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First published online 16 November 2004
doi: 10.1242/jcs.01532

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The lamin CxxM motif promotes nuclear membrane growth

Division of Electron Microscopy, Biocenter of the University of Würzburg, Am Hubland, 97074 Würzburg, Germany

View larger version (24K): [in a new window]   Fig. 1. Schematic of GFP-lamin fusion proteins. These are wild-type human lamin A, Xenopus lamin B2, Drosophila lamins Dm0 and C, mutants derived from these lamins and an N-terminal deletion mutant of zebrafish lamin B2 (zB2-N3). The position of the GFP, the helical region (1A, 1A, 2), the nuclear localization signal (NLS) and the CxxM-motif are indicated. Three N-terminal deletion mutants (B2-N1, B2-N2, B2-N3) and a point mutation (B2-SxxM) where the cysteine in the CxxM-motif had been replaced by a serine have been generated from the Xenopus lamin B2. C-CxxM is a Drosophila lamin C mutant containing the CxxM motif of lamin Dm0 and C-CxxM-N1, a Drosophila lamin C mutant with the CxxM motif lacking the N-terminal 228 amino acids. The numbers of amino acids (AA) of each lamin contained in the GFP fusion protein are listed.

View larger version (22K): [in a new window]   Fig. 2. Characterization of GFP-lamin fusion proteins. Total protein of COS-7 cells (lanes 1-12), Xenopus A6 cells (lanes 13, 14) and zebrafish embryos (lane 15) expressing the GFP-lamin fusion proteins listed in Fig. 1 were separated by SDS-PAGE and immunoblotted with GFP antibodies. COS-7 and A6 cells were transfected and zebrafish embryos were microinjected with in vitro synthesized lamin mRNA at the 2-4 cell stage. Embryos were analyzed 24 hours post fertilization. Lane 1, non transfected cells; lane 2, human lamin A (hA); lane 3, Drosophila lamin Dm0 (dDm0); lane 4, Drosophila lamin C (dC); lanes 5 and 11, Drosophila lamin C-CxxM (dCCxxM); lane 6, Xenopus lamin B2 (xB2); lane 7, Xenopus lamin B2-N1 (xB2-N1); lane 8, Xenopus lamin B2-N2 (xB2-N2); lane 9, Xenopus lamin B2 mutant B2-SxxM (xB2SxxM); lane 12, Drosophila lamin CCxxM-N1 (dCCxxM-N1); lane 13, Xenopus lamin B2-N3 (xB2-N3); lanes 14 and 15, zebrafish lamin B2-N3 (zB2-N3). Molecular masses of reference proteins (lanes M) are marked and are from top to bottom: 116, 97, 66 and 45 kDa.

View larger version (45K): [in a new window]   Fig. 3. Light microscopy of Xenopus A6 cells overexpressing GFP fusion proteins of wild-type lamins. (A-E') Cells overexpressing wild-type Xenopus lamin B2 (A,A') human lamin A (B,B'), Drosophila lamin Dm0 (C,C',E,E') and Drosophila lamin C (D,D') were examined for GFP fluorescence (left panels) and lamin immunofluorescence (right panels). Lamin antibodies specific for lamins A (antibody X94; A',C'), B2 (antibody X223; B',D') and Drosophila lamin Dm0 (mixture of antibodies ADL195 and ADL84; E') were used. Digital images taken by confocal laser-scanning microscopy are shown. Bars, 10 µm.

View larger version (161K): [in a new window]   Fig. 4. Electron microscopy of ultrathin sections through microinjected Xenopus A6 cells. (A-E) A6 cells overexpressing Xenopus lamin B2 (A,D), human lamin A (B), Drosophila lamin Dm0 (C) and lamin C (E). Note the highly lobulated nuclear envelopes (A-C), small (B,C, arrows) and large (D, arrow and inset) intranuclear membrane assemblies. (D, inset) A dot-like membrane assembly is shown at higher magnification. (E) Drosophila lamin C forms membrane-free electron-dense aggregates at the nuclear envelope (arrow) and in the nucleoplasm (arrowhead) that have a banded substructure when sectioned parallel to the longitudinal axis of the lamin aggregates (inset). cy, cytoplasm; no, nucleolus; nu, nucleoplasm. Bars 1 µm (A-E); 500 nm (insets in D,E).

View larger version (182K): [in a new window]   Fig. 5. Intranuclear membranes are formed in cells overexpressing wild-type lamins. (A-I) High magnification electron micrographs of ultrathin sections through microinjected Xenopus A6 cells expressing Xenopus lamin B2 (A,F,H), human lamin A (C-E,I) and Drosophila lamin Dm0 (B,G). Intranuclear membranes form flat cisternae that are separated from the inner nuclear membrane by a 15-25 nm-thick layer similar in electron density to the lamina (B, arrow). Examples where only a few locally restricted intranuclear membranes were found are shown in A and B, and a nuclear envelope nearly completely bordered by an intranuclear membrane cisterna in C. (D) Nuclear envelope bordered in neighboring areas by one (arrowhead 1), two (arrowhead 2) and three (arrowhead 3) membrane cisternae. (E) Part of a highly lobulated nucleus showing an area where the nucleoplasm is nearly excluded between the multi-membrane-layered nuclear membranes. (F-H) Lobulated intranuclear membrane cisternae of increasing complexity. (I) Irregular vesicle-like intranuclear membranes. Nuclear pore complexes are indicated by arrowheads in A-C, I. cy, cytoplasm; ne, areas of the nuclear envelope with an unaltered morphology; nu, nucleoplasm. Bars, 500 nm.

View larger version (110K): [in a new window]   Fig. 6. The influence of the CxxM motif on the formation of intranuclear membranes. (A-D) Electron micrographs of ultrathin sections through microinjected Xenopus A6 cells expressing Xenopus lamin B2 mutant B2-SxxM (A,B) and Drosophila lamin mutant C-CxxM (C,D). (A,B) Nuclei of cells expressing mutant lamin B2-SxxM contain proteinaceous aggregates that are associated with the nuclear envelope (asterisks) but no intranuclear membranes. (C,D) Protrusions in the nuclear envelope (arrows) are seen in cells expressing the lamin C-CxxM mutant. The protrusions are filled with protein aggregates similar in electron density to those formed by Drosophila lamin C (see Fig. 4E). A pore complex is indicated with an arrowhead in B. cy, cytoplasm; no, nucleolus. Bars, 1 µm (A,C); 500 nm (B,D).

View larger version (116K): [in a new window]   Fig. 7. Intranuclear membranes formed by lamin mutants containing a CxxM motif and N-terminal deletions. (A-C) Electron micrographs of ultrathin sections through microinjected (A,C) or transfected (B) Xenopus A6 cells expressing Drosophila lamin C-CxxM-N1 (A), Xenopus lamin B2-N1 (B) and Xenopus Lamin B2-N2 (C). Lamin mutants containing the complete helix 2 (A) or the C-terminal half of helix 2 (B) can induce the formation of multi-layered nuclear envelopes. (C) Mutant B2-N2 induced the formation of intranuclear spherical assemblies of multiple membrane cisternae that were in contact with the nuclear envelope. Note that the layers between adjacent cisternae (A,B) are less electron dense than in cells expressing wild-type lamins. cy, cytoplasm; ne, nuclear envelope; nu, nucleoplasm. Bars, 500 nm.

View larger version (101K): [in a new window]   Fig. 8. Expression of GFP-lamin fusion proteins in COS-7 cells and zebrafish embryos. (A-E) Electron micrographs of ultrathin sections through transfected (A) and microinjected COS-7 cells (D,E) and zebrafish embryos at 9 (B) and 36 (C) hours post fertilization. Cells express Xenopus lamin B2-N3 (A) and human lamin A (D,E). Zebrafish embryos were microinjected at the 2-4 cell stage with mRNA encoding zebrafish lamin B2 mutant zB2-N3 (B,C). Multi-layered nuclear membranes (A,C) and nuclear lobulations (B, arrow) were seen in cells overexpressing this lamin B2 mutant. The arrowhead in B indicates an unaltered area of the nuclear envelope containing pore complexes. (D,E) COS-7 cells expressing human lamin A possess an electron-dense layer of irregular thickness underneath the inner nuclear membrane (D) or spherical aggregates attached to the inner nuclear membrane (E). Intranuclear membranes are attached to some spherical aggregates (E, arrow). cy, cytoplasm; nu, nucleoplasm. Bars, 500 nm (A,C-E); 1 µm (B).

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