PMC

(a,b) Examples of early and late aborting cargos. Kymographs showing motion along the transport axis, X, as a function of time (grey, raw data; red, 0.1 s running mean filtered). (c) An example of a successfully importing trajectory showing early confinement at an internal constriction. (d) A second example of a successful trajectory without visible early confinement. (e) Ensemble averaged Mean Squared Displacement (MSD) plots showing anomalous sub-diffusion in the transverse and transport axes of the central channel. Fits to a power law 〈r²〉= Γt^α are shown as solid lines (). Raw frame-to-frame displacements versus channel position are provided as . Errors are standard error of the mean (n_success = 23, n_early = 11, n_late = 22).

(a) Left, Schematic of the nuclear pore complex showing approximate sizes from the literature. The QD-IBB cargo is shown as a red circle to scale. Right, Three examples each of aborted and successful import trajectories (0.1 s running mean filtered) and coloured according to sub-steps: Docking events are coloured in blue, motion in the central channel in green and undocking in red. Raw, unfiltered data of cargos showing rapid cargo exit into the nucleus are provided as . (b) Contour maps of the density of found cargo positions in the central channel for all successful, all aborted, early aborts, and late aborts. The contours are coloured according to the normalised density of observations, with red representing the highest density of found cargo positions and blue the lowest (See for details of trajectory alignment). (c–e) Positional histograms along the transport axis, for successful, early and late aborting cargos.

(a) Schematic of QD based cargo. The Snurportin-1 IBB/Z-domain fusion protein is coupled via a bifunctional SMCC crosslinker to the amino-PEG polymer coat of a fluorescent QD. The three helix Z-domain acts as a spacer to correctly present the IBB for biological function. Not to scale. (b) Dynamic Light Scattering size distributions of QD-IBB cargos in the presence and absence of importin-β. (c) Dwell time distribution of all QD interactions with the NPC. The time axis is truncated at 300 s. (d) Brightfield image of a nucleus with a quantum dot fluorescence image (with background subtraction applied) overlaid in red. A single quantum dot cargo at the nuclear envelope is highlighted. (e) Individual consecutive frames from a single-molecule experiment showing the arrival (first frame) from the cytoplasm and departure (final frame) of the cargo into the nucleus. The centroids determined from fitting of the PSF are overlaid as red crosses. Frame numbers are in the bottom left hand corner of each frame. Movies were captured at 40 Hz.

(a) Detail of a crossing event within the central channel (Grey, raw data; blue line, 0.1s running average). The cargo dwells at the cytoplasmic and nuclear faces of the channel for various durations. The crossing time is the time between leaving the cytoplasmic face and arriving at the nuclear face and vice versa. (b) Cargo dwell times with 50% or 100% IBB. The mean/median dwell time increases for the 50% labelled cargo. The distributions (p<0.05, Kolmogorov Smirnov test) and medians (p<0.05, two-tailed Mann-Whitney test) are significantly different (). (c) Crossing time distributions for cargos with 50% or 100% IBB. QB-IBB^50% cargos take significantly longer to cross the channel (p<0.05, Mann-Whitney test). (d,e) Position histograms of early (d) and late (e) aborts with and without Ran. Inset: import statistics with and without Ran. The presence of Ran significantly increases the probability of successful import. (f) Results summary. Cargos arriving from the cytoplasm (white circles) may dock on the cytoplasmic filaments or directly enter the NPC. Once inside the central channel, cargos exhibit anomalous subdiffusion. There is a size selective constriction (blue) within the first 30nm of the channel. Efficient cargo exit into the nucleus requires Ran (red bar). (g) Probabilities of cargos being rejected versus position, highlighting the sequence of steps in cargo translocation. Many cargos interact with the cytoplasmic filaments, but most (75%) immediately rebound into the cytoplasm. The remaining 25% interact with the NPC (grey box) for longer times. Of those, 20% abort early due to a size gate and 80% reach the central channel. Once inside the channel, in the presence of Ran, 50% of cargos ultimately enter the nucleus and the remaining 50% abort. (h) In the absence of Ran, cargos do not enter the nucleus (>99% abort) and return to the cytoplasm.