The EMBO Journal Peer Review Process File - EMBO-2012-82292
Manuscript EMBO-2012-82292
The Mitotic Exit Network and Cdc14 phosphatase initiate
cytokinesis by counteracting CDK
Alberto Sanchez-Diaz, Pedro Nkosi, Stephen Murray, Karim Labib
Corresponding author: Karim Labib, Cancer Research U.K.
Review timeline:
Submission date:
Editorial Decision:
Additional correspondence
Additional correspondence
Re-submission:
Accepted:
3 November 2011
22 December 2011
23 December 2011
7 February 2012
07 June 2012
17 July 2012
Transaction Report:
(Note: With the exception of the correction of typographical or spelling errors that could be a source of ambiguity,
letters and reports are not edited. The original formatting of letters and referee reports may not be reflected in this
compilation.)
1st Editorial Decision
22 December 2011
We have now finally heard back from all three referees who agreed to take a look at your
CDK/MEN/cytokinesis manuscript (EMBOJ-2011-80031). I apologize that it took so
embarrassingly long to have it reviewed, but it was initially quite difficult to find a sufficient
number of suitable experts ready to review the study at this time.
The feedback we have now received, I am afraid to say, provides insufficient support for us to
consider this study further for publication. While referee 1 is cautiously leaning to a more favorably
recommendation, both referees 2 and 3 indicate major reservations. These are based on a number of
specific concerns with the experimental evidence and its presentation, which I will not repeat in
detail here, but importantly also on overriding conceptual issues. In particular, referee 3 considers
your present results in budding yeast in many aspects merely confirmatory of previously reported
data from other systems, thus not offering a sufficient advance for publication in a broad general
journal. Referee 2 furthermore points out serious concerns with the employed experimental settings,
and questions the wider relevance of the conclusions derived from its use.
With these critical opinions and recommendations of trusted and knowledgable referees in this field,
I hope you understand that we will not be able to offer publication of a revised manuscript - in light
of our high submission number we can really only move forward with those manuscripts that are
met with above-average referee enthusiasm already upon initial review. I am sorry that I cannot be
more positive on this occasion but do hope that you will at least find the detailed comments and
suggestions of the expert reviewers helpful.
With best regards,
Editor
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The EMBO Journal
Referee reports:
Referee #1 (Remarks to the Author):
Review
In this study the authors set out to investigate how Clb-CDK inactivation affects cytokinesis in the
budding yeast Saccharomyces cerevisiae.
This reviewer finds the paper well organized, clearly written and containing novel findings.
However few additional controls should be provided to fully support the authors' conclusions.
In detail:
1. Supplementary figure 1: the authors state that "the bulk population of Cdc14 remains in the
nucleolus" in GAL-SIC1 cells arrested in nocodazole. The authors should document this finding
with pictures, similarly to what was done for the wild-type cells.
2. The authors should include western blot analyses to assess changes in protein levels for both Sic1
and Myo1 in experiments where they are overexpressed or depleted, respectively. This is especially
important as the authors talk about a delay between Sic1 accumulation and cytokinesis.
3. Throughout the manuscript, the authors talk about Clb-CDK inactivation, however kinase levels
were never measured by kinase assays but inferred by looking at the localization of various
substrates. As different substrates are de-phosphorylated at different times, this reviewer believes
that it would be appropriate to show at least in one figure a correlation between CDK inactivation
and de-phosphorylation or re-localization of the proteins the authors use to infer Clb-CDK kinase
activity.
4. At page 11 of the manuscript authors interpret their results in the following manner: " this
suggested that cytokinesis might be occurring more slowly or less efficiently for some reason, when
driven by inactivation of mitotic CDK in the absence of the normal events of anaphase". That kinase
inactivation per se does not suffice when an appropriate, counteracting phosphatase is not activated
is a very well established concept. As such, this reviewer was expecting a comment on phosphatase
activity and to see whether Cdc14 inactivation affected the observed phenotype. More specifically,
Figure 1C should include a panel with images of GAL-SIC1 cdc14 mutant cells.
5. Figure 7A: 20% of cells of the control strain have already divided cytoplasms at time point zero.
How was the nocodazole arrest assessed in these strains? Taking this into consideration, this
reviewer wishes to see this experiment in cells that are completely arrested. Otherwise conclusions
could be mis-leading.
6. A possibility is that MEN activity is required for Cdc14 localization at the bud neck. Hence, it
would be feasible to assess the consequences on cytokinesis in their mutant phenotype in the
presence of a fusion of Cdc14 with bud neck components.
Referee #2 (Remarks to the Author):
In their manuscript, Sanchez-Diaz et al. describe a system of forced cytokinesis in budding yeast.
Metaphase-arrested cells undergo cytokinesis due to the premature inactivation of mitotic CDK,
although full cell separation does not occur because of a failure to activate the Ace2 transcription
factor. Cytokinesis is slow and is correlated with relocation of actin to the new bud site. Finally, this
relocation is prevented by expression of Cdc14, and is accompanied by more efficient cytokinesis.
After careful examination of the manuscript I feel that the relevance of the presented work is
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limited. The paper is based on the over-expression of a CDK inhibitor and on the consequent mitotic
exit and cytokinetic events that occur. The main conclusions of the paper (the failure to activate the
Ace2 transcription factor properly; the 'confusion' of the actin cytoskeleton; the potential role of
Cdc14 in promoting efficient cytokinesis) cannot be easily translated to what happens in a normal
cell. For instance, in a normal cell cycle actin will never be confused as bud site appearance occurs
well after contraction of the actomyosin-ring. Also, the absence of nuclear division that underlies the
lack of nuclear Ace2 accumulation is normally never observed in a cell cycle. Therefore in my
opinion these conclusions remain a description of an artificial system.
In addition to issues with its relevance, several of the experiments are inconclusive. For a number of
important claims in this paper, the necessary controls are missing. For instance, the 'confusion' of
actin as an explanation for impaired cytokinesis upon GAL-SIC1 NT-espression is merely based on
a correlation between the appearance of a bud and impaired cytokinesis. However, in the performed
experiments, bud formation is presumably always accompanied by the induction of Cln-CDK
activity, which could provide an alternative explanation for the cytokinetic defects. In addition, an
important role for cytoplasmic Cdc14 in cytokinesis is suggested, but again critical controls are
missing for the experiments on which this is based (see below for my specific comments).
Major points:
1) In the manuscript it is suggested that prolonged expression of SIC1 NT leads to 'confusion' of the
actin cytoskeleton, and that this could (help) explain the defects observed in cytokinesis. However,
the link between actin appearance around the site of the new bud, and the cytokinetic defects is
purely correlative. Bud appearance requires Cln-CDK activity, and it might very well be that this
inhibits cytokinesis, explaining the correlation. As this is one of the major claims of the paper, more
work needs to be done to show that limiting the amount of actin patches around the bud neck
inhibits cytokinesis, and that this also happens when actin relocalisation is uncoupled from an
increase in Cln-CDK activity. In my view the block in bud formation by UV illumination (figure 5c)
does not help here. Cells are obiously damaged under these conditions and it will be hard to reach
firm conclusions from the study of cytokinesis in these damaged cells. In addition, when comparing
figure 4a and 4b, it is hard to judge whether there is less actin patches around the bud neck in SIC1
NT-expressing cells. Quantification of this phenotype would have been helpful. Is the eventual
Myo-ring contraction correlated with re-appearance of actin patches?
2) The statement that 'cytoplasmic Cdc14 is a key feature of the mechanism by which the Mitotic
Exit Network promotes the onset of cytokinesis in budding yeast', should be backed up with more
data. For instance, one should show that under the experimental conditions the cdc14-BP1,2A allele
is indeed solely expressed in the cytoplasm, and that another allele that does not display this
localization (such as the cdc14-PS1,2A allele) does not restore cytokinesis.
3) A careful characterisation of the NLS-NES-GFP marker is required, as it is not clear whether this
marker switches localization during early or late anaphase. For instance, the timing of appearance of
binucleates and of the nuclear form of this marker is similar (figure 3A), and this could be
interpreted as NLS-NES-GFP being a marker of early anaphase. If this were true, one possibility
would be that in GAL-SIC1 NT cells CDK activity does not drop sufficiently (beyond the point
required for the localisation switch of the marker) to allow efficient cytokinesis. In this regard it
would be helpful to correlate the localization of this marker to spindle length and mitotic kinase
activity in the context of a normal cell cycle.
4) The results of improved cytokinesis upon co-expression of SIC1 NT and CDC14 can be
explained by increased stability of the SIC1 NT protein, as it still contains a number of CDKphosphorylation sites and it can still bind Cdc4. Can the authors exclude that increased expression of
the SIC1-mutant is causing the increased cytokinetic efficiency? Also for other experiments (for
instance, in the experiments with the cdc14-BP1,2A allele, and with the MEN ts mutants), controls
for Sic1-stability should have been included.
5) I do not share the conclusion that cytokinesis is slower in cdc14-1 cdc15-2 than in control cells,
as the entire difference in the number of divided cytoplasms could be explained by a higher
background in the control cells. If the control curve in figure 7a were to be shifted down to have a
comparable background to the cdc14-1 cdc15-2 cells at time point 0, the curves look identical. One
could also argue that the same is true for the dbf2-2 dbf20 cells, since the difference there is small
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also, and the difference between experiments is significant (compare 7a and 7b). In addition, the
authors observe a difference in the nuclear accumulation of the NLS-NES-GFP marker that could
(partially) explain delayed Myo1-ring contraction in this context. It remains unclear why the NLSNES-GFP assay is discounted on this occasion. Instead the authors resort to images of Inn1-GFP,
but this assay remains underdeveloped and conclusions based on one photograph without assessing
the timing of Inn1-relocalization seem unfounded.
Minor points:
6) It is not unexpected that cells co-expressing CDC14 do not form a new bud, as it has been shown
previously that expression of Cdc14 on its own arrests cells in G1.
7) How representative are the EM pictures of GAL-SIC1 NT-expressing cells in figure 1C and
Figure S2? In both cases pictures are chosen that do not display the premature appearance of a new
bud site, in contrast to most other pictures of these cells. Do budded cells with a new bud site (i.e.
cells as displayed in figure 1A, lower photograph) also always contain 1 nucleus only?
8) On page 10, the authors conclude from the experiments in figure 1 that 'inactivation of mitotic
CDK before anaphase is sufficient to induce actomyosin-ring dependent cytokinesis in almost all
cells, despite the apparent failure of cell division'. However, while cytokinesis is clearly myosindependent, the contribution of the actomyosin-ring is not clear. An alternative explanation for their
observations is that no ring contraction occurs, and that instead the smaller diameter of the Myo1ring is due to the deposition of septal material, ingression of the cell wall, and therefore a release of
the tension from the ring.
9) Page 21: In the Zhai et al, 2010 paper, that the authors refer to, the contribution of cytoplasmic
Cdc14 to relocalisation of relicensing factors is not addressed. This questions the validity of the
authors assumption that cytoplasmic Cdc14 is required for regulation of the NLS-NES-GFP cassette.
10) On page 11, the authors probably want to refer to figure S2B rather than figure 2B (2nd line).
Referee #3 (Remarks to the Author):
This paper makes several interesting points about the control of cytokinesis by CDK and its
antagonizing phosphatase Cdc14 in budding yeast. The rationale for these studies was to determine
if CDK inhibits cytokinesis in budding yeast as in human cells and other model eukaryotes, and
whether Cdc14 promotes cytokinesis by dephosphorylating Cdk1 targets involved directly in
cytokinesis or indirectly participates in cytokinesis by antagonizing Cdk1 activity. The first
observation is that CDK inactivation in nocadazole arrested budding yeast cells induces cytokinesis
and septation without cell separation. Cell separation does not occur because the targets of the
anaphase-specific Ace2 transcription factor including glucanases are not produced, septa are not
digested and daughter cells remain stuck together. Similar findings on this point have been reported
previously in fission yeast. Second, it is demonstrated that when cytokinesis occurs in Cdk1depleted SAC-activated cells, it is slower and abnormal, partly due to reactivation of Cln-CDK and
also partly due to the lack of mitotic exit network activity and cytoplasmic Cdc14 activity.
Overall, the findings reported in this paper are mostly novel for budding yeast and nicely knit
together observations from numerous labs, emphasizing the necessity of CDK inactivation to
cytokinesis in this organism and the role of Cdc14 in cytokinesis apart from CDK inactivation. This
may be a significant advance to the budding yeast cytokinesis field. However, it has been generally
accepted that cytoplasmic Cdc14 is required for cytokinesis in budding yeast based on the finding
that preventing Cdc14 from exiting the nucleus generates cytokinesis defects (Bembenek et al.,
2005), the finding that preventing Cdc14 from entering the nucleus partially rescued mutations in
the mitotic exit network (Molt et al., 2009), and the work in fission yeast on the same topic. I would
expect therefore that further mechanistic insight would be required to constitute a significant
advance. The idea that there is potential competition between actin structures at the end of the cell
cycle is interesting but I found the data on this point preliminary.
Major comments:
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1. The first results in the paper, presented in figures 1 and 2, could be inferred following work in
fission yeast and the results of previous studies in budding yeast. Specifically, the major
experimental paradigm, inducing cytokinesis out of spindle checkpoint arrested cells by inhibiting
Cdk1, was pioneered in fission yeast (He X, Patterson TE, and Sazer, S., PNAS, 1997; Guertin et
al., EMBO J., 2000; Dischinger et al., J Cell Sci, 2008). And, similar findings regarding the
independence of cytokinesis and resolution of the division septa via absence of Ace2 target gene
expression were also reported previously in fission yeast (Alonso-Nunez et al., MBC, 2005; Petit et
al., J Cell Science, 2005). Relocalization of Cdc14 out of the nucleolus requires Cdk1 activity in
budding yeast (Azzam et al., Science, 2004) so it would be expected that inhibition of Cdk1 activity
in SAC-arrest would not cause Cdc14 "release". Also, the MEN, analogous to the fission yeast SIN,
maintains the Cdc14 out of the nucleolus once it has been released (Molt et al., 2009; Trautmann et
al., 2001; Chen et al., 2008) so therefore without MEN activation, steady state Cdc14 localization
would be nucleolar. Although these first experiments are necessary to describe the experimental
paradigm, they could be introduced with more background and simplified. The experiments
following up septum persistence (Figure 2) seem tangential to the major points being developed and
were expected so at least placed in supplemental. In terms of the experimental paradigm, I found it
curious that overproduction of Sic1 was chosen as the sole means of inactivating CDK in all
experiments. It seems that the use of the analog-sensitive mutant or a temperature-sensitive mutant
would have simplified some of these experiments and in the former case especially, allowed more
time-lapse imaging with no need to monitor NLS-NES-GFP. Other approaches to CDK inactivation
would also be a more direct means of eliminating all cyclin-CDK activities that was the objective in
some of the later experiments. Why was only Sic1 overproduction used?
2. More live cell imaging in some experiments (figures 3-5) would be preferable so that the exact
times of ring constriction and nuclear localization of NLS-NES-GFP could be determined and
compared between various backgrounds. Also, I could not find mention in the paper of how many
cells were examined for protein localization (points on the graphs) or how many replicas of the
experiments were performed.
3. The idea that cell re-polarization might compete with the completion of cytokinesis is quite
interesting. However, I am not convinced by the data that re-polarization inhibits cytokinesis. To
inactivate CDK-Cln, which induces re-polarization of actin to a new bud site, cells were treated with
alpha factor, which apparently rescued partly the defect in cytokinesis. However, schmoo formation,
like bud formation, is dependent upon re-polarization of the actin cytoskeleton. Thus, repolarization
of the actin cytoskeleton or "confusion of the actin cytoskeleton" does not seem to be a good
explanation for the cytokinesis defects. Rather it could be just inactivation of Cln-CDK that is
important to allow cytokinesis to complete. In these experiments, more straightforward and direct
approaches of inactivating CDK or re-directing polarization would clarify. It is also possible that
cytokinesis stalls because a CDK substrate is not dephosphorylated on schedule and that it might
also be a Cln-CDK substrate, especially under these conditions. Proteomic screens have been
performed to detect Cdc14 substrates. Are there any candidates at the ring that could be examined
for phosphostatus in this experimental paradigm?
Minor comments:
1. The description of the Cdc14 mutant on page 21 could be improved to make clear that this mutant
protein is located in the cytoplasm rather than being defective in phosphorylation.
2. On page 13, the "length" of Myo1 rings is presented. Do the authors mean width?
3. The meaning of the section heading "Release of cytoplasmic Cdc14 is central to the mechanism
...." on page 18 is not understandable. Do the authors mean release of Cdc14 into the cytoplasm?
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Additional correspondence
23 December 2011
Thanks for your letter regarding our paper 'Cdc14 phosphatase induces
cytokinesis and blocks polarised growth by counteracting effects of CDK'
(EMBOJ-2011-80031). I'm sure you understand that I take the review process
very seriously. But on this occasion I feel very strongly that the key objections made by the
reviewers are either unreasonable, unfounded, or else can easily be
addressed by additional experiments, and so I'm writing to try and persuade
you to consider a revised version of our manuscript.
I¹ll firstly summarise my reply to the general comments of the reviewers,
and then I¹ll provide below a plan for new experiments that would address
each of the specific concerns raised by the three reviewers:
(i) Reviewer 1 was positive about our work and raised a series of specific
points that we would address with new experiments in a revised version, as
discussed in detail below.
(ii) Reviewer 2¹s main argument was that the relevance of our work is
limited as we are using an experimental system to drive cytokinesis by
inactivation of mitotic CDK:
³The main conclusions of the paper (the failure to activate the Ace2
transcription factor properly; the 'confusion' of the actin cytoskeleton;
the potential role of Cdc14 in promoting efficient cytokinesis) cannot be
easily translated to what happens in a normal cell. For instance, in a
normal cell cycle actin will never be confused as bud site appearance occurs
well after contraction of the actomyosin-ring. Also, the absence of nuclear
division that underlies the lack of nuclear Ace2 accumulation is normally
never observed in a cell cycle. Therefore in my opinion these conclusions
remain a description of an artificial system.²
I¹m sorry to say that this argument completely misses the point of our paper
for two reasons.
- firstly, we wanted to distinguish the role of mitotic CDK from the role of
the mitotic exit network (MEN), and this issue could only possibly be
addressed via the kind of experimental system described in our manuscript
(the previous literature argues that MEN is required for actomyosin ring
dependent cytokinesis independently of CDK inactivation, and we show that
reversing CDK phosphorylations is central to the action of the MEN during
cytokinesis). It makes little sense to dismiss our system as Oartificial¹,
therefore, when such an experimental system was essential if we were to
address the question.
- secondly, we wanted to understand why polarised growth and budding never
normally occurs before cytokinesis, when mitotic CDK is inactivated during
late mitosis. Previous work showed that experimental inactivation of
mitotic CDK causes reactivation of G1-CDK and re-budding, and we show that
cytokinesis also occurs under such conditions, but is slow as budding
competes with ring contraction. Obviously we agree with the reviewer that
cells normally manage to delay budding until after cytokinesis, but the
interesting and unanswered question is Ohow?¹, and this can only be
addressed with an experimental system such as the one we employ. Our
manuscript provides the answer Cdc14 counteracts G1-CDK until cytokinesis
has been completed, when growth controls take over and limit accumulation of
Cln cyclins until late G1-phase. Therefore, the reviewer¹s criticism makes
no sense whatsoever and completely misses the point we are not studying a
phenomenon particular to our experimental system, but rather are providing
an experimental system to address the issue of how cells normally prevent
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premature polarisation of growth before cytokinesis.
The reviewer also comments on our Ace2 data, but in this case the data are
only being used to validate our system, illustrating that CDK-inactivation
drives cytokinesis but not cell separation. On this point we agree with
reviewer 3 that the Ace2 data could go to Supplementary Information.
Reviewer 2 also had two main objections to experiments that he/she thought
were inconclusive:
- the reviewer argues that accumulation of Cln-CDK following inactivation of
mitotic CDK might interfere with cytokinesis independently of premature
repolarisation of the actin cytoskeleton. However, our data already show
that this is very unlikely. As discussed in the last paragraph of page 19,
inactivation of mitotic CDK at 37{degree sign}C still causes re-accumulation of Cln-CDK
(Amon et al, 1993), but the key point is that re-budding is inhibited by the
mild heat-shock (Figure 7A), and this improves the efficiency of cytokinesis
as predicted by the rest of our data. We realise that we did not make this
point sufficiently clear (as reviewer 3 raised the same issue), and so we
would emphasise the point more clearly in the revised version.
(iii) Reviewer 3 thought that our manuscript ³makes several interesting
points about the control of cytokinesis by CDK and its antagonizing
phosphatase Cdc14 in budding yeast², but then noted:
³it has been generally accepted that cytoplasmic Cdc14 is required for
cytokinesis in budding yeast based on the finding that preventing Cdc14 from
exiting the nucleus generates cytokinesis defects (Bembenek et al., 2005),
the finding that preventing Cdc14 from entering the nucleus partially
rescued mutations in the mitotic exit network (Molt et al., 2009), and the
work in fission yeast on the same topic. I would expect therefore that
further mechanistic insight would be required to constitute a significant
advance.²
The key point here is that the literature is dominated by the idea that the
Mitotic Exit Network drives cytokinesis independently of CDK inactivation.
For example, the recent paper by Meitinger et al (2011) in Genes and
Development (25, 875-888), states in the abstract that ³The mitotic exit
network (MEN) pathway controls both the timely initiation of mitotic exit
and cytokinesis in budding yeast.², and the main conclusion of that paper is
that ³phosphorylation of Hof1 by Dbf2Mob1 and a functional SH3 domain are
required for AMR constriction.² (the last lines of the Results section on
page 884). In contrast, our data indicate that Cdc14 is the major effector
of the MEN for cytokinesis, and provides the key mechanistic insight that
Cdc14 acts during cytokinesis by counteracting CDK activity, both mitotic
CDK (to allow initiation of cytokinesis) and G1-CDK (to prevent premature
polarisation of growth).
Reviewer 3 also commented:
³The idea that there is potential competition between actin structures at
the end of the cell cycle is interesting but I found the data on this point
preliminary.²
As discussed in detail below, we think we can address the reviewers concerns
on this point in a revised version.
In summary, Reviewer 1 was positive and Reviewer 2¹s main criticisms were largely unfounded
(and the specific points can be addressed experimentally).
Reviewer 3 was positive, but had one major concern about mechanistic insight that in fact is
addressed by our work (the MEN controls initiation of cytokinesis largely by allowing Cdc14 to
counteract mitotic and G1 forms of CDK), and then also raised specific concerns that can be
addressed experimentally.
To consider the bigger picture and the broader significance of our work, previous studies indicated
that the initiation of cytokinesis was fundamentally different in budding yeast to animal cells, as it
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was driven directly by the MEN, and only indirectly by inactivation of mitotic CDK.
Our study shows that the situation is the other way around, and this means that future studies will be
able to focus on using the power of yeast genetics and biochemistry to characterise systematically
which steps of actomyosin-ring dependent cytokinesis are inhibited by CDK. Such work is
likely to be of relevance to studies of cytokinesis in other eukaryotes including animal cells, in
which almost all attention so far has just focused on one step that is inhibited by CDK, namely the
recruitment and activation of RhoA at the cleavage site.
For all these reasons, I very much hope that you might consider a revised version, containing
additional experimental work as discussed below, to address the specific concerns of the three
reviewers.
Additional correspondence (editor)
07 February 2012
Thanks for your patience with our reconsiderations on your manuscript, EMBOJ- 2011-80031. We
have now received feedback on referee 2's criticisms from one of the other referees, who I first
invited to take an unbiased look at these comments, and then in a follow-up communication also
confronted with your response. Based on the extra feedback we got, we realize that the overriding
criticisms of the experimental approach raised by referee 2 may not have been fully warranted, and
agree with your arguments why the approach you took would be potentially valuable to reconcile
the views on CDK inactivation and mitotic exit/cytokinesis in budding yeast.
What concerns remain however are those brought up initially by referee 3 - that your results may be
helpful to correct the confusing picture currently dominating the budding yeast literature, but may
not bring forward major new concepts beyond the budding yeast system, since similar mechanisms
have already been described in other systems/organisms. Bernd and I discussed this now in some
depth, and concluded that we could offer the following proceedings: we would grant your request
to resubmit a revised version of this manuscript, and we would agree to not send it back to referee
2. However, to gauge the general interest level, we would instead have it judged by an additional
arbitrating referee not involved initially; this replacement referee would be asked to not raise new
experimental concerns, but to provide input especially on the conceptual issues and on your
responses to them.
Should you be happy with this way of proceeding, then please go ahead and submit a carefully
revised manuscript and detailed point-by-point response whenever you are ready. We would then
turn this new submission into a revision of the earlier one.
With best regards,
Editor
The EMBO Journal
Re-submission
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We thank the reviewers for their suggestions and comments. The revised
version includes a large amount of new data that address the various points
that were raised (the new data are located in Figure 1A (i); Figure 1B (i);
Figure 3A (i); Figure 3B (i) and (ii); Figure 4B; Figure 7A; Figure 8A (ii); Figure
S1; Figure S2B; Figure S3; Figure S4C; Figure S6; Figure S7; Figure S8;
Figure S9), and we hope that the reviewers might now agree that the present
version is appropriate for publication in The EMBO Journal.
Reviewer 1
This reviewer thought that our paper was “well organized, clearly written
and containing novel findings”, but thought that “additional controls
should be provided to fully support the authors' conclusions”.
1. “Supplementary figure 1: the authors state that "the bulk population
of Cdc14 remains in the nucleolus" in GAL-SIC1 cells arrested in
nocodazole. The authors should document this finding with pictures,
similarly to what was done for the wild-type cells.”
We now provide these pictures in Supplementary Figure 2B (GALSIC1∆NT without alpha factor) and Supplementary Figure 2C (GAL-SIC1∆NT
plus alpha factor).
2. “The authors should include western blot analyses to assess
changes in protein levels for both Sic1 and Myo1 in experiments where
they are overexpressed or depleted, respectively.”
We now show an immunoblot for Sic1∆NT in Figure 1A (i). We also
provide additional blots in Supplementary Figure 7 to show that induction of
Sic1∆NT is not altered by co-expression of GAL-CDC14, GAL-cdc14BP1,2A,
or by expression in cdc14-1 cdc15-2 or dbf2-2 dbf20∆.
3.
“Throughout the manuscript, the authors talk about Clb-CDK
inactivation, however kinase levels were never measured by kinase
assays but inferred by looking at the localization of various substrates.
1 As different substrates are de-phosphorylated at different times, this
reviewer believes that it would be appropriate to show at least in one
figure a correlation between CDK inactivation and de-phosphorylation or
re-localization of the proteins the authors use to infer Clb-CDK kinase
activity.”
A previous study by Zhai et al, J. Cell Sci. (2010) 123, 3933-3943,
already showed that induction of the exact same GAL-SIC1∆NT construct that
we use in our paper leads to essentially complete inhibition of detectable ClbCDK activity in vitro, and the kinetics of Clb-CDK inhibition mirrored the
kinetics of Sic1∆NT expression that is observed in immunoblots (e.g. Figure
2A of that paper). We now show the kinetics of expression of Sic1∆NT in
Figure 1A or our paper, which correlates very well with accumulation of the
NLS-NES-GFP cassette in the nucleus (Figure 2B of our paper). We also
confirm that transient expression of Sic1∆NT is sufficient to reset replication
origins and allow a new round of DNA replication (Supplementary Figure 1;
see Noton and Diffley, 2000), confirming independently the efficiency of ClbCDK inactivation.
Desdouetts et al (EMBOJ., (1998), 17, 4139-4146) showed that
expression of Sic1∆NT correlates well with dephosphorylation of the Clb-CDK
target Pol12 (Figure 2B of their paper), and we reproduce this in our own
study (Figure 1A of our paper). We think that new data make a useful
addition to our paper, and so we are grateful to the reviewer for the comments
and hope that he/she might agree that the point is now clear.
4.
“At page 11 of the manuscript authors interpret their results in the
following manner: " this suggested that cytokinesis might be occurring
more slowly or less efficiently for some reason, when driven by
inactivation of mitotic CDK in the absence of the normal events of
anaphase". That kinase inactivation per se does not suffice when an
appropriate, counteracting phosphatase is not activated is a very well
established concept. As such, this reviewer was expecting a comment
on phosphatase activity and to see whether Cdc14 inactivation affected
the observed phenotype. More specifically, Figure 1C should include a
panel with images of GAL-SIC1 cdc14 mutant cells.”
2 We have re-written the relevant section of text (top of page 12) and
now provide the requested pictures in Supplementary Figure 8.
5.
“Figure 7A: 20% of cells of the control strain have already divided
cytoplasms at time point zero. How was the nocodazole arrest assessed
in these strains? Taking this into consideration, this reviewer wishes to
see this experiment in cells that are completely arrested. Otherwise
conclusions could be mis-leading.”
Having done these experiments many times, it is clear that almost the
entire population of cells arrests nicely in G2-M phase at 24°C or 30°C upon
treatment with nocodazole. We monitored this in various ways, always
counting at least 100 cells: for example by determining the proportion that are
large-budded without a new bud (an extra bud indicates leakage); by directly
monitoring cell division using GFP-RAS2, or by using a DNA-binding dye to
show that cells remain uni-nucleate. Very few of the arrested cells have
divided cytoplasm before inactivation of Clb-CDK, for example see the
experiment in Figure 1A (ii). Cell division upon expression of Sic1∆NT is very
specific to inactivation of CDK, as it is not seen upon equivalent incubation of
control cells (Figure 1A (ii), Control).
However, a small proportion of cells leaks past the nocodazole block
when the arrested cells are shifted to 37°C (we now quantify this
phenomenon in Supplementary Figure 3A, Control). The proportion is usually
between 10-20%, but does not increase above 20% even after prolonged
incubation at 37°C (e.g. Supplementary Figure 8). This is why a background
of 10-20% of cells with divided cytoplasm is seen in Figures 7 and 8 before
expression of Sic1∆NT, for nocodazole-arrested control cells shifted to 37°C.
This background of divided cells is not seen when MEN mutants are shifted to
37°C, as they are defective in CDK inactivation and so arrest in G2-M phase
very stably, until expression of Sic1∆NT.
The observed background of divided cells in the control strain is not a
big problem, however, as the large majority of cells only divide at 37°C if we
inactivate Clb-CDK (compare Control and cdc28-td in Supplementary Figure
3A, or Control and GAL-SIC1∆NT in Supplementary Figure 8).
In the original Figure 7A, the percentage of divided cells before expression of
3 Sic1∆NT was at the higher end of the range in such experiments, and so we
repeated this experiment and have used our new data in the revised version
of Figure 7A.
6.
“A possibility is that MEN activity is required for Cdc14 localization
at the bud neck. Hence, it would be feasible to assess the consequences
on cytokinesis in their mutant phenotype in the presence of a fusion of
Cdc14 with bud neck components.”
We agree that this would be an interesting thing to test in the next
phase of our work, but we simply didn’t have time until now. We’ve done a
number of analogous fusions involving other proteins and they are useful
when they work but un-interpretable when they don’t (as they can fail for
technical reasons, particularly with a protein like Cdc14 with highly regulated
localization to various sub-cellular locations). We have been very busy over
several months with the other experiments requested by all the reviewers, and
so we hope that reviewer 1 might agree that this particular experiment can be
kept for the future.
Reviewer 2
1. “Bud appearance requires Cln-CDK activity, and it might very well be
that this [i.e. Cln-CDK not repolarisation of actin] inhibits cytokinesis,
explaining the correlation. As this is one of the major claims of the
paper, more work needs to be done to show that limiting the amount of
actin patches around the bud neck inhibits cytokinesis, and that this
also happens when actin relocalisation is uncoupled from an increase in
Cln-CDK activity.”
Two lines of evidence argue that re-polarisation of the actin
cytoskeleton to a new bud is the key factor perturbing cytokinesis, following
inactivation of Clb-CDK and re-activation of Cln-CDK:
(a) As discussed in the last paragraph of page 19, inactivation of mitotic CDK
at 37°C still causes re-accumulation of Cln-CDK (Amon et al, 1993), but rebudding is inhibited by the mild heat-shock (Dahmann et al, 1995; also see
phase contrast images of GAL-SIC1∆NT strain in Figure 7A). Despite re 4 activation of Cln-CDK, inactivation of Clb-CDK in the absence of re-budding
induces rapid disappearance of the Myo1 ring from the bud-neck (Figure 7C,
D) and rapid completion of cytokinesis (Figure 7A, B). These data indicate
that re-activation of Cln-CDK does not impede cytokinesis under conditions
where re-budding is largely absent or greatly delayed.
(b) Whereas inactivation of Clb-CDK at 30°C in G2-M phase leads to
inefficient cytokinesis, since re-polarisation of the actin cytoskeleton to a new
bud occurs contemporaneously with actin ring formation, we now include new
data in Figure 4B to show that blocking both Clb-CDK and Cln-CDK allows
actin ring formation to occur without re-polarisation of the actin cytoskeleton
(Figure 4B (iii)), mimicking the normal situation at the end of the cell cycle
(shmoo formation only occurs post-cytokinesis and is thus too slow to inhibit
actin ring function). As discussed above, cytokinesis is rapidly completed
under such conditions (Figure 5B) and the Myo1 ring disappears rapidly from
the bud-neck (Figure 5A).
These findings raise the key question of how cells normally prevent
premature re-activation of Cln-CDK and re-polarisation of the actin
cytoskeleton, when they inactivate Clb-CDK at the end of mitosis and
assemble the actomyosin ring.
Our data in the rest of the manuscript argue
that MEN-dependent release of cytoplasmic Cdc14 is a key feature of this
mechanism.
“In addition, when comparing figure 4a and 4b, it is hard to judge
whether there is less actin patches around the bud neck in SIC1∆NTexpressing cells. Quantification of this phenotype would have been
helpful. Is the eventual Myo-ring contraction correlated with reappearance of actin patches?”
Figure 4 from the original version has now become Figure 3 in the
revised version. Following expression of Sic1∆NT and inactivation of ClbCDK, disappearance of the actin ring is indeed associated with the transient
appearance of actin patches at the bud-neck, as seen during normal cell
division. We now include a better example to illustrate this fact in Figure 3B
(ii) (75’ timepoint), and also quantify the appearance of actin patches as
requested by the reviewer (Figure 3A (i) and 3B (i)).
5 During normal cell division, cells form actin rings, then the rings
contract and disappear and are replaced subsequently by actin patches.
Following expression of Sic1∆NT in G2-M phase, actin rings persist for longer
and contract poorly due to contemporaneous re-polarisation of the actin, as
shown in the first version of our manuscript. The eventual disappearance of
actin rings is associated with the subsequent formation of actin patches at the
bud-neck, as expected.
2. “The statement that 'cytoplasmic Cdc14 is a key feature of the
mechanism by which the Mitotic Exit Network promotes the onset of
cytokinesis in budding yeast', should be backed up with more data.”
We now include new data in Figure 8A (ii), to show that expression of
the Cdc14-BP1,2A allele (Mohl et al, 2009 show this is largely cytoplasmic) is
more effective than expression of wild type Cdc14, at rescuing the cytokinesis
defect of dbf2-2 dbf20∆ cells upon inactivation of Clb-CDK.
3. “A careful characterisation of the NLS-NES-GFP marker is required,
as it is not clear whether this marker switches localization during early
or late anaphase.””In this regard it would be helpful to correlate the
localization of this marker to spindle length and mitotic kinase activity in
the context of a normal cell cycle.”
It has already been reported by others (Liku et al, 2005), that the NLSNES-GFP cassette switches localization in a similar fashion to the
endogenous Mcm2-7 proteins (Labib et al, 1999; Nguyen et al, 2000), which
only enter the nucleus at the end of anaphase upon inactivation of Clb-CDK
(we discuss this issue on page 12, lines 7-18). We now include new timelapse data in Supplementary Figure 6, directly confirming in live cells released
from Nocodazole arrest that the NLS-NES-GFP cassette only enters the
nucleus at the end of anaphase.
4. “The results of improved cytokinesis upon co-expression of SIC1∆NT
and CDC14 can be explained by increased stability of the SIC1∆NT
protein, as it still contains a number of CDK-phosphorylation sites and it
can still bind Cdc4. Can the authors exclude that increased expression
6 of the SIC1-mutant is causing the increased cytokinetic efficiency? Also
for other experiments (for instance, in the experiments with the cdc14BP1,2A allele, and with the MEN ts mutants), controls for Sic1-stability
should have been included.”
New data in Supplementary Figure 7A now demonstrate that
expression of Sic1∆T is not affected by co-expression of Cdc14 or Cdc14BP1,2A.
New data in Supplementary Figure 7B show that induction of Sic1∆NT
is equally efficient in G2-M phase at 37°C in control cells, cdc14-1 cdc15-2, or
dbf2-2 dbf20∆. Moreover, co-expression of Cdc14-BP1,2A does not increase
expression of Sic1∆NT in dbf2-2 dbf20∆ cells. The ability of Cdc14-BP1,2A to
suppress the cytokines defects of dbf2-2 dbf20∆ is thus via some other
mechanism, most likely by dephosphorylation of key CDK target proteins for
cytokinesis.
5. “I do not share the conclusion that cytokinesis is slower in cdc14-1
cdc15-2 than in control cells, as the entire difference in the number of
divided cytoplasms could be explained by a higher background in the
control cells. If the control curve in figure 7a were to be shifted down to
have a comparable background to the cdc14-1 cdc15-2 cells at time
point 0, the curves look identical. One could also argue that the same is
true for the dbf2-2 dbf20 cells, since the difference there is small also,
and the difference between experiments is significant (compare 7a and
7b).”
With respect, the reviewer’s point is simply not true. If the control curve
in the original Figure 7A had been superimposed upon that of cdc14-1 cdc152, it would have been apparent at the 60’ time-point that about 10% of cdc141 cdc15-2 cells had divided, compared to about 35% of control cells. So our
conclusion that cytokinesis is slower in cdc14-1 cdc15-2 was valid.
To emphasise this point, we have now repeated the experiment and
included the new data in Figure 7A of the revised version. Once again, it is
clear that cytokinesis is slower in cdc14-1 cdc15-2 (Figure 7A), despite
comparable expression of Sic1∆NT (Supplementary Figure 7B).
7 “In addition, the authors observe a difference in the nuclear
accumulation of the NLS-NES-GFP marker that could (partially) explain
delayed Myo1-ring contraction in this context. It remains unclear why
the NLS-NES-GFP assay is discounted on this occasion. Instead the
authors resort to images of Inn1-GFP, but this assay remains
underdeveloped and conclusions based on one photograph without
assessing the timing of Inn1-relocalization seem unfounded.”
The point is that re-localisation of Inn1 to the bud-neck during late
anaphase requires inactivation of Clb-CDK, but is independent of MEN and
Cdc14 (Meitinger et al, 2010). Consistent with this fact, we have inactivated
Clb-CDK in G2-M phase and found that re-localisation of Inn1-GFP to the
bud-neck does not require MEN or Cdc14. The data are now included in
Supplementary Figure 9, including quantification as requested by the
reviewer, and the data show that Inn1-GFP actually accumulates at the budneck in more cells in the cdc14-1 cdc15-2 strain compared to the control, due
to delayed progression through cytokinesis. This is a very important control
for our study, as it indicates that inactivation of Clb-CDK per se is not
defective in the cdc14-1 cdc15-2 strain.
In contrast to the behavior of Inn1, the nuclear accumulation of the
NLS-NES-GFP cassette is delayed in the cdc14-1 cdc15-2 strain at 37°C
upon inactivation of Clb-CDK, indicating that the defect in cytokinesis upon
the inactivation of Clb-CDK correlates with defective regulation of Cdc14
targets (Zhai et al, 2010, previously showed that nuclear localization of Mcm27 upon inactivation of Clb-CDK by Sic1∆NT requires Cdc14).
In conclusion, therefore, we didn’t discount the NLS-NES-GFP data (it’s
discussed on page 21, lines 10-22), and our point was precisely that defective
accumulation of NLS-NES-GFP in the MEN mutants after Clb-CDK
inactivation suggests that defects in dephosphorylating Cdc14 targets are
likely to underlie the cytokinesis defects.
6. “It is not unexpected that cells co-expressing CDC14 do not form a
new bud, as it has been shown previously that expression of Cdc14 on
its own arrests cells in G1.”
Visintin et al (1998) reported that expression of GAL-CDC14 in
8 asynchronous cell cultures inhibited budding, and we now cite this work on
page 18, line 2. Our data indicate that one important role of cytoplasmic
Cdc14 during cytokinesis is to block re-budding upon inactivation of Clb-CDK,
thus allowing efficient progression through cytokinesis.
7. “How representative are the EM pictures of GAL-SIC1∆NT-expressing
cells in figure 1C and Figure S2? In both cases pictures are chosen that
do not display the premature appearance of a new bud site, in contrast
to most other pictures of these cells. Do budded cells with a new bud
site (i.e. cells as displayed in figure 1A, lower photograph) also always
contain 1 nucleus only?”
We arrested control and GAL-SIC1∆NT in G2-M phase with
nocodazole, and used electron microscopy to examine serial sections across
the bud-neck of 30 control cells and 30 cells expressing Sic1∆NT. All these
cells were seen to have a single nucleus (we now make this clear on page 10,
lines 21-22). The EM data showed that 13 of the GAL-SIC1∆NT cells also had
a new bud that could be seen in some of the same EM sections as the original
bud-neck (mentioned on page 11, lines 1-2), and examples are now shown in
Supplementary Figure 4C. The other cells probably had new buds too, but we
focused our analysis of the EM data on serial sections going across the
original bud-neck. This meant that we would have missed any new buds that
were located above or below the level of the original bud-neck in our z-series
(we looked at contiguous series of z-sections across the old bud-neck, but
these did not always go across the whole body of the cell).
8. “On page 10, the authors conclude from the experiments in figure 1
that 'inactivation of mitotic CDK before anaphase is sufficient to induce
actomyosin-ring dependent cytokinesis in almost all cells, despite the
apparent failure of cell division'. However, while cytokinesis is clearly
myosin-dependent, the contribution of the actomyosin-ring is not clear.
An alternative explanation for their observations is that no ring
contraction occurs, and that instead the smaller diameter of the Myo1ring is due to the deposition of septal material, ingression of the cell
wall, and therefore a release of the tension from the ring.”
9 Our data in Figures 2-5 show that cytokinesis upon inactivation of ClbCDK in G2-M phase cells is impeded by re-polarisation of the actin
cytoskeleton to a new bud, dependent upon re-activation of Cln-CDK.
Cytokinesis is faster if Clb-CDK is inactivated under conditions where rebudding cannot occur, and the time-lapse data in Figure 5C and Figure 7C
show that this is associated with contraction of the Myo1 ring. The literature
already shows clearly that ring contraction and septum formation are
intimately connected – our point is that inactivation of mitotic CDK drives
both.
9. “Page 21: In the Zhai et al, 2010 paper, that the authors refer to, the
contribution of cytoplasmic Cdc14 to relocalisation of relicensing
factors is not addressed. This questions the validity of the authors
assumption that cytoplasmic Cdc14 is required for regulation of the
NLS-NES-GFP cassette.”
Zhai et al, 2010 showed that nuclear localization of Mcm4 requires
Cdc14, upon inactivation of Clb-CDK by Sic1∆NT (we cite this on page 21,
lines 20-22). The NLS-NES-GFP cassette is derived from Mcm2 and Mcm3
(Liku et al, 2005), which are responsible for the nuclear localization of Mcm4
upon inactivation of Clb-CDK (Labib et al, 1999; Nguyen et al, 2000), and the
localization of NLS-NES-GFP mirrors endogenous Mcm2-7 (Liku et al, 2005
and our new data in Supplementary Figure 6).
We show in Figure 7C that nuclear accumulation of the NLS-NES-GFP
cassette is delayed in cdc14-1 cdc15-2, upon inactivation of Clb-CDK,
probably reflecting the role of Cdc14 in dephosphorylating key targets of ClbCDK. The re-localisation of NLS-NES-GFP upon Clb-CDK inactivation is also
delayed in dbf2-2 dbf20∆, suggesting a role for cytoplasmic Cdc14
(presumably the small pool of Cdc14 not present in the nucleolus in G2-M
phase) as Dbf2-20 are required for accumulation of Cdc14 in the cytoplasm
(Mohl et al, 2009)
10. “On page 11, the authors probably want to refer to figure S2B rather
than figure 2B (2nd line).”
No! Figure 2B showed that nuclear division and nuclear accumulation
10 of Ace2-GFP do not occur when mitotic CDK is inactivated in G2-M phase,
and this was what we were referring to.
Following a suggestion of reviewer 1 (see above), we have now moved the
relevant data to Supplementary Figure 5. The relevant part of the text in the
revised version can be found on page 11, line 15 (referring to Supplementary
Figure 5B).
Reviewer 3
This reviewer thought that our paper “makes several interesting points
about the control of cytokinesis by CDK and its antagonizing
phosphatase Cdc14 in budding yeast”, “the findings reported in this
paper are mostly novel for budding yeast and nicely knit together
observations from numerous labs, emphasizing the necessity of CDK
inactivation to cytokinesis in this organism and the role of Cdc14 in
cytokinesis apart from CDK inactivation”, but felt that “further
mechanistic insight would be required to constitute a significant
advance. The idea that there is potential competition between actin
structures at the end of the cell cycle is interesting but I found the data
on this point preliminary”.
Major comments: 1. “The first results in the paper, presented in figures 1 and 2, could be
inferred following work in fission yeast and the results of previous
studies in budding yeast.”
“Although these first experiments are necessary to describe the
experimental paradigm, they could be introduced with more background
and simplified. The experiments following up septum persistence
(Figure 2) seem tangential to the major points being developed and were
expected so at least placed in supplemental.”
Previous work with fission and budding yeasts showed that aspects of
cell division occur when mitotic CDK is inactivated (e.g. deposition of septal
material), and our data go further by showing that cell division is completed
11 under such conditions in budding yeast, and the only thing missing is cell
separation via Ace2. We agree with the reviewer that the main role of these
data in our story is to establish the experimental paradigm, which we then use
to address aspects of the underlying mechanism of cytokinesis following ClbCDK inactivation, such as role of the MEN and Cdc14, and the need to
prevent premature resumption of polarized growth. Following the reviewer’s
suggestion, we moved the original Figure 2 to Supplementary Information (as
Supplementary Figure 5) and have made appropriate changes to the text. We
are grateful to the reviewer for this suggestion as it helps to focus our
manuscript on the main points.
“I found it curious that overproduction of Sic1 was chosen as the sole
means of inactivating CDK in all experiments.” “Why was only Sic1
overproduction used?”
To initiate cytokinesis at the end of mitosis, cells have to inactivate
mitotic CDK (Clb-CDK in budding yeast). Sic1 is a specific inhibitor of ClbCDK, and there is a well-established literature to show that that expression of
GAL-SIC1 (or GAL-SIC1∆NT) inactivates mitotic CDK and thus causes
reactivation of G1-cyclins and re-budding, as well as the relicensing of
replication origins (Dohman et al (1995); Noton and Diffley (2000); Zhai et al,
2010). It was only through the use of a specific inhibitor of Clb-CDK that we
were able to discover that cells must normally have a mechanism to prevent
the premature polarisation of growth at the end of mitosis in response to the
re-activation of Cln-CDK, in order to allow efficient progression through
cytokinesis. Our data indicate that release of Cdc14 into the cytoplasm by the
MEN is a key feature of this mechanism, and we could only have made this
discovery by using a specific inhibitor of Clb-CDK.
To confirm that inactivation of total CDK by another means would drive
efficient completion of cytokinesis, as predicted by our work, we now include
new data in Supplementary Figure 3 to show that degradation in G2-M phase
of Cdc28 (the budding yeast CDK) drives the rapid onset and completion of
cell division.
2. “More live cell imaging in some experiments (figures 3-5) would be
12 preferable so that the exact times of ring constriction and nuclear
localization of NLS-NES-GFP could be determined and compared
between various backgrounds.”
We now include time-lapse data in Supplementary Figure 6, confirming
that NLS-NES-GFP only enters the nucleus at the end of anaphase, and ring
constriction begins around ten minutes later. This illustrates that the timing of
nuclear accumulation of NLS-NES-GFP is very similar to the endogenous
Mcm2-7 proteins that are also regulated by Clb-CDK (Labib et al, 1999;
Nguyen et al, 2000).
The corresponding live cell data in Figure 5C show that ring
constriction also begins around 10-12 minutes after entry of NLS-NES-GFP
into the nucleus (inactivation of Clb-CDK) following expression of Sic1∆NT in
cells arrested in G2-M phase by nocodazole, under conditions where rebudding has been inhibited. These experiments validate the use of the NLSNES-GFP in the rest of our experiments.
The other experiments in the original Figures 3-5 (Figures 2-5 in the
revised version) involved the analysis of multiple markers in single cells, and
to study the actin cytoskeleton we had to use fixed cells. For every time-point
we analysed a minimum of 100 cells, many more cells than would have been
possible in time-lapse experiments.
“Also, I could not find mention in the paper of how many cells were
examined for protein localization (points on the graphs) or how many
replicas of the experiments were performed.”
In the Methods section on page 28, lines 6-7, we state “for
quantification we examined 100 cells per sample unless specified otherwise in
the text.” The key experiments were performed multiple times over a five-year
period.
3. “The idea that cell re-polarization might compete with the completion
of cytokinesis is quite interesting. However, I am not convinced by the
data that re-polarization inhibits cytokinesis. To inactivate CDK-Cln,
which induces re-polarization of actin to a new bud site, cells were
treated with alpha factor, which apparently rescued partly the defect in
13 cytokinesis. However, schmoo formation, like bud formation, is
dependent upon re-polarization of the actin cytoskeleton. Thus,
repolarization of the actin cytoskeleton or "confusion of the actin
cytoskeleton" does not seem to be a good explanation for the
cytokinesis defects. Rather it could be just inactivation of Cln-CDK that
is important to allow cytokinesis to complete.”
Two lines of evidence argue that re-polarisation of the actin
cytoskeleton to a new bud is the key factor perturbing cytokinesis, following
inactivation of Clb-CDK and re-activation of Cln-CDK:
(a) In response to the reviewer’s concern, we now include new data in Figure
4B to show that blocking both Clb-CDK and Cln-CDK allows actin ring
formation to occur without re-polarisation of the actin cytoskeleton, mimicking
the normal situation at the end of the cell cycle. The key point is that shmoo
formation only occurs post-cytokinesis and is thus too slow to inhibit actin ring
function (we examined 287 cells with actin rings and saw that none of them
had started to polarize the actin cytoskeleton to the site of a shmoo). As
discussed above, cytokinesis is rapidly completed under such conditions
(Figure 5B) and the Myo1 ring disappears rapidly from the bud-neck (Figure
5A).
(b) As discussed in the first paragraph of page 20, inactivation of mitotic CDK
at 37°C still causes re-accumulation of Cln-CDK (Amon et al, 1993), but rebudding is inhibited by the mild heat-shock (Dahmann et al, 1995; also see
phase contrast images of GAL-SIC1∆NT strain in Figure 7A). Despite reactivation of Cln-CDK, inactivation of Clb-CDK in the absence of re-budding
induces rapid disappearance of the Myo1 ring from the bud-neck (Figure 7C,
D) and rapid completion of cytokinesis (Figure 7A, B). These data indicate
that re-activation of Cln-CDK does not impede cytokinesis under conditions
where re-budding is largely absent or greatly delayed.
These findings raise the key question of how cells normally prevent
premature re-activation of Cln-CDK, when they inactivate Clb-CDK at the end
of mitosis.
Our data in the rest of the manuscript argue that MEN-dependent
release of cytoplasmic Cdc14 is a key feature of this mechanism.
“It is also possible that cytokinesis stalls because a CDK substrate is
14 not dephosphorylated on schedule and that it might also be a Cln-CDK
substrate, especially under these conditions. Proteomic screens have
been performed to detect Cdc14 substrates. Are there any candidates
at the ring that could be examined for phosphostatus in this
experimental paradigm?”
We agree with the reviewer that de-phosphorylation of key CDK
substrates is likely to be central to the regulation of cytokinesis by the MEN
and Cdc14 (this is the main point of our paper). This is very likely to be a
highly complex and multi-faceted business, and the experimental paradigm
described in this work provides an ideal way of testing the many candidates
one-by-one, to distinguish whether their recruitment to the bud-neck is
regulated by the MEN or by CDK inactivation (or both). Key cytokinesis
proteins such as the formin Bni1 and the IQGAP protein Iqg1 are known to be
CDK targets, and our work also points to the need to study how Cln-CDK
activity is either restrained or antagonised by Cdc14 activity, in order to
prevent the premature resumption of polarized growth. Several labs are
currently extending further the list of potential substrates for Cdc14 during
cytokinesis, and we hope that the reviewer might agree that the use of our
experimental system to analyse such targets will be a very interesting future
extension of our work, but is also something that is well beyond the scope of
the present manuscript.
Minor comments:
1. “The description of the Cdc14 mutant on page 21 could be improved
to make clear that this mutant protein is located in the cytoplasm rather
than being defective in phosphorylation.”
We now make clear on page 22, lines 1-3, that the cdc14-BP1,2A allele
contains mutations of basic amino acids that are located within the NLS and
are important for its function.
2. “On page 13, the "length" of Myo1 rings is presented. Do the authors
mean width?”
15 We have now changed the two occurrences of the word “length” on page 13
to “width” (lines 11 and 14).
3. “The meaning of the section heading "Release of cytoplasmic Cdc14
is central to the mechanism...." on page 18 is not understandable. Do
the authors mean release of Cdc14 into the cytoplasm?”
We have now changed the section heading to “Release of Cdc14 into the
cytoplasm…” (bottom of page 18).
16 The EMBO Journal Peer Review Process File - EMBO-2012-82292
Pre-acceptance letter
13 July 2012
Thank you for submitting a new version of your manuscript, following our earlier consultations and
discussions. The revised manuscript and your responses to the initial reports have now been assessed
by the original referees 1 and 3, as well by an additional arbitrating referee 4 (see comments
below). In light of their positive feedback, I am happy to inform you that there are no further
objections towards publication in The EMBO Journal!
There are a few minor issues to be addressed before we shall be able to send you a formal letter of
acceptance:
- please consider referee 3's remaining comments/suggestions and modify the manuscript text (and
running title?) accordingly
- please add a brief Conflict of Interest statement at the end of the manuscript text (after the Author
Contributions)
- please remove all supplementary text (legends) from the main manuscript text, and instead
combine it with all supplementary tables and figures into a single Supplementary Information PDF.
Please send us this PDF simply via email
Once we will have received and uploaded these modified files, we should then be able to swiftly
proceed with formal acceptance and production of the manuscript!
Yours sincerely,
Editor
The EMBO Journal
___________________________________
Referee #1
(Remarks to the Author)
The authors satisfactorily addressed most of mine and the other reviewers' concerns. The manuscript
has improved greatly and is suitable for publication with no additional changes. With this work the
authors have added an interesting new dimension to our understanding of how cytokinesis is
regulated.
Referee #3
(Remarks to the Author)
My concerns with the first version of the manuscript have been satisfactorily addressed. The data
concerning premature re-polariziation of the actin cytoskeleton to the new bud having an adverse
affect on cytokinesis is particularly interesting.
I have a few minor textual changes to recommend.
1) The last line of the Results. I believe the authors would prefer to say that "These data indicate that
the retention of Cdc14 in the cytoplasm is a key feature of the mechanism by which the MEN . . .
.etc. The sentence as is has the same problem of the corrected subtitle. Also, it is important to state
that it is retention, not release into.
2) Last paragraph of introduction, line 20. , "analogous to the regulation of cytokinesis by mitotic
CDK in animal cells and fission yeast."
© European Molecular Biology Organization
9
The EMBO Journal Peer Review Process File - EMBO-2012-82292
3) First sentence of Discussion, line 16 should include animal cells, fission yeast, and most other
eukaryotes.
Referee #4
(Remarks to the Author)
I am acting here as an arbitrating reviewer, and will therefore not go into the technical details. I will
focus on the two questions that I am more specifically asked.
1- Is the advance significant, also in comparison to other systems?
I personally believe that the idea that actin repolarization confuses and thereby affects cytokinesis is
an interesting and relatively new idea. Some pombe people might believe that they already showed
it, but this is actually not the case. Would it be so, pombe and cerevisiae grow in such different
manners that I believe that working this out in more than one organism would be very important to
establish the generality of the concept. Clearly, previous work in other organisms have emphasized
the importance of Cdk inactivation for cytokinesis and have rather focused on Cdk1 as an inhibitor
of the cytokinesis machinery itself. They have not so much asked how and why it is important to
inactivate Cdk1 to complete cytokinesis. The point of the results presented here is to go one clear
step further, suggesting that the problem is not only to release of the cytokinesis machinery from
inhibition, but also to inhibit other actin-based processes normally occurring at G1/S, and which
"confuse" the cytokinetic machinery. I am not sure that the reviewer 3 got that point, which is
actually very novel, interesting and potentially very important.
Thus, I have myself no hesitation about whether this work represents a significant advance. At the
conceptual level, the response is yes.
2- In my opinion, are the conceptual problems of reviewer 2 valid? Are the responses of the authors
closing the case?
In my opinion, the reviewer 2 makes one major point: Are the authors truly separating the issue of
Cln/Cdk activation on its own, from the activation of new budding events? Because this is actually
the important question. Is it really bud emergence that confuses the cytokinetic machinery, or is bud
emergence simply a correlation, the actual inhibition coming from Cln/Cdk activity acting directly
on the cytokinesis machinery independently of budding. This is actually an important and very valid
question. The risk being, though, that the truth is not either/or but that both processes, Cln activation
AND bud emergence contribute each in their own way to inhibiting cytokinesis. The main argument
of the authors in favor of budding inhibiting cytokinesis is the fact that at 37{degree sign}C bud
emergence is inhibited and cytokinesis is more efficient, while, they assume, Cln activation is not
delayed. Based on Amon et al., 1993, they might very well be right. It would be much more
convincing if one could more directly inhibit budding despite of Cln activation, without stressing
the cells as much. However, I have to admit that I do not know how this could be done. The
mechanisms by which Cln's induce budding is after all not that well understood, yet. Therefore, we
have for now to satisfy ourself with the arguments currently available, which are provided by the
authors. For the rest, the authors give quite thorough responses to the criticisms of the reviewer, and
I am satisfied with their answers.
In summary, I believe that the authors bring up an important and interesting possibility, i.e., that Cdk
inactivation may serve to protect the cytokinesis machinery from other, confusing actin polarization
signals. I find the idea very interesting and very likely, and I agree that the authors have started to
accumulate data supporting it. I do not believe that an autonomous role of Cln/Cdk1 in cytokinesis
inhibition has been excluded, and I am not convinced either that it has to be excluded, as expressed
above. To conclude, I think that this paper is innovative and deserves publication.
© European Molecular Biology Organization
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