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.

Figures at a glance

Primary authors

1. These authors contributed equally to this work.

   • Alan R. Lowe & 
   • Jake J. Siegel

Affiliations

1. Department of Physics, University of California, Berkeley, California 94720, USA

   • Alan R. Lowe &
    
   • Jan T. Liphardt

2. QB3, University of California, Berkeley, California 94720, USA

   • Alan R. Lowe,
    
   • Jake J. Siegel,
    
   • Karsten Weis &
    
   • Jan T. Liphardt

3. Bay Area Physical Sciences–Oncology Center, University of California, Berkeley, California 94720, USA

   • Alan R. Lowe &
    
   • Jan T. Liphardt

4. Biophysics Graduate Group, University of California, Berkeley, California 94720, USA

   • Jake J. Siegel,
    
   • Merek Siu &
    
   • Jan T. Liphardt

5. Department of Molecular and Cellular Biology, University of California, Berkeley, California 94720, USA

   • Petr Kalab &
    
   • Karsten Weis

6. Physical Biosciences Division, Lawrence Berkeley National Laboratory, California 94720, USA

   • Alan R. Lowe &
    
   • Jan T. Liphardt

7. Present addresses: Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892, USA (P.K.); Illumina Inc., Hayward, California 94545, USA (M.S.).

   • Petr Kalab &
    
   • Merek Siu

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to: 

• Karsten Weis (kweis@berkeley.edu)

Movies

1. Supplementary Movie 1 (24.4M)

   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.

2. Supplementary Movie 2 (691K)

   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).

PDF files

1. Supplementary Information (4.4M)

   This file contains Supplementary Information 1-8, Supplementary Figures 1-13 with legends, Supplementary Tables 1-3 and additional references.