Lamin B1 and Neural Tube Defects Figure 1. Lamin B1 shows differential protein migration by two-dimensional gel electrophoresis in curly tail and wild-type embryo samples. Protein profiles of the caudal region of stage-matched wild-type (A) and curly tail (B) embryos analysed by 2-DE (representative gels shown encompass pH 3.0–5.6 on the x-axis, basic pH to the right). A series of silver-stained protein spots exhibits a different migration pattern (C: enlarged area of gel corresponds to dashed box in A and B). On 2-DE of ct/ct samples, spots (pink arrows and pink spots on merged image) migrate to a more basic position than the corresponding spots on +ct/+ct gels (green arrows and green spots on merged image). Spots whose migration does not differ between samples appear black on the merged image. Western blot (D–E) and qRT-PCR (F) of protein and mRNA samples from the caudal region of +ct/+ct and ct/ct embryos at E10.5 show no significant difference between strains in relative abundance of either lamin B1 protein (normalised to beta- tubulin; arbitrary units) or mRNA (normalised to Gapdh with one wild-type sample chosen as calibrator; value set to 1.0). Number of samples, n, is shown on graphs. (G, H) The distribution of Lmnb1 mRNA at E10.5, as determined by whole mount in situ hybridisation is comparable in wild-type and ct/ct embryos (scale bar represents 1 mm). doi:10.1371/journal.pgen.1003059.g001 migration difference of lamin B1 spots on 2D gels generated from ct/ct and +ct/+ct samples. The Glu repeat in the lamin B1 tail domain is predicted to form an alpha-helix (PSIPRED secondary structure prediction [36]). Loss of a residue would impose a hundred degree rotation on the C-terminal region of the protein. The helix is likely to be capable of interacting with the inner nuclear phospholipid membrane [37]. Given that this region contains another strong membrane interactor, the C-terminal farnesylcysteine, we hypothesised that the interaction of lamin B1 with the nuclear membrane could be affected by variation in the number of Glu residues. We therefore used fluorescence loss in photobleaching (FLIP) to investigate possible functional effects on the stability of the lamin B1 tail domain within the nuclear envelope. Full length laminB1-YFP fusion proteins appeared to be stably integrated into the nuclear lamina without apparent difference between variants. We also tested truncated forms of the protein as these have previously been found to provide greater sensitivity to altered properties in this assay [26]. Fusion proteins comprising a nuclear localisation sequence, YFP and the forty C-terminal residues of lamin B1 were expressed in primary mouse embryonic fibroblasts (MEFs) and subjected to FLIP, as previously performed for human lamin B1 [26]. The decline in fluorescence intensity in the unbleached area of membrane was much more rapid in cells expressing Lmnb18E compared with Lmnb19E (Figure 2C). After 100 seconds, there was an approximately 43% decline in intensity in cells expressing Lmnb18E compared with only a 21% decline with Lmnb19E (p,0.001, t-test). This significant difference between variants persisted throughout the analysis, and is indicative of increased mobility, and hence decreased stability of interaction of Lmnb18E within the nuclear envelope. Sequencing of exon 10 of lamin B1 in a series of mouse strains showed that the wild-type (Lmnb19E) variant of lamin B1 is found in the majority of strains including C57BL/6, C3H/HeJ, SWR, DBA/2J, BALB/c, LPT/Le and CAST/EiJ. However, the Lmnb18E variant occurs in CBA/Ca, a sub-strain of which (CBA/Gr) contributed to the genetic background of the curly tail strain [38]. The variant was also present in the 101 strain and hence in mice harbouring the splotch (Sp2H; Pax3) mutation, which arose in a mutagenesis experiment on a mixed CBA/101 genetic background [39]. The 18: 56868078 SNP and Deletion 18: 56909394 were found to be in linkage disequilibrium. Thus, the Lmnb18E variant in ct/ct is characteristic of this particular genetic background. Embryos of the CBA/Ca strain do not exhibit developmental abnormalities under normal laboratory conditions, indicating that the Lmnb18E variant alone is insufficient to cause NTDs. Nevertheless, given the possible effect on stability of the lamina, we speculated that this variant could represent one of the modifier genes that are major determinants of penetrance of the curly tail PLOS Genetics | www.plosgenetics.org 3 November 2012 | Volume 8 | Issue 11 | e1003059