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Silencing nuclear pore protein Tpr elicits a senescent-like phenotype in cancer cells.

David-Watine B - PLoS ONE (2011)

Bottom Line: We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153.In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, CNRS URA2582, Groupe E3 Biologie Cellulaire du Noyau, Paris, France. brigitte.david-watine@pasteur.fr

ABSTRACT

Background: Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human.

Methodology/principal findings: Here we show that depletion of Tpr by RNA interference triggers G0-G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.

Conclusions: Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

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Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.
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pone-0022423-g003: Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.

Mentions: To test this hypothesis we first used the construct pSTAT1-NES-GFP, which encodes residues 367–427 of human STAT1 that confers NES activity [35], to prevent additional regulatory events interfering with the inhibition of Crm1-dependent nuclear export. This construct was co-transfected with Tpr, Crm1 and control siRNAs to probe for NES-dependent export. As shown in Fig. 3A, the distribution of the GFP signal in live cells 48 hours after transfection showed that Crm1- and Tpr-depletion led to substantial nuclear retention of the NES-GFP construct whereas GFP remained mainly cytosolic in mock-treated cells. The cells were then permeabilized and labeled with Crm1 and Tpr antibodies to check depletion efficiency (Fig. 3B).


Silencing nuclear pore protein Tpr elicits a senescent-like phenotype in cancer cells.

David-Watine B - PLoS ONE (2011)

Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3139644&req=5

pone-0022423-g003: Analysis of Crm1 dependent NES-proteins nuclear export in Tpr depleted cells.3A–3B. Distribution of GFP in cells co-transfected with STAT1-NES-GFP and Tpr, Crm1 or control (mock) siRNAs. 3A: GFP distribution was analyzed in live cells. Nuclei were labeled with Hoechst 33342. Scale bar: 10 µm. 3B: Cells were then permeabilized and labeled with antibodies specific for Tpr and Crm1 and DAPI for the DNA. Top panel: Tpr depletion and control; lower panel: Crm1 depletion and control. Scale bar: 10 µm. 3C–3D: Tpr depletion delays nuclear export of NFκB. HeLa cells were transfected with Tpr or mock siRNAs 2 days prior to NFκB induction. Transfected and control cells were then incubated with TNF 100 IU/mL for 40 min. TNF was then removed from the medium and the cells were either fixed or kept for another 1h30 in fresh medium before fixation. 3C: Quantification of the NFκB signal: All images were acquired at the same magnification and exposure and the NFκB signal was quantified using the same surface area in the nuclei and cytoplasm of cells under the different experimental conditions using OpenLab3.1.2. The cytosolic signal was plotted against the nuclear signal and standard deviation was calculated using Microsoft Excel. Error bars represent the standard deviation. Note that the cytosolic to nuclear signal ratio is very similar in control cells before TNF treatment and 1h30 after TNF removal. This was as expected and is due to NFκB fully returning to the cytoplasm at this time. 1h30 after TNF removal the cytosolic to nuclear signal ratio was about 25% lower in the Tpr-depleted cells than in the control cells. 3D: Immunofluorescent labeling of NFκB and Tpr 1h30 after TNF removal in Tpr-depleted and control cells. Fixed cells were labeled with a rabbit anti-NFκB antibody and anti-Tpr mAb 203-37.
Mentions: To test this hypothesis we first used the construct pSTAT1-NES-GFP, which encodes residues 367–427 of human STAT1 that confers NES activity [35], to prevent additional regulatory events interfering with the inhibition of Crm1-dependent nuclear export. This construct was co-transfected with Tpr, Crm1 and control siRNAs to probe for NES-dependent export. As shown in Fig. 3A, the distribution of the GFP signal in live cells 48 hours after transfection showed that Crm1- and Tpr-depletion led to substantial nuclear retention of the NES-GFP construct whereas GFP remained mainly cytosolic in mock-treated cells. The cells were then permeabilized and labeled with Crm1 and Tpr antibodies to check depletion efficiency (Fig. 3B).

Bottom Line: We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153.In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

View Article: PubMed Central - PubMed

Affiliation: Institut Pasteur, CNRS URA2582, Groupe E3 Biologie Cellulaire du Noyau, Paris, France. brigitte.david-watine@pasteur.fr

ABSTRACT

Background: Tpr is a large coiled-coil protein located in the nuclear basket of the nuclear pore complex for which many different functions were proposed from yeast to human.

Methodology/principal findings: Here we show that depletion of Tpr by RNA interference triggers G0-G1 arrest and ultimately induces a senescent-like phenotype dependent on the presence of p53. We also found that Tpr depletion impairs the NES [nuclear export sequence]-dependent nuclear export of proteins and causes partial co-depletion of Nup153. In addition Tpr depletion impacts on level and function of the SUMO-protease SENP2 thus affecting SUMOylation regulation at the nuclear pore and overall SUMOylation in the cell.

Conclusions: Our data for the first time provide evidence that a nuclear pore component plays a role in controlling cellular senescence. Our findings also point to new roles for Tpr in the regulation of SUMO-1 conjugation at the nuclear pore and directly confirm Tpr involvement in the nuclear export of NES-proteins.

Show MeSH
Related in: MedlinePlus