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. 2004 Aug 31;101(35):12887-92.
doi: 10.1073/pnas.0403675101. Epub 2004 Aug 11.

Imaging of single-molecule translocation through nuclear pore complexes

Weidong Yang  1 , Jeff Gelles, Siegfried M Musser
Affiliations

Imaging of single-molecule translocation through nuclear pore complexes

Weidong Yang et al. Proc Natl Acad Sci U S A. .

Abstract

Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoprotein complexes across the double-membrane nuclear envelope. In vitro studies with purified transport cofactors have revealed a general scheme of cofactor-dependent transport energetically driven by the G protein Ran. However, the size and complexity of NPCs have made it difficult to clearly define the loci and kinetics of the cofactor-NPC interactions required for transport. We now report the use of single-molecule fluorescence microscopy to directly monitor a model protein substrate undergoing transport through NPCs in permeabilized cells. This substrate, NLS-2xGFP, interacts with NPCs for an average of 10 +/- 1 ms during transport. However, because the maximum nuclear accumulation rate of NLS-2xGFP was measured to be at least approximately 10(3) molecules per NPC per s, NPCs must be capable of transporting at least approximately 10 substrate molecules simultaneously. Molecular tracking reveals that substrate molecules spend most of their transit time randomly moving in the central pore of the NPC and that the rate-limiting step is escape from the central pore.

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Figures

Fig. 1.
Fig. 1.
The NLS-2xGFP model substrate and its transport into permeabilized HeLa cells. (a) Schematic diagram of the substrate. Two identical GFP domains, represented here by structural models (PDB ID code 1C4F), have N- and C-terminal extensions and are linked by the peptide shown. The identified cysteines are each labeled with a molecule of Alexa-555 maleimide. The N-terminal extension contains an NLS (red) that targets the protein for Im α/Im β-dependent transport. Control experiments used protein with the indicated K → T NLS mutation, which blocks recognition by Im α (16). (b–e) Extent of nuclear accumulation of NLS-2xGFP in permeabilized HeLa cells measured by epifluorescence. Substrate (0.44 μM) was incubated with cells for 10 min and then washed away. (Scale bar, 10 μm.) (b) Normal nuclear accumulation in the presence of GTP, Ran, NTF2, Im α and Im β. Successful nuclear transport is detected as bright fluorescence (red) in oval nuclei. (c) Control experiment with NTF2, Im α, and Im β but without Ran and GTP. Substrate is localized at the NE. Under these conditions, the substrate is expected to remain bound to the NPCs as an Im α/β–substrate complex, because there is no RanGTP available on the nucleoplasmic side of the NE to dissociate this complex (20). (d) Control experiment with WGA, an inhibitor of nucleocytoplasmic transport (18). When the cells are preincubated with WGA before addition of substrate and cofactors as described for b, no transport occurs because IC binding is blocked by WGA. (e) Control experiment with substrate containing a mutant NLS. The K → T NLS mutant substrate is not transported under the conditions of b, presumably because the mutant NLS is not recognized by Im α. (f) Dependence of initial nuclear accumulation rates determined by confocal microscopy on substrate concentration. Initial transport rates in μM/s were converted to molecules per NPC per s by assuming 3,000 NPCs per nucleus (17, 32). (Error bars, SDs over five nuclei.)
Fig. 2.
Fig. 2.
Binding and release of single-substrate molecules at the NE visualized by SMF microscopy. (a) Consecutive video frames (3-ms duration) showing the appearance and disappearance of an Alexa-555-labeled NLS-2xGFP molecule at the NE. The position of the NE (dashed line) was determined by bright-field microscopy (see Methods). The nuclear (N) and cytoplasmic (C) compartments are indicated. Numbers denote time (ms). (Scale bar, 2 μm.) (b) Time course of the fluorescence intensity at the position of the NE-localized spot shown in a. Intensity of a 2 × 2-pixel area was quantified over the entire video sequence. Two NE-interaction events are observed, the first of which is shown in a.(Insets) Expanded views of the interaction events. (c) Histogram of NE residence times for 231 NE-interaction events (3-ms bins). The exponential fit yields a time constant of τ = 8.8 ± 0.6 ms. (d) Time course of the fluorescence intensity for substrate molecules immobilized on a glass surface. For comparison, the time-dependent fluorescence intensity observed for two isolated substrate molecules is shown under the same conditions and on the same intensity scale as described for b. The blue trace shows a single-step photobleach event and thus is a substrate molecule with one Alexa-555-dye label. The red trace shows two-step photobleaching, indicating a doubly labeled substrate molecule. Photobleaching (tave = 820 ± 30 ms) occurs on a time scale much longer than the NE-interaction time.
Fig. 3.
Fig. 3.
NE-interaction sites. (a) Distribution of substrate-interaction sites (red dots) on the NE detected under the conditions described for Fig. 2a in 33 s of video. Nuclear (N) and cytoplasmic (C) compartments are indicated. Green, illumination area; blue, NE position (see Methods). (Scale bar, 1 μm.) (Inset) Lower-magnification view showing the entire nucleus; the area of the main image is enclosed by the red box. (b) Distribution of nearest-neighbor spacings between interaction sites (n = 21).
Fig. 4.
Fig. 4.
Single-molecule tracking precision and transport trajectories. (a) Tracking precision measured for immobilized substrate molecules with different S/N ratios. The line is an exponential intended only to guide the eye. (b) Control: successive position measurements for three immobilized substrate molecules (S/N ratio = 7.8–10.3). A schematic (gray; to scale) of an NPC showing the cytoplasmic filaments (Upper), nuclear basket (Lower), and NE (horizontal lines; each line is a membrane bilayer) is underlaid for size comparison; the shape of the pore is adapted from figure 1 of Fahrenkrog and Aebi (2). Precisions in the vertical and horizontal directions are 32.0 ± 2.3 and 28.8 ± 1.2 nm, respectively. Acquisition conditions are as described for Fig. 2a.(c) Overlay of trajectories for three substrate molecules that interacted successively with the same NPC. The vertical position of the trajectories relative to the NE of the NPC schematic was determined by the NE-localization procedure (see Methods). The horizontal centroid from all the trajectories for this NPC was aligned with the horizontal center of the NPC schematic. For each trajectory, the points are numbered in sequence. (d) Superimposed plots of 17 trajectories (64 points) from 11 NPCs. The vertical position of the trajectories relative to the NPC schematic was aligned as described for b; the horizontal centroid from all the trajectories for each NPC was aligned with the horizontal center of the NPC schematic. By using the centroid of the trajectories for each NPC as reference points, the SDs of the position measurements in the plane of and perpendicular to the NE are 33 ± 4 and 50 ± 5 nm, respectively. The average position perpendicular to the NE is 0.2 ± 5.4 nm.
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