In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358⁄ RanBP2 within individual NPCs increases during mus-cle diff
Trang 1Nup358, a nucleoporin, functions as a key determinant of the nuclear pore complex structure remodeling during
skeletal myogenesis
Munehiro Asally1, Yoshinari Yasuda2, Masahiro Oka1,2, Shotaro Otsuka3, Shige H Yoshimura3, Kunio Takeyasu3and Yoshihiro Yoneda1,2,4
1 Japan Science and Technology Agency, Core Research for Evolutional Science and Technology, Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Japan
2 Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Japan
3 Department of Responses to Environmental Signals and Stresses, Graduate School of Biostudies, Kyoto University, Japan
4 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, Osaka University, Japan
Introduction
Nuclear-cytoplasmic trafficking regulates the
move-ment of molecules into and out of the nucleus and is
necessary for the survival of eukaryotic cells The
pas-sage of molecules such as RNA, proteins and ions
across the nuclear envelope occurs through the nuclear
pore complex (NPC), a huge protein complex
embed-ded in the nuclear envelope Small molecules (< 9 nm
in diameter) pass through the NPC by passive diffu-sion, whereas larger molecules are transported in a facilitated manner [1] The selective nuclear transport
of proteins is directed by specific signal sequences: nuclear localization signals (NLS) for import and nuclear export signals (NES) for export To achieve selective transport, soluble transport factors are
Keywords
NPC remodeling; nuclear pore complex;
nuclear transport; Nup358 ⁄ RanBP2;
skeletal myogenesis
Correspondence
Y Yoneda, Department of Frontier
Biosciences, Graduate School of Frontier
Biosciences, Osaka University, 1–3
Yamada-oka Suita Osaka 565-0871, Japan
Fax: +81 6 6879 4609
Tel: +81 6 6879 4606
E-mail: yyoneda@anat3.med.osaka-u.ac.jp
(Received 23 August 2010, revised 26
November 2010, accepted 3 December
2010)
doi:10.1111/j.1742-4658.2010.07982.x
The nuclear pore complex (NPC) is the only gateway for molecular traf-ficking across the nuclear envelope The NPC is not merely a static nuclear-cytoplasmic transport gate; the functional analysis of nucleoporins has revealed dynamic features of the NPC in various cellular functions, such as mitotic spindle formation and protein modification However, it is not known whether the NPC undergoes dynamic changes during biological processes such as cell differentiation In the present study, we evaluate changes in the expression levels of several nucleoporins and show that the amount of Nup358⁄ RanBP2 within individual NPCs increases during mus-cle differentiation in C2C12 cells Using atomic force microscopy, we dem-onstrate structural differences between the cytoplasmic surfaces of myoblast and myotube NPCs and a correlation between the copy number
of Nup358 and the NPC structure Furthermore, small interfering RNA-mediated depletion of Nup358 in myoblasts suppresses myotube formation without affecting cell viability, suggesting that NUP358 plays a role in myogenesis These findings indicate that the NPC undergoes dynamic remodeling during muscle cell differentiation and that Nup358 is promi-nently involved in the remodeling process
Abbreviations
AFM, atomic force microscopy; DM, differentiation medium; EDMD, Emery–Dreifuss muscular dystrophy; EGFP, enhanced green
fluorescent protein; GM, growth medium; MyHC, myosin heavy chain; NES, nuclear export signal; NLS, nuclear localization signal;
NPC, nuclear pore complex.
Trang 2required to interact with both cargo substances and
the NPC One of the best characterized nuclear protein
import pathways is mediated by the importin-a⁄ b
hete-rodimer, which imports basic NLS-containing proteins
Conversely, the nuclear export of leucine-rich
NES-containing proteins is mediated by CRM1 (also known
as exportin-1) [1–3]
A gradient of the small GTPase, RanGTP⁄ GDP,
across the nuclear envelope regulates the binding and
release of cargo by transport factors RanGTP is
abun-dant in the nucleus as a result of the activity of RCC1,
a guanine nucleotide exchange factor for Ran
Con-versely, the concentration of RanGDP is high in the
cytoplasm as a result of the action of RanGAP, which
promotes cytoplasmic GTP hydrolysis by Ran in
con-junction with RanBP1 and⁄ or Nup358 (also known as
RanBP2) RanGTP interacts with transport factors
and regulates the formation of transport complexes
GTP hydrolysis by Ran in the cytoplasm drives
disso-ciation of the export cargo from the export complex,
whereas RanGTP promotes disassembly of the import
complex in the nucleus [1,2]
The NPC is composed of approximately 30 distinct
proteins, known as nucleoporins [4], whose functional
analyses have recently attracted much attention The
general architecture of the NPC is evolutionarily
con-served in all eukaryotes and consists of filaments on
the cytoplasmic surface and spoke rings and a
basket-like structure on the nuclear side of the envelope [5]
In recent years, nucleoporins have been found to play
dynamic roles in a variety of cellular functions, in
addition to their well known function as structural
components of the nuclear pore For example,
nucleo-porins have been shown to be involved in protein
modification and the regulation of mitotic spindle
for-mation [6] Nup358 and the Nup107–160 subcomplex
are localized to kinetochores during mitosis and play a
role in spindle assembly [7] It has long been suggested
that nucleoporins are ubiquitously expressed in all cell
types and at all developmental stages, although recent
studies indicate that some nucleoporins, such as gp210,
are expressed in a cell type- and tissue-specific manner
[8,9] In addition, the structural composition of the
NPC changes during cell differentiation [10], although
it is still unknown whether alterations in NPC
archi-tecture play an important role in cellular
differentia-tion
C2C12 cells are a well established model system for
skeletal muscle differentiation In high-serum growth
medium (GM), these cells grow as mononuclear
myo-blasts, although they fuse to form multinuclear
myotu-bes when cultured in low-serum differentiation
medium (DM) Dynamic remodeling of the nuclear
envelope was reported during C2C12 skeletal muscle differentiation [11], including changes in the distribu-tion of lamins, which are components of the nuclear envelope lamina Mutations within A-type lamin are known to cause several muscle diseases, including Emery–Dreifuss muscular dystrophy (EDMD), and the over-expression of a lamin A⁄ C mutant in C2C12 cells has been shown to reliably mimic the features of EDMD [12] Inner nuclear envelope proteins are also known to regulate NPC dynamics [13] Understanding NPC dynamics during muscle differentiation will not only provide novel information regarding NPC func-tion, but also will contribute to a better understanding
of the pathogenesis of muscular disorders such as EDMD
In the present study, we examine NPC remodeling and its associated functional changes during skeletal muscle differentiation in a C2C12 murine myoblast cell line We compare the composition, architecture and nuclear-cytoplasmic transport activity of NPCs in myoblasts and myotubes We find that dynamic remodeling of the NPC occurs during muscle cell differentiatin
Results
Expression patterns of nucleoporins are altered from myoblasts to myotubes
To determine the expression levels of NPC compo-nents (nucleoporins) during skeletal muscle cell differ-entiation, we used real-time PCR to quantify the mRNA levels of each nucleoporin in C2C12 cells before and after the induction of differentiation (Fig 1) Approximately half of the nucleoporin mRNAs (i.e Nup107, Nup85, Nup160, Nup43, Nup37, Nup35, Nup205, Nup188, Pom121, Ndc1, Nup155, Nup54, Nup62, NupL1, Nup153 and Nup50) were down-regulated after the induction of myogenesis By contrast, the expression levels of some nucleoporin mRNAs (i.e Nup214, Nup88, NupL2 (CG1), Nup133, Seh1, Nup93, Gp210, Nup98, Rae1 and Tpr) remained largely unchanged, and the remaining nucleoporin mRNAs (i.e Nup358 and Sec13) were slightly up-regu-lated These results indicate that the relative expression levels of individual nucleoporins change during muscle cell differentiation
To examine the protein levels of specific nucleopo-rins, we prepared C2C12 cell lysates at different stages
of muscle cell differentiation and immunoblotted with available antibodies against several nucleoporins Lysates from C2C12 cells grown in GM and from cells differentiated for either 2 or 5 days in DM were
Trang 3analyzed As shown in Fig 2A, the protein level of
Nup358, a component of the cytoplasmic filaments of
the NPC, gradually increased as muscle cell
differentia-tion proceeded All other nucleoporins tested (Nup160,
Nup98, Nup93 and Nup62) showed no obvious
changes in protein expression levels during
differentia-tion Furthermore, immunofluorescence analysis of
Nup358 revealed a stronger signal in myotubes
com-pared to myoblasts, whereas the Nup98 signal
appeared to be unaltered (Figs 2B and S1) By
con-trast, although anti-Nup358 staining also showed an
intracellular signal (Fig 2B), we considered the
intra-nuclear signal to be nonspecific (Doc S1A) We next
focused on the nuclear envelope signal of anti-Nup358
staining To definitely verify the increased expression
of Nup358 at the NPCs of myotubes, we examined the
Nup358 binding partners, RanGAP and CRM1, which
localize to the nuclear rim in a Nup358-dependent
manner [2] RanGAP staining at the nuclear envelope
was much stronger in myotubes than in myoblasts
(Fig 2C) Although CRM1 staining in myoblasts and
myotubes was comparable when the cells were
permea-bilized after formaldehyde fixation, the differences
became more evident when the cells were fixed with
formaldehyde containing Triton X-100, with stronger
CRM1 signals being observed for myotubes than for
myoblasts (Fig 2D) These immunostaining patterns
of Nup358, CRM1 and RanGAP clearly reveal that
Nup358 expression is up-regulated during skeletal myogenesis in C2C12 cells
To determine whether this increase in Nup358 on the nuclear envelope corresponded to an increase in the number of Nup358 proteins in each NPC, we mea-sured NPC density Consistent with a recent study [14], nuclear pore density was similar in myoblasts and myotubes (Fig 2E), indicating that the composition of the myoblast NPC must differ from that of the myo-tube NPC Specifically, the number of Nup358 pro-teins within individual NPCs increase during skeletal muscle differentiation
An architectural change in the NPC occurs during differentiation from myoblasts to myotubes Because the composition of the NPC changes during differentiation (Figs 1 and 2), we attempted to visual-ize the structure of NPCs at the nanoscale level using atomic force microscopy (AFM) As shown in Fig 3A, AFM images of the NPCs in myoblasts and myotubes were successfully captured It was previ-ously observed that the centers of some NPCs are plugged by complexes passing through the NPC [15–17] Consistent with these studies, we observed that 29.3% of the NPCs in myoblasts were plugged, whereas 54.4% of NPCs in myotubes were plugged (Fig 3B)
Fig 1 The relative mRNA expression levels of nucleoporins differ during muscle differentiation C2C12 cells were grown in GM (blue bars)
or for 2 days in DM (red bars) Nucleoporin mRNA levels were analyzed by real-time PCR (n = 3, PCR reaction; n = 2, RNA extraction) and normalized to tubulin Error bars represent the SEM.
Trang 4The NPC characteristics measured in the AFM
profiles were: outer diameter (O), height (H) and upper
rim diameter (Fig 3C) The values were statistically
analyzed (U) using the Mann–Whitney U-test
(P < 0.05 was considered statistically significant) The
outer diameter of NPCs did not differ significantly
between myoblasts and myotubes (Figs 3D and S2A)
Myotubes contain greater amounts of Nup358 than do
myoblasts (Figs 1 and 2) and, because Nup358 is
local-ized in the cytoplasmic filaments of NPCs, we
sus-pected that the heights of myotube NPCs might be
greater than those of myoblasts NPC height appeared
to not be significantly different in myotubes and
myo-blasts (P > 0.05) when unplugged and plugged NPCs
were analyzed separately (Fig 3F)
By contrast, the mean ± SEM upper rim diameter
increased significantly, from 72.5 ± 1.3 nm (n = 99)
in myoblasts to 81.6 ± 1.3 nm (n = 68) in myotubes
(Fig 3E) This trend was observed even when
unplugged and plugged NPCs were analyzed separately
(Fig 3D, E) and the different distributions of NPC
upper rim diameters in myoblasts and myotubes are clearly demonstrated in a histogram (Fig S2C) The upper rim diameter of the NPC increased by approxi-mately 9 nm during myotube formation, whereas the outer diameter and the height of the NPC were not significantly changed Thus, the shape of the NPCs in myoblasts differs from that in myotubes
Nup358 is a cytoplasmically exposed component of the NPC [18] and was increased within individual NPCs during muscle differentiation Thus, we hypothe-sized that increased Nup358 in individual NPCs was responsible for the change in NPC upper rim diameter
To test this, we used AFM to visualize Nup358-depleted NPCs AFM analysis of NPCs revealed that the upper rim diameter of Nup358-depleted NPCs was smaller than for NPCs in control small interfering RNA (siRNA)-treated cells (Fig 4B) By contrast, the height and outer diameter of Nup358-depleted NPCs did not differ significantly from those of the control NPCs (Fig 4A, C) These results indicate that the Nup358 copy number within individual NPCs is
Fig 2 The level of Nup358 protein increases during skeletal myogenesis (A) C2C12 cells were grown in GM (0 days) or in DM for 2 or
5 days Immunoblotting analysis was performed with antibodies against a-tubulin, MyHC (muscle marker protein), Nup160, Nup98, Nup93, Nup62 and mAb414 (B–D) C2C12 myoblast cells (MB) and myotube cells (MT) were cultured on coverslips The Nup358 cells were fixed and stained with antibodies against Nup358, Nup98 or RanGAP1 (C) The cells were fixed with (D, lower panels) or without (D, upper panels) Triton X-100 and then permeabilized and stained with an antibody against CRM1 Cells were observed with confocal microscopy (LSM510) Scale bars = 5 lm (E) Myoblasts and myotubes were stained for RL1 NPC density was determined by counting the dots on the nuclear envelope (mean ± SD).
Trang 5closely correlated with the upper rim diameter We
conclude that the architecture of the NPC changes
during muscle differentiation and that Nup358 is a
critical determinant of the NPC architecture
Passive diffusion rate is the same in myoblasts
and myotubes
We next compared molecular trafficking through the
NPCs in myoblasts and myotubes because the NPC is
the only gateway for nuclear-cytoplasmic traffic We
observed both compositional and architectural
differ-ences between NPCs in these two cell types First, we
used fluorescence recovery after photobleaching analy-sis to examine the passive diffusion rate in C2C12 cells stably expressing enhanced green fluorescent protein (EGFP), a protein small enough to passively diffuse through the NPC The nuclear EGFP was photo-bleached, fluorescence recovery was monitored (Fig 5A) and the relative intensity of fluorescence in the nucleus was plotted over time (Fig 5B) In the early phase, the fluorescence recovery curves for myo-blasts and myotubes overlapped with each other, indi-cating that the passive diffusion rate through the NPC
is the same in myoblasts and myotubes We observed apparent differences in the fluorescence intensity at
Fig 3 Structural differences between myoblast and myotube NPCs (A) Denuded nuclei from myoblasts and myotubes visualized by AFM The area shown is 3 · 3 lm Scale bar = 0.5 lm (B) The percentage of unplugged and plugged NPCs (C) The NPC profile was taken from each NPC image and three measurements were obtained for each NPC profile O, I, and H represent the outer diameter, upper rim diameter and height, respectively Outer and upper rim diameters indicate the distance between two points on the NPC ring base and on the cyto-plasmic ring, respectively Height indicates the vertical distance between the NPC cytocyto-plasmic surface and the base Scale bar = 50 nm (D) The outer diameter was measured and plotted [mean ± SEM; 145.4 ± 2.4 nm, n = 58 (MB); 144.8 ± 3.7 nm, n = 23 (MT) for unplugged and 143.9 ± 2.2 nm, n = 29 (MB); 145.7 ± 2.5 nm, n = 37 (MT) for plugged] (E) The upper rim diameter was measured and plotted [mean ± SEM; 70.2 ± 1.9 nm, n = 58 (MB); 78.1 ± 2.3 nm, n = 23 (MT) for unplugged, and 76.1 ± 1.5 nm, n = 29 (MB); 83.2 ± 1.7 nm, n = 37 (MT) for plugged] (F) The height was measured and plotted [mean ± SEM; 5.5 ± 0.16 nm, n = 116 (MB); 5.43 ± 0.31 nm, n = 46 (MT) for unplugged, and 5.2 ± 0.22 nm, n = 58 (MB); 4.8 ± 0.17 nm, n = 74 (MT) for plugged] Data were statistically compared by Mann–Whitney U-tests.
Trang 6steady state, although this is likely a result of a
difference in the ratio of the bleached nuclear area to
the total cell volume (Doc S1B)
Nuclear export appears to be more active in
myotubes than in myoblasts
We next compared the nuclear transport activities
before and after muscle differentiation, using
microin-jection to examine the rate of active
nuclear-cytoplas-mic protein transport Either GST-SV40NLS-GFP
(NLS-GFP) or GST-GFP-RevNES-SV40NLS
(GFP-NES-NLS) recombinant protein was microinjected into
the cytoplasm of C2C12 cells, and the rate of nuclear
transport was observed using time-lapse fluorescence
microscopy The nuclear import efficiency of
NLS-GFP did not differ significantly in myoblasts and
myo-tubes (Fig 6A and Videos S1 and S2), suggesting that
nuclear import activity is almost identical in myoblasts
and myotubes
By contrast, the subcellular localization of
microin-jected GFP-NES-NLS was clearly different in the two
cell types In myoblasts, GFP-NES-NLS was primarily
localized to the nucleus, whereas, in myotubes, it was
evenly distributed between the nucleus and the
cyto-plasm (Fig 6B and Videos S3 and S4) It is important
to note that myotubes are polykaryons, whereas
myo-blasts have one nucleus To address the possible effect
of this difference on the subcellular distribution of
GFP-NES-NLS, myoblasts were fused using
polyethyl-ene glycol and GFP-NES-NLS was microinjected into
the cytoplasm of the fused cells (Fig 6D) The
locali-zation in the fused cells was very similar to that in
nor-mal myoblasts, indicating that the differential
distribution of GFP-NES-NLS observed in myoblasts
and myotubes is not simply a result of the difference
in nuclear number Additionally, treatment of myotu-bes with leptomycin B, a specific inhibitor of CRM1-mediated nuclear export, caused the accumulation of GFP-NES-NLS proteins in the nucleus (Fig 6C), showing that GFP-NES-NLS protein is actively shut-tled across the nuclear envelope in myotubes Taken together with the data from the NLS injections, these results strongly suggest that nuclear export efficiency is increased in myotubes relative to myoblasts
siRNA-mediated Nup358 depletion suppresses myotube formation
To determine whether Nup358 is necessary for C2C12 differentiation, we performed siRNA experiments using two different sets of siRNA duplexes against Nup358 As indicated, both Nup358 siRNAs effec-tively reduce Nup358 expression (Fig 7A) Immunoflu-orescence staining demonstrated a marked reduction in the nuclear rim signal for Nup358 in siNup358-treated cells (Fig 7B) Furthermore, the nuclear rim signal of RanGAP, which localizes to the nuclear envelope in a Nup358-dependent manner, was clearly diminished (Fig 7B) Other nucleoporins (Nup214, Nup98 and Nup62) remained unaltered at the nuclear rim in Nup358-depleted cells (Fig 7B), indicating that the depletion of Nup358 did not affect all NPC compo-nents
Microinjection analysis of the GFP-NES-NLS trans-port substrates suggested that nuclear imtrans-port and export of proteins was globally unaffected in Nup358-depleted cells (Figs 7C and S3) Under these condi-tions, the siRNA-treated C2C12 myoblasts were cultured in DM and then stained for myosin heavy chain (MyHC; green) and DNA (blue) As shown in Fig 7D, the efficiency of multinuclear myotube forma-tion was clearly decreased in Nup358-depleted cells, whereas the control cells (left two panels) efficiently underwent myotube formation These results indicate that Nup358 is specifically involved in muscle cell differentiation
Discussion
In the present study, we have demonstrated that both the composition and the architecture of NPCs are altered during the differentiation of C2C12 cells Our real-time PCR data indicate that the mRNA expres-sion levels of several nucleoporins differ between myo-blasts and myotubes (Fig 1) The mRNA levels of a large proportion of nucleoporins decreased during dif-ferentiation (i.e Nup107, Nup85, Nup160, Nup43, Nup37, Nup35, Nup205, Nup188, Pom121, Ndc1,
Fig 4 Nup358 depletion decreases the upper rim diameter of the
NPC C2C12 cells were transfected with nontargeting siRNA or
Nup358 siRNA oligonucleotides (siNup358-2) and incubated for
48 h The outer diameter (A), upper rim diameter (B) and height (C)
of the NPC were measured and shown in a graph as in Fig 3.
Trang 7Nup155, Nup54, Nup62, NupL1, Nup153 and Nup50).
Notably, most of the nucleoporins for which mRNA
expression was maintained during differentiation (e.g.,
Nup358, Nup214, Nup88 and NupL2 (CG1), Nup98,
Rae1 and Tpr) are involved in the nuclear export of
proteins or mRNA, indicating that the relative
expres-sion of the nucleoporins involved in nuclear export
increased during myogenesis Consistent with this, we
found that nuclear export in myotubes was more active
than that in myoblasts (Fig 6), whereas the rates of
nuclear import (Fig 6) and passive diffusion (Fig 5)
remained constant
We also showed protein expression levels of some
nucleoporins by immunoblotting (Fig 2) Although
real-time PCR showed decreased expression of Nup107
and Nup62, their protein expression appeared to be
equivalent, as indicated by immunoblotting An
expla-nation for such discrepancies of nucleoporin expression
levels between qPCR and immunoblot results was
offered by a recent study [14] demonstrating that some scaffold nucleoporins, such as members of the Nup107-160 subcomplex, have extremely long protein half-lives compared to other nucleoporins Thus, pro-teins levels remain stable, even if mRNA expression levels are decreased We specifically demonstrated that the copy number of Nup358 within individual NPCs increases during C2C12 muscle differentiation, at both mRNA (Fig 1) and protein (Fig 2) levels Nup358 plays a supportive role in CRM1-mediated nuclear export by stimulating CRM1 recycling [2] Nup358 provides a platform for the disassembly of CRM1 export complexes and the re-import of free CRM1 to the nucleus [19] As shown in Fig 2D, CRM1 signals
on the NPC were increased in myotubes compared to myoblasts, coinciding with the increase of Nup358
in myotube NPCs Although we were unable to demonstrate the direct effects of Nup358 on protein export in myotubes (Fig 7C), it is possible that the
Fig 5 The efficiency of passive diffusion though the NPC is the same in myoblasts and myotubes (A) C2C12 cells stably expressing EGFP were photobleached for 4 s and fluorescence recovery was monitored immediately after bleaching (B) Data were normalized to the pre-bleaching value of 100% and plotted against time [mean ± SEM; n = 5 (myoblasts), n = 12 (myotubes)].
Trang 8relative increase in several nucleoporins, which are
known to function in protein export, rather than the
increase of Nup358 alone, may cause the activation of
CRM1 in myotubes It is therefore likely that an
increase in Nup358 during myogenesis supports the
CRM1 activity and is involved in the activation of
nuclear protein export in myotubes
It is highly possible that a selective increase in the
efficiency of nuclear protein export triggers the
redistri-bution of a number of key molecules important for cell
differentiation and that the difference in nuclear export
activity between myotubes and myoblasts affects cell
differentiation from myoblasts to myotubes For
exam-ple, HDAC5 (histone deacetylase 5) is known to move
from the nucleus to the cytoplasm during myotube
for-mation in a differentiation-dependent manner and is
involved in the control of cellular differentiation in
C2C12 [20,21] Similarly, the regulation of
nuclear-cytoplasmic transport by importin-a subtype switching
was shown to trigger cell differentiation [22] Further
studies will be required to elucidate this possibility
The structure of the NPC changes to allow
adapta-tion to different cellular environments during
differen-tiation [8–10] In the present study, using AFM, we
found that the architecture of the NPC changes during
muscle differentiation and that Nup358 is a key
deter-minant of NPC architecture In addition, we showed
that the depletion of Nup358 prevented myotube for-mation (Fig 7) These results indicate that Nup358 affects the structure of the NPC and plays a role in muscle cell differentiation Recent evidence suggests that the NPCs are closely related to the transcriptional machinery [6] and thus it is also possible that the structural changes induced by Nup358 modulate the transcription of genes required for cell differentiation Furthermore, Nup358 is known to be a
multifunction-al protein that acts as both an multifunction-allosteric activator for kinesin [23] and a SUMO E3 ligase [24] Nup358 may therefore contribute to muscle differentiation in C2C12 cells in various ways
In conclusion, in the present study, we demonstrate that the NPC is functionally and structurally regulated during muscle cell differentiation and that Nup358 is required for muscle cell differentiation and also is likely involved in the remodeling of the NPC
Materials and methods
Cells and antibodies
C2C12 myoblast cells were cultured in GM consisting of DMEM (D5796; Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum The cells were maintained in an incubator at 5% CO2and 37C To induce differentiation,
Fig 6 Active nuclear export through NPCs
is facilitated in myotubes
GST-SV40NLS-GFP (A) or GST-GST-SV40NLS-GFP-RevNES-SV40NLS (B)
recombinant protein was microinjected with
an injection marker into the cytoplasm of
C2C12 cells and then incubated for 10 min
at 37 C (for NLS substrate, see Videos S1
and 2; for NES-NLS substrate, see Videos
S3 and 4) Scale bars = 50 lm (C)
GST-GFP-RevNES-SV40NLS protein was
microin-jected with an injection marker into the
cytoplasm and then incubated in
LMB-con-taining medium for 10 min at 37 C Scale
bar = 20 lm (D) C2C12 myoblast cells
were fused using polyethylene glycol (PEG).
Recombinant GST-GFP-RevNES-SV40NLS
protein was microinjected with an injection
marker into the cytoplasm of PEG-fused
myoblasts The cells were incubated in
DMEM for 10 min at 37 C Scale
bar = 20 lm.
Trang 9GM was replaced with DM consisting of DMEM with 3%
horse serum for 2–5 days For poly(ethylene glycol) fusion,
cells were incubated in 50% PEG-8000 in DMEM (D5796;
Sigma) for 1 min and washed with NaCl⁄ Pi Cells were
then cultured for 2 h in an incubator at 5% CO2 and
37C
Anti-Nup98 sera were kindly provided by Tachibana
et al [25] Antibodies purchased from commercial sources were: mAb414 (MMS-120p; Covance, Princeton, NJ, USA), anti-Nup214 (sc-26055; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Nup160 (sc-27401; Santa Cruz Biotechnology), anti-Nup107 (A301-787A; Bethyl,
Mont-Fig 7 Depletion of Nup358 from C2C12 cells by siRNA C2C12 cells were mock-transfected (no siRNA) or transfected with nontargeting siRNA or Nup358 siRNA oligonucleotides (siNup358-1, -2) and incubated for 48 h (A) Immunoblotting analysis with mAb414 was performed
48 h after transfection Nup62 was used as a loading control (B) Immunofluorescence was performed with antibodies against Nup358, Ran-GAP1, Nup214, Nup98 and Nup62 Scale bars = 10 lm (C) C2C12 cells were transfected with nontargeting siRNA or Nup358 siRNA oligo-nucleotide and incubated for 48 h Recombinant GST-GFP-RevNES-SV40NLS protein was microinjected with an injection marker into the cytoplasm of C2C12 cells transfected with siRNA oligonucleotides (D) C2C12 cells were mock-transfected (no siRNA) or transfected with nontargeting siRNA or Nup358 siRNA oligonucleotides (siNup358-1, -2) The cells were induced to differentiate 2 days after transfection and cultured in DM without transfection reagents for 2.5 days The cells were fixed and stained with anti-MyHC (green) and Hoechst 33342 (blue) Scale bar = 100 lm.
Trang 10gomery, TX, USA), anti-Nup358 (PA1-082; Affinity
BioRe-agents, Golden, CO, USA), anti-Nup93 (551976; BD
Pharmingen, San Diego, CA, USA), anti-Nup62 (N43620;
Transduction Lab, Lexington, KY, USA), anti-skeletal
myosin heavy chain (M4276; Sigma) and anti-a-tubulin
(T9026; Sigma)
Plasmids and recombinant proteins
For recombinant GST-GFP-NES-NLS, synthesized
oligo-nucleotides for RevNES (5¢-GATCTCCTCTTCAGCTA
CCACCGCTTGAGAGACTTACTCTTGATTGTAACGA
GGATA-3¢ and 5¢-AGCTTATCCTCGTTACAATCAA
GAGTAAGTCTCTCAAGCGGTGGTAGCTGAAGAGG
A-3¢) were annealed and inserted into the BglII and HindIII
sites of pEGFP-NLS The oligonucleotide fragment for
NES-NLS was flanked with BglII and BamHI, and then
inserted into the BamHI site of pGEX-hGFP Recombinant
GST-GFP-NES-NLS and GST-NLS-GFP proteins were
expressed and purified as described previously [3]
Real-time PCR
Total RNA from C2C12 cells was purified using the RNeasy
kit (Qiagen, Valencia, CA, USA) and cDNA was synthesized
using SuperScript III reverse transcriptase (Invitrogen,
Carlsbad, CA, USA) Quantitative real-time PCR was
per-formed using FastStart SYBR Green Master (Roche, Basel,
Switzerland) with an ABI PRISM 7900HT (Applied
Biosys-tems, Foster City, CA, USA) Primer sets were designed by
Primer Bank [26] and are listed in Table S1
RNA interference
Cells were either mock-transfected or transfected with
syn-thesized siRNAs [nontargeting,
5¢-GCAGCAUCUUUAAU-GAAUAdTdT-3¢ and 5¢-AUAAGUAAUUUCUACGACG
dTdT-3¢; Nup358, 5¢-CCAGUCACUUACAAUUAAAd
TdT-3¢ and 5¢-UUUAAUUGUAAGUGACUGGdTdT-3¢
(siNup358-1), 5¢-UGAAGCACAUGCUAUAAAAdTdT-3¢
and 5¢-UUUUAUAGCAUGUGCUUCAdTdT-3¢
(siNup-358-2)] Transfection with a specific siRNA was performed
using RNAi Max (Invitrogen) in accordance with the
manu-facturer’s instructions The transfected cells were harvested
48 h (for Nup358) or 72 h (for Nup107) after transfection
and fixed for immunofluorescence, lysed for immunoblotting
or used for induced myotube formation in DM without
siR-NA reagents
Immunoblotting and immunofluorescence
For immunoblotting, C2C12 cells were lysed in NP-40
buf-fer (150 mm sodium chloride, 1% NP-40 and 50 mm Tris,
pH 8.0), analyzed by SDS⁄ PAGE and immunoblotted
using Immobilon-P (Millipore, Billerica, MA, USA) with
standard semidry methods The ECL detection reagent (GE Healthcare, Milwaukee, WI, USA) was used for pro-tein visualization
For immunofluorescence, C2C12 cells were grown on glass coverslips (Matsunami, Osaka, Japan) The cells were washed twice in NaCl⁄ Pi, fixed with 3.7% formaldehyde in NaCl⁄ Pi for 10 min, and permeabilized with 0.5% Triton X-100 in NaCl⁄ Pifor 5 min For Nup358 staining, the cells were permeabilized simultaneously with fixation After incu-bation with 5% skim milk in NaCl⁄ Pi, antibodies were incubated with the cells for 1 h The cells were washed with NaCl⁄ Piand incubated with Alexa 488- or 546-conjugated antibodies for 1 h Samples were then thoroughly washed with NaCl⁄ Pi and mounted in NaCl⁄ Pi-glycerol plus 1,4-diazabicyclo[2.2.2]octane DNA was counterstained with Hoechst 33342 All procedures for immunofluorescence were performed at room temperature The stained cells were observed with a laser-scanning LSM510 microscope (Carl Zeiss, Oberkochen, Germany)
Calculation of NPC density
For the measurement of NPC density, methanol⁄ acetone-fixed cells were stained with mAb414 NPC number in a
2· 2 lm area was counted for both myoblast and myotube nuclei The fluorescence intensity of a spot was used to deter-mine how many pores existed in a diffraction-limited area
AFM
AFM was performed in contact mode with a Molecular Force Probe 3D (MFP-3D; Asylum Research, Santa Bar-bara, CA, USA) using a microcantilever OMCL TR-400 PSA (Olympus, Tokyo, Japan) To prepare the AFM sam-ple, myoblast and myotube cells were cultured on an eight-well slide glass (MP Biomedicals, LLC, Santa Ana, CA, USA) The cells were treated with hypotonic buffer (40 mm NaCl, 5.4 mm KCl, 0.8 mm MgCl, 1 mm NaH2PO4 and
10 mm Hepes, pH 7.4) for 3 min and then treated with buf-fer X (1% Triton X-100, 75 mm KCL, 15 mm NaCl and
20 mm Mops, pH 7.4) for 6 min Denuded nuclei were fur-ther washed with buffer W (15.5 mm NaCl, 70 mm KCl, 6.5 mm K2HPO4and 1.5 mm NaH2PO4) and fixed with 1% glutaraldehyde and 3.7% formaldehyde in NaCl⁄ Pi for
15 min Finally, the nuclei were rinsed with Milli-Q water (Millipore) and air-dried Data were analyzed with Igor-Pro (Wavemetrix Inc., Portland, OR, USA) and statistical anal-yses were performed with GraphPad Prism 4 (GraphPad Software Inc., San Diego, CA, USA)
Fluorescence recovery after photobleaching
To establish stable cell lines, transfection was carried out with Effectene Transfection Reagent (Qiagen) Ssp1-digested pIRES-puro3-EGFP was transfected into C2C12