The Function of Drosophila Lamins Phosphorylation 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. doi:10.1371/journal.pone.0032649.g005 pulse. Next, the recovery of the fluorescent signal in the bleached regions was measured and expressed as the recovery. Figure 7 shows a graphical presentation of the collected data. All recombination proteins had a lower mobility than free EGFP. The wild type lamin Dm, and the majority of pseudopho- sphorylated mutants as well as human lamins showed similar mobility regardless of localization to the NE, nuclear interior structures or blebs of NE. What is worth noting, the lamin Dm T435E fusion protein showed higher mobility than other tested mutants or wild-type lamins and only about two times lower than nuclear EGFP alone. Discussion In order to keep the experimental system as simple as possible we decided to analyze only single phosphorylation sites, although in vivo several sites may be phosphorylated on a single molecule [37] and they may act synergically [21]. For similar reasons we did not analyze the negative mutants (e.g. S or T into A). The potential phosphosites lay very close, so the mutation of a single phospho-acceptor may not prevent phosphorylation on the neighboring one – e.g. in vivo lamin Dm can be phosphorylated on S25 and S595 [41] but also on S19 and T597 [42]. Thus to have a ‘‘truly’’ unphosphorylated control for a single positive phosphor- ylation we would have had to mutate all serines and threonines within the entire region or protein itself. Since most of our studies are performed in vitro and no phosphorylation is possible, wild type lamins seem to be an adequate control. Besides, for sterical reasons, threonine should have been substituted by an amino acid with an identical side chain, which does not exist. Moreover, substitution of serine or threonine for alanine may affect the structure or interaction of such a mutant. A recent example might be the identical effect of mutations T435D and T435A in a 30 kDa lamin Dm fragment interacting in vitro with histones [34]. Polymerization of Drosophila lamins Since no data concerning mapping of in vivo phosphorylation sites on lamin C have been published, in our study we used only lamin C S37E mutant, representing the putative N-terminal cdc2 site. Based on the structure and the sequence of lamins, S37 seems to be the equivalent of the identified in vivo S22 in mammalian lamin A, S28 from goldfish lamin A [44] and S45 in lamin Dm. The lamin C region containing the C-terminal cdc2 site did not show such a high level of similarity to other lamins, as the N-terminal region shows (Figure 1). In vertebrates, mitotic lamin phosphorylation is localized at two cdc2 (cdk1) sites flanking the rod domain [20–21,45]. D. melanogaster lamins have only an N-terminal typical cdc2 kinase site (Figure 1). The potential C-terminal cdc2 sites are located differently in lamin Dm and lamin C as well as the vertebrate lamins. In vitro lamin Dm can be either depolymerized or its polymerization inhibited by cdc2 kinase, PKC or PKA [11]. Also single point mutants of lamin Dm (R64C, R64H, I396V) showed decreased polymerization ability [8,11,13,43,46–47], demonstrat- ing that single amino acid substitution itself can affect D. melanogaster lamin polymerization in vitro. The lack of the effect of modification of the N-terminal cdc2 kinase site at lamin Dm on the polymerization is supported by the similar data obtained for vertebrate B-type lamins where C- terminal in vivo phosphorylation sites were detected for mitotic lamin B [48–49]. This confirms that lamin Dm is a typical B-type lamin in this respect. Similarly, A-type lamins from vertebrates were reported to be phosphorylated on N-terminal or both N- and C-terminal sites during mitosis or while solubilized in vitro [21,50]. This in turn is reflected by our data demonstrating that S37 pseudophosphoryla- tion is sufficient for inhibition of polymerization of lamin C in vitro. Our discovery that phosphorylation of lamin C on the N terminal cdc 2 site is sufficient (to the same extent as lamin Dm phosphorylation on the C terminal cdc 2 site) for increasing solubility in vitro (Figure 2) may explain different behaviour of A and B-type lamins and may reflect the evolutionary adaptation to different functions of both types of lamins in the cell. The presented FRAP data also indicated that pseudopho- sphorylation of T435 is sufficient for increasing solubility and mobility of lamin Dm in vivo (Figure 7). Preliminary experiments using fluorescence correlation spectroscopy (FCS) in an in vitro experimental setup with bacterially expressed lamins with Alexa 488 (data not shown) also support our thesis. The FCS data indicated that increased mobility of lamin Dm mutant T435E in vivo is the result of decreased polymerization since in vitro diffusion times for lamin Dm T435E and Dm R64H were similar (2.2 ms and 3.57 ms respectively; P,0.05) and ten times lower than for lamin Dm (32.15 ms; P,0.05). Similar differences in diffusion were obtained in FRAP experiments. The last report on the in vivo phosphorylation on lamin Dm in fly embryos suggested S45 (N-terminal cdc2 site) and T432 or T435 (C-terminal cdc2 site) as phosphosites during embryogenesis with molar stoichiometry of about 0.75 and 0.2 mol phosphate/mol protein respectively [41]. Phosphorylated S45 was identified in the soluble lamin Dm fraction in early (0–5 h old) embryos. The C- terminal cdc2 site (T432/435) was identified in the late embryos in the lamin Dm fraction, which was insoluble. These results seem, at the first look, to be in contradiction to what should be expected about the function of B-type lamin phosphorylation at the cdc2 site (e.g. S405 is a predominant ‘‘mitotic’’ phosphorylation site on human soluble lamin B1 in vivo) [48]. Our data also indicate that single phosphorylation of the C-terminal cdc 2 site (T435), but not the N-terminal site (S45) is sufficient for lamin Dm solubilization. There may be several plausible explanations for such differences. Typically, lamin Dm in somatic cells is phosphorylated on two to three sites [37,41] so identification of only a single site at the submolar ratio does indicate that other phosphosites had to be present on lamin Dm protein (and had not been identified), which might affect lamin polymerization. Note that not only phosphor- PLoS ONE | www.plosone.org 8 February 2012 | Volume 7 | Issue 2 | e32649