increased solubility of the protein. This indicates that phosphor- ylation of the C-terminal alone, but not the N-terminal conserved cdc2 site on lamin Dm may be sufficient for solubilization of lamin Dm in vitro. Only lamin C S37E mutant mimicking phosphorylation on the N-terminal cdc2 site does not bind to chromatin in vitro Another expected feature of lamin phosphorylation in vivo should be the loss of interaction between lamins, chromatin and nuclear envelope components. To test this assumption for Drosophila lamins, we used isolated Xenopus sperm chromatin as a target for lamin binding in vitro. Only lamin C S37E mutant failed to bind to chromatin in vitro (Figure 3), while the other tested pseudophosphorylated lamins and control proteins were able to bind to both condensed and decondensed chromatin, with similar efficiency, as revealed by IF studies. It is worth mentioning that lamin Dm R64H and T435E mutants, which had the same solubility as lamin C S37E, did bind to both forms of chromatin. Similar data were obtained on isolated HeLa interphase The Function of Drosophila Lamins Phosphorylation chromatin, permeabilized, interphase HeLa nuclei and chromatin isolated from late Drosophila embryos (not shown) as well as when chromatin was decondensed using interphase extracts from unfertilized Xenopus eggs. All lamin Dm proteins were able to associate with Xenopus pronuclei (Figure S3) while lamin C S37E protein did not (Figure S4). Lamin Dm S45E and T435E show different distribution in transfected Drosophila S2 and human HeLa cells In order to test the in vivo functions of lamin phosphorylation, we constructed wild type lamin Dm and pseudophosphorylated mutants as EGFP (enhanced green fluorescent protein) fusion proteins. As a control, we used human wild type lamin A, B and C also as EGFP fusion proteins (Figure S5). Overexpression of lamin Dm, lamin Dm S25E and S595E in S2 cells resulted in location of these proteins in the nucleus and in the nuclear lamina, where they colocalized with endogenous lamin Dm (Figure 4A, B, C). Lamin Dm mutants S45E and T435E showed similar location to each other but different from the rest of the mutants. Although lamin Dm S45E and T435E mutants 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. doi:10.1371/journal.pone.0032649.g003 PLoS ONE | www.plosone.org 4 February 2012 | Volume 7 | Issue 2 | e32649