Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking

Abstract

The nuclear pore complex (NPC) mediates all exchange between the cytoplasm and the nucleus. Small molecules can passively diffuse through the NPC, whereas larger cargos require transport receptors to translocate1. How the NPC facilitates the translocation of transport receptor/cargo complexes remains unclear. To investigate this process, we tracked single protein-functionalized quantum dot cargos as they moved through human NPCs. Here we show that import proceeds by successive substeps comprising cargo capture, filtering and translocation, and release into the nucleus. Most quantum dots are rejected at one of these steps and return to the cytoplasm, including very large cargos that abort at a size-selective barrier. Cargo movement in the central channel is subdiffusive and cargos that can bind more transport receptors diffuse more freely. Without Ran GTPase, a critical regulator of transport directionality1, cargos still explore the entire NPC, but have a markedly reduced probability of exit into the nucleus, suggesting that NPC entry and exit steps are not equivalent and that the pore is functionally asymmetric to importing cargos. The overall selectivity of the NPC seems to arise from the cumulative action of multiple reversible substeps and a final irreversible exit step.

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Figure 1: Experimental design.
Figure 2: Examples of single-molecule trajectories and a functional map of the NPC interior.
Figure 3: Cargo motion in the NPC central channel.
Figure 4: Effect of IBB density on cargo motion, location of Ran action, and data summary.

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Acknowledgements

We thank H. Agarwal for help with initial experiments; H. Aaron, A. Fischer and B. Cohen for use of facilities and discussions; the Bustamante, Chu and Krantz laboratories for use of equipment; and C. Bustamante, M. Welch and D. Grünwald for discussions and comments on the manuscript. This work was partially funded by the NIH (GM058065 to K.W. and GM77856 to J.T.L.) and the NCI (U54CA143836 to J.T.L.).

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Contributions

A.R.L., J.J.S., P.K., M.S., K.W. and J.T.L. designed the experiments. A.R.L., J.J.S. and P.K. prepared materials. A.R.L. and J.J.S. performed the QD optimization and functionalization, the single-molecule experiments, and the data analysis. A.R.L., J.J.S., K.W. and J.T.L. wrote the manuscript.

Corresponding author

Correspondence to Karsten Weis.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Information 1-8, Supplementary Figures 1-13 with legends, Supplementary Tables 1-3 and additional references. (PDF 4589 kb)

Supplementary Movie 1

A typical movie showing imported QDs in a single nucleus after 20 minutes of import. 2 QDs are seen to diffuse inside the confined volume of the cell nucleus. The fitted outline of the nuclear envelope taken from a brightfield image taken immediately after this movie is overlaid in green. (AVI 25084 kb)

Supplementary Movie 2

An example of single QD cargo successfully translocating through a NPC. The nuclear envelope bisects the field of view, running from top to bottom. The QD is observed arriving from the cytoplasm (left), residing at the NPC, and then leaving into the nucleus (right). (AVI 691 kb)

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Lowe, A., Siegel, J., Kalab, P. et al. Selectivity mechanism of the nuclear pore complex characterized by single cargo tracking. Nature 467, 600–603 (2010). https://doi.org/10.1038/nature09285

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