The nucleoporin MEL-28 promotes RanGTP-dependent γ-tubulin recruitment and microtubule nucleation in mitotic spindle formation.

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 γ-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.

Nuclear inclusion of nontargeted and chromatin-targeted polystyrene beads and plasmid DNA containing nanoparticles.

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 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 polyethyleneglycolated (PEGylated) ones) of the 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 postdivision 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 from being present near the chromatin at the moment of cell division.

The Nup107-160 nucleoporin complex promotes mitotic events via control of the localization state of the chromosome passenger complex.

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.

MEL-28/ELYS is required for the recruitment of nucleoporins to chromatin and postmitotic nuclear pore complex assembly.

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.

Circular permutation as a tool to reduce surface entropy triggers crystallization of the signal recognition particle receptor beta subunit.

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 beta subunit of the eukaryotic signal recognition particle (SRbeta), bearing a long and flexible internal loop. Native SRbeta 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.