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-LAP2α (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-LAP2β antibodies. Molecular weights in kDa are indicated on the left.

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 LAP2α, LBR or LAP2β (red). DNA was stained with Hoechst (blue). Bars, 10 μm in (A) and 5 μm in (B).

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-xLaminB1Δ2+; 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), LAP2α (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 LAP2α, and bound proteins were detected by autoradiography.

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.

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.

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.

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.