Linked for life: temporal and spatial coordination of
late mitotic events
Anupama Seshan and Angelika Amon1
Establishing the temporal order of mitotic events is critical to
ensure that each daughter cell receives a complete DNA
complement. The spatial co-ordination of the cytokinetic ring site
with the axis of chromosome segregation is likewise crucial.
Recent studies in fungi indicate that regulators of chromosome
segregation also participate in promoting mitotic exit and that the
proteins that initiate mitotic exit, in turn, additionally regulate
cytokinesis. These findings suggest that late mitotic events are
coupled by employing one pathway to control multiple events.
The regulatory mechanisms that ensure the spatial co-ordination
of the mitotic spindle apparatus with the division site have also
been elucidated recently in the asymmetrically dividing budding
yeast. Interestingly, the spatial co-ordination of late mitotic
events seems also to be important in higher eukaryotes.
Addresses
Center for Cancer Research, Howard Hughes Medical Institute,
Massachusetts Institute of Technology, E17-233, 40 Ames Street,
Cambridge MA 02139, USA
1
e-mail: angelika@mit.edu
Current Opinion in Cell Biology 2004, 16:41–48
This review comes from a themed issue on
Cell structure and dynamics
Edited by John A Cooper and Margaret A Titus
0955-0674/$ – see front matter
ß 2003 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.ceb.2003.11.003
Abbreviations
CDK cyclin-dependent kinase
FEAR cdc14 early anaphase release
GEF guanine exchange factor
MEN mitotic exit network
MC mother centriole
PAK p21-activated protein kinase
SIN septation initiation network
SPB spindle pole body
Introduction
Preserving the sequence of events during cell division is
essential for the maintenance of genomic integrity and
hence the viability of an organism. Order is established in
two ways: first, by the coupling of cell-cycle events such
that a later step cannot be carried out until a previous one
has occurred, and second, by surveillance mechanisms.
Surveillance mechanisms, also known as checkpoints,
sense whether an event has been completed correctly.
If not, cell-cycle progression is halted until the proper
completion of this stage in the cell cycle.
www.sciencedirect.com
During mitosis, the onset of chromosome segregation, the
exit from mitosis and cytokinesis are all under the control
of surveillance mechanisms and coupled to one another to
achieve temporal and spatial coordination. This review
will focus on recent findings in budding and fission yeast,
where the mechanisms that establish the order of events
during mitosis are best understood. We will also describe
recent discoveries in Drosophila melanogaster and mam-
mals that provide insights into how order is established
in metazoans.
Temporal controls
Coupling chromosome segregation and exit from
mitosis
The mechanism whereby chromosome segregation is
initiated at the metaphase–anaphase transition is con-
served from yeast to human ([1]; Figure 1). A protease
known as Separase cleaves a component of the cohesin
complex (Scc1/Mcd1 in Saccharomyces cerevisiae, Rad21 in
S. pombe and D. melanogaster and Scc1 in human), which
holds sister chromatids together. In this process, Separase
is aided by Polo kinase. Polo kinase phosphorylates the
Separase target in the cohesin complex, thereby promot-
ing its cleavage [2–4].
Exit from mitosis occurs after the completion of chromo-
some segregation and involves the disassembly of the
mitotic spindle and the decondensation of chromosomes.
The inactivation of mitotic cyclin-dependent kinases
(CDKs), the protein kinases that promote entry into
mitosis earlier in the cell cycle, appears to be required
for this transition to occur in all species examined to date
(Figure 1). Mitotic CDK inactivation is predominantly
achieved by the ubiquitin-dependent degradation of the
regulatory cyclin subunit.
In budding yeast, mitotic CDK inactivation, and hence
exit from mitosis, requires two regulatory networks: the
Cdc14 early anaphase release (FEAR) network and the
mitotic exit network (MEN; see Figure 1). Both signal-
ing networks control the activity of the protein phospha-
tase Cdc14, which initiates mitotic CDK inactivation by
reversing CDK-dependent phosphorylation (reviewed
in [5]). The newly identified FEAR network activates
Cdc14 during early anaphase, whereas the MEN, which
resembles a Ras-like signaling cascade, stimulates Cdc14
activity during late stages of anaphase and telophase
[5,6,7,8].
Budding yeast ensures that mitotic exit does not occur
before chromosome segregation by utilizing the same
Current Opinion in Cell Biology 2004, 16:41–48
42 Cell structure and dynamics

Figure 1

FEAR network MEN

Bub2/Bfa1 Lte1
Securin
(Pds1)
Tem1

Nud1
Spo12/Bns1 Cdc15
Slk19
Separase Polo
(Esp1) (Cdc5) Dbf2/Dbf20–Mob1

Cohesin Cdc14 Mitotic CDKs

Metaphase Anaphase G1

Current Opinion in Cell Biology

Components of the FEAR network regulate both the metaphase–anaphase transition and the exit from mitosis in yeast. The metaphase–anaphase
transition is triggered by the destruction of cohesin, the glue that holds sister chromatids together. Securin inhibits Separase, which cleaves the
cohesin subunit Scc1/Mcd1, thereby allowing sister chromatids to separate. Polo kinase aids in the dissolution of sister chromatid cohesion by
phosphorylating Scc1/Mcd1. Separase and Polo also promote exit from mitosis by initiating the release of Cdc14 from the nucleolus during early
anaphase as part of the FEAR network. Additional components of the FEAR network are the kinetochore protein Slk19, and Spo12/Bns1,
proteins of unknown function. A pathway known as MEN promotes and maintains Cdc14 in the released state during late anaphase. The MEN
components Tem1 (a GTPase), Bub2/Bfa1 (a two-component GTPase-activating complex), Cdc15 (a protein kinase), Dbf2 and Dbf20 (homologous
protein kinases) and Mob1 (a Dbf2-associated factor) are anchored at the SPB by a scaffold protein Nud1. The putative GEF Lte1 is sequestered in
the newly forming daughter cell. Components of the FEAR network are highlighted in blue and MEN pathway components in pink. As some FEAR
network components are known to regulate sister-chromatid separation, the FEAR network may ensure that exit from mitosis is not initiated
before sister-chromatid separation.

proteins for both processes. Separase (budding yeast
Esp1) and polo kinase (Cdc5) both trigger chromosome
segregation and are components of the FEAR network
([1,6,8,9]; Figure 1). Whether or not other FEAR
network components are also involved in coupling aspects
of chromosome segregation with exit from mitosis is
unknown. It is, however, tempting to speculate that
the FEAR network component Slk19, which belongs to
the family of passenger proteins [10], may be involved in
ensuring that kinetochores are engaged on the mitotic
spindle before exit from mitosis has occurred. Passenger
proteins localize to kinetochores during metaphase and to
the spindle midzone during anaphase and may hence, we
propose, be involved in signaling between these two
structures. In mammalian cells, chromosome passenger
proteins such as inner centromere protein (INCENP) and
aurora-B kinase also exhibit this same dynamic localization
during the cell cycle and mutants lacking these proteins
have defects in both chromosome segregation and cyto-
kinesis (reviewed in [11]). Thus, they may function sim-
ilarly to the FEAR network in coordinating accurate
chromosome segregation with later mitotic events.
Coupling exit from mitosis and cytokinesis
The mechanism whereby cells ensure that mitosis has
been completed before undergoing cytokinesis is best
understood in fission yeast. The septation initiation net-
work (SIN), a pathway homologous to the MEN, pro-
motes cytokinesis in S. pombe and its inhibition by mitotic
CDKs ensures that the pathway is not active until mitotic
CDKs have been inactivated (reviewed in [12]). Mitotic
CDKs inhibit SIN activation by preventing the localiza-
tion of the SIN kinase Sid1p to the spindle pole body
(SPB), the yeast functional equivalent of the centrosome
([13,14]; Figure 2a). As a result, the protein kinase Sid2p,
which functions downstream of Sid1p, fails to translocate
from the SPB to the site of septum formation and cyto-
kinesis is inhibited [13,14].
The essential role of the SIN is to initiate septum for-
mation after mitotic CDK inactivation has occurred,
although the SIN is also capable of inhibiting mitotic
CDKs under some circumstances [15,16]. By contrast, the
MEN is essential for both exit from mitosis and cytokin-
esis [5,12]. Until recently, however, it was difficult to
determine whether the absence of cytokinesis in MEN
mutants was an indirect consequence of cells arresting
before exit from mitosis. Recent findings, however, indi-
cate that the MEN plays a direct and essential role in the
separation of daughter cells following the completion of
mitosis. Several groups previously reported that the MEN
protein kinases Cdc5, Cdc15, Dbf2 and Dbf20 and the

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 Linked for life: temporal and spatial coordination of late mitotic events Seshan and Amon 43

Figure 2

(a) SIN (b) MEN

Lte1
Spg1p Byr4p/Cdc16p
Tem1 Bub2/Bfa1

Cdc7p

Cdc11p

Nud1
Cdc15
SPB Mitotic
Sid1pCdc14p
CDKs Dbf2/ Mob1 Cdc14
20
Sid2p Mob1p
Mitotic Mitotic
exit CDKs

Mitotic
Dbf2/ Mob1 Dbf2/ Mob1 exit
Sid2p Mob1p Sid2p Mob1p 20 20
Septum
Sid2p Mob1p Sid2p Mob1p Dbf2/ Mob1 Dbf2/ Mob1
20 20

Cytokinesis Cytokinesis

Current Opinion in Cell Biology

CDK inactivation promotes MEN and SIN function at the site of septation. (a) The SIN in S. pombe regulates septum formation. It is composed of
the scaffold protein Cdc11p, the GTPase Spg1p, the two-component GAP Byr4p/Cdc16p, the kinases Cdc7p, Sid1p and Sid2p and the Sid1p- and
Sid2p-associated proteins Cdc14p and Mob1p, respectively. SIN components localize to SPBs but shortly before septum formation Sid2p and
Mob1p associate with the site of septum formation. Mitotic CDKs inhibit the localization of Sid1p to the SPB, thereby preventing the translocation
of Sid2p/Mob1p to the septum site. Mitotic CDKs may also prevent the activation of Spg1p. (b) The MEN regulates mitotic exit in S. cerevisiae,
but components of the MEN may also regulate cytokinesis. Mitotic CDK inactivation is a prerequisite for the relocation of the MEN kinases Dbf2
and Dbf20 and perhaps Mob1 from the SPB to the mother–bud neck, the site of septum formation. Homologous MEN and SIN components are
presented in similar colors.

Dbf2-associated factor Mob1 localize to the SPB during
mitotic exit and at the bud neck during cytokinesis
[17–20]. Evidence for the functional importance of these
proteins in cytokinesis came from the observation that
mob1-77 mutants that overexpress the CDK inhibitor
SIC1, hence alleviating the need for MOB1 in mitotic
exit, are still impaired in cytokinesis [21]. The MEN has
also been implicated in the regulation of septin ring
dynamics. Dobbelaere et al. [22] observed that the septin
ring is ‘fluid’ during bud emergence and telophase, with
mobility of ring components within the ring structure.
The fluidity of the ring during telophase is required for a
late event in cytokinesis to occur and also requires MEN
activity [22]. These data imply a direct mitotic-exit-
independent role for the MEN in cytokinesis. Thus, it
appears that utilizing the same proteins to complete
sequential events is a recurring theme in cell-cycle reg-
ulation in budding yeast.
MEN proteins are required to initiate cytokinesis, but is
budding yeast like S. pombe in that mitotic CDK inactiva-
tion is a prerequisite for cytokinesis? A recent study by
Lim et al. [23] suggests that this is the case. Dbf2 and a
related protein Dbf20 (both homologs of Sid2p) localize
to the mother–bud neck concomitant with mitotic CDK
inactivation (Figure 2b). Furthermore, mitotic kinase
inactivation is required for the translocation of Dbf2
and Dbf20 to the bud neck. Conversely, ectopic inactiva-
tion of CDKs is sufficient to promote the association of
Dbf2 and Dbf20 with the bud neck [23]. The relocation
of Dbf2/Sid2p from the SPB to the site of cytokinesis is
likely to be an important step in the execution of cytokin-
esis as it is conserved between yeasts. Furthermore, the
dependence of this translocation on CDK inactivation
suggests an elegant mechanism for ensuring that cytokin-
esis follows exit from mitosis. However, proof of this
model will require the analysis of Dbf2/Sid2p mutants
that are immune to inhibition by CDKs and others that
fail to localize to the site of septation.
In higher eukaryotes, mitotic CDK inactivation also
appears to be a prerequisite for cytokinesis. In the fruit
fly D. melanogaster, Pebble, a RhoA GEF, initiates cyto-
kinetic furrow formation by activating the RhoA pathway

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44 Cell structure and dynamics
and inducing actin reorganization at the division site.
Echard and O’Farrell [24] found that two mitotic CDKs,
cyclin-B-CDK and cyclin-B3-CDK, must be inactivated
to allow Pebble activation and furrow formation. Mam-
malian cells expressing forms of mitotic CDKs that can-
not be inactivated also fail to undergo cytokinesis,
pointing to the possibility that this mechanism of linking
cytokinesis to the inactivation of mitotic CDKs is con-
served across the eukaryotic kingdom [25].
Spatial controls
Cell division not only requires events to occur in a
precise temporal order, but also requires precision in
the placement of the division site. To ensure that each
daughter cell receives exactly one DNA complement,
the division site must bisect the mitotic spindle. In
fission yeast and higher eukaryotes, the division site is
determined by the position of the mitotic spindle and
signaling between the cell membrane and the mitotic
spindle apparatus is likely to be important to coordinate
chromosome segregation and cytokinesis in space. In
S. cerevisiae, the division site is pre-determined and
therefore the proper orientation of the spindle apparatus
through the mother–bud neck before mitotic spindle
disassembly and cytokinesis is crucial for the fidelity of
chromosome segregation.
Coupling mitotic exit with nuclear position
Because of its unusual division pattern (budding), S.
cerevisiae faces the unique challenge that the nucleus
needs to be threaded through the mother–bud neck
before exit from mitosis. It thus comes as no surprise
that mechanisms exist that prevent exit from mitosis
until the bud, the future daughter cell, receives a com-
plete DNA complement. The fact that nuclear position is
under surveillance was first discovered in yeast cells
defective in assembling the microtubule-capture
machinery at the bud cortex, which orients the mitotic
spindle along the mother–daughter axis. Mutations in
components of this complex (such as the yeast dynein
homolog DYN1/DHC1) lead to spindle orientation
defects causing spindle elongation to take place in the
mother cell [26,27]. The activity of a surveillance
mechanism termed the ‘spindle orientation checkpoint’
blocks exit from mitosis until the spindle is correctly
oriented ([26,28]; Figure 3a).
One mechanism that helps prevent exit from mitosis in
cells with a mispositioned mitotic spindle is the spatial
segregation of components of the MEN under these
conditions. Lte1, which functions as an activator of
Tem1 and resembles a guanine exchange factor (GEF),
becomes sequestered at the bud cortex concomitant with
bud formation while Tem1 localizes specifically to the
daughter-bound SPB [29,30]. Tem1 and Lte1 only come
into contact when the daughter-bound SPB moves into
the bud during anaphase [29,30]. The spatial restriction of
Current Opinion in Cell Biology 2004, 16:41–48
Figure 3
(a) dyn1∆ dyn1∆ + GAL–LTE1
or sep7∆
No exit Exit
(b) arp1∆ arp1∆ cdc10∆
Bub2/Bfa1 Bub2/Bfa1
No exit Exit
Microtubules Active Tem1 Inactive Tem1
Microtubule sensor Lte1 protein
Current Opinion in Cell Biology
Mechanisms sensing nuclear position in S. cerevisiae. (a) In the
absence of dynein (dyn1D), 10% of cells have mispositioned spindles
such that spindle elongation takes place entirely within the mother
cell. These cells do not exit from mitosis until the spindle has been
properly re-oriented due to the activity of the spindle position
checkpoint. The spatial segregation of the mitotic exit activators Tem1
and Lte1 until the migration of the Tem1-bearing SPB into the bud, or
daughter cell, is important for preventing inappropriate mitotic exit in cell
with misaligned spindles. Cells with misoriented spindles that are
defective for Lte1 localization (sep7D or overexpression of LTE1
[GAL–LTE1]) exit from mitosis and accumulate anucleate and multi-
nucleate cells. (b) Astral microtubules traversing the mother–bud neck
may act as a signal for misaligned spindles and activate a checkpoint
that blocks inappropriate mitotic exit. This checkpoint, we speculate,
could function by activating the Tem1 GAP Bub2/Bfa1 and thereby
inhibit MEN activity. The septin CDC10 may act as a scaffold for a
sensor of astral microtubules passing through the mother–bud neck
as in the absence of CDC10 such cells are able to exit from mitosis
inappropriately in an LTE1-independent manner.
Tem1 and Lte1 is important for preventing exit from
mitosis in cells with misaligned spindles. This idea is
consistent with the finding that overexpression of LTE1,
which causes the protein to be present in the mother cell
as well as the daughter cell, allows cells with misoriented
spindles to exit from mitosis [29]. Importantly, a recent
study indicates that modulation of Lte1 localization by
means other than overexpression also results in inap-
propriate exit from mitosis. Castillon et al. [31] found
that disruption of the septin ring using a SEP7 deletion
allows Lte1 to leak into the mother cell. In sep7D cells
with misaligned spindles, inappropriate exit from mitosis
occurred in an LTE1-dependent manner [31], suggest-
ing that the leakage of Lte1 into the mother cell allows
cells with mispositioned spindles to exit from mitosis.
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 Linked for life: temporal and spatial coordination of late mitotic events Seshan and Amon 45
The sequestration of Lte1 in the bud not only appears to
be important to ensure that mitotic exit does not occur
when the nucleus is not properly positioned, but also
seems to be needed for efficient exit from mitosis. Several
recent studies identified cell polarity proteins such as the
Rho-like GTPase Cdc42 and the p21-activated protein
kinase (PAK)-like kinase Cla4 as well as Ras as being
required for Lte1 anchorage at the bud cortex [32–36].
Interestingly, in the absence of these factors exit from
mitosis is delayed, suggesting that a certain amount of
Lte1 in the bud is important for exit from mitosis to occur
in a timely manner. However, in the absence of activity
assays for Lte1 we cannot exclude the possibility that
these factors are also important for Lte1 function.
Whether Lte1 stimulates Tem1 function remains con-
troversial. Genetic experiments indicate that Lte1 func-
tions upstream of Tem1 [37,38] and the two proteins
interact physically [33]. However, whether Lte1 func-
tions as a GEF for Tem1 is not known. Recent studies
report conflicting results concerning the necessity of
Lte1’s GEF homology domain in exit from mitosis
[34,36]. It will be important to resolve this discrepancy
to elucidate the mechanism of Lte1 activity.
The spatial restriction of Lte1 and Tem1 is not the only
mechanism that prevents cells with misaligned spindles
from inactivating mitotic CDKs. Adames et al. [39] first
reported that certain mutants with spindle position
defects inappropriately exited mitosis even when LTE1
was deleted. The authors suggested that astral microtu-
bules interacting with the bud neck act as a sensor for
spindle position, as inappropriate exit of cells with mis-
aligned spindles correlated with loss of cytoplasmic
microtubules from the bud neck. Castillon et al. [31]
further corroborate this hypothesis with the finding that
arp1D mutants, in which the mitotic spindle is frequently
mispositioned, inappropriately exit mitosis when septin
ring function at the mother–bud neck is compromised as a
result of inactivation of the septin CDC10. In arp1D
cdc10D cells, inappropriate mitotic exit was only partially
prevented by deleting LTE1. Thus CDC10 plays an
LTE1-dependent and independent role in monitoring
spindle position [31]. The presence of microtubules
in the bud neck could act as a signal to indicate that the
nucleus is not correctly positioned along the mother–bud
axis, which in turn prevents exit from mitosis (Figure 3b).
The nature of such a signaling pathway remains elusive,
but it may impinge on Tem1’s GTPase-activating pro-
tein complex Bub2/Bfa1 (Figure 3b). Deletion of BUB2
allows cells with a misaligned mitotic spindle to exit from
mitosis [29,30]. Perhaps signals sensing indicators of nu-
clear position such as astral microtubules promote Bub2-
Bfa1 activity, thus preventing activation of Tem1.
The spindle-position checkpoint also appears to function
in fission yeast. Oliferenko and Balasubramanian [40]
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found that a mutant defective in astral microtubule for-
mation, mia1D, exhibits defects in mitotic spindle orien-
tation and arrests in metaphase until the spindle orients
correctly along the longitudinal axis of the cell. Evidence
for a spindle position checkpoint also exists in mamma-
lian cells. Rat epithelial cells that have been micro-
manipulated so that the mitotic spindle is mispositioned
delay anaphase onset until the spindle is reoriented along
the long axis of the cell [41]. In the absence of dynein,
spindle repositioning does not occur and the cell-cycle
delay imposed by spindle misorientation also seems to be
abolished [41]. This suggests that in contrast to budding
yeast dynein, which serves as a trigger of the spindle
position checkpoint, dynein in mammalian cells not only
functions to correct spindle position defects but is also
required for checkpoint activation.
A spindle position checkpoint, however, does not appear
to exist in the one-celled C. elegans embryo. The one-
celled embryo divides asymmetrically to produce a larger
anterior and a smaller posterior cell, which requires asym-
metric positioning of the mitotic spindle (reviewed in
[42]). The positioning of the spindle requires cytoplasmic
dynein — RNAi-mediated loss of the C. elegans dynein
homolog dhc-1 causes spindle orientation defects [43].
However, cell-cycle progression does not seem to be
delayed in cells with mispositioned spindles, resulting
in the occasional occurrence of missegregated chromo-
somes [43]. Perhaps a checkpoint in this instance is
superfluous because the constraints of the embryo’s egg-
shell force the spindle to re-position properly in most
cases [43]. It is also possible that the spindle position
checkpoint, like other checkpoints, is not active during
embryonic cell cycles [44,45].
Conclusions and perspectives
It is clear that the FEAR network, MEN and SIN are
essential for ensuring temporal and spatial controls during
mitosis. However, whether such signaling pathways exist
in metazoans is still unknown. Two components of the
FEAR network, Separase and Polo kinase, are conserved
across species. A role for Polo kinase in triggering sister
chromatid segregation and cytokinesis is well established
in multicellular organisms [46,47]. Additionally, there are
hints in the literature that Separase may also be needed
for cell-cycle steps after cohesin removal. RNAi-
mediated knock down of Separase function in C. elegans
not only prevents sister chromatid separation but also
appears to slow down subsequent cell-cycle events [48].
Mammalian homologs of MEN and SIN components have
also been identified. Remarkably, in at least one instance
the subcellular localization of the homolog as well as its role
in regulating late mitotic events appears to be conserved.
Centriolin encodes the mammalian homolog of the S.
cerevisiae Nud1 protein and S. pombe Cdc11p protein,
which act as scaffolds for MEN and SIN components,
Current Opinion in Cell Biology 2004, 16:41–48
46 Cell structure and dynamics
respectively, at the SPB. Centriolin localizes specifically
to the mother centriole, which displays the unique prop-
erty of moving towards the midbody at the end of mitosis,
triggering abscission of the daughter cells. Centriolin is
likely to be important for this abscission event to occur, as
reducing centriolin expression using siRNA disables cell
separation [49]. Interestingly, one isoform of the human
homolog of S. cerevisiae CDC14, hCdc14A, localizes to
centrosomes during telophase and cells lacking hCdc14A
have cytokinesis defects [50]. An attractive hypothesis is
that hCdc14A localizes specifically to the mother cen-
triole and plays a role in regulating the events that lead to
cell separation. Further insights into the function of
mammalian MEN/SIN homologs will elucidate whether
mammalian cells use similar mechanisms to coordinate
late mitotic events.
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review, have been highlighted as:
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18. Song S, Grenfell TZ, Garfield S, Erikson RL, Lee KS: Essential
function of the polo box of Cdc5 in subcellular localization and
induction of cytokinetic structures. Mol Cell Biol 2000,
20:286-298.
19. Yoshida S, Toh-e A: Regulation of the localization of Dbf2 and
Mob1 during cell division of Saccharomyces cerevisiae.
Genes Genet Syst 2001, 76:141-147.
20. Xu S, Huang HK, Kaiser P, Latterich M, Hunter T: Phosphorylation
and spindle pole body localization of the Cdc15p mitotic
regulatory protein kinase in budding yeast. Curr Biol 2000,
10:329-332.
21. Luca FC, Mody M, Kurischko C, Roof DM, Giddings TH, Winey M:
Saccharomyces cerevisiae Mob1p is required for cytokinesis
and mitotic exit. Mol Cell Biol 2001, 21:6972-6983.
22. Dobbelaere J, Gentry MS, Hallberg RL, Barral Y: Phosphorylation-
 dependent regulation of septin dynamics during the cell cycle.
Dev Cell 2003, 4:345-357.
A novel role for the MEN in regulating septin dynamics and cytokinesis
in S. cerevisiae is described. The authors have discovered that the septin
ring exists in a fluid and immobile state. The immobile state is character-
istic of cells in S, G2, and M phases and is achieved by the phosphoryla-
tion of the septin subunit Sep7/Shs1 by the Gin4 kinase. The mobile state
is present at bud emergence and during telophase and is essential
for cytokinesis to occur. The PP2A phosphatase regulatory subunit,
Rts1, is required for dephosphorylation of Sep7/Shs1, an event, which
is believed to trigger septin ring mobility. The activity of the MEN, in turn, is
required for Rts1 function, thereby coupling mitotic exit and cytokinesis
temporally.
23. Lim HH, Yeong FM, Surana U: Inactivation of mitotic kinase
 triggers translocation of MEN components to mother–
daughter neck in yeast. Mol Cell Biol 2003, 14:4734-4743.
This paper provides evidence that mitotic CDK inactivation is required for
cytokinesis in budding yeast. The authors find that cells mutant for the
MEN kinase CDC15 are capable, when grown at a semi-permissive
temperature, of exiting from mitosis, but cannot form a septum. In
addition, the authors find that mitotic CDK inactivation is necessary
and sufficient for the relocation of Dbf2/20 to the site of septum formation
and suggest that this relocation is a key event in the initiation of cytokin-
esis. However, the localization of Dbf2 and Dbf20 to the septum does not
allow cytokinesis to ensue anachronistically, indicating that controls that
are independent of CDK inactivation are also important.
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 Linked for life: temporal and spatial coordination of late mitotic events Seshan and Amon 47
24. Echard A, O’Farrell PH: The degradation of two mitotic cyclins
 contributes to the timing of cytokinesis. Curr Biol 2003,
13:373-383.
A new link between mitotic CDK inactivation and cytokinesis is pre-
sented here. By examining the consequences of expressing non-
degradable cyclin B or cyclin B3 in D. melanogaster, the authors have
discovered that cytokinetic furrow formation requires the inactivation of
cyclin-B–Cdk and cyclin-B3–CDK complexes. The degradation of cyclin
B seems to be required for both mitotic exit and cytokinesis, whereas
cyclin B3 degradation is required only for mitotic exit. However, the
degradation of both cyclins is important, to different extents, for activation
of the Rho GEF Pebble at the site of cytokinetic furrow formation. Since
Pebble is an important activator of furrow formation, these findings
provide a temporal link between the onset of cytokinesis and cyclin B
and cyclin B3 destruction.
25. Wheatley SP, Hinchcliffe EH, Glotzer M, Hyman AA, Sluder G,
Wang Y: CDK1 inactivation regulates anaphase spindle
dynamics and cytokinesis in vivo. J Cell Biol 1997, 138:385-393.
26. Yeh E, Skibbens RV, Cheng JW, Salmon ED, Bloom K: Spindle
dynamics and cell cycle regulation of dynein in the budding
yeast, Saccharomyces cerevisiae. J Cell Biol 1995, 130:687-700.
27. Carminati JL, Stearns T: Microtubules orient the mitotic spindle
in yeast through dynein-dependent interactions with the cell
cortex. J Cell Biol 1997, 138:629-641.
28. Muhua L, Adames NR, Murphy MD, Shields CR, Cooper JA:
A cytokinesis checkpoint requiring the yeast homologue of
an APC-binding protein. Nature 1998, 393:487-491.
29. Bardin AJ, Visintin R, Amon A: A mechanism for coupling exit
from mitosis to partitioning of the nucleus. Cell 2000, 102:21-31.
30. Pereira G, Hofken T, Grindlay J, Manson C, Schiebel E: The Bub2p
spindle checkpoint links nuclear migration with mitotic exit.
Mol Cell 2000, 6:1-10.
31. Castillon GA, Adames NR, Rosello CH, Seidel HS, Longtine MS,
 Cooper JA, Heil-Chapdelaine RA: Septins have a dual role in
controlling mitotic exit in budding yeast. Curr Biol 2003,
13:654-658.
This paper describes the identification of two septin ring components as
being important to prevent exit from mitosis in cells with misaligned
spindles. In cells lacking SEP7, exit from mitosis occurs in an LTE1-
dependent manner indicating that, in this mutant, delocalization of Lte1
into the mother cell allows exit from mitosis to occur. In cells lacking
CDC10, however, exit from mitosis occurs at least to some extent even in
the absence of LTE1, suggesting that other mechanisms are responsible
for preventing inappropriate exit from mitosis. The authors suggest that
this inappropriate exit is caused by the failure of cytoplasmic microtu-
bules to interact with the bud neck in the cdc10D mutant.
32. Seshan A, Bardin AJ, Amon A: Control of Lte1 localization by cell
 polarity determinants and Cdc14. Curr Biol 2002, 12:2098-2110.
The authors report that Cdc42 and Cla4 are required for Lte1 to localize to
the bud cortex and that Cdc14 mediates its dephosphorylation and
delocalization. In addition, the septins and the Kelch-repeat-containing
Kel1 protein are implicated in maintaining Lte1 at the bud cortex.
33. Hofken T, Schiebel E: A role for cell polarity proteins in mitotic
 exit. EMBO J 2002, 21:4851-4862.
This paper describes a role for Cla4 in targeting Lte1 to the bud. The
authors additionally find that another PAK kinase homolog in S. cerevi-
siae, Ste20, regulates mitotic exit in an LTE1-independent manner, and
may function in a pathway parallel to the MEN in promoting mitotic CDK
inactivation.
34. Jensen S, Geymonat M, Johnson AL, Segal M, Johnston LH:
 Spatial regulation of the guanine nucleotide exchange
factor Lte1 in Saccharomyces cerevisiae. J Cell Sci 2002,
115:4977-4991.
This paper identifies a role for the Rho-like GTPase Cdc42 in Lte1
localization. The authors suggest that phosphorylation by Cln/Cdc28
regulates Lte1 localization and that Cdc14 dephosphorylation mediates
the delocalization of Lte1 during telophase. This paper also shows that
the GEF homology domain of LTE1 may be required for its function in
promoting mitotic exit.
35. Chiroli E, Fraschini R, Beretta A, Tonelli M, Lucchini G, Piatti S:
 Budding yeast PAK kinases regulate mitotic exit by two
different mechanisms. J Cell Biol 2003, 160:857-874.
This paper implicates the PAK-like kinase Cla4, best known for its role in
establishing and maintaining cell polarity, in the regulation of Lte1 loca-
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lization. The authors isolate a dominant-negative allele of CLA4, which,
when overproduced, prevents both APC-Cdc20 activation and cyclin B
Clb2 degradation. The authors show that APC–Cdc20 activation is
restored by deleting the WEE1 homolog SWE1 in these cells, as judged
by degradation of Pds1/securin. However, Clb2 levels remain high, and
the MEN phosphatase Cdc14 is not activated. This could be due to the
fact that the MEN activator Lte1 is delocalized from the bud in cells
overexpressing dominant-negative CLA4. The localization of Lte1 to the
bud may thus be necessary for its activity, or, alternatively, CLA4 may be
required for Lte1 activity.
36. Yoshida S, Ichihashi R, Toh-e A: Ras recruits mitotic exit
 regulator Lte1 to the bud cortex in budding yeast. J Cell Biol
2003, 161:889-897.
The authors find that Ras is important for restricting Lte1 to the bud
cortex, which may be important for its mitotic exit function. They also
report that the GEF domain of Lte1 is not important for the protein’s ability
to promote mitotic CDK inactivation. This raises the possibility that Lte1 is
not a GEF for the MEN GTPase Tem1 but stimulates mitotic exit by an
unknown mechanism.
37. Shirayama M, Matsui Y, Tanaka K, Toh-e A: Isolation of a CDC25
family gene, MSI2/LTE1, as a multicopy suppressor of ira1.
Yeast 1994, 10:451-461.
38. Jaspersen SL, Charles JF, Tinker-Kulberg RL, Morgan DO: A late
mitotic regulatory network controlling cyclin destruction in
Saccharomyces cerevisiae. Mol Biol Cell 1998, 9:2803-2817.
39. Adames NR, Oberle JR, Cooper JA: The surveillance mechanism
of the spindle position checkpoint in yeast. J Cell Biol 2001,
153:159-168.
40. Oliferenko S, Balasubramanian MK: Astral microtubules monitor
 metaphase spindle alignment in fission yeast. Nat Cell Biol 2002,
4:816-820.
Astral microtubules are implicated in orienting the mitotic spindle in
budding yeast. This paper provides evidence that astral microtubules
also regulate spindle position in S. pombe and that spindle orientation is
under checkpoint control in fission yeast. Cells that lack astral micro-
tubules and that are also mutant for the Atf1 transcription factor do not
arrest in metaphase in response to misaligned spindles and proceed
through an aberrant mitosis, producing anucleate or multi-nucleate cells,
implicating astral microtubules in preservation of the fidelity of chromo-
some transmission.
41. O’Connell CB, Wang YL: Mammalian spindle orientation and
position respond to changes in cell shape in a dynein-
dependent fashion. Mol Biol Cell 2000, 11:1765-1774.
42. Ahringer J: Control of cell polarity and mitotic spindle
positioning in animal cells. Curr Opin Cell Biol 2003, 15:73-81.
43. Gonczy P, Pichler S, Kirkham M, Hyman AA: Cytoplasmic dynein
is required for distinct aspects of MTOC positioning, including
centrosome separation, in the one cell stage Caenorhabditis
elegans embryo. J Cell Biol 1999, 147:135-150.
44. Minshull J, Sun H, Tonks NK, Murray AW: A MAP kinase-
dependent spindle assembly checkpoint in Xenopus egg
extracts. Cell 1994, 79:475-486.
45. Dasso M, Newport JW: Completion of DNA replication is
monitored by a feedback system that controls the initiation of
mitosis in vitro: studies in Xenopus. Cell 1990, 61:811-823.
46. Weitzer S, Uhlmann F: Chromosome segregation: playing polo in
prophase. Dev Cell 2002, 2:381-382.
47. Nigg EA: Polo-like kinases: positive regulators of cell division
from start to finish. Curr Opin Cell Biol 1998, 10:776-783.
48. Siomos MF, Badrinath A, Pasierbek P, Livingstone D, White J,
Glotzer M, Nasmyth K: Separase is required for chromosome
segregation during meiosis I in Caenorhabditis elegans.
Curr Biol 2001, 11:1825-1835.
49. Gromley A, Jurczyk A, Sillibourne J, Halilovic E, Mogensen M,
 Groisman I, Blomberg M, Doxsey S: A novel human protein of the
maternal centriole is required for the final stages of cytokinesis
and entry into S phase. J Cell Biol 2003, 161:535-545.
The separation of daughter cells upon the completion of mitosis, called
abscission, is regulated by centrosomal dynamics in mammalian cells.
The mother centriole (MC) moves from the cell cortex toward the midbody
of dividing cells and the cells separate as soon as the MC leaves the
midbody. The signaling event(s) initiated by this translocation are
Current Opinion in Cell Biology 2004, 16:41–48
48 Cell structure and dynamics
unknown. Gromley et al. report that a mammalian homolog of the MEN
scaffold protein Nud1 called centriolin localizes to the MC and plays a role
in mediating daughter cell abscission. This paper lends support to the
idea that homologs of MEN and SIN components regulate cytokinesis in
higher eukaryotes.
50. Kaiser BK, Zimmerman ZA, Charbonneau H, Jackson PK:
 Disruption of centrosome structure, chromosome segregation,
and cytokinesis by misexpression of human Cdc14A
phosphatase. Mol Biol Cell 2002, 13:2289-2300.
The function of MEN homologs in mammalian cells has thus far
remained largely unexplored. In this study, Kaiser et al. examine the
Current Opinion in Cell Biology 2004, 16:41–48
properties of the two human homologs of budding yeast Cdc14,
hCdc14A and hCdc14B both in vitro and in vivo. They find that like
its budding yeast counterpart, hCdc14A can dephosphorylate CDK
Substrates in vitro. Interestingly, hCdc14A localizes to the centro-
somes, whereas hCdc14B is found at the nucleolus, and the localization
of both proteins is dynamic throughout the cell cycle. The overproduc-
tion of hCdc14A is associated with cytokinesis and centrosomal
defects, whereas depletion of hCdc14A using targeted siRNA causes
anaphase defects and prevents cells from completing cytoplasmic
abscission. These findings support the hypothesis that the MEN (and
possibly the FEAR) components function to regulate mitotic exit and
cytokinesis in higher eukaryotes.
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