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First published online December 9, 2005
doi: 10.1242/10.1242/jcs.02701

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LEM2 is a novel MAN1-related inner nuclear membrane protein associated with A-type lamins

¹ Department of Medical Biochemistry, Max F. Perutz Laboratories, Vienna Biocenter, Medical University of Vienna, Dr Bohrgasse 9/3, A-1030 Vienna, Austria
² Department of Molecular Cell Biology, University of Vienna, Max F. Perutz Laboratories, Vienna Biocenter, Dr Bohrgasse 9/3, A-1030 Vienna, Austria

View larger version (50K): [in a new window]   Fig. 1. LEM2 is a novel ubiquitously expressed member of the MAN1 subfamily within the LEM-domain proteins. (A) Comparison of the domain organization of human LEM2 (hLEM2) with orthologues of mouse (mLEM2) and C. elegans (Ce-LEM2), as well as with MAN1 proteins from human (hMAN1), Xenopus laevis (XMAN1), Drosophila (dmCG3167) and Saccharomyces cerevisiae (scSRC1p). Percentage identity of aa residues relative to human LEM2 are indicated within defined domains (intersected by small black bars), including the N-terminus, the lumenal part and the C-terminus. The MAN1-specific region is additionally marked by a black line. LEM/SAP domains are shown in red, transmembrane domains in blue, the MSC domain in green and the MAN1-specific RRM domain in yellow. (B) Cladogram displaying predicted phylogenetic divergence between members of a proposed LEM2-MAN1 family. The domain organization of the proteins is schematically indicated by the coloured boxes, as in (A). (C) Multiple sequence alignment of the LEM domains of hLEM2, MAN1, LAP2ß, emerin and of the LEM-like motif in LAP2ß. Invariant residues are in red, highly conserved in blue. Numbers indicate respective aa positions in the full-length proteins. (D) LEM2 mRNA levels in human and mouse tissues determined by semiquantitative RT-PCR. Actin mRNA levels were used for normalization. Agarose gels of PCR fragments are shown; B. Marrow, bone marrow; Sk. Muscle, skeletal muscle.

View larger version (62K): [in a new window]   Fig. 2. Human LEM2 is a lamina protein of the INM. HeLa cells stably expressing V5-tagged hLEM2 were processed for immunofluorescence using anti-V5 (green) and anti-LAP2 (red) antibodies. DNA was stained with Hoechst (blue). Cells were either extracted with (A) Triton X-100 or (B) digitonin after fixation. Arrows point to mitotic cells, which serve as positive control for antibody reactivity. Bars, 10 µm. (C) Subcellular fractionation of lysates of stable HeLa clones expressing V5-tagged hLEM2. Nuclei were extracted with Triton X-100 and/or salt, or with 7M urea, and soluble supernatant and insoluble pellet fractions were subjected to western blot analysis using anti-V5 and anti-LAP2ß antibodies. Molecular weights in kDa are indicated on the left.

View larger version (96K): [in a new window]   Fig. 3. Dynamics of LEM2 during the cell cycle. (A) HeLa cells stably expressing hLEM2 were processed for indirect immunofluorescence using anti-phospho-histone-H3 antibody (pHistone3; red) and anti-V5 antibody (green). DNA was stained using Hoechst dye (blue). Representative images of defined cell-cycle stages are shown. (B) Cells at anaphase or different telophase stages, were stained for exogenous LEM2 with anti-V5 antibody (green) and for either LAP2, LBR or LAP2ß (red). DNA was stained with Hoechst (blue). Bars, 10 µm in (A) and 5 µm in (B).

View larger version (65K): [in a new window]   Fig. 4. Localization of LEM2 at the NE depends on the presence of A-type lamins. (A-C) Mouse embryonic fibroblasts (MEFs) from wild-type (A) or lamin A/C-knockout (B,C) mice were transfected with V5-tagged hLEM2 plasmids. In (C), cells expressing V5-tagged LEM2 were co-transfected with GFP-tagged pre-lamin A (GFP-LaminA) plasmid and were stained for LEM2 using anti-V5 antibody (A-D, red) and for DNA with Hoechst dye (C-E, blue). Ectopically expressed lamin A was detected by GFP fluorescence (C, green). (D,E) HeLa cells stably expressing V5-tagged hLEM2 were transfected with a construct encoding a GFP-tagged version of a dominant-negative Xenopus Lamin B1 (GFP-xLaminB12+; green) and stained either for LEM2 using anti-V5 antibody (D, red) or endogenous lamin A (E, red). Arrows depict mislocalization of LEM2 to the ER in cells expressing the dominant-negative lamin B mutant. Note, that V5-tagged LEM2 decorates only the nuclear rim in untransfected cells. Bars, 10 µm. (F) Recombinant GST alone (lanes 5), LAP2 (lanes 4) and GST-lamin C head domain (lanes 1), rod domain (lanes 2) and tail domain (lanes 3) were transblotted to nitrocellulose membranes. PonceauS staining detects recombinant proteins (asterisks). Membranes were probed with 35S-labelled full-length LEM2 (LEM2-FL), the N-terminus of LEM2 (LEM2-NT) and LAP2, and bound proteins were detected by autoradiography.

View larger version (50K): [in a new window]   Fig. 5. The N-terminus of LEM2 is essential and sufficient for lamin A-mediated nuclear targeting. Confocal immunofluorescence images of HeLa cells or Lmna–/– MEFs transiently expressing different V5-tagged truncation mutants of hLEM2, as indicated in the schematic drawing below each image. The LEM domain is shown in red, transmembrane domains in blue, and MSC domain in green. Numbers depict aa that border each deletion. LEM2 polypeptides were detected by immunofluorescence using anti-V5 antibody (red). DNA was stained with Hoechst (blue). Arrows in the lower right image mark cytoplasmic staining of LEM2-NT in Lmna–/– MEFs. Bars, 10 µm.

View larger version (70K): [in a new window]   Fig. 6. Overexpressed LEM2 accumulates in peripheral patches. (A) HeLa or C2C12 cells expressing high levels of LEM2-V5 were processed for indirect immunofluorescence microscopy using anti-V5 antibody (red) and DNA was stained with Hoechst dye (blue). Bars, 10 µm. (B) Confocal images of cells in xy and xz optical sections. White line indicates location of xz section. Bars, 2 µm. (C) Transmission electron microscopic images of thin sections of embedded wild-type HeLa cells (left) or hLEM2-overexpressing HeLa cells (right). Bars, 5 µm.

View larger version (113K): [in a new window]   Fig. 7. A subset of NE proteins is recruited to LEM2 patches. HeLa cells constitutively expressing high levels of V5-tagged hLEM2 were processed for immunofluorescence microscopy using anti-V5 antibody (green) and antibodies to different NE proteins (red). Bottom row shows images of HeLa cells stably expressing V5-tagged MAN1 (red) and co-transfected with human LEM2-GFP (green)-encoding plasmid. Images on the right show untransfected HeLa cells stained for the NE proteins indicated (control; red) or cells expressing V5-tagged MAN1 alone (bottom row). Arrowheads in left image (row 4) depicts lamin A patches. Bars, 10 µm.

View larger version (109K): [in a new window]   Fig. 8. LEM2-induced tubules are stable connections, persisting throughout several cell cycles. HeLa cells stably overexpressing V5-tagged hLEM2 were processed for immunofluorescence microscopy using antibodies to indicated proteins. Images depict LEM2 stained with anti-V5 (green) and actin (A, red) or phospho-histone H3 (C, red) or BAF (F, red), and DNA (A-E, blue). Arrows in (C) indicate cells shown at higher magnification in (E). Arrows in (E) depict LEM2 patches from which tubular structures emerge. Single colour images corresponding to the merged images in A and B are shown. Bars, 10 µm.

View larger version (95K): [in a new window]   Fig. 9. LEM2-containing tubular structures between adjacent nuclei also contain lamin A/C-associated proteins. HeLa cells stably overexpressing V5-tagged hLEM2 were processed for immunofluorescence microscopy using antibodies to indicated proteins. Cells were stained for LEM2-V5 (green), DNA (blue), and either lamin A, emerin, BAF, NUP62, lamin B1, LBR, -tubulin or -calnexin (red). Bars, 10 µm.

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