XB-ART-55738
Sci Rep
2019 Feb 27;91:2861. doi: 10.1038/s41598-019-39500-y.
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Nuclear import of Xenopus egg extract components into cultured cells for reprogramming purposes: a case study on goldfish fin cells.
Chênais N, Lorca T, Morin N, Guillet B, Rime H, Le Bail PY, Labbé C.
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Reprogramming of cultured cells using Xenopus egg extract involves controlling four major steps: plasma membrane permeabilization, egg factors import into the nucleus, membrane resealing, and cell proliferation. Using propidium iodide to assess plasma membrane permeability, we established that 90% of the cultured fin cells were permeabilized by digitonin without any cell losses. We showed that egg extract at metaphase II stage was essential to maintain nuclear import function in the permeabilized cells, as assessed with a fusion GFP protein carrying the nuclear import signal NLS. Moreover, the Xenopus-egg-specific Lamin B3 was detected in 87% of the cell nuclei, suggesting that other egg extract reprogramming factors of similar size could successfully enter the nucleus. Lamin B3 labelling was maintained in most cells recovered 24 h after membrane resealing with calcium, and cells successfully resumed cell cycle in culture. In contrast, permeabilized cells that were not treated with egg extract failed to proliferate in culture and died, implying that egg extract provided factor essential to the survival of those cells. To conclude, fish fin cells were successfully primed for treatment with reprogramming factors, and egg extract was shown to play a major role in their survival and recovery after permeabilization.
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Species referenced: Xenopus laevis
Genes referenced: kpna1 kpnb1 lmnb3
GO keywords: nuclear import
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Figure 1: Digitonin-induced permeabilization of fin cells in suspension. Mesenchymal fin cells were tested for their ability to respond to various digitonin treatments. (A) Permeabilization rates and cell recovery rates after 2 min incubation. (B) Response over time of digitonin-induced permeabilization (2 to 24 min). Cells were incubated at digitonin concentrations ranging from 5 to 30 µg/mL and labelled with the non membrane permeant propidium iodide (PI). Cell permeabilization rates are expressed as a percentage of PI-positive cells to the total cell number in the sample. Cell recovery rates are expressed as a percentage of the cell number after digitonin treatment to the initial cell number before treatment. Bars represent means ± SD (n = 3 to 13 independent fin cell suspension). In (A), different letters indicate significant differences (p < 0.01) for a given concentration. |
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Figure 2: Digitonin-induced permeabilization of adherent fin cells in relation to cell density. Cells were permeabilized by digitonin at 7.5 µg/mL (Dig7.5) and 30 µg/mL (Dig30). After 2 min exposure at 4 °C, cells were labelled with Hoechst 33243 (upper picture) and the non membrane permeant propidium iodide (PI) (lower pictures). The permeabilized cells were identified by a red PI fluorescence observed in low and high density areas. Note that no red fluorescence was detected in non-permeabilized cells (Control cells) included in this experiment. These pictures are representative of seven independent replicates. Scale bar = 20 µm. |
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Figure 3: Cell morphology (A) and loss of cytoplasmic proteins (B) after digitonin-induced permeabilization. The cells were permeabilized for 2 min with 30 µg/mL digitonin at 4 °C. (A) The morphology of permeabilized (Dig30) and non-permeabilized (Control) cells was observed by phase-contrast microscopy. Note the nuclear envelope that was more contrasted (arrow) in permeabilized cells than in control cells. Scale bar = 20 µm. (B) Digitonin-permeabilized (Dig30) and non-permeabilized (Control) cells were lysed with RIPA buffer added to the adherent cells. Protein lysates were submitted to SDS-PAGE then silver stained. The loss of a protein band at about 60 kDa was observed (arrow) in the permeabilized cells. |
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Figure 4: Labelling of permeabilized cells with a large 70 kDa dextran. Cells permeabilized with 30 µg/mL digitonin (Dig30, A) and non-permeabilized cells (Control, B) were labelled with Texas red-conjugated 70 kDa dextran. The red fluorescence was present in the cytoplasm of permeabilized cells and excluded from nuclei. Most permeabilized cells were strongly labelled, some cells showed lower fluorescence pattern (arrowheads) and only few cells contained no fluorescence (not shown). No fluorescence was detected in the non-permeabilized cells. These pictures are representative of three independent replicates. Scale bar = 20 µm. |
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Figure 5: Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) (B) and Karyopherin beta-1 (KPNB1–97 kDa) (C) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti-Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min). |
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Figure 6: Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. (A) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti-Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). (B) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm. |
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Figure 7: Morphology of the permeabilized cells over the time post-resealing. Permeabilized cells (Dig30) were treated with Xenopus egg extract (+Extract) or left without (−Extract) for 1 h at 25 °C. Cells were then incubated in the resealing medium for 2 h. Permeabilized cell behavior was assessed by phase contrast microscopy after the 2 h resealing and 24 h post-resealing. The permeabilized cells that were egg-extract treated exhibited a round and refracting morphology (arrows) that progressively spread. By contrast, the permeabilized cells that were not exposed to the Xenopus egg extract did not survive in the resealing medium (arrow heads). These pictures are representative of six independent replicates. Non-permeabilized cells incubated in culture medium were included as control cells. Scale bar = 20 µm. |
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Figure 8: Detection of Xenopus Lamin B3 in treated cells 24 h post-resealing. Permeabilized cells were treated with Xenopus egg extract (Dig30 + Extract) for 1 h at 25 °C and incubated in resealing medium for 2 h. At 24 h post-resealing, Lamin B3 was detected by immunofluorescence in the nuclei of recovered cells. Nuclei were counterstained with Hoechst 33243. Several representative fields are shown including an enlarged view. Non-permeabilized cells were incubated in absence of Xenopus egg extract (Control – Extract). These pictures are representative of three independent replicates. Scale bar = 20 µm. |
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Supplementary Fig. S1 DNA fragmentation of the permeabilized cells maintained in culture without Xenopus egg extract. Cells in suspension were permeabilized by 30 µg/mL digitonin (Dig30) for 2 min at 4°C and were then maintained at 25°C in the presence (+ Extract) or absence (- Extract) of Xenopus egg extract. After 60 min, cells were labelled with Hoechst 33243. Note the presence of nuclear fragmentation (arrows) when the extract was absent. Scale bar = 20 µm. |
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Supplementary Fig. S2 Morphology of permeabilized cells treated with Xenopus egg-extract. The morphology of permeabilized egg-extract treated (Dig30 + Extract) and non-permeabilized (Control) cells was observed by phase-contrast microscopy. Note that the cytoplasm is no longer distinguishable in treated cells. Only the nuclei remained strongly visible with a white appearance (arrows). Inset: magnification of one nucleus showing nucleoli very dark. Scale bar = 20 µm. |
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Supplementary Fig. S3 Successful plasma membrane recovery of extract-treated permeabilized cells. Cells permeabilized with 30 µg/mL digitonin and treated with Xenopus egg-extract were incubated in resealing medium containing 2mM CaCl2 for 2 h at 25°C. After 24h, the cells were labelled with the non membrane permeant propidium iodide (PI) and Hoechst 33243 according the labelling condition described in Fig 2. No fluorescent PI signal was detected, indicating a successful plasma membrane recovery of the treated cells. These pictures are representative of 3 independent cultures in which 3 different egg extract batches were used |
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Figure 1. Digitonin-induced permeabilization of fin cells in suspension. Mesenchymal fin cells were tested for their ability to respond to various digitonin treatments. (A) Permeabilization rates and cell recovery rates after 2 min incubation. (B) Response over time of digitonin-induced permeabilization (2 to 24 min). Cells were incubated at digitonin concentrations ranging from 5 to 30 µg/mL and labelled with the non membrane permeant propidium iodide (PI). Cell permeabilization rates are expressed as a percentage of PI-positive cells to the total cell number in the sample. Cell recovery rates are expressed as a percentage of the cell number after digitonin treatment to the initial cell number before treatment. Bars represent means ± SD (n = 3 to 13 independent fin cell suspension). In (A), different letters indicate significant differences (p < 0.01) for a given concentration. |
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Figure 2. Digitonin-induced permeabilization of adherent fin cells in relation to cell density. Cells were permeabilized by digitonin at 7.5 µg/mL (Dig7.5) and 30 µg/mL (Dig30). After 2 min exposure at 4 °C, cells were labelled with Hoechst 33243 (upper picture) and the non membrane permeant propidium iodide (PI) (lower pictures). The permeabilized cells were identified by a red PI fluorescence observed in low and high density areas. Note that no red fluorescence was detected in non-permeabilized cells (Control cells) included in this experiment. These pictures are representative of seven independent replicates. Scale bar = 20 µm. |
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Figure 3. Cell morphology (A) and loss of cytoplasmic proteins (B) after digitonin-induced permeabilization. The cells were permeabilized for 2 min with 30 µg/mL digitonin at 4 °C. (A) The morphology of permeabilized (Dig30) and non-permeabilized (Control) cells was observed by phase-contrast microscopy. Note the nuclear envelope that was more contrasted (arrow) in permeabilized cells than in control cells. Scale bar = 20 µm. (B) Digitonin-permeabilized (Dig30) and non-permeabilized (Control) cells were lysed with RIPA buffer added to the adherent cells. Protein lysates were submitted to SDS-PAGE then silver stained. The loss of a protein band at about 60 kDa was observed (arrow) in the permeabilized cells. |
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Figure 4. Labelling of permeabilized cells with a large 70 kDa dextran. Cells permeabilized with 30 µg/mL digitonin (Dig30, A) and non-permeabilized cells (Control, B) were labelled with Texas red-conjugated 70 kDa dextran. The red fluorescence was present in the cytoplasm of permeabilized cells and excluded from nuclei. Most permeabilized cells were strongly labelled, some cells showed lower fluorescence pattern (arrowheads) and only few cells contained no fluorescence (not shown). No fluorescence was detected in the non-permeabilized cells. These pictures are representative of three independent replicates. Scale bar = 20 µm. |
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Figure 5. Nuclear import of the fusion protein GST-GFP-NLS in the permeabilized cells. Cells permeabilized with 30 µg/mL digitonin (Dig30) and non-permeabilized cells (Control) were incubated with GST-GFP-NLS in the absence (−Extract) or presence (+Extract) of Xenopus egg extract supplemented with ATP for 1 h at 25 °C. The incorporation of GST-GFP-NLS was assessed by green fluorescence. Cell nuclei were counterstained with Hoechst 33243. Note that without egg extract, the nuclei are not labelled (arrows). Due to light scattering of the fluorescence in the cytoplasm, the nuclei appear smaller than they are. The egg extract restored nuclear import in the permeabilized cells. These pictures are representative of five independent replicates. Scale bar = 20 µm. Detection of importin alpha-1 (55 kDa) (B) and Karyopherin beta-1 (KPNB1–97 kDa) (C) in Xenopus egg extract by western blot in the presence (+) or in absence (−) of the anti-Xenopus importin alpha-1 antibody (clone 15) and the anti-rat KPNB1 antibody (KPNB1-clone 23). Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gels and were analyzed with the same exposure times (B:1 sec; C:1 min). |
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Figure 6. Incorporation of Lamin B3 from Xenopus eggs into the nuclei of permeabilized cells. (A) Detection of Lamin B3 (68 kDa) in Xenopus egg extract (Extract) by western blot in the presence (+) or absence (−) of anti-Xenopus Lamin B3. Beta actin was used as a loading control. M: size markers. The cropped blots came from the same gel and were analyzed with the same exposure times (Lamin B3:2 sec; beta actin: 1 min). (B) Nuclear import of Lamin B3 detected by immunofluorescence in permeabilized (Dig30) and non-permeabilized (Control) cells incubated for 60 min in the presence (+Extract) of Xenopus egg extract under energy supplementation at 25 °C. Lamin B3-positive cells presenting a detectable signal (from weak to strong staining) were observed. A high proportion of nuclei were labelled. Inset: magnification of one nucleus showing a strong labelling of the nuclear lamina. These pictures are representative of five independent replicates. Scale bar = 20 µm. |
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Figure 7. Morphology of the permeabilized cells over the time post-resealing. Permeabilized cells (Dig30) were treated with Xenopus egg extract (+Extract) or left without (−Extract) for 1 h at 25 °C. Cells were then incubated in the resealing medium for 2 h. Permeabilized cell behavior was assessed by phase contrast microscopy after the 2 h resealing and 24 h post-resealing. The permeabilized cells that were egg-extract treated exhibited a round and refracting morphology (arrows) that progressively spread. By contrast, the permeabilized cells that were not exposed to the Xenopus egg extract did not survive in the resealing medium (arrow heads). These pictures are representative of six independent replicates. Non-permeabilized cells incubated in culture medium were included as control cells. Scale bar = 20 µm. |
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Figure 8. Detection of Xenopus Lamin B3 in treated cells 24 h post-resealing. Permeabilized cells were treated with Xenopus egg extract (Dig30 + Extract) for 1 h at 25 °C and incubated in resealing medium for 2 h. At 24 h post-resealing, Lamin B3 was detected by immunofluorescence in the nuclei of recovered cells. Nuclei were counterstained with Hoechst 33243. Several representative fields are shown including an enlarged view. Non-permeabilized cells were incubated in absence of Xenopus egg extract (Control – Extract). These pictures are representative of three independent replicates. Scale bar = 20 µm. |
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