Drummond SP, Wilson KL.

R01-GM48646 NIGMS NIH HHS , R01 GM048646 NIGMS NIH HHS , Wellcome Trust

	Figure 1. Partial Xenopus gp210 sequence and purification and testing of gp210 tail polypeptide. (A) Sequence alignment of rat gp210 (residues 1636–1886) and the 251-residue polypeptide encoded by the partial Xenopus cDNA. The transmembrane domain (TMD) and the 16-residue peptide (antigen) used to generate rabbit serum 3860 are underlined. Also indicated is the tail polypeptide of Xenopus gp210 that was recombinantly expressed, purified, and used to inhibit nuclear assembly. (B) SDS-PAGE gel showing the GST–gp210 tail fusion protein before (GST:tail) and after (GST, tail) cleavage by thrombin (see Materials and methods). (C) Effects of purified gp210 tail on nuclear assembly in Xenopus egg extracts. Purified gp210 tail was added to nuclear assembly reactions (final concentrations indicated) at time zero. 2 h later, nuclei were visualized by phase contrast microscopy and Hoescht staining for DNA. Bar, 10 μm.
	Figure 2. Purified gp210 tail polypeptide inhibits nuclear growth and NPC assembly. (A) Time course of nuclear assembly in the presence of 10 μM tail polypeptide. Nuclei were assembled in the presence (10 μM) or absence (buffer control) of purified gp210 tail. Aliquots were removed at the times indicated and imaged by phase contrast microscopy to visualize nuclear membranes, and by fluorescence microscopy to visualize Hoescht-stained DNA. Bar, 10 μm. (B and C) Western blot analysis of tail-inhibited nuclei. Nuclei assembled in the presence of buffer or 10 μM gp210 tail were isolated after 5, 10, 20, 30, 60, and 90 min of assembly, run on SDS-PAGE, blotted, and probed with (B) mAb 414 to identify incorporated FG repeat nucleoporins, and (C) mAb S49F to detect lamin B3. Each blot was stripped and reprobed with antibodies against Xenopus LAP2 to confirm recruitment of nuclear-specific membranes (bottom). The blots in (C) were lastly restripped and probed with antibodies against gp210 (bottom). Lanes marked LSS (low speed supernatant) were loaded with 1 μl of crude Xenopus egg extract.
	Figure 3. Polyclonal anti–tail peptide serum 3860 recognizes a 195-kD membrane protein that colocalizes with NPCs. (A) SDS-PAGE of Xenopus egg cytosol (cyto) and membrane (memb) proteins stained with Coomassie blue (C) or transferred to polyvinylidene difluoride membrane and probed with preimmune serum (P), immune serum (I), or peptide-blocked immune serum (+). (B) Immunofluorescence of Xenopus A6 cells stained with Hoescht to localize DNA, mAb 414 to localize NPCs, and either preimmune (pre) or immune (im) serum 3860 against Xgp210. Bar, 10 μm.
	Figure 4. Antibodies to the gp210 tail inhibit nuclear growth. (A) Nuclei were assembled in the presence of immune or preimmune serum 3860 antibodies against the gp210 tail (see Materials and methods). Aliquots were removed at the times indicated, fixed, and visualized by phase contrast microscopy to show membranes and fluorescence microscopy to show Hoescht-stained DNA. Bar, 10 μm. (B and C) Western blot analysis of antibody-inhibited nuclei. Nuclei assembled in the presence of preimmune or immune serum 3860 were isolated after 5, 10, 20, 30, 60, and 90 min of incubation, run on SDS-PAGE, blotted, and probed with (B) mAb 414 to identify incorporated FG repeat nucleoporins and (C) mAb S49F to detect lamin B3. Each blot was stripped and reprobed with antibodies against Xenopus LAP2 to confirm the recruitment of nuclear-specific membranes (bottom).
	Figure 5. SEM of tail- and antibody-inhibited nuclei coated with a 2.5-nm layer of chromium. Top row shows nuclei at low magnification to demonstrate the growth arrest caused by exogenous gp210 tails and immune antiserum. (Left) Nuclei were assembled for 2 h in the presence of buffer (+ buffer) or 10 μM purified gp210 tail (+ tail). Control nuclei were enclosed by flattened membranes with mature NPCs, best seen at higher magnification in the bottom. Nuclei assembled with 10 μM gp210 tails were enclosed by fused and flattened nuclear membranes that lacked mature NPCs, but did have tiny pores (+ tail, bottom, arrows). (Right) Nuclei were assembled with either preimmune (+ preimmune) or immune (+ immune) antisera against the gp210 tail. Preimmune-treated control nuclei had typical nuclear envelopes and NPCs (+ preimmune, bottom). Nuclei assembled with immune antibodies were enclosed by flattened membranes that lacked any surface-detectable pore-related structures. Scale bars (left) apply to each row.
	Figure 6. TEM of tail- and antibody-inhibited nuclei. Nuclei were assembled for 2 h in the presence of (A) buffer alone or (B, C, and D) 10 μM purified gp210 tail polypeptides. Control nuclei had decondensed chromatin enclosed within a typical flattened nuclear envelope with NPCs (arrows). Nuclear membranes assembled in the presence of 10 μM gp210 tail (B–D) accumulated novel arrested structures (B, arrows) consistent with a late stage of fusion or arrested dilation of nascent pores. C shows an enlarged region of B, which is reproduced in D with dashed white lines tracing the inferred position of each membrane. Dotted white lines indicate ambiguities, including places where the plane of the lipid bilayer was not perpendicular to the TEM section; such “slanted” membranes appear to split into two lines. (E–H) Antibody inhibition. Nuclei were assembled in the presence of (E) preimmune or (F–H) immune serum 3860 for 2 h. Positive control nuclei had typical nuclear envelopes with NPCs (E, arrows). Treatment with immune serum produced arrested electron-dense structures (F, arrows). G and H show an enlargement of two arrested nascent pores, with dashed white lines in H tracing the inferred position of each membrane. (I and J) Tangential section through mature NPC, with membranes traced by dashed white lines in J. Bars: (A, B, E, and F) 200 nm; (C, D, G, H, and J) 10 nm; (I) refer to bar in J.
	Figure 7. Quantitation of pore-related nuclear membrane structures. We (A) identified and (B) quantitated four morphological features termed dimples, twinned membranes, mature NPCs, and arrested structures on TEM micrographs of arrested and control nuclei. Results are graphed in C as the average number of each feature per micron for nuclei arrested by 10 μM gp210 tail (or buffer control), and for nuclei treated with preimmune (preimm) or immune (im) antibodies against the gp210 tail. For each positive control, the number (n) of individual nuclei from which these measurements were taken was 10; for tail polypeptide–inhibited nuclei, n = 14; for antibody-arrested nuclei, n = 15.

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