The metazoan nuclear envelope (NE) breaks down and re-forms during each cell cycle. Nuclear pore complexes (NPCs), which allow nucleocytoplasmic transport during interphase, assemble into the re-forming NE at the end of mitosis. Using in vitro NE assembly, we show that the vertebrate homologue of MEL-28 (maternal effect lethal), a recently discovered NE component in Caenorhabditis elegans, functions in postmitotic NPC assembly. MEL-28 interacts with the Nup107-160 complex (Nup for nucleoporin), an important building block of the NPC, and is essential for the recruitment of the Nup107-160 complex to chromatin. We suggest that MEL-28 acts as a seeding point for NPC assembly.

The human Nup107-160 nucleoporin complex plays a major role in formation of the nuclear pore complex and is localized to kinetochores in mitosis. Here we report that Seh1, a component of the Nup107-160 complex, functions in chromosome alignment and segregation by regulating the centromeric localization of Aurora B and other chromosome passenger complex proteins. Localization of CENP-E is not affected by Seh1 depletion and analysis by electron microscopy showed that microtubule kinetochore attachments are intact. Seh1-depleted cells show impaired Aurora B localization, which results in severe defects in biorientation and organization of the spindle midzone and midbody. Our results indicate that a major function of the Nup107 complex in mitosis is to ensure the proper localization of the CPC at the centromere. INTRODUCTIONThe nuclear envelope (NE) forms the interface between the nucleus and the cytoplasm of the interphase eukaryotic cell and is essential to maintain the unique identity of each compartment. Transport between the two compartments takes place via the nuclear pore complexes (NPCs) of which there are several thousand in vertebrate somatic cells (Allen et al., 2000;Conti and Izaurralde, 2001). Each NPC contains multiple subunits of ϳ30 proteins called nucleoporins (Nups;Cronshaw et al., 2002). In higher eukaryotes NPCs are stable throughout interphase (Daigle et al., 2001), but during mitosis both the nuclear envelope and NPC undergo major structural reorganization. Starting early in prometaphase, breakdown of the nuclear envelope occurs, including disassembly of the nuclear lamina and NPCs. The nuclear envelope membrane proteins and the transmembrane nucleoporins relocalize to the endoplasmic reticulum (ER), whereas the rest of the nuclear envelope and NPC components become distributed throughout the mitotic cytoplasm (Antonin et al., 2008).During mitosis, two complete sets of chromosomes are delivered to a pair of daughter cells. Segregation of each sister chromatid pair is achieved by a highly orchestrated process that requires attachment of the sister kinetochores of each chromosome to microtubules emanating from opposite spindle poles. Kinetochores are protein structures that assemble on chromosome regions known as centromeres and mediate microtubule attachment, mitotic checkpoint signaling, and force generation (Maiato et al., 2004;Tanaka et al., 2005;Cheeseman and Desai, 2008). Electron microscopy (EM) has provided information regarding the structure of vertebrate kinetochores and has led to the division of kinetochores into three distinct regions: the inner kinetochore that associates with chromatin, the outer kinetochore that interacts with spindle microtubules, and the less dense middle kinetochore region (McEwen et al., 2007). Multiple different MT-associated proteins function at kinetochores to form a core attachment site between kinetochore and MTs, with the KMN network (KNL-1/Mis12/NDC80 complex) being the major structural component (Cheeseman et al., 2006). Functional analysis of the NDC8...

The GTP-bound form of the Ran GTPase (RanGTP), produced around chromosomes, drives nuclear envelope and nuclear pore complex (NPC) re-assembly after mitosis. The nucleoporin MEL-28/ELYS binds chromatin in a RanGTP-regulated manner and acts to seed NPC assembly. Here we show that, upon mitotic NPC disassembly, MEL-28 dissociates from chromatin and re-localizes to spindle microtubules and kinetochores. MEL-28 directly binds microtubules in a RanGTP-regulated way via its C-terminal chromatin-binding domain. Using Xenopus egg extracts, we demonstrate that MEL-28 is essential for RanGTP-dependent microtubule nucleation and spindle assembly, independent of its function in NPC assembly. Specifically, MEL-28 interacts with the g-tubulin ring complex and recruits it to microtubule nucleation sites. Our data identify MEL-28 as a RanGTP target that functions throughout the cell cycle. Its cell cycle-dependent binding to chromatin or microtubules discriminates MEL-28 functions in interphase and mitosis, and ensures that spindle assembly occurs only after NPC breakdown.

ABSTRACT. The nuclear membrane is one of the major cellular barriers in the delivery of plasmid DNA (pDNA). Cell division has a positive influence on the expression efficiency since at the end of mitosis, pDNA or pDNA containing complexes near the chromatin are probably included by a random process in the nuclei of the daughter cells. However, very little is known about the nuclear inclusion of nanoparticles during cell division. Using the Xenopus nuclear envelope reassembly (XNER) assay, we found that the nuclear enclosure of nanoparticles was dependent on size (with 100 nm and 200 nm particles being better included than the 500 nm ones) and charge (with positively charged particles being better included than negatively charged or poly-ethyleneglycolated (PEG-ylated) ones) of the 2 beads. Also, coupling chromatin-targeting peptides to the polystyrene beads or pDNA complexes improved their inclusion by 2-to 3-fold. Upon microinjection in living HeLa cells, however, nanoparticles were never observed in the nuclei of cells post-division but accumulated in a specific perinuclear region, which was identified as the lysosomal compartment. This indicates that nanoparticles can end up in the lysosomes even when they were not delivered through endocytosis. To elucidate if the chromatin binding peptides also have potential in living cells, this additional barrier first has to be tackled, since it prevents free particles to be present near the chromatin at the moment of cell division.

The production of diffraction-quality crystals remains a difficult obstacle on the road to high-resolution structural characterization of proteins. This is primarily a result of the empirical nature of the process. Although crystallization is not predictable, factors inhibiting it are well established. First, crystal formation is always entropically unfavorable. Reducing the entropic cost of crystallizing a given protein is thus desirable. It is common practice to map boundaries and remove unstructured regions surrounding the folded protein domain. However, a problem arises when flexible regions are not at the boundaries but within a domain. Such regions cannot be deleted without adding new restraints to the domain. We encountered this problem during an attempt to crystallize the ␤ subunit of the eukaryotic signal recognition particle (SR␤), bearing a long and flexible internal loop. Native SR␤ did not crystallize. However, after circularly permuting the protein by connecting the spatially close N and C termini with a short heptapeptide linker GGGSGGG and removing 26 highly flexible loop residues within the domain, we obtained diffraction-quality crystals. This protein-engineering method is simple and should be applicable to other proteins, especially because N and C termini of protein domains are often close in space. The success of this method profits from prior knowledge of the domain fold, which is becoming increasingly common in today's postgenomic era.Keywords: X-ray crystallography; protein engineering; circular permutation; G-proteins Because an ultimate understanding of a protein's function is impossible without knowledge of its structure, the structural determination of proteins is at the forefront of biological and medical research. X-ray crystallography remains the most powerful technique for solving the three-dimensional structure of proteins.A prerequisite for such studies, and often the rate-limiting step in the process, is the production of protein crystals of suitable quality. Technological advances have made it possible to easily screen a wide range of crystallization conditions within a day. However, statistical data from structural genomics centers reveal that increasing the number of crystallization trials does not correlate with the success rate of crystallizing a particular protein (Service 2002). It turns out that a certain fraction (about 10%) of soluble proteins crystallizes relatively easily in many conditions, whereas the remaining 90% are recalcitrant (Claverie et al. 2002;Ding et al. 2002;Sulzenbacher et al. 2002). As a consequence, the protein itself should be regarded as the key variable in crystallization trials Dale et al. 2003).Crystallization is still a largely empirical process, and there is no evidence that this situation will change in the near future. However, although one cannot predict which proteins will crystallize, some obvious factors clearly hamper the effort. Article published online ahead of print. Article and publication date are at