vivo with DNA at all. In vitro invertebrate and vertebrate lamins bind scaffold associated or matrix attachment regions of DNA (SAR/MAR DNA) [54–55] and centromeric regions [56–57]. Lamins bind in vitro to chromatin or nucleosomes [58–63]. In the case of mammalian lamins A/C, the DNA binding domain was localized in the C-terminal domain [64], similar to D. melanogaster lamin Dm, which binds to chromatin through histones H2A and H2B [43]. A recent report demonstrated that the C terminal fragment (aa: 425–473) of lamin Dm protein binds in vitro to chromatin through histones H2A and H2B [34]. This peptide, containing a C-terminal cdc2 site and NLS, did bind to mitotic chromatin, while substitution of T432D, T435D but also T432A and T435A inhibited binding. The authors suggested the conservative sequence T432RAT is essential for binding. Deletion of the NLS sequence also decreased binding to chromosomes similarly to human lamin A binding to DNA [64]. In our experiments with full length Drosophila lamins, only lamin C mutant S37E did not bind to any chromatin. All other proteins did bind to chromatin from Xenopus (Figure 3; Figures S3, S4), HeLa cells and Drosophila embryos (not shown), which suggested that other regions of lamins may also be involved in interactions with chromatin. This binding ability is not simply dependent on polymerization since lamin Dm R64H and T435E did bind to chromatin. Lamins interact with chromatin in at least two modes, through nucleic acids and through histones, and each way of binding requires different lamin fragments for interaction. Thus phosphorylation of the C-terminal cdc2 site on lamin Dm, while possibly affecting interactions through histones, may not affect interactions with nucleic acids or other protein(s) associated with nucleic acids. The lamin Dm sequence flanking from the C-end the C- terminal cdc2 site is the site which is necessary for binding to the chromatin through histones [34]. The TRAT (SRAT/S) motif is only present in lamin Dm and other B-type lamins, being absent from lamin C (GRVT) and A-type lamins from humans (GRAS) and Xenopus (ARTI) (Figure 1). Surprisingly, a similar motif to lamin Dm (RNST432RATPS) is present in the N-terminal fragment of lamin C (RVS13RASTSTP). Phosphorylation of S37 on lamin C may affect a hairpin-like structure of the head domain of lamin C and ‘‘hide’’ this potential histone binding motif. The best example of such a sterical change may be the report that in vitro phosphorylation of S25 on lamin Dm inhibits binding of monoclonal antibody ADL84 to its epitope RPPS25AGP. But also single point mutations in lamin Dm protein distant to this epitope or on the rod domain (e.g. A40V, K146M) abolish antibody binding [8–13]. This suggests that S37 phosphorylation on lamin C may also induce conformational change in the head domain of lamin C, which in turn prevents binding to chromatin through histones. Experimental data indicate that in vertebrates, lamins A/C reassociate with the decondensing chromatin and reforming nuclear envelope later than B-type lamins [65–66]. Our data demonstrate that lamin Dm and lamin C properties such as polymerization, chromatin binding, mobility and nuclear import may be regulated by phosphorylation. Moreover, phosphorylation regulates the mentioned properties of lamin Dm and lamin C in different ways. We suggest that the different effect of pseudopho- sphorylation on B-type and A-type lamin in flies observed by us may, at least partially, be responsible for the unique properties of both types of lamins during interphase and mitosis. Materials and Methods Protein sequences used for Clustal W analysis Lamin Dm (AAF52262); Lamin C (NP_523742); Lamin-1 (CeLam-1) (Ce-lamin) (Q21443); Xenopus laevis Lamin B1 The Function of Drosophila Lamins Phosphorylation (AAC31543); Lamin B2 (AAC31544); Lamin-L (Lamin-B3) (P10999); Lamin-A (P11048). Gallus gallus (chicken) lamin B2 (NP 990616); Homo sapiens (human): Lamin B1 (AAA36162). Constructs The expression vector pET23a xLAP2 1–165 for the N- terminal 165 aa fragment of Xenopus laevis LAP2 (xLAP2) protein was a kind gift of Prof. Kathrin Wilson (Baltimore, USA) [67]. The cDNA sequence of lamin Dm point mutant lamin Dm R64H in expression vector pET20b(+) was a kind gift of Prof. Yosef Gruenbaum (Jerusalem, Israel) [13]. Cloning and mutagenesis The full length Drosophila melanogaster lamin C coding sequence from previously cloned pET17xblamC vector [13] was subcloned into pET29b vector (Novagen) using PCR. Lamin Dm cDNA was subcloned into pET29b (Novagen) vector from pET3a vector [68]. Mutations were introduced by using the Stratagene QuikChange XL mutagenesis kit, according to the manufacturer’s instructions. The mutagenic primers were: LamDmS25E: 59-ccggccgccagaggcggg- tccgcagc-39; 5-gctgcggacccgcctctggcggccgg-39; LamDmS45E: 59- ctccagccccctcgagcccacccggcactcg-39; 59-cgagtgccgggtgggctcgagggg- gctggag-39; LamDmT435E: 59ctccacgcgagccgagccatcgcgtcgcac39; 59gtgcgacgcgatggctcggctcgcgtggag39; LamDmS595E: 59ctgagtcgtc- gtcgcgaggtgaccgccgtggacgg39; 59ccgtccacggcggtcacctcgcgacgacgact- cag39; LamCS37-E: 59cctcacccaccgagcccacgcgcaccagc39; 59gctggtg- cgcgtgggctcggtgggtgagg39. Sequences of the mutants were confirmed by DNA sequencing. Protein expression and purification For protein overexpression in bacteria, the E. coli BL21DE3(- pLysS) (Stratagene) strain was transfected with derived expression vectors. Protein expression was induced with 1 mM IPTG for 6 h. Bacteria were harvested by centrifugation, washed with PBS and suspended in PBS buffer containing 0.1% Tween 20. Bacterial cells were lysed by sonication, extracted on ice for 10 min followed by centrifugation at 12000 g for 15 min. Resultant supernatant was collected for analysis. The obtained pellet was extracted successively with PBS buffer containing 1% Triton X-100, 3 M urea and 6 M urea. His-tagged lamin fusion proteins were purified on Ni-NTA-agarose columns (Qiagen). Lamin polymerization Proteins were incubated in PBS buffer at room temperature for 60 min at the concentration of 100 mg/ml. After incubation, samples were centrifuged at 12 000 g for 20 minutes. Pellet fractions were solubilized for SDS PAGE. Supernatant fractions were TCA precipitated (10% final concentration) for 10 min on ice and proteins collected by centrifugation (16 000 g; 20 min; 4uC). Resultant pellets were washed twice with ice cold acetone and solubilized for SDS PAGE. Pellets and supernatants from the sedimentation assays were analyzed in immunoblotting. Immuno- blots were digitalized and analyzed in gel documentation equipment with a CCD camera and using Bio-Profil V99 software (Vilber Lourmat). Quantitative data were obtained from 3 independent experiments and were expressed as the ratio of the amount of a particular protein in the pellet or supernatant fraction compared to the total amount of protein in both fractions. Isolation of Xenopus egg extract and demembranated sperm chromatin Egg collection and extract preparation were according to Salpingidou et al. [69]. For sperm chromatin isolation male frogs PLoS ONE | www.plosone.org 11 February 2012 | Volume 7 | Issue 2 | e32649