Zaremba-Czogalla M , Piekarowicz K , Wachowicz K , Kozioł K , Dubińska-Magiera M , Rzepecki R .

	Figure 1. 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.A schematic view of the lamin monomer with in vivo 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 Drosophila 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 Drosophila 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 Drosophila melanogaster 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 Xenopus lamin B1, LB3-X Xenopus lamin B3, LA-X Xenopus lamin A.
	Figure 2. Phosphorylation of lamins changes their solubility in vitro.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 D. melanogaster lamins. Panel B shows the diagram from the data obtained after quantification of the sedimentation assays. Data were obtained from three independent experiments.
	Figure 3. All bacterially expressed lamin proteins except lamin C mutant S37E bind Xenopus chromatin in vitro.Isolated Xenopus laevis sperm chromatin in condensed and decondensed form was used to assess the binding properties of lamin proteins and N-terminal fragment of Xenopus 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.
	Figure 4. All lamin Dm mutants localize to nuclear lamina in transfected Drosophila S2 cells but lamin Dm S45E and T435E mutants show significantly different distribution.Localization of fusion GFP-lamin Dm and mutant proteins after 48 h (C) post-transfection into Drosophila 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.
	Figure 5. 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.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.
	Figure 6. Farnesylation incompetent lamin Dm mutants do not localize efficiently to nuclear lamina and nuclear envelope.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.
	Figure 7. Graphical presentation of the data collected during FRAP experiments with farnesylation incompetent lamin Dm mutants in HeLa cells.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 D. melanogaster 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 Drosophila lamins show similar mobility as human lamins in HeLa cells. Only specific mutation (T435E) can increase protein mobility, indicating lower polymerization ability in vivo. 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).

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