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Single-Molecule Imaging of Nuclear Transport
Published on: June 9, 2010
Quantification of nuclear protein transport using induced heterodimerization
Albert Busch1, Tilman Kiel, Stefan Hübner
1University of Würzburg, Institute of Anatomy and Cell Biology, Würzburg, Germany.
Traffic (Copenhagen, Denmark)
|July 16, 2009
Summary
Researchers developed a novel assay to measure protein movement between the cytoplasm and nucleus in living cells. This method uses a specific agent to control nuclear localization sequence (NLS) import and nuclear export sequence (NES) export.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Nucleocytoplasmic transport is essential for eukaryotic cell function.
- Protein import and export are regulated by specific sequences like NLS and NES.
- Kinetically measuring these processes in real-time has been challenging.
Purpose of the Study:
- To adapt a protein heterodimerization assay for kinetic measurement of Crm1-mediated nuclear export.
- To investigate the use of AP21967 as a heterodimerizing agent for studying nucleocytoplasmic trafficking.
- To establish a non-invasive, time-saving method for quantitative analysis of protein transport.
Main Methods:
- Developed a protein heterodimerization assay using AP21967 and fusion proteins with NLS/NES.
- Utilized HeLa cells for live-cell imaging and kinetic analysis.
- Measured Crm1-mediated nuclear export and NLS-mediated nuclear import.
Main Results:
- Observed rapid nuclear export of NLS-containing fusion proteins induced by AP21967.
- Demonstrated that the extent of export correlated with the number of AP21967-binding motifs.
- Showed AP21967-induced export was inhibited by leptomycin B and half-maximal export occurred in ~10 min.
- Successfully studied induced NLS-mediated nuclear import using the same assay.
Conclusions:
- Induced protein heterodimerization is a versatile tool for quantitative study of nucleocytoplasmic protein trafficking.
- The developed assay allows for non-invasive, real-time kinetic measurements.
- This method offers a significant advancement in studying protein transport dynamics.
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Proteins targeted to the nucleus carry nuclear localization signals or NLS recognized by import receptors in the cytosol. Similarly, proteins with nuclear export signals are recognized by export receptors. Import and export receptors are...
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01:45Regulation of Nuclear Protein Sorting
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01:42Nuclear Export
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