|
FIGURE 1:. Nup93 is essential for NPC formation. (A) Western blot analysis of untreated, mock, Nup93-depleted (Î93) and Nup93-depleted extracts with full-length recombinant Nup93 (addback), respectively. The Nup93 antibody recognizes a slightly slower migrating cross-reactivity by Western blotting (asterisk), which is neither immunoprecipitated nor depleted. The recombinant Nup93 migrates slightly more slowly than the endogenous protein probably due to absence of eukaryotic posttranslational modifications. (B) Nuclei were assembled in mock, Nup93-depleted extracts (Î93) or Nup93-depleted extracts supplemented with full-length recombinant Nup93 (addback) for 90 min, respectively, fixed with 2% paraformaldehyde (PFA) and 0.5% glutaraldehyde, and analyzed for chromatin and membrane staining (blue, 4â²,6-diamidino-2-phenylindole [DAPI]; red, DiIC18; bar, 10 μm). (C) Quantitation of chromatin substrates with a closed nuclear envelope of reactions done as in B. More than 100 randomly chosen chromatin substrates were counted per reaction. The average of three independent experiments are shown; error bars represent the total variation. (D) Transmission electron micrography of a nucleus assembled in mock, Nup93-depleted extracts or depleted extracts supplemented with full-length Nup93 as in B. Bar is 2 μm.
|
|
FIGURE 2:. Nup188 and Nup205 together are not essential for NPC formation. Nuclei were assembled in mock, Nup93-depleted extracts (Î93) or Nup93-depleted extracts supplemented with full-length Nup93 (addback) for 90 min, fixed with 4% PFA, and analyzed with Nup93-, Nup188-, or Nup205-specific antibodies, respectively (green), and the monoclonal antibody mAb414, which recognizes FG repeat nucleoporins (red). Chromatin is stained with DAPI; scale bar, 10 μm.
|
|
FIGURE 3:. Nuclei lacking Nup188 and Nup205 have a normal NPC composition. Nuclei were assembled in mock, Nup93-depleted extracts (Î93) or Nup93-depleted extracts supplemented with full-length Nup93 (addback) for 90 min, fixed with 4% PFA, and stained with the respective antibodies. Scale bar, 10 μm.
|
|
FIGURE 4:. The C-terminal Nup93 fragment supports formation of a closed nuclear envelope. (A) Schematic representation of the domain structure of Xenopus Nup93 and the fragments used. The N-terminal coiled-coil region is marked in red, the α-helical region in blue. Numbers indicate the amino acids of the respective constructs. (B) Nuclei were assembled in Nup93-depleted extracts supplemented as indicated either with full-length recombinant Nup93 (1â820) or the respective fragments for 90 min, fixed with 2% PFA and 0.5% glutaraldehyde, and analyzed for chromatin and membrane staining (blue, DAPI; red, DiIC18; bar, 20 μm). (C) Quantitation of chromatin substrates with a closed nuclear envelope of reactions done as in B. More than 100 randomly chosen chromatin substrates were counted per reaction. The average of three independent experiments is shown; error bars represent the total variation.
|
|
FIGURE 5:. The C-terminal Nup93 fragment supports formation of the structural part of the NPC but not incorporation of the Nup62 complex. (A) Transmission electron micrographs of nuclei assembled in Nup93-depleted extracts supplemented with the C-terminal Nup93 fragment containing aa 608â820. Note the presence of a closed nuclear envelope. Bar, 2 μm. (B) Nuclei were assembled in mock, Nup93-depleted extracts (Î93) or Nup93-depleted extracts supplemented with either full-length recombinant Nup93 (1â820) or the C-terminal fragment (608â820) for 90 min, fixed with 4% PFA, and analyzed with the antibody mAB414 (top), an antibody against Nup62 (middle), or one against Nup58 (bottom). Overlays with DAPI staining (blue) are shown. Scale bar, 10 μm. (C) Nuclei were assembled in Nup93-depleted extracts supplemented with the C-terminal fragment (aa 608â820) for 90 min, fixed with 4% PFA, and stained with the respective antibodies. Scale bar, 10 μm.
|
|
FIGURE 6:. The N-terminal coiled-coil region of Nup93 is required to assemble import-competent nuclei. (A) Nuclei were assembled in mock or Nup93-depleted extracts supplemented with the either full-length Nup93 (1â820), a fragment lacking the N-terminal coiled-coil region (183â820), or the C-terminal fragment (608â820). After 50 min an enhanced green fluorescent proteinâfused nuclear import substrate was added. After 120 min nuclei were isolated and analyzed by confocal microscopy. Membranes are stained with DiIC18 (red). Bars, 20 μm. (B) Quantitation of import reactions performed as in A. More than 100 randomly chosen chromatin substrates were counted per reaction. The average of three independent experiments is shown; error bars represent the total variation.
|
|
FIGURE 7:. The N-terminal-coiled coil region of Nup93 is required to assemble exclusion-competent nuclei. (a) Nuclei were assembled in mock or Nup93-depleted extracts supplemented with the either full-length Nup93 (1â820) or the C-terminal fragment (608â820). After 120 min fluorescein-labeled, 70-kDa dextran was added. DNA was stained with DAPI to identify nuclei. Samples were analyzed by confocal microscopy. Representative images of the dextran staining are shown. Scale bar, 10 μm. (B) Quantitation of the size exclusion assays with 70-kDa dextran performed as in A. For each condition more than 40 nuclei from at least two independent experiments were analyzed.
|
|
FIGURE 8:. The C-terminus of Nup93 stabilizes the Nup53âNup155 interaction. (A) GST fusions of the nucleoplasmic domain of gp210 (control) or Nup53 were incubated with cytosol from Xenopus egg extracts alone or, where indicated, in the presence of SUMO fusions of the Nup93 middle fragment (183â582) or Nup93 C-terminal fragment (608â820), respectively. Eluates were analyzed by Western blotting with antibodies against SUMO to detect the Nup93 fragments or antibodies against Nup155, Nup153, and Nup98 to detect the respective protein. In the left three lanes 5% of the input of the Nup93 fragments and 2% of the cytosol were loaded. (B) Purified recombinant GST or GST-Nup53 fusion protein was incubated with lysates from bacteria expressing recombinant Xenopus Nup155 and supplemented with the C-terminal fragment Nup93 (608â820) where indicated. Eluates and 2% of the input were analyzed by Western blotting using antibodies against the His6 tag. Because of the removal of the His6 tag from the Nup93 fragment during TEV protease elution, it is not possible to detect this protein in the eluate. (C) Model for Nup93 function in postmitotic NPC assembly. The C-terminal region of Nup93 stabilizes the Nup155âNup53 interaction, allowing the assembly of the structural part of the NPC. This function of Nup93 can be substituted by a fragment comprising amino acids 608â820 (right). The N-terminal coiled-coil region of Nup93 recruits the Nup62 complex to the structural part of the NPC. This requires in the assembly reaction full-length Nup93 and leads to the association of the central channel and formation of functional NPCs (left). Note that for the sake of simplicity, transmembrane nucleoporins and the Nup107â160 complex are not shown, although our data indicate that they are present in both assembly lines.
|
