The Function of Drosophila Lamins Phosphorylation 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 mm2/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). doi:10.1371/journal.pone.0032649.g007 ylation of a particular mitotic site but also dephosphorylation of interphase sites may be essential for lamin solubilization [24]. An interesting feature of the syncytial embryo is that mitosis is very fast, synchronous and propagates in waves from one pole of the developing embryo. An additional explanation is that early embryos contain vast quantities of maternally provided lamin Dm as supplies for fast syncytial mitotic divisions. Such lamin Dm is gradually used up and recruited into newly created syncytial nuclei in the developing embryo. Besides, mitosis in syncytial embryos is ‘‘semi-closed’’, which means that the nuclear envelope is depolymerized only at spindle poles at metaphase and disassembles almost completely only briefly at anaphase, forming a structure called the ‘‘nuclear spindle envelope’’ [51]. So lamin Dm modification in Drosophila early embryos on S45 may serve either as a storage form of maternally provided lamin Dm or as a necessary component of semi-closed mitosis in syncytial Drosophila embryos (with other unidentified phosphosites) or both. Moreover, early embryo soluble lamin Dm named Dmmit was found to be copurified with a 90 kDa protein [52] or 90 kDa and 84 kDa proteins [42]. Interaction with these proteins may keep the lamin Dm fraction soluble. Additional support for soluble lamin inducible by interactions with protein comes from the reports about interaction of lamin A phosphorylated on S404 interacting with nuclear 14-3-3 protein [53]. This thesis is supported by an in vitro experiment in which interphase insoluble lamin Dm can be made soluble by addition of anti-lamin Dm IgGs [52]. Thus, lamin Dm S45 is probably not the only early embryo (syncytial) site and definitively not the only site in somatic cells because mutant S45E has the same properties as wild type lamin Dm. Instead, T435 residue seems to be the best available candidate, since mutant T435E is soluble. Among all tested lamins, only lamin C S37E perfectly fits the ideal model of the effect of mitotic phosphorylation. This single site mutant was soluble, was not able to bind to chromatin or the nuclear envelope, and was unable to associate with the reforming cell nucleus in in vitro nuclear assembly. This indicates a much stronger effect of the N-terminal cdc2 site phosphorylation than in vertebrate A-type lamins, where both cdc2 sites have to be modified to observe the full effect [21]. Perhaps, the reason why S45 mutant and T435 mutant show only partial mislocalization in S2 cells is that farnesylation, by providing the mechanism for nuclear lamina retention as well as the possibility for ER lateral transport into the nucleus, ‘‘hides’’ the effect of phosphorylation on lamin location (Figure 4 versus Figure 5). Lamins and nuclear import Transient transfection studies on HeLa cells with farnesylation incompetent lamin mutants demonstrated that all lamin Dm proteins, except T435E mutant, localized to the nuclear envelope and the cell nucleus (Figures 5, 6). The most probable explanation for T435E mutant location in cytoplasm is the inhibition of the nuclear import of this protein. This suggestion is supported by a previous report on chicken lamin B2 indicating that phosphory- lation on C-terminal PKC sites (S410, S411) in response to phorbol ester treatment significantly reduced the import of lamin B2 into the nucleus [27]. Chromatin binding Lamins bind chromatin components in vivo directly or indirectly. Lamin Dm interacts directly with DNA in vivo during interphase but not during mitosis [39]. Drosophila lamin C does not interact in PLoS ONE | www.plosone.org 10 February 2012 | Volume 7 | Issue 2 | e32649