Research Article
1201
Cell-cycle regulation and dynamics of cytoplasmic
compartments containing the promyelocytic leukemia
protein and nucleoporins
Åsne Jul-Larsen1,*, Amra Grudic1,*, Rolf Bjerkvig1 and Stig Ove Bøe1,2,‡
1
Department of Biomedicine, University of Bergen, Bergen, Norway
Institute of Clinical Biochemistry, University of Oslo, Rikshospitalet Medical Centre, Oslo, Norway
2
*These authors contributed equally to this work
‡
Author for correspondence (e-mail: [email protected])
Journal of Cell Science
Accepted 2 December 2008
Journal of Cell Science 122, 1201-1210 Published by The Company of Biologists 2009
doi:10.1242/jcs.040840
Summary
Nucleoporins and the promyelocytic leukemia protein (PML)
represent structural entities of nuclear pore complexes and PML
nuclear bodies, respectively. In addition, these proteins might
function in a common biological mechanism, because at least
two different nucleoporins, Nup98 and Nup214, as well as PML,
can become aberrantly expressed as oncogenic fusion proteins
in acute myeloid leukemia (AML) cells. Here we show that PML
and nucleoporins become directed to common cytoplasmic
compartments during the mitosis-to-G1 transition of the cell
cycle. These protein assemblies, which we have termed CyPNs
(cytoplasmic assemblies of PML and nucleoporins), move on
the microtubular network and become stably connected to the
nuclear membrane once contact with the nucleus has been made.
The ability of PML to target CyPNs depends on its nuclear
Introduction
The promyelocytic leukemia protein (PML) has been implicated in
multiple cellular functions including, apoptosis, differentiation,
DNA repair, senescence, angiogenesis and virus defense (Bernardi
and Pandolfi, 2007; Borden, 2008; Everett and Chelbi-Alix, 2007;
Salomoni et al., 2008). In addition, this protein is linked to the
pathogenesis of a subtype of acute myelogenous leukemia (AML)
termed acute promyelocytic leukemia (APL), because PML is
almost invariably expressed as an oncogenic fusion protein (PMLRARα) in APL blasts as a result of the t(15;17) translocation (de
The et al., 1990; Kakizuka et al., 1991).
PML is the defining unit of the subnuclear compartments termed
PML nuclear bodies (PML-NBs). The ability of PML to assemble
into these structures is, in part, stimulated by intermolecular
interactions between SUMOylated residues and a SUMO-interaction
domain present on the PML protein (Shen et al., 2006). PML also
recruits a multitude of other proteins (with diverse functions) to
PML-NBs, many of which contain SUMOylated residues or a
SUMO-interacting motif (Lin et al., 2006). In APL blasts, the PMLNBs become disrupted as a result of expression of the PML-RARα
fusion protein (Dyck et al., 1994; Koken et al., 1994; Weis et al.,
1994).
In addition to the PML-NBs that are present within interphase
nuclei, PML can also be detected in subcellular mitotic structures,
termed MAPPs (mitotic accumulations of PML proteins) (Dellaire
et al., 2006; Everett et al., 1999) and in cytoplasmic subdomains
that appear during the G1 phase (Dellaire et al., 2006; Everett et
localization signal, and loss of PML causes an increase in
cytoplasmic-bound
versus
nuclear-membrane-bound
nucleoporins. CyPNs are also targeted by the acute
promyelocytic leukemia (APL) fusion protein PML-RARα and
can be readily detected within the APL cell line NB4. These
results provide insight into a dynamic pool of cytoplasmic
nucleoporins that form a complex with the tumor suppressor
protein PML during the G1 phase of the cell cycle.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/122/8/1201/DC1
Key words: AML, CBP, Cytoplasm, Nuclear pore complex,
Nucleoporins, PML
al., 1999) or in response to virus infection (Borden et al., 1998;
McNally et al., 2008; Turelli et al., 2001). Whereas the nuclear PMLcontaining bodies have been intensely studied over the past 15 years,
comparatively little information is available regarding the
biochemical composition or function of MAPPs and the cytoplasmic
PML-containing particles.
Nuclear pore complexes, the protein assemblies that form
channels through the nuclear envelope, might also contribute to the
pathogenesis of certain types of AML. This is suggested by the
identification of recurrent chromosomal translocations in AML
blasts that involve the genes encoding the nucleoporins Nup98 or
Nup214/CAN (Borrow et al., 1996a; Dash and Gilliland, 2001;
Graux et al., 2004; Kraemer et al., 1994; Nakamura et al., 1996).
In addition, the transcriptional co-activator CREB-binding protein
(CBP), which also represents a recurrent target for chromosomal
translocation in AML blasts (Borrow et al., 1996b; Dash and
Gilliland, 2001), has been shown to functionally interact with
nucleoporin-specific FG-repeat sequences (Kasper et al., 1999).
Nuclear pore complexes have long been known to have a major
role in bidirectional transport across the nuclear membrane (Tran
and Wente, 2006; Weis, 2003), but have in recent years also been
linked to other cellular functions, such as gene expression regulation
(Brown et al., 2008; Faria et al., 2006; Kurshakova et al., 2007;
Mendjan et al., 2006). The precise function of nucleoporins in AML
pathogenesis, however, has not yet been determined.
In addition to their presence in the nuclear pore complexes of
the nuclear membrane, nucleoporins can also be detected in the
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cytoplasm. In infected cells, as well as in oocytes and embryos of
various organisms, fully assembled nuclear pore complexes can be
detected at the electron microscopic level as membranous cisternae
termed annulate lamellae (Kessel, 1992). In addition, a large excess
of soluble, membrane-free nucleoporins in the cytoplasm has also
been reported (Onischenko et al., 2004). Although the existence of
nucleoporins and nuclear pore complexes in the cytoplasm has been
known for several decades, their functions remain largely unknown.
In the present study, we identified a cytoplasmic compartment
that contains high concentrations of nucleoporins, as well as the
tumor-suppressor proteins PML and CBP. These structures, which
we refer to as CyPNs (cytoplasmic accumulations of PML and
nucleoporins), form immediately following exit from mitosis, move
on microtubule filaments and have the ability to dock at the nuclear
membrane. We also find that the cytoplasmic distribution of
nucleoporins becomes affected by siRNA-mediated depletion of
PML and by expression of the PML-RARα fusion protein,
suggesting a functional interaction between PML and nucleoporins
that might contribute to APL pathogenesis.
Results
Journal of Cell Science
Targeting of PML and nucleoporins to CyPNs
By performing immunofluorescence labeling of HaCaT cells using
antibodies against PML in combination with nucleoporin-specific
antibodies, we found that PML and components of the nuclear pore
complex localize to common cytoplasmic compartments that we
have termed CyPNs. Among the nucleoporins that we detected in
CyPNs were Nup153, Nup98 and Nup214. In addition, these
cytoplasmic compartments were effectively labeled by Mab414, a
monoclonal antibody that is known to react with several
nucleoporins through the nucleoporin-specific FG-repeat motif
(Davis and Blobel, 1986). CyPNs were detected in 8-10% of the
cells in an asynchronously growing population of HaCaT cells, and
the number of CyPNs that could be detected within a single cell
generally ranged from 1 to 20. Notably, colocalization between PML
and nucleoporins was detected exclusively in the cytoplasm and
not in the nuclear membrane or within the PML-NBs. We also noted
that CyPNs frequently contacted the nuclear membrane, indicating
an affinity of these structures for the nucleus (Fig. 1A). The
formation of CyPNs was not specific for HaCaT cells, because these
structures could also be detected in HeLa, MCF7, U2OS and
primary fibroblasts in approximately 4%, 5%, 3% and 2% of the
cells, respectively. Additionally, in several cells that we examined,
the cytoplasmic accumulations of PML were found to be restricted
to a subset of nucleoporin-positive structures, suggesting differences
in the biochemical composition between different cytoplasmic
domains enriched in nucleoporins (Fig. 1B). We were not able to
convincingly detect cytoplasmic annulate lamellae in cells that
contained CyPNs (e.g. HaCaT cells) by electron microscopy and
could therefore not determine whether these two types of structure
are related.
CyPNs form during the mitosis-to-G1 transition of the cell cycle
Previous studies have reported a cell-cycle dependent appearance
of cytoplasmic PML particles during the G1 phase (Dellaire et al.,
2006; Everett et al., 1999). This is consistent with the results
described above, which show that only a fraction of asynchronously
growing cells contain CyPNs. To verify that CyPN formation is
cell-cycle dependent, we analyzed HaCaT cells that had been
arrested by culturing in serum-free medium for 3 days and
subsequently released into the cell cycle by serum stimulation.
Fig. 1. PML colocalizes with nucleoporins in the cytoplasm. (A) HaCaT cells
were labeled with anti-PML antibodies (shown in green) in combination with
one of the indicated nucleoporin specific antibodies (red). Merged images
reveal colocalization (yellow) between PML and nucleoporins exclusively
within cytoplasmic subdomains. (B) Immunofluorescence labeling of a
primary human fibroblast cell using anti-PML (red) and the nucleoporinspecific Mab414 antibody (green). PML is detected in a subset of nucleoporinpositive cytoplasmic domains.
Analysis by laser-scanning cytometry revealed that these cells
synchronously entered S-phase at approximately 13 hours after
serum stimulation, and, following progression through G2 and
mitosis, reached a second G1 phase approximately 30 hours after
cell-cycle release (Fig. 2A). To determine the level of CyPNs at
different cell-cycle stages we scored cytoplasmic foci that stained
positive for PML and the nucleoporin Nup153 at different time
points after cell-cycle release. By estimating the percentage of
CyPN-containing cells, as well as the average number of CyPNs
per cell, we found that the level of CyPNs was consistently low
during the first 24 hours after cell-cycle release. At 24-30 hours,
Journal of Cell Science
Nucleoporins and PML in cytoplasm
1203
however, concomitant with exit from mitosis and entry
into G1, we observed a considerable increase in the
number of these cytoplasmic compartments (Fig. 2A).
A similar result was obtained by analyzing HaCaT
cells that had been synchronized by nocodazole-induced
mitotic arrest and subsequent release from the cell-cycle
block by drug removal. In this case too, we observed an
increase of CyPNs as the cells traversed from mitosis to
G1 (Fig. 2B). Together, these results show that CyPNs
primarily form in G1 cells, and that their assembly is
stimulated by a preceding mitosis.
That formation of CyPNs primarily occurs during the
mitosis-to-G1 transition was also supported by highresolution confocal microscopy of HaCaT cells
fluorescently labeled with anti-PML and the nucleoporinspecific antibody Mab414. During all stages of mitosis,
PML was mainly found to accumulate within MAPPs,
whereas the nucleoporins exhibited a dispersed
distribution in prophase, metaphase and telophase, and
tethered to condensed chromatin during cytokinesis.
Immediately after mitosis, concomitant with the
formation of two relatively small closely paired daughter
nuclei with decondensed chromatin, colocalization
between nucleoporins and PML within CyPNs was
almost invariably detected (Fig. 2C). These results show
that complex formation between nucleoporins and PML
is prevented during mitosis but occurs immediately after
its completion.
CyPNs move on the microtubular network
We next studied the dynamics of CyPNs using live-cell
imaging. For this purpose we monitored the movements
of YFP-tagged PML-III transiently expressed in U2OS
cells. As shown in Fig. 3A, YFP-PML-III colocalizes
with nucleoporins in the cytoplasm and exhibits a
cytoplasmic distribution that is consistent with that of
endogenous CyPNs. By using a spinning disc confocal
microscope, we collected a Z-stack of images every
second for a total imaging time of 2 minutes. This
Fig. 2. Cell-cycle-dependent formation of CyPNs. (A) HaCaT cells were grown in serumexperiment revealed that some YFP-PML-III-containing
free medium for 3 days and subsequently stimulated into the cell cycle by adding serum.
particles move rapidly from one cytoplasmic location to
Cell-cycle stages were determined by laser-scanning cytometry of TO-PRO-3-labeled cells
another in short bursts, whereas others remained
(upper panels). The percentage of cells containing CyPNs and the average number of
stationary during the recording period (supplementary
CyPNs per cell was determined by scoring cytoplasmic foci that stained positive for PML
and the nucleoporin Nup153 (bottom panels). (B) HaCaT cells were synchronized by a
material Movie 1). By increasing the intervals between
nocodazole block and subsequently released from the block by drug removal. At the
Z-stack acquisitions to 30 seconds and by using a total
indicated time points, cells were analyzed as in A. All results are mean±s.d. (C) Analysis
recording period of 40 minutes, we found that the
of HaCaT cells by confocal laser-scanning microscopy at different stages of mitosis and
majority of YFP-labeled CyPNs were capable of moving
early G1 phase. Nucleoporins stained by the Mab414 antibody are shown in green, PML in
extensively within the cytoplasm (supplementary material
red and DAPI nuclear staining is blue.
Movie 2). In these longer movies, we also observed
several examples of fusions between YFP-labeled
cytoplasmic bodies (supplementary material Movie 2).
microtubule filaments. For this purpose, we pretreated transfected
Interestingly, most CyPNs appeared to be highly motile as long as
U2OS cells with the microtubule-disrupting drug nocodazole for 2
they did not come into contact with the nuclear surface, but as soon
hours and subsequently recorded the movements of YFP-PML-III
as contact with the nuclear envelope had been made, they remained
by collecting Z-stacks at intervals of 30 seconds for a total imaging
stably connected (Fig. 3B; supplementary material Movie 3). This
period of 40 minutes. The drug treatment caused a considerable
demonstrates an affinity of CyPNs for the nucleus and might explain
reduction in YFP-PML-III motility (compare supplementary
why these structures are frequently detected at or within the nuclear
material Movies 2 and 4). A similar result was also obtained by
envelope (see Fig. 1A).
treating transfected cells with the microtubule-stabilizing drug
Owing to the general involvement of the microtubular network
vinblastine (data not shown). To obtain a graphic representation of
in active cytoplasmic transport in mammalian cells, we wanted to
the data presented in supplementary material Movie 2 (untreated
determine whether the observed motility of CyPNs is facilitated by
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Journal of Cell Science
Fig. 3. Dynamics of CyPNs in living cells.
(A) Immunofluorescence labeling of U2OS cells
transiently expressing YFP-PML-III (shown in green).
The nucleoporins labeled with the Mab414 antibody are
shown in red. (B) Selected still images of living U2OS
cells transiently expressing YFP-PML-III. Z-stacks were
collected at 30 second intervals for a total period of 40
minutes. The cytoplasmic particles indicated by arrows
become stably associated with the nuclear membrane
once they come into contact with the nuclear surface.
The complete movie is presented as supplementary
material Movie 2. (C) The motility of cytoplasmic PML
is inhibited by the microtubule-disrupting drug
nocodazole. U2OS cells transiently expressing YFPPML-III (shown in yellow) and mCherry (shown in gray
to visualize nuclear and cytoplasmic boundaries) were
subjected to live-cell imaging. Z-stacks were collected at
30 second intervals for a total period of 40 minutes. A
number was assigned to each of the cytoplasmic particles
in the two cells, and for each time point the shortest
distance for each of these particles to the nuclear
membrane was determined. The data demonstrate
decreased velocity of CyPNs upon nocodazole treatment.
These data were derived from supplementary material
Movies 2 and 4. (D) U2OS cells transiently coexpressing
CFP-PML-III and YFP-α-tubulin were recorded at 1
frame per second for 2 minutes. The selected still
pictures show a CFP-PML-III-containing particle that
moves along microtubule filaments. The top panels show
the merged images (CFP-PML-III in green and YFP-αtubulin in blue). The bottom panels show YFP-α-tubulin
only (arrow indicates the filament attachment sites).
cells) and Movie 4 (nocodazole-treated cells), we determined the
shortest distance from each of the individual YFP-PML-IIIcontaining particles to the nuclear membrane at each of the time
points collected during imaging. These data are presented in Fig.
3C and illustrate reduced motility of CyPNs in nocodazole-treated
cells compared with that in untreated cells.
To confirm the association between cytoplasmic PML and
microtubule filaments, we coexpressed CFP-PML-III and YFP-αtubulin in U2OS cells. By using a capture rate of 1 frame per second
and a total imaging period of 2 minutes, we were able to visualize
CyPNs that moved on microtubule filaments. One example is
shown in supplementary material Movie 5 and as still images in
Fig. 3D. In this particular example, an YFP-PML-III-containing
particle is observed to move in two consecutive short bursts along
two microtubule filaments running in different directions.
The nuclear localization sequence of PML is required for CyPN
targeting
Several different isoforms of PML are expressed in human cells as
a result of alternative splicing (Jensen et al., 2001). The different
splice variants contain a tripartite TRIM domain (comprising a
RING-finger, two B-boxes and a coiled-coil) in their N-terminus
and a variable C-terminus that might contribute to isoform-specific
functions (Fig. 4A). To assess the ability of different PML isoforms
to target CyPNs, we studied the subcellular localization of PML-I,
PML-II, PML-III, PML-IV, PML-V and PML-VII that were
transiently expressed in U2OS cells. All isoforms, except the
cytoplasmic PML-VII splice variant, were found to target CyPNs
(Fig. 4B). Notably, the transiently expressed PML-III isoform
appeared to be able to target CyPNs more effectively than the other
nuclear isoforms (Fig. 4B).
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Nucleoporins and PML in cytoplasm
Fig. 4. The nuclear-targeting sequence and the RING-finger domain of PML are required for a functional interaction with cytoplasmic nucleoporins.
(A) Schematics of PML isoforms and mutants. The upper bar indicates the functional domains, including the RING finger (R), the two B boxes (B1 and B2), the
coiled-coil domain (CC) and the nuclear-targeting sequence (N). SUMOylation sites (S) are also indicated. Mutated residues are shown in red. (B) Transient
expression of His-tagged PML isoforms in U2OS cells. Cells were fixed 20 hours following transfection and subsequently processed for immunofluorescence
labeling by anti-His and anti-Nup153 antibodies. 100 cells that showed positive staining of the His tag were examined for the presence of CyPNs, and the
percentage of successfully transfected cells containing CyPNs is indicated below the panels. These numbers represent the average of two independent experiments
± s.d. (C) Transient expression of the different mutated forms of PML-III in U2OS cells. Cells were labeled and examined as in B. (D) Living U2OS cells
expressing YFP-PML-III or YFP-PML-IIIringS. The dotted circle indicates the nuclear boundary.
To further examine a link between nuclear import and the ability
of PML to form complexes with CyPNs, we mutated Arg486 and
the Lys487, two amino acids known to be crucial for nuclear import
(Duprez et al., 1999), to Ala (a mutant that we termed PML-IIInls)
(Fig. 4A). This import-defective mutant was detected exclusively
in the cytoplasm following transfection into U2OS cells, but no
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colocalization between PML-IIInls and cytoplasmic nucleoporins
was detected in any of the cells examined (Fig. 4C). This suggested
that the capacity of PML to target CyPNs is linked to its nuclearimport capabilities.
We also mutated the RING-finger domain of PML, because this
motif is thought to be crucially important for several PML functions.
To achieve this, we substituted two of the zinc-coordinating
cysteines (Cys57 and Cys60) with Ser (Fig. 4A) (Shen et al., 2006).
This mutant (that we termed PML-IIIringS) retained its capacity to
target CyPNs, but induced an altered morphology and distribution
of these structures (Fig. 4C,D). Most notably the PML-IIIringS
mutant induced CyPNs that were generally larger in size, fewer in
number and were commonly detected in cytoplasmic regions more
distal to the nuclear envelope compared with the wild-type protein.
Furthermore, live-cell analysis of YFP-tagged PML-IIIringS
revealed that this mutant was completely impaired in its ability to
form stable interactions with the nuclear membrane (Fig. 4D). Since
a RING-finger domain containing Cys-to-Ser mutations might retain
some zinc-binding activity, we also constructed a less conserved
ring finger mutant (PML-IIIringA) where Cys57 and Cys60 were
replaced by Ala (Fig. 4A). The subcellular distribution of PMLIIIringA was found to be indistinguishable from that of PMLIIIringS (Fig. 4C).
Finally, we analyzed the cytoplasmic distribution of a
SUMOylation-defective PML-III protein that contained Lys-to-Arg
substitutions at Lys65, Lys160 and Lys490 (a mutant that we termed
PML-IIIsumo) (Fig. 4A). This mutant colocalized with the
cytoplasmic CyPNs in a manner that was comparable with that seen
for wild-type PML-III (Fig. 4C). Thus, PML SUMOylation appears
to be dispensable for CyPN formation.
Loss of PML causes increased levels of nucleoporins in the
cytoplasm
We next assessed the effect of PML loss on nucleoporin distribution.
For this purpose, we transfected U2OS cells with two different
siRNAs specific for PML and a siRNA designed to target the firefly
luciferase gene as a control. The two PML-specific siRNAs, but
not the control siRNA, caused downregulation of PML expression
and a concomitant increase of nucleoporins in the cytoplasm (Fig.
5). This result suggests a functional role of PML in regulating the
cytoplasmic levels of nucleoporins. The increase of cytoplasmic
nucleoporins was not due to alteration in cell-cycle distribution,
because analysis of PML-depleted and control cells by laserscanning cytometry revealed similar cell-cycle profiles (Fig. 5, right
panels). Notably, the phenotype of PML-depleted U2OS cells
reported here is reminiscent of that previously seen for Nup98negative cells (Wu et al., 2001).
CBP targets CyPNs and MAPPs
To determine whether other proteins, in addition to nucleoporins
and PML, have the capacity to target CyPNs, we tested a number
of different antibodies against nuclear proteins for their ability to
react with these cytoplasmic compartments. Among the antibodies
tested were antibodies specific for MDM2, CyclinB1 (CCNB1),
CyclinE (CCNE1), SUMO1, cdc2, Daxx, BLM, CBP, p21, CyclinA
(CCNA2), OGG1, INI1/SF5, Rad51, RARα and MLL. In agreement
with previous reports (Dellaire et al., 2006; Everett et al., 1999),
the PML-NB-resident portions Daxx and SUMO1 were readily
detected in PML-NBs but were not observed to colocalize with
MAPPs or with PML-containing cytoplasmic particles (data not
shown). The majority of the other antibodies that we tested did not
Fig. 5. Loss of PML causes subcellular redistribution of nucleoporins. U2OS
cells were transfected with the indicated siRNAs and fixed 3 days later. PML
and nucleoporins were visualized in transfected cells by immunofluorescence
labeling using anti-PML and the nucleoporin-specific Mab414 antibody.
Panels to the right show cell-cycle distribution of the transfected cells as
determined by laser-scanning cytometry of TO-PRO-3-stained nuclei.
reveal any affinity for CyPNs, suggesting that the ability to target
CyPNs is restricted to a small group of proteins and is not a general
characteristic of proteins containing a nuclear-targeting signal. The
only protein in addition to PML and nucleoporins that we were able
to detect within CyPNs by using the antibodies mentioned above
was the transcription coactivator CBP. Interestingly, this protein was
also readily detected within MAPPs (Fig. 6). A cytoplasmic
association between PML and CBP was also noted in a previous
study in which CBP and an exogenously expressed nuclear-importdefective mutant of PML were found to colocalize within
cytoplasmic subdomains (Bellodi et al., 2006).
CyPNs are targeted by the PML-RARα fusion protein
Previous studies have revealed a more dispersed distribution of PML
and other PML-NB constituents in APL cells compared with normal
cells, an effect that was attributed to expression of the oncogenic
PML-RARα fusion protein (Dyck et al., 1994; Koken et al., 1994;
Weis et al., 1994). To determine whether this protein chimera also
has the capacity to alter the morphology of CyPNs, we
overexpressed PML-RARα in U2OS cells and subsequently
analyzed the distribution of nucleoporins and the expressed fusion
protein by immunofluorescence. Using antibodies targeted to both
the PML and the RARα moiety of PML-RARα, we detected
cytoplasmic clusters of nucleoporins and the expressed fusion
protein in 15-20% of the transfected cells. Notably, the number of
CyPNs was frequently observed to be markedly higher in PMLRARα-transfected cells compared with untransfected cells (Fig.
7A).
We also examined the APL cell line NB4 for the presence of
CyPNs. This cell line is known to exhibit a dispersed PML
distribution in the nucleus because of expression of PML-RARα,
and cells regain prominent PML-NBs in response to differentiationinduced treatment with all-trans retinoic acid (ATRA) (Dyck et al.,
Nucleoporins and PML in cytoplasm
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Journal of Cell Science
Fig. 6. CBP and PML colocalize with MAPPs and CyPNs in mitotic and
interphase HaCaT cells, respectively. Asynchronous HaCaT cells were fixed in
paraformaldeyhyde and subsequently fluorescently labeled using antibodies
against PML (red) and CBP (green).
1994; Koken et al., 1994; Weis et al., 1994). In untreated NB4 cells,
we detected prominent CyPNs in more than 30% of the cell
population. Moreover, by culturing these cells in the presence of
ATRA, which is known to cause degradation of the PML-RARα
fusion protein, we observed a gradual decrease in the number of
cells containing detectable CyPNs. In addition, CyPNs in ATRAtreated cells appeared smaller than those detected in unstimulated
cells (Fig. 7B). Thus, in contrast to the nuclear PML bodies, the
morphology of CyPNs is not disrupted by PML-RARα expression.
Discussion
The data presented in this study show that two proteins – PML and
CBP – which have roles in regulation of gene expression, become
stably associated with cytoplasmic nucleoporins during the mitosisto-G1 transition. These cytoplasmic protein assemblies, the CyPNs,
have the ability to move on the microtubular network, and they
become stably associated with the nuclear membrane if they come
into contact with the nucleus. Furthermore, CyPN formation is not
supported by the cytoplasmic PML-VII splice variant, and the ability
of PML to target these structures depends on its nuclear-localization
sequence. Thus, despite the cytoplasmic localization of CyPNs, they
might contribute to nuclear functions.
The formation of CyPNs might be transitionally linked to
MAPPs, which represent a cellular compartment specific for the
mitotic phase of the cell cycle (Dellaire et al., 2006; Everett et al.,
1999). This is suggested by the finding that CBP, the only nonnucleoporin protein in addition to PML that we so far have been
able to detect within CyPNs, also accumulates within MAPPs during
mitosis. In addition, the formation of CyPNs during early G1 of
the cell cycle was greatly stimulated by a preceding mitosis,
suggesting the involvement of mitotic proteins and/or structures for
their formation. Thus, PML might form a complex with CBP (and
possibly other proteins) within MAPPs during the mitotic phase of
the cell cycle, and subsequently, following completion of mitosis,
might become stably attached to nucleoporins. A contributing role
of MAPPs to the formation of cytoplasmic PML particles has also
been proposed in previous reports (Chen et al., 2008; Dellaire et
al., 2006).
The ability of PML to become linked to cytoplasmic nucleoporins
in early G1 cells appears to be critically dependent on its nucleartargeting domain, because both the cytoplasmic PML-VII isoform,
Fig. 7. PML-RARα targets CyPNs. (A) U2OS cells were transfected with a
PML-RARα-expressing plasmid and fixed in paraformaldehyde 20 hours later.
Immunofluorescence was then detected using the antibodies indicated. An
expanded view of these images showing untransfected neighboring cells is
presented in supplementary material Fig. S1. (B) NB4 cells were fixed in
paraformaldehyde and fluorescently labeled using anti-PML (red) and the
nucleoporin-specific antibody Mab414 (green) at the indicated time points
following addition of ATRA to the cell culture medium. Some CyPNs are
indicated by white arrows. The fraction of cells containing CyPNs and the
average number of CyPNs per cell was determined (lower panels). Results are
means±s.d.
as well as the nuclear import defective PML-IIInls mutant, failed
to accumulate within CyPNs. This argues that PML is capable of
associating stably with cytoplasmic nucleoporins by interacting
directly with components of the nuclear pore complexes that
mediate its nuclear import. The PML protein could therefore
potentially become targeted to these cytoplasmic compartments by
interacting with import adaptor proteins, such as importin-α or
importin-β. Consistent with this notion, previous studies show that
both of these import receptors are present in nuclear pore complexes
of cytoplasmic annulate lamellae in Xenopus egg extracts (Cordes
et al., 1997; Miller and Forbes, 2000). However, since PML contains
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an unconventional nuclear localization signal that does not match
the consensus of any of the nuclear localization motifs that currently
are known to bind import factors (Duprez et al., 1999), it might be
equally possible that the nuclear-targeting sequence of PML contacts
other components of the nuclear pore complex. In this respect it is
of interest to note that CBP, the other CyPN constituent discovered
in this study, has been reported to interact directly with nucleoporinspecific FG repeats (Kasper et al., 1999). Further studies should
determine the precise protein interactions that take place between
the nuclear-targeting domain of PML and components of the
nuclear pore complexes.
The dependency of the PML nuclear-targeting sequence for
association with cytoplasmic nucleoporins might also indicate that
CyPNs represent nonfunctional complexes that form because of
fortuitous interactions between PML and nuclear-import receptors
present on fully assembled cytoplasmic nuclear pore complexes.
However, several findings made in the present study suggest that
cotargeting of PML and nucleoporins to common cytoplasmic
compartments reflects a functional interaction between PML and
constituents of the nuclear pore complexes. First, the PML-IIIring
mutant, which contains a disrupted RING-finger domain, induced
the formation of morphologically deformed CyPNs, which, in
contrast to CyPNs containing wild-type PML-III, were impaired
in their ability to dock at the nuclear membrane. Second, siRNAmediated depletion of PML caused a considerable increase in the
levels of cytoplasmic nucleoporins, suggesting a functional role
of PML in regulating the balance between cytoplasmic versus
membrane-bound nucleoporins. Last, during the course of this
study, more than 15 different proteins were examined for their
ability to target CyPNs, and only two of these (PML and CBP)
could be detected within these compartments. Thus, although it
is expected that other proteins in addition to those identified in
the present study are present within CyPNs, these structures appear
to be highly selective for a relatively small group of nuclear
proteins and do not generally attract proteins that contain a nuclear
localization signal.
Since both PML and CBP are known to be involved in various
aspects of gene expression, this might indicate a role of CyPNs in
transcriptional regulation. This idea is consistent with several
studies demonstrating a role of nuclear pore complexes in the
regulation of gene expression. Most notably several studies in yeast
have demonstrated activation of gene expression following the
recruitment of genetic elements from the interior of the nucleus to
nuclear pore proximal sites at the nuclear periphery (Casolari et al.,
2004; Menon et al., 2005; Schmid et al., 2006; Taddei et al., 2006).
In a recent study performed on human cells, several binding sites
for the nucleoporin Nup93 were mapped to regions of transcriptional
regulation on chromosomes 5, 7 and 16, indicating a similar role
of nuclear pore complexes in mammalian gene expression (Brown
et al., 2008). An intriguing possibility is that some protein complexes
that participate in transcriptional regulation might be routed to the
nucleus by a mechanism that involves preassembly of nucleoporins
and transcription complexes in the cytoplasm and subsequent
exposure of these transcription factors to the nuclear environment
following insertion of new nuclear pore channels into the nuclear
envelope.
It is also possible that the observed colocalization between PML
and cytoplasmic nucleoporins reflects a role of these proteins in
nucleocytoplasmic transport. In agreement with this, PML has been
implicated in the export of selected mRNA species involved in cellcycle regulation through an interaction with the translation initiation
factor eIF4E (Cohen et al., 2001; Culjkovic et al., 2005; Culjkovic
et al., 2006; Lai and Borden, 2000).
A third possibility could be that CyPNs represent sites of protein
degradation and that the presence of nucleoporins within these
structures in early G1 phase reflects an excess of nuclear pore
complex constituents that were not incorporated into the progeny
nuclei following completion of mitosis. This theory is consistent
with previous studies supporting a role of PML in protein
degradation (Chai et al., 1999; Janer et al., 2006). Furthermore, a
role of PML in degrading excessive nucleoporins is in agreement
with the observed increase of cytoplasmic nucleoporins in PMLdepleted cells.
Although it has long been known that the cytoplasm contains
PML and nucleoporins that are readily visualized by fluorescence
microcopy, very little functional information exists regarding the
cytoplasmic life of these proteins. The data presented here reveal
a dynamic interplay between nucleoporins, the tumor suppressor
proteins PML and CBP, and the nuclear membrane. Furthermore,
several of the CyPN-resident proteins, including Nup98, Nup214,
PML and CBP are encoded by genes that are at the break points of
chromosomal translocations that recurrently are identified in AML
blasts. Further research into the composition and function of CyPNs
might therefore provide new insight into the molecular mechanisms
that determine the development of this type of cancer.
Materials and Methods
Cell-lines and cell-cycle synchronization
The cell lines U2OS (human osteosarcoma) and HaCaT (human keratinocyte), HeLa
(human cervical cancer) and NB4 (human promyelocytic leukemia) were maintained
in Iscove’s modified Dulbecco’s medium (IMDM) (BioWhittaker, Belgium)
containing 10% fetal calf serum (PAA, Austria). MCF7 cells were grown in RPMI
medium (BioWhittaker) containing 10% fetal calf serum. Human embryonic (HE)
fibroblast cells were obtained from the National Institute of Public Health (Oslo,
Norway) and grown in Dulbecco’s modified Eagle’s medium (BioWhittaker) with
10% fetal calf serum. Synchronization by serum starvation was performed by culturing
HaCaT cells on coverslips for 72 hours in serum-free medium followed by release
into the cell cycle by adding fresh pre-warmed medium containing 10% serum.
Synchronization of cells in the mitotic phase of the cell cycle was achieved by culturing
HaCaT cells on coverslips for 16 hours in the presence of 60 ng/ml nocodazole
(Calbiochem, CA) and subsequent cell-cycle release by adding pre-warmed drugfree medium. ATRA was purchased from Sigma and used at 1 μM.
Antibodies and immunofluorescence
The antibodies used were mouse anti-PML (PG-M3), rabbit anti-PML (H238) and
rabbit anti-CBP (A-22) from Santa Cruz, mouse anti-Nup153 (QE5), Mab414, mouse
anti-His (HIS.H8) rat anti-Nup98 (2H10) and rabbit anti-Nup214 (Ab40797) from
Abcam and mouse anti-HA (12CA5) from Roche. Fixation and immunofluorescence
labeling of cells was performed as previously described (Jul-Larsen et al., 2004).
Plasmids, siRNAs and transfection
His-tagged PML isoforms I to V cloned into pcDNA3 were kindly provided by KunSang Chang at the University of Texas (Xu et al., 2005). His-tagged PML-VII was
derived from pcDNA-PML-V by first introducing a ClaI site at nucleotide positions
1345-1350 by silent mutagenesis and subsequently placing a synthetic ClaI-XbaI linker
containing the PML-VII specific C-terminal sequence into the ClaI-XbaI site.
Plasmids expressing YFP-PML-III or CFP-PML-III were kindly provided by Karien
Wiesmeijer and Roeland Dirks at Leiden University Medical Centre in the Netherlands.
The plasmid expressing YFP-tagged α-tubulin was kindly provided by Michaël Marie
at the University of Bergen, Norway. The pCNA-PML-RARα was a kind gift from
Pier G. Pelicci at the European Institute of Oncology, Milan, Italy. The mutants PMLIIInls (R486A; K487A), PML-IIIringS (C57S; C60S), PML-IIIringA (C57A;C60A)
and PML-IIIsumo (K65R; K160R; K490R) were generated by using the QuikChange
kit from Stratagene.
Plasmid transfections were performed using the FuGENE6 transfection reagent from
Roche. In all experiments, cells were analyzed or fixed at 12-24 hours following
transfection. Transfections with siRNAs were performed using Oligofectamine
(Invitrogen, CA) according to the manufacturer’s instructions. Cells were fixed and
processed for immunofluorescence at day 3 after transfection. siRNA oligos used were:
PML siRNA1, 5⬘-GUUUCUGCGCUGCCAGCAAtt-3⬘ (Eurogentec, Belgium); PML
siRNA2, 5⬘-AGAUGCAGCUGUAUCCAAGtt-3⬘ (Ambion, TX); control siRNA
(against fire fly luciferase), 5⬘-UCGAAGUACUCAGCGUAAGtt-3⬘ (Eurogentec).
Nucleoporins and PML in cytoplasm
Laser-scanning cytometry (LSC) and quantification of CyPNs
To determine the cell-cycle profile of asynchronous and synchronously gowning cells
on coverslips DNA was stained with TO-PRO-3 (Invitrogen), and subjected to laserscanning cytometry (CompuCyte, Cambridge, MA). CyPN quantification was
performed by immunofluorescence labeling using antibodies against Nup153 and
PML. For each sample 6-8 randomly selected microscopic fields were captured by
using a Leica DMRXA fluorescent microscope equipped with a ⫻63 oil-immersion
objective and a DC500 camera. Quantification was performed on merged Z-stacks
of 8-12 images comprising the entire cell, and CyPNs were scored positive if the
localization of Nup153 and PML labeled particles overlapped. Both the percentage
of cells containing CyPNs as well as the average number of CyPNs per cell was
calculated. Quantification of CyPNs in transfected cells was performed by analyzing
a total of 100 randomly selected transfected cells (identified by His-tag-positive
staining) that had been labeled with anti-His and anti-Nup153 antibodies. Cells were
scored positive if His- and Nup153-positive cytoplasmic particles colocalized.
Journal of Cell Science
Confocal microscopy and live-cell imaging
Confocal microscopy images were obtained using a Zeiss LSM 510 Meta laser
confocal microscope (Jena, Germany) equipped with a ⫻63 oil-immersion lens (Zeiss).
Live cell imaging was performed using an Ultraview spinning disc confocal
microscope from Perkin Elmer (Wellesly, MA) attached to a ⫻63 oil-immersion lens.
Cells were grown in 6 cm glass bottom dishes from MatTek (Ashland, MA) and
transfected 1 day before imaging. During imaging, cells were maintained at 37°C
and 5% CO2 using an environmental chamber. For short sequences, Z-stacks of 1-3
images were collected every second for 2 minutes. For longer periods of imaging
(30 to 40 minutes total imaging time) Z-stacks of 8-12 images were collected at
intervals of 30 seconds. Exposure times for each of the images varied from 30
mseconds to 60 mseconds. Velocity diagrams were obtained by determining the
shortest distance between a YFP-labeled particle to the nuclear membrane using the
measuring tool in Adobe Photoshop.
This work was supported by the Cancer Gene Therapy Program
funded by the Norwegian Health Department and Helse-Vest. Support
from the Norwegian Cancer Society and the Norwegian Research
Council is also acknowledged. Live-cell imaging was performed at the
National Technology Platform Molecular Imaging Center, supported
by the functional genomics program (FUGE) in the Research Council
of Norway.
References
Bellodi, C., Kindle, K., Bernassola, F., Dinsdale, D., Cossarizza, A., Melino, G., Heery,
D. and Salomoni, P. (2006). Cytoplasmic function of mutant promyelocytic leukemia
(PML) and PML-retinoic acid receptor-alpha. J. Biol. Chem. 281, 14465-14473.
Bernardi, R. and Pandolfi, P. P. (2007). Structure, dynamics and functions of promyelocytic
leukaemia nuclear bodies. Nat. Rev. Mol. Cell. Biol. 8, 1006-1016.
Borden, K. L. (2008). Pondering the puzzle of PML (promyelocytic leukemia) nuclear
bodies: Can we fit the pieces together using an RNA regulon? Biochim. Biophys. Acta.
1783, 2145-2154.
Borden, K. L., Campbell Dwyer, E. J. and Salvato, M. S. (1998). An arenavirus RING
(zinc-binding) protein binds the oncoprotein promyelocyte leukemia protein (PML) and
relocates PML nuclear bodies to the cytoplasm. J. Virol. 72, 758-766.
Borrow, J., Shearman, A. M., Stanton, V. P., Jr, Becher, R., Collins, T., Williams, A.
J., Dube, I., Katz, F., Kwong, Y. L., Morris, C. et al. (1996a). The t(7;11)(p15;p15)
translocation in acute myeloid leukaemia fuses the genes for nucleoporin NUP98 and
class I homeoprotein HOXA9. Nat. Genet. 12, 159-167.
Borrow, J., Stanton, V. P., Jr, Andresen, J. M., Becher, R., Behm, F. G., Chaganti, R.
S., Civin, C. I., Disteche, C., Dube, I., Frischauf, A. M. et al. (1996b). The
translocation t(8;16)(p11;p13) of acute myeloid leukaemia fuses a putative
acetyltransferase to the CREB-binding protein. Nat. Genet. 14, 33-41.
Brown, C. R., Kennedy, C. J., Delmar, V. A., Forbes, D. J. and Silver, P. A. (2008).
Global histone acetylation induces functional genomic reorganization at mammalian
nuclear pore complexes. Genes Dev. 22, 627-639.
Casolari, J. M., Brown, C. R., Komili, S., West, J., Hieronymus, H. and Silver, P. A.
(2004). Genome-wide localization of the nuclear transport machinery couples
transcriptional status and nuclear organization. Cell 117, 427-439.
Chai, Y., Koppenhafer, S. L., Shoesmith, S. J., Perez, M. K. and Paulson, H. L. (1999).
Evidence for proteasome involvement in polyglutamine disease: localization to nuclear
inclusions in SCA3/MJD and suppression of polyglutamine aggregation in vitro. Hum.
Mol. Genet. 8, 673-682.
Chen, Y. C., Kappel, C., Beaudouin, J., Eils, R. and Spector, D. L. (2008). Live cell
dynamics of promyelocytic leukemia nuclear bodies upon entry into and exit from mitosis.
Mol. Biol. Cell 19, 3147-3162.
Cohen, N., Sharma, M., Kentsis, A., Perez, J. M., Strudwick, S. and Borden, K. L.
(2001). PML RING suppresses oncogenic transformation by reducing the affinity of
eIF4E for mRNA. EMBO J. 20, 4547-4559.
Cordes, V. C., Rackwitz, H. R. and Reidenbach, S. (1997). Mediators of nuclear protein
import target karyophilic proteins to pore complexes of cytoplasmic annulate lamellae.
Exp. Cell Res. 237, 419-433.
1209
Culjkovic, B., Topisirovic, I., Skrabanek, L., Ruiz-Gutierrez, M. and Borden, K. L.
(2005). eIF4E promotes nuclear export of cyclin D1 mRNAs via an element in the 3⬘UTR.
J. Cell Biol. 169, 245-256.
Culjkovic, B., Topisirovic, I., Skrabanek, L., Ruiz-Gutierrez, M. and Borden, K. L.
(2006). eIF4E is a central node of an RNA regulon that governs cellular proliferation.
J. Cell Biol. 175, 415-426.
Dash, A. and Gilliland, D. G. (2001). Molecular genetics of acute myeloid leukaemia.
Best Pract. Res. Clin. Haematol. 14, 49-64.
Davis, L. I. and Blobel, G. (1986). Identification and characterization of a nuclear pore
complex protein. Cell 45, 699-709.
de The, H., Chomienne, C., Lanotte, M., Degos, L. and Dejean, A. (1990). The t(15;17)
translocation of acute promyelocytic leukaemia fuses the retinoic acid receptor alpha
gene to a novel transcribed locus. Nature 347, 558-561.
Dellaire, G., Eskiw, C. H., Dehghani, H., Ching, R. W. and Bazett-Jones, D. P. (2006).
Mitotic accumulations of PML protein contribute to the re-establishment of PML nuclear
bodies in G1. J. Cell Sci. 119, 1034-1042.
Duprez, E., Saurin, A. J., Desterro, J. M., Lallemand-Breitenbach, V., Howe, K., Boddy,
M. N., Solomon, E., de The, H., Hay, R. T. and Freemont, P. S. (1999). SUMO-1
modification of the acute promyelocytic leukaemia protein PML: implications for nuclear
localisation. J. Cell Sci. 112, 381-393.
Dyck, J. A., Maul, G. G., Miller, W. H., Jr, Chen, J. D., Kakizuka, A. and Evans, R.
M. (1994). A novel macromolecular structure is a target of the promyelocyte-retinoic
acid receptor oncoprotein. Cell 76, 333-343.
Everett, R. D. and Chelbi-Alix, M. K. (2007). PML and PML nuclear bodies: implications
in antiviral defence. Biochimie 89, 819-830.
Everett, R. D., Lomonte, P., Sternsdorf, T., van Driel, R. and Orr, A. (1999). Cell
cycle regulation of PML modification and ND10 composition. J. Cell Sci. 112, 45814588.
Faria, A. M., Levay, A., Wang, Y., Kamphorst, A. O., Rosa, M. L., Nussenzveig, D.
R., Balkan, W., Chook, Y. M., Levy, D. E. and Fontoura, B. M. (2006). The
nucleoporin Nup96 is required for proper expression of interferon-regulated proteins
and functions. Immunity 24, 295-304.
Graux, C., Cools, J., Melotte, C., Quentmeier, H., Ferrando, A., Levine, R., Vermeesch,
J. R., Stul, M., Dutta, B., Boeckx, N. et al. (2004). Fusion of NUP214 to ABL1 on
amplified episomes in T-cell acute lymphoblastic leukemia. Nat. Genet. 36, 1084-1089.
Janer, A., Martin, E., Muriel, M. P., Latouche, M., Fujigasaki, H., Ruberg, M., Brice,
A., Trottier, Y. and Sittler, A. (2006). PML clastosomes prevent nuclear accumulation
of mutant ataxin-7 and other polyglutamine proteins. J. Cell Biol. 174, 65-76.
Jensen, K., Shiels, C. and Freemont, P. S. (2001). PML protein isoforms and the
RBCC/TRIM motif. Oncogene 20, 7223-7233.
Jul-Larsen, A., Visted, T., Karlsen, B. O., Rinaldo, C. H., Bjerkvig, R., Lonning, P.
E. and Boe, S. O. (2004). PML-nuclear bodies accumulate DNA in response to
polyomavirus BK and simian virus 40 replication. Exp. Cell Res. 298, 58-73.
Kakizuka, A., Miller, W. H., Jr, Umesono, K., Warrell, R. P., Jr, Frankel, S. R., Murty,
V. V., Dmitrovsky, E. and Evans, R. M. (1991). Chromosomal translocation t(15;17)
in human acute promyelocytic leukemia fuses RAR alpha with a novel putative
transcription factor, PML. Cell 66, 663-674.
Kasper, L. H., Brindle, P. K., Schnabel, C. A., Pritchard, C. E., Cleary, M. L. and van
Deursen, J. M. (1999). CREB binding protein interacts with nucleoporin-specific FG
repeats that activate transcription and mediate NUP98-HOXA9 oncogenicity. Mol. Cell.
Biol. 19, 764-776.
Kessel, R. G. (1992). Annulate lamellae: a last frontier in cellular organelles. Int. Rev.
Cytol. 133, 43-120.
Koken, M. H., Puvion-Dutilleul, F., Guillemin, M. C., Viron, A., Linares-Cruz, G.,
Stuurman, N., de Jong, L., Szostecki, C., Calvo, F., Chomienne, C. et al. (1994).
The t(15;17) translocation alters a nuclear body in a retinoic acid-reversible fashion.
EMBO J. 13, 1073-1083.
Kraemer, D., Wozniak, R. W., Blobel, G. and Radu, A. (1994). The human CAN protein,
a putative oncogene product associated with myeloid leukemogenesis, is a nuclear
pore complex protein that faces the cytoplasm. Proc. Natl. Acad. Sci. USA 91, 15191523.
Kurshakova, M. M., Krasnov, A. N., Kopytova, D. V., Shidlovskii, Y. V., Nikolenko,
J. V., Nabirochkina, E. N., Spehner, D., Schultz, P., Tora, L. and Georgieva, S. G.
(2007). SAGA and a novel Drosophila export complex anchor efficient transcription
and mRNA export to NPC. EMBO J. 26, 4956-4965.
Lai, H. K. and Borden, K. L. (2000). The promyelocytic leukemia (PML) protein
suppresses cyclin D1 protein production by altering the nuclear cytoplasmic distribution
of cyclin D1 mRNA. Oncogene 19, 1623-1634.
Lin, D. Y., Huang, Y. S., Jeng, J. C., Kuo, H. Y., Chang, C. C., Chao, T. T., Ho, C. C.,
Chen, Y. C., Lin, T. P., Fang, H. I. et al. (2006). Role of SUMO-interacting motif in
Daxx SUMO modification, subnuclear localization, and repression of sumoylated
transcription factors. Mol. Cell 24, 341-354.
McNally, B. A., Trgovcich, J., Maul, G. G., Liu, Y. and Zheng, P. (2008). A role for
cytoplasmic PML in cellular resistance to viral infection. PLoS ONE 3, e2277.
Mendjan, S., Taipale, M., Kind, J., Holz, H., Gebhardt, P., Schelder, M., Vermeulen,
M., Buscaino, A., Duncan, K., Mueller, J. et al. (2006). Nuclear pore components are
involved in the transcriptional regulation of dosage compensation in Drosophila. Mol.
Cell 21, 811-823.
Menon, B. B., Sarma, N. J., Pasula, S., Deminoff, S. J., Willis, K. A., Barbara, K. E.,
Andrews, B. and Santangelo, G. M. (2005). Reverse recruitment: the Nup84 nuclear
pore subcomplex mediates Rap1/Gcr1/Gcr2 transcriptional activation. Proc. Natl. Acad.
Sci. USA 102, 5749-5754.
Miller, B. R. and Forbes, D. J. (2000). Purification of the vertebrate nuclear pore complex
by biochemical criteria. Traffic 1, 941-951.
1210
Journal of Cell Science 122 (8)
Journal of Cell Science
Nakamura, T., Largaespada, D. A., Lee, M. P., Johnson, L. A., Ohyashiki, K., Toyama,
K., Chen, S. J., Willman, C. L., Chen, I. M., Feinberg, A. P. et al. (1996). Fusion of
the nucleoporin gene NUP98 to HOXA9 by the chromosome translocation
t(7;11)(p15;p15) in human myeloid leukaemia. Nat. Genet. 12, 154-158.
Onischenko, E. A., Gubanova, N. V., Kieselbach, T., Kiseleva, E. V. and Hallberg, E.
(2004). Annulate lamellae play only a minor role in the storage of excess nucleoporins
in Drosophila embryos. Traffic 5, 152-164.
Salomoni, P., Ferguson, B. J., Wyllie, A. H. and Rich, T. (2008). New insights into the
role of PML in tumour suppression. Cell Res. 18, 622-640.
Schmid, M., Arib, G., Laemmli, C., Nishikawa, J., Durussel, T. and Laemmli, U. K.
(2006). Nup-PI: the nucleopore-promoter interaction of genes in yeast. Mol. Cell 21,
379-391.
Shen, T. H., Lin, H. K., Scaglioni, P. P., Yung, T. M. and Pandolfi, P. P. (2006). The
mechanisms of PML-nuclear body formation. Mol. Cell 24, 331-339.
Taddei, A., Van Houwe, G., Hediger, F., Kalck, V., Cubizolles, F., Schober, H. and
Gasser, S. M. (2006). Nuclear pore association confers optimal expression levels for
an inducible yeast gene. Nature 441, 774-778.
Tran, E. J. and Wente, S. R. (2006). Dynamic nuclear pore complexes: life on the edge.
Cell 125, 1041-1053.
Turelli, P., Doucas, V., Craig, E., Mangeat, B., Klages, N., Evans, R., Kalpana, G. and
Trono, D. (2001). Cytoplasmic recruitment of INI1 and PML on incoming HIV
preintegration complexes: interference with early steps of viral replication. Mol. Cell 7,
1245-1254.
Weis, K. (2003). Regulating access to the genome: nucleocytoplasmic transport throughout
the cell cycle. Cell 112, 441-451.
Weis, K., Rambaud, S., Lavau, C., Jansen, J., Carvalho, T., Carmo-Fonseca, M.,
Lamond, A. and Dejean, A. (1994). Retinoic acid regulates aberrant nuclear localization
of PML-RAR alpha in acute promyelocytic leukemia cells. Cell 76, 345-356.
Wu, X., Kasper, L. H., Mantcheva, R. T., Mantchev, G. T., Springett, M. J. and van
Deursen, J. M. (2001). Disruption of the FG nucleoporin NUP98 causes selective
changes in nuclear pore complex stoichiometry and function. Proc. Natl. Acad. Sci. USA
98, 3191-3196.
Xu, Z. X., Zou, W. X., Lin, P. and Chang, K. S. (2005). A role for PML3 in centrosome
duplication and genome stability. Mol. Cell 17, 721-732.