (green boxes) and pores (red circle) overlaid on nuclear pore from l. Bars = 2 μm, ‘n/c’ = nucleus/cytoplasm, ‘max’ =maximum intensity projection, i) & o) axis pixels (= 64 nm). h–o LoG filtered (ImageJ, D. Sage).

Fig. 2

Fig. 2

Dwell times of β-actin mRNA at the NPC. mRNA co-localized with NPCs, no. frames as milliseconds. Histogram = observed mRNAs per time bin of 20 ms. a) Fit of dwell time of cumulative trace length distribution (black circles). First bin = total number of observed traces. Fast transport events (<0.8 s) show mono-exponential decay (red circles). Dwell time = 172±3 ms, (red line, first component black line). Second time constant = 2000±120 ms is needed to fit complete data set (black line). mRNA in the nucleoplasm (grey line), dwell time = 15±1 ms (90%) and 104±6 ms (10%). Data normalized. b) Data from a) (black circles) re-plotted as trace duration histogram (black bars). Cut-off (SI &Table S3, adjacent averaging width = 5 bins). Inset = unprocessed raw data. Two-step convolution model (red line) reveals two kinetic rates, dwell times kfast = 43±1 ms and kslow = 139±10 ms. Identifying export = two observations = 40 ms. Result consistent with multistep process containing at least two rate constants, total time = 180ms.

Fig. 3

Fig. 3

‘Binding Sites’ of mRNAs at Nuclear Pores. Distances between mRNA and POM121-tdT (zero position) bin widths = 25 nm. (−) = cytoplasmic (blue C), (+) = nucleoplasmic position (green N). Red lines are global fits, green and blue lines are fits to cytoplasmic and nucleoplasmic binding distributions. a) Histogram of all observed transport events at NPCs (b + c). b) Histogram for fast transported mRNAs (90% translocation). c) Histogram for slow mRNAs, observed for extended times at NPC.

Fig. 4

Fig. 4

NPC Topography of mRNA Export. Results from Fig. 3B&C (hatched & open bars) plotted (a) to scale with known NPC dimensions (b). mRNA export timescale (red = kslow; orange = kfast) along NPC axis combined with single molecule data (grey bars) of Nup358, import factors and import release site. Nuclear peak position of slow transporting mRNAs located between binding sites for import factors and import release site (Fig. 4 & Table 1). Length of grey bars = FWHM of binding site distributions.

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