Fig 1.
Colocalization of Nemp1 and lamins through region A.
(A) The diagram of Xl_Nemp1 and Mm_Nemp1. Xl_Nemp1 but not Mm_Nemp1 contains the KR sequence. Blue, magenta, green, and yellow boxes represent signal peptides (SP), transmembrane domains (TMs), KR sequence, and region B, respectively. (B,C) Confocal analysis was performed using transfected COS-7 cells. (B) Mm_Nemp1-HA with lamin or Nup153. Transfected cells were stained with anti-HA (red) and anti-lamin or anti-Nup153 (green) antibody. Scale bars, 5 μm. (C) Xl_Nemp1-HA or its deletion mutants with lamin. Transfected cells were stained with anti-HA (red) and anti-lamin (green) antibodies. Scale bars, 5 μm.
Fig 2.
Oligomerization of Nemp1 through the TMs.
A. Co-IP of Xl_Nemp1 with Xl_Nemp1 itself, MAN1, or Emerin. Xl_Nemp1-HA mRNA was coinjected into the animal pole region of two cell stage Xenopus embryos with mRNA for Xl_Nemp1-Myc, XMAN1-Myc, or Hs_emerin-Myc. Injected embryos were collected at the late blastula stage (stage 9) and lysed with lysis buffer A. Black arrowheads, expected product bands; white arrowheads, immunoglobulin bands; asterisks, shifted bands of Emerin due to phosphorylation [40]. B. Co-IP of Nemp1 with its deletion constructs. mRNA for Xl_Nemp1-HA was injected into Xenopus embryos with mRNA for deletion constructs of Xl_Nemp1-Myc. Deletion constructs of Nemp1, ΔN, ΔA, ΔTM, ΔB, N, SP+A, SP+B, and Ct (Δ, deleted; N, the N-terminal region; A, region A; TM, transmembrane domains; B, region B; SP, signal peptide; Ct, the C-terminal region; S2 Fig for diagrams) were used (see [10] for more detail). After immunoprecipitation against Myc, western blotting was performed with anti-Myc or-HA antibody as indicated below each panel.
Fig 3.
Interaction of region B with RanGTP.
A. Yeast two hybrid screening. Left panel, schematic representation of the bait, the Bt region of Mm_Nemp1 (DBD-Mm_Bt). Right panels, colony formation (in duplicate) of yeast AH109 cells transformed with DBD (upper) or DBD-Mm_Bt (lower) with AD-Mm_Ran on plates lacking tryptophan, leucine, and adenine. DBD, the DNA binding domain of Gal4; AD, the activation domain of Gal4. B. Co-IP of region B with Ran or its mutants using Xenopus embryos. mRNA for HA-tagged Mm_Bt was coinjected into Xenopus embryos with mRNA for a Myc-tagged construct of Mm_Ran, the RanGDP form mutant T24N, the RanGTP form mutant Q69L, or EGFP. Experimental conditions were the same as in Fig 2. C. GST pulldown assays using Xenopus embryo lysates. Purified GST or GST-Mm_Bt protein absorbed onto glutathione-Sepharose beads were incubated with lysates of Xenopus embryos, which had been injected with mRNA for HA-Mm_Ran, HA-T24N, or HA-Myc-Q69L (500 pg/embryo). Proteins bound to the beads were analyzed by western blotting. D. In vitro binding assays with recombinant proteins, Myc-Mm_RanQ69L(GTP) and GST-Mm_Bt. Purified GST-Mm_Bt or GST (2.8 μg) was incubated with purified Myc-RanQ69L (5 μg), which had been loaded with 2 mM GTP in the binding buffer. E. Co-IP of Mm_Ran mutants T42A and ΔC with Mm_Bt-HA using Xenopus embryos. mRNA for HA-tagged Mm_Bt was coinjected into Xenopus embryos with mRNA for Myc-tagged Mm_Ran, or its mutants (T42A or ΔC). Unnecessary lanes were removed from a single blot. F. GST pulldown assays of GST-Mm_RanQ69L, importin β and Mm_Bt. Xenopus embryos were injected with mRNA for FLAG-tagged importin β. Lysates were added with 0.1, 1, 10 μg of recombinant Mm_Bt and glutathione beads absorbed 10 μg of GST-RanQ69L. Western blotting was performed with antibodies as indicated below each panel. Arrowheads, expected product bands.
Fig 4.
The binding region of region B for Ran.
A. Co-IP of Mm_Ran with deletion constructs of Mm_Bt. Left panel, schematic structures of Mm_Bt deletion constructs. Right panels, western blotting of immunoprecipitated proteins or lysates as indicated. mRNA for HA-tagged Mm_Bt constructs was injected with mRNA for Myc-tagged Mm_Ran into Xenopus embryos. Arrowheads, expected bands; B. Co-IP of Xl_Ran with Xl_Bt or Xl_Bt_ΔBBS. Left panel, schematic structures of Xl_Bt and Xl_Bt_ΔBBS constructs. Right panels, western blotting of immunoprecipitated proteins or lysates as indicated. Experimental conditions (A, B) were the same as in Fig 3.
Fig 5.
The interaction of Nemp1 with Ran at the NE.
(A) Co-IP of Nemp1 with Ran or its mutants using Xenopus embryos. mRNA for HA-tagged Mm_Nemp1 was coinjected into Xenopus embryos with mRNA for a Myc-tagged construct of Mm_Ran or its mutants (T24N, Q69L, T42A, ΔC). Injected embryos were collected at the mid blastula stage (stage 9) and lysed with lysis buffer B. Black arrowheads, modified forms of Nemp1. This data is the same as lanes 1–6 shown in S3 Fig (B) Colocalization of Ran with Nemp1 at the nuclear periphery. COS-7 cells were transfected with Myc-Mm_Ran (red) with or without Mm_Nemp1-HA (green), and analyzed by confocal analysis. DNA was counterstained with SytoxGreen (blue). Scale bars, 5 μm.
Fig 6.
A. Developmental analysis for modified Xl_Nemp1. Xenopus embryos were injected with mRNA for Xl_Nemp1-HA and collected at the indicated stages (St.). Lysates were subjected to western blotting with anti-HA or β tubulin antibody (loading control). uc, uninjected control. B. In vitro alkaline phosphatase assay of Xl_Nemp1. Lysates were prepared at the late blastula stage (stage 9). Xl_Nemp1-HA was immunoprecipitated by anti-HA antibody and treated with (+) or without (-) calf intestinal alkaline phosphatase (CIAP). C. In vitro alkaline phosphatase assay of Mm_Nemp1. Lysates were prepared at the mid blastula stage (stages 8–8.5), and treated with (+) or without (-) λ protein phosphatase (λPP). D. Co-IP of Mm_Ran with phosphorylation site mutants of Mm_Nemp1. mRNA for Myc-tagged Mm_Ran was injected into Xenopus embryos with mRNA for HA-tagged Mm_Nemp1, its alanine mutant (5SA), or its glutamic acid mutant (5SE). Injected embryos were collected at the mid blastula stage and lysed with lysis buffer B. This data is the same as lanes 1, 2, 7, and 8 shown in S3 Fig Black arrowheads, modified forms.
Fig 7.
Cooperativity of Nemp1 and Ran in early eye development.
A. Spatiotemporal expression of Xenopus ran in the early development. Developmental stages are indicated. (a) Lateral view. (b, c) Dorsal view with the anterior side up. (d) Lateral view with the dorsal side up. opv, optic vesicles; otv, otic vesicles; ba, branchial arches. B. Eye defect phenotypes by knockdown of nemp1 and ran. nemp1MOs (5–20 ng) and n-βxgal mRNA as a tracer (blue) were injected into the animal pole region of a dorsal blastomere at the four cell stage with ranMO or standard control MO (stdMO). Upper panels show eye-defect phenotypes at the tailbud stage (around stage 35) as indicated. The lower bar graph shows percentages of eye defects at tailbud stages. n, the number of embryos examined; exp, the number of independent experiments.
Fig 8.
Reduction of cell densities by co-knockdown of nemp1 and ran.
(A) A. Effects of nemp1MOs on cell densities. Dorsoanterior views (a, b, c, d, e, f) of embryos are shown. Yellow boxes in a, b, d, and e correspond to enlarged areas a’, b’, d’, and e’, respectively. Upper panels, stdMO (40 ng/embryo); lower panels, nemp1MOs and stdMO (20 ng each/embryo) (the same experiment as in panel B). Embryos were injected with MOs and FITC-dextran as a tracer, fixed at stages 12.5–13, and immunostained with anti-phospho histone H3 antibody (red). DAPI was used for nuclear staining. White arrowheads, positions of blastopores; magenta scale bars, 500 μm; white scale bars, 100 μm. (B)B. Synergistic effects of nemp1MOs and ranMOs on cell densities. Combinations of MOs and amounts (ng/embryo) are as indicated. Experiments were repeated three times and similar results were obtained, one of which is presented here. DAPI-stained nuclei were counted in FITC-positive areas. C,D. Rescue of reduced cell density in morphants by mRNA injection. Combinations of MOs and mRNAs as well as amounts of MO (ng/embryo) and mRNA (pg/embryo) are as indicated. Injected embryos were fixed and immunostained using anti–HA antibody. DAPI-stained nuclei were counted in EGFP-HA positive areas. (E) E. Reduction of cell densities by overexpression of Nemp1. Injected mRNA and amount (pg/embryo) are as indicated. DAPI-stained nuclei were counted in EGFP-HA positive areas. *, P<0.05; ***, P<0.005; error bars, standard deviation.
Fig 9.
Evolutionary conservation of Ran binding of region B in Arabidopsis.
A. Co-IP of At_Nemp and At_Ran. Because region B of At_Nemp proteins are not well defined by comparison to vertebrate Nemp1, which is attributed to low amino acid conservation, the entire C-terminal regions downstream of the last TM (named Ct, see S7 Fig C) were used for co-IP experiments. mRNA for GFP-tagged At_Ct constructs were injected with mRNA for Myc-tagged At_Ran2 into Xenopus embryos. B. Co-IP of At_Nemp with Mm_Ran or Mm_Nemp1 with At_Ran2. Xenopus embryos were coinjected with combinations of mRNAs as indicated. Experimental conditions were the same as in Fig 3. Black arrowhead, expected product bands; white arrowheads, cross-reacted bands. After immunoprecipitation against HA, western blotting was performed with anti-Myc or HA antibody as indicated.