Tor and LEM domain proteins such as emerin, MAN1 and Lap2. Membrane recruitment involves the nucleoplasmic domains of many of these INM proteins, which bind multiple chromatin-associated targets in vitro. For LBR, these potential interactors include DNA, histones and HP1. LEM domain proteins can interact with DNA but their DNA binding activity is not sufficient for chromatin association, which requires an adapter protein, BAF. BAF itself can bind to DNA and histones in vitro. Consistent with the idea of redundancy between INM proteins, single knockdowns do not cause strong NE formation phenotypes, and co-depletions have additive effects. In vitro, INM proteins can recruit purified membranes to DNA in the absence of other co-factors. However, because many of these proteins also bind histones or histone interactors, it was not clear whether the interaction with DNA is sufficient under physiological conditions. The recent data from nucleosome-free DNA in egg extracts and mouse oocytes demonstrated that, under physiological conditions, nucleosomes are indeed not required for membrane recruitment. Furthermore, despite its potential association with histones, BAF does not require nucleosomes for chromatin association. Therefore, NE membranes appear to primarily be targeted directly to DNA. Once bound to chromatin, membrane TG-101348 fusion in egg extract was suggested to require GTPase activity, thought to be due to Ran. Furthermore, Ran-coated micron-scale beads can assemble nuclear import-compatible NEs in the absence of chromatin in Xenopus egg extracts. In C. elegans embryos, interfering with Ran itself or the balance between RanGDP and RanGTP caused defects in NE formation. The mechanism by which Ran promotes NE assembly remains largely unclear, but at least one INM protein, LBR, interacts with importin , suggesting that Ran may regulate the interaction of some INM proteins with chromatin by releasing them from importin. However, more recent studies indicate fusion of membranes to require RanGTP only when NE assembly is reconstituted with vesicular precursors in the absence of a functional ER network, a situation occurring in an egg extract based system that is sometimes used to study NE assembly. Strikingly, nucleosome-free pronuclei in mouse oocytes can form closed NEs without 
enrichment of RCC1, and thus, of RanGTP. Thus, similar to the situation in spindle assembly, the requirement for RanGTP in NE formation may be context dependent. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nuclear pore complex assembly is initiated by nucleosomes Two major mechanisms PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19853339 ensure that NPCs and the lamina are formed at chromosomeassociated membranes, and not generally at the ER, the Golgi or the plasma membrane. First, similarly to mitotic spindle assembly, NPC formation requires RanGTP. Second, lamina assembly only occurs once nuclear import has been established. Analysing the consequences of the absence of nucleosomes also revealed that nucleosomes are required for NPC assembly. The NPC defect get 606143-89-9 observed in the absence of nucleosomes could only slightly be improved by forcing RCC1 to DNA, suggesting the existence of a second NPC assembly step, independently of the generation of RanGTP, Bioessays. Author manuscript; available in PMC 2016 October 01. Zierhut and Funabiki Page 9 that is regulated by nucleosomes. Indeed, it was found that this factor is ELYS, the initiator of NPC assembly , and that NPC formation on nucleosome-free DNA cou.Tor and LEM domain proteins such as emerin, MAN1 and Lap2. Membrane recruitment involves the nucleoplasmic domains of many of these INM proteins, which bind multiple chromatin-associated targets in vitro. For LBR, these potential interactors include DNA, histones and HP1. LEM domain proteins can interact with DNA but their DNA binding activity is not sufficient for chromatin association, which requires an adapter protein, BAF. BAF itself can bind to DNA and histones in vitro. Consistent with the idea of redundancy between INM proteins, single knockdowns do not cause strong NE formation phenotypes, and co-depletions have additive effects. In vitro, INM proteins can recruit purified membranes to DNA in the absence of other co-factors. However, because many of these proteins also bind histones or histone interactors, it was not clear whether the interaction with DNA is sufficient under physiological conditions. The recent data from nucleosome-free DNA in egg extracts and mouse oocytes demonstrated that, under physiological conditions, nucleosomes are indeed not required for membrane recruitment. Furthermore, despite its potential association with histones, BAF does not require nucleosomes for chromatin association. Therefore, NE membranes appear to primarily be targeted directly to DNA. Once bound to chromatin, membrane fusion in egg extract was suggested to require GTPase activity, thought to be due to Ran. Furthermore, Ran-coated micron-scale beads can assemble nuclear import-compatible NEs in the absence of chromatin in Xenopus egg extracts. In C. elegans embryos, interfering with Ran itself or the balance between RanGDP and RanGTP caused defects in NE formation. The mechanism by which Ran promotes NE assembly remains largely unclear, but at least one INM protein, LBR, interacts with importin , suggesting that Ran may regulate the interaction of some INM proteins with chromatin by releasing them from importin. However, more recent studies indicate fusion of membranes to require RanGTP only when NE assembly is reconstituted with vesicular precursors in the absence of a functional ER network, a situation occurring in an egg extract based system that is sometimes used to study NE assembly. Strikingly, nucleosome-free pronuclei in mouse oocytes can form closed NEs without enrichment of RCC1, and thus, of RanGTP. Thus, similar to the situation in spindle assembly, the requirement for RanGTP in NE formation may be context dependent. Author Manuscript Author Manuscript Author Manuscript Author Manuscript Nuclear pore complex assembly is initiated by nucleosomes Two major mechanisms PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19853339 ensure that NPCs and the lamina are formed at chromosomeassociated membranes, and not generally at the ER, the Golgi or the plasma membrane. First, similarly to mitotic spindle assembly, NPC formation requires RanGTP. Second, lamina assembly only occurs once nuclear import has been established. Analysing the consequences of the absence of nucleosomes also revealed that nucleosomes are required for NPC assembly. The NPC defect observed in the absence of nucleosomes could only slightly be improved by forcing RCC1 to DNA, suggesting the existence of a second NPC assembly step, independently of the generation of RanGTP, Bioessays. Author manuscript; available in PMC 2016 October 01. Zierhut and Funabiki Page 9 that is regulated by nucleosomes. Indeed, it was found that this factor is ELYS, the initiator of NPC assembly , and that NPC formation on nucleosome-free DNA cou.