Journal: The Journal of Cell Biology

Article Title: UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope

doi: 10.1083/jcb.202310006

Figure Lengend Snippet: UBAP2L localizes to the NE and NPCs. (A and B) Representative images of the localization of UBAP2L and Nups in HeLa cells with/without NaAsO2 treatment shown by immunofluorescence microscopy with UBAP2L and mAb414 antibodies. Nuclei were stained with DAPI. The arrowheads indicate the NE localization of endogenous UBAP2L. The magnified framed regions are shown in the corresponding numbered panels. Scale bars, 5 μm (A). The colocalization (EPCV, events per cell view) of UBAP2L and mAb414 in A was measured by CellProfiler (mean ± SD, ****P < 0.0001, unpaired two-tailed t test; 175 cells for NaAsO 2 treatment and 110 cells without NaAsO 2 treatment were counted) (B). (C) Representative immunofluorescence images depicting the localization of UBAP2L in HeLa cells after chemical pre-extraction of the cytoplasm using 0.01% of Triton X-100 for 90 s in indicated cell cycle stages and visualized by UBAP2L antibody. Nuclei and chromosomes were stained with DAPI. Scale bar, 5 μm. (D–F) Representative super-resolution immunofluorescence images of Nup96-GFP KI U2OS cells acquired using multicolor SMLM with a dichroic image splitter (splitSMLM) show NPCs on the nuclear surface (top view) and in the cross-section of the NE (side view). Nup96 signal labels the cytoplasmic and nuclear ring of the NPC and the localization of the central channel NPC component is analyzed by Nup62 antibody. The nuclear (Nuc) and cytoplasmic (Cyt) sides of the NE are indicated in the side view. The magnified framed regions are shown in the corresponding numbered panels. Note that UBAP2L can localize to both structures within the NPCs (framed regions 1 and 2 in the top view) and is found preferentially at the nuclear ring labeled with Nup96 (double arrowheads in framed region 3 in the side view). Scale bars, 300 and 100 nm, respectively (D). Radial distribution of localizations of Nup62, Nup96, and UBAP2L in D was obtained by averaging 1932 NPC particles (E). Averaged “side view” profiles of Nup62, Nup96, and UBAP2L in D were obtained by alignment of 83 individual NPCs (F). Orientation bars point to the NPC center (central channel middle point) as well as the cytoplasmic and nuclear sides (E and F).

Article Snippet: UBAP2L KO in Nup96-GFP KI U2OS (CLS Cell Line Service, 300174; a generous gift of Arnaud Poterszman, IGBMC [ ]) cell lines were generated using CRISPR/Cas9 genome editing system ( ).

Techniques: Immunofluorescence, Microscopy, Staining, Two Tailed Test, Extraction, Labeling

Journal: The Journal of Cell Biology

Article Title: UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope

doi: 10.1083/jcb.202310006

Figure Lengend Snippet: UBAP2L may facilitate the assembly of the NPC scaffold elements and the biogenesis of NPCs. (A) Representative splitSMLM images depicting several NPC components on the nuclear surface (top view) and in the cross-section of the NE (side view) in WT and UBAP2L KO HeLa cells synchronized in early interphase by DTBR at 12 h. Nup133 signal labels the cytoplasmic and nuclear rings of the NPC, the localization of the central channel is visualized by Nup62 and mAb414 antibodies, and the cytoplasmic filaments are labeled by RanBP2. The magnified framed regions are shown in the corresponding numbered panels. The nuclear (Nuc) and cytoplasmic (Cyt) side of the NE are indicated in the side view. The arrowheads indicate the disrupted localization of Nup62 or mAb414 at NE in UBAP2L KO HeLa cells and the numbers point to the individual identified spokes of the NPC. Scale bars, 300 and 100 nm, respectively. (B and C) The nuclear density of NPCs (mAb414 and RanBP2) in cells shown in A was quantified (B) (mean ± SD, ****P < 0.0001, unpaired two-tailed t test; counted 32 cells per cell line). The nuclear density of NPCs (mAb414) in HeLa cells expressing Flag alone or Flag-UBAP2L for 35 h and synchronized in interphase by DTBR at 12 h was quantified (C) (mean ± SD, *P < 0.05, unpaired two-tailed t test; counted 35 cells for Flag and 32 cells for Flag-UBAP2L). The corresponding representative images are shown in . (D) The rotational symmetry of NPCs in A was quantified by alignment of Nup133 particles and segmentation analysis (mean ± SD, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test; counted 851 NPCs for WT HeLa cell line and 559 NPCs for UBAP2L KO HeLa cell line). (E and F) Representative SMLM immunofluorescence images of FG-Nups (mAb414) at the nuclear surface in Nup96-GFP KI U2OS WT and UBAP2L KO cells in interphase cells synchronized by DTBR at 12 h. Scale bars, 1 μm (E). The nuclear density of NPCs (mAb414) in cells shown in E was quantified in F (mean ± SD, ****P < 0.0001, unpaired two-tailed t test; counted 60 cells per cell line). (G) Lysates of interphase WT and UBAP2L KO HeLa cells expressing GFP alone or 3XGFP-Nup85 for 27 h were immunoprecipitated using agarose GFP-Trap A beads (GFP-IP), analyzed by western blot, and signal intensities were quantified (shown a mean value, **P < 0.01, ***P < 0.001, unpaired two-tailed t test; n = 3 independent experiments). The asterisk indicates a non-specific, faster migrating band. (H) Lysates of interphase U2OS cells expressing GFP alone for 27 h and Nup96-GFP KI U2OS WT and UBAP2L KO cells were immunoprecipitated using agarose GFP-Trap A beads (GFP-IP), analyzed by western blot, and signal intensities were quantified (shown a mean value, *P < 0.05, **P < 0.01, unpaired two-tailed t test; n = 3 independent experiments) (H). Source data are available for this figure: .

Article Snippet: UBAP2L KO in Nup96-GFP KI U2OS (CLS Cell Line Service, 300174; a generous gift of Arnaud Poterszman, IGBMC [ ]) cell lines were generated using CRISPR/Cas9 genome editing system ( ).

Techniques: Labeling, Two Tailed Test, Expressing, Immunofluorescence, Immunoprecipitation, Western Blot

Journal: The Journal of Cell Biology

Article Title: UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope

doi: 10.1083/jcb.202310006

Figure Lengend Snippet: UBAP2L may inhibit formation of cytoplasmic AL or AL-like Nup assemblies. (A) Representative splitSMLM immunofluorescence images depicting the localization of NPC components corresponding to the central channel (Nups labeled by mAb414) and cytoplasmic filaments (RanBP2) at the NE and in the cytoplasm, as well as the localization of NPC components corresponding to the central channel (FG-Nup Nup62) and the outer ring (Y-complex Nup133) in the cytoplasm in WT and UBAP2L KO HeLa cells synchronized in interphase by DTBR at 12 h. Note that unlike at the NE where RanBP2 can localize exclusively to the cytoplasmic side of the NPCs , deletion of UBAP2L leads to the accumulation of the Nup assemblies in the cytoplasm with a symmetric distribution of RanBP2. Moreover, similar to the nuclear surface, in the cytoplasm, Nup62 signal is surrounded by Nup133 ring-like structures in both WT and UBAP2L KO cells. The magnified framed regions are shown in the corresponding numbered panels. Scale bars, 1,000, 300, and 150 nm, respectively. (B and C) Representative SMLM immunofluorescence images of FG-Nups (mAb414) at the nuclear surface and in the cytoplasm in interphase HeLa cells expressing Flag alone or Flag-UBAP2L for 35 h and synchronized by DTBR at 12 h. The magnified framed regions are shown in the corresponding numbered panels and corresponding quantification is shown in . The arrowheads indicate the cytoplasmic co-localization of FLAG-UBAP2L and mAb414-reactive Nups, which were highlighted in the corresponding magnified regions. Scale bars, 1,000 and 500 nm, respectively (B). The colocalization (EPCV, events per cell cytoplasmic view) of cytoplasmic mAb414 with Flag and Flag-UBAP2L in B was measured by CellProfiler (mean ± SD, ****P < 0.0001, unpaired two-tailed t test; counted 35 cells for Flag and 32 cells for Flag-UBAP2L) (C). (D and E) Validation of CRISPR/Cas9-mediated UBAP2L KO Nup96-GFP KI U2OS cell clones by western blot (D) and Sanger sequencing (E). (F–H) Representative immunofluorescence images of the localization of Nups (GFP-Nup96 and mAb414) and FXR1 in WT and in two UBAP2L KO Nup96-GFP KI U2OS clonal cell lines in interphase cells synchronized by DTBR at 15 h. Nuclei were stained with DAPI. Scale bar, 5 μm (F). The percentage of cells with cytoplasmic granules of Nups (mAb414) (G) and of FXR1 (H) shown in F were quantified. At least 200 cells per condition were analyzed (mean ± SD, **P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t test, n = 3 independent experiments). (I) Lysates of WT and UBAP2L KO Hela cells expressing GFP alone or 3XGFP-Nup85 for 27 h and synchronized in G1/S phase by Thymidine 16 h were immunoprecipitated using agarose GFP-Trap A beads (GFP-IP), analyzed by western blot, and signal intensities were quantified (shown a mean value, *P < 0.05, unpaired two-tailed t test; n = 3 independent experiments). (J) HeLa cells lysates of cells synchronized in interphase (Thymidine 16 h) and of cells synchronized in mitosis (STLC 16 h) were immunoprecipitated using Nup85 antibody or IgG, analyzed by western blot, and signal intensities were quantified (shown a mean value, ***P < 0.001, unpaired two-tailed t test; n = 3 independent experiments). (K) Lysates of interphase WT and UBAP2L KO HeLa cells expressing GFP alone or GFP-FXR1 for 27 h were immunoprecipitated using agarose GFP-Trap A beads (GFP-IP), analyzed by western blot, and signal intensities were quantified (shown a mean value, *P < 0.05, **P < 0.01, unpaired two-tailed t test; n = 3 independent experiments). Source data are available for this figure: .

Article Snippet: UBAP2L KO in Nup96-GFP KI U2OS (CLS Cell Line Service, 300174; a generous gift of Arnaud Poterszman, IGBMC [ ]) cell lines were generated using CRISPR/Cas9 genome editing system ( ).

Techniques: Immunofluorescence, Labeling, Expressing, Two Tailed Test, Biomarker Discovery, CRISPR, Clone Assay, Western Blot, Sequencing, Staining, Immunoprecipitation

Journal: Small methods

Article Title: Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

doi: 10.1002/smtd.202301497

Figure Lengend Snippet: Figure 1. MINFLUX imaging of PaX560- and PaX+560-labeled NPCs. Rep- resentative MINFLUX images of U-2 OS-CRISPR-NUP96-Halo cells la- beled (live) with A,B) PaX560-Halo and D,E) PaX+560-Halo, fixed and mounted in phosphate-buffered saline (PBS) prior to imaging; B,E) show zoom-ins of NPCs marked in A and D. Data properties can be found in Figure S6 and Table S4 (Supporting Information). Normalized occu- pancy histograms of HaloTagged NPCs labeled with C) PaX560-Halo and F) PaX+560-Halo. A fit to a probabilistic model is included in each his- togram (bars: experimental data, circles: fit), including the residuals in the top plots, considering eight labeling sites and four protein copies at each one (Equation S1, Supporting Information). The fitted ELE value (Figure S9, Supporting Information), the mean occupancy (Occ), and the number of analyzed NPCs (N) are indicated. Scale bars: overviews 200 nm, zoom- ins 50 nm.

Article Snippet: The authors thank the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany) for providing the U-2 OS-CRISPR-NUP96Halo clone #252 (330448, CLS) and the U-2 OS-CRISPR-NUP96-mMaple clone #16 (300461, CLS), and Dr. S. Jakobs (MPI-NAT, Goettingen, Germany) for providing the U-2 OS-Vim-Halo cell line.

Techniques: Imaging, Labeling, CRISPR, Saline

Journal: Small methods

Article Title: Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

doi: 10.1002/smtd.202301497

Figure Lengend Snippet: Figure 2. MINFLUX imaging of mMaple-tagged NPCs. Representative MINFLUX images of U-2 OS-CRISPR-NUP96-mMaple, fixed, mounted in 50 mM Tris buffer (pH 8) in 95% D2O and imaged with A,B) seq_PaX and D,E) seq_mMaple; B,E) show zoom-ins of NPCs marked in A and D. Data properties can be found in Figure S6 and Table S4 (Supporting In- formation). Normalized occupancy histograms of mMaple-tagged NPCs imaged with C) seq_PaX and with F) seq_mMaple. A fit to a probabilis- tic model is included in each histogram (bars: experimental data, circles: fit), considering eight labeling sites and four protein copies at each one (Equation S1, Supporting Information). The fitted ELE value (Figure S9, Supporting Information), the mean occupancy (Occ), and the number of analyzed NPCs (N) are indicated. Scale bars: overviews 200 nm, zoom-ins 50 nm.

Article Snippet: The authors thank the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany) for providing the U-2 OS-CRISPR-NUP96Halo clone #252 (330448, CLS) and the U-2 OS-CRISPR-NUP96-mMaple clone #16 (300461, CLS), and Dr. S. Jakobs (MPI-NAT, Goettingen, Germany) for providing the U-2 OS-Vim-Halo cell line.

Techniques: Imaging, CRISPR, Labeling

Journal: Small methods

Article Title: Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

doi: 10.1002/smtd.202301497

Figure Lengend Snippet: Figure 3. Dual color 561 nm MINFLUX imaging with PaX560 and PaX595 based on spectral classification. A) Absorption and B) emission spectra of solutions of PaX560 and PaX595 in methanol after complete activation with 405 nm light. The excitation wavelength (561 nm) and the detection windows used for MINFLUX imaging are indicated in the corresponding plots. The inset shows the color of the solutions under A) normal and B) UV (366 nm) illumination. C) Histogram of the DCR value of all localizations (grey) and the ones after spectral classification (green/magenta). D) Dual color MIN- FLUX image of NPCs in fixed U-2 OS-CRISPR-NUP96-Halo cells labeled with PaX560-Halo (Nup96, green), and with a primary antibody against Nup62 (magenta) in combination with a secondary antibody conjugated with PaX595. The inset shows a selected NPC. The spectral decomposition of the im- age is shown in Figure S13 (Supporting Information). Data properties can be found in Figure S14 and Table S5 (Supporting Information). Scale bars: overview 200 nm, zoom-in 50 nm.

Article Snippet: The authors thank the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany) for providing the U-2 OS-CRISPR-NUP96Halo clone #252 (330448, CLS) and the U-2 OS-CRISPR-NUP96-mMaple clone #16 (300461, CLS), and Dr. S. Jakobs (MPI-NAT, Goettingen, Germany) for providing the U-2 OS-Vim-Halo cell line.

Techniques: Imaging, Activation Assay, CRISPR, Labeling

Journal: Small methods

Article Title: Photoactivatable Xanthone (PaX) Dyes Enable Quantitative, Dual Color, and Live-Cell MINFLUX Nanoscopy.

doi: 10.1002/smtd.202301497

Figure Lengend Snippet: Figure 4. Live-cell MINFLUX imaging of PaX560-labeled NPCs. Representative MINFLUX images of living U-2 OS-CRISPR-NUP96-Halo cells labeled with compound PaX560-Halo and imaged in complete cell medium without any special additives. Total imaging times were A) 120, B) 50, and C) 80 s. The image generation over time of A is shown in Figure S16 (Supporting Information). Data properties can be found in Figure S17 and Table S6 (Supporting Information). Scale bar: 50 nm.

Article Snippet: The authors thank the European Molecular Biology Laboratory (EMBL; Heidelberg, Germany) for providing the U-2 OS-CRISPR-NUP96Halo clone #252 (330448, CLS) and the U-2 OS-CRISPR-NUP96-mMaple clone #16 (300461, CLS), and Dr. S. Jakobs (MPI-NAT, Goettingen, Germany) for providing the U-2 OS-Vim-Halo cell line.

Techniques: Imaging, Labeling, CRISPR