Live imaging system for visualizing nuclear pore complex
(NPC) formation during interphase in mammalian cells
Haruki Iino1, Kazuhiro Maeshima1a*, Reiko Nakatomi2, Shingo Kose1,
Tsutomu Hashikawa2, Taro Tachibana3 and Naoko Imamoto1,*
1
Cellular Dynamics Laboratory, RIKEN Advanced Science Institute, Wako, Saitama 351-0198, Japan
Support Unit for Neuromorphological Analysis, Brain Science Institute, RIKEN, Wako, Saitama 351-0198, Japan
3
Department of Bioengineering, Graduate School of Engineering Osaka City University, Osaka 558-8585, Japan
2
Nuclear pore complexes (NPCs) are ‘supramolecular complexes’ on the nuclear envelope
assembled from multiple copies of approximately 30 different proteins called nucleoporins
(Nups) that provide aqueous channels for nucleocytoplasmic transport during interphase.
Although the structural aspects of NPCs have been characterized in detail, NPC formation and
its regulation, especially during interphase, are poorly understood. In this study, using the
temperature-sensitive RCC1 mutant tsBN2, a baby hamster kidney 21 cell line, we found that
a lack of RCC1 activity inhibited NPC formation during interphase, suggesting that RanGTP
is required for NPC formation during interphase in mammalian cells. Utilizing the reversible
RCC1 activity in tsBN2 cells, we established a live-cell system that allows for the inhibition or
initiation of NPC formation by changes in temperature. Our system enables the examination
of NPC formation during interphase in living cells. As a lack of RCC1 decreased some Nups
containing unstructured phenylalanine–glycine repeats in the NPC structure, we propose that
RCC1 is also involved in maintaining NPC integrity during interphase in mammalian cells.
Introduction
In eukaryotic cells, genomic DNA and the cytoplasm
are physically separated by a lipid bilayer known as
the nuclear envelope (NE) (Hetzer et al. 2005). This
enables eukaryotic cells to regulate gene expression in
a spatio-temporal manner, which may have contributed to the diversification of eukaryotes. Nuclear
pore complexes (NPCs) are embedded in the NE at
sites of fusion between the outer and inner nuclear
membranes (ONM and INM, respectively) (Tran &
Wente 2006; Antonin et al. 2008; D’Angelo & Hetzer 2008). NPCs provide aqueous channels for
nucleocytoplasmic transport during interphase (Feldh-
Communicated by : Hiroyuki Araki
*Correspondence: [email protected] or nimamoto@
riken.jp
a
Present address : Biological Macromolecules Laboratory,
Structural Biology Center, National Institute of Genetics,
Yata 1111, Mishima, Shizuoka 411-8540, Japan.
err et al. 1984; Görlich & Kutay 1999; Imamoto
2000; Stewart 2007).
The NPC is a large protein complex with an estimated molecular mass of 60–125 MDa in vertebrates
and 40–60 MDa in yeast. Despite the difference in
their molecular masses, the fundamental architecture
of NPCs is well conserved among eukaryotes. In the
plane of the NE, an eightfold symmetrical perpendicular central framework, consisting of a cytoplasmic
ring, spokes, and nuclear ring, form a central channel
where bidirectional transport occurs (Antonin et al.
2008; D’Angelo & Hetzer 2008; Beck & Medalia
2008). Eight filaments attached to the cytoplasmic
ring, called cytoplasmic fibrils, have loose ends, while
eight filaments extending from the nuclear ring are
attached to the distal ring forming a structure known
as a nuclear basket (Antonin et al. 2008; D’Angelo &
Hetzer 2008; Beck & Medalia 2008). Proteomic analyses have identified most of the components of vertebrate and yeast NPCs, and they indicate that NPCs
are composed of multiple copies of approximately 30
different proteins, known as nucleoporins (Nups)
DOI: 10.1111/j.1365-2443.2010.01406.x
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2010)
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H Iino et al.
(Rout et al. 2000; Cronshaw et al. 2002). Biochemical
studies have shown that Nups form various subcomplexes, which are considered to be the building
blocks of NPCs (Alber et al. 2007a,b). The central
channel in a NPC is filled with unstructured polypeptides containing hydrophobic phenylalanine–glycine (FG) repeats, and it serves as a selective transport
barrier (Tran & Wente 2006; Antonin et al. 2008;
D’Angelo & Hetzer 2008). In vertebrates, the central
channel is surrounded by a framework consisting of
the Nup107-160 complex, which plays an essential
role in NPC architecture (Harel et al. 2003b; Walther
et al. 2003a). Components of the Nup107-160 complex, including Nup107, are retained stably in the
NPC with a resident time of tens of hours (Rabut
et al. 2004). Vertebrate NPCs contain three integral
membrane proteins (Ndc1, gp210, and Pom121),
which anchor the NPC scaffold to the nuclear membrane (Antonin et al. 2005; Stavru et al. 2006; Madrid
et al. 2006; Funakoshi et al. 2007; Mansfeld et al.
2006).
Although the structure of the NPC has been studied intensively, the mechanism of assembly remains
largely unknown. In higher eukaryotic cells, which
have open mitosis, NPC assembly occurs twice during the cell cycle: postmitosis and during interphase
(Antonin et al. 2008; D’Angelo & Hetzer 2008).
Postmitotic NPC assembly is a stepwise process, triggered by the recruitment of Nup107-160 complexes
and integral membrane Nups, including Pom121 and
Ndc1, onto mitotic chromosomes shortly after the
onset of anaphase (Bodoor et al. 1999; Dultz et al.
2008). NPC assembly is completed upon addition of
the nuclear basket and cytoplasmic fibrils (Rabut et al.
2004). Compared to postmitotic NPC assembly,
much less is known about the molecular mechanism
of NPC formation during interphase.
The number of NPCs doubles during interphase,
both in mammals and in yeast, suggesting that NPC
formation on the NE is an evolutionarily conserved
process (Maul et al. 1972; Maeshima et al. 2006;
Winey et al. 1997). Using a Xenopus egg extract system, Angelo et al. showed that NPC formation occurs
during interphase by a de novo mechanism (D’Angelo
et al. 2006). The small GTPase Ran is a common factor that reportedly plays a key role in NPC assembly
both in yeast and vertebrates. In Xenopus eggs, the
production of RanGTP was shown to be required for
both postmitotic NPC assembly and NPC formation
during interphase (Walther et al. 2003b; D’Angelo
et al. 2006). The exact role of Ran in NPC formation
during interphase is currently unknown; however, it
2
Genes to Cells (2010)
is likely that Ran operates in NPC formation by
reversing the negative effect of importin b (D’Angelo
et al. 2006; Harel et al. 2003a; Ryan et al. 2007).
The RanGTPase cycle is regulated by two Ranspecific regulators: RCC1 and RanGTPase-activating
protein (RanGAP) (Görlich & Kutay 1999; Nishimoto 2000; Stewart 2007). RCC1 is the guanine
nucleotide exchange factor (GEF) for Ran and a
chromatin-binding protein. RCC1 localizes within
the nucleus, while RanGAP is a cytoplasmic protein.
As a result, the concentration of RanGTP is high in
the nucleus, whereas it is low in the cytoplasm (Kalab
et al. 2006). The distinct RanGTP concentrations
between the nucleus and cytoplasm were discovered
through the study of importin-based nucleocytoplasmic transport. However, the role of Ran in NPC
formation during interphase was suggested to be independent of its role in transport, because cytoplasmic
RanGTP is thought to play a direct role in NPC formation (D’Angelo et al. 2006; Ryan et al. 2007).
In this study, we demonstrated that RanGTP
production is required for NPC formation during
interphase in mammalian cells using the temperaturesensitive RCC1 mutant tsBN2, a baby hamster kidney (BHK)21 cell line (Nishimoto et al. 1978). In
tsBN2 cells, RCC1 was degraded immediately upon
an upward temperature shift to a non-permissive temperature, while a downward temperature shift to a
permissive temperature restored RCC1 activity
(Nishitani et al. 1991; Arnaoutov et al. 2005). Immunofluorescence and photobleaching assays showed that
a loss of RCC1 inhibited NPC formation during
interphase. We took advantage of the reversible
RCC1 activity in tsBN2 cells to establish a live-cell
system that allows for the inhibition or initiation of
NPC formation by a change in temperature. Furthermore, as the loss of RCC1 caused a decrease in some
FG-Nups in NPCs, we propose that RCC1 is
involved in the maintenance of NPC integrity during
interphase. Finally, the possible roles of RCC1 in
NPC maintenance during interphase are discussed.
Results
RCC1 is necessary for NPC formation during
interphase in mammalian cells
To elucidate the role of RCC1 in NPC formation
during interphase, we used tsBN2, a temperature-sensitive RCC1 mutant BHK21 cell line (Nishimoto
et al. 1978). Because of a point mutation in RCC1
(Uchida et al. 1990), RCC1 is degraded immediately
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Visualizing nuclear pore complex formation
at non-permissive temperatures (Fig. S1A in Supporting
Information and Fig. 3D) (Nishitani et al. 1991; Tachibana et al. 1994). The tsBN2 cells grow normally at
permissive temperatures, whereas at non-permissive
temperatures, they arrest in G1 phase (Figs S1B and
S3D in Supporting Information) or undergo premature chromosome condensation in S-G2 phase. These
characteristics of tsBN2 cells are useful for elucidating
(A)
the mechanism of formation of NPCs, which double
in number during the transition from G1 to S phase.
First, we tested the effect of RCC1 depletion on
NPC formation during interphase in fixed cells. To
synchronize tsBN2 cells in G1 phase, cells arrested in
mitosis by nocodazole were released and incubated
for 2 h at a permissive temperature (32.0 C). The
cells were then incubated at either the permissive or a
Non-permissive
39.7 °C
Permissive
Mitotic arrest
mAb414 (NPCs)
mAb414 (NPCs)
Non-permissive
(39.7 °C)
Non-permissive
(39.7 °C)
(D)
(NPCs/µm2)
(C)
Permissive
(32.0 °C)
RCC1
Permissive
(32.0 °C)
(B)
32.0 °C
18 h
4h
NPC Density
(E)
(µm3)
Nuclear Volume
4000
Enhanced
(F)
Total Number of NPCs
6000
6
5000
3000
5
4000
2000
4
3000
3
1000
Permissive Non-permissive
(32.0 °C)
(39.7 °C)
2000
Permissive Non-permissive
(32.0 °C)
(39.7 °C)
Permissive Non-permissive
(32.0 °C)
(39.7 °C)
Figure 1 Loss of RCC1 in tsBN2 cells affects NPC formation during interphase. (A) Schematic of the temperature-shift experiment. Synchronized mitotic tsBN2 cells were released and incubated at a permissive temperature (32.0 C) for 4 h. The cells were
then cultured for 18 h at the permissive or non-permissive temperature (39.7 C). For the cells at the permissive temperature,
5 lg ⁄ ml aphidicolin was added to arrest the cells in G1 ⁄ S phase (Pedrali-Noy et al. 1980). (B) RCC1 (left) and NPCs (right) in
the cells grown at the permissive (top) and non-permissive temperatures (bottom) were visualized by immunostaining. The images
shown are of the same intensity. The RCC1 signal in the cells cultured at the non-permissive temperature (bottom) was much
lower than that in the cells cultured at the non-permissive temperature (top). Bar, 20 lm. (C) High-magnification images of the
mAb414-labeled cells. The signal intensity of the image for the non-permissive cells (bottom) was enhanced to improve visualization. The insets show magnified images of single NPCs. Numerous cytoplasmic foci were observed in the cells cultured at the
non-permissive temperature. (D–F) Scatter plots of the measured NPC density (Examined nuclei number, N = 10), nuclear
volume (N = 20), and total number of NPCs per nucleus (N = 20) are shown (for details of the calculations, see Experimental
procedures). Bars indicate the mean values.
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Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
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H Iino et al.
non-permissive temperature (39.7 C) for 18 h, and
then fixed with formaldehyde (FA) (Fig. 1A). Aphidicolin, an inhibitor of DNA polymerase a ⁄ d, was
added to control cells at the permissive temperature
to inhibit further cell cycle progression (Pedrali-Noy
et al. 1980). The NPCs in the cells were then visualized by immunostaining with mAb414, which recognizes the FG repeats in some Nups. Interestingly, the
signal intensity of the NPCs from the cells incubated
at the non-permissive temperature was significantly
decreased (Fig. 1B). Moreover, many mAb414labeled foci were detected in the cytoplasm of the
cells grown at the non-permissive temperature,
although a few were detected in the cells grown at
the permissive temperature (Fig. 1B,C). Cytoplasmic
foci were observed in 91.9% of tsBN2 cells grown at
the non-permissive temperature, whereas only 3.4%
of the cells grown at the permissive temperature had
such foci (Fig. S2A in Supporting Information). As a
control, the effect of temperature on the existence of
cytoplasmic foci and NPC staining was examined in
BHK21 cells (the parental line of tsBN2). A few foci
were detected in the cytoplasm of BHK21 cells
grown at 32.0 and 39.7 C; however, neither temperature-specific cytoplasmic foci nor weak NPC
staining was observed (Figs S2B and S3C in Supporting Information).
Next, using signal-enhanced images, the NPC
density in cells grown at the permissive or non-permissive temperature was examined (Fig. 1C; for
details on how the NPC density was quantified, see
the Experimental procedures). By comparison, NPC
density in the cells grown at the non-permissive temperature was lower than that in the cells grown at the
permissive one (Fig. 1C and Inset). The average
NPC densities at the permissive and non-permissive
temperatures were 5.373 ± 0.248 and 3.943 ± 0.487
[standard deviation (SD)] NPCs ⁄ lm2, respectively
(Fig. 1D). The average NPC density at the permissive
temperature was significantly higher than that at the
non-permissive temperature (P-value <0.0001). The
average measured nuclear volumes at the permissive
and non-permissive temperatures were similar
(P-value = 0.0982): 2385 ± 228 and 2204 ± 572
lm3, respectively (Fig. 1E). The estimated average
total numbers of NPCs at the permissive temperature
(4635 ± 296 NPCs) were thus significantly higher
than that at the non-permissive temperature
(3213 ± 552 NPCs) (P-value <0.0001) (Fig. 1F).
These findings indicate that RCC1 is necessary for
NPC formation during interphase in mammalian
cells.
4
Genes to Cells (2010)
Visualization of newly formed NPCs during
interphase
As described above, we manually counted the number
of NPCs to determine the NPC density; however, this
procedure is laborious and can lead to such errors as
underestimation, both because the relatively low
resolution of light microscopes can be insufficient for
distinguishing adjacent NPCs and because nuclear
volume increases as the cell cycle progresses. We thus
attempted to establish a live-cell system for visualizing
NPC formation during interphase so as to obtain
additional spatio-temporal information (Fig. 2B,C). Our
system utilizes fluorescently labeled Nup107, a component of NPC scaffolds, which is retained stably in
NPCs and which has the longest resident time among
all Nups examined thus far (>40 h) (Rabut et al. 2004;
D’Angelo et al. 2009). Nup107 fused with the bright
yellow fluorescent protein Venus (Nagai et al. 2002)
was stably expressed in tsBN2 cells (tsBN2Venus-NUP107
cells; see Experimental procedures). On the nuclear
surface in the tsBN2Venus-NUP107 cells, NPCs were
observed as individual dots of Venus fluorescence. To
distinguish newly formed NPCs from existing ones,
the existing NPC signals from about half of the nuclear
surface were photobleached using a 488-nm laser
(Fig. 2A). Several hours later, newly formed dots corresponding to Venus fluorescence were detected in the
bleached region (Fig. 2A). These dots likely represent
new NPCs that were formed during interphase,
because Nup107 is a scaffold protein and one of the
most immobile NPC components (Rabut et al. 2004).
It was previously shown in a myoblast fusion system
that Nup107 does not exchange once incorporated
into NPCs (D’Angelo et al. 2009). To analyze NPC
formation, we systematically defined the Venus fluorescence signals (Fig. S3A in Supporting Information;
see Experimental procedures) and examined their density every 3 h (Fig. 2B). The number of Venus dots
increased in a time-dependent manner (Fig. 2D). To
confirm that the formation of the fluorescent dots
indeed represented NPC formation during interphase,
the bleached nuclear surfaces were immunolabeled
with anti-Nup153 antibodies. The number of new
yellow fluorescent dots was clearly decreased compared
to the Nup153 signal, which reflects the total
number of NPCs on the nuclear surface (Fig. S3B in
Supporting Information). Thus, our system may be
used to observe NPCs formed during interphase.
We next tested the effect of RCC1 depletion on
NPC formation using the live-cell system described
above. Mitotically synchronized tsBN2Venus-NUP107
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Visualizing nuclear pore complex formation
(A)
Photobleach
Mitotic arrest
Pre-bleach
Post-bleach
Permissive
12 h
4h
Post-bleach
12 h after bleach
3
2
1
(B)
4h
Post-bleach
(C)
12 h
9h
6h
3h
(D)
Photobleach
Non
NonNon-permissive
(NPCs/µm 2 )
Mitotic arrest
4h
Pre-bleach
1
4h
12 h
Post-bleach
2
NPC density
3
Permissive
12 h after bleach
2
3
1
0
Nonpermissive
+3 h +6 h +9 h +12 h
Figure 2 Visualization of newly formed NPCs and the involvement of RCC1 in NPC formation during interphase. (A) Design
of the photobleaching experiment (upper panel). On the nuclear surface, existing NPCs were observed as bright dots of Venus fluorescence in tsBN2Venus-Nup107 cells (Prebleach). NPC signals in the region of interest in the G1 cells were photobleached (Postbleach). (B) Fluorescent dot formation in the bleached region was observed over time. The insets are magnified images of NPCs.
(C) Involvement of RCC1 activity in NPC formation. The upper illustration shows our experimental design. Mitotic tsBN2 cells
were released and a region of interest on the nuclear surface was photobleached at the non-permissive temperature. After photobleaching, Venus dots were not detected in the bleached areas in the cells incubated at the non-permissive temperature. (D) The
results given in (B) are shown as bar graphs, with the standard deviation indicated by error bars.
cells were released, incubated for 4 h at a permissive
temperature, and then shifted to a non-permissive
one (Fig. 2C). Photobleaching was performed at 4 h
after the upward temperature shift. The bleached cells
were then further incubated for 12 h at the non-permissive temperature. Almost no new NPC formation
was detected in the bleached areas under these
conditions (Fig. 2C). In contrast, when the cells were
incubated at the permissive temperature, many fluorescent dots corresponding to NPCs were detected in
the bleached areas (Fig. 2A). This indicates that
RCC1 is required for NPC formation during interphase in living mammalian cells.
Manipulation of NPC formation during interphase
by a temperature shift
Using the live-cell system described above, we
observed the inhibition of NPC formation at the
non-permissive temperature. Thus, we examined
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2010)
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H Iino et al.
of newly formed NPCs increased in a time-dependent manner (Fig. 3B,C) as seen in Fig. 2D. Thus,
we concluded that NPC formation began again after
the shift from a non-permissive temperature to a permissive one.
To confirm these data, the recovery rate of RCC1
activity was examined after the temperature shifts
described above. We first examined the amount of
active RCC1 after a downward temperature shift
(Fig. 3D–F). After mitotic release, synchronized
tsBN2 cells were incubated at the non-permissive
temperature for 12 h, and then the temperature was
whether if the temperature was shifted down to the
permissive value from the non-permissive one, NPC
formation would resume. To test this possibility, after
their release from mitosis, synchronized tsBN2 cells
were incubated at the non-permissive temperature for
12 h (Fig. 3A). Existing NPC signals on the nuclear
surface of interest were then bleached, and the temperature was shifted down to the permissive value
(Fig. 3A). Twelve hours after photobleaching, newly
formed NPCs were detected in the bleached area as
bright fluorescent dots (Fig. 3A). Time-lapse monitoring of the bleached area showed that the number
(A)
Photobleach
Non
NonNon-permissive
Pre-bleach
Post-bleach 12 h after bleach
3
2
1
Permissive
Mitotic arrest
4h
12 h
12 h
Post-bleach
+3 h
Pe
Pre-bleach
rm
i
No ssiv
e
npe
rm
+4
is
h
si
ve
+8
h
+1
2
h
(D)
(B)
RCC1
β-actin
+6 h
+12 h
+9 h
(E)
NonPermissive permissive
(C)
(NPCs/µm 2 )
NPC density
3
+6h
+1h
+12h
(F)
Temperature-shift up Temperature-shift down
(ratio)
1.2
2
Immunofluorescence
Immunoblotting
1
0.8
0.6
1
0.4
0.2
0
0
Nonpermissive
+3 h
+6 h
+9 h +12 h
5
10 0 3
Non-permissive
8
13
(h)
Permissive
Figure 3 Manipulation of NPC formation by a temperature shift. (A) Experimental design for the temperature-shift experiment.
Mitotic tsBN2Venus-Nup107 cells were released and incubated at the non-permissive temperature for 12 h. The existing fluorescent
dots on the nuclear surface of interest were bleached. Cells were incubated at the permissive temperature for an additional 12 h
and then observed. NPC formation was detected after 12 h at the permissive temperature (right image). (B) The number of newly
formed NPCs increased in a time-dependent manner. (C) The NPC density was examined and plotted. Error bars show the standard deviation. (D) and (E) The amount of RCC1 was examined after the temperature shift. After mitotic release, synchronized
tsBN2 cells were incubated at the non-permissive temperature for 12 h. After a downward shift to the permissive temperature
(0 h), the amount of RCC1 was monitored over time by immunoblotting (D) and immunofluorescence staining (E). Whole-cell
lysates (equivalent to 2 · 104 cells) prepared at the indicated time points were loaded in each lane. (F) Graphical representation of
(D) and (E). The amount of RCC1 was plotted as the ratio to the control value. The standard deviation is indicated by error bars.
6
Genes to Cells (2010)
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Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Visualizing nuclear pore complex formation
shifted down to the permissive value (0 h). The
amount of RCC1 activity was monitored over time
by immunofluorescence staining and immunoblotting.
Both analyses showed that RCC1 signals increased
after the downward temperature shift (Fig. 3F),
confirming that new NPC formation after the shift
was dependent on the recovery of RCC1 in tsBN2
cells. In addition, we verified that the cell cycle
arrest at G1 phase was also released upon the
downward temperature shift (Fig. S3D in Supporting
Information).
Our results suggest that NPC formation during
interphase could be manipulated by a temperature shift
in tsBN2 cells. To demonstrate the inhibition and
recovery of NPC formation (Fig. S4A in Supporting
Information), tsBN2Venus-NUP107 cells synchronized in
G1 phase and grown at the non-permissive temperature were photobleached. The temperature was then
kept at the non-permissive value for 10 h before being
shifted down to the permissive value and maintained
for another 12 h. Newly formed NPCs were
quantified manually by counting of the NPC number
(Fig. S4C in Supporting Information). At the
non-permissive temperature, few NPCs were detected
[Fig. S4B,C in Supporting Information; NPC density:
0.248 ± 0.227 (SD) NPCs ⁄ lm2], but after the shift
down to the permissive temperature, the NPC density
increased to 2.861 ± 0.412 NPCs ⁄ lm2 after 12 h
(Fig. S4B,C in Supporting Information). Thus, our
tsBN2 system makes it possible to inhibit and restart
NPC formation during interphase by a shift in
temperature.
Structural integrity of NPCs is affected by RCC1
activity
In tsBN2 cells incubated at the non-permissive temperature, the signal intensity of NPCs labeled with
mAb414 was decreased significantly (Fig. 1B),
whereas this impairment was not observed in BHK21
cells (Fig. S3C in Supporting Information). We thus
tested whether the signal intensity could be recovered
after a downward shift to the permissive temperature.
After 12 h at the non-permissive temperature, a significant decrease in the intensity of mAb414 staining
was observed (P-value <0.0001) (Fig. 4A). However,
when the temperature was shifted down to the permissive value, its intensity was almost completely
recovered (Fig. 4B). The signal intensity of NPCs
labeled with anti-Nup153 antibodies was also examined. Nup153 is a constituent of the NPC nuclear
basket, and FG-Nups have a very high turnover rate
(Rabut et al. 2004). The staining intensity of Nup153
declined at the non-permissive temperature (P-value
<0.0001), but recovered upon a downward shift to
the permissive temperature, as was observed using
mAb414 (Fig. 4B).
To examine this finding in living cells, tsBN2 cells
stably expressing Venus-Nup153 were established
(tsBN2Venus-Nup153). The change in intensity of
Venus-Nup153 upon the temperature shift was analyzed by live-cell imaging. Consistent with our previous results, the intensity of Nup153 decreased at the
non-permissive temperature (Fig. S6B in Supporting
Information), suggesting that RCC1 maintains the
structural integrity of NPCs during interphase.
To further investigate NPC structure at the nonpermissive temperature, we next stained tsBN2Venus-Nup107
cells with mAb414 (Fig. 5A). Although mAb414
staining at the non-permissive temperature was
reduced by 35.5 ± 11.9 (SD) % (Fig. 5A,B), the
intensity of Venus-Nup107 was decreased by only
11.9 ± 22.6% (Fig. 5A,B). This suggests that significant fractions of scaffold Nups were retained in the
NPCs even at the non-permissive temperature. To
confirm this idea, we observed NPC structure
directly using cryo-scanning electron microscopy
(SEM). Synchronized tsBN2 cells incubated for 12 h
at the permissive and non-permissive temperatures
were freeze-fractured, and the nuclear surfaces in the
fractured cells were observed by SEM. Although a
temperature-dependent difference in NPC density
was observed (Fig. 5C, lower images), no notable
difference in overall NPC structure was detected
(Fig. 5C, upper images). This suggests that RCC1
depletion does not significantly affect the organization
of scaffold Nups in NPCs.
Discussion
tsBN2 cells: a novel system for studying NPC
formation during interphase
In this study, a novel live-cell system was established
for analyzing NPC formation during interphase. In
this system, we took advantage of the unique characteristics of tsBN2 cells, a temperature-sensitive
mutant of RCC1(Nishimoto et al. 1978). A key feature of tsBN2 cells is the rapid degradation of
RCC1 at non-permissive temperatures (Fig. S1A in
Supporting Information) (Nishitani et al. 1991).
Although several methods may be used to eliminate
RCC1 function, the rapid depletion of RCC1 in
tsBN2 cells is noteworthy. Based on this feature, we
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2010)
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H Iino et al.
(A)
Non-permissive
Non-permissive Permissive 2 h
Nup153
mAb414 (NPC)
Permissive
mAb414
Nup153
1.4
Relative intensity
Relative intensity
(B)
1
0.6
0.2
1.4
1
0.6
0.2
NonPermissive Nonpermissive permissive
NonPermissive Nonpermissive permissive
permissive
2h
permissive
2h
Figure 4 Rapid recovery of the signal intensity of mAb414
and Nup153 upon the shift from a non-permissive to a permissive temperature. (A) Immunofluorescence staining for
mAb414 (top) and Nup153 (bottom) in the cells at the permissive temperature (left), non-permissive temperature (center), and following a shift to the permissive temperature from
the non-permissive value (right) is shown. The images are
shown using the same intensity scale for comparison. Although
the intensities of the mAb414 and Nup153 signals at the
non-permissive temperature were much lower than those at
the permissive one, their intensities were increased significantly
after incubation at the permissive temperature for 2 h. Scale
bar, 10 lm. (B) Relative changes in intensity upon the temperature shift were quantified. The intensity of each NPC
labeled with mAb414 (left) or Nup153 (right) in the cells
(Examined nuclei number, N = 10) was measured and plotted
(upper panels). Each circle in the graph indicates the mean
intensity on a single nuclear surface. The bars indicate
the mean values. The mean values of relative intensities of
mAb414 at the permissive, non-permissive, and non-permissive-to-permissive temperatures were 1 ± 0.138 standard
deviation (SD), 0.645 ± 0.119, and 0.958 ± 0.220, respectively. Those of Nup153 at the permissive, non-permissive,
and non-permissive-to-permissive temperatures were 1 ±
0.121, 0.530 ± 0.126, and 0.857 ± 0.137, respectively. The
same data sets are shown as bar graphs for comparison (bottom
panel). SD is indicated by error bars.
Relative intensity
mAb414
Nup153
1.2
Live-cell system for observing NPC formation
during interphase
0.8
Compared to our understanding of postmitotic NPC
assembly, our knowledge of NPC formation during
interphase is limited (Antonin et al. 2008; D’Angelo
& Hetzer 2008), presumably because of the lack of a
practical system for analysis. The most serious obstacle
to visualizing NPC formation during interphase is
that NPCs already exist after NE formation, and these
existing NPCs usually interfere with the observation
of newly formed NPCs. To distinguish new NPCs
from existing ones, we applied a photobleaching
technique to the tsBN2Venus-Nup107 cell line. The signals from the existing NPCs were almost completely
abrogated by photobleaching. Several hours later, we
detected newly formed dots of Venus fluorescence in
the bleached region (Fig. 2). If Nup107 from
unlabeled NPCs can be exchanged for free Venus–
Nup107, then the labeling of an NPC would not
necessarily mean that it is newly formed. However,
this is unlikely. First, Nup107 is a component of
NPC scaffolds, which are retained stably in NPCs
with a residence time exceeding 40 h (Rabut et al.
2004); moreover, it was shown that Nup107 could
not be exchanged once incorporated into NPCs
0.4
0
Permissive
Nonpermissive
Nonpermissive
permissive
2h
found defects in NPC formation during interphase
as a result of the loss of RCC1, not only in the
fixed cells but also in living cells (Figs 1 and 2).
Another notable finding is the reversibility of RCC1
activity in tsBN2 cells (Fig. 3D–F); the amount of
RCC1 was restored following a shift in temperature.
Under these conditions, NPC formation could be
restarted. Using these unique characteristics of tsBN2
cells, we were able to establish a live-cell system for
the observation of NPC formation during interphase
(Fig. 3).
8
Genes to Cells (2010)
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Visualizing nuclear pore complex formation
(C)
(A)
mAb414
Nup107+mAb414
Permissive
Non-permissive
Non-permissive
Permissive
Nup107
10 µm
400 nm
mAb414
Nup107
Relative intensity
(B)
1.4
1
0.6
Permissive
NonNonpermissive permissive
permissive
2h
Figure 5 Most Nup107 is retained in the NPCs at a non-permissive temperature. (A) tsBN2Venus-Nup107 cells were incubated at a
permissive (top rows) or non-permissive temperature (bottom rows) for 12 h and immunostained with mAb414. The images are
shown on the same intensity scale for comparison. The fluorescence intensity of Nup107 was maintained at the non-permissive
temperature, whereas the intensity of mAb414 staining decreased. (B) The signal intensities of Venus-Nup107 and mAb414 in ten
cells were quantified and are shown as bar graphs; error bars show standard deviation. (C) Direct observation of NPCs in cells
grown at the permissive or non-permissive temperature by cryo-SEM. Synchronized tsBN2 cells incubated at the permissive (left)
or non-permissive temperature (right) for 12 h were freeze-fractured to expose their nuclear surfaces. The upper images were
collected at a high magnification. The NPC density at the non-permissive temperature was lower than that at the permissive
one (lower images), but overall the structure of the NPCs was similar between the cells incubated at the permissive (left) and
non-permissive temperatures (right).
(D’Angelo et al. 2009). Second, if the dots of Venus
fluorescence in the bleached areas are derived from
an exchange for free Venus–Nup107 as in conventional fluorescence recovery after photobleaching
(Houtsmuller 2005), the fluorescence recovery of
bleached NPCs would occur uniformly and gradually,
and NPC density in the bleached regions would be
the same as in the unbleached areas. However, we
observed the Venus signals as discrete dots instead of
as uniform ones. The NPC density in the bleached
areas was much less than that in the unbleached ones
(Fig. 2D), and this result was confirmed by immunostaining (Fig. S3B in Supporting Information). We
therefore concluded that the Venus fluorescent dots
that appeared in the bleached areas were newly
formed NPCs and that we successfully created a new
live-cell system for observing newly formed NPCs
during interphase, although the observed fluorescent
dots might contain an intermediate or incomplete
form of NPCs.
RCC1 function in NPC formation during
interphase
How does RCC1 activity contribute to NPC formation during interphase? RCC1 is the GEF for Ran.
Previously, Ryan et al. reported that the RanGTPase
cycle was required for NPC assembly in yeast (Ryan
et al. 2003). Angelo et al. suggested that RanGTP
production is essential in Xenopus eggs (D’Angelo
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2010)
9
H Iino et al.
et al. 2006). Consistent with this, our results suggest
that RanGTP production is essential for NPC formation during interphase in mammalian cells. However,
we do not know exactly how the Ran system is
involved in this process. Our system, which was
established using tsBN2Venus-Nup107 cells, will enable
us to investigate the mechanism in detail because we
can manipulate the activity of RCC1 in living cells
and observe newly formed NPCs on their nuclear
surfaces. In this study, we obtained several clues concerning how RCC1 functions in NPC formation
during interphase.
An obvious possibility is the inactivation of Randependent transport systems. As shown in Fig. S5A,B
in Supporting Information, when tsBN2 cells were
incubated at the non-permissive temperature, Randependent transport systems became impaired [also
see, (Tachibana et al. 1994, 2000)]. Approximately
6 h after the shift to the non-permissive temperature,
nuclear import and export were both inactivated
(Fig. S5C,D in Supporting Information). If the
nuclear import of some Nups is essential for NPC
formation during interphase, the formation process
would be inhibited. The cytoplasmic foci that we
observed in the cells at the non-permissive temperature (Fig. 1B) may correspond to Nups retained in
the cytoplasm. In fact, cycloheximide, a translation
inhibitor, inhibited the formation of cytoplasmic foci
(Fig. S2C in Supporting Information). The requirement for Ran-dependent transport activity is similar
to that for postmitotic NPC assembly. Postmitosis,
RCC1 binds to chromatin, and high RanGTP concentrations are created around the chromatin. This
high RanGTP concentration is essential for the dissociation of importin b–Nups complexes and their
incorporation into assembling NPCs on chromatin
(Walther et al. 2003a; Harel et al. 2003a). It is tempting for us to think that a similar Nup recruitment
mechanism might be necessary for NPC formation
during interphase.
A second possibility is that RCC1 functions in
NPC formation through cell cycle regulation. The
tsBN2 cells arrested in G1 phase at the non-permissive temperature (Fig. S1B in Supporting Information). If NPC formation is tightly linked to cell cycle
regulation, the G1 arrest itself could inhibit NPC
formation during interphase. In fact, whereas cyclinD1
accumulated in the cells, cyclinA, an S phase
regulator (Morgan 2007), was downregulated at the
non-permissive temperature (data not shown). Further
investigation is required to determine the function of
RCC1 in NPC formation during interphase.
10
Genes to Cells (2010)
Structural maintenance of NPCs by RCC1 activity
during interphase
By taking advantage of the properties of tsBN2 cells,
we detected weak NPC staining with mAb414 in cells
grown at the non-permissive temperature (Figs 1B,
4A and 5A). Surprisingly, the weak signal was rapidly
recovered after a downward shift to the permissive
temperature (Fig. 4B). Interestingly, the inhibition of
Ran-dependent nucleocytoplasmic transport at the
non-permissive temperature was also quickly reversed
after a shift to the permissive temperature (Fig. S5D in
Supporting Information). mAb414 is a monoclonal
antibody that recognizes FG repeats in some Nups,
including Nup153. A similar result was obtained using
anti-Nup153 antibodies (Fig. 4), and also in living
tsBN2Venus-Nup153 cells (Fig. S6B in Supporting Information), suggesting that a loss of RCC1 directly or
indirectly affects the behavior of some FG-Nups in
NPCs. In fact, at the non-permissive temperature,
leakage of the Nup62-Venus signal from the cytoplasm
to the nucleoplasm was detected in tsBN2Nup62-Venus
cells (Fig. S6A in Supporting Information). Nup62 is
one of the most abundant FG-Nups and a major constituent of the central channel, which functions as a
permeable transport barrier (Hu et al. 1996). On the
other hand, most Nup107, a scaffold Nup, was
retained in the NPCs even at the non-permissive
temperature (Fig. 5A,B). The overall structure of the
NPCs as revealed by SEM was not different between
the cells grown at permissive and non-permissive
temperatures (Fig. 5C).
In conclusion, our results produced using a new
live-cell system clearly suggest that RCC1 is involved
not only in NPC formation, but also in maintaining
the structural integrity of NPCs during interphase.
Active transport systems regulated by RCC1 may be
necessary for the maintenance of NPC structures,
especially FG-Nups.
Experimental procedures
Cell culture
Baby hamster kidney 21 and tsBN2 cells were generously provided by Dr T. Nishimoto. The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine
serum in a humidified incubator under 5% CO2. The BHK21
cells were maintained at 37 C, whereas the tsBN2 cells
were cultured at 32.0 (permissive temperature) or 39.7 C
(non-permissive temperature).
To synchronize the BHK21 and tsBN2 cells in metaphase,
the cells were incubated with 0.1 lg ⁄ ml nocodazole (Sigma)
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Visualizing nuclear pore complex formation
for 4 h. Mitotic cells were collected by shaking the culture
dishes, washed three times with phosphate-buffered saline
(PBS), and seeded again onto poly-L-lysine-coated coverslips
or glass-bottom dishes (Iwaki, Japan). To avoid forming binucleate cells after cytokinesis, the released tsBN2 cells were
pre-incubated for 4 h at 32.0 C to ensure the completion of
cytokinesis. After incubation, the cells were used in our
temperature-shift experiments.
Cell cycle profiles of the synchronized cells were produced
using a laser-scanning cytometer (LSC2; Olympus, Tokyo,
Japan) to quantify the DNA after treatment of the fixed cells
with 100 lg ⁄ ml propidium iodide and 1 mg ⁄ ml RNase (both
Sigma) in PBS at 37.0 C for 30 min.
Immunofluorescence
Cells grown on poly-L-lysine-coated coverslips were rinsed
once with PBS and fixed with 2% FA for 15 min. Immunofluorescence was carried out at room temperature as described
previously ((Maeshima & Laemmli 2003; Maeshima et al.
2006)), with minor modifications. The primary antibodies and
dilutions used in our experiments were as follows: goat polyclonal anti-RCC1 antibodies (Santa Cruz Biotechnology),
1 : 100; the mouse monoclonal antibody mAb414 (abcam),
1 : 2000; and a rat monoclonal anti-Nup153 antibody (prepared as described previously in (Fukuhara et al. 2006)), 1 : 20.
The secondary antibodies used were Alexa 594 donkey antigoat (Molecular Probes), Alexa 488 goat anti-mouse, and
Alexa 594 goat anti-rat antibodies at a 1 : 1000 dilution.
NPC density, nuclear volume, and the total
number of NPCs per nucleus
NPCs on the nuclear surfaces were visualized by immunolabeling using mAb414. Images of the nuclear surfaces adjacent
to the coverslips were recorded as Z-stacks using a DeltaVision
restoration microscope (Applied Precision) with a 100· objective lens (Olympus). Maximum nuclear surface areas, which
show clear fluorescent NPC signals (Fig. S1C in Supporting
Information), were measured using IMAGE J software (http://
rsb.info.nih.gov/ij/). The number of NPCs in such areas was
counted manually to obtain the NPC density (pores ⁄ lm2)
(counted nuclear areas, N = 10). An LSM510 Meta confocal
microscope (Zeiss) was used to measure the nuclear volumes,
which were calculated from the Z-stack data collected for the
nuclei (N = 20) using IMAGE J software (Sync measure 3D
1.7). The nuclear surface areas were estimated from the
Z-stacks and multiplied by the NPC density to obtain the
total number of NPCs per nucleus. Statistical analyses were
performed by using Kaleida Graph (Synergy Software, USA).
Plasmid construction
PEF-1a-Venus-hNUP107 was created as follows. The coding
region of Venus was amplified by PCR from Venus cDNA [a
gift from Dr A. Miyawaki (RIKEN, Wako, Japan)]. The
amplified fragment was then subcloned into pEGFP-C3
(Clontech) to replace the EGFP region (Venus-C3 vector).
The coding region of human NUP107 was amplified from its
cDNA (a gift from Dr V. Doye [Curie Institute, Paris,
France]) and inserted into Venus-C3 to generate VenushNUP107. For moderate expression, the cytomegalovirus
(CMV) promoter in Venus-hNUP107 was replaced with the
human EF1a promoter to produce PEF-1a-Venus-hNUP107.
PEF-1a-Venus-NUP153 and PEF-1a-NUP62-Venus were constructed using the multisite Gateway system (Invitrogen) as
described previously (Yahata et al. 2007). To stably establish
tsBN2 cells expressing Venus-hNUP107, PEF-1a-VenushNUP107 (100 ng) was transfected into tsBN2 cells using an
Effecten Transfection Reagent Kit (Qiagen), and the transformants were selected using 1.5 mg ⁄ ml G418 (Gibco). The
localization of Venus-Nup107 in the tsBN2Venus-Nup107 cells
was confirmed by fluorescence microscopy. The Flp-In System
(Invitrogen) was used to create the tsBN2Venus-Nup153 and
tsBN2Nup62-Venus lines. A pFRT-bla construct was transfected
into tsBN2 cells. The cells containing Flp recombination target
(FRT) sites were selected with 5 lg ⁄ ml blasticidin (Invitrogen)
and used for the isolation of stable transformants. The isolation
procedure was described previously in (Yahata et al. 2007).
Live-cell imaging and photobleaching
For live-cell imaging and our photobleaching experiments,
tsBN2 cells on glass-bottom dishes were set in a live-cell
chamber (MI-IBC; Olympus) on the stage of a DeltaVision
microscope. Nuclear surface images were recorded before and
after photobleaching with a 60· objective lens (Olympus).
Photobleaching was performed in the region of interest using
a quantifiable laser module (50 mW, 488 nm solid-state laser
with a ten spacing pattern) on a DeltaVision microscope
(Tahara et al. 2008). Images were acquired several hours after
photobleaching (Fig. 2A–C).
For quantification of the newly formed NPCs, the mean
fluorescence intensity in an unbleached area was measured and
used as a threshold value to define the newly formed fluorescent dots. In a bleached area (Fig. S3A in Supporting Information, left image), regions whose intensity exceeded the
threshold value were labeled (Fig. S3A in Supporting Information, center image). The number of labeled regions, which
were regarded as newly formed NPCs, was counted manually.
With this procedure, the NPC density in the bleached areas
was obtained (Figs 2D and 3C; Fig. S4C in Supporting Information).
Quantification of RCC1 recovery after a
temperature shift
For immunofluorescence, after incubation at 39.7 C (nonpermissive temperature) for 12 h, tsBN2 cells were subjected
to the permissive temperature (32.0 C). Cells were fixed at
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.
Genes to Cells (2010)
11
H Iino et al.
the indicated time points in Fig. 3F, and immunolabeled
with an anti-RCC1 antibody. The tsBN2 cells were also
incubated at 32 C with 5 lg ⁄ ml aphidicolin as a control.
The mean intensities of the nuclear RCC1 signals in a total
of ten cells were determined, and their averaged values were
compared with those of the control cells. For immunoblot
analysis, tsBN2 cells were manipulated as described above.
Whole-cell lysates were prepared every 4 h after the downward temperature shift (Fig. 3D). Whole-cell lysates of
unsynchronized tsBN2 cells were used as a control. Signal
quantification was done using LAS3000 (Fujifilm, Tokyo,
Japan).
Analysis of nuclear import and export
Nucleocytoplasmic transport in tsBN2 cells was examined
based on the diffusible sizes of EGFP-M9 and EGFP-RevNES. EGFP-M9 and EGFP-Rev-NES (Yokoya et al. 1999)
were transiently expressed in tsBN2 cells as described above.
Two days after transfection, the incubation temperature was
shifted up to 39.7 C (non-permissive temperature) and maintained for 10 h. After fixation with 2% FA, transport activity
was evaluated by EGFP localization in the cells (Fig. S5A,B in
Supporting Information). In our live-cell experiments, after
the incubation, temperature was shifted to the non-permissive
value, localization of the EGFP signals in the cells was monitored every hour (Fig. S5C in Supporting Information). When
the temperature was shifted from 39.7 (non-permissive temperature) to 32.0 C (permissive temperature), EGFP signals
were monitored every 10 min (Fig. S5D in Supporting Information).
Cryo-SEM
Synchronized mitotic tsBN2 cells were seeded onto poly-Llysine-coated metal specimen holders and cultured for 12 h
at the permissive or non-permissive temperature. The cells
were then rapidly frozen in liquid propane ()184 C) and
fractured in a cryo-transfer system (Alto-2500; Oxford,
Oxon, UK) at )120 C. The fractured cells were then
coated with chromium and observed using a field emission
scanning electron microscope (LE01530; LEO, Oberkochen,
Germany).
Acknowledgements
We are grateful to Ms S. Hihara for providing technical assistance. We thank Drs T. Nishimoto, Y. Yoneda, K. Wilson,
A. Miyawaki, T. Nagai, and V. Doye for their generous gifts
of materials. We also thank the members of the Cellular
Dynamics Lab at RIKEN and Dr Nishijima at NIG for many
helpful discussions. This work was supported by a grant-in-aid
from MEXT, Special Project Funding for Basic Science
(Bioarchitect Project) from RIKEN, and RIKEN R&D (President’s Discretionary Fund).
12
Genes to Cells (2010)
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Received: 4 February 2010
Accepted: 6 March 2010
Supporting Information ⁄ Supplementary
material
The following Supporting Information can be found in the
online version of the article:
Figure S1 (A) RCC1 in tsBN2 cells was degraded immediately
after incubation at a non-permissive (39.7 C) temperature.
(B) The tsBN2 cells incubated at the non-permissive temperature were arrested in G1 phase. (C) Highmagnification images
of the nuclear surfaces in tsBN2 cells incubated at the permissive (i) or non-permissive temperature (ii).
Figure S2 (A) The number of cells with mAb414-labeled
cytoplasmic foci was determined. (B) Temperature-specific
14
Genes to Cells (2010)
cytoplasmic foci were not detected in the parental
line BHK21, both at 32.0 (left image) and 39.7 C (right
image). (C) Cycloheximide, a translation inhibitor, suppressed
the formation of cytoplasmic foci at the non-permissive
temperature.
Figure S3 (A) Quantification of newly formed NPCs during
interphase, based on the formation of Venus dots in bleached
areas. (B) Verification that the new Venus dots in the bleached
region were newly formed NPCs. (C) The effect of temperature on the NPC signals was examined in BHK21 cells (the
parental line of tsBN2). (D) The cell cycle resumed after the
shift from the non-permissive to the permissive temperature.
Figure S4 NPC formation resumed just after the shift from a
non-permissive to a permissive temperature.
Figure S5 (A) and (B) Nucleocytoplasmic transport (import
and export) in tsBN2 cells incubated at a permissive or nonpermissive temperature was examined using transiently
expressed EGFP-M9 or EGFP-Rev-NES. (C) Nucleocytoplasmic transport in tsBN2 cells upon the temperature shift
was monitored by time-lapse imaging. (D) Recovery of
nucleocytoplasmic transport following a downward temperature shift.
Figure S6 (A) Nuclear leakage of Nup62 at the non-permissive temperature. tsBN2Nup62-Venus cells were monitored at
the permissive temperature and then at the non-permissive
temperature by time-lapse imaging. (B) Attenuation of VenusNup153 signals in tsBN2 Venus-Nup153 cells upon the shift
to a non-permissive temperature.
Additional Supporting Information may be found in the online
version of this article.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by
the authors. Any queries (other than missing material) should
be directed to the corresponding author for the article.
2010 The Authors
Journal compilation 2010 by the Molecular Biology Society of Japan/Blackwell Publishing Ltd.