Characterization of the role of the tumor marker Nup88 in mitosis
© Hashizume et al; licensee BioMed Central Ltd. 2010
Received: 7 January 2010
Accepted: 24 May 2010
Published: 24 May 2010
Nuclear pore complexes are massive multiprotein channels responsible for traffic between the nucleus and cytoplasm, and are composed of approximately 30 proteins, termed nucleoporins (Nup). Our recent studies indicated that the nucleoporins Rae1 and Tpr play critical roles in maintaining the spindle bipolarity during cell division. In the present study, we found that another nucleoporin, Nup88, was localized on the spindles together with Nup214 during mitosis. Nup88 expression is linked to the progression of carcinogenesis, Nup88 has been proposed as a tumor marker. Overexpression of Nup88 enhanced multinucleated cell formation. RNAi-mediated knockdown of Nup88 disrupted Nup214 expression and localization and caused multipolar spindle phenotypes. Our data indicate that proper expression of Nup88 is critical for preventing aneuploidy formation and tumorigenesis.
The nuclear envelope forms a physical selective barrier between the nucleus and cytoplasm, and controls protein, RNA and ribonucleoprotein transportations in eukaryotic cells. Nucleocytoplasmic transport is exclusively mediated by nuclear pore complexes (NPCs), which are large proteinaceous channels that span the nuclear envelope. Vertebrate NPCs are composed of about 30 proteins, termed nucleoporins (Nups), which are present in multiple copies. Despite differences in the overall sizes in different species, the basic architecture of NPCs is well conserved among species. NPCs/Nups localization is very dynamics. In higher eukaryotes, NPCs are disassembled during cell division. We found that nucleoporins (Rae1 and Tpr) play critical roles in maintaining the spindle bipolarity during mitosis [1–4]. On the other hand, during interphase, pore proteins or nucleoporins [5, 6] (designated Nup followed by their predicted molecular weight) are modular in that a limited number of structural motifs (coiled-coils, α-solenoids and β-propellers) are used repeatedly to build the symmetrical NPC channels on the nuclear membrane . Approximately one-third of nucleoporins contain a domain of phenylalanine-glycine (FG) motifs interspersed with spacer sequences. These repeat domains are natively unstructured and serve as interaction sites for transport receptors (karyopherins) that escort cargos through the pore. For more information on NPC structure and function, a number of excellent reviews are available [7, 8].
In the past few years, several components of NPCs have been revealed to play important roles during mitosis [8–14]. In particular, we demonstrated that a nucleoporin, RNA export factor 1 (Rae1), interacted with NuMA  and cohesin subunit SMC1 [1, 2] during mitosis, and played crucial roles in proper spindle formation. Interestingly, a recent report showed that during Vesicular stomatitis virus (VSV) infection or in the presence of M protein alone, cells can undergo death during mitosis after inhibiting spindle assembly and nuclear formation, which involves disruption of Rae1 functions .
To further clarify the specific role of Nup88 in mitosis, we analyzed the composition of purified Nup88 complexes in mitotic HeLa cells. Nup88 was reported to interact with the FG repeat nucleoporin CAN/Nup214, another nucleoporin and a proto-oncogene implicated in leukemia during interphase . In immunoprecipitation experiments (Additional file 1), an anti-Nup88 antibody immunoprecipitated Nup214 but not Nup153 (Figure 1B). Conversely, an anti-Nup214 antibody immunoprecipitated Nup88 but not Nup153 (Figure 1B). These data suggest that Nup88 and Nup214 interact during mitosis. Consistent with the immunoprecipitation data, we found that Nup88 and Nup214 colocalized in HeLa cells during the cell cycle (Figure 1D). These experiments revealed a stable association of Nup214 and Nup88 during the whole cell cycle. To examine the Nup88 topography with respect to the mitotic apparatus, we used specific antibodies against Nup88, α-tubulin (spindle marker) and CENP-E (kinetochore marker) to examine their localizations at different stages of the cell cycle. Immunofluorescence microscopy (Additional file 1) of HeLa cells during interphase revealed that Nup88 was predominantly distributed on the nuclear envelope, with typical nuclear rim staining (Figure 1D and 1E, upper panel), whereas α-tubulin was mainly localized in the cytoplasm (Figure 1E, upper panel). Moreover, we could not immunoprecipitate CENP-E nor co-localize with this kinetochore marker, our data indicated that Nup88 was not mainly localized on the kinetochores during mitosis (Figure 1C). Although, colocalization of Nup88 and α-tubulin in the interphase cytoplasm was relatively weak (Figure 1E, yellow areas in merged images); we found that at early prophase, Nup88 and microtubules were concentrated at the vertices of the developing spindle poles (Figure 1E). From late prophase through anaphase, Nup88 and α-tubulin were enriched in a crescent-shaped area and intensely stained at the spindle and spindle poles (Figure 1E, metaphase, anaphase). At telophase, Nup88 was detected in the cytoplasm as well as in the newly developed nuclear envelope membrane, whereas α-tubulin was mainly associated with midzone microtubules (Figure 1E). No staining was apparent when primary antibodies were replaced by pre-immune rabbit or mouse IgG (data not shown). We examined endogenous Nup88 in ≈100 interphase cells and ≈100 cells in each stage of mitosis in three separate experiments. Consequently, these results suggest that Nup88 partially colocalizes with α-tubulin in the cytoplasm of interphase cells and spindles during mitosis.
We thank Günter Blobel (Rockefeller University), in whose laboratory this project was initiated. This work was supported by the Program for Improvement of the Research Environment for Young Researchers from the Special Coordination Funds for Promoting Science and Technology (SCF), Grants-in-Aid for Young Scientists (B) and Scientific Research on Innovative Areas from MEXT Japan, and also by grants from the Takeda Science Foundation, the Astellas Foundation for research on metabolic disorders and the Novartis Foundation (Japan) to RW.
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