Enhanced intracellular mobility and nuclear accumulation of DNA plasmids associated with a karyophilic protein.
The use of synthetic gene delivery systems in human gene transfer is hampered by poor transfection efficiencies, largely because of the inability of DNA to translocate across the nuclear pore complex. A means to overcome this barrier is to bind the DNA to nuclear localization signals (NLSs), which are recognized by shuttling receptors of the nuclear import machinery. Here, we studied the intracellular transport of plasmid DNA microinjected into HeLa cell cytoplasm, alone or as a complex with intact or NLS-deleted NFkappaB p50, using confocal microscopy imaging. We found that association of NLS-carrying p50 with DNA facilitated not only nuclear entry of the DNA but also its migration through the cytoplasm toward the nucleus. Facilitated transport of p50-DNA complexes in the cytoplasm proceeded along microtubules in a dynein-dependent manner and is mediated by the heterodimeric nuclear transport receptor that recognizes the p50-born NLS.
Mesika
Adi
A
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Kiss
Vladimir
V
Brumfeld
Vlad
V
Ghosh
Gourisankar
G
Reich
Ziv
Z
D021581:Active Transport, Cell Nucleus; D001665:Binding Sites; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004398:Dyneins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008845:Microinjections; D018613:Microscopy, Confocal; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D052002:NF-kappa B p50 Subunit; D019913:Nuclear Localization Signals / Q000502:physiology*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D028961:beta Karyopherins / Q000378:metabolism
D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit; D019913:Nuclear Localization Signals; D011498:Protein Precursors; D028961:beta Karyopherins; D004247:DNA; D004398:Dyneins