Research ArticleCELL BIOLOGY

JLP-centrosome is essential for the microtubule-mediated nucleocytoplasmic transport induced by extracellular stimuli

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaav0318
DOI: 10.1126/sciadv.aav0318
  • Fig. 1 JLP focus.

    (A) Induction of JLP foci by the arsenite treatment (0.5 mM for 30 min) in H1299 cells. They were fixed and stained with a control antibody (Ab) or the JLP antibody together with the DNA-intercalating dye 4′,6-diamidino-2-phenylindole (DAPI). The percentages of cells showing the JLP foci were scored and presented as mean ± SD % (n = 3). ***P < 0.0005 (as compared with untreated cells). (B) Time course of JLP localization after arsenite treatment. (C) Various cell lines were treated with or without arsenite (0.5 mM) for 30 min. (D) H1299 cells were treated with various stress stimuli or proinflammatory cytokines as indicated. Proliferation cytokine IL-3 served as a negative control. Scale bars, 20 μm.

  • Fig. 2 p38MAPK regulates the formation of JLP focus.

    (A) Effects of kinase inhibitors (2.5 and 20 μM) on the arsenite-induced JLP focus formation. Scale bar, 10 μm. Bottom panel shows the Western blot analysis of the JLP and α-tubulin levels in those treated cells. (B) Quantification of the average arbitrary JLP focus intensity per cell using ImageJ in (A) at 2 μM of the inhibitors. Error bars represent SD (n = 3). **P < 0.02 (as compared with the arsenite-treated vehicle control). DMSO, dimethyl sulfoxide. (C) Concentration effects of the kinase inhibitors. The average arbitrary JLP focus intensity per cell was calculated as percentages of their vehicle-treated controls. Error bars represent SD (n = 4). *P < 0.04 and **P < 0.005 (as compared with the corresponding untreated control). (D) Effects of the wild-type (WT) or dominant-negative (DN) mutant of FLAG-p38MAPK on the JLP focus formation. The percentages of cells showing the JLP foci were scored and presented as mean ± SD % (n = 4). ***P < 0.0001 (as compared with stimulated p38MAPK wild type–expressing cells). Scale bar, 20 μm.

  • Fig. 3 Dynein regulates the retrograde movement of JLP in its focus formation.

    (A) Effects of dynein inhibitor (EHNA; 1 mM) or kinesin-1 inhibitor (RBL; 40 μM) on the JLP focus formation. The relative arbitrary JLP focus intensity per cell was calculated as in Fig. 2B. Error bars represent SD (n = 4). ***P < 0.0001 (as compared with arsenite-treated vehicle control). Scale bar, 20 μm. (B) Association between exogenous JLP and DCTN1. (C) Mapping of the domain of DCTN1 involved in association with JLP. WB, Western blotting. (D) Association between endogenous JLP and DCTN1 in cells treated with or without arsenite (0.5 mM) for 30 min. IP, immunoprecipitation; ppt, precipitate.

  • Fig. 4 Formation of JLP focus and MT array is interdependent.

    (A) Induction of JLP foci and MT arrays by arsenite (0.5 mM), NaCl (500 mM), or TGFβ (5 ng/ml) for 30 min. Scale bar, 10 μm. (B) Arsenite does not affect the β-actin network. H1299 cells were treated with or without arsenite (0.5 mM) for 30 min. Scale bar, 10 μm. (C) Influx of α-tubulin to the nucleus induced by arsenite and its dependence on JLP. shC and shJLP denote the control and JLP-ablated cells, respectively. Ablation of JLP also reduces the nuclear translocation of NF-κB p65. RhoGDIα is a marker for the cytoplasmic fraction. The relative nuclear NF-κB p65 levels are quantified and presented as mean ± SD (n = 4). *P < 0.02, **P < 0.005, and ***P < 0.001. (D) MT-modulating agents inhibit the formation of JLP foci. The percentages of cells showing the JLP foci and the MT arrays were scored and presented as mean ± SD % (n = 4). ***P < 0.0001 (as compared with arsenite-treated control cells). Scale bar, 20 μm. (E) ΔCLD mutant of JLP inhibits the formation of the MT arrays. The percentages of cells showing the JLP foci and the MT arrays were scored and presented as mean ± SD % (n = 4). ***P < 0.0001 (as compared with arsenite-treated FLAG-JLP wild-type cells). Scale bar, 10 μm. (F) The top surface and medial views of lamin A/C and MTs in cells treated with or without arsenite (0.5 mM) for 40 min. The radiating MTs creating troughs on the nuclear membrane are indicated by arrows. Scale bars, 2 μm. Confocal z-stack images are shown in fig. S4.

  • Fig. 5 Stimulated nuclear translocation of the endogenous NF-κB p65 depends on the JLP focus.

    (A) The JLPΔCLD mutant inhibits the nuclear translocation of NF-κB p65 in H1299 cells. The percentages of cells showing the nuclear NF-κB p65 were scored and presented as mean ± SD % (n = 3). *P < 0.0003 (as compared with arsenite-treated FLAG-JLP wild-type cells). (B) Interaction between endogenous JLP and NF-κB p65. HEK293T cells were serum-deprived for 16 hours before they were stimulated with arsenite (0.5 mM) or tumor necrosis factor–α (TNFα; 30 ng/ml) for 25 min. The cell lysates were precipitated with the JLP antibody. (C) Colocalization of JLP and NF-κB p65. H1299 cells were stimulated with arsenite (0.5 mM) or TNFα (30 ng/ml) for 10 min before they were stained for immunofluorescence studies. Puncta of the colocalized JLP–NF-κB p65 appear orange. (D) Ablation of JLP reduces the transactivation of NF-κB in H1299 cells stimulated with arsenite (top) or TNFα (bottom). shC and shJLP denote the control and JLP-ablated cells, respectively. The relative NF-κB transactivation activities are presented as mean ± SD (n = 3). *P < 0.02, **P < 0.01, and ***P < 0.005. (E) Association of the S-tagged JLP domains with NF-κB p65 after arsenite stimulation (0.5 mM for 45 min). (F) Binding of the S-tagged Domain IIIΔCLD with NF-κB p65 after arsenite stimulation (0.5 mM for 45 min). (G) Reduction of NF-κB p65 nuclear translocation by IIIΔCLD in immunofluorescence studies in H1299 cells treated with or without TNFα (10 ng/ml) for 25 min. The percentages of cells showing the nuclear NF-κB p65 were scored and presented as mean ± SD % (n = 3). *P < 0.002 (as compared with the treated V cells). (H) Reduction of NF-κB p65 nuclear translocation by IIIΔCLD in subcellular fractionation studies in COS-7 cells. Transiently transfected COS-7 cells were stimulated with or without arsenite (0.5 mM) for 45 min, followed by fractionation. The relative nuclear NF-κB p65/β-actin levels are quantified and presented as mean ± SD (n = 4). *P < 0.01 and **P < 0.001. Scale bar, 10 μm. RhoGDIα is a marker for the cytoplasmic fraction. (I) Top surface and medial views of Nup133 and MTs on the nuclear membrane in the cells treated with or without arsenite (0.5 mM) for 40 min. Scale bars, 2 μm.

  • Fig. 6 FRET confocal microscopy of JLP and NF-κB p65.

    TagCFP-JLP and NF-κB p65-Venus (NF-κB–Venus) were expressed in H1299 cells. They were treated with or without TNFα (10 ng/ml) for the indicated period of time. Confocal images were taken and analyzed for FRET. The FRET signals were corrected for the bleed-through signals. Some images were enhanced to show the trace amount of JLP and FRET signals in the nucleus 20 min after stimulation. Scale bars, 5 μm.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaav0318/DC1

    Fig. S1. Specificity of the JLP antibody.

    Fig. S2. Colocalization of JLP focus and the MTOC.

    Fig. S3. Nuclear JLP.

    Fig. S4. The MTOC and nuclear membrane.

    Fig. S5. The role of the CLD in the association of JLP focus with centrosome.

    Fig. S6. The CLD does not bind centrosome.

    Fig. S7. The role of the CLD in the formation of JLP focus.

    Fig. S8. PLK1 is a part of JLP focus.

    Fig. S9. JLP focus concentrates the nuclear pore protein Nup358.

    Fig. S10. Models for the formation of JLP focus and its role in nuclear translocation of NF-κB p65.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Specificity of the JLP antibody.
    • Fig. S2. Colocalization of JLP focus and the MTOC.
    • Fig. S3. Nuclear JLP.
    • Fig. S4. The MTOC and nuclear membrane.
    • Fig. S5. The role of the CLD in the association of JLP focus with centrosome.
    • Fig. S6. The CLD does not bind centrosome.
    • Fig. S7. The role of the CLD in the formation of JLP focus.
    • Fig. S8. PLK1 is a part of JLP focus.
    • Fig. S9. JLP focus concentrates the nuclear pore protein Nup358.
    • Fig. S10. Models for the formation of JLP focus and its role in nuclear translocation of NF-κB p65.

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