Nucleoporin Nup188 is required for chromosome alignment in mitosis

Abstract

Most cancer cells are aneuploid, which could be caused by defects in chromosome segregation machinery. Nucleoporins (Nup) are components of the nuclear pore complex, which is essential for nuclear transport during interphase, but several nucleoporins are also known to be involved in chromosome segregation. Here we report a novel function of Nup188, one of the nucleoporins regulating chromosome segregation. Nup188 localizes to spindle poles during mitosis, through the C‐terminal region of Nup188. In Nup188‐depleted mitotic cells, chromosomes fail to align to the metaphase plate, which causes mitotic arrest due to the spindle assembly checkpoint. Both the middle and the C‐terminal regions were required for chromosome alignment. Robust K‐fibers, microtubule bundles attaching to kinetochores, were hardly formed in Nup188‐depleted cells. Significantly, we found that Nup188 interacts with NuMA, which plays an instrumental role in focusing microtubules at centrosomes, and NuMA localization to spindle poles is perturbed in Nup188‐depleted cells. These data suggest that Nup188 promotes chromosome alignment through K‐fiber formation and recruitment of NuMA to spindle poles.

CLIP-170 is required to recruit PLK1 to kinetochores during early mitosis for chromosome alignment

“…S4). To test whether CLIP-170 phosphorylation on T287 might play a role in KT-MT attachment, we examined the stability of K-fibers during cold treatment using an approach described previously (Itoh et al, 2011;Itoh et al, 2013). Immunofluorescence data showed that cells expressing mCherry-CLIP-170-T287A had unstable K-fibers and unattached chromosomes, whereas cells expressing mCherry-CLIP-170-WT had robust and stable K-fibers after cold treatment (Fig.…”

“…The primary antibodies were detected by using Alexa-Fluor-labeled secondary antibodies at a dilution of 1:500 (Molecular Probes), and DNA was counterstained with 1 mg/ml DAPI. Analyses of cold-stable microtubules were performed as reported previously (Itoh et al, 2011;Itoh et al, 2013).…”

“…Western blotting was performed as described previously (Itoh, et al, 2013). For immunoprecipitation assays, pre-cleared native protein extracts (1 mg of total protein) were incubated overnight with mouse anti-mCherry at a dilution of 1:100 (Clontech) or mouse nonspecific IgG at a dilution of 1:100, followed by incubation with 40 ml of anti-mouseIgG magnetic Dynabeads (Invitrogen) for 2 hours at 4˚C.…”

The NIMA-like kinase Nek2 is a key switch balancing cilia biogenesis and resorption in the development of left-right asymmetry

“…Moreover, forced dimerization of Nup62 perturbs the directional trafficking of cytosolic proteins into the cilium, but has no effect on membrane proteins (Takao et al, 2014). Studies by other groups have shown functional roles for the nucleoporins Nup62 and Nup188 at the centrosome, which contributes its mother centriole to form the ciliary basal body (Hashizume et al, 2013;Itoh et al, 2013). Tantalizing evidence that RanGTP localizes to the axoneme and that nuclear import factor importin-β2 (also known as transportin 1) is required to import multiple ciliary proteins is consistent with the hypothesis that nucleoporins at the cilium might regulate directional transport of proteins into and out of the ciliary matrix, in addition to functioning as a general diffusion barrier (Dishinger et al, 2010;Hurd et al, 2011).…”

“…Furthermore, overexpression of a mutant form of Nup98, which cannot be phosphorylated by the Nek kinases, stabilizes the NPC and delays mitotic entry (Laurell et al, 2011). Noting that several NPC components have now been reported to localize to the base of the cilium or to the centrosome (Hashizume et al, 2013;Itoh et al, 2013;Kee et al, 2012), we hypothesized that Nup98 plays a role in the integrity of the cilium downstream of Nek2, similar to the way it plays a role in the integrity of the NE.…”