The Function of Drosophila Lamins Phosphorylation 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. doi:10.1371/journal.pone.0032649.g004 generally localized to the nucleus, a significant amount of cells demonstrated nucleocytoplasmic and cytoplasmic locations (about 40%) of these proteins. This suggests that lamin Dm S45E and T435E mutants have impaired ability to be incorporated into the cell nucleus and the nuclear lamina compared to lamin Dm and other mutants. Since all lamin Dm mutants bind to chromatin in vitro and lamin Dm T435E does not polymerize while lamin Dm S45E polymerizes in vitro, this suggests that for lamin Dm location in the nuclear lamina in vivo, at least one property other than polymerization ability and chromatin binding ability is important as well. This also indicates that phosphorylation of S45 (N-terminal cdc2 site) alone is not sufficient for solubilization of lamin Dm in vivo, at least in S2 cells. Similarly, the phosphorylation of T435, which affects polymerization in vitro, has only a moderate effect on location of this mutant in vivo. In order to test the generality of the lamin Dm interactions and due to the use of Xenopus sperm chromatin and HeLa chromatin in our in vitro studies, we overexpressed the same lamin Dm mutants in HeLa and Xenopus XTC cells. As a control, we used human wild type lamin A, B and C. Figure S5 demonstrates typical localization of wild-type human lamins after transfection of HeLa cells. Depending on the level of expression, human lamin-GFP proteins localize to the nucleus only, or the nucleus and surrounding space, forming nuclear envelope blebs and infrequently decorating ER structures. Wild type lamin Dm-GFP and mutants S25E and S595E demonstrated predominant nuclear location while S45E and T435E mutants were located both in the nucleus and cytoplasm. Over 20% of cells showed, beside nuclear, also cytoplasmic location of these mutants (Figure 5A, B, C). All lamin Dm proteins showed lower ability to be incorporated into nuclear lamina in HeLa cells when compared to Drosophila S2 cells but T435E mutant was the least effectively incorporated into the nuclear lamina. This mutant also showed the highest frequency of cytoplasmic location. One should note that mutant S595E, although it showed nuclear location, showed similarly decreased localization at the nuclear lamina as S45E and T435E mutants. These data support our hypothesis that factors other than polymerization ability and chromatin binding participate in affecting lamin Dm location in vivo both in Drosophila and in human cells. Surprisingly, S45 and T435 (also S595E) modification had a greater effect on location of lamin in HeLa cells than in Drosophila cells. This may indicate the additional role played by interacting protein partners of lamins in their nuclear lamina retention. In Drosophila, abnormal location is less frequent, probably due to the interactions, while in human cells lamin Dm may not interact or interact with lower affinity. Observed differences can also be connected with different physiological temperatures of human and insect cells lines, which can affect the protein properties (e.g. synthesis, folding, interac- tions). It should be pointed out that transfection of wild type human lamin A, B1, C as well as lamin Dm and mutants into HeLa cells similarly decreased the mitotic index of cells (below 0.5%) and induced apoptosis. The highest level of apoptosis (about 35%) was shown by lamin Dm T435E, the lowest by lamin A (about 10%). Preliminary analyses of cell cycle progression in connection with cytoplasmic/nuclear location of lamins do not show any specific correlation. There is, however, a correlation between the level of overexpression of exogenous protein and apoptosis. An approximately 50% decrease is observed in the number of cells highly overexpressing exogenous lamin between 24 h and 48 h. This effect is due to the increased rate of apoptosis in such cells and is characteristic for all lamins, both human and fly. Detailed analyses of distribution of lamin Dm and endogenous lamins in HeLa cells demonstrated that most lamin Dm does not colocalize with endogenous lamins (A, B1 and C) at the nuclear envelope. Lamin Dm proteins localize at the nuclear envelope as small ‘‘domains’’ or form bigger fragments up to ‘‘budding’’ fragments of nuclear envelope (not shown). Lamin Dm T435E is not imported into the cell nucleus and shows higher dynamics in vivo In order to test the hypothesis that the effect of lamin Dm pseudophosphorylation can be moderated or hidden by the effect of farnesylation of the C-terminal CAIM motif and nuclear or nuclear lamina retention may be the effect of nuclear envelope targeting by the farnesyl moiety, we prepared the same set of mutants (as EGFP fusion proteins) but with additional mutation in the C-terminus which prevents farnesylation. Results of statistical analyses of transfected HeLa cells after 24 and 48 h are shown in Figure 6A. The amount of cells with nuclear or both nuclear and cytoplasmic location is similar for lamin Dm-GFP and lamin A-GFP transfection after 24 and 48 h and their nuclear location increases over time. The most profound effect was observed for lamin Dm T435E mutant, which was predominantly cytoplasmic after 24 and 48 h post-transfection (Figure 6B). This indicates that lamin Dm T435E mutation affects nuclear import of lamin Dm and/or its retention in the nuclear lamina. This effect was undetected when the mechanism of nuclear transport and retention in the nuclear envelope through farnesylated C-terminus was active. In order to gain insight into mobility of transfected fusion proteins and polymerization ability of lamins in vivo, we performed FRAP (fluorescence recovery after photobleaching) analysis. Experiments on transfected S2 cells and transgenic flies (embryos and salivary glands from larvae) expressing wild type lamin Dm- GFP demonstrated that lamin Dm in transgenic flies and all mutants in S2 cells have the same mobility. The mobility was similar to mobility of human lamin B-GFP transfected into HeLa cells. It is noteworthy that our previous experiments indicated that lamin Dm mutants transfected into S2 cells were predominantly nuclear and that farnesylation of lamin Dm facilitates its targeting to the nuclear envelope. This may affect mobility independently of polymerization ability of a particular lamin Dm mutant in vivo. Therefore we decided to measure the mobility of farnesylation incompetent lamin Dm mutants, using the FRAP method. We expressed lamins as fusion proteins with EGFP in HeLa cells. In FRAP experiments, fluorescent molecules were irreversibly photobleached in a defined area by a single, high-powered laser PLoS ONE | www.plosone.org 6 February 2012 | Volume 7 | Issue 2 | e32649