Matyas Gorjanacz1, Elke Klerkx2, Peter Askjaer2, Rachel Santarella1, Vincent Galy3, Iain W. Mattaj1 The nuclear envelope (NE) of eukaryotic cells provides an essential barrier that separates the genome from the cytoplasm. In metazoan organisms the seemingly stable structure of the interphase NE is dynamically re-arranged during the cell division. In mitotic prophase the NE breaks down, while in anaphase and telophase it reassembles around the segregated chromatids. Nevertheless, the precise molecular mechanisms and regulations of the NE reformation are still not known. In order to identify novel candidates potentially involved in NE assembly we analyzed a group of maternal-effect embryonic lethal mutations in Caenorhabditis elegans that show nuclear appearance defects. This analysis discovered a point-mutation in
baf-1 gene that encodes the C. elegans homologue of human Barrier-to-autointegration factor (BAF). BAF is a small and essential protein that is highly conserved among the metazoan species. BAF can bind simultaneously to DNA and a group of integral nuclear membrane proteins and by doing this it was proposed to have a structural role in NE. By structural and functional studies we found that both
baf-1 mutation and BAF down-regulation by RNA-interference (RNAi) results in strong NE assembly defects. Genetic studies showed that the
baf-1 mutation is temperature sensitive. At permissive temperature (15C) the
baf-1 homozygous mutant embryos are alive and have a functional NE that is indistinguishable from the wild-type NE. At restrictive temperature (20C and higher) the embryos die and their NE structure resembles that of the BAF RNAi NE. Biochemical studies showed that the mutant BAF protein fail to be phosphorylated at restrictive temperature, while at permissive temperature it is properly phosphorylated. Furthermore we identified the VRK-1 protein kinase as one of the kinases that phosphorylates BAF. Our studies therefore suggested that the role of BAF in NE formation is regulated by VRK-1 kinase. The temperature sensitivity of the
baf-1 mutation is one of the most appropriate tools to investigate the role of BAF during NE assembly in vivo. Shifting up the temperature from permissive to restrictive during time-lapse recordings of the
baf-1 homozygous mutant embryos expressing different GFP-tagged NE marker molecules may answer when and how BAF is involved in this complex mitotic process. In order to do these experiments we developed a novel fast response heating-cooling device suitable for confocal microscopy. Using this device our novel discoveries in this field would be discussed.