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Fig. 1. Experimental uncoupling of nuclear membrane formation and NPC assembly by using two different membrane fractions in the nuclear reconstitution assay. (A) Isolation of light and heavy membranes from mitotic egg extract. (B) Incubation of sperm chromatin in cytosol supplemented with ATP-regenerating system together with either the light (left panel) or heavy (middle panel) membrane fraction. The light membranes do not bind to chromatin in contrast to the heavy membranes, which form a closed double nuclear membrane around the chromatin without recognizable NPC, as shown by thin section EM (upper row). Consistent with the EM observations, fluorescence microscopy (bottom row) reveals the absence of DiOC18 pre-labeled light membranes (green) from chromatin, but shows association of the DilC18 pre-labeled heavy membranes (red) with the chromatin surface in a rim-like pattern, indicative of membrane fusion. DNA is counterstained with Hoechst 33258 (blue). When pore-free nuclei were first allowed to assemble by incubating chromatin with cytosol and heavy membranes for 1 hour, followed by addition of light membranes, NPC were recognized by EM (right panel, NPCs are indicated by arrows in the insert). Under these conditions, the differentially labeled membrane fractions both bind to the chromatin surface (bottom row). (C) Incubation of chromatin as outlined above (indicated on the left-hand side of the figure), followed by immunofluorescent detection of NPCs with antibodies against Nup62 and nuclear import with antibodies against fibrillarin. In the presence of functional NPCs, the nucleolar protein fibrillarin is imported into the reconstituted nuclei where it forms distinct nuclear bodies (Ewald et al., 1997 ). Corresponding phase contrast images and Hoechst fluorescence are also shown. Functional NPC are recognized only after sequential addition of the two membrane fractions (bottom row). Scale bars of the EM micrographs (upper part of B), 1 µm and 0.1 µm (inserts), all other bars 10 µm.
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104.000) is slightly higher than its calculated Mr (95.670). (B) Proteins of a vault-enriched fraction from Xenopus eggs were separated by SDS-PAGE and visualized by silver staining (lane 1). A sample run in parallel was blotted and incubated with anti-xMVP (lane 2) or anti-human MVP antibodies (lane 3). MVP is present as a distinct band in the vault fraction. (C) Proteins of the indicated egg fractions were separated by SDS-PAGE and stained with Coomassie blue (left). The MVP band is indicated by the arrowhead in lane 5. A gel run in parallel was probed with antibodies against xMVP (right). MVP is present in egg extract (lane 1), the washed membrane fraction (P200, lane 2) and the light membranes (lane 5), but is absent from the cytosol (S200, lane 3) and the heavy membranes (lane 4). Molecular mass standards are indicated in kDa.
