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The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the nuclear envelope. Association of lamins with the inner nuclear membrane is mediated by specific modifications in the CaaX motif at their C-termini. B-type lamins are permanently isoprenylated whereas lamin A loses its modification by a lamin A-specific processing step after incorporation into the lamina. Lamins are differentially expressed during development and tissue differentiation. Here we show that an increased synthesis of lamins B1 and B2 in amphibian oocytes induces the formation of intranuclear membrane structures that form extensive arrays of stacked cisternae. These ''lamin membrane arrays'' are attached to the inner nuclear membrane but are not continuous with it. Induction of this membrane proliferation depends on CaaX-specific posttranslational modification. Moreover, in transfected HeLa cells, chimeric GFP containing a nuclear localization signal and a C-terminal CaaX motif of N-Ras induces intranuclear membrane stacks that resemble those induced by lamins and ER-like cisternae that are induced in the cytoplasm upon increased synthesis of integral ER membrane proteins. Implications for the synthesis of CaaX-containing proteins are discussed and the difference from intranuclear fibrous lamina annulate lamellae formations is emphasized.
Fig.1. Lamins synthesized in oocytes associate with the nuclear envelope. (A-E') Lamins were expressed in Xenopus oocytes by RNA injection. 16 hours after injection nuclei were manually isolated and either processed directly (GV, lane 1) or separated into nuclear content (NC, lane 2) and nuclear envelope (NE, lane 3). Fractions were separated by SDS-PAGE and lamins were detected by immunoblotting using chemiluminescence. Material from three nuclei was loaded in each lane. Lamin B1 was detected with mAb L7-4A2 (A), lamin B2 with mAb L7-8C6 (B,E), lamin Flag-LIII and Flag-A with mAb M2 (C and D, respectively). Control fractions of uninjected oocytes (n.i.) were processed in parallel (lanes 4-6). All blots were reprobed with lamin LIII-specific mAbs, mAb L6-5D5 (A'-D') or mAb NUC195 (E'). Note that blots were not stripped before reprobing, therefore, residual chemiluminescence signal from the first immunoreaction was still detectable, as seen in A'-E'.
Fig.2. Indirect immunofluorescence analysis of oocytes synthesizing nuclear lamins. Cryostat sections (A-E) and nuclear envelope spread preparations (F-L) are shown. The type of RNA injected is indicated at the margins of each panel. n.i., non-injected control oocytes. Oocyte lamin LIII was detected with mAb NUC195 (A) or mAb L6-5D5 (F), Flag-A, Flag-B1 and Flag-LIII with mAb M2 (B-E,H,K), lamin B1 and non-injected control envelopes were reacted with mAb L7-4A2 (I,L); myc-LIII with mAb 9E10 (G); lamin B2 with mAb L7-8C6 (J). Cy3-conjugated goat anti-mouse IgG was used as a secondary antibody. Brighter staining along folds of the nuclear envelope in F-H, K is due to folding of the nuclear envelope during the spreading procedure. Bars, 20 μm.
Fig.3. Electron microscopy of lamin-induced intranuclear membrane structures. (A-E) EM sections of isolated oocyte nuclei are shown. Oocytes were injected with RNA encoding lamin B2 (A-D) or lamin B2-SaaX (E). Nuclei were isolated and processed for electron microscopy. Sections were stained with uranyl acetate and lead citrate (A-C,E) or processed for pre-embedding immunoelectron microscopy (D). Lamin B2 was detected with mAb L7-8C6 and nanogold-coated secondary antibody. Arrows in A indicate membrane arrays attached to the nuclear envelope. Arrows in D indicate gold particles decorating the outer membrane cisternae of a lamin B2-induced membrane array. C, cytoplasm; NC, nucleoplasm; NE, nuclear envelope; NPC, nuclear pore complex. Bar, 5 μm (A); 1 μm (B,E); and 0.5 μm (C,D).
Fig.4. Nuclear GFP containing the membrane targeting motifs of N-Ras induces intranuclear vesicular structures. HeLa cells transiently transfected with NLS-MT-GFP-N-Ras (A-C) or NLS-MT-GFP (D) chimeras were fixed 24 hours after transfection and examined by fluorescence confocal laser scanning microscopy. In A an overlay of the GFP fluorescence and differential interference contrast picture is shown. Note the presence of brightly fluorescing vesicular structures inside nuclei of cells expressing high levels of the chimeric GFP in C. Bar, 10 μm (A); 20 μm (B-D).
Fig.5. NLS-MT-GFP-N-Ras chimeras induce formation of intranuclear membrane arrays in HeLa cells. (A-D) HeLa cells transiently transfected with NLS-MT-GFP-N-Ras were processed for EM 24 hours after transfection. Sections were stained with uranyl acetate and lead citrate. Note the formation of stacked membrane cisternae aligned with the nuclear envelope as well as the formation of membrane arrays within the nucleoplasm. NE, nuclear envelope; PM, plasma membranes. Bar, 5 μm (A); 1 μm (B-D).