All lamin Dm mutants localize to nuclear lamina in transfected <i>Drosophila</i> S2 cells but lamin Dm S⁴⁵E and T⁴³⁵E mutants show significantly different distribution.

<p>Localization of fusion GFP-lamin Dm and mutant proteins after 48 h (C) post-transfection into <i>Drosophila</i> S2 cells visualized under a confocal microscope and quantitative analyses of appearance of the particular phenotypes (A and B). Cells were stained for DNA with DAPI, for endogenous lamin Dm with mouse monoclonal antibodies ADL67. Exogenous lamin Dm proteins were visualized by eGFP fluorescence. Panel A demonstrates statistical analyses of distribution of lamin fusion proteins in nucleus only in nucleus and cytoplasm and in cytoplasm only. Panel B demonstrates statistical analyses of fusion proteins' localization to nuclear envelope and nuclear lamina (membrane), diffused phenotype, inclusion bodies phenotype and mixed phenotype respectively. Single confocal sections through the center of nuclei are shown. 200 cells were analyzed.</p

Comparison of the conserved amino acid sequences located in the N-terminal and C-terminal fragment of lamins containing the cdc2 kinase site, using Clustal W.

<p>A schematic view of the lamin monomer with <i>in vivo</i> identified embryonic phosphorylation sites as well as NLS, Ig-fold and CaaX motif are shown. Identical amino acids are marked by black boxes, similar by shadowed boxes. Four conserved regions were identified in vertebrate lamins and three in <i>Drosophila</i> lamins. The first region in all lamins also contains the N-terminal cdc2 site (SPTR motif). The second region located at the very beginning of the tail domain is present in vertebrates only and contains their C-terminal cdc2 site (SPXXR motif). The <i>Drosophila</i> C-terminal cdc2 site is located partially in the third conservative region (T/SRAT/S sequences) – TPSR motif for lamin Dm and TPSGR motif in lamin C. There is also an alternative C-terminal cdc2 site in lamin C (S⁴⁰⁵ in SPGR motif ). LDm-D <i>Drosophila melanogaster</i> lamin Dm, LC-D lamin C from fruit fly, LA/C-H human lamin A/C, LB1-H human lamin B1, LB-M mouse lamin B, LB2-G chicken lamin B2, LB1-X <i>Xenopus</i> lamin B1, LB3-X <i>Xenopus</i> lamin B3, LA-X <i>Xenopus</i> lamin A.</p

Graphical presentation of the data collected during FRAP experiments with farnesylation incompetent lamin Dm mutants in HeLa cells.

<p>Lamin Dm and all pseudophosphorylated mutants except lamin Dm T⁴³⁵E show the same low diffusion rate. Lack of recovery after photobleaching was observed for control human lamins (A, C and B1) (data not shown) as well as <i>D. melanogaster</i> wild type lamin Dm and the majority of lamin Dm mutants (S²⁵E, S45E, S⁵⁹⁵E), excluding lamin Dm T⁴³⁵E. They showed no measurable recovery after several minutes. In contrast, lamin Dm mutant with threonine 435 substituted by glutamic acid to mimic stable, permanent phosphorylation displayed increased dynamics (D = 2.7 µm²/s; Mf = 20%). The presented fluorescence recovery curve shows that <i>Drosophila</i> lamins show similar mobility as human lamins in HeLa cells. Only specific mutation (T⁴³⁵E) can increase protein mobility, indicating lower polymerization ability <i>in vivo</i>. For control experiments we used HeLa cells expressing free EGFP. EGFP expression was seen to be evenly distributed within the nucleus and cytoplasm. Cytoplasmic fraction of EGFP shows a slower recovery (t1/2 = 2.05 seconds) versus that observed in the nucleus (t1/2 = 1.2 seconds).</p

All bacterially expressed lamin proteins except lamin C mutant S³⁷E bind <i>Xenopus</i> chromatin <i>in vitro</i>.

<p>Isolated <i>Xenopus laevis</i> sperm chromatin in condensed and decondensed form was used to assess the binding properties of lamin proteins and N-terminal fragment of <i>Xenopus</i> LAP2 protein. Lamin proteins were incubated with condensed and decondensed sperm chromatin for 15 min at room temperature. Preparations were then fixed in PBS buffer containing 1 mM EGS followed by spin down through a glycerol cushion onto a glass coverslip and processed for immunofluorescence microscopy analysis.</p

Farnesylation incompetent lamin Dm mutants do not localize efficiently to nuclear lamina and nuclear envelope.

<p>Localization of fusion GFP-lamin Dm and mutant proteins after 24 h (A) and 48 h (B) post-transfection into HeLa cells visualized under a confocal microscope and quantitative analyses of appearance of the particular phenotypes. Cells were stained for DNA with DAPI, for endogenous lamin A/C with mouse monoclonal antibodies Jol-2 and for lamin C alone with rabbit affinity purified antibodies. Staining with secondary antibodies was with goat anti-mouse secondary antibodies conjugated with TRITC and goat anti-rabbit secondary antibodies conjugated with Cy-5 respectively. Lamin Dm S²⁵E and T⁴³⁵E were visualized by eGFP fluorescence. Single confocal Z-sections are shown through the center of nuclei. All typically observed phenotypes are shown for lamin Dm T⁴³⁵E mutant. Note the localization of lamin Dm T⁴³⁵E in the cytoplasm only.</p

Phosphorylation of lamins changes their solubility <i>in vitro</i>.

<p>Panel A shows the typical result of sedimentation assay demonstrating polymerization ability of lamins. Proteins were allowed to polymerize for 60 min and centrifuged at 12 000×g for 20 min. Equal amounts of pellet and supernatant fractions were resolved onto 10% SDS PAGE followed by western blot with antibodies against <i>D. melanogaster</i> lamins. Panel B shows the diagram from the data obtained after quantification of the sedimentation assays. Data were obtained from three independent experiments.</p

All lamin Dm mutants localize less efficiently to nuclear lamina in transfected HeLa cells but lamin Dm S⁴⁵E, T⁴³⁵E and S⁵⁹⁵E mutants show significantly the lowest association with nuclear envelope.

<p>Localization of fusion GFP-lamin Dm and mutant proteins after 48 h (C) post-transfection into HeLa cells visualized under confocal microscope and quantitative analyses of appearance of the particular phenotypes (A and B). Exogenous lamin Dm proteins were visualized by eGFP fluorescence. Panel A demonstrates statistical analyses of distribution of lamin fusion proteins in nucleus only, in nucleus and cytoplasm, and in cytoplasm only. Panel B demonstrates statistical analyses of fusion proteins' localization to nuclear envelope and nuclear lamina (membrane), diffused phenotype, inclusion bodies phenotype and mixed phenotype respectively. Single confocal sections through the center of nuclei are shown. 200 cells were analyzed from at least 10 observation fields. Cells were stained for DNA with DAPI, for endogenous lamin A/C with mouse monoclonal antibodies Jol-2 and for lamin C alone with rabbit affinity purified antibodies. Staining with secondary antibodies was with goat anti-mouse secondary antibodies conjugated with TRITC and goat anti-rabbit secondary antibodies conjugated with Cy-5 respectively.</p

Additional file 2: of Laminopathies: what can humans learn from fruit flies

Figure S1. Multiple sequence alignment of BAF proteins from different species: Caenorhabditis elegans (NP_499085.1 Barrier-to-autointegration factor 1), Drosophila melanogaster (NP_001260220.1 barrier to autointegration factor), Xenopus laevis: factor A (NP_001084558.1 barrier-to-autointegration factor A) and factor B (NP_001087314.1 barrier-to-autointegration factor B), Mus musculus (NP_001033320.1 barrier-to-autointegration factor), and Homo sapiens (NP_003851.1 barrier-to-autointegration factor). The darker blue color indicates higher similarity. The numbering above the multiple sequence alignment is for D. melanogaster. All sequences were aligned with CLUSTALX v. 2.0. Each alignment was edited in JALVIEW v. 2.8. and individually corrected for inaccurate fragments. (PNG 33 kb

Additional file 3: of Laminopathies: what can humans learn from fruit flies

Figure S2. Multiple sequence alignment of MAN1 proteins from different species: Caenorhabditis elegans (NP_496944.1 LEM protein 2), Drosophila melanogaster (NP_001286812.1 MAN1), Xenopus laevis: (NP_001082578.1 LEM domain containing 3 S homeolog), Mus musculus (NP_001074662.2 inner nuclear membrane protein Man1), and Homo sapiens: isoform 1 (NP_055134.2 inner nuclear membrane protein Man1 isoform 1) and isoform 2 (NP_001161086.1 inner nuclear membrane protein Man1 isoform 2). The darker blue color indicates higher similarity. The numbering above the multiple sequence alignment is for D. melanogaster. All sequences were aligned with CLUSTALX v. 2.0. Each alignment was edited in JALVIEW v. 2.8. and individually corrected for inaccurate fragments. (PNG 309 kb

Additional file 4: of Laminopathies: what can humans learn from fruit flies

Figure S3. Multiple sequence alignment of LBR proteins from different species: Drosophila melanogaster: isoform A (NP_611608.1 lamin B receptor, isoform A), isoform B (NP_726115.1 lamin B receptor, isoform B), isoform C (NP_726114.1 lamin B receptor, isoform C), Xenopus laevis (NP_001079301.1 lamin B receptor S homeolog), Mus musculus (NP_598576.2 lamin-B receptor), and Homo sapiens (NP_002287.2 lamin-B receptor). Isoforms A and B of LBR proteins in D. melanogaster are shown on one alignment because they have the same amino acid sequence. The darker blue color indicates higher similarity. The numbering above the multiple sequence alignment is for D. melanogaster. All sequences were aligned with CLUSTALX v. 2.0. Each alignment was edited in JALVIEW v. 2.8. and individually corrected for inaccurate fragments. (PNG 217 kb