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First published online 30 March 2004
doi: 10.1242/jcs.01052

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The lamin B receptor of Drosophila melanogaster

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

View larger version (48K): [in a new window]   Fig. 1. Amino acid sequence and scheme of the D. melanogaster lamin B receptor (dLBR) encoded by gene CG17952. (A) Amino acid sequence comparison of the lamin B receptor of human (hLBR), Xenopus laevis (XLBR) and dLBR. The membrane-spanning domains are underlined. The sequence used for the generation of polyclonal antibodies against the Drosophila protein is marked by a dotted line. Amino acids identical in all three species are printed in bold and marked by asterisks. Amino acids that are identical in the two vertebrates are also printed in bold. (B) Schematic drawings of the dLBR, hLBR and XLBR. The positions of individual amino acids are marked by numbers. Boxes depict transmembrane segments (black) and two globular domains (G1, G2; gray) in the N-terminal half of the human and Xenopus LBR; these globular domains are not predicted for the dLBR sequence. The nucleotide sequence of the D. melanogaster gene CG17952 (dLBR) is available under accession number CG17952 of the Berkeley Drosophila Genome Project (BDGP) Database. The corrected sequence of the dLBR shown here is available under accession number AJ606680.

View larger version (24K): [in a new window]   Fig. 2. Characterization of the dLBR protein. (A-C) Total larval proteins of Drosophila melanogaster second (A, lane 1) and third (A, lane 2; B, lane 1) instar, and pupa (B, lane 2) were separated by SDS-PAGE and immunoblotted with affinity-purified dLBR antibodies (A) or non-purified dLBR antibodies (B,C). In each lane, total proteins of one animal were loaded. (C) Aliquots of S2 cells were incubated with 8 M urea and fractionated by 100,000 g centrifugation into a supernatant (S) and a pellet containing predominantly membranes (P). Proportional amounts of proteins of both fractions were separated by SDS-PAGE and immunoblotted with dLBR antibodies. A polypeptide of Mr 66,000 is detected by the dLBR antibodies (A-C, arrowheads). dLBR does form aggregates in SDS sample buffer resulting in a smear close to the top of the gel (A-C; Fig. 7A, Fig. 8A). Molecular masses of reference proteins (in kDa) are marked.

View larger version (34K): [in a new window]   Fig. 3. The dLBR is localized to the inner nuclear membrane. Indirect immunofluorescence microscopy (A-C; A'-C') of S2 cells after staining with antibodies against the dLBR (A,A'), lamin Dm0 (B,B') and -tubulin (C,C'). Cells were fixed with formaldehyde and then permeabilized with Triton X-100 (A-C) or digitonin (A'-C'). The inner nuclear membrane and nuclear interior are only accessible to antibodies in Triton-treated cells. Scale bars, 10 µm.

View larger version (58K): [in a new window]   Fig. 4. The dLBR is targeted to the nuclear membrane of vertebrate and insect cells. (A) Expression of amino acids 16-334 of the dLBR as a GFP fusion protein (dLBR-GFP) in Xenopus A6 cells (A,B), COS-7 cells (C) and Drosophila S2 cells (D). This fusion protein comprises the N-terminal domain and the first membrane-spanning segment. The fluorescence of the GFP fusion protein in the transfected cells is shown (A-D) with the staining of endogenous lamin B2 by indirect immunofluorescence microscopy with antibody X223 (A',B') or a phase-contrast image (C',D',A''-D''; merge – overlays). Digital images taken by CLSM are shown. Scale bars, 10 µm. (E). Biochemical properties of the fusion protein dLBR-GFP in COS-7 cells. Aliquots of transfected COS-7 cells were incubated with buffered 8 M urea and fractionated by 100,000 g centrifugation into a supernatant (S) and a pellet fraction (P). Proteins were separated by SDS-PAGE and immunoblotted with antibodies against GFP (lanes 1, 2) or lamin B2 (lanes 3, 4). The position of dLBR-GFP is marked by an arrow and lamin B2 is marked by an arrowhead. The two polypeptide bands with higher mobility than the dLBR-GFP that were reacting with the GFP antibodies (lane 1) represent degradation products. Molecular masses of reference proteins (in kDa) are marked.

View larger version (48K): [in a new window]   Fig. 5. (A) Co-immunoprecipitation of the dLBR and lamins from the 13,000 g supernatant of Drosophila Kc167-cells extracted with immunoprecipitation buffer. Immunoprecipitations were performed with polyclonal guinea pig (gp-dLBR; lanes 1, 3) and mouse (m-dLBR; lane 2) antibodies against dLBR that were bound to protein-A/Sepharose. As a control (control; lane 4), proteins of the extract bound to the protein-A/Sepharose in the absence of antibodies were analyzed. Proteins of immunoprecipitates were separated by SDS-PAGE and immunoblotted with mouse monoclonal antibodies against lamin Dm0 (lanes 1, 4; Blot lamin Dm0) and lamin C (lane 3; Blot lamin C), and with guinea pig antibodies against lamin Dm0 (lane 2; Blot lamin Dm0). The position of lamin Dm0 is marked by an arrow (Dm0) and the heavy chains (HC) of the antibodies by an arrowhead. (B) Co-immunoprecipitation of [35S]-methionine-labeled dLBR (amino acids 17-262) and lamin Dm0 from reticulocyte lysates with guinea pig antibodies against dLBR (gp-dLBR) that were bound to protein-A/Sepharose. Both proteins had been translated in the reticulocyte lysate [lane 1; lamin Dm0/dLBR (17-262)]. Total proteins of the reticulocyte lysate (lane 1), proteins remaining in the supernatant after immunoprecipitation (lane 2, supernatant) and immunoprecipitated proteins (lane 3; IP, gp-dLBR) were separated by SDS-PAGE and visualized by fluorography. The positions of the dLBR (arrow) and lamin Dm0 (arrowhead) are marked. (C,D) In vitro binding of [35S]-methionine-labeled lamin Dm0 to the immobilized N-terminal domain of the dLBR (C; amino acids 17-262 of the dLBR) and of [35S]-methionine-labeled dLBR to the immobilized lamin Dm0 (D). Wells of ELISA plates that had been coated with the dLBR (C; lanes 1-3; coating, dLBR), lamin Dm0 (D, lanes 1-3) or BSA (lanes 4 in C,D; coating, BSA) were incubated with [35S]-methionine-labeled lamin Dm0 (C; lanes 1, 2, 4; Inc. Dm0) or with [35S]-methionine-labeled dLBR (D; lanes 1, 2, 4; Inc. dLBR). As controls [35S]-methionine-labeled lamin Dm0 was preincubated with the dLBR in solution (C; lane 3; Inc. Dm0 + dLBR) or [35S]-methionine-labeled dLBR was preincubated with lamin Dm0 in solution (D; lane 3; Inc. dLBR + Dm0) and then added to the wells. Proteins bound to the wells were separated by SDS-PAGE. X-ray films of both gels are shown. Quantification of the bound radioactively labeled proteins are shown in Tables 1 and 2. Molecular masses of reference proteins (in kDa) are marked in A-D.

View larger version (68K): [in a new window]   Fig. 6. In vitro binding of the bacterially expressed N-terminal domain of Drosophila LBR (amino acids 17-262 of the dLBR) and Xenopus LBR (amino acids 4-210 of the XLBR) to sperm chromatin. Soluble proteins of heat-treated Xenopus egg extract (S200) supplemented with dLBR (A) or XLBR (B) were incubated in the presence (+Sp) (lanes 2, 4) or absence (–Sp) (lanes 1, 3) of demembranated sperm chromatin, then fractionated into supernatants (S) and pellets (P) by centrifugation. Proteins of each fraction and of sperm chromatin that had not been incubated (SP; lane 5) were separated by SDS-PAGE and analyzed by immunoblotting with antibodies against the dLBR (A) and XLBR (B). The distinct polypeptide bands (A, lane 4) labeled by the dLBR antibodies in the relative molecular weight range Mr 60,000 to Mr 200,000 represent oligomeric complexes of the dLBR that had been formed at the sperm chromatin during incubation. The XLBR also forms some oligomeric complexes in the presence of sperm chromatin. Arrows (A,B) mark non-aggregated LBR. Molecular masses of reference proteins (in kDa) are marked.

View larger version (37K): [in a new window]   Fig. 7. Expression of the dLBR in the S. cerevisiae erg24 mutant and analysis of synthesized sterols. (A) erg24 mutant cells were transformed with plasmids containing the coding region of dLBR (lane 1), the wild-type ERG24 gene (lane 2) or the plasmid without gene (lane 3). Total proteins of these yeast strains were separated by SDS-PAGE and immunoblotted with dLBR antibodies. The dLBR expressed in yeast forms aggregates in SDS sample buffer; the position of unaggregated dLBR is marked by an arrow. The dLBR antibodies cross-react in addition with a low molecular weight yeast protein present in all three strains. Molecular masses of reference proteins (in kDa) are marked. (B-G) Mass spectrometric analysis of sterols synthesized in S. cerevisiae erg24 mutant cells that had been transformed with a plasmid containing cDNAs of the following genes: S. cerevisiae ERG24 (C); Arabidopsis thaliana FACKEL (D); a plasmid without gene (F, control); and the Drosophila LBR (G, dLBR). Sterols were extracted from cells that had been grown for 24 hours in YPAD medium. The mass spectra of ergosterol (B) and ignosterol (E) are shown as standards.

View larger version (66K): [in a new window]   Fig. 8. Downregulation of the Drosophila proteins dLBR and lamin Dm0 in cultured Drosophila cells by RNAi (A-G). Drosophila S2 (A-E) and Kc167 cells (F,G) were transfected with dsRNA specific for the dLBR gene (A,C) and the lamin Dm0 gene (B,D-G), and analyzed by immunoblotting (A,B) and immunofluorescence microscopy (C-G). (A) RNAi of the dLBR. Total proteins of identical numbers of untreated control S2 cells (lanes 1, 2; control) and of S2 cells 3 days after transfection with dLBR dsRNA (lanes 3,4; RNAi) were separated by SDS-PAGE and immunoblotted with antibodies against the dLBR (A), lamin Dm0 (A') and -tubulin (A''). (B) RNAi of lamin Dm0. Total proteins of identical numbers of untreated control S2 cells (lane 1; control) and of S2 cells 3 days after transfection with dsRNA for the lamin Dm0 gene (lane 2; RNAi) were separated by SDS-PAGE and immunoblotted with antibodies against lamin Dm0 (B), the dLBR (B') and -tubulin (B''). Arrows (A,B') mark unaggregated dLBR. Molecular masses of reference proteins (in kDa) are marked. (C-C'') Immunofluorescence microscopy of S2 cells 72 hours after transfection with dLBR dsRNA. Cells were stained with antibodies against the dLBR (C, dLBR) and lamin Dm0 (C', lamin Dm0; C'', merge: overlay of C and C'). (D-G) Immunofluorescence microscopy of S2 (D,E) and Kc167 cells (F,G) 72 hours after transfection with lamin Dm0 dsRNA. Cells were stained with antibodies against lamin Dm0 (D-G, lamin Dm0), the dLBR (E',G', dLBR), and antibody mab414 that is specific for nuclear pore complex proteins (D',F', nucleoporin). The corresponding overlays are shown (D''-G'', merge). Digital images were taken by CLSM. Scale bars, 20 µm (in E'' for C-E''; in G'' for F-G'').

View larger version (115K): [in a new window]   Fig. 9. Depletion of dLBR and lamin Dm0 in Drosophila embryos by the microinjection of dsRNA. Embryos at 30 minutes old were microinjected with dsRNA specific for the dLBR gene (B-B'', dLBR; A-A'', uninjected control embryo) or lamin Dm0 (D-D'', lamin Dm0; C-C'', uninjected control embryo). Squash preparations of 24-hour-old control and microinjected embryos were stained with antibodies against dLBR (dLBR, A,A',B,B',C',D') or lamin Dm0 (A'',B'',C,D) and the chromatin was stained by Hoechst 33258 (C'',D'', DNA). To demonstrate the degree of the reduction of the nuclear staining of embryos microinjected with dLBR dsRNA, digital images were recorded with a signal enhancement of 647 V (A,B) and 774 V (A',B'). Weak nuclear staining by dLBR antibodies of embryos microinjected with dLBR dsRNA was first visible at a signal enhancement of 774 V (B'). Most of the nuclei shown in D-D'' were ruptured during the squash preparation owing to the depletion of lamin Dm0 (for quantitative data, see Table 3). Digital images were taken by CLSM (A-B'',C,C',D,D') and with a Zeiss Axiophot (C'',D''). Scale bars, 10 µm.

View larger version (118K): [in a new window]   Fig. 10. Electron microscopy of Drosophila embryos 48 hours after the microinjection with dsRNA specific for lamin Dm0. Electron micrographs of ultrathin sections show two nuclei with altered morphology (A,B) showing clustered pore complexes in the nuclear envelope (brackets) and areas where the outer and inner nuclear membrane has been separated (arrowheads) or where the nuclear envelope is ruptured (A, arrows). Scale bars, 0.5 µm.

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