EXPERIMENTAL CELL RESEARCH
ARTICLE NO.
229, 174–180 (1996)
0356
SPECIAL ARTICLE
Dynamic Changes in Nuclear Architecture during Mitosis:
On the Role of Protein Phosphorylation in Spindle
Assembly and Chromosome Segregation
ERICH A. NIGG,*,†,1 ANNE BLANGY,†
AND
HEIDI A. LANE†
*Department of Molecular Biology, Sciences II, University of Geneva, 30, Quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland; and
†Swiss Institute for Experimental Cancer Research (ISREC), 155, Chemin des Boveresses, CH-1066 Epalinges, Switzerland
presence of functional centrosomes.
During mitosis, the vertebrate cell nucleus undergoes profound changes in architecture. At the onset of
mitosis, the nuclear envelope breaks down, the nuclear lamina is depolymerized, and interphase chromatin is condensed to chromosomes. Concomitantly,
cytoplasmic microtubules are reorganized into a mitotic spindle apparatus, a highly dynamic structure
required for the segregation of sister chromatids.
Many of the above events are controlled by reversible
phosphorylation. Hence, our laboratory is interested
in characterizing the kinases involved in promoting
progression through mitosis and in identifying their
relevant substrates. Prominent among the kinases responsible for regulating entry into mitosis is the Cdc2
kinase, the first member of the cyclin dependent kinase (Cdk) family. Recently, we found that Cdc2 phosphorylates HsEg5, a human kinesin-related motor protein associated with centrosomes and the spindle apparatus. Our results indicate that phosphorylation
regulates the association of HsEg5 with the mitotic
spindle and that the function of this plus-end directed
motor is essential for centrosome separation and bipolar spindle formation. Another kinase implicated in
regulating progression through mitosis is Plk1 (pololike kinase 1), the human homologue of the Drosophila
gene product ‘‘polo.’’ By antibody microinjection we
have found that Plk1 is required for the functional
maturation of centrosomes and hence for entry into
mitosis. Furthermore, we found that microinjected
anti-Plk1 antibodies caused a more severe block to cell
cycle progression in diploid fibroblasts than in immortalized tumor cells. This observation hints at the existence of a checkpoint linking Cdc2 activation to the
Data presented at a Nobel Symposium on ‘‘The Functional Organization of the Eukaryotic Cell Nucleus,’’ Saltsjöbaden and Stockholm,
September 3–6, 1996.
1
To whom correspondence and reprint requests should be addressed. Fax: /41 22 702 6868. E-mail: [email protected].
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INTRODUCTION
In the life of a vertebrate nucleus, mitosis is a dramatic experience. At the very onset of mitosis, the nuclear envelope breaks down and, as a consequence, nuclear and cytoplasmic contents mix. Concomitantly,
chromatin is condensed to chromosomes, and the separating centrosomes nucleate a set of highly dynamic
microtubules. These assemble to form a bipolar spindle
apparatus, which is necessary for the segregation of
sister chromatids to daughter cells. Considering that
the survival of all organisms depends on the fidelity of
transmission of genetic information during cell division, it is not surprising that checkpoint mechanisms
have evolved to monitor the correct formation and functioning of the spindle apparatus. For example, one of
these checkpoints prevents anaphase onset until all
chromosomes display an appropriate bipolar attachment to the spindle [e.g. 1–3, for reviews see 4, 5].
The failure of such surveillance systems will inevitably
result in genetic instability, which in turn is expected
to lead to cell death in unicellular organisms and to
cancer in multicellular species [reviewed in 6].
Recent years have seen major advances in our understanding of cell cycle regulation. Thus, it is now well
established that key transitions in the cell cycle are
controlled by a family of structurally related protein
kinases, known as the cyclin-dependent kinases
(Cdks). These kinases function in association with
cyclin regulatory subunits, and their activities are further regulated by reversible phosphorylation and by
the binding of inhibitory polypeptides [for reviews see
7–10]. In brief, different Cdk/cyclin complexes are activated at different times during the cell cycle, and they
act on distinct substrates. Complexes of Cdk4 or Cdk6
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FIG. 1. Regulation of entry into mitosis. This scheme illustrates
the central role of the Cdc2/cyclin B complex in promoting the onset
of mitosis. When the Cdc2/cyclin B complex assembles during the
later stages of interphase, the Cdc2 subunit becomes phosphorylated
on three sites: phosphorylation of T161 is essential for kinase activity, whereas phosphorylation of T14 and Y15, by kinases known as
Wee1 and Myt1, inhibits kinase activity. Thus, activation of the Cdc2/
cyclin B complex at the G2-to-M transition requires dephosphorylation of T14 and Y15 by an isoform of the Cdc25 family of dualspecificity phosphatases. In addition, it is possible that activation of
Cdc2/cyclin B may require the destruction or inactivation of an as
yet poorly defined inhibitor. Finally, the functions of other kinases,
notably Plk1 and a NIMA-related kinase (Nek), may also be required
for entry into mitosis. (For references and further discussion see
text).
and D-type cyclins are required for the exit from quiescence and the traverse of G1 phase, and one important
substrate for these kinases is the retinoblastoma protein pRb. Subsequently, the activity of Cdk2/cyclin E is
required for the onset of DNA replication, while Cdk2/
cyclin A is required for progression through S phase.
The definitive identification of physiologically relevant
substrates for Cdk2/cyclin E and Cdk2/cyclin A remains an important task. Finally, complexes of Cdc2
with both A- and B-type cyclins are responsible for promoting the entry of cells into mitosis. As discussed below, Cdc2/cyclin complexes phosphorylate multiple proteins, including kinesin-related motors involved in
spindle assembly and chromosome segregation.
HOW TO GET INTO MITOSIS AND
HOW TO GET OUT AGAIN
Binding of a B-type cyclin is not sufficient to activate
Cdc2 at the G2-to-M transition. Kinase activity also
requires phosporylation of an evolutionarily conserved
threonine (T161 in the case of human Cdc2) located
within the so-called T-loop [reviewed in 9, 11]. Furthermore, Cdc2 is subject to negative regulation by phosphorylation (see Fig. 1). As a consequence, its activation at the G2-to-M transition requires dephosphorylation of threonine 14 (T14) and tyrosine 15 (Y15), two
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neighboring residues located within the ATP binding
site of the kinase [12, 13]. The identities of the kinases
and phosphatases acting on T161 remain a subject of
some debate [for review see 11], but it is well established that phosphorylation of T14 and Y15 is mediated
by kinases known as Wee1 and Myt1 and that dephosphorylation of the same residues is brought about by
a dual-specificity phosphatase termed Cdc25 [for references see 14–19]. Whether dephosphorylation of T14
and Y15 is sufficient to activate Cdc2 under physiological conditions is not entirely clear. It is possible that, in
addition to dephosphorylation, a Cdc2-specific inhibitor
may need to be destroyed or inactivated; however, the
molecular identity of this inhibitor has not yet been
established [20].
Inactivation of mitotic Cdc2/cyclin complexes is
brought about by the proteolytic destruction of the
cyclin subunits. The pathway leading to the activation
of the destruction machinery remains to be elucidated,
but it seems clear that actived Cdc2/cyclin B complexes
themselves initiate a cascade of events which will eventually lead to their inactivation. Cyclin destruction
occurs by a ubiquitination-dependent mechanism [21,
reviewed in 22, 23] and involves the proteasome
multiprotease [24]. How this ubiquitin-dependent destruction machinery is activated at the right time during mitosis and how it is targeted to the appropriate
substrates remain to be understood. However, it is becoming clear that a multiprotein complex, termed APC
(for anaphase-promoting complex; [25]) or ‘‘cyclosome’’
[26], plays a key role in the regulation of anaphase
onset. This multiprotein complex corresponds to the
E3 enzymatic component of the ubiquitin-dependent
proteolytic pathway [for reviews see 24, 27], and its
activity appears to be regulated by cell cycle-dependent
phosphorylation [25]. Since the destruction of cyclin B
coincides temporally with the beginning of sister chromatid separation, it has initially been postulated that
the inactivation of Cdc2 might constitute the trigger
for the onset of anaphase. However, more recent experiments have revealed that proteolysis of proteins other
than cyclin B may be critical for the timing of anaphase
onset [28, 29]. Among the potentially important targets
for proteolysis are the Pds1p gene product from Saccharomyces cerevisiae [30, 31] and the distantly related
Cut2 protein from Schizosaccharomyces pombe [32]
(see Fig. 2). Other proteins of great interest in this
context are the products of the Drosophila genes ‘‘pimples’’ and ‘‘three rows’’; these proteins are essential for
sister chromatid separation, and at least the pimples
protein is rapidly degraded after the metaphase-toanaphase transition [33]. It is possible that pimples
and three rows are involved in targeting kinetochoreassociated proteins for destruction.
Compared to our fairly advanced understanding of
the mechanisms that lead to the activation and inacti-
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NIGG, BLANGY, AND LANE
vide a satisfactory explanation for the regulation of at
least one hallmark event of vertebrate mitosis, i.e., the
disassembly of the nuclear lamina. Furthermore, it
supports the notion that Cdc2-dependent phosphorylation may cause structural changes by rather direct
mechanisms.
KINESIN-RELATED MOTOR PROTEINS AS MITOTIC
TARGETS OF Cdc2 KINASE
FIG. 2. Regulation of exit from mitosis. This scheme emphasizes
the key role of proteolysis in promoting the metaphase-to-anaphase
transition. Activated Cdc2/cyclin B is thought to cause, either directly
or indirectly, the phosphorylation of one or several components of
the APC (i.e., the E3 component of a ubiquitin-dependent proteolytic
degradation system). Activated APC is then thought to target several
proteins for destruction by the proteasome. These proteins include
cyclin B, Pds1p (from S. cerevisiae), and Cut2 (from S. pombe).
Whereas degradation of cyclin B results in the inactivation of Cdc2
kinase, the destruction of Pds1p and Cut2 is essential for sister chromatid separation. (For references and further discussion see text).
vation of Cdc2, much remains to be learned about the
mechanisms by which Cdc2 kinase brings about the
drastic changes in cellular architecture that are characteristic of mitosis. On the one hand, Cdc2 is likely to
activate other kinases. One intriguing example of this
mode of action is provided by the NIMA kinase, a gene
product required for the G2-to-M transition in Aspergillus nidulans [34, reviewed in 35]. In the case of NIMA,
maximal activation has in fact been shown to require
phosphorylation of the C-terminus by Cdc2 [36]. However, although kinases structurally related to NIMA
have been identified in mammals, the functions of these
NIMA related kinases (or Neks) remain to be determined. As yet, no bona fide functional homologue of
NIMA has definitively been identified in vertebrates,
and the precise function(s) of NIMA remains unknown
even in fungi [for reviews see 35, 37]. On the other
hand, the available evidence indicates that Cdc2 kinase
may directly phosphorylate several of the structural
proteins that are important for reorganizing cellular
architecture during mitosis [for review see 38]. One
striking illustration of this point is provided by the
nuclear lamins, a family of proteins whose polymerization state is regulated directly by Cdc2: in the interphase nucleus, these intermediate filament type
proteins form a karyoskeleton underlying the inner nuclear membrane, but at the onset of mitosis, the lamina
depolymerizes, at least to a large extent due to the
direct phosphorylation of lamin proteins by Cdc2 [reviewed in 38]. This latter finding would appear to pro-
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The mechanisms and regulation of spindle assembly
and chromosome segregation have fascinated cell biologists for more than 100 years [e.g., 39]. As is readily
apparent from the microscopic examination of chromosomes, centrosomes, and spindle microtubules in living
cells, spindle assembly and chromosome segregation
represent highly dynamic processes. Many of the forces
involved in orchestrating the various movements (e.g.,
centrosome separation during bipolar spindle formation, congression of chromosomes to a metaphase plate,
chromosome to pole movement during anaphase A, and
spindle pole separation during anaphase B) may be
attributed to mechanochemical motor proteins that associate with different elements of the mitotic spindle
apparatus. These microtubule-dependent ATPases belong to the families of both kinesin related proteins
(KRPs) and cytoplasmic dyneins [reviewed in 40–44].
In vertebrates, cytoplasmic dynein and at least seven
kinesin-related proteins have been found in the mitotic
spindle, and it will be an important but formidable task
to unravel the function and regulation of each of these
motors.
Considering that phosphorylation has been implicated in regulating many different aspects of mitosis
(see above), we decided to investigate whether KRPs
might be direct physiological substrates of the Cdc2
kinase. Our initial studies were focused on HsEg5, a
human motor protein belonging to the BimC subfamily
of KRPs [for review see 40, 42]. By microinjection of
antibodies against HsEg5 we were able to show that
this motor is required for the separation of centrosomes
at the onset of mitosis and hence for the formation of
a bipolar spindle [45]. These results corroborate and
extend genetic studies on BimC family members in
lower eukaryotes [46–48] and in vitro studies on the
frog homologue of HsEg5 [49]. By immunofluorescent
staining of cultured HeLa cells with anti-HsEg5 antibodies, we could further show that HsEg5 associates
with centrosomes just prior to the onset of mitosis, i.e.,
at about the time when Cdc2 is activated, and then
stays associated with elements of the spindle apparatus throughout mitosis. Concomitant with the onset of
mitosis, HsEg5 became hyperphosphorylated [45].
Most intriguingly, while HsEg5 was phosphorylated
exclusively on serine residues during interphase of the
cell cycle, our analysis of HsEg5 isolated from mitoti-
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CONTROL OF SPINDLE ASSEMBLY AND CHROMOSOME SEGREGATION
cally arrested cells revealed additional phosphorylation
of threonine(s). Examination of the sequences of HsEg5
and BimC family members showed that all these proteins, with the noticeable exception of the S. cerevisiae
motors Cin8p and Kip1p [50, 51], contain a short (about
40-amino-acid) conserved stretch in the tail domain
(the BimC box), which in turn contains a highly conserved Cdc2 consensus motif (TPxK/RR). Direct biochemical analysis confirmed that this threonine residue (Thr927) is indeed phosphorylated by Cdc2 kinase
in vitro and most likely in vivo [45]. Expression of
HsEg5 mutants lacking this phosphorylation site did
not affect cell cycle progression, but, remarkably, replacements of this critical threonine by nonphosphorylatable residues abolished the ability of both
HsEg5 and Xenopus Eg5 to associate with centrosomes
in their respective species [45, 52]. Hence, phosphorylation by Cdc2 kinase appears to be essential for promoting a strong association of HsEg5 with the spindle apparatus.
The available evidence suggests that BimC motor
proteins may function as tetramers [53]. Thus, it is
possible that phosphorylation might regulate the oligomerization state of HsEg5. Alternatively, phosphorylation might regulate the interaction between HsEg5 and
other proteins. One protein that interacts with HsEg5
in a phosphorylation-dependent manner has recently
been identified in our laboratory, using the yeast 2hybrid screen; at present, work is in progress to determine whether this protein is a likely physiological interactor of HsEg5 (A. Blangy, L. Arnaud, and E. A.
Nigg, unpublished results). How exactly HsEg5 and
other BimC family members function to promote
centrosome separation remains to be determined. Two
possible models for HsEg5 function are illustrated in
Fig. 3 [for further explanation see 42, 49]. As discussed
previously, we consider it attractive to think that not
only BimC family members but several other spindleor chromosome-associated motor proteins may also be
direct substrates of Cdc2 kinase [45].
Plk-1, A NEWLY EMERGING KINASE INVOLVED IN
CELL CYCLE REGULATION
While the key role of Cdc2 kinase in triggering entry
into mitosis is well established and widely appreciated,
there is growing evidence pointing to the involvement
of yet other protein kinases in mitotic progression. In
our laboratory, particular interest is focused on potential human homologues of the NIMA protein kinase
from A. nidulans, which is required for the G2-to-M
transition [34, 35], and on homologues of the polo kinase from Drosophila melanogaster, which is implicated in various aspects of mitosis and cytokinesis [54–
56]. Kinases structurally related to NIMA have been
identified in several species including mammals, where
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FIG. 3. Models for the function of the kinesin-related motor protein HsEg5. HsEg5 displays a tripartite structure, characteristic of
kinesin-related motor proteins. The N-terminal head domain contains the microtubule-dependent motor (ATPase) activity (indicated
by a star), the central domain contains heptad repeats favoring
multimerization via coiled-coil interactions, and the C-terminal tail
domain is believed to interact with as yet unidentified proteins.
Based on results obtained for other BimC family members, HsEg5
is likely to function as a tetramer [53]. To describe the function of
HsEg5 in relation to spindle poles, two distinct models have been
proposed [42, 49]. According to model A, HsEg5 tetramers crosslink
microtubules; in those cases where crosslinked microtubules are nucleated from distinct poles, plus-end directed movement of HsEg5
would cause spindle pole separation. According to model B, HsEg5
interacts with an as yet undefined structural elements associated
with spindle poles. By moving toward the plus-end of microtubules,
it would pull their minus-ends toward the pole during pole separation
and thereby establish a dynamic link between these microtubules
and the pole.
they are called Neks [for review see 37, 57], but a bona
fide homologue of NIMA has so far been found only in
Neurospora crassa [58], and the functions of the mammalian Neks remain unknown. Our understanding is
somewhat further advanced in the case of the polo-like
kinases (or Plk’s) [for review see 59]. Likely functional
homologues of Drosophila polo have in fact been characterized in both budding yeast (where the polo-like
kinase is called Cdc5p; [60]) and fission yeast (where it
is called plo1; [61]). Furthermore, at least three kinases
structurally related to Drosophila polo (termed Plk1
(or Plk), Snk, and Fnk) have been identified in mammals [62–67]. Of these, Plk1 most probably represents
a functional homologue of Drosophila polo.
Strong support for a mitotic function of human Plk1
comes from recent antibody microinjection experiments
performed in our laboratory [68]. When injected into
HeLa cells or human diploid fibroblasts (Hs68 cells),
anti-Plk1 antibodies did not detectably interfere with
either entry into S phase or exit from S phase, at variance with previous sense/antisense RNA injection stud-
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NIGG, BLANGY, AND LANE
ies suggesting a role of Plk1 in DNA replication [65].
Instead, anti-Plk1 antibodies had a profound effect on
mitotic progression and cell division. One particularly
striking phenotypical consequence of anti-Plk1 antibody injection became manifest at the level of
centrosomes: in normal cells, centrosomes undergo
drastic changes at the onset of mitosis, when they acquire the ability to nucleate large numbers of highly
dynamic microtubules [reviewed in 69]. These changes
are accompanied by the recruitment of multiple cytoplasmic proteins to centrosomes, which results in a substantial increase in the size of these organelles [70–
73]. As visualized by immunostaining of cells with antibodies against several centrosome-associated antigens,
these centrosomal changes were abolished when Plk1
function was impaired by microinjection of anti-Plk1
antibodies, and, as a consequence, most of the injected
cells were unable to construct a bipolar spindle [68].
Another interesting observation was made when
comparing the effects produced by microinjected antiPlk1 antibodies in either human diploid fibroblasts or
HeLa cells. Although qualitatively similar aberrant mitotic phenotypes were observed in both cell types, a
majority of the injected diploid fibroblasts never attempted entering mitosis in the presence of anti-Plk1
antibodies [68]. This result is reminiscent of the observation that microsurgical removal of centrosomes from
cultured monkey kidney cells blocks not only spindle
formation, but other manifestations of mitosis such as
chromosome condensation and nuclear envelope breakdown [74]. Taken together, these data suggest that normal cells require Plk1 and/or a functionally mature
centrosome for activating Cdc2. Specifically, we propose that normal cells use a checkpoint mechanism to
monitor the status of centrosomes before they activate
Cdc2. Conceptually, and perhaps mechanistically (see
below), this centrosome-dependent checkpoint may resemble the checkpoints which ensure that Cdc2 is not
activated in the presence of either DNA damage or unreplicated DNA (Fig. 4).
In contrast to Hs68 diploid fibroblasts, almost all
HeLa cells injected with anti-Plk1 antibodies made
some attempt at entering mitosis, although, in the absence of Plk1 function, most of them failed to execute
a proper cell division [68]. These findings suggest that
HeLa cells, and presumably other tumor cells, have
lost the centrosome checkpoint. We consider it plausible that loss of this checkpoint may result in spindle
anomalies and give rise to aneuploidies.
Recently, Kumagai and Dunphy have uncovered an
exciting link between a putative Xenopus homologue of
Plk1 and Cdc2 [75]. These authors have in fact shown
that Xenopus Plk is able to activate Cdc25, the phosphatase that in turn activates Cdc2 (see Fig. 1). Considered in the light of these results, our data suggest that
HeLa cells are able to activate Cdc2 in spite of impaired
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FIG. 4. A centrosome maturation checkpoint for Cdc2 activation?
Prior to the onset of mitosis, centrosomes recruit several proteins and
hence grow in size. Throughout this paper, this process is referred
to as ‘‘maturation.’’ Based on the results of our anti-Plk1 antibody
injection experiments [68], and data from Maniotis and Schliwa [74],
we propose that cells monitor the proper maturation of centrosomes
before they activate Cdc2 at the G2-to-M transition. In addition to
a positive signal emanating from mature centrosomes, it is possible
that immature centrosomes may emit an inhibitory signal. As seems
to be the case for the DNA replication and the DNA damage checkpoints, it is possible that the proposed centrosome maturation checkpoint acts via controlling the phosphorylation state of Cdc2. (For
further discussion see text).
Plk1 function. This appears plausible, since tumor cells
frequently display deregulated expression of cell cycle
regulatory proteins [76], including Cdc25 isoforms [77].
Phosphorylation of Cdc25 by Xenopus Plk generates
phosphoepitopes that can be recognized by the MPM2 monoclonal antibody [75]. This finding is particularly
interesting since several other cell cycle regulators also
acquire MPM-2 immunoreactivity at the onset of mitosis. Although multiple kinases have previously been
shown to be able to generate MPM-2 reactivity [78], it
will clearly be interesting to investigate the possibility
that MPM-2 antigens other than Cdc25 may also represent Plk1 substrates. Prominent among the known
MPM-2 antigens are Wee1 and Myt1, two kinases implicated in the negative regulation of Cdc2 (via phosphorylation of T14 and Y15 (see Fig. 1). Thus, it is
conceivable that Plk1 may not only activate a Cdc2stimulatory pathway, via its demonstrated ability to
activate Cdc25 [75], but may also block a Cdc2-inhibitory pathway, via inhibition of Wee1 and/or Myt1. Furthermore, Plk1 has been observed to localize to several
cellular substructures that are known to acquire MPM2 immunoreactivity during mitosis [79, 80]. Thus,
MPM-2 antigens associated with mitotic chromosomes
and/or the spindle apparatus would also seem to qualify as candidate Plk1 substrates.
The phenotypes caused by mutations of putative
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CONTROL OF SPINDLE ASSEMBLY AND CHROMOSOME SEGREGATION
Plk1 homologues in invertebrates and lower eukaryotes (Drosophila, S. cerevisiae and S. pombe) strongly
suggest that Plk’s may perform multiple functions [54–
56, 60, 61]. Although direct experimental evidence for
late mitotic functions of mammalian Plk1 is lacking,
the observed changes in subcellular localization of this
kinase would seem consistent with the idea of multiple
functions during mitosis [79]. We consider it attractive
to propose, therefore, that Plk1 might serve to integrate spindle function with the activity of Cdk/cyclin
complexes throughout cell division.
Work in the authors’ laboratory was supported by grants from
the Swiss National Science Foundation (31-33615.92) and the Swiss
Cancer League (SKL 267-1-1996). H.A.L. acknowledges a postdoctoral fellowship from the Schering Research Foundation, Berlin, and
A.B. acknowledges a postdoctoral fellowship from the Roche Foundation, Basel.
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Received September 4, 1996
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