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Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Dhanoya A, Wang T, Keshavarz-Moore E, Fassati A, Chain BM - Traffic (2012)

Bottom Line: Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls.Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response.Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

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Related in: MedlinePlus

Imp7 stimulates nuclear import of plasmid DNANuclear import assay in permeabilized-HeLa cells in the presence of labelled plasmid DNA and: buffer; 1× energy mix (+Energy); 1× energy mix + 1× Ran mix (+Energy + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (+Energy + Ran + imp7); 1 µm imp7 + Ran mix (+Ran + imp7); 1× energy mix + 1× Ran mix and 1 µm impβ (+Energy + Ran + impβ). Nuclear import was quantified by ImageJ software and the signal level in the buffer samples is given an arbitrary value of 100. Bars represent mean ± SD from 40–50 cells. Results are representative of four independent experiments.
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fig01: Imp7 stimulates nuclear import of plasmid DNANuclear import assay in permeabilized-HeLa cells in the presence of labelled plasmid DNA and: buffer; 1× energy mix (+Energy); 1× energy mix + 1× Ran mix (+Energy + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (+Energy + Ran + imp7); 1 µm imp7 + Ran mix (+Ran + imp7); 1× energy mix + 1× Ran mix and 1 µm impβ (+Energy + Ran + impβ). Nuclear import was quantified by ImageJ software and the signal level in the buffer samples is given an arbitrary value of 100. Bars represent mean ± SD from 40–50 cells. Results are representative of four independent experiments.

Mentions: To directly test if imp7 promotes nuclear import of DNA, we fluorescently labelled a 9.3-kb plasmid DNA 24 and used it in an in vitro nuclear import assay. In this assay, cells are permeabilized with digitonin, which selectively solubilizes cholesterol and hence affects the plasma membrane but not the nuclear envelope. Cytosolic contents are removed by washing and nuclear import is reconstituted by the addition of recombinant factors, the Ran system and an energy regenerating system 25. This assay allows to study the contribution of individual components to the import of specific substrates and is widely used in the nuclear import field 12,13,18,19,22. Figure 1 shows that incubation of labelled DNA with an energy regenerating system and with the Ran mix stimulated DNA nuclear accumulation above background, consistent with previous reports 11,12. This background signal is most likely caused by cytosolic factors remaining in the permeabilized cells in sufficient quantity to stimulate low level of nuclear import. However, maximal stimulation of DNA import was observed only when imp7, energy and Ran mix were present together. Energy was required for the process because omitting the energy regenerating system reduced nuclear fluorescence even in the presence of the Ran system and imp7. Impβ on its own did not stimulate DNA nuclear import significantly above that obtained in the presence of energy and Ran (Figure 1). Interestingly, most of the fluorescent signal was detected in nucleoli, however this is not unusual and has been observed before 11,26. Similar results were obtained with different plasmids, thus this effect was not vector specific (not shown). These data are entirely consistent with the lower transfection efficiency seen in imp7 KD cells 14 and indicated that imp7 promoted plasmid-DNA nuclear import in a Ran- and energy-dependent way.


Importin-7 mediates nuclear trafficking of DNA in mammalian cells.

Dhanoya A, Wang T, Keshavarz-Moore E, Fassati A, Chain BM - Traffic (2012)

Imp7 stimulates nuclear import of plasmid DNANuclear import assay in permeabilized-HeLa cells in the presence of labelled plasmid DNA and: buffer; 1× energy mix (+Energy); 1× energy mix + 1× Ran mix (+Energy + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (+Energy + Ran + imp7); 1 µm imp7 + Ran mix (+Ran + imp7); 1× energy mix + 1× Ran mix and 1 µm impβ (+Energy + Ran + impβ). Nuclear import was quantified by ImageJ software and the signal level in the buffer samples is given an arbitrary value of 100. Bars represent mean ± SD from 40–50 cells. Results are representative of four independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3672689&req=5

fig01: Imp7 stimulates nuclear import of plasmid DNANuclear import assay in permeabilized-HeLa cells in the presence of labelled plasmid DNA and: buffer; 1× energy mix (+Energy); 1× energy mix + 1× Ran mix (+Energy + Ran); 1× energy mix + 1× Ran mix + 1 µm imp7 (+Energy + Ran + imp7); 1 µm imp7 + Ran mix (+Ran + imp7); 1× energy mix + 1× Ran mix and 1 µm impβ (+Energy + Ran + impβ). Nuclear import was quantified by ImageJ software and the signal level in the buffer samples is given an arbitrary value of 100. Bars represent mean ± SD from 40–50 cells. Results are representative of four independent experiments.
Mentions: To directly test if imp7 promotes nuclear import of DNA, we fluorescently labelled a 9.3-kb plasmid DNA 24 and used it in an in vitro nuclear import assay. In this assay, cells are permeabilized with digitonin, which selectively solubilizes cholesterol and hence affects the plasma membrane but not the nuclear envelope. Cytosolic contents are removed by washing and nuclear import is reconstituted by the addition of recombinant factors, the Ran system and an energy regenerating system 25. This assay allows to study the contribution of individual components to the import of specific substrates and is widely used in the nuclear import field 12,13,18,19,22. Figure 1 shows that incubation of labelled DNA with an energy regenerating system and with the Ran mix stimulated DNA nuclear accumulation above background, consistent with previous reports 11,12. This background signal is most likely caused by cytosolic factors remaining in the permeabilized cells in sufficient quantity to stimulate low level of nuclear import. However, maximal stimulation of DNA import was observed only when imp7, energy and Ran mix were present together. Energy was required for the process because omitting the energy regenerating system reduced nuclear fluorescence even in the presence of the Ran system and imp7. Impβ on its own did not stimulate DNA nuclear import significantly above that obtained in the presence of energy and Ran (Figure 1). Interestingly, most of the fluorescent signal was detected in nucleoli, however this is not unusual and has been observed before 11,26. Similar results were obtained with different plasmids, thus this effect was not vector specific (not shown). These data are entirely consistent with the lower transfection efficiency seen in imp7 KD cells 14 and indicated that imp7 promoted plasmid-DNA nuclear import in a Ran- and energy-dependent way.

Bottom Line: Poor DNA nuclear import in cells depleted of imp7 directly correlated with lower gene expression levels in these cells compared to controls.Inefficient nuclear import of transfected DNA induced greater upregulation of the interferon pathway, suggesting that rapid DNA nuclear import may prevent uncontrolled activation of the innate immune response.Our results provide evidence that imp7 is a non-redundant component of an intrinsic pathway in mammalian cells for efficient accumulation of exogenous and endogenous DNA in the nucleus, which may be critical for the exchange of genetic information between mitochondria and nuclear genomes and to control activation of the innate immune response.

View Article: PubMed Central - PubMed

Affiliation: The Advanced Centre for Biochemical Engineering, University College London, Torrington Place, London, WC1E 7JE, UK.

Show MeSH
Related in: MedlinePlus