In both mutants the nuclei were multilobed and large parts of their chromatin were not covered with NE membranes.21,24 These phenotypic similarities prompted us to speculate that these two proteins might either function together or (-)-Catechin gallate that one might (-)-Catechin gallate regulate the other. mutation in gene, also known as resulted in nuclear shape and NE membrane business defects very similar to those caused by inactivation of the gene. In both mutants the nuclei were multilobed and large parts of their chromatin were not covered with NE membranes.21,24 These phenotypic similarities prompted us to speculate that these two proteins might either function together or that one might regulate the other. While we found that BAF experienced no effect on LEM?4, we found that LEM?4 was an essential regulator of BAF?1 localization during mitotic exit in worm and human cells.24 BAF shows a very dynamic localization pattern throughout the cell cycle, and this is conserved from worms21 to humans.22 During interphase, BAF is mainly enriched at the INM due to its specific interactions with the LEM Rabbit Polyclonal to ARTS-1 domains of LEM?2, Emerin21,25 and other proteins of the NE.8 Inactivation of these proteins consequently results in the loss of BAF from the INM. During mitotic entry BAF is released from the NE and the chromatin and is uniformly distributed throughout the cytoplasm. During mitotic exit however, BAF is very rapidly recruited to the segregated chromatids. It is strongly enriched in the transient dense structures around the anaphase chromatin called core regions. The appearance of these structures coincides with the reformation of the closed NE and is hypothesized to be essential for the organization of the membranes around the chromatin.21,22 Inactivation of LEM?4, but not other LEM domain proteins, completely abolished the recruitment of BAF to the chromatin surface and to the core region in worm and human cells.24 Consequently, this resulted in abnormal nuclear structure and defects in NE membrane organization. We hypothesized that LEM?4 localizes BAF during mitosis in a way other than via direct interaction. We based this assumption on the facts that; surprisingly, BAF (-)-Catechin gallate cannot bind to the LEM domain of the human LEM-4 protein as found by immunoprecipitation experiments and GST pulldown assays and its worm ortholog does not possess a recognizable LEM domain. Second, because BAF is recruited to the core region prior to any LEM domain protein.21,22 The question was then; how does LEM?4 regulate BAF localization and function during mitosis? To address this question we turned to genetics and performed a suppressor screen on the temperature sensitive mutant worm line. We identified a suppressor mutation in the gene, which suppressed not only the embryonic lethality, but also the nuclear defects seen in temperature sensitive mutant worms at restrictive temperature. is an essential gene; its inactivation by dsRNA-interference (RNAi) or by the suppressor mutation results in embryonic lethality, but not when it is combined with the co-inactivation of results in robust hyper-accumulation of BAF on the mitotic chromosomes and the NE membrane remnants throughout the entire mitosis.21 This effect of VRK?1 on BAF localization is the precise opposite of the effect of LEM?4.24 While VRK?1 is required to release BAF from its binding partners during mitosis, LEM?4 is required to (-)-Catechin gallate recruit BAF to the chromatin surface during mitotic exit. Given the opposing effects of VRK?1 and LEM?4 on BAF localization and given the fact that VRK?1 regulates BAF localization through phosphorylation; we hypothesized that LEM?4 might also regulate BAF localization through regulating its phosphorylation state. Indeed, analyses of BAF phosphoisoforms in worms that were either mutant for these components or had been treated with RNAi to reduce their concentration revealed that, while inactivation of resulted in hypo-phosphorylation of BAF, inactivation of resulted in its hyper-phosphorylation. The phosphorylation balance changed back to normal in the suppressor conditions, also suggesting that neither nor was completely inactivated in these mutant conditions. The subcellular localization of BAF is thus determined by its phosphorylation state, where LEM?4 is responsible for dephosphorylation of BAF and for its recruitment to the chromatin surface at the end of mitosis. The intriguing question arising from this result is; how does LEM?4 regulate the dephosphorylation of BAF? One possible explanation might be that LEM?4 is a new phosphatase. However, we found that, at least in vitro, LEM?4 cannot dephosphorylate BAF, and thus hypothesized that LEM?4 might either inhibit the kinase activity of VRK?1 or activate a so far unidentified phosphatase to dephosphorylate BAF. Our experiments testing the first idea showed that worm and human LEM?4 can indeed directly bind to VRK? 1 and completely inhibit.