Systematic kinetic analysis of mitotic dis- and reassembly of the nuclear pore in living cells
During mitosis in higher eukaryotes, nuclear pore complexes (NPCs) disassemble in prophase and are rebuilt in anaphase and telophase. NPC formation is hypothesized to occur by the interaction of mitotically stable subcomplexes that form defined structural intermediates. To determine the sequence of events that lead to breakdown and reformation of functional NPCs during mitosis, we present here our quantitative assay based on confocal time-lapse microscopy of single dividing cells. We use this assay to systematically investigate the kinetics of dis- and reassembly for eight nucleoporin subcomplexes relative to nuclear transport in NRK cells, linking the assembly state of the NPC with its function. Our data establish that NPC assembly is an ordered stepwise process that leads to import function already in a partially assembled state. We furthermore find that nucleoporin dissociation does not occur in the reverse order from binding during assembly, which may indicate a distinct mechanism.
THE JOURNAL OF CELL BIOLOGY
© The Rockefeller University Press $30.00
The Journal of Cell Biology, Vol. 180, No. 5, March 10, 2008 857–865
E. Dultz, E. Zanin, and C. Wurzenberger contributed equally to this paper.
Correspondence to J. Ellenberg: email@example.com
Abbreviations used in this paper: IBB, importin ␤ – binding domain of importin ␣ ;
LBR, lamin B receptor; NE, nuclear envelope; NPC, nuclear pore complex;
The online version of this paper contains supplemental material.
Nuclear pore complexes (NPCs) mediate all traf c of macro-
molecules across the nuclear envelope (NE). They are large pro-
tein assemblies composed of multiple copies of ⵑ 30 different
proteins, the nucleoporins (Nups), which are organized in about
10 subcomplexes and arranged with eightfold symmetry. In meta-
zoa, NPCs are stable throughout interphase ( Daigle et al., 2001 )
but disassemble into their subcomplexes during mitosis. When
the NE breaks down in pro/metaphase, most Nups become cyto-
plasmic and transmembrane Nups relocalize to the ER together
with other nuclear membrane proteins ( Ellenberg et al., 1997 ;
Yang et al., 1997 ; Daigle et al., 2001 ; Beaudouin et al., 2002 ).
Reassembly occurs during anaphase and telophase when the NE
is rebuilt around chromatin.
In live cells, NE disassembly has been shown to start by
partial disassembly of NPCs, with Nup98 leaving the NE early
followed by dissociation of Nup153 and Nup214 before the NE is
completely permeabilized. The membrane Nup POM121 disso-
ciates from NE fragments only after permeabilization ( Beaudouin
et al., 2002 ; Lenart et al., 2003 ). In xed cells, the nuclear bas-
ket Nup Tpr dissociates from the NE before Nup107 but later
than Nup98 and Nup50 ( Hase and Cordes, 2003 ).
More is known about the mechanism of postmitotic NPC
assembly. In vitro studies of nuclear assembly in Xenopus laevis
egg extracts have shed light on the essential role of the Ran –
importin system, which regulates the release of several Nups from
importin in proximity to chromatin, enabling them to reassoci-
ate and form NPCs ( Harel et al., 2003a ; Walther et al., 2003b ).
Several Nups bind to chromatin in early anaphase before membrane
association ( Belgareh et al., 2001 ; Walther et al., 2003a ), where
they have been postulated to form a prepore ( Suntharalingam
and Wente, 2003 ; Wozniak and Clarke, 2003 ; Rabut et al., 2004b ).
The mechanism of subsequent insertion into the membrane and
full assembly of the NPC remains to be understood.
For some Nups, the order of reassociation with the reform-
ing NE was investigated in various experimental systems, xed
cells of different mammalian species, or nuclei assembled in
X . laevis egg extracts. Together, these data predict that the Nup107 –
160 complex, Nup153, Nup98, and POM121 bind during ana-
phase, followed by the Nup62 and Nup93 complexes, Nup358,
and Nup214 in telophase, whereas Tpr and gp210 reassemble
only in early G1 (for review see Burke and Ellenberg, 2002 ).
Evidence for structural disassembly and reassembly inter-
mediates has been provided by eld emission scanning electron
microscopy. Porelike structures of different levels of complex-
ity could be visualized in egg extract nuclei ( Goldberg et al.,
1997 ; Wiese et al., 1997 ; Kiseleva et al., 2001 ) and a rough time
course of the formation of these structures could be established
in Drosophila melanogaster embryos ( Kiseleva et al., 2001 ).
Their protein composition remained, however, unclear.
Our current knowledge predicts that NPC disassembly
and reassembly are ordered processes that proceed via a de ned
set of intermediates formed by sequential interactions of NPC
uring mitosis in higher eukaryotes, nuclear pore
complexes (NPCs) disassemble in prophase and
are rebuilt in anaphase and telophase. NPC forma-
tion is hypothesized to occur by the interaction of mitotically
stable subcomplexes that form deﬁ ned structural inter-
mediates. To determine the sequence of events that lead to
breakdown and reformation of functional NPCs during mi-
tosis, we present here our quantitative assay based on con-
focal time-lapse microscopy of single dividing cells. We use
this assay to systematically investigate the kinetics of dis-
and reassembly for eight nucleoporin subcomplexes rela-
tive to nuclear transport in NRK cells, linking the assembly
state of the NPC with its function. Our data establish that
NPC assembly is an ordered stepwise process that leads
to import function already in a partially assembled state.
We furthermore ﬁ nd that nucleoporin dissociation does not
occur in the reverse order from binding during assembly,
which may indicate a distinct mechanism.
Systematic kinetic analysis of mitotic dis- and
reassembly of the nuclear pore in living cells
Elisa Dultz , Esther Zanin , Claudia Wurzenberger , Marion Braun , Gw é na ë l Rabut , Lucia Sironi , and Jan Ellenberg
Gene Expression Unit, European Molecular Biology Laboratory, D-69117 Heidelberg, Germany
JCB • VOLUME 180 • NUMBER 5 • 2008 858
complexes ( Rabut et al., 2004a ): Nup133, Nup107, Seh1, and
Nup43 (all from the Nup107 – 160 complex); the cytoplasmic
Nup Nup214, Nup98, Nup58 (Nup62 complex), Nup93 (Nup93
complex); the nucleoplasmic Nups Nup50 and Nup153; and the
transmembrane Nup POM121. In triple color time-lapse se-
quences of individual dividing cells, we recorded each GFP-Nup
together with a red uorescent nuclear import marker (importin
␤ – binding domain of importin ␣ [IBB]; Gorlich et al., 1996 )
and vital DNA staining ( Fig. 1, A and B ). DNA was used as
spatial reference to quantify nuclear (envelope) intensities (for
details see Materials and methods) and to monitor mitotic pro-
gression. The import marker IBB was ef ciently imported into
the nucleus during interphase, released into the cytoplasm at
NEBD, and reimported in telophase, providing a functional ref-
erence for the import competence of the NPCs. In addition, we
used the reimport/release of IBB to temporally align the assem-
bly time series of the different Nups ( Fig. 1, C and D ). In sum-
mary, this assay allowed us to analyze the kinetics of NPC
disassembly and reassembly in detail and to determine the im-
port competence of the nucleus in different states of NPC as-
sembly in living cells.
The Nup107 – 160 subcomplex binds to
chromatin in early anaphase
Members of the Nup107 – 160 complex were the rst to bind to
chromatin in early anaphase. During mitosis, a small subpopu-
lation of the complex localized to kinetochores as described
subcomplexes. However, the precise order in which the differ-
ent subcomplexes bind, the kinetics of the assembly events, and
the functional state of the different intermediates are unknown.
To address this, we systematically investigated the kinetics of
mitotic NPC disassembly and reassembly by time lapse confocal
microscopy in single dividing cells. Simultaneously, we moni-
tored import competence of the nucleus. We analyzed a set of
GFP-tagged Nups ( Rabut et al., 2004a ) representing eight dif-
ferent NPC subcomplexes. Our results show that NPC assembly
is indeed a highly ordered process that proceeds in a stepwise
fashion. Partially assembled NPCs were already import compe-
tent, which indicates that several Nups may not be required to
reestablish import function. Regarding NPC disassembly, we
found it to occur more rapidly than assembly and not simply in
the reverse order, which could indicate a distinct mechanism.
Based on our data, we present the rst comprehensive model for
the order, composition, and functional state of NPC disassembly
and reassembly intermediates in living cells.
Results and discussion
A functional and quantitative assay
for the kinetics of NPC disassembly
The kinetics of Nup dissociation from and reassociation with
the NE during mitosis was monitored in live NRK cells express-
ing 11 GFP-tagged Nups representative of eight different sub-
Figure 1. Quantiﬁ cation of Nup and import marker ﬂ uorescence intensities. (A and B) Regions of interest (outlines) were obtained from the Hoechst chan-
nel automatically (whole nucleus) or interactively (NE) and mean intensities were measured in the IBB and Nup channels. Time stamps give min:s relative
(import). (C and D) Normalized Nup intensities over time extracted from sequences shown in A and B after alignment to t
(import) (green). Black
curves represent the mean of ﬁ ve independent experiments (error bars indicate SD). Red curves, IBB mean.
859MITOTIC NPC DIS/REASSEMBLY • DULTZ ET AL.
and Nup43 are less stably associated with the complex and, in-
deed, this has been reported for Seh1 although not for Nup43
( Loiodice et al., 2004 ).
To test whether the binding of members of the Nup107 –
160 complex to chromatin represented formation of NPCs rather
than a general “ coating ” of chromatin, we analyzed early
assembly stages by high resolution microscopy of living cells.
Binding of GFP-tagged members of the Nup107 – 160 complex
to chromatin occurred in discrete patches and small dots of the
appearance of single pores ( Fig. 3 A ). If these structures truly
represent partially assembled NPCs, they should also contain
Nups from other subcomplexes. We tested this by simultane-
ously imaging GFP-tagged Nup107 – 160 complex members and
mCherry-tagged POM121. Indeed, POM121 rst accumulated
in patches around chromatin that also showed a strong localiza-
tion of Nup107 – 160 complex members ( Fig. 3 B ). To rule out
that this re ected the inability of the ER to contact other regions
of chromatin in anaphase, we also analyzed the localization of
mCherry-tagged lamin B receptor (LBR), a protein of the inner
nuclear membrane known to bind to chromatin ( Ye and Worman,
1994 ). In contrast to POM121, the localization of LBR was rela-
tively smooth and did not show a bias for sites of Nup107 – 160
labeling ( Fig. 3 C ). Our data therefore suggest that Nup binding
previously ( Belgareh et al., 2001 ). General association of Nup133
with chromatin was detected shortly after the metaphase –
anaphase transition or 8.5 ± 0.5 min ( n = 5) before the time point of
half maximal IBB intensity in the nucleus (t
[import]; Figs. 2 and
S1 A; and Video 1, available at http://www.jcb.org/cgi/content/
full/jcb.200707026/DC1). Nup133 had already reached its
maximal concentration at t
(import). These observations are in
line with the essential function of the Nup107 – 160 complex in
NPC assembly observed in vitro ( Boehmer et al., 2003 ; Harel
et al., 2003b ; Walther et al., 2003a ; D ’ Angelo et al., 2006 ).
We analyzed the assembly of three additional proteins of
this subcomplex (Nup107, Seh1, and Nup43). NPC subcomplexes
are thought to be stable throughout the cell cycle ( Matsuoka
et al., 1999 ; Belgareh et al., 2001 ; Loiodice et al., 2004 ) and
should thus bind to the reforming NE as a unit with identical
kinetics. Indeed, we found Nup107 to faithfully recapitulate the
assembly kinetics of Nup133 (Fig. S2 B, available at http://
www.jcb.org/cgi/content/full/jcb.200707026/DC1). This suggests
that stable subcomplexes are well represented by one member
in our assay. Although the assembly of Seh1 and Nup43 also
started early and was completed before t
(import), their ki-
netics were slightly but consistently delayed relative to Nup107
and Nup133 during early anaphase. This could indicate that Seh1
Figure 2. Time series representing the assembly of four Nups. The contrast of the image series was normalized to a common maximal mean intensity
reached on the nuclear rim at the last time point of each series. Plots on the right show the data obtained from the series shown (green) and the mean of
n series (black). As a reference, Nup133 (red) and IBB (dark red) intensity means are shown in all plots. Time stamps give min:s relative to t
(import). Video 1
(available at http://www.jcb.org/cgi/content/full/jcb.200707026/DC1) shows representative full-image sequences for Nup133. Error bars indicate SD.
JCB • VOLUME 180 • NUMBER 5 • 2008 860
times in interphase ( Rabut et al., 2004a ). In our assay, both Nup153
and Nup50 were detected at the periphery of the chromatin
as early as 7.9 ± 1.4 ( n = 4) and 6.6 ± 0.8 min ( n = 6) before
(import), respectively ( Figs. 2 and S2 A; and Video 2, avail-
able at http://www.jcb.org/cgi/content/full/jcb.200707026/DC1).
However, this early pool accounted for < 10% of the nal nu-
clear intensity for Nup153 and only ⵑ 20% for Nup50 ( Figs. 2
and S2 A, blue shading). The major pools of these Nups associ-
ated with the NE considerably later and reached their half maxi-
mal intensity at the NE only 1.0 ± 0.3 (Nup153) or 1.1 ± 0.5 min
(Nup50) before t
(import) (see Fig. 5 D).
The biphasic assembly behavior we observed is consistent
with the interphase dynamics and reinforces the interpretation
that both proteins have two distinct modes of binding at the pore.
Because both proteins are bound on the nucleoplasmic side of
the pore, the early association of a small pool to chromatin
could be involved in the formation of functional pores. The sec-
ond phase of assembly paralleled initiation of nuclear import
and transport through the rst functional NPC assembly inter-
mediates may therefore add the full complement of Nup50 and
Nup153 to the complex.
POM121 accumulates at the NE after
several soluble Nups
In interphase cells, the vertebrate-speci c membrane Nup POM121
localizes almost exclusively to the NE, whereas it disperses in
the ER during mitosis ( Daigle et al., 2001 ). In metaphase, the
ER is largely excluded from chromatin and spindle regions.
However, ER membranes come close to the poleward face of
the separating chromosomes early in anaphase ( Fig. 3, B and C ).
The resulting early increase of POM121 signal around chroma-
tin does therefore not re ect a speci c accumulation ( Fig. 3 B
and not depicted). Accumulation in the NE over ER background
became visible at 5.9 ± 1.0 min ( n = 5) before t
(import) and then
rapidly reached its maximal intensity at t
(import) ( Fig. 2 ).
Together with the colocalization with the Nup107 – 160
complex, our kinetic data suggest that POM121-binding sites
on chromatin become available only in late anaphase. At this
time point, ER membranes come into physical contact with the
separated chromosome masses from all sides and POM121 as-
sociates with chromatin at sites where Nup107 – 160 components
are already bound.
Nup93, Nup98, and Nup58 assemble after
The Nup93 as well as the Nup62 complex are thought to local-
ize to central positions of the pore. In our assay, the Nup93 and
Nup62 complexes (represented by Nup58) accumulated at the
NE starting at 3.8 ± 0.4 ( n = 5) and 3.3 ± 1.4 min ( n = 11) before
(import), respectively. The more peripheral Nup98 was rst
detected 3.8 ± 0.6 min ( n = 6) before t
(import) ( Figs. 2 and S2 A).
All three Nups reached their maximal intensity at the NE shortly
Binding of these three complexes occurred only after sev-
eral other Nups were already present on chromatin. Their addition
may be the last step for the formation of an import competent NPC
assembly intermediate because IBB import initiated concomitant
to chromatin in anaphase is caused by the formation of pore com-
plexes and is consistent with the hypothesis that prepores form
already on the naked chromatin before the attachment of nuclear
membranes ( Suntharalingam and Wente, 2003 ; Wozniak and
Clarke, 2003 ; Rabut et al., 2004b ).
Reassociation of Nup153 and Nup50 to
the NE is biphasic
Nup153 and Nup50 localize to the nuclear basket and have been
shown to exchange dynamically from the NPC with two residence
Figure 3. Localization pattern of Nups on chromatin during anaphase.
Cells were followed from metaphase and single images were taken at deﬁ ned
time points. Images were ﬁ ltered with an anisotropic diffusion ﬁ lter. Boxes
indicate regions of enlargements. Intensity proﬁ les measured along a 0.45- m-
wide line as indicated by the white outlines were plotted after subtraction
of cytoplasmic background. Time stamps indicate minutes after anaphase
onset. (A) Cells expressing GFP-tagged Nup107, Nup133, and Nup37.
(B) Cells expressing GFP-Nup107, GFP-Nup133, and POM121-mCherry.
(C) Cells expressing GFP- Nup107, GFP-Nup133, and LBR-mCherry.
861MITOTIC NPC DIS/REASSEMBLY • DULTZ ET AL.
rst and export only later when nuclear biosynthesis has re-
started. This would explain the late assembly time of factors not
required for import such as Nup214.
NPC disassembly in prophase occurs
rapidly and synchronously
The same set of eight representative Nups was followed during
dissociation from the NE in prophase ( Figs. 4 , S1 B, and S3 A,
available at http://www.jcb.org/cgi/content/full/jcb.200707026/
DC1). Disassembly proceeded more rapidly than assembly
and more synchronously for the different Nups so that distinct
steps in the disassembly process could not be clearly resolved
(compare Fig. 5, A and B ). This could be caused by insuf -
cient time resolution of the assay or simply the fact that dis-
assembly occurs in fewer steps than assembly. Disintegration
of a large part of the pore could be triggered in a single step.
Also, recent EM data suggest that the disassembly of individual
pores within one nucleus in X . laevis egg extract is asynchronous,
leading to pore intermediates in different states of disassembly
at the same time ( Cotter et al., 2007 ). If this occurs in live
mammalian cells, it would compromise our ability to detect
the order of the process because we measure the mean of many
with their assembly (see Fig. 5, B and D). At this time, the
Nup107 – 160 complex and POM121 were assembled already to
ⵑ 80%, whereas only the minor early fractions of Nup50 and
Nup153 were present.
Nup214 association with the NE lasts well
Nup214 is a peripheral cytoplasmic Nup with a residence time
of several hours at interphase NPCs ( Rabut et al., 2004a ).
We rst detected Nup214 at the NE 0.8 ± 0.2 min ( n = 4) before
(import) (Fig. S2). It was thus the last Nup to associate with
the newly forming NPC investigated in this study. Its rst ap-
pearance was concomitant with the regaining of nuclear import
activity but its concentration continued to increase over cyto-
plasmic background long after the maximal IBB intensity in the
nucleus was reached. High import rates were reached already
when Nup214 had only reached 50% of its maximal intensity at
the NE (see Fig. 5 D). These kinetics suggest that Nup214 may
not be required for IBB import, which is consistent with previous
ndings that show no role of Nup214 in protein import via clas-
sical import routes but rather suggest an activity in protein export
( Walther et al., 2002 ; Hutten and Kehlenbach, 2006 ). A newly
assembled nucleus will likely have to establish import function
Figure 4. Time series representing the dissociation of four Nups from the NE during prophase. The contrast of the image series was normalized to a
common maximal mean intensity on the nuclear rim at the ﬁ rst time point of each series. Plots show the data obtained from the series shown (red) and the
mean of n series (black). As a reference, mean intensities of Nup98 (cyan) and POM121 (green) are shown in all plots. Time stamps give min:s relative
(import). Videos 3 and 4 (available at http://www.jcb.org/cgi/content/full/jcb.200707026/DC1) show representative full-image sequences for
Nup98 and POM121. Error bars indicate SD.
JCB • VOLUME 180 • NUMBER 5 • 2008 862
Figure 5. Summary of NPC disassembly and reassembly kinetics. (A and B) Overview over all means of disassembly (A) and assembly (B) kinetics.
(C) Time points of ﬁ rst visible nuclear accumulation over background for all analyzed Nups. (D) Time points of 50% assembly of Nups relative to the ﬁ rst
derivative of IBB intensity as a measure for import rate. Because of the change in concentration distribution of IBB between cytoplasm and nucleus during
the import phase, the ﬁ rst derivative of IBB intensity systematically underestimates true instantaneous import rates. The maximum reached at time point 0
therefore does not reﬂ ect the true maximal import rates, which may be reached later. (E and F) Models for mitotic NPC disassembly and reassembly. Fila-
ment structures are included in the model in gray on the basis of previous data. The precise positions of the Nups in the NPC are unknown and thus drawn
schematically. Because the different Nup-expressing cell lines showed some variability in the timing of mitotic progression (10.6 ± 1.5 min from anaphase
onset to t
[import]; not depicted), the time between anaphase onset and t
(import) was normalized to 10 min in B to D. Error bars indicate SD.
863MITOTIC NPC DIS/REASSEMBLY • DULTZ ET AL.
Electron microscopy of D. melanogaster embryos has re-
vealed disassembly intermediates similar to assembly; however,
one intermediate dominated all prophase nuclei, indicating that
other intermediates may be very transient ( Kiseleva et al., 2001 ).
This ts well with our observation in living mammalian cells
that disassembly is very rapid. The similar ultrastructural ap-
pearance of NPC intermediates lead to the hypothesis that dis-
assembly could be the reversal of assembly. Despite the limitations
of our assay, our data indicate that this may not be the case.
For example, the Nups that assembled earliest and latest during
anaphase, i.e., Nup133 and Nup214, dissociated from the NE in
the middle of the disassembly process. Nup98, which assembles
at an intermediate time point in anaphase, was clearly the rst
Nup to dissociate from the nuclear periphery in prometaphase,
which is in agreement with data from star sh oocytes ( Lenart
et al., 2003 ). Finally, Pom121, which is assembled after the
Nup107 – 160 complex in anaphase, also dissociated clearly after
the Nup107 – 160 complex during disassembly.
Interestingly, the Nup107 – 160, Nup93, and Nup214 com-
plexes, which are the most stable NPC subcomplexes during
interphase ( Rabut et al., 2004a ), dissociated early and rapidly,
whereas Nup50 and Nup58 (Nup62 complex) together with
POM121 remained longest in fragments of the NE ( Figs. 4 and
S3 A; and Videos 3 and 4, available at http://www.jcb.org/cgi/
content/full/jcb.200707026/DC1). Thus, the NE identity of
POM121-containing membranes appears to be lost only gradu-
ally in prometaphase, which is in agreement with previous ob-
servations ( Beaudouin et al., 2002 ).
The persistence of Nup50 at the NE might be caused by
chromatin rather than NPC association because we found Nup50
to coat chromatin throughout mitosis from prophase until ana-
phase (Fig. S3 B). It formed a dynamic coat, which rapidly ex-
changed with the cytoplasmic pool as assayed by photobleaching
(unpublished data). This localization is consistent with the pres-
ence of the Aspergillus nidulans homologue of Nup50 on mitotic
chromatin ( Osmani et al., 2006 ) and could indicate a conserved
mitotic function. However, it could also be caused by an inher-
ent chromatin af nity of Nup50 because the yeast Nup50 homo-
logue has been implicated in NPC associated gene regulation
( Schmid et al., 2006 ).
In summary, our systematic study allows us to propose the rst
comprehensive model for mitotic NPC disassembly and re-
assembly ( Fig. 5, E and F ). Disassembly occurs in mammalian
cells in a similar manner to star sh oocytes ( Lenart et al., 2003 )
but with faster kinetics ( Fig. 5 A ). The composition of dis-
assembly intermediates appears to differ from assembly inter-
mediates, which suggests a distinct mechanism.
Our data provide detailed insight into the kinetics of pore
assembly with high time resolution. Consistent with previous
studies, we nd NPC assembly to be a highly ordered process
( Fig. 5 C ). For the rst time, we can relate the composition of
the different assembly intermediates to import function. Our data
supports the model that assembly starts with formation of a
prepore on chromatin and indicates that such a structure con-
tains the Nup107 – 160 complex as well as substoichiometric
amounts of Nup153 and Nup50 ( Fig. 5 F ). These may provide
the binding platform for additional components like the trans-
membrane Nup POM121.
In our live cell assay, we measure the mean concentration
of Nups over all NPCs in the imaging plane to determine their
assembly kinetics. We therefore cannot formally decide whether
the fact that the association kinetics of individual Nups stretch
over several minutes re ects asynchronous assembly of differ-
ent NPCs in the nucleus, the sequential addition of multiple
copies of the same Nup to NPCs in the same state of assembly,
or a mixture of the two processes. However, our high-resolution
imaging data showed similar concentration of Nups in adjacent
pores at single time points during assembly ( Fig. 3 ). Further-
more, electron microscopic data from D. melanogaster indicate
that speci c assembly intermediates dominate at any stage of
mitosis ( Kiseleva et al., 2001 ). We therefore assume that our ki-
netics re ect at least to a large extent the synchronous assembly
process of many NPCs after mitosis.
What then is the rst assembly intermediate that is com-
petent for nuclear import? Comparing the time of half maximal
concentration for each Nup with the rate of import ( Fig. 5 D ),
our data show that the assembly intermediate containing mainly
the Nup107 – 160 complex and POM121 does not support protein
import ( Fig. 5, B and D ). Only upon association of Nup93,
Nup58 (Nup62 complex), and Nup98 does IBB import initiate,
which suggests that these complexes add transport activity to
the new pore, possibly by providing many phenylalanine-glycine
repeats. At this time point, at least a fraction of the pores in the
nucleus contain all subunits necessary to support protein import
function. In addition, the presence of a sealed or nearly sealed
membrane around the nuclear compartment is likely required for
IBB to accumulate in the nucleus. In contrast, the nucleoplasmic
Nup50 and Nup153 as well as the cytoplasmic Nup214 are prob-
ably not required for import activity in stoichiometric amounts.
In the future, it will be very interesting to analyze the be-
havior of additional Nups, especially the membrane-bound Ndc1
and ELYS/Mel28, which have very recently been reported to play
crucial roles in NPC assembly ( Galy et al., 2006 ; Mansfeld et al.,
2006 ; Rasala et al., 2006 ; Stavru et al., 2006 ; Franz et al., 2007 ).
In addition, similar data obtained for interphase assembly will
allow to test whether the insertion of NPCs into an intact inter-
phase NE follows the same mechanism as postmitotic assembly.
Our assay using IBB as a functional and temporal marker
should furthermore prove very useful to study additional as-
pects of NEBD and NE assembly. Besides a detailed kinetic
understanding, the assay can also yield mechanistic insight when
combined with molecular perturbations by RNAi or the ex-
pression of dominant-negative proteins.
Materials and methods
DNA constructs and cell lines
pIBB-DiHcRed was generated by ligating the fragment of the IBB domain from
the plasmid pQE60-IBB-GFP ( Ribbeck and Gorlich, 2002 ) into pDiHcRed-N1
( Gerlich et al., 2003 ) with a 5 – amino acid linker (GPVAT) between the IBB
domain and DiHcRed.
pPOM121-mCherry was cloned by exchanging 3EGFP in pPOM121-
3EGFP ( Rabut et al., 2004a ) with mCherry ( Shaner et al., 2004 ). pLBR1TM-
mCherry contains the N terminus of LBR and its ﬁ rst transmembrane domain.
JCB • VOLUME 180 • NUMBER 5 • 2008 864
Submitted: 3 July 2007
Accepted: 5 February 2008
Beaudouin , J. , D. Gerlich , N. Daigle , R. Eils , and J. Ellenberg . 2002 . Nuclear en-
velope breakdown proceeds by microtubule-induced tearing of the lam-
ina. Cell . 108 : 83 – 96 .
Belgareh , N. , G. Rabut , S.W. Bai , M. van Overbeek , J. Beaudouin , N. Daigle ,
O.V. Zatsepina , F. Pasteau , V. Labas , M. Fromont-Racine , et al . 2001 .
An evolutionarily conserved NPC subcomplex, which redistributes in part
to kinetochores in mammalian cells. J. Cell Biol. 154 : 1147 – 1160 .
Boehmer , T. , J. Enninga , S. Dales , G. Blobel , and H. Zhong . 2003 . Depletion of
a single nucleoporin, Nup107, prevents the assembly of a subset of nu-
cleoporins into the nuclear pore complex. Proc. Natl. Acad. Sci. USA .
100 : 981 – 985 .
Burke , B. , and J. Ellenberg . 2002 . Remodelling the walls of the nucleus. Nat.
Rev. Mol. Cell Biol. 3 : 487 – 497 .
Cotter , L. , T.D. Allen , E. Kiseleva , and M.W. Goldberg . 2007 . Nuclear mem-
brane disassembly and rupture. J. Mol. Biol. 369 : 683 – 695 .
Daigle , N. , J. Beaudouin , L. Hartnell , G. Imreh , E. Hallberg , J. Lippincott-
Schwartz , and J. Ellenberg . 2001 . Nuclear pore complexes form immo-
bile networks and have a very low turnover in live mammalian cells.
J. Cell Biol. 154 : 71 – 84 .
D ’ Angelo , M.A. , D.J. Anderson , E. Richard , and M.W. Hetzer . 2006 . Nuclear
pores form de novo from both sides of the nuclear envelope. Science .
312 : 440 – 443 .
Ellenberg , J. , E.D. Siggia , J.E. Moreira , C.L. Smith , J.F. Presley , H.J. Worman ,
and J. Lippincott-Schwartz . 1997 . Nuclear membrane dynamics and re-
assembly in living cells: targeting of an inner nuclear membrane protein
in interphase and mitosis. J. Cell Biol. 138 : 1193 – 1206 .
Franz , C. , R. Walczak , S. Yavuz , R. Santarella , M. Gentzel , P. Askjaer , V. Galy ,
M. Hetzer , I.W. Mattaj , and W. Antonin . 2007 . MEL-28/ELYS is required
for the recruitment of nucleoporins to chromatin and postmitotic nuclear
pore complex assembly. EMBO Rep. 8 : 165 – 172 .
Galy , V. , P. Askjaer , C. Franz , C. Lopez-Iglesias , and I.W. Mattaj . 2006 . MEL-28,
a novel nuclear-envelope and kinetochore protein essential for zygotic
nuclear-envelope assembly in C. elegans . Curr. Biol. 16 : 1748 – 1756 .
Gerlich , D. , J. Beaudouin , B. Kalbfuss , N. Daigle , R. Eils , and J. Ellenberg .
2003 . Global chromosome positions are transmitted through mitosis in
mammalian cells. Cell . 112 : 751 – 764 .
Goldberg , M.W. , C. Wiese , T.D. Allen , and K.L. Wilson . 1997 . Dimples, pores,
star-rings, and thin rings on growing nuclear envelopes: evidence for
structural intermediates in nuclear pore complex assembly. J. Cell Sci.
110 : 409 – 420 .
Gorlich , D. , P. Henklein , R.A. Laskey , and E. Hartmann . 1996 . A 41 amino acid
motif in importin-alpha confers binding to importin-beta and hence tran-
sit into the nucleus. EMBO J. 15 : 1810 – 1817 .
Harel , A. , R.C. Chan , A. Lachish-Zalait , E. Zimmerman , M. Elbaum , and D.J.
Forbes . 2003a . Importin beta negatively regulates nuclear membrane fusion
and nuclear pore complex assembly. Mol. Biol. Cell . 14 : 4387 – 4396 .
Harel , A. , A.V. Orjalo , T. Vincent , A. Lachish-Zalait , S. Vasu , S. Shah , E. Zimmerman ,
M. Elbaum , and D.J. Forbes . 2003b . Removal of a single pore subcomplex
results in vertebrate nuclei devoid of nuclear pores. Mol. Cell . 11 : 853 – 864 .
Hase , M.E. , and V.C. Cordes . 2003 . Direct interaction with nup153 mediates
binding of Tpr to the periphery of the nuclear pore complex. Mol. Biol.
Cell . 14 : 1923 – 1940 .
Hutten , S. , and R.H. Kehlenbach . 2006 . Nup214 is required for CRM1- dependent
nuclear protein export in vivo. Mol. Cell. Biol. 26 : 6772 – 6785 .
Kiseleva , E. , S. Rutherford , L.M. Cotter , T.D. Allen , and M.W. Goldberg . 2001 .
Steps of nuclear pore complex disassembly and reassembly during mito-
sis in early Drosophila embryos. J. Cell Sci. 114 : 3607 – 3618 .
Lenart , P. , G. Rabut , N. Daigle , A.R. Hand , M. Terasaki , and J. Ellenberg . 2003 .
Nuclear envelope breakdown in star sh oocytes proceeds by partial NPC
disassembly followed by a rapidly spreading fenestration of nuclear
membranes. J. Cell Biol. 160 : 1055 – 1068 .
Loiodice , I. , A. Alves , G. Rabut , M. Van Overbeek , J. Ellenberg , J.B. Sibarita ,
and V. Doye . 2004 . The entire Nup107-160 complex, including three new
members, is targeted as one entity to kinetochores in mitosis. Mol. Biol.
Cell . 15 : 3333 – 3344 .
Mansfeld , J. , S. Guttinger , L.A. Hawryluk-Gara , N. Pante , M. Mall , V. Galy ,
U. Haselmann , P. Muhlhausser , R.W. Wozniak , I.W. Mattaj , et al . 2006 .
The conserved transmembrane nucleoporin NDC1 is required for nuclear
pore complex assembly in vertebrate cells. Mol. Cell . 22 : 93 – 103 .
Matsuoka , Y. , M. Takagi , T. Ban , M. Miyazaki , T. Yamamoto , Y. Kondo , and
Y. Yoneda . 1999 . Identi cation and characterization of nuclear pore
It was cloned by exchanging YFP in pLBR1TM-YPF ( Daigle et al., 2001 )
NRK cells were grown in standard medium. NRK cell lines stably
expressing Nups tagged with EGFP (Nup50, Nup58, Nup93, Nup98,
Nup133, Nup153, Nup214, Pom121, Nup43, and Seh1) as described
previously ( Rabut et al., 2004a ) were maintained at 0.5 mg/ml G418.
Some experiments were performed by transient transfection with the same
plasmids used for generation of the stable cell lines. Transient transfections
with pIBB-DiHcRed and Nup plasmids were performed with FuGene 6
(Roche) 24 – 72 h before imaging. For dual-color high-resolution imaging
( Fig. 3 ), cells coexpressing GFP-tagged members of the Nup107 – 160
complex and LBR- or POM121-mCherry were enriched by FACS.
Live cell microscopy
For live cell microscopy, cells were grown in Lab-Tek chambered cover-
glasses (Thermo Fisher Scientiﬁ c). 30 min before imaging, the medium was
exchanged for prewarmed CO
-independent medium without phenol red
supplemented with 20% FCS, 2 mM glutamine, 100 mg/ml penicillin and
streptomycin, and 0.2 μ g/ml Hoechst 33342. The chambers were sealed
with silicone grease. Time lapse sequences of 2 – 4- m thick confocal slices
were recorded at 37 ° C on confocal microscope systems (LSM 510) using
a 63 × 1.4 NA Plan Apochromat objective (Carl Zeiss, Inc.). Fluorescent
chromatin was automatically tracked and focused during imaging using in-
house developed macros ( Rabut and Ellenberg, 2004 ). High-resolution im-
aging for Fig. 3 was performed with a 100 × Plan Apochromat NA 1.4
objective (Carl Zeiss, Inc.).
Quantiﬁ cation and image analysis
Images were segmented on the chromatin channel in Image J (http://rsb.
info.nih.gov/ij/) by successive application of a Gaussian and an anisotro-
pic diffusion ﬁ lter and thresholding of the ﬁ ltered image with an in-house-
developed macro. The segmentation was applied to the raw images of the
IBB channel and the mean nuclear ﬂ uorescence intensities were quantiﬁ ed.
For the assembly of most Nups, the same segmentation was used to quan-
tify the mean intensity of the Nups on the chromatin region. During inter-
phase, the soluble pools of both Nup50 and Nup153 localize to the
nucleoplasm and a clear discrimination between nuclear rim association
and nuclear import in later stages of mitosis could therefore not be achieved
with the assay. However, the quantiﬁ cation on the nuclear rim region alone
as compared with the complete chromatin region did not yield signiﬁ cantly
different results for any of the two proteins, which suggests that the contri-
bution of import to the measured kinetics is minor.
Manual rim segmentation was applied for all disassembly series to
avoid folded regions of the NE. The apparent decrease in Nup133 ﬂ uorescence
in the nuclear region after t
(import) is caused by dilution of the signal
during growth of the nuclear surface area in telophase upon chromatin
decondensation. Mean intensities were background subtracted and nor-
malized. Different time series were aligned according to the time of the half
maximal IBB intensity (t
[import]) in the nucleus (set to zero). Temporal
alignment of assembly series along the metaphase – anaphase transition
gave similar overall results but yielded consistently higher SDs and was
therefore not pursued. The time point of ﬁ rst accumulation of signal over
cytoplasmic background in the chromatin region was scored visually. For
presentation purposes, images shown in Figs. 1, 2, 4 , S1, and S2 were ﬁ l-
tered with a Gaussian blur ﬁ lter (Image J), kernel size 1. Error bars in all
ﬁ gures represent the SD.
Online supplemental material
Fig. S1 shows all individual disassembly/reassembly curves used to de-
rive the mean kinetics shown in Fig. 5 (A and B) . Fig. S2 shows rep-
resentative image series for the assembly of Nup50, Nup98, Nup93,
and Nup214 and mean assembly curves for all analyzed members of
the Nup107 – 160 complex. Fig. S3 shows a representative image series
for the disassembly of Nup133, Nup153, Nup93, and Nup214 and the
localization of Nup50 on chromatin during mitosis. Videos 1 and 2 show
representative assembly series forNup133 and Nup93, respectively.
Videos 3 and 4 show disassembly series for POM121 and Nup98. Online
supplemental material is available at http://www.jcb.org/cgi/content/
We would like to thank Katharina Ribbeck and Dirk G ö rlich for the IBB construct.
J. Ellenberg acknowledges funding by the Deutsche Forschungsgemein-
schaft priority program SPP1175 (DFG EL 246/3-1). E. Dultz was supported
by a fellowship from the European Molecular Biology Laboratory International
865MITOTIC NPC DIS/REASSEMBLY • DULTZ ET AL.
subcomplexes in mitotic extract of human somatic cells. Biochem. Biophys.
Res. Commun. 254 : 417 – 423 .
Osmani , A.H. , J. Davies , H.L. Liu , and S.A. Osmani . 2006 . Systematic deletion
and mitotic localization of the nuclear pore complex proteins of Aspergillus
nidulans . Mol. Biol. Cell . 17 : 4946 – 4961 .
Rabut , G. , and J. Ellenberg . 2004 . Automatic real-time three-dimensional cell
tracking by uorescence microscopy. J. Microsc. 216 : 131 – 137 .
Rabut , G. , V. Doye , and J. Ellenberg . 2004a . Mapping the dynamic organization
of the nuclear pore complex inside single living cells. Nat. Cell Biol.
6 : 1114 – 1121 .
Rabut , G. , P. Lenart , and J. Ellenberg . 2004b . Dynamics of nuclear pore complex
organization through the cell cycle. Curr. Opin. Cell Biol. 16 : 314 – 321 .
Rasala , B.A. , A.V. Orjalo , Z. Shen , S. Briggs , and D.J. Forbes . 2006 . ELYS is a
dual nucleoporin/kinetochore protein required for nuclear pore assembly
and proper cell division. Proc. Natl. Acad. Sci. USA . 103 : 17801 – 17806 .
Ribbeck , K. , and D. Gorlich . 2002 . The permeability barrier of nuclear pore
complexes appears to operate via hydrophobic exclusion. EMBO J.
21 : 2664 – 2671 .
Schmid , M. , G. Arib , C. Laemmli , J. Nishikawa , T. Durussel , and U.K. Laemmli .
2006 . Nup-PI: the nucleopore-promoter interaction of genes in yeast.
Mol. Cell . 21 : 379 – 391 .
Shaner , N.C. , R.E. Campbell , P.A. Steinbach , B.N. Giepmans , A.E. Palmer , and
R.Y. Tsien . 2004 . Improved monomeric red, orange and yellow uorescent
proteins derived from Discosoma sp. red uorescent protein. Nat. Biotechnol.
22 : 1567 – 1572 .
Stavru , F. , B.B. Hulsmann , A. Spang , E. Hartmann , V.C. Cordes , and D. Gorlich .
2006 . NDC1: a crucial membrane-integral nucleoporin of metazoan nu-
clear pore complexes. J. Cell Biol. 173 : 509 – 519 .
Suntharalingam , M. , and S.R. Wente . 2003 . Peering through the pore: nuclear
pore complex structure, assembly, and function. Dev. Cell . 4 : 775 – 789 .
Walther , T.C. , H.S. Pickersgill , V.C. Cordes , M.W. Goldberg , T.D. Allen , I.W.
Mattaj , and M. Fornerod . 2002 . The cytoplasmic laments of the nuclear
pore complex are dispensable for selective nuclear protein import. J. Cell
Biol. 158 : 63 – 77 .
Walther , T.C. , A. Alves , H. Pickersgill , I. Loiodice , M. Hetzer , V. Galy , B.B.
Hulsmann , T. Kocher , M. Wilm , T. Allen , et al . 2003a . The conserved
Nup107-160 complex is critical for nuclear pore complex assembly. Cell .
113 : 195 – 206 .
Walther , T.C. , P. Askjaer , M. Gentzel , A. Habermann , G. Grif ths , M. Wilm , I.W.
Mattaj , and M. Hetzer . 2003b .
RanGTP mediates nuclear pore complex
assembly. Nature . 424 : 689 – 694 .
Wiese , C. , M.W. Goldberg , T.D. Allen , and K.L. Wilson . 1997 . Nuclear envelope
assembly in Xenopus extracts visualized by scanning EM reveals a transport-
dependent ‘ envelope smoothing ’ event. J. Cell Sci. 110 : 1489 – 1502 .
Wozniak , R. , and P.R. Clarke . 2003 . Nuclear pores: sowing the seeds of assembly
on the chromatin landscape. Curr. Biol. 13 : R970 – R972 .
Yang , L. , T. Guan , and L. Gerace . 1997 . Integral membrane proteins of the nu-
clear envelope are dispersed throughout the endoplasmic reticulum dur-
ing mitosis. J. Cell Biol. 137 : 1199 – 1210 .
Ye , Q. , and H.J. Worman . 1994 . Primary structure analysis and lamin B and DNA
binding of human LBR, an integral protein of the nuclear envelope inner
membrane. J. Biol. Chem. 269 : 11306 – 11311 .