(2019) 14:7
Hartono et al. Cell Div
https://doi.org/10.1186/s13008-019-0050-z

Cell Division
Open Access

SHORT REPORT

Nucleoporin Nup58 localizes to centrosomes
and mid‑bodies during mitosis
Hartono1,2*, Masaharu Hazawa3,4, Kee Siang Lim4, Firli R. P. Dewi3,5, Akiko Kobayashi3 and Richard W. Wong1,3,4*Abstract
Background: Nuclear pore complexes (NPCs) act as nano-turnstiles within nuclear membranes between the cytoplasm and nucleus of mammalian cells. NPC proteins, called nucleoporins (Nups), mediate trafficking of proteins andRNA into and out of the nucleus, and are involved in a variety of mitotic processes. We previously reported that Nup62
localizes to the centrosome and mitotic spindle during mitosis, and plays a role in centrosome homeostasis. However,whether Nup58, a Nup62 subcomplex protein, also localizes to spindle poles is unknown.
Result: Herein, we show that Nup58 localizes to the nuclear rim during interphase, and to mitotic spindles, centrosomes, and midbodies during mitosis. Our confocal microscopy, live-cell imaging, and stimulated emission depletion nanoscopy results also demonstrated that Nup58 localized to the centrosomes during metaphase and relocalized
to midbodies during abscission. Depletion of Nup58 resulted in centrosomal abnormalities and delayed abscission.
Conclusion: Nup58 localized at the centrosomes and mitotic spindle during metaphase and relocalized at midbodies during abscission. This study highlights the important role of Nup58 in mitosis.
Keywords: Nup58, Centrosome, Midbody, Mitosis, Cytokinesis
Background
Nuclear pore complexes (NPCs) control trafficking ofmacromolecules between the cytoplasm and nucleus ininterphase cells [1–3]. NPCs consist of approximately 30
proteins (called nucleoporins/Nups), which form a central nano-turnstile with filaments extending into both thenucleus and cytoplasm [4, 5]. Macromolecular transport
across the pore occurs rapidly by an energy-dependent
process. Nuclear trafficking pathways are regulated byan intracellular gradient of the small GTPase Ran, witha high concentration of RanGTP in the nucleus and highconcentration of RanGDP in the cytoplasm. In addition
to macromolecular trafficking, Nups also contribute to
the control of gene expression, chromatin maintenance,and mitotic progression [6, 7]. Indeed, as mammaliancells enter mitosis, the nuclear envelope pulls apart during its breakdown process, which involves the disassembly of NPCs and nuclear lamina, as well as release*Correspondence: hartono@unm.ac.id; rwong@staff.kanazawa‑u.ac.jp
1
School of Natural System, Institute of Natural Science and Technology,
Kanazawa University, Kanazawa, Japan
Full list of author information is available at the end of the articleof the nuclear envelope membrane from chromatin [8,9]. An increasing number of Nups have now been demonstrated to function at kinetochores, centrosomes, andspindles during mitosis [10–13]. NPC components areactively involved at various stage during mitosis. However, precise roles of individual Nups, both spatially andtemporally, during cell cycle progression are still largely
unknown (for review see: [14–17]). Using conventional
confocal microscopy and live-cell imaging techniques,
we previously demonstrated the mitotic functions [16] of
nucleoporin Rae1 [18–21], Nup88 [22], Tpr [23–26], andNup358 [27]; more recently, we also revealed that Nup62
plays a novel role in centrosome integrity during mitosis[28, 29].
Nup62 is a constituent of the Nup62 complex that also
includes Nup58 and Nup54 [30, 31], which are anchoredby Nup93 at the pore and are critical for NPC function[32]. Although Nup62 localizes to the centrosome duringmitosis, little is known about the spatiotemporal localization and functions of Nup58 [33, 34]. Depletion of
Nup58 by RNA interference (RNAi) has been shown to
reduce viability of colorectal cancer cell lines (DLD1 andSW480) [35]. Moreover, knockdown of Nup58 by RNAi

© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License(http://creat​iveco​mmons​.org/licen​ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat​iveco​mmons​.org/
publi​cdoma​in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Hartono et al. Cell Div(2019) 14:7

in MDCK cell lines elicited cell polarity defects [36]. Further, Nup58 also facilitates metastasis and epithelial-mesenchymal transition (EMT) of lung adenocarcinoma via
the GSK-3β/Snail signaling pathway during interphase[37]. However, the functional consequences of Nup58 in
the cell cycle of cancer cells remains unknown.
In this report, we address whether Nup58 plays a role
in regulating cell cycle progression. We demonstrate that
Nup58 localized to the centrosomes and midbody at themetaphase-telophase-cytokinesis transition during cell
division. In addition, Nup58 depletion further influenced
the abscission process, resulting in defective cytokinesis.

Results
Nup58 localized to mitotic spindles and spindle poles
during mitosis

To further clarify specific roles of Nup58 during mitosis,
we sought to investigate the detailed subcellular localization of Nup58 during the cell cycle in HeLa cells. We
first synchronized HeLa cells using a double-thymidine
block (Fig. 1a) and examined Nup58 localization during cell division. HeLa cells were coimmunostained for
endogenous Nup58 and α-tubulin. We observed dynamic
localization of Nup58 within different mitotic and cytokinetic subcompartments. During interphase, Nup58 waspresent in the cytoplasm and on the nuclear envelope,
thereby forming a typical nuclear rim pattern in HeLa
cells (Fig. 1b). However, during prometaphase and metaphase, Nup58 gradually accumulated into spindle-likestructures, which were identified by co-staining with an
α-tubulin antibody; Nup58 was also detectable at mitotic
spindle poles or centrosomal regions during metaphase.
As cells progressed into late telophase or cytokinesis,
Nup58 was no longer detectable at the centrosome, butinstead ephemerally localized at the midzone (Fig. 1b,
Additional file 1: Fig. S1). Thus, our immunofluorescencedata indicated that Nup58 has dynamic localization
during the cell cycle and is associated with the cytokinesis apparatus during the late telophase-cytokinesis
transition.
Nup58 localized to centrosomes during the metaphase–
anaphase transition

The metaphase observations described above led us tosurmise that Nup58 contributes to centrosome assembly during cell division. Indeed, we showed that a fraction of Nup62, the bona fide binding partner of Nup58,

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localized to centrosomes during mitosis [28]. To investigate whether Nup58 also localized to centrosomes, weused specific antibodies against centrosome markers
Nup58, γ-tubulin, and SAS-6 to examine their localization at different cell cycle stages. Deconvoluted confocal
imaging of HeLa cells indicated colocalization of Nup58,
SAS-6, and γ-tubulin (Fig. 2a, b). Moreover, a series ofmaximum projections of the z-plane were captured torule out potential staining artifacts (Additional file 1:
Fig. S2a-c). Given the colocalization of Nup58 with
γ-tubulin at the centrosome, we sought to investigate
whether Nup58 physically interacts with centrosomal
proteins. Thus, we next examined whether Nup58 was
associated with centrosomal proteins in HeLa cells during interphase or mitosis. Using immunoblotting of antiNup58 immunoprecipitates, we detected coprecipitating
γ-tubulin and SAS-6, but not the coiled-coil centrosomal
protein ninein during mitosis (Fig. 2c). To confirm thebinding specificity and sensitivity of Nup58 in our immunoprecipitation assay, we used ninein as negative control
because it has a major role in microtubule minus-end
anchorage and also acts as a docking site for γ-tubulincontaining complexes at centrosomes [38]. Similarly, Tpr,
which acts as another negative control Nup, localized to
the nuclear basket region [25]. Moreover, we found that
Nup58 did not pull down any centrosomal proteins during interphase (Fig. 2d). These data suggest that Nup58,
along with Nup62, interact with γ-tubulin and SAS-6 at
the spindle pole/centrosome during mitosis. Thus, ourstaining data indicated that endogenous Nup58 localized
at centrosomal regions during the metaphase–anaphasetransition, although the biological function of the interaction between Nup58 and centrosome markers during
cell division needs further exploration.
Nup58 localized to the dark zone region of midbodies
during telophase

We next isolated and purified midbody extracts and confirmed that Nup58 and Nup62 were indeed accumulated
in the biochemical midbody fraction. Moreover, to confirm the specificity of fractionation, we blotted Tpr asa negative control and Aurora B, α-tubulin, γ-tubulin,
and KIF4 as positive controls (Fig. 3a). Midbody protein localization was further categorized into two maingroups: bulge and dark zone-flanking regions (Fig. 3b).
We compared Nup58 midbody localization by counterstaining for two different midbody marker proteins,(See figure on next page.)
Fig. 1 Localization of Nup58 on mitotic spindles during mitosis, as acquired with a confocal microscope. a Schedule of collecting mitotic HeLa cells
for confocal microscopy imaging. b Deconvoluted confocal images of HeLa cells at different cell cycle stages. Green, anti-Nup58; red, anti-α-tubulin;
blue, chromatin (DAPI). Scale bars, 5 µm

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Fig. 2 Localization of Nup58 on the centrosome and its interaction with centrosome protein during cell division. a, b Deconvoluted confocal
images of HeLa cells at different stages in the cell cycle. Green, anti-Nup58; red, anti-γ-tubulin (a) or anti-SAS-6 (b); blue, chromatin (DAPI). Scalebars, 5 µm. c, d Immunoprecipitation of HeLa cell extracts with nonspecific rabbit antibodies (IgG) or anti-Nup58, as analyzed by SDS-PAGE andimmunoblotting with antibodies against Nup58, Nup62, γ-tubulin, SAS-6, ninein, and Tpr during mitosis (c) and interphase (d)such as γ-tubulin (bulge protein marker) and KIF4 (darkzone protein marker) (Fig. 3b, Additional file 1: Fig. S1).
To further refine and validate Nup58 midbody localization, we performed stimulated emission depletion(STED) nanoscopy. We co-stained for Nup58 and KIF4 in
mitotic HeLa cells and found that KIF4 associated with
Nup58 at midbodies, consistent with immunofluorescence results. In addition, our STED data also revealed
that mitotic Nup58 completely colocalized with KIF4 atthe dark zone-flanking region, but not at the bulge (withγ-tubulin) at midbodies during cytokinesis (Fig. 3c–f ).
Altogether, during the telophase-cytokinesis transition,
Nup58 signal was detected in two disks on either side of
the midbody. These data suggest that Nup58 first associates with the centrosome in metaphase, and remains
accumulated at the finest portions surrounding the darkflanking zone in the midbody during abscission.
Nup58 relocalized to the midbody during telophase

To prevent non-specificity in the expression patternestablished using a Nup58 antibody and further ratify
the centrosomal or midbody-like localization of Nup58
during mitosis, we performed real-time live imagingof Nup58. An N-terminal green fluorescent protein(GFP)-tagged Nup58 was generated to detect expression of the GFP-tag (Fig. 4a). Live-cell imaging analysis
was used to determine Nup58-GFP cell cycle localization. A representative series from time-lapse images of
GFP-Nup58 during interphase–anaphase transitions isshown in Additional file 2: Video 1. We observed a GFPNup58 staining pattern around the nuclear rim during
interphase, and then at the centrosome during metaphase, which dynamically relocalized back to the newly
developed chromatin boundary during late anaphase(Additional file 2: Video 1). Consistent with immunofluorescence data (Figs. 1, 2 and 3, Additional file 1:
Fig. S1), we also found that some GFP-Nup58 partiallylocalized to midbody regions during late telophase andcytokinesis (Fig. 4b, c; Additional files 4, 5: Video 3a, 3b).
Notably, with live-cell imaging, we found that Nup58GFP signals were obviously detectable at the midbody,where they remained until the abscission process finished [Fig. 4c (red arrowheads) and Additional files 4, 5:
Video 3a, 3b] compared with GFP vector alone (Fig. 4b,
Additional file 3: Video 2). Thus, our live-cell imaging
data suggest that, in addition to its well-defined nuclear

envelope localization, Nup58 also partially relocalizes atthe centrosome during metaphase and midbody during
abscission.
Nup58 depletion triggered centrosomal abnormalities
and delayed abscission

To further analyze the mitotic role of Nup58, we used a
Nup58 siRNA-knockdown approach (Fig. 5a). Immunoblotting analysis of HeLa cells subjected to Nup58
siRNA treatment for 3 days revealed ~ 77% reduction in
Nup58 compared with controls (Fig. 5b, Additional file 1:
Fig. S3). Moreover, there were no significant changes in
γ-tubulin levels. Notably, Nup62 and SAS-6 levels werereduced compared with mock-treated (control siRNA)cells (Fig. 5b). Moreover, we did not observe significant
changes in mRNA levels of Nup62 or SAS-6 by qPCR,
apart from potential Nup58 siRNA off-target effects(Fig. 5c). To confirm that mitotic phenotypes were specific to Nup58 and not a secondary effect arising fromother problems with nuclear transport, we also examined
the level of importin-β, a major protein in nuclear transport, in cytoplasmic and nuclear fractions of Nup58knockdown cells and controls. Our results revealed that
there was no significant difference in protein levels ineither cell type (Additional file 1: Fig. S4). This result is
in line with a study by Rodriguez-Berriguete et al. [39]reporting that knockdown of Nup58 does not impair
nuclear import. Furthermore, we found that centrosomal abnormalities were increased three-fold comparedwith control siRNA mitotic cells (n = 500 mitotic cells).
In contrast with centrosomal abnormalities induced by
Nup62 knockdown, which mostly involve the formationof multipolar spindles [28], Nup58 depletion induced
monopolar spindles including spindles with poorly
separated poles (34.96% vs. 7.93%; p < 0.0001; Fig. 5d).
Moreover, compared with the staining pattern in control siRNA cells, downregulation of Nup58 also induced
a marked ~ 25% increase in the formation of monopolar spindle (co-staining with γ-tubulin, as a centrosome
marker, n = 500 mitotic cells) (Fig. 5d, e). Next, we analyzed cytokinesis in Nup58-depleted cells in detail. In
fixed-cell images, siNup58-treated cells exhibited a considerably shifted distribution of midbody widths together
with elongated and thin midbodies (Fig. 5f, g). Midbody
maturation to abscission was also significantly slower
when Nup58 was depleted (Fig. 5g, p < 0.01). On the basis

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Fig. 3 Colocalization of Nup58 with midbody protein markers. a Total cell extracts of interphase cells and extracts of purified midbodies were
examined by immunoblotting with the indicated antibodies. b Categorization of midbody proteins according to localization on the midbody. c, dDeconvoluted confocal images of HeLa cells during stages of cytokinesis. e, f Super-resolution images of Nup58 and KIF4 acquired with confocal
and STED microscopy, respectively. Green, anti-Nup58 ab; red, anti-γ-tubulin (c) or anti-KIF4 (d–f). Line scans show the positions of proteins inrelation to each otherof observed late-stage midbody defects, we further examined whether loss of Nup58 induced delays or failures inabscission by carrying out live-cell time-lapse imaging
with siRNA-treated HeLa cells stably expressing GFP-αtubulin. Figure 5h shows a typical example of time-lapse
images of abscission collected via confocal microscopy(Fig. 5h, Additional file 6: Video 4). We recorded significant delays in the abscission process (> 180 min) in
Nup58-depleted cells compared with control siRNAmitotic cells (~ 70 min; Fig. 5h, Additional file 6: Video
4). Further, in response to Nup58 depletion, cell division
was halted primarily in metaphase, and those Nup58depleted monopolar spindle cells exhibited severe mitotic
catastrophe and cell death (Additional file 7: Video 5).

Discussion
In recent years, Nups have been shown to perform a great
variety of alternative functions, many of which are apparently unrelated to nuclear transport [2, 4, 7]. Herein, we
found evidence for an additional role of Nup58, a NPC
member required for nuclear pore biogenesis [3], incytokinesis; namely, temporal regulation of telophase,cytokinesis, and abscission. Using live-cell imaging and
STED nanoscopy, we revealed that Nup58 transiently
localized to the centrosomes (Additional file 2: Video 1)
and midbody (Additional file 4, 5: Video 3a, 3b), and wasespecially enriched near the midbody core and arounddark zone/flanking regions; these patterns are distinct
from other Nups.
Indeed, during cytokinesis, contraction of the actomyosin ring alters the cellular cortex to form a cleavage
furrow and midbody, which serves as a platform for thegathering of the abscission apparatus that governs the
final separation of daughter cells [40]. The midbody, initially described by Walther Flemming in the 19th century,
forms from the midzone—a bipolar microtubule arraythat assembles between separating sister chromatids during anaphase [41]. Recently, it has become clear that the
midbody acts as a polarity cue during spindle orientation,
asymmetric cell division, and cell polarization by orchestrating vesicular transport, cytoskeletal organization, andlocalized cortical cues [41–44].
Although the overall program of cytokinesis is welldocumented, many questions remain, particularly at themolecular level, in part because of the high spatiotemporal complexity of cytokinesis accompanied by conditions

for significant force during cleavage [40, 45, 46]. We
demonstrated here that Nup58 depletion changed midbody widths. This may arise from the role of Nup58 inbundling and anchoring microtubules at the center of the
midzone. Our finding that Nup58 depletion induces cells
to be stuck in abscission for hours also raises the ideathat Nup58 may contribute to effectual abscission during
cytokinesis.
Previously, we reported that Nup62 knockdown
induced significantly higher numbers of multipolar spindles compared with controls [28]. In contrast, our present
study revealed that Nup58 depletion primarily enhanced
monopolar spindle formation. Our live-cell imaging also
revealed that Nup58-depleted monopolar spindle cells
induce mitotic catastrophe, aneuploidy, and eventuallycell death; as these delayed processes (~ 300 min) happened before cytokinesis, midbody formations were
often not found (Fig. 5d, e, Additional file 7: Video 5). In
mammals, aneuploidy has been linked to cancer progression, which, like cancer development, is a complex process involving functional and genetic abnormalities [27].
Moreover, Nup58 aids metastasis and EMT in lung cancer [37].
Another attractive option is that Nup58 stabilizes
microtubules in a specific arrangement to enable the formation of dark zone and/or flanking regions with othermidbody proteins (such as KIF4, PLK1, or ESCRT). In aprevious study, we reported that Nup62 localized on the
mitotic spindles and centrosomes during cell division[28]. In line with this observation, the centrosome has
been shown to contribute to abscission and many centrosomal proteins have been found to localize to the midbody ring. Indeed, Nup62 also transiently localizes to themidbody ring at the end of abscission, whereby it interacts with the filamentous actin-capping protein CapG
[47]. This study is consistent with our result (Fig. 3a).
Assuming that such a Nup58-Nup62-Nup54 protein subcomplex is required for the correct progression of cytokinesis, then a missing partner might
dissolve the complex and result in cytokinetic defects,
as observed with Nup58 depletion (Fig. 5h, Additional file 6: Video 4). Given this perspective, we postulate that Nup54 may also localize to centrosomesor midbodies during mitosis. Several organelles, suchas the mitotic spindle, centrosome, and midbody, use
microtubules as a structural constituent. Interactions

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Fig. 4 Localization of Nup58 on the midbody during cytokinesis. a Cell lysates of HeLa cells transfected with Nup58–GFP expression plasmid or
GFP-vector were analyzed by immunoblotting for GFP or Nup58–GFP. b Live-cell images of GFP-vector-expressing HeLa cells during cytokinesis
from midbody maturation to final abscission (Additional file 3: Video 2). c Live-cell images of Nup58–GFP-expressing HeLa cells during cytokinesis
from midbody maturation to final abscission showing localization of Nup58 on the midbody. Insets are magnifications of midbody areas inobserved cells (Additional files 4, 5: Video 3a, 3b). Arrowheads indicate HeLa cell midbodies during cytokinesis. Scale bars, 5 µm

between microtubule-dependent organelles and theactin cytoskeleton can be categorized as regulatory
or structural [47]. However, the nature of regulatory
effects elicited by Nup58 and Nup62 on centrosomes

and midbodies is complex, and remains to be resolvedin future studies. Indeed, future experiments should
also examine the detailed interplay between molecularmechanisms of Nup58, Nup62, Nup54, and other midbody protein interactions.

Hartono et al. Cell Div(2019) 14:7

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Fig. 5 Nup58 depletion induced centrosomal abnormalities and delayed cytokinesis. a Brief agenda of collecting cells after Nup58 knockdown
via siRNA transfection. b HeLa cells were transfected with control or Nup58 siRNA, then cell lysates were collected 72 h after transfection andanalyzed by immunoblot of Nup58, Nup62, SAS-6, γ-tubulin, and β-actin expression. c qRTPCR analysis of Nup58, Nup62, and SAS-6 mRNA in HeLa
cells after transfection with siRNA Nup58 for 72 h. Data show mean ± SD of three separate experiments. Significance difference was assessed witha Student’s t-test, **p < 0.01. d Quantification (relative %) of abnormal centrosome phenotypes in control or Nup58 siRNA-transfected HeLa cells.
Values are based on three independent experiments (n = 500 cells). Mean values ± SD (error bars) are shown. Significance difference was assessedwith a Student’s t-test, ****p < 0.0001. e Deconvoluted confocal images of synchronous HeLa cells, transfected with control or Nup58 siRNA, 72-h
post-transfection. Green, anti-Nup58; red, anti-α-tubulin; blue, chromatin (DAPI). Scale bars, 5 µm. White arrows indicate bipolar and monopolar
spindles as centrosome abnormalities when Nup58 was completely depleted. f Deconvoluted confocal images of HeLa cells 72-h post-transfection
with control or Nup58 siRNA. Green, anti-Nup58; red, anti-acetylated-γ-tubulin; blue, chromatin (DAPI). Scale bars, 5 µm. Insets are magnificationsof the midbody areas in observed cells. g Midbody maturation to abscission time in cells transfected with Nup58 siRNA (n = 30 cells) comparedwith control (n = 30 cells). Mean values ± SD (error bars) are shown, and significant differences were assessed with a Mann–Whitney U test,****p < 0.0001. h Confocal live-cell images of stable GFP-α-tubulin expression in HeLa cells transfected with Nup58 siRNA shows delayed cytokinesis
expression compared with the control. Insets are magnifications of microtubules areas in observed cells (Additional file 6: Video 4). Arrowheads
indicate microtubules abscission. Scale bars, 5 µm

Conclusions
We revealed the presence of Nup58 at the centrosome
during metaphase and its localization in the midbody
during cytokinesis. We propose that mitotic Nup58 localization favors abscission during cytokinesis.
Methods
Antibodies

Primary antibodies used for western blot analysis, immunostaining, and immunoprecipitation (IP) were as follows: anti-Nup58 (R36031, Atlas Antibodies, Bromma,
Sweden), anti-SAS-6 (sc-82360, Santa Cruz Biotechnology, Dallas, TX, USA), anti-α-tubulin (T9026, clone
DM1A, Sigma-Aldrich, St. Louis, MO, USA), anti-γtubulin (T5326, clone GTU-88, Sigma-Aldrich), antiKIF4 (K1765, clone 3E2, Sigma-Aldrich), anti-acetylated
α-tubulin (ab24610, clone 6-11B-1, Abcam, Cambridge,UK), anti-Nup62 (N1163, Sigma-Aldrich), anti-Tpr(sc-101294, Santa Cruz Biotechnology), anti-ninein(sc-376420, Santa Cruz Biotechnology), anti Aurora B(ab2254, Abcam), anti-β-actin (sc-47778, Santa Cruz Biotechnology), and GFP tag polyclonal antibody (A11122,
Thermofisher, Rockford, IL, USA). Secondary antibodies (Alexa Fluor- or rhodamine-conjugated) were from
Molecular Probes (Life Technologies, Carlsbad, CA,USA).
Cell culture

HeLa cells were obtained from American Type Culture Collection (Manassas, VA, USA). Cell lines were
propagated in Dulbecco’s Modified Eagle’s Medium(Nacalai Tesque, Kyoto, Japan) supplemented with 10%(v/v) fetal bovine serum (Life Technologies) and 50 U/
ml penicillin–streptomycin (Nacalai Tesque). HeLa cells
were maintained in a humidified incubator at 37 °C with
5% ­CO2 [23]. The HeLa EGFP-α-tubulin cell line, usedin live cell imaging, was generated by transfection of

EGFP-α-tubulin cDNA to HeLa cells and maintained in
G418 (600 µg/ml).
Immunostaining

Procedures used for immunostaining was described
previously [23]. Briefly, M-phase-synchronized HeLa
cells cultured on coverslips were washed in phosphatebuffered saline (PBS) and fixed for 10 min in ice-coldmethanol absolute. Cells were then permeabilized with
0.3% Triton X-100 in phosphate-buffered saline for10 min at room temperature. After incubation with primary and secondary antibody, samples were mounted
onto coverslips with ProLong Gold Antifade reagent (Invitrogen) and were examined on an Olympus
FV10i-LIV laser-scanning confocal microscope with a
60× PlanApo/1.45NA DIC objective (Olympus, Tokyo,
Japan).
Immunoprecipitation

Procedures used for IP and western blotting were previously described [23, 26]. Briefly, Mitotic HeLa cells werecollected, washed with PBS, spun at 400×g for 10 min,and lysed in 1 ml of cold lysis buffer (50 mM Tris–HCl(pH 7.2), 250 mM NaCl, 0.1% Nonidet P-40, 2 mM EDTA,
10% glycerol) containing 1× protease inhibitor mixture(Roche Applied Science) and 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged for 30 min at 4 °C
at 14,000×g. The resulting lysate supernatants were precleared with 50 μl of protein A/G bead slurry (Santa Cruz
Biotechnology), mixed with 10 μl of various antibodies as
specified, and incubated for 1 h at 4 °C with rocking. The
beads were then washed five times with 500 μl of lysis
buffer. After the last wash, 50 μl of 1× SDS-PAGE blue
loading buffer (New England Biolabs) was added to thebead pellet before loading. Signals were detected with anenhanced chemiluminescence system (GE Healthcare,
Chicago, IL) and quantified using a LAS-4000 image

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Hartono et al. Cell Div(2019) 14:7

analyzer (Fuji Film, Tokyo, Japan) according to the manufacturer’s specifications.
Midbody isolation

Midbodies were isolated from HeLa cells as described
previously [48, 49]. Briefly, HeLa cells were collected
after synchronized by thymidine/nocodazole followedby mitotic shake-off. Midbodies were isolated in a buffer(2 mM PIPES, pH 6.9, 0.25% Triton X-100, and 20 µg/
ml Taxol) supplemented with protease inhibitor mixture(Roche Applied Science). The final midbody pellet waschilled on ice, washed, and subjected to centrifugation
through a cushion of 40% glycerol.
Plasmids, RNA interference, and transfection

An expression plasmid containing Nup58-GFP was
generated by inserting 1800 bp of the Nup58 isoform
A gene into the pEGFP-N1 vector (Takara, Kusatsu,
Japan). The construct was confirmed by DNA sequencing (PRISM3100-AvantGenetic Analyzer, Applied Biosystems, Foster City, CA, USA). Small interfering (si)RNA
duplexes targeting Nup58-specific siRNA (sc-75984) andcontrol siRNA (sc-37007) were purchased from Santa
Cruz Biotechnology. Procedures used for transfection
was previously described [26].
Confocal live‑cell imaging

Time-lapse analysis of Nup58 dynamics during themetaphase–cytokinesis transition in live cells wasrecorded from α-tubulin-GFP-, Nup58-GFP- and GFP
vector-expressing HeLa cells. Cells were placed into
glass-bottomed dished on the stage of an Olympus
FV10i-LIV laser-scanning confocal microscope with a
60× PlanApo/1.45NA DIC objective.
Confocal microscopy image processing

Confocal microscopy images were processed using CellSens (ver.2.1) software (Olympus) to produce deconvoluted confocal images, which were then converted to
16-bit images for processing with Adobe Photoshop CS6
and ImageJ software (imagej.nih.gov/ij/). Procedures
used for immunostaining and image processing were previously described [50–52].
STED nanoscopy, image deconvolution, and analysis

For STED, cells were imaged with a 100× NA 1.4 oilobjective on a Leica SP8 gated STED microscope (Wetzlar, Germany). Alexa Fluor 488-labeled probes were
excited with a 488-nm wavelength of pulsed white light(WL) using an 80-MHz laser and subsequently depletedwith a CW 592-nm STED laser with a typical maximum
power of 260–300 mW at the back aperture of the objective (corresponding to ~ 150 MW/cm2 in the focal plane).

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The settings optimized for a maximum gain in lateral resolution, corresponding to full laser depletion on an internal Leica GaAsP HyD hybrid detector with a time gate of
1.5 ≤ tg ≤ 6.5 ns.
Deconvolution of STED data was carried out using the
STED module in Huygens Professional Deconvolution
software (version 14.10; Scientific Volume Imaging, Hilversun, the Netherlands), which contains a theoreticalestimation of the STED PSF based on values calculated
from metadata of the acquired image. For deconvoluted
images presented here, we used the calculated Huygens
default deconvolution settings that were estimated fromthe metadata with the exception of a STED immunity
fraction of 5%.
cDNA preparation and quantitative real‑time RT‑PCR assay

Procedures for cDNA preparation and qRT-PCR were
performed as previously described [23, 29]. Total RNA
was isolated using a NucleoSpin RNA isolation kit(Macherey–Nagel), and then 500 ng total RNA wasused for cDNA preparation using a cDNA synthesis
kit (ThermoScript RT-PCR system, Takara). Quantitative real time PCR (qPCR) was performed using a Thermal Cycler Dice Real Time System with SYBR Premix
Ex Taq II (Takara). The primer sets were as follows: for
Nup58, forward: 5′-CAC​AGC​CAT​CTC​TGG​GAG​TT-3′
and reverse: 5′-GCC​AAA​GCC​TGC​ACT​AAG​AC-3′ for
Nup62, forward: 5′- ACA​TCG​ATG​CAC​AGC​TCA​AG-3′
and reverse: 5′-ACT​GCA​GTG​AGT​CCA​TGT​GC -3′; forSAS-6, forward: 5′-CGC​AGG​CTG​TTT​GAA​ATG​TA-3′
and reverse: 5′-TGT​ATG​TGA​CGC​CCA​TTC​AT-3′; for
GAPDH, forward: 5′-GTC​AGT​GGT​GGA​CCT​GAC​
CT-3′ and reverse: 5′-AGG​GGT​TCT​ACA​TGG​CAA​
CTG-3′. Relative mRNA expression levels of target genes
were calculated using GAPDH as an internal control.
Statistics

Statistical analyses were performed using Prism 7(GraphPad Software, San Diego, CA, USA). Data are presented as mean ± standard deviation (SD). Statisticallysignificant differences in mean or median values between
respective groups were tested by a Student’s t test or a
Mann–Whitney U test. P values < 0.05 with a confidence
interval of 95% were considered to indicate a statisticallysignificant difference.

Additional files
Additional file 1: Figure S1. Colocalization of Nup58 with midbody protein markers γ-tubulin (a) and KIF4 (b) acquired with confocal microscopy.
Figure S2. z-plane confocal images of HeLa cells during metaphase andits maximum projections showing colocalization of Nup58 with midbody

Hartono et al. Cell Div(2019) 14:7

protein markers α-tubulin (a), γ-tubulin (b) and SAS-6 (c). Figure S3.
Depletion of Nup58 in HeLa cells. HeLa cells were transfected with control
or Nup58 siRNA, then cell lysates were collected 72 h after transfection,
analyzed for knockdown efficiency by immunoblot of Nup58 and β-actin
expression (upper panel) and for expression of Nup62 by confocal images(lower panel) Green, anti- γ-tubulin; red, anti-Nup62; blue, chromatin(DAPI). Scale bars, 5 µm. Figure S4. Expression of importin-β after depletion of Nup58 in HeLa cells.
Additional file 2: Video 1. Localization of Nup58 on the centrosome in
Nup58–GFP-expressing HeLa cells (right) during mitosis compared with
GFP-vector-expressing HeLa cells (left) acquired with live-cell imaging
Additional file 3: Video 2. Live-cell imaging of GFP-vector-expressing
HeLa cells during cytokinesis from midbody maturation to final abscission.
Additional file 4: Video 3a. Localization of Nup58 on the midbody in
Nup58–GFP-expressing HeLa cells during cytokinesis from midbody
maturation to final abscission acquired with live-cell imaging.
Additional file 5: Video 3b. Magnification of Nup58–GFP-expressing
HeLa cells from video 3a. The inset shows enlarged detail of midbody
areas in observed cells. Arrowheads indicate HeLa cell midbodies during
cytokinesis.
Additional file 6: Video 4. Delayed cytokinesis of Nup58 siRNA transfected GFP-α-tubulin expressing HeLa cells (right) compared with thecontrol (left) acquired with live-cell imaging.
Additional file 7: Video 5. A halt in cell division of Nup58 siRNA transfected GFP-α-tubulin expressing HeLa cells (right) compared with thecontrol (left) acquired with live-cell imaging.
Acknowledgements
We are grateful to Dini Kurnia Ikliptikawati for technical assistance. We also
thank all our colleagues for sharing their reagents. We thank Edanz Group forediting a revision manuscript.
Authors’ contributions
RW designed the study, wrote the manuscript. H and MH performed theexperiments and prepared figures. KL, FRPD and AK analyzed the data. All
authors read and approved the final manuscript.
Funding
Research was funded by the Grant from the MEXT/JSPS KAKENHI Grant
Number 17H05874, 17K08655 (to R.W.) from MEXT Japan, by Grants from the
Kobayashi International Scholarship Foundation (to R.W), the Shimadzu Science Foundation (to R. W.), and by Indonesia Endowment Fund for education(LPDP) and Ministry of Research, Technology and Higher Education of the
Republic of Indonesia (BUDI-LN Scholarship) (to H).
Data availability
All data generated or analyzed during this study are included in the article(and its additional files).
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1
School of Natural System, Institute of Natural Science and Technology,
Kanazawa University, Kanazawa, Japan. 2 Biology Department, Faculty of Mathematics and Natural Science, Universitas Negeri Makassar, Jl. Dg Tata Raya,
Makassar 90224, Indonesia. 3 Cell‑Bionomics Research Unit, INFINITI, Kanazawa
University, Kanazawa, Japan. 4 WPI‑Nano Life Science Institute, Kanazawa
University, Kakuma‑machi, Kanazawa, Japan. 5 Biology Department, Facultyof Science and Technology, University of Airlangga, Jl. Airlangga No. 2‑4,
Gubeng, Surabaya, East Java 60115, Indonesia.

Page 12 of 13

Received: 1 April 2019 Accepted: 3 July 2019

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