Department of Biology, University College London, Gower Street, London WC1E 6BT, UK
* Author for correspondence (e-mail: d.mulvihill{at}ucl.ac.uk)
Accepted 25 June 2002
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Summary |
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Key words: S. pombe, cytokinetic actin ring, microtubules, spindle assembly checkpoint
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Introduction |
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In the fission yeast Schizosaccharomyces pombe, the CAR contains
two type II myosins, Myo2 and Myp2 (Marks
and Hyams, 1985; Bezanilla et
al., 1997
; Kitayama et al.,
1997
; May et al.,
1997
; Mulvihill et al.,
2000
) (reviewed in Win et al.,
2002
), as well as a number of other proteins required for its
correct placement and function (reviewed in
Le Goff et al., 1999a
;
Balasubramanian et al., 2000
).
Although other classes of myosins localise to the division plane in S.
pombe (Win et al., 2001
;
Win et al., 2002
), only the
type II myosins appear to have a role in CAR contraction. Myo2 is essential
for cytokinesis (Kitayama et al.,
1997
; May et al.,
1997
), whereas Myp2 is dispensable under normal growth conditions
(Bezanilla et al., 1997
;
Motegi et al., 1997
;
Mulvihill et al., 2000
). Why
fission yeast should have two type II myosins for cytokinesis (their only
known function) when other organisms get by perfectly well with just one
(Watts et al., 1987
;
De Lozanne and Spudich, 1987
;
Knecht and Loomis, 1987
)
remains to be determined. This is particularly puzzling given that
Dictyostelium cells lacking myosin II can undergo cytokinesis in the
complete absence of the motor protein
(Gerisch and Weber, 2000
), as
can certain strains of the budding yeast, Saccharomyces cerevisiae
(Bi et al., 1998
).
Myo2 localises to the CAR early in mitosis, but CAR contraction only begins
at the end of anaphase B/telophase when the two daughter nuclei have been
segregated to opposite ends of the cell and then returned to what will become
midpoints of the two daughter cells
(Kitayama et al., 1997).
However, the exact timing of the recruitment of Myo2 to the division plane has
yet to be determined. This is due, at least in part, to the fact that
metaphase is extremely brief in S. pombe
(Nabeshima et al., 1998
) and,
hence, the precise relationship between chromosome alignment and segregation
and CAR formation and function is difficult to establish. The onset of
anaphase chromosome separation is regulated by the anaphase-promoting complex
(APC), which determines both the loss of sister chromatid cohesion and cyclin
B degradation (reviewed in Morgan,
1999
; Zacchariae and Nasmyth,
1999
). The APC in turn receives signals that can inhibit its
activity from the spindle assembly checkpoint (SAC). This provides a mechanism
to inhibit anaphase until all sister chromatids are attached to the spindle
and properly aligned at the metaphase plate. In S. pombe, microtubule
depolymerisation also delays the onset of cytokinesis
(Alfa et al., 1990
), as well as
inhibiting the activation of MPF, an event generally considered to be
essential for cytokinesis to occur
(Satterwhite and Pollard,
1992
). The relationship between anaphase onset and CAR formation
remains to be established, as does the trigger for CAR contraction. However,
several molecules have roles in both processes. One such protein is Plo1, the
S. pombe polo-like kinase (Ohkura
et al., 1995
). Perturbing the cellular level of Plo1 can
dramatically effect both mitosis and cytokinesis, even driving CAR and septum
formation in interphase cells (Ohkura et
al., 1995
). In the budding yeast, S. cerevisiae, the
polo-like kinase Cdc5p has an essential role in activating the onset of
anaphase by both activating the APC and phosphorylating APC substrates
(Shirayama et al., 1998
;
Alexandru et al., 2001
). It
also provides the signal to activate the mitotic exit network (MEN) and thus
the eventual onset of cytokinesis (Hu et
al., 2001
; Stegmeier et al.,
2002
). The MEN equivalent in the fission yeast, the septation
initiation network (SIN), is a signal transduction pathway that lies
downstream of Plo1 (Mulvihill et al.,
1999
; Tanaka et al.,
2001
). Both Plo1 and all the components of the SIN localise to the
spindle pole body (SPB), and this association is essential for the correct
regulation of cytokinesis (reviewed in
Balasubramanian et al., 2000
;
Bardin and Amon, 2001
). The SIN
is activated by a G protein, Spg1 (Schmidt
et al., 1997
), which in its GTP-bound form activates a downstream
protein kinase, Cdc7 (Fankauser and Simanis, 1994;
Sohrmann et al., 1998
). The
GAP complex, which restores the GDP form of Spg1 is made up of two proteins,
Byr4 and Cdc16, the latter being a homologue of the budding yeast spindle
checkpoint protein, Bub2 (Fankhauser et
al., 1993
; Furge et al.,
1998
). Cdc7 function is required for the stable recruitment of
Myo2 to the CAR, but whether it directly phosphorylates Myo2 heavy chain
remains to be demonstrated (Mulvihill et
al., 2001
). Cdc7 also activates downstream kinases in the SIN,
leading to cytokinesis and the eventual formation of a septum. Previous
studies have demonstrated the existence of checkpoint mechanisms that act
through the SIN proteins to prevent septation from occurring in mitotic cells
if its progression is inhibited (Murone
and Simanis, 1996
;
Beltraminelli et al., 1999
;
Le Goff et al., 1999b
). Thus,
the SIN provides a mechanism in which communication can occur between spindle
formation, CAR formation and cytokinesis. Here we present evidence to show
that the formation of a stable CAR in S. pombe is coincident with the
onset of anaphase A. Activation of the SAC delays CAR formation by inhibition
of the localisation of Plo1 to the SPB. This in turn prevents downstream
events such as the Cdc7 localisation to the SPB and the recruitment of Myo2 to
the CAR.
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Materials and Methods |
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Fluorescence microscopy
For GFP autofluorescence microscopy, cells were fixed in 3.7% formalin for
10 minutes. DAPI staining of DNA and calcofluor staining to visualise septa
were performed according to Moreno et al.
(Moreno et al., 1991). Actin
localisation was carried out using rhodamine-conjugated phalloidin according
to Marks and Hyams (Marks and Hyams,
1985
). Cells were visualised using a Zeiss Axiophot microscope,
and images were captured via a Hamamatsu C2400-08 digital camera with C2400
controller using Openlab software (Improvision, Coventry, UK). Plo1-GFP
fluorescence intensity measurements were determined using Openlab software by
comparing GFP fluorescence at the SPB with the cytoplasmic background
signal.
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Results |
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These findings do not clarify whether the CAR forms prior to, or subsequent
to, the onset of anaphase A. To address this question the myo2-gc
allele was introduced into a genetic background in which the
cen1+ locus was tagged with GFP
(Nabeshima et al., 1998). In
asynchronous cultures, Myo2 rings were only seen in cells in which it was
possible to see two Cen1-GFP dots (data not shown). When cells were
synchronised using the cdc25-22 allele, separated centromeres just
preceded the appearance of Myo2 rings (Fig.
2A), and cells were often seen with separated Cen1 loci but no CAR
(Fig. 2B). We therefore
conclude that stable Myo2 rings form immediately following the onset of
anaphase A.
|
To confirm that Myo2 ring formation was an anaphase event and to explore
the possibility that ring formation was dependent on APC activity, cells were
arrested in mitosis by overexpressing the mad2+ gene.
Overproduction of Mad2 results in the accumulation of cells with short
metaphase spindles but which do not initiate cytokinesis
(He et al., 1997).
mad2+ was overexpressed in myo2-gc cells using
the full-strength nmt1+ promoter for 16 hours. Aseptate
cells with condensed chromatin, typical of a metaphase arrest
(Fig. 2C arrow heads), did not
possess a ring of Myo2 (Fig.
2C, left panel), demonstrating that a stable CAR forms post
metaphase. We confirmed these findings using a different method of removing
functional APC from myo2-gc cells by shut off of the essential APC
component, Lid1 (Chang et al.,
2001
). Cells lacking Lid1 arrested without a CAR (data not
shown).
Myo2 recruitment to the incipient division site does not require
actin
We next addressed the dependency of Myo2 ring formation on the actin and
microtubule cytoskeletons. First, we examined the requirement for actin by
using the actin depolymerising drug latrunculin B
(Gachet et al., 2001).
myo2-gc cdc25-22 cells were synchronised by temperature block and
released in the presence or absence of 10 µM latrunculin B, a concentration
that completely depolymerises actin (Fig.
3A,C). Whereas Myo2 was recruited to the cell equator in the
presence of latrunculin B at the same time as in control cells, it accumulated
as a diffuse punctate band at the medial cell cortex rather than forming a
distinct ring (Fig. 3D),
consistent with previous reports (Naqvi et
al., 1999
; Motegi et al.,
2000
). Interestingly, the deposition of septal material continued
in latrunculin-B-treated cells, also in a punctate manner, with the spots of
septal material corresponding to foci of Myo2
(Fig. 3E). Thus, the timing of
Myo2 recruitment to the cell equator and subsequent deposition of septal
material is actin independent but CAR assembly is actin dependent. These
results also further emphasise the intimate relationship between the CAR and
the positioning of the cytokinetic septum.
|
Myo2 recruitment to the incipient division site is delayed following
microtubule depolymerisation
We next examined the effect of depolymerising microtubules on Myo2 ring
formation. Synchronised myo2-gc cdc25-22 cells were arrested and
released in either latrunculin B, TBZ, MBC or DMSO as a solvent control. As
described above, Myo2 was recruited to the cell equator after 40 minutes in
both the control and latrunculin B cultures (albeit the rings were not formed
in latrunculin B) (Fig. 4A,B).
By contrast, microtubule depolymerisation resulted in a delay of 90 minutes in
the appearance of Myo2 rings (Fig.
4C,D). The fact that no such delay was observed in latrunculin,
together with the fact that a similar delay was observed in both MBC and TBZ,
demonstrate that this is a microtubule-specific phenomenon. The delay in the
appearance of binucleate cells in Fig.
4D is caused by the activation of the spindle orientation
checkpoint (Gachet et al.,
2001).
|
To eliminate the possibility that the delay in Myo2 ring formation was a consequence of the cdc25-22 mutation, we attempted to repeat the experiment in a strain possessing the wild-type copy of the cdc25+ gene. However, size-selected wild-type G2 cells released into TBZ do not attain the critical mass required for entry into mitosis. As an alternative, myo2-gc cells were transiently arrested in S phase using the DNA synthesis inhibitor hydroxyurea (HU) and then washed into fresh medium in the presence or absence of TBZ (Fig. 6A). Cells continue to grow in HU and therefore attain the critical mass for mitotic entry prior to release into TBZ. HU-blocked cells released into TBZ exhibited a 40 minute delay in CAR formation, as visualised by the accumulation of Myo2-GFP rings. Thus the delay in CAR formation and subsequent septation is not an effect of the cdc25-22 mutation but, rather, indicates the existence of a checkpoint mechanism acting at an early stage of mitosis.
|
The delay in CAR formation requires Mad2
There are two possible reasons why a delay in CAR formation is induced when
microtubules are depolymerised. The first is that components of the CAR are
delivered to the cell equator along cytoplasmic microtubules
(Bezanilla et al., 2000). Thus,
in the absence of microtubules, components of the CAR are localised to the
cell equator by a less efficient mechanism, such as diffusion. Alternatively,
the delay could be caused by a checkpoint mechanism that prevents CAR
formation from occurring if spindle formation is compromised. One way to
distinguish between these possibilities would be to identify proteins required
to maintain the delay. As CAR formation occurs early in anaphase, obvious
candidates are proteins involved in the spindle assembly checkpoint. We
therefore created a myo2-gc cdc25-22 strain in which the gene
encoding for the spindle assembly checkpoint protein Mad2 was replaced with
the ura4+ gene (myo2-gc cdc25-22 mad2
).
When these cells were synchronously released into mitosis in the presence of
DMSO, cytological events such as CAR formation, nuclear separation and septum
formation occurred with the same timing as in mad2+
strains (Fig. 5A-C). However,
when cells were released into TBZ (Fig.
5D) or MBC (Fig.
6E), the delay was alleviated, thus demonstrating the existence of
a Mad2-dependent checkpoint. Synchronised mad2
strains showed
a marked reduction in viability in TBZ compared with the equivalent
mad2+ strain (Fig.
5B,D). This is explained by the earlier appearance of cells with
septa and unseparated nuclei, the typical `cut' phenotype
(Hirano et al., 1986
;
Yanagida, 1998
).
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Having established that the CAR assembly checkpoint is Mad2 dependent, we
next determined whether the checkpoint was also disrupted in cells lacking
other spindle checkpoint components. The experiment was therefore repeated in
a strain bearing a mutation in the cdc16 gene, which encodes an
essential component of the GAP complex that regulates the onset of cytokinesis
and subsequent septum formation. In the absence of functional Cdc16, cells go
through multiple rounds of unregulated septum formation
(Minet et al., 1979;
Fankhauser et al., 1993
).
Cells bearing the cdc16.116 temperature-sensitive mutation were
arrested in interphase using hydroxyurea for 3 hours, then washed in fresh
medium lacking hydroxyurea and raised to the restrictive temperature of
36°C in the presence or absence of TBZ. In contrast to the delay observed
in cells incubated at the permissive temperature (data not shown), a reduced
delay was seen at 36°C, and cells went on to accumulate multiple septa
with similar timing to that observed in controls
(Fig. 6B). Thus, both Mad2 and
Cdc16 contribute to the observed delay in both CAR and septum formation upon
microtubule depolymerisation. Using similar strategies we determined that the
CAR formation checkpoint is also dependent on Zfs1, which is involved in the
fission yeast septation checkpoint
(Beltraminelli et al., 1999
)
(Fig. 6D). To determine whether
these proteins were functioning in a single pathway, we created a
myo2-gc strain lacking both zfs1+ and
mad2+. This strain demonstrated cytokinetic defects even
at 25°C and failed to synchronise normally using the cdc25-22
allele and therefore the delay in CAR formation could not be compared with
other strains. Further examination revealed synthetic lethality between the
mad2
and zfs1
alleles, suggesting that Mad2
and Zfs1 function on separate pathways (data not shown).
Actin shows a similar delay in medial recruitment in the absence of
microtubules
We next investigated whether the relocalisation of actin from the cell ends
to the cell equator occured normally in TBZ. myo2-gc cdc25-22 cells
were synchronised and released in the presence of DMSO or TBZ, and actin
imaged using rhodamine-phalloidin. Medial recruitment of actin showed a delay
similar to that seen with Myo2 - actin remaining at the cell tips longer when
microtubules were absent - with no observable Myo2 localisation
(Fig. 7A).
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We were interested to examine whether components of the medial ring, which
recruit to the cell equator prior to Myo2, were also effected by microtubule
depolymerisation. One such protein is Dmf1/Mid1, which precedes actin at the
division site and is required for the correct position and orientation of the
CAR (Chang et al., 1996;
Sohrmann et al., 1996
). A
strain was created bearing a gfp+-tagged copy of the
mid1 gene in combination with the cdc25-22 allele. Cells
were synchronised by temperature arrest and release and allowed to enter
mitosis in the presence or absence of TBZ
(Fig. 7B) or MBC (data not
shown). In control cells, Dmf1/Mid1 was exported from the nucleus to the
medial ring early in mitosis and persisted there until septation
(Fig. 7C) (Sohrmann et al., 1996
). In
the presence of the anti-microtubule drugs, however, Mid1-GFP remained in the
nucleus in
81% of cells (Fig.
7B). In those minority of cells in which Mid1-GFP did exit the
nucleus, the protein failed to form a distinct ring but rather remained as a
diffuse band at the cell cortex (Fig.
7D).
Full Plo1 SPB recruitment requires an intact microtubule
cytoskeleton
Having established that the recruitment of not only Myo2 but also actin and
Dmf1/Mid1 to the medial cortex is subject to a microtubule checkpoint, we
examined the effect of microtubule drugs on the localisation of possible
regulators of these proteins. One such protein is the polo protein
kinase Plo1, which controls both actin and Dmf1/Mid1 ring formation
(Ohkura et al., 1995;
Bähler et al., 1998
). Plo1
localises to the SPB in a cell-cycle-specific manner. SPB staining is first
visible at the start of mitosis, the intensity reducing as anaphase progresses
(Bähler et al., 1998
;
Mulvihill et al., 1999
).
plo1-gfp cdc25-22 cells
(Bähler et al., 1998
) were
synchronised by temperature shift and allowed to enter mitosis either in the
presence or absence of TBZ (Fig.
8A). Plo1 recruited to the SPB with similar timing in both
conditions. However, in TBZ a reduced amount of Plo1 was recruited onto the
SPB's, the Plo1-GFP signal being
20% of that seen in control cells
(Fig. 8B,C). Whereas in
wild-type cells Plo1 localised transiently to the SPBs, with the protein
becoming less visible as anaphase progressed, in TBZ the reduced Plo1-GFP
signal persisted long after the control signal had disappeared
(Fig. 8A). As cells began to
leak through the microtubule checkpoint and septate, this reduced level of
Plo1 persisted at the SPBs.
|
As Plo1 is a true SPB component and binds directly to SPBs rather than the
minus ends of microtubules (Mulvihill et
al., 1999), we next determined whether the recruitment of the
remaining population of Plo1 to the SPB was influenced by the spindle
checkpoint mechanism. The above experiment was therefore repeated in a
plo1-gfp mad2
cdc25-22 strain. In contrast to the situation in
a mad2+ background, Plo1 intensity in cells lacking
microtubules increased to a similar amount to the control
(Fig. 8E,F). Interestingly,
Plo1 seemed to be associated with the SPB for longer in the absence of Mad2
than in its presence and was seen to persist for longer at the SPB in DMSO
control cells, even those which had completed mitosis
(Fig. 8E), suggesting that Mad2
may have a role in regulating Plo1's association with the SPB. Consistent with
this, in plo1-gfp cells overexpressing mad2+,
Plo1 failed to recruit fully to the SPB
(Fig. 8G), with only
10%
of the normal SPB associated Plo1 intensity being observed. Plo1 is recruited
prematurely to the SPB in strains bearing the stf1.1 allele, a
dominant mutation in the gene encoding for the essential SPB component Cut12
and which suppresses the cdc25-22 mutation
(Bridge et al., 1998
;
Mulvihill et al., 1999
). No
effect on the length of the delay of CAR formation was observed in
stf1-1 (data not shown).
Finally, we investigated whether the localisation of components of the
septation initiation network, which act downstream of Plo1
(Mulvihill et al., 1999;
Tanaka et al., 2001
), were
effected by microtubule disruption. A cdc25-22 strain containing a
GFP-tagged copy of the gene encoding the Cdc7 protein kinase was synchronised
in G2 and released either in the presence of absence of TBZ. As originally
described by Sohrmann et al., Cdc7 localised to both SPBs early in mitosis but
was associated with only one pole during anaphase
(Sohrmann et al., 1998
). At
the end of mitosis, no SPB-associated Cdc7 was visible. Interestingly,
Cdc7-GFP was seen to recruit to the SPB coincident with Myo2 ring formation
(Fig. 9A). However, in TBZ
(Fig. 9B) or MBC (data not
shown), Cdc7 recruitment to the SPB was delayed to the same extent as Myo2
ring formation (i.e. SPB bound Cdc7 became visible coincident with the
appearance of Myo2 rings). These results demonstrate that the SIN components,
which act downstream of Plo1 and upstream of Myo2 activity, are also affected
by the CAR formation checkpoint.
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Discussion |
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In higher eukaryotes, regulation of type II myosins is by
post-translational modification of their light chains
(Satterwhite et al., 1992).
This does not appear to be the case in S. pombe. Both myosin II light
chains are essential for cytokinesis. Cdc4, the essential light chain
(Naqvi et al., 1999
), is a
phosphoprotein but the timing of cytokinesis unchanged in cdc4
mutants in which the phosphorylation sites are mutated
(McCollum et al., 1999
). Cells
lacking the regulatory light chain, Rlc1, grow normally at higher temperatures
but display cytokinetic defects at lower temperatures. These can be almost
totally suppressed by removing the second IQ domain within the Myo2 neck
(Naqvi et al., 2000
;
Le Goff et al., 2000
). Whether
post-translational modifications of Rlc1 are important for Myo2 function (as
is the case of myosin IIs in most non-muscle cells and in smooth muscle)
remains an open question. However, S. pombe contains no obvious
myosin light chain kinase (MLCK), and cells are insensitive to the MLCK
inhibitor ML-7 (D.P.M. and J.S.H., unpublished). Rather, recruitment of Myo2
to the CAR depends upon phosphorylation of residues within the tail domain
(Mulvihill et al., 2001
).
Proteins essential for the correct timing of CAR formation in S.
pombe are components of the SIN pathway
(McCollum and Gould, 2001).
Spg1 and Plo1 inappropriately drive ring formation when overproduced in
interphase cells (Ohkura et al.,
1995
; Schmidt et al.,
1997
). Recruitment of Myo2 to the CAR is dependent on functional
Cdc7 protein kinase (Mulvihill et al.,
2000
; Mulvihill et al.,
2001
), a key component of the SIN, which acts downstream of both
Plo1 and Spg1 (Sohrmann et al.,
1998
; Mulvihill et al.,
1999
; Tanaka et al.,
2001
). Another component of the SIN is Cdc16, the fission yeast
homologue of the spindle assembly checkpoint protein Bub2. Inactivation of
Cdc16, which forms part of the Spg1 GAP, drives CAR formation and septation
(Fankhauser et al., 1993
;
Furge et al., 1998
;
Sohrmann et al., 1998
;
Cerutti and Simanis, 1999
;
Mulvihill et al., 2000
).
In this report we have carried out a detailed analysis of the timing of
Myo2 ring formation and the factors that regulate it. Previous studies of the
timing of Myo2 ring formation (Kitayama et
al., 1997; Bezanilla et al.,
2000
; Motegi et al.,
2000
) were carried out using Myo2 driven from a heterologous
promoter, in the presence of the native protein. Using the myo2-gc
strain in which Myo2-GFP is under the control of its own promoter and is the
sole source of Myo2 in the cell we have established that Myo2 forms a stable
ring at the cell equator at the onset of anaphase. CAR formation is delayed in
response to microtubule depolymerisation, and this delay is dependent on a
functional spindle assembly checkpoint. Finally, we provide evidence to
suggest that the recruitment of regulators of CAR formation to the SPB may
provide the signal for Myo2 ring formation.
Myo2 ring formation normally occurs at anaphase onset is independent
of MPF activity
The timing of Myo2 ring formation was examined by introducing GFP-tagged
SPB and centromere markers into myo2-gc. An intact Myo2 ring was only
observed in cells that had a mitotic spindle and separated centromeres. Thus
CAR formation is coupled to anaphase onset. This finding was further supported
by the fact that myo2-gc cells arrested in metaphase by
overexpressing the SAC component Mad2 accumulated as aseptate cells lacking a
Myo2 ring, further linking CAR formation to anaphase commitment. The CAR
persists in an uncontracted state throughout the remainder of mitosis and only
contracts at the end of telophase, marking the onset of cytokinesis.
The timing of Myo2 ring formation was dependent upon the presence of a
functional microtubule cytoskeleton. Cells allowed to enter mitosis in the
presence of the antimicrotubule drugs TBZ or MBC showed a significant delay in
CAR formation. No such delay was observed in latrunculin B, although Myo2 was
not incorporated into a ring in the absence of actin. It has previously been
demonstrated that execution of the G2/M transition in the presence of TBZ has
no effect on the recruitment of Cdc2 and Cdc13 (which together form MPF in
fission yeast) to the SPB nor on chromosomes condensation
(Alfa et al., 1990). The same
study also showed that TBZ delayed the activation of MPF activity, normally
associated with mitotic entry, by 2 hours. Myo2 ring formation is also delayed
by TBZ but to a lesser extent, suggesting that CAR formation is not dependent
upon Cdc2 activity. This is not unexpected as overexpressing the polo-like
kinase, Plo1, induces CAR formation and eventual septation without an
associated increase in Cdc2 activity
(Ohkura et al., 1995
).
Using cold-sensitive tubulin mutants, CAR formation has been demonstrated
to occur independently of the presence of a mitotic spindle
(Chang et al., 1996). However,
these experiments were carried out in asynchronous cultures, so the delay in
CAR formation described in this study would not have been observed.
Beltraminelli et al. have shown that the appearance of actin rings and septa
were delayed when synchronous populations of nda2-KM52 and
nda2-KM52 zfs1
cells were incubated at the restrictive
temperature, thus demonstrating that the CAR checkpoint exists in
cold-sensitive tubulin mutants
(Beltraminelli et al., 1999
).
Thus, CAR formation is independent of Cdc2 activity and can occur in the
absence of spindle formation.
Removing microtubules effects Plo1 and Cdc7 SPB recruitment
What provides the signal for CAR formation and subsequent septation? Data
presented here suggest that the localisation of the polo kinase Plo1 as well
as Cdc7 (an essential component of the SIN pathway) to the SPBs are key
events. This is consistent with the findings that Plo1 has an essential role
in promoting actin ring formation (Ohkura
et al., 1995). In a normally dividing culture, Plo1 is recruited
to the SPB at the onset of mitosis and is, indeed, the earliest mitotic event
in fission yeast demonstrated to date
(Mulvihill et al., 1999
).
However, when cells enter M phase in TBZ, Plo1 is recruited to the SPB but to
only
20% of its normal level. Hence, a subfraction of Plo1 localises to
the SPB in the absence of MPF activity, further suggesting that this may be
one of the earlier steps in the commitment to mitosis. As with Plo1, Cdc2 and
Cyclin B localise to the SPB at the normal time in the presence of TBZ but
fail to delocalise until MPF activity increases
(Alfa et al., 1990
). Thus, MPF
may provide the signal to delocalise regulators of CAR formation from the
SPB.
Intriguingly, recruitment of Cdc7 to the SPB always precedes Myo2 ring
formation. This occurs in normally dividing cells, as well as cells in which
CAR formation has been delayed by microtubule depolymerisation. Thus,
localisation of Cdc7 to the SPB may also be an essential prerequisite for CAR
formation. We have previously shown that Cdc7 activity is required for the
recruitment of Myo2 to the CAR but whether SPB-bound Cdc7 is the active
protein remains to be discovered
(Mulvihill et al., 2001).
Importantly, Cdc7 localisation is downstream of Plo1 activity
(Mulvihill et al., 1999
).
The CAR formation checkpoint is specifically triggered by a defective
microtubule cytoskeleton
Microtubule depolymerisation in pre-mitotic cells caused a delay in the
appearance of not only Myo2 but also actin and Mid1/Dmf1. This is consistent
with the fact that, like Myo2, Mid1 rings form after centromere separation. No
Mid1 rings were observed in cells overexpressing Mad2, rather Mid1 formed a
diffuse band at the cell cortex adjacent to the nucleus (data not shown). We
were initially concerned that microtubule depolymerisation with TBZ or MBC
might inhibit CAR formation indirectly by affecting the actin cytoskeleton.
However, parallel experiments with latrunculin B showed that CAR components
appeared at the cell equator with normal timing, albeit that the CAR was not
assembled in the absence of actin. Further, actin is not depolymerised by TBZ
under our experimental conditions but remains at the cell tips. Thus, the
observed delay in CAR formation is a microtubule-specific phenomenon. An
interesting aside of the latrunculin experiments was the observation in
latrunculin-treated cells that patches of septal material formed on the
outside of the cell membrane coincident with the location of patches of Myo2
on the inside of the cell membrane. Thus, independent of actin, Myo2 appears
to link to the cell membrane and, more specifically, to the intacellular
domain of the glucan synthases that lay down the division septum.
Microtubule depolymerisation induces a Mad2-dependent delay in CAR
formation
To further characterise the TBZ-induced delay in CAR formation we attempted
to identify proteins required to maintain the delay. Because of the intimate
relationship between mitosis and cytokinesis, we looked initially at
components of the spindle assembly checkpoint. The TBZ delay was reduced in
cells lacking Mad2. However, since Mad2 is involved in microtubule-based
processes other than spindle assembly in S. pombe
(Petersen et al., 1998), we
also investigated the situation in mutants of Cdc16 which provides the link
between spindle integrity and the SIN
(Fankhauser et al., 1993
). As
with Mad2, functional Cdc16 was required to maintain the full TBZ delay. This
suggests that the delay is not caused by problems in the recruitment of CAR
components to the cell equator along microtubule tracks
(Bezanilla et al., 2000
) but,
rather, that CAR formation is downstream of the spindle assembly checkpoint.
Consistent with these findings, PtK cells injected with anti-Mad2 antibodies
are capable of forming a cytokinetic furrow independently of a pre-anaphase
spindle (Canman et al.,
2000
).
Mad2 regulates CAR formation via Plo1 recruitment to the SPB
To better understand the relationship between the SAC and CAR formation, we
examined the effect of TBZ on Plo1 recruitment in the presence and the absence
of Mad2. Strikingly, the reduction in recruitment of Plo1 to the SPBs in
response to TBZ was abolished in mad2 cells. Increasing the
cellular level of Mad2 also reduced the SPB recruitment of Plo1. The
localisation of Plo1 to the SPB is influenced by more than one SPB protein
(Mulvihill et al., 1999
), and
our findings suggest that checkpoint proteins play a direct role in this
process. Whether the TBZ-sensitive/Mad2-dependent and
TBZ-insensitive/Mad2-independent Plo1 subpopulations serve different roles
remains to be explored. However, it is interesting to note that two distinct
peaks of Plo1 activity, the first being
20% of the activity of the later,
major peak, are observed when highly synchronised cells enter M phase
(Tanaka et al., 2001
). From
the results presented here, we conclude that the Plo1 subpopulation is
involved in signalling to the CAR, the latter Plo1 being required for other
functions. The TBZ-induced delay in the association of Cdc7 with the SPB is
consistent with such a view. Cdc7 lies downstream of Plo1
(Mulvihill et al., 1999
) and
upstream of Myo2 assembly into the CAR
(Mulvihill et al., 2001
). A
diagrammatic representation of the relationship between the various elements
discussed here is shown in Fig.
10. Although there is no direct evidence to support the idea that
SPB-association equates to Plo1 activity, we see localisation as necessary to
initiate the pathway leading to both CAR formation and septation. A feedback
loop from the SIN proteins back to Plo1 as suggested by Tanaka et al. provides
a way of downregulating the pathway
(Tanaka et al., 2001
). Hence,
TBZ only induces a delay and not a true cell cycle arrest. Evidence of a link
between the Pololike kinases, the spindle assembly checkpoint and cytokinesis
was recently forthcoming from studies in budding yeast showing that the
polo-like kinase Cdc5p phosphorylates and activates Bfa1, the partner of Bub2
(Hu et al., 2001
). An as yet
unexplained feature of our model is that changes in the localisation of both
Plo1 and Cdc7 occur early in mitosis, as indeed does CAR formation (this
report). CAR contraction and septum formation on the other hand occur at the
end of mitosis, a gap of some 30 minutes in cells growing at 25°C. Further
studies are required to determine what other events must occur during this
window to terminate the sequence of events set in train by Plo1's
redeployment.
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References |
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