[Cell Cycle 7:3, 1-4; 1 February 2008]; ©2008 Landes Bioscience
During anaphase the mitotic spindle extends dramatically,
promoting the segregation of the chromosomes into the two
daughter cells. The spindle midzone, assembled at the onset of
anaphase, is critical for this extension. How this assembly is linked
to progression through the cell cycle is not fully understood. Our
data show that in budding yeast the conserved phosphatase Cdc14,
activated in early anaphase, regulates the formation of the spindle
midzone. Cdc14 dephosphorylates residues of a core midzone
component, the conserved microtubule bundling factor Ase1, that
were previously phosphorylated by the cyclin-dependent kinase
complex. In addition, Cdc14 activation is also indirectly respon-
sible for midzone localization of the separase-Slk19 complex. This
dual control of midzone assembly by Cdc14 is necessary for the
formation of the focused and centered spindle midzone that drives
the continuous and full elongation of the anaphase spindle. The
identification of Ase1 as a key Cdc14 substrate elucidates how
spindle midzone assembly is coordinated with the metaphase to
The spindle midzone: organization and function. The mitotic
spindle is a microtubule (MT)-based machine responsible for chro-
mosome segregation in eukaryotes. It is composed of three sets of
MTs: kinetochore MTs that attach the chromosomes to the spindle
poles, astral MTs that are responsible for interactions between the
spindle and the cell cortex and, finally, the interpolar MTs that inter-
digitate between the two spindle poles to form an antiparallel MT
array that is known as the spindle midzone (Fig. 1A). The spindle
midzone is required to maintain the integrity of the anaphase spindle
of organisms as diverse as yeast and humans. Moreover, without a
stable overlap of antiparallel interpolar MTs the kinetics of pole sepa-
ration and chromosome segregation is disturbed. In Caenorhabditis
elegans embryos, where spindle elongation results from cortical
pulling forces acting on the spindle poles, interfering with the spindle
midzone either genetically or by laser ablation accelerates the rate
of spindle elongation.1,2 In contrast, in Schizosaccharomyces pombe
and Saccharomyces cerevisiae spindle elongation is driven mostly, if
not solely, by forces created within the spindle midzone. Therefore,
genetic or physical perturbations of the spindle midzone result in
deficient separation of the spindle poles and collapse of the anaphase
spindle.3-6 In addition, in all eukaryotes the midzone regulates
different aspects of cytokinesis.5,7-10
Ase1, a conserved MT bundler. Budding yeast Ase1 is the
founding member of a conserved family of midzone-specific proteins
with MT bundling activity that includes the human PRC1, SPD-1
in C. elegans, Feo in Drosophila melanogaster and MAP65 in plant
cells.2,3,7,9,11 Although the degree of sequence identity is low, all
family members are required for midzone formation, promoting
spindle extension and stability in anaphase and, later, play a role in
cytokinesis.3,12-14 It was recently suggested that in budding yeast
a functional midzone is also necessary for cytokinesis.10 However,
experiments performed in our laboratory could not confirm the
published observations, namely, no major cytokinetic defects were
detected in cells deleted of ASE1 (ase1Δ, Roostalu J, Schiebel E).
Spindle extension is particularly prominent in S. cerevisiae. In
approximately 20 minutes the anaphase spindle elongates about five
fold, from 1.5 μm in metaphase to reach 8–10 μm before disas-
sembly and mitotic exit (Fig. 1B).3,15,16 Yeast cells without ASE1
assemble a normal bipolar spindle in metaphase but severe defects
arise with progression into anaphase. Shortly after extension begins
the spindle of ase1Δ cells collapses,3 resulting in a 10-fold increase
in the rate at which chromosomes are lost during cell division (our
Regulation of anaphase. As soon as all of the chromosomes (sister
chromatid pairs) have bound microtubules emanating from either
spindle pole to achieve bi-orientation on the metaphase spindle, the
spindle assembly checkpoint is switched off, promoting the activa-
tion of the anaphase promoting complex. This in turn activates the
protease separase which then cleaves the cohesin complex holding the
sister chromatids together, thereby initiating anaphase.17 Whereas
metaphase is characterized by high cyclin-dependent kinase (Cdk1)
activity, anaphase is marked by a decline in Cdk1 activity and an
increase in phosphatase activity. In budding yeast, it is mainly the
phosphatase Cdc14 that counteracts Cdk1 activity in anaphase.
During most of the cell cycle Cdc14 is kept inactive by entrapment
in the nucleolus.18,19 However, with anaphase onset Cdc14 is released
and activated in two phases. The cdc Fourteen Early Anaphase Release
(FEAR) pathway triggers a partial and transient release of Cdc14 from
Assembling the spindle midzone in the right place at the right time
Anton Khmelinskii and Elmar Schiebel*
Zentrum für Molekulare Biologie der Universität Heidelberg; Heidelberg, Germany
Abbreviations: Cdk1, cycling-dependent kinase; FEAR, cdc fourteen early anaphase release; MEN, mitotic exit network; MT, microtubule
Key words: ASE1, PRC1, CDC14, midzone, separase
*Correspondence to: Elmar Schiebel; ZMBH; Im Neuenheimer Feld 282; Heidelberg
69120 Germany; Tel.: 49.6221.546814; Fax: 49.6221.545892; Email: e.schiebel@
Submitted: 11/15/07; Accepted: 11/21/07
Previously published online as a Cell Cycle E-publication:
Coordinating spindle midzone assembly with cell cycle progression
the nucleolus. The FEAR-activated Cdc14 regulates processes such
as the drastic drop in MT dynamics and spindle stabilization that
occur as cells enter anaphase. Known spindle-associated targets of
Cdc14 involved in these processes are the chromosomal passenger
protein Sli15 (a homolog of the mammalian inner centromere protein
INCENP), the DASH complex component Ask1 and the spindle
protein Fin1.20-22 However, the FEAR-controlled Cdc14 is insuffi-
cient to promote mitotic exit, that is the transition from mitosis into
G1 phase of the cell cycle. Only the full release of Cdc14, promoted
midway through anaphase, allows exit from mitosis by conteracting
and inactivating Cdk1 activity.23 This full release is not controlled by
FEAR, rather a distinct regulatory network known as the mitotic exit
network (MEN), that comprises a GTPase driven signaling cascade.
Ase1 Defines the Spindle Midzone
Through extensive analysis of the dependency relationships
between the localization of midzone components we were able to
establish that Ase1 functions as a core component of the spindle
midzone in budding yeast. Midzone localiza-
tion of Ase1 was independent of most other
midzone proteins tested. Only minor defects
(broader and not centered Ase1 region) were
observed in mutants of separase and SLK19,
two FEAR components with midzone local-
ization. Conversely, the correct localization
of all midzone proteins depended on Ase1.
In cells deleted of ASE1, most midzone
components, such as the kinesin-5 Cin8 or
the CLASP homolog Stu1, bound uniformly
along the interpolar MTs. In contrast, separase
and Slk19 never associated with the interpolar
MTs of ase1Δ cells. At present it is not clear
whether separase and Slk19 have MT-binding
activity or whether their midzone localization
is directly dependent on Ase1.
It is important to note that Ase1 localized
to the spindle midzone in the triple kinesin
motor mutant cin8-3 kip1Δ kar3Δ.24 The only
kinesin left in the nuclei of these cells is the
kinesin-8, Kip3. Kip3 is a plus-end directed
motor protein with plus-end depolymerase
activity in vitro and affects MT dynamics
in vivo.25,26 In agreement with previously
published data describing normal anaphase
spindle extension in kip3Δ cells,27 deletion
of KIP3 had no effect on midzone organiza-
tion. Thus, budding yeast Ase1 accumulates
in the spindle midzone independently of any
MT-based motor activity. This is in contrast
to its human homolog PRC1 that requires a
member of the kinesin-4 family, KIF4, for its
efficient accumulation in the midzone.28,29
This difference could be a reflection of the
distinct functional spindle organization in the
two organisms. In budding yeast the length of
the spindle in metaphase and of the midzone
in anaphase are the same (Fig. 1B). As Ase1 is
spread along the metaphase spindle, crosslinking the few interpolar
MTs present already in metaphase30 would allow midzone formation
in early anaphase without assistance from motor proteins. In HeLa
cells, PRC1 uniformly covers the 10 μm long metaphase spindle and
concentrates into a region of 2–3 μm in early anaphase.13 The need
to drastically relocalize PRC1 with anaphase onset probably accounts
for the reliance upona kinesin to assemble a midzone in human cells.
Furthermore, we observed that in budding yeast the spindle midzone
length was maintained during spindle extension, in a kinesin-inde-
pendent fashion, and no Ase1-GFP signal could be detected outside
the midzone region. It seems thus likely that budding yeast Ase1 can
selectively bundle antiparallel MTs, similar to the bundling of inter-
phase MTs by Ase1 in fission yeast.31 This increased affinity of Ase1
towards antiparallel MTs, combined with the simplicity of the yeast
spindle (composed only of 3–6 interpolar MTs and 32 kinetochore
MTs, one per chromosome),30 could explain how the midzone is
formed and maintained throughout anaphase merely as a result of
Figure 1. (A) Organization of the mitotic spindle. Shown are the spindle poles (red), chromosomes
(blue) and different sets of MTs: kinetochore (1), astral or cytoplasmic (2) and interpolar (3). With the
onset of anaphase the spindle midzone (4) is formed in the overlap of interpolar MTs. (B) Time-lapse
analysis of Ase1-GFP localization. Spc42 (a component of the spindle pole body, the yeast equivalent
of the centrosome) fused to the red fluorescent protein eqFP61142 was used to visualize the spindle
poles. Shown are maximum z-series projections every 2 minutes. Scale bar = 5 μm. (C) Midzone
organization and function depend on the phosphorylation state of Ase1. The figure shows cartoons
of late-anaphase spindles in cells of indicated genotypes (spindle poles are in red, Ase1 is in green).
The panels below show the pole-to-pole distance over time in corresponding representative cells. The
time of anaphase onset (arrowheads) and spindle disassembly (arrows) are indicated. (D) Cdc14 con-
trols spindle midzone assembly. FEAR-activated Cdc14 directly dephosphorylates Ase1. In an Aurora
B-dependent manner, Cdc14 directs Esp1 together with the Esp1-processed C-terminal fragment of
Slk19 to the developing spindle midzone. There, Esp1 and Slk19 assist Ase1 in midzone formation
and stabilize the anaphase spindle.
284 Cell Cycle 2008; Vol. 7 Issue 3
Coordinating spindle midzone assembly with cell cycle progression
Cdc14 Phosphatase Links Midzone Assembly to
How is the assembly of the spindle midzone coupled to the initia-
tion of anaphase? The central role of Ase1 in organizing the midzone
makes it a plausible target for cell cycle-dependent regulation. In
fact, the MT bundling activity of the human PRC1 and its interac-
tion with KIF4 are both negatively regulated via Cdk1-dependent
phosphorylation prior to anaphase.13,29,32 This might be a conserved
regulatory scheme since budding yeast Ase1 is a phosphorylation
target of Cdk1,33 and we could observe dephosphorylation of Ase1
in early anaphase. Therefore, we sought to identify the phosphatase
responsible for this dephosphorylation, and the functions of this
regulation. Our experiments show that the conserved phosphatase
Cdc14 directly regulates Ase1. In a conditional cdc14 mutant, Ase1
was no longer dephosphorylated upon entry into anaphase, and,
together with other midzone components, covered a broader region
on the interpolar MTs than in wild type cells. In addition, this
abnormal midzone region was frequently shifted towards one of the
spindle poles. When the seven Cdk1 consensus phosphorylation sites
in Ase1 (S/T-P-X-K/R) were mutated to aspartate (Ase17D) in a wild
type CDC14 background, to mimic constitutive phosphorylation,
a mislocalization of Ase17D and other midzone components was
observed, as in cdc14 cells. Consequently, ASE17D spindles frequently
stalled during elongation and, on average, extended to a lesser extent
than in wild type cells (Fig. 1C). In contrast, the non-phosphorylat-
able Ase17A mutant was able to promote the formation of a normal
midzone. Although the midzone was centered and focused in ASE17A
cells, spindles frequently collapsed during anaphase extension, which
was on average faster than in wild type cells. These results imply that
phosphorylation of Ase1 in metaphase is required for spindle stability
in anaphase. With anaphase onset Ase1 has to be dephosphorylated
in order to assemble a spatially restricted spindle midzone. The
effects of this phosphorylation cycle on the properties of Ase1 are
currently under investigation.
Data from a number of groups show that FEAR-activated Cdc14
dephosphorylates several MT-associated proteins that are required
for the stabilization of MTs that is observed in anaphase cells.20-
22 Regulating midzone assembly by Cdc14 release enables the cell
to link this process, in time, with the onset of anaphase and the
concomitant changes in MT dynamics that are required for the
persistence of a stable midzone.21
A regulatory mechanism similar to that described by us for
Ase1 was recently reported for the centralspindlin complex in
C. elegans.34 Present only in metazoans, the centralspindlin complex
is composed of the kinesin-6 ZEN-4/MKLP1 and the RhoGAP
CYK-4/MgcRacGAP, and is required for cytokinesis.35 It can bundle
MTs in vitro and cooperates with SPD-1, the C. elegans homolog
of Ase1, in midzone formation.2,36 Phosphorylation of ZEN-4 by
Cdk1-cyclin B inhibits its MT binding activity, preventing midzone
assembly in metaphase. Moreover, in contrast to wild type ZEN-4,
a non-phosphorylatable ZEN-4 mutant localizes normally in cdc-14
RNAi embryos,34 suggesting that CDC-14 regulates spindle midzone
assembly in C. elegans at least via the centralspindlin complex. Thus
in different organisms several analogous processes prevent MT
bundling and midzone assembly in metaphase, ensuring genome
stability.13,16,32,34 It will be important to further understand the role
of Cdc14 phosphatase in this context, namely its role in regulating
the metazoan Ase1 homologs.
Separase and Slk19 in Midzone Assembly
With the onset of anaphase, budding yeast separase/Esp1 localizes
to the spindle midzone. It does so together with its inhibitor securin/
Pds1 and the C-terminal fragment of Slk19, a kinetochore protein
that is cleaved by Esp1.37,38 Esp1 and Slk19 are interdependent
in their binding to the midzone.16,38 Results from our laboratory
show that midzone localization of Esp1 and Slk19 depends on
spindle localization of the Aurora B kinase complex, which has to
be dephosphorylated by Cdc14 in early anaphase (Fig. 1D).16,20
Taken together, these data suggest that Esp1, Pds1 and Slk19 arrive
at the developing midzone in early anaphase as a complex. How this
binding is regulated by Aurora B kinase is not known. Moreover,
Esp1 and Slk19 do not localize to the anaphase spindles of ase1Δ
cells, raising the possibility that they might interact directly with
Ase1. Accordingly, we have observed interactions between the three
proteins in the yeast two-hybrid assay (our unpublished data).
So what are their functions at the midzone? The absence of Slk19
from anaphase spindles renders them unstable.38 We observed that
Slk19 remained at the midzone after Esp1 left in mid-anaphase, and
midzone localization of Pds1 was previously observed to be very
transient.37 These data suggest that Esp1 and Pds1 may serve only to
deliver Slk19 to the midzone.
In slk19Δ and conditional esp1 mutants the midzone was broader
and frequently shifted towards one of the poles, indicating a function
in assembly or maintenance of the midzone. Since Ase1 is regulated
by Cdc14, and Esp1 and Slk19 are part of the FEAR network that
activates Cdc14 in early anaphase, the observed midzone defects
could be solely indirect. Several lines of evidence argue against this
hypothesis. First, induced activation of Cdc14 in the esp1 mutant did
not restore normal midzone localization of Ase1.16 Second, cells that
lack the FEAR component SPO12 have normal midzone localization
of Ase1 (our unpublished data). It is important to note that spo12Δ
cells have defective Cdc14 release similar to that seen in the slk19Δ
mutant.39 Thus, Esp1 and Slk19 must have a FEAR-independent
role in midzone assembly. A hint as to the function of these two
proteins comes from our observation that Ase17A can rescue the
midzone assembly but not the spindle stability defects of slk19Δ cells
(our unpublished data). We therefore propose that midzone-associ-
ated Esp1 and Slk19 ensure local efficient dephosphorylation of Ase1
by Cdc14 and, thus, contribute to proper midzone assembly.
Similar to budding yeast, fission yeast separase/Cut1 localizes to
the anaphase spindle during initial extension, in a securin/Cut-2-
dependent manner.40 In a recent report separase was observed to
localize to the anaphase spindle in C. elegans embryos.41 It is possible
that the role of separase in regulating anaphase spindle function is
The spindle midzone, defined by the overlap of interpolar MTs
in anaphase, is critical for spindle elongation and cytokinesis. The
midzone forms in early anaphase, in parallel with the initiation of
chromosome segregation and the reduction in MT dynamics. Cdc14
phosphatase, activated through the FEAR network with anaphase
onset, promotes the coordination of these different events. Direct
www.landesbioscience.comCell Cycle 285
Coordinating spindle midzone assembly with cell cycle progression
dephosphorylation of the MT bundling factor Ase1 by Cdc14 is
required to define a centered and focused midzone that can drive
continuous spindle elongation. Separase and Slk19, two FEAR
components with Cdc14-dependent midzone localization, cooperate
with Ase1 in midzone formation. How separase and Slk19 partici-
pate in midzone assembly is not clear, but their contribution is, at
least in part, FEAR-independent. Future studies will be important to
gain further insight into the molecular details and conserved aspects
of the described regulatory mechanisms.
We thank Ambra Bianco, Bengu Sezen and Ian Hagan for critical
reading of the manuscript and Johanna Roostalu for helpful discus-
sions. This work was supported by a Marie Curie Training Network
grant to A. Khmelinskii.
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286 Cell Cycle 2008; Vol. 7 Issue 3