Mechanisms of PP2A-Ankle2 dependent nuclear reassembly after mitosis
- Institute for Research in Immunology and Cancer, Université de Montréal, Canada
- Département de biochimie et médecine moléculaire, Université de Montréal, Canada
- Department of Biochemistry, McGill University, Canada
- Département de chimie, Université de Montréal, Canada
Figures
Figure 1 with 1 supplement
Ankle2 interacts with Protein Phosphatases 2A (PP2A) through its Ankyrin repeats region.
(A) Ankle2 functions with PP2A to allow the dephosphorylation of Barrier-to-Autointegration Factor (BAF) and its recruitment to segregated chromosomes. BAF promotes the formation of a single nucleus …
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Figure 1—source data 1
Proteins identified from affinity purifications of Ankle2-GFP from embryos.
Numbers of total spectral counts and unique peptides for Ankle2-GFP or Flag-GFP (control) are indicated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-data1-v1.xlsx
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Figure 1—source data 2
Proteins identified from affinity purifications of PP2A-29B-GFP from embryos.
Numbers of total spectral counts and unique peptides for PP2A-29B-GFP or GFP (control) are indicated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-data2-v1.xlsx
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Figure 1—source data 3
Figures with uncropped western blots and gels annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-data3-v1.zip
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Figure 1—source data 4
Original files for western blots and gels.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-data4-v1.zip
Figure 1—figure supplement 1
Ankle2 interacts with Protein Phosphatases 2A (PP2A) through its Ankyrin repeats region.
(A, B) Proteins obtained after purification of Ankle2-GFP, GFP-Ankle2, or Flag-GFP from D-Mel cells. (A) Silver-stained gel showing a fraction of the purification products. (B) Proteins specifically …
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Figure 1—figure supplement 1—source data 1
Proteins identified from affinity purifications of GFP-fused Ankle2 from D-Mel cells.
Numbers of total spectral counts and unique peptides for GFP-Ankle2, Ankle2-GFP, or Flag-GFP (control) are indicated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-figsupp1-data1-v1.xlsx
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Figure 1—figure supplement 1—source data 2
Proteins identified from affinity purifications of GFP-fused PP2A-29B from D-Mel cells.
Numbers of total spectral counts and unique peptides for GFP-PP2A-29B, PP2A-29B-GFP, or GFP (control) are indicated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-figsupp1-data2-v1.xlsx
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Figure 1—figure supplement 1—source data 3
Figures with uncropped western blots and gels annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-figsupp1-data3-v1.zip
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Figure 1—figure supplement 1—source data 4
Original files for western blots and gels.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig1-figsupp1-data4-v1.zip
Figure 2
Protein Phosphatases 2A (PP2A)-Ankle2 promotes Barrier-to-Autointegration Factor (BAF) dephosphorylation.
(A) Phosphoproteomic analysis identified BAF as being hyperphosphorylated at Thr4 and Ser5 upon Ankle2 depletion. D-Mel cells were transfected with dsRNA against Ankle2 or non-target dsRNA (against …
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Figure 2—source data 1
Phosphopeptide identification and quantification from cells after Ankle2 or control RNAi.
Results are listed for four replicates in each condition, as well as the average fold-changes and p-values.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig2-data1-v1.xlsx
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Figure 2—source data 2
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig2-data2-v1.xlsx
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Figure 2—source data 3
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig2-data3-v1.zip
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Figure 2—source data 4
Original files for western blots.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig2-data4-v1.zip
Figure 3 with 1 supplement
Ankle2 co-localizes with Vap33.
(A) Video images of a D-Mel cell co-expressing Ankle2-RFP and GFP-Vap33 through different stages of cell division. (B) Video images of a syncytial embryo co-expressing Ankle2-GFP and RFP-Vap33 …
Figure 3—figure supplement 1
The localization of Ankle2 depends on Vap33 in egg chambers.
(A) Schematic representation of different stages of egg chamber maturation. (B) Egg chambers expressing Ankle2-GFP from the matα4-GAL-VP16 driver, with or without expression of Vap33 RNAi from the …
Figure 4 with 1 supplement
Ankle2 interacts with Vap33 through FFAT motifs.
(A) Cells were transfected with the indicated proteins and used in GFP affinity purifications. Products were analyzed by western blots. WCL: Whole cell extracts. (B, C) GST pulldown mapping …
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Figure 4—source data 1
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blots.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig4-data2-v1.zip
Figure 4—figure supplement 1
A Vap33-Ankle2-PP2A complex may promote nuclear reassembly in a localized manner.
(A) Hypothetical model discussed in the text. ER: Endoplasmic Reticulum; NE: Nuclear Envelope. (B) AlphaFold3 predicted model of a complex between Drosophila Ankle2, PP2A-29B, Mts, and Vap33. …
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Figure 4—figure supplement 1—source data 1
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig4-figsupp1-data1-v1.zip
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Figure 4—figure supplement 1—source data 2
Original files for western blots.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig4-figsupp1-data2-v1.zip
Figure 5 with 3 supplements
Ankle2 interaction with Protein Phosphatases 2A (PP2A) but not with Vap33 is required for nuclear reassembly.
(A) RNAi depletion of endogenous Ankle2 and simultaneous expression of RNAi-insensitive Ankle2-GFP. D-Mel cells expressing RNAi-insensitive Ankle2-GFP or not were transfected with dsRNA against …
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Figure 5—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-data1-v1.xlsx
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Figure 5—source data 2
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-data2-v1.zip
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Figure 5—source data 3
Original files for western blots.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-data3-v1.zip
Figure 5—figure supplement 1
Results of rescue experiments in D-Mel cells in culture.
Endogenous Ankle2 was depleted by RNAi and RNAi-insensitive Ankle2-GFP variants were simultaneously expressed as in Figure 6. D-Mel cells expressing RNAi-insensitive Ankle2-GFP variants or not were …
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Figure 5—figure supplement 1—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-figsupp1-data1-v1.xlsx
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Figure 5—figure supplement 1—source data 2
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Original files for western blots.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig5-figsupp1-data3-v1.zip
Figure 5—figure supplement 2
Summary of results for various deletion mutants of Ankle2-GFP assessed for their ability to rescue abnormal Lamin phenotypes.
Regions of interest discussed in the text are indicated with colored boxes. Red Xs: mutations of FFAT and FFAT-Like 1 Vap33 binding motifs.
Figure 5—figure supplement 3
Localization of Ankle2-GFP variants during the cell cycle in D-Mel cells.
Cells were imaged on a confocal microscope. Representative images of different stages of the mitotic cycle are shown. Int: Interphase; Pro: Prophase; Promet: Prometaphase; Met: Metaphase; Ana: …
Figure 6 with 2 supplements
Ankle2 interactions with Protein Phosphatases 2A (PP2A) and Vap33 promote BAF recruitment at reassembling nuclei.
(A) Syncytial embryos depleted of endogenous Ankle2 and expressing RNAi-insensitive Ankle2WT-GFP (top), Ankle2ΔANK-GFP (middle), or Ankle2Fm+FL1m-GFP (bottom) along with RFP-BAF were imaged through …
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Figure 6—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Female germline expression of Ankle2-GFP variants in transgenic lines.
Expression of UASp-Ankle2-GFP WT, ΔANK, or Fm +FL1 m (RNAi insensitive) was induced using the matα4-GAL-VP16 driver. Ankle2 RNAi (line BDSC 77437) was also induced using by the same driver. Western …
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Figure 6—figure supplement 1—source data 1
Figures with uncropped western blots annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig6-figsupp1-data1-v1.zip
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Figure 6—figure supplement 1—source data 2
Original files for western blots and gels.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
Quantifications in embryos.
(A) Illustration of the quantification of fluorescence intensities at the inner core region of the reassembling nuclear envelope (NE) in embryos expressing Ankle2-GFP and RFP-BAF. Inner core regions …
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Figure 6—figure supplement 2—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig6-figsupp2-data1-v1.xlsx
Figure 7 with 1 supplement
Ankle2 interaction with Protein Phosphatases 2A (PP2A) but not with Vap33 is required for development.
(A) Number of embryos laid over three days per female of the indicated genotypes. (B) Percentage of embryos hatching per female of the indicated genotypes. In A-B, the Ankle2 RNAi insertion from …
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Figure 7—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig7-data1-v1.xlsx
Figure 7—figure supplement 1
Phenotypes resulting from RNAi depletion of Ankle2 during oogenesis.
Ankle2 RNAi (line BDSC77437) was using the matα4-GAL-VP16 driver. (A) Western blot from ovaries showing Ankle2 depletion. The asterisk indicates a non-specific band used as the loading control. (B) …
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Figure 7—figure supplement 1—source data 1
Numerical data is used to make graphs.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig7-figsupp1-data1-v1.xlsx
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Figure 7—figure supplement 1—source data 2
Figure with uncropped western blot annotated.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig7-figsupp1-data2-v1.zip
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Figure 7—figure supplement 1—source data 3
Original file for western blot.
- https://cdn.elifesciences.org/articles/104233/elife-104233-fig7-figsupp1-data3-v1.zip
Videos
Video 1
Localization of Ankle2-RFP and GFP-Vap33 during mitosis and cytokinesis in a D-Mel cell.
Orthogonal maximum intensity projection of 4 z-sections spaced by 1 μm. Images were taken every 205 s. Scale bar: 5 μm.
Video 2
Localization of Ankle2-GFP and RFP-Vap33 during the cell cycle in a syncytial embryo.
A single plane is shown. Images were taken every 10 s. Scale bar: 5 μm.
Video 3
Localization of Ankle2Fm+FL1m-GFP and RFP-Vap33 during the cell cycle in a syncytial embryo.
A single plane is shown. Images were taken every 10 s. Scale bar: 5 μm.
Video 4
Localization of Ankle2WT-GFP and RFP-BAF during the cell cycle in a syncytial embryo where endogenous is depleted by RNAi.
A single plane is shown. Images were taken every 10 s. Scale bar: 5 μm.
Video 5
Localization of Ankle2ΔANK-GFP and RFP-BAF during the cell cycle in a syncytial embryo where endogenous is depleted by RNAi.
A single plane is shown. Images were taken every 10 s. Scale bar: 5 μm.
Video 6
Localization of Ankle2Fm+FL1m-GFP and RFP-BAF during the cell cycle in a syncytial embryo where endogenous is depleted by RNAi.
A single plane is shown. Images were taken every 10 s. Scale bar: 5 μm.
